2015: Laparoscopic artificial insemination with frozen-thawed semen in a bitch Laparoscopic artificial insemination with frozen-thawed semen in a bitch Mushtaq A. Memon, Boel A. Fransson, Harmon A. Rogers Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Washington State University, Pullman, WA Summary Laparoscopic artificial insemination (L-AI) in a bitch is described. Breeding management of a six-year-old Rottweiler bitch was accomplished by blood progesterone and luteinizing hormonal assays. For L-AI, the bitch was anesthetized, and the surgical site prepared under aseptic techniques. Two incisions about 1.5 cm in length were made, one for the laparoscope and the other for the instrument portal. The uterus was elevated to the ventral abdomen where a 20g indwelling catheter was threaded into the body of the uterus and fed into each horn individually delivering 2.5ml of frozen-thawed semen. The incisions were closed, and the bitch discharged two hours after L-AI. The bitch’s pregnancy was confirmed by transabdominal ultrasonography. On day 60 after insemination the bitch vaginally delivered two stillborn puppies. Because of abdominal contractions without delivery of remaining fetuses, the bitch was taken to a veterinary clinic where a cesarean section was performed. Five live and three dead puppies were delivered. Laparoscopic artificial insemination provides another option for depositing frozen-thawed semen in the canine uterus. The main advantage of L-AI compared to traditional surgical AI is the quick recovery. Keywords: Laparoscope, artificial insemination, frozen-thawed semen, bitch Case presentation A six year-old female Rottweiler was presented for pre-breeding examination and artificial insemination using frozen thawed semen. Three years earlier the bitch was inseminated by transcervical insemination (TCI) but pregnancy did not occur. Two years earlier the bitch was bred by natural service resulting in four puppies delivered via cesarean section These breedings were with different sires. The bitch was current on vaccination and was negative for Brucella canis. The bitch was not on any medication and did not have any previous medical conditions. On physical examination, the bitch was bright, alert and responsive. She was in desirable body condition (47 kg; 3.5/5 BCS), her hair coat was soft and shiny, no musculoskeletal conformation abnormalities were noticed. Ears, eyes, nose and mouth were clean, clear and free of any discharge or debris. No lymph nodes were palpably abnormal. Her rectal temperature, respiratory rate and heart rate were within normal limits. Her mucous membranes were pink and moist with a capillary refill time of <2 seconds. Mammary chains were normal. A mild clear, viscous discharge from vulva was noted. The vulva was swollen and felt warm to the touch. Vaginal examination revealed a moderate stricture at the vestibule vaginal junction approximately 3 cm. from the vulvar opening. The stricture was not considered to be sufficient to prevent normal whelping. No pain was elicited with flexion and extension of all joints. Abdominal palpation was within normal limits. Neurologic examination of the cranial nerves and proprioception were within normal limits. Breeding management Blood samples were collected for progesterone (P4) and luteinizing hormone (LH) assays. Progesterone was assayed by radioimmunoassay, whereas the LH was detected by enzymeimmunoassay(Witness LH test kits, Synbiotics, Kansas City, MO). Cotton-tipped swabs were used to collect vaginal cells which were smeared on to microscope slides, stained with a commercial Romanowsky stain variant (Diff Quik, Jorgensen Labs, Loveland, CO) and evaluated microscopically. Transcervical insemination was attempted using an endoscope (Karl Storz, Tuttingen, Germany). Many vaginal folds were visualized, further characterizing estrus activity. Several attempts were made over a 15 minute period to pass a catheter through the cervix into the uterus but were unsuccessful. Clinical Theriogenology • Volume 7, Number 4 • December 2015431 Laparoscopic artificial insemination The bitch was premedicated with hydromorphone (0.1 mg/kg) and dexmedetomidine (2 mcg/kg) as a mixed intramuscular injection. Anesthesia was induced with intravenous injections of midazolam (0.2 mg/kg), ketamine (3.6 mg/kg), and dexmedetomidine (1 mcg/kg), and maintained by isoflurane. The surgical site, including the ventral abdomen from the xyphoid to the pubis was prepared using routine aseptic techniques and draped. Two ventral midline incisions were made, one for the laparoscope and one for an instrument portal, both approximately 1.5 cm in length. The visual portal was located immediately caudal to the umbilicus and the caudal instrument portal 3 cm cranial to the pubic rim. The uterus was grasped with an atraumatic tissue forceps, elevated to the ventral abdominal wall where a 5F central silicon catheter (V-Cath Picc, Neomedical, Mila International Inc, Erlanger, KY) was placed transabdominally, utilizing the 18 g over-the-needle peel-away introducer of the catheter kit. The introducer entered the body of the uterus in an oblique angle, aiding cranial advancement of the catheter. The catheter was advanced 10-15 centimeters, and 2.5ml of properly thawed and prepared semen delivered into each horn. The abdomen and its organs were free of any adhesions or obvious lesions. The incisions were closed in three layers including the abdominal wall, subcutis and intradermal. The bitch recovered uneventfully, and was free of any outward signs of pain including posturing, vocalizing and general attitude. She was discharged two hours postoperatively. No further pain medication was deemed necessary. Outcome On day 60 after insemination the bitch vaginally delivered two stillborn puppies. Because of abdominal contractions without delivery of remaining fetuses, the bitch was taken to a veterinary clinic where a cesarean section was performed. Five live and three dead puppies were delivered. Discussion To our knowledge, this is the first report on successful outcome of a laparoscopic AI (L-AI) with frozen-thawed semen in the bitch. Laparoscopic AI with fresh canine semen was reported by Silva, et al.1 In dogs, laparoscopic surgery has been associated with less pain and faster recovery, compared to open procedures.2-4 In addition, intraoperative oxidative stress,5 C-reactive protein levels,6 and hemodynamic changes7 are minimized with laparoscopy. Therefore, the minimally invasive nature of the surgery in this case may have contributed to the successful outcome. However, laparoscopic surgery entails limitations including the requirement for specialized equipment and skills. The unsuccessful attempts to catheterize the cervix in our case may have been due to nonpatency of the cervix as a result of a previous cesarian section. In addition to challenging canine breeding management, AI requires additional efforts, especially AI with frozen-thawed semen. Frozen-thawed sperm is viable for a short time, therefore intrauterine insemination is recommended. Pregnancy rates of 10-60% are reported with vaginal insemination with frozen-thawed semen.8-11 Semen deposition into the uterus via the vagina is difficult due to the unique cervical anatomy which is unfavorable for catheterization without visualization of the cervical os.1,8,9,12,13 The long canine vagina, which can be up to 29cm long in large breeds of dogs13 is another difficulty in reaching the cervix through the vagina. Due to these reasons, endoscopic catheterization or TCI is commonly used for canine artificial insemination with frozen-thawed semen. Numerous TCI techniques are reported.15-18 However, TCI requires practice and sometimes can be challenging.14,19 Surgical insemination in the uterus is another technique to deposit semen directly into the uterus.20 The main disadvantage of surgical insemination is the time required for surgical preparation and post-surgical recovery of the patient. Laparoscopic artificial insemination offers quick recovery. In the case reported, the bitch was released from the hospital two hours after L-AI. Learning points • Laparoscopic artificial insemination provides one more technique for depositing frozen-thawed semen into the canine uterus. Clinical Theriogenology • Volume 7, Number 4 • December 2015 432 • The main advantage of L-AI compared to traditional surgical AI is rapid recovery. Acknowledgements The authors wish to thank Alpine Animal Clinic, Helena, MT for referring the case to the Veterinary Teaching Hospital at Washington State University, and to Dakota Woodward for attending the case. References 1. Silva LDM, Onclin K, Snaps F, et al: Laparoscopic intrauterine insemination in the bitch. Theriogenology 1995;43:615-623. 2. Hancock RB, Lanz OI, Waldron DR, et al: Comparison of postoperative pain after ovariohysterectomy by harmonic scalpel-assisted laparoscopy compared with median celiotomy and ligation in dogs. Vet Surg 2005;34:273-282. 3. Davidson EB, Moll HD, Payton ME: Comparison of laparoscopic ovariohysterectomy and ovariohysterectomy in dogs. Vet Surg 2004;33:62-69. 4. Culp WT, Mayhew PD, Brown DC: The effect of laparoscopic versus open ovariectomy on postsurgical activity in small dogs. Vet Surg2009;38:811-817. 5. Lee JY, Kim MC: Comparison of oxidative stress status in dogs undergoing laparoscopic and open ovariectomy. J Vet Med Sci 2014;76:273-276. 6. Kjelgaard-Hansen M, Strom H, Mikkelsen LF, et al: C-reactive protein as a quantitative marker of the inflammatory stimulus of aseptic elective soft tissue surgery. Vet Clin Pathol 2013;42:342-345. 7. Hoglund OV, Olsson K, Hagman R, et al: Comparison of haemodynamic changes during two surgical methods for neutering female dogs. Res Vet Sci 2011;91:159-163. 8. Silva LDM, Onclin K, Snaps F, et al: Comparison of intravaginal and intra-uterine insemination of bitches with fresh or frozen semen. Vet Rec 1996;17:154-157. 9. Linde-Forsberg C, Strom HB, Govette G: Comparison of data from vaginal vs. uterine insemination of frozen thawed dog semen. A retrospective study. Theriogenology 1999;52:11-13. 10. Rota A, Iguer-Ouada M, Verstegen J, et al: Fertility after vaginal or uterine deposition of dog semen frozen in a tris extender with or without Equex STM paste. Theriogenology 1999;52:1045-1058. 11. Thomassen R, Farstad W, Krogenaes A, et al: Artificial insemination with frozen semen in dogs: a retrospective study. J Reprod Fertil Suppl 2001;57:341-346. 12. Wilson MS: Non-surgical intrauterine artificial insemination in bitches using frozen semen. J Reprod Fertil Suppl 1993;47:307-311. 13. Linde-Forsberg C: Intrauterine insemination in the dog using the Scandinavian transcerival catheter and a comparison with other methods. In: Concannon PW, England G, Verstegan J, et al, editors. Recent advances in small animal reproduction. Itaca (NY): International Veterinary Information Services; 2001. http://www.ivis.org/ 14. Wilson MS: Endoscopic transcervical insemination in the bitch. In: Concannon PW, England G, Verstegan J, et al, editors. Recent advances in small animal reproduction. Ithaca (NY): International Veterinary Information Services; 2003 http://www.ivis.org/ 15. Thomassen R, Sanson G, Krogenaes A, et al: Artificial insemination in dogs: a retrospective study of 10 years using a non-surgical approach. Theriogenology 2006;66:1645-1650. 16. Hayashi K, Morita R, Takuya A, et al: Evaluation of transcervical insemination using frozen semen by flexible endoscope in dogs. J Vet Med Sci 2013;75:315-318. 17. Kustritz MVR, Rice R: Retrospective evaluation of breeding management data and breeding type and correlation with successful breeding of bitches. Clin Therio 2013;5:213-219. 18. Mason SJ, Rous NR: Comparison of endoscopic-assisted transcervical and laparotomy insemination with frozen- thawed dog semen: a retrospective clinical study. Theriogenology 2014;82:844-850. 19. Pretzer SD, Lillich RK, Althouse GC: Single, transcervical insemination using frozen-thawed semen in the Greyhound: a case series study. Theriogenology 2006;65:1029-1036. 20. Brittain D, Concannon PW, Flanders JA, et al: Use of surgical intrauterine insemination to manage infertility in a colony of research German shepherd dogs. Lab Anim Sci 1995;45:404-407. Clinical Theriogenology • Volume 7, Number 4 • December 2015433 Clinical Theriogenology • Volume 7, Number 4 • December 2015 434 OMNIBLANK: