Evolution of synchronization protocols and use of fixed time artificial insemination in beef cattle in South America Evolution of synchronization protocols and use of fixed time artificial insemination in beef cattle in South America Gabriel Bó,1 Pietro Baruselli,2 Alejo Menchaca,3 Reuben Mapletoft4 1Instituto de Reproducción Animal Córdoba and Universidad Nacional de Villa Maria Cordoba, Argentina 2Department of Animal Reproduction, University of São Paulo, São Paulo, Brazil 3Instituto de Reproducción Animal Uruguay Fundación IRAUy, Montevideo, Uruguay 4WCVM, University of Saskatchewan, Saskatoon, SK, Canada Abstract As our understanding of ovarian function in cattle improved, our ability to control it also increased. Although prostaglandin F2α (PGF2α) has been used to synchronize estrus for several years, development of fixed time artificial insemination (FTAI) protocols permitted widespread application of AI for breeding management and genetic improvement in beef herds in South America. Over the last 20 years, a 10 fold increase in AI was largely due to use of FTAI. Main reasons for this growth were the possibility of obtaining acceptable pregnancy rates without estrus detection and induction of cyclicity in lactating cows that were anestrus and heifers that were prepubertal at the beginning of the breeding season. Most FTAI treatments for beef cattle in South America are based on use of progesterone (P4) releasing devices and estradiol to synchronize follicle wave emergence and ovulation, with pregnancies per AI (P/AI) ranging from 40 - 60%. These protocols are adapted for use with sexed semen with acceptable P/AI and several protocols have been developed for re-synchronization of ovulation for a second FTAI in nonpregnant animals. Most of these protocols are implemented on a regular basis, allowing producers to access high quality genetics through bull selection and to increase overall pregnancy rates during a breeding season. Furthermore, it provided practitioners involved in those programs a new source of income and diversification of their practices to activities other than regular clinical work. Keywords: Estrus synchronization, ovulation, fixed time artificial insemination, beef cattle, pregnancy rates Introduction Increasing knowledge of ovarian physiology in cattle, mainly through application of real time ultrasonography in the late 1980’s, provided approaches for effective manipulation of ovarian function. Protocols designed to control both luteal and follicular function permitted synchronization of ovulation and efficient implementation of fixed time artificial insemination (FTAI).1 Bovine practitioners around the world are now using these protocols, especially in South America.2 Over the last 20 years, use of artificial insemination (AI) in South America increased 10 fold, due largely to the use of FTAI.2 This presentation will briefly review the FTAI protocols currently used and describe evolution of this technology in South America. Synchronization of ovulation and fixed time artificial insemination protocols in beef cattle Estradiol/progesterone based treatments for fixed time artificial insemination Estradiol and progesterone (P4) treatments involve insertion of a P4 releasing device and administration of 2 mg of estradiol benzoate (EB) on random days of the cycle (day 0; to induce follicle atresia and synchronize follicular wave emergence 4 days later), PGF2α at the time of P4 device removal on days 7, 8 or 9 (to ensure luteolysis) and the subsequent application of 1 mg EB 24 hours later, GnRH or pLH 54 hours later, or 0.5 or 1 mg of estradiol cypionate (ECP) at the time of P4 device removal to synchronize ovulation.1 Another treatment instrumental in successful application of FTAI in South America is administration of equine chorionic gonadotropin (eCG) at the time of removal the P4 device,1,3 as this improves growth of dominant follicle and increases ovulation rate4,5 and circulating P4 concentrations in the subsequent luteal 255 Clinical Theriogenology • Volume 11, Number 3 • September 2019 phase in cows with postpartum anestrus.5,6 In South America, 80% of suckled cows and 50% of yearling heifers are not cycling at the beginning of breeding season in most commercial herds.7 Therefore, use of eCG has permitted increases in P/AI from 30 - 35% to 45 - 60% in Bos indicus1,4 and Bos taurus herds5,7 with high incidences of postpartum anestrus. GnRH based treatments GnRH based protocols are used widely for beef cattle in North America and Europe. Although GnRH use is limited in South America because of cost and availability of less expensive estradiol esters, there is increased interest in GnRH based protocols among practitioners, due to political pressure from the European Union to avoid estradiol treatments in EU certified herds. Most commonly used GnRH protocol is called CoSynch; GnRH is administered at the time of FTAI to synchronize ovulation.8 In general, CoSynch protocols have included insertion of a P4 device to overcome poor ovulation rates after first GnRH in heifers9 and in anestrus postpartum suckled beef cows.10 Data on the addition of eCG to GnRH/P4 based treatment protocols are more controversial, with reported improvements in P/AI in Bos indicus11 and Bos taurus cows in postpartum anestrus12 and in primiparous Bos taurus cows that were not presynchronized.13 However, no improvement in P/AI has been reported in Bos taurus cows with low incidences of postpartum anestrus and moderate to high body condition scores (BCS).14 Extending the proestrus period in FTAI protocols Protocols for FTAI that prolong the period from P4 device removal to ovulation were developed with the objective of lengthening the interval of preovulatory estradiol exposure and improving uterine function and early embryo development.15,16 This protocol is referred to as the 5 day CoSynch + P4 and reported to result in higher P/AI than 7 day CoSynch + P4 in beef cows.15,17 Main changes in this protocol are a reduced interval of presence of P4 device from 7 to 5 days, to avoid adverse effects of persistent follicles on fertility in cows not ovulating to first GnRH, and a prolonged interval from P4 device removal to GnRH treatment to lengthen proestrus and increase exposure to elevated circulating estradiol concentrations prior to ovulation.18 Higher estradiol concentrations in the preovulatory period have been associated with an increased ability of the uterus to support conceptus development19,20 and lower embryonic losses in the interval between maternal recognition of pregnancy and placental attachment.21 Because of the shorter interval between the first GnRH and induction of luteolysis in the 5 day CoSynch + P4 protocol, a single treatment of PGF2α was not effective in inducing luteolysis in beef cows that ovulated in response to GnRH;22 2 doses of PGF2α 8 -12 hours apart were required for high P/AI.23 In a large field trial with 2465 postpartum beef cows, P/AI was greater (p < 0.05) in cows receiving two PGF2α treatments, 8 hours apart (55%) than those receiving only one PGF2α (48%), with those receiving two PGF2α at the same time being intermediate (51%).16 Hence, double PGF2α given 8 - 24 hours apart seemed necessary to maximize fertility with the 5 day protocol. If farm conditions do not permit the extra handling, a double dose of PGF2α given at device removal would be an acceptable alternative, or the first GnRH may not be administered, especially in heifers (see below). Use of the 5 day CoSynch + P4 protocol has also been investigated in Bos indicus breeds in South America, with lower P/AI in suckled Nelore cows than with the conventional 8 day estradiol/P4 based protocol24. However, an important difference in this study was that 400 IU eCG was used in the estradiol/P4 based protocol but it was not used in the 5 day CoSynch + P4 protocol. To confirm the importance of eCG, we found no differences in P/AI in cycling cows treated with either 5 day CoSynch + P4 or estradiol/P4 based protocol without the use of eCG, but P/AI was higher in cows in postpartum anestrus that received 400 IU eCG at P4 device removal (5 day CoSynch + P4: 46.3%, 120/259; estradiol/P4 based: 54.5%, 151/277) than in cows treated with 5 day CoSynch + P4 but without eCG (26.8%, 71/265; p < 0.05).12 The 5 day CoSynch + P4 protocol was examined in heifers,25 with some modifications; for example, Colazo and Ambrose26 and Cruppe et al.27 reported that P/AI did not differ in heifers that did not receive GnRH at the time of insertion of a P4 device. The important issue with this 256Clinical Theriogenology • Volume 11, Number 3 • September 2019 approach is that heifers are not highly responsive to GnRH administered at random stages of the follicular wave and the 5 days of P4 are not sufficient for development of a persistent follicle; by not administering the first GnRH, a single injection of PGF2α is all that is required to induce luteal regression. Controversy still exists concerning the necessity of using one or two doses of PGF2α, when the first GnRH is administered in the 5 day protocol, with no reported differences28 and higher pregnancy rates when two doses of PGF2α were used, with intervals ranging from 6 - 24 hours.25,29 Optimal timing of FTAI remains controversial; Kasimanickam et al.28 reported higher P/AI in heifers inseminated at 56 hours after P4 device removal than at 72 hours, whereas Day25 suggested FTAI 60 - 66 hours after P4 device removal or insemination 12 hours after estrus observed using tail patches or tail paint and FTAI/GnRH to all those not in estrus by 72 hours. Expression of estrus influences P/AI in cows30 and Colazo et al.31 have reported similar findings in heifers inseminated with sexed semen, suggesting the possibility of splitting insemination times based on expression of estrus (i.e. delaying insemination in animals not detected in estrus by 72 hours after removal of a P4 device). Estradiol/P4 based protocols with extended proestrus We recently conducted a series of experiments to evaluate an estradiol/P4 based protocol with a prolonged proestrus interval, which has been named JSynch.32 The treatment consists of administration of 2 mg EB at the time of insertion of a P4 device that is removed 6 days later. A single dose of PGF2α and sometimes 300 (heifers) or 400 (cows) IU of eCG are given at device removal. All cattle receive GnRH and are FTAI, without estrus detection, 72 hours later (day 9). If estrus detection aids, such as tail paint or patches are used, the time of AI in heifers in estrus can be advanced to 60 or 66 hours after P4 device removal and only those not displaying estrus by 72 hours receive GnRH and FTAI at that time.33 Studies using real time ultrasonography and hormone determinations have shown that the growth rate of the ovulatory follicle and serum P4 concentrations in the ensuing luteal phase were greater in heifers treated with the 6-day JSynch protocol than in those treated with the conventional 7 day estradiol/P4 based protocol. Furthermore, immunohistochemistry and real-time PCR of biopsies taken on day 6 after ovulation suggested that heifers treated with the prolonged proestrus protocol had a more mature uterine environment for embryo development.34 In most studies conducted in beef Bos taurus heifers, P/AI was higher with the JSynch protocol than with the conventional estradiol/P4 protocol (Table 1).33,35 Although P/AI did not differ in a study in Bos indicus heifers,36 P/AI was higher in Nelore heifers treated with the JSynch protocol than those treated with the conventional protocol in a more recent study (Table 1).37 Table 1. Pregnancy rates in beef heifers treated with the JSynch or with the conventional estradiol based treatment protocols. Experiments - Reference JSynch Conventional p value 1 - Bó et al. 2016 (n = 583) 59.7% (175/293) 53.1% (154/290) 0.12 2 - Bó et al. 2016 (n = 208) 67.9% (70/103) 46.6% (49/105) 0.01 3 - Bó et al. 2016 (n = 555) 37.8% (104/275) 49.3% (138/280) 0.01 4 - Bó et al. 2016 (n = 2.349) 56.1% (631/1,125) 50.7% (620/1,224) 0.01 5 - Motta et al. 2016 (n = 785) 52.9% (207/391) 53.6% (211/394) 0.8 6 - Pincinato et al. 2018 (241) 62.9% (68/108) 48.1% (64/133) 0.01 Total 54.7% (1,255/2,295) 50.1% (1,236/2,426) 0.01 In Experiments 1, 2 and 3, heifers did not receive eCG at device removal. In Experiments 4, 5 and 6, all heifers received 200 (Experiment 4) or 300 IU eCG at P4 device removal. In Experiment 2, heifers were in low body condition score (4 - 6 in a 1 - 9 scale) and losing weight, whereas in all other studies, heifers were in good body condition score (6 - 7) and gaining weight. 257 Clinical Theriogenology • Volume 11, Number 3 • September 2019 Fixed time artificial insemination using sexed semen Improvements in sorting and freezing procedures with sexed semen and increasing number of sperm from 2.1 x 106 - 4 x 106 sperm per straw has created interest in the application of this technology in beef herds. The new product is called SexedULTRATM.38 Experiments with SexedULTRATM semen have been performed in beef heifers and suckled cows in South America. The overall recommendation with the conventional estradiol/P4 based protocols is to delay insemination time with sexed semen in both heifers and cows by 12 hours and to inseminate only those that have shown estrus by the time of FTAI.33,39 We performed 2 studies to evaluate P/AI in beef heifers (n = 850) and suckled cows (n = 877) treated with the JSynch protocol and inseminated with SexedULTRATM or nonsorted (conventional) semen from split ejaculates of the same Angus sires.33,40 All cattle were treated with the JSynch protocol as described above and were tail painted for estrus determination. Cattle with the paint rubbed off by 60 or 72 hours after device removal were inseminated at 72 hours, whereas those not displaying estrus by 72 hours received GnRH at that time and were inseminated at 84 hours. In heifers, the overall P/AI were 49.3% for those inseminated with SexedULTRATM and 58.3% for those inseminated with conventional semen (p < 0.05, Table 2).33 In suckled cows, the overall P/AI was 45.3% for those receiving SexedULTRATM semen and 68.3% for those inseminated with conventional semen (p < 0.05, Table 2).40 In summary, protocols designed for FTAI in cows and heifers can be adapted for sexed semen. Although, P/AI are lower than those obtained with conventional semen, delaying the time of AI or limiting AI to those showing estrus would result in P/AI ranging from 40 and 50% and often higher. Table 2. Pregnancy rates in Angus heifers and suckled cows inseminated with sexed (SexedULTRATM) or conventional semen according to the expression of estrus (tail paint reading) and time of insemination (AI) after removal of the progesterone releasing device. n Estrus 60 hours AI 72 hours Estrus 72 hours AI 72 hours No estrus at 60 or 72 hours AI 84 hours Total Beef heifers SexedULTRA (4 x 106 sperm) 426 59.0% (104/176) 45.5%a (61/134) 38.8% (45/116) 49.3%a (210/426) Conventional (25 x 106 sperm) 424 69.2% (119/172) 58.0%b (79/136) 42.2% (49/116) 58.3%b (247/424) Beef suckled cows SexedULTRA (4 x106 sperm) 435 40.8%a (93/228) 43.0%a (34/79) 56.5%a (70/124) 45.3%a (197/435) Conventional (25 x 106 sperm) 442 65.8%b (154/234) 67.9%b (57/84) 74.2%b (92/124) 68.6%b (303/442) ab Denotes differences in P/AI between sexed or conventional semen (p < 0.05). Resynchronization treatments With P/AI ranging from 40 - 60% after the first AI, producers are asking researchers and practitioners for development of protocols that would achieve higher P/AI in a breeding season or even the possibility of developing protocols in which 85 - 95% of the cows get pregnant only by AI, without the necessity of cleanup bulls. Several resynchronization protocols were investigated over the years; however, most require estrus observations or an interval of approximately 40 days between the first and second FTAI.35,41 In order to be able to inseminate nonpregnant cows as early as possible, resynchronization treatments must start earlier than pregnancy diagnosis. One protocol developed involves insertion of a used P4 device (i.e. previously used in the first synchronization) on days 14 - 16 after the first AI and its removal on day 23 when GnRH is administered. Ultrasonographic pregnancy diagnosis is performed on day 258Clinical Theriogenology • Volume 11, Number 3 • September 2019 30 and those that are non-pregnant receive PGF2α combined with either 0.5 mg ECP at that time or GnRH at the time of FTAI on day 32. P/AI for the first and second FTAI at an interval of 32 days and the overall P/AI in a field trial involving 6431 beef cows and heifers were 57, 51 and 79%, respectively.35 Two other protocols for resynchronization are called Resynch 22 and Resynch 14.41 In the Resynch 22, cows receive 2 mg EB and heifers 1 mg EB at the time of P4 device insertion on day 22 after FTAI. Pregnancy diagnosis is performed at device removal on day 30 when nonpregnant cattle receive PGF2α and ECP and are inseminated on day 32. In a recent field trial, the use of three consecutive FTAI with the Resynch 22 protocol had a similar overall pregnancy rate (87.8%, 663/755) as achieved using cleanup bulls after two FTAI using Resynch 22 (87.7%, 263/300) and greater pregnancy rate than one FTAI followed by bull exposure (77.1%, 347/450).42 In Resynch 14, the initial treatment starts 14 days after FTAI with reinsertion of a used P4 device and administration of 100 mg P4 IM.41 Pregnancy assessment is done by the evaluation of the vascularity of the CL using Doppler ultrasonography at the time of P4 device removal on day 22. Nonpregnant cattle receive PGF2α and ECP at that time and are inseminated on day 24. In a recent study, similar P/AI were observed for Resynch 22 and Resynch 14 groups following the first FTAI (48% versus 53%; p = 0.57) and resynchronization (56 versus 51%; p = 0.37), respectively. However, Resynch 14 reduced the interval between FTAI, which resulted in a 21-day pregnancy rate of 87.5% compared to 66% with Resynch 22.43 In conclusion, with the existing resynchronization programs it is now possible to breed beef cows exclusively with FTAI, eliminating the need for estrus detection and cleanup bulls. Application of fixed time artificial insemination in South America South American countries represent the largest beef herd in the world. In 2016, Latin American countries had approximately 359 M cattle (accounting for 24% of the world's cattle population) and produced 15.1 M tons of beef.2 Of the main beef producing countries in South America, Brazil (60.8% of the total), Argentina (14.7%) and Uruguay (3.3%) are also prominent in development and application of assisted reproductive biotechnologies. Through training and continuing education programs, many of which one or more of the authors have organized or participated in, these technologies are being used increasingly in other South American countries. It is through these efforts, in partnership with the pharmaceutical industry, that the ruminant reproductive revolution in South America has become widespread. Brazil and Argentina, in particular, are not only very active in research but also in continuing education and postgraduate student training in reproductive biotechnology. In just one program, the Specialization in Bovine Reproduction at the Institute of Animal Reproduction in Cordoba Argentina (IRAC), more than 1200 practitioners from most South American countries have taken postgraduate training since 2002. Most beef herds in Brazil and Paraguay are composed of Bos indicus or Bos indicus crosses, whereas Argentina and Uruguay often have more Bos taurus breeds. Bos indicus-influenced synthetic breeds have increased in Argentina due to the recent expansion of agriculture that has pushed cattle breeding to less favorable environments. It is noteworthy that Bos indicus breeds tend to have prolonged postpartum anestrus and low body condition scores on pasture, with an increased interval from calving to conception and low pregnancy rates.45 However, in a study involving 21,329 suckled Bos taurus cows and heifers examined by ultrasonography at the beginning of the breeding season in commercial beef farms in Uruguay, 80 - 90% of cows and 40% of heifers are not cycling (i.e. without a CL) at the beginning of the breeding season.7 Thus, in these pasture-based cow calf production systems, synchronization protocols are not only necessary to produce pregnancies by AI during a short breeding season, but also to induce cyclicity in cows in anestrus that would likely not get pregnant if not treated.46 These are the main reasons why FTAI has been incorporated into the breeding schemes of most progressive producers.1,35 Breeding objectives have been to inseminate early in the breeding season, early ultrasonographic pregnancy diagnosis and re-insemination of nonpregnant cattle as soon as possible.41 As was mentioned previously, because estradiol preparations have been available in South America, most FTAI protocols include estradiol as a means of synchronizing follicle 259 Clinical Theriogenology • Volume 11, Number 3 • September 2019 wave emergence and ovulation.1 As herd cyclicity and body condition scores are usually low, progestin devices and eCG are usually included in the synchronization protocols.1,3,6,35 Protocols of 7, 8 or 9 days progestin treatment have resulted in similar pregnancy rates, and shortened progestin protocols with a lengthened proestrus involving either estradiol or GnRH have been used with success.41 FTAI is increasing the use of AI, especially with Bos taurus sires on Bos indicus cows in Brazil, Paraguay and the northern part of Argentina. In Brazil, the use of FTAI increased from ~ 1 M protocols in 2005 (11% of all AI) to 10.5 M protocols in 2015 (77% of all AI), and a further increase to > 11 M FTAI in 2016 (Figure 1).2 This represents more than a 10 fold increase in the use of AI in the Brazilian beef herd. In Argentina, there were < 200 x 103 FTAI protocols in 2002 and this has increased to ~ 2.5 M in 2015 and > 3 M in the last breeding season. This accounts for > 90% of inseminations done in beef cattle in Argentina, which is about 15% of the beef breeding females in the country (Figure 1). Uruguay and Paraguay have experienced similar trends, with 300 x 103 FTAI in Uruguay and 500 x 103 FTAI in Paraguay, representing ~ 10% of their breeding females. In total, > 15 M cattle were inseminated by FTAI in these four countries over the past year. The adoption of this technology has been suggested to be an excellent example of a technological change in the production sector emerging from scientific developments in the academic sector.2 Figure 1. Evolution of the application of FTAI in Uruguay, Brazil and Argentina. Data were calculated based on official semen and hormone sales in the three countries. Although the evolution figures for Paraguay are not available, the number of cows FTAI in the 2017 breeding season is included for reference and comparison. Impacts of use of fixed time artificial insemination in commercial beef herds One of the main advantages of implementing FTAI programs in a beef herd is that more cows can be impregnated earlier in the breeding season to genetically improved bulls, resulting in heavier weaning weights (reviewed46). Between 40 and 60% of the cows could become pregnant on the first day of the breeding season, resulting in a higher number of cows calving at the beginning of the calving season. Their calves will be older and heavier at weaning, and the use of genetically improved bulls will also result in heavier calves at weaning. Impact of FTAI is equally effective in various management systems in Argentina and Brazil.45,46 A recent study47 compared weaning weights and kilograms of beef per cow using estradiol/P4 based protocols with FTAI or bull exposure versus traditional natural service. At the beginning of the breeding season, cows were randomly allocated to three treatment groups. Cows in two groups received the conventional estradiol/P4 based treatment with eCG at device removal and then half of those cows were FTAI and the other half directly exposed to 5% bulls for 90 days immediately after P4 device removal. Cows in the control group were exposed directly to 5% bulls without synchronization treatments at the same time that devices were removed in two synchronized groups. Cows in FTAI group were also exposed to 5% bulls 10 days after FTAI for the remainder of the breeding season. Pregnancy diagnosis was performed by four sequential ultrasonographic examinations every 30 days. Pregnancy rates after the first 25 days and at the end of the breeding season were significantly higher in cows in 260Clinical Theriogenology • Volume 11, Number 3 • September 2019 the two synchronized groups than in those in natural service (p < 0.05; Table 3). However, the most important finding was the difference in weaning weights. Calves in FTAI group were 27.6 kg heavier and those in the synchronized group that were directly exposed to bulls were 19.1 kg heavier than those in the natural service group. Furthermore, cows in the FTAI group produced 69 kg more of beef at weaning than those exposed to natural service (Table 3). Changes in calving patterns and weaning weights have been considered the main reason why the most progressive cow/calf producers in South America have adopted this technology to improve profits.2,45,46 Table 3. Pregnancy rates during the first 25 days and at the end of a 90 day breeding season and weaning weights of calves produced, in beef cows that were synchronized with an estradiol/P4 protocol and exposed to bulls (Synch with bulls), or FTAI and then exposed to bulls for the remainder of the breeding season (FTAI plus bulls), or directly exposed to bulls without any synchronization treatment (natural service). 25 day Pregnancy rate (%) Overall Pregnancy rate (%) Calf weaning weights (kg) Synch with bulls 54.1% (60/111)b 94.6% (105/111)b 204.8 ± 28.8b FTAI plus bulls 58.1% (61/105)b 96.2% (101/105)b 216.8 ± 19.8c Natural Service 25.5% (35/137)a 83.9% (115/137)a 191.2 ± 18.2a abc Denotes differences within pregnancy rates or weaning weights (p < 0.05). Conclusion Ability to control follicular wave emergence and ovulation in cycling and noncycling cattle has allowed successful implementation of FTAI in beef herds in South America. Treatments with GnRH or estradiol and P4 releasing devices made FTAI possible in beef cattle. These treatments are practical and easy to perform by the farm staff and are efficient and useful for inducing cyclicity at the beginning of the breeding season. Practitioners in most South American countries are using these protocols, which has facilitated the use of AI in beef herds and application of other assisted reproductive technologies. Currently, FTAI procedures account for > 85% of all AI done in beef cattle in Argentina, Brazil, Uruguay, and Paraguay. Successful application of FTAI in South America is resulting in the dissemination of new and improved genetics and increased reproductive performance in all classes of cattle, with a corresponding increase in economic activity. Conflict of interest There are no conflicts of interest to declare. Acknowledgement Research was supported by FONCYT (PICT 2017-4550), Universidad Nacional de Villa María, Zoetis and Sexing Technologies from Argentina, and ANII, Syntex Uruguaya SA and Fundaciba from Uruguay. References 1. Bó GA, Baruselli PS, Mapletoft RJ: Synchronization techniques to increase the utilization of artificial insemination in beef and dairy cattle. Anim Reprod 2013;10:137-142. 2. Mapletoft RJ, Bó GA, Baruselli PS et al: Evolution of knowledge on ovarian physiology and its contribution to the widespread application of reproductive biotechnologies in South American cattle. Anim Reprod 2018;15 (Suppl 1):1003-1014. 3. Baruselli PS, Reis EL, Marques MO, et al: The use of treatments to improve reproductive performance of anestrus beef cattle in tropical climates. Anim Reprod Sci 2004;82-83:479-486. 4. Sá Filho MF, Ayres H, Ferreira RM, et al: Equine chorionic gonadotropin and gonadotropin-releasing hormone enhance fertility in a norgestomet-based, timed artificial insemination protocol in suckled Nelore (Bos indicus) cows. Theriogenology 2010;73:651-658. 261 Clinical Theriogenology • Volume 11, Number 3 • September 2019 5. Núñez-Olivera R, de Castro T, García-Pintos C, et al: Ovulatory response and luteal function after eCG administration at the end of a progesterone and estradiol-based treatment in postpartum anestrous beef cattle. Anim Reprod Sci 2014;146:111-116. 6. Baruselli PS, Sá Filho MF, Ferreira RM, et al: Manipulation of follicle development to ensure optimal oocyte quality and conception rates in cattle. Reprod Dom Anim 2012;47 (Suppl 4):134-141. 7. Menchaca A, Núñez R, Wijma R, et al: How fertility can be improved in fixed-time AI programs in beef cattle. Proc X International Symposium on Animal Reproduction, IRAC, Cordoba, Argentina, 2013; p. 103- 134. 8. Geary TW, Whittier JC, Hallford DM, et al: Calf removal improves conception rates to the Ovsynch and Co- synch protocols. J Anim Sci 2001;79:1-4. 9. Martinez MF, Kastelic JP, Adams GP, et al: The use of progestins in regimens for fixed-time artificial insemination in beef cattle. Theriogenology 2002;57:1049-1059. 10. Lamb GC, Stevenson JS, Kesler DJ, et al: Inclusion of an intravaginal progesterone insert plus GnRH and prostaglandin F2α for ovulation control in postpartum suckled beef cows. J Anim Sci 2001;79:2253-2259. 11. Pincinato D: 2012. Follicular dynamics and fertility in beef suckled cows synchronized with progesterone releasing devices and GnRH. Master of Science Thesis. Faculty of Agriculture Sciences, National University of Cordoba, Cordoba, Argentina 2012; p. 45. 12. Huguenine E, Peracchia S, Benitez R, et al: Effect of the utilization of 5-day CO-Synch protocols combined or not with eCG in suckled cows in postpartum anoestrus. Proc X Symposium on Animal Reproduction, IRAC, Córdoba, Argentina, 2013; p. 313 (abstract). 13. Small JA, Colazo MG, Kastelic JP, et al: Effects of progesterone presynchronization and eCG on pregnancy rates to GnRH-based, timed-AI in beef cattle. Theriogenology 2009;71:698-706. 14. Marquezini GHL, Mercadante VRG, Olson KC, et al: Effects of equine chorionic gonadotropin on follicle development and pregnancy rates in suckled beef cows with or without calf removal. J Anim Sci 2013;91:1216-1224. 15. Bridges GA, Helser LA, Grum DE, et al: Decreasing the interval between GnRH and PGF2α from 7 to 5 days and lengthening proestrus increases timed-AI pregnancy rates in beef cows. Theriogenology 2008;69:843-851. 16. Bridges GA, Ahola JK, Brauner C, et al: Determination of the appropriate delivery of prostaglandin F2α in the five-day CO-Synch + controlled intravaginal drug release protocol in suckled beef cows. J Anim Sci 2012;90:4814-4822. 17. Whittier WD, Currin JF, Schramm H, et al: Fertility in Angus cross beef cows following 5-day CO-Synch + CIDR or 7-day CO-Synch + CIDR estrus synchronization and timed artificial insemination. Theriogenology 2013:80:963-969. 18. Bridges GA, Mussard ML, Hesler LA, et al: Comparison of follicular dynamics and hormone concentrations between the 7-day and 5-day CO-Synch + CIDR program in primiparous beef cows. Theriogenology 2014:81:632-638. 19. Bridges GA, Day ML, Geary TW, et al: Deficiencies in the uterine environment and failure to support embryonic development. J Anim Sci 2013;91:3002-3013. 20. Binelli M, Pugliesi G, Hoeck VV, 2014: The role of proestrus on fertility and postovulatory uterine function in the cow. Animal Reproduction 2014;11:246-253. 21. Madsen CA, Perry GA, Mogck CL, et al: Effects of preovulatory estradiol on embryo survival and pregnancy establishment in beef cows. Anim Reprod Sci 2015;158:96-103. 22. Souto LA, Maquivar M, Mussard ML, et al: Fertility and luteal regression with 5-d CIDR synchronization programs in postpartum beef cows using differing luteolytic treatments J Animal Sci 2009:87 (Suppl 2):372 (abstract). 23. Kasimanickam R, Day ML, Rudolph JS, et al: Two doses of prostaglandin improve pregnancy rates to timed-AI in a 5-day progesterone based synchronization protocol in beef cows. Theriogenology 2009;71:762-767. 24. Ferraz Jr MVC, Pires AV, Biehl MV, et al: Comparison of two timed artificial insemination system schemes to synchronize estrus and ovulation in Nelore cattle. Theriogenology 2016;86:1939-1943. 25. Day ML: State of the art of GnRH-based timed AI in beef cattle. Anim Reprod 2015;12:473-478. 26. Colazo MG, Ambrose DJ: Neither duration of progesterone insert nor initial GnRH treatment affected pregnancy per timed-insemination in dairy heifers subjected to a Co-synch protocol. Theriogenology 2011:76:578-588. 27. 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Anim Reprod Sci 2016;166:133-140. 262Clinical Theriogenology • Volume 11, Number 3 • September 2019 31. Colazo MG, Macmillan K, Mapletoft RJ. Modifications of a 5-d GnRH-based timed-AI protocol to optimize fertility in Holstein heifers inseminated with sex-selected semen. Anim Reprod 2018;15(Suppl 1):1111 (abstract). 32. de la Mata JJ, Bó GA. Estrus synchronization and ovulation using protocols with estradiol benzoate and GnRH and reduced periods of insertion of a progesterone releasing device in beef heifers. Taurus 2012;55:17-23. 33. Bó GA, Huguenine E. de la Mata JJ, et al: Programs for fixed-time artificial insemination in South American beef cattle. Anim Reprod 2018; 15 (Suppl 1):952-962. 34. de la Mata JJ, Núñez-Olivera F, Cuadro F, et al: Impact of extending the proestrus length in an oestradiol and progesterone-based oestrus synchronization program on follicular and luteal development, uterine gene expression, and protein localization, and pregnancy rates in beef heifers. Reprod Fertil Dev 2018; 30:1541–1552 35. Bó GA, de la Mata JJ, Baruselli PS, et al: Alternative programs for synchronizing and resynchronizing ovulation in beef cattle. Theriogenology 2016;86:388-396. 36. Motta JCL, Colli MHA, Penteado L, et al: Pregnancy rate to FTAI in Nelore and crossbreed heifers submitted to J-Synch protocol (6 days). Anim Reprod 2016;13:401. (abstract). 37. Pincinato D, Peres LC, Lorentz L, et al: Pregnancy rates in Nelore heifers using a shortened estradiol/progesterone-based protocol that provides for a lengthened proestrus (J-Synch). Anim Reprod 2018;15:350 (abstract) 38. Vishwanath R: Sexed sperm vs conventional sperm – a comparative discussion. Proceedings, Applied Reproductive Strategies in Beef Cattle 2015; p. 250-256. 39. Baruselli PS, Campos Filho EP, Crepaldi GA, et al: New strategies to improve pregnancy rate at TAI using sex-sorted semen. Anim Reprod 2017;14:711 (abstract). 40. Bó GA, Huguenine E, de la Mata JJ, et al: Pregnancy rates in suckled beef cows synchronized with a shortened progesterone/estradiol-based protocol (J-Synch) and inseminated with conventional or sexed- sorted semen. Reprod Fertil Dev 2019;31:129 (abstract). 41. Baruselli PS, Ferreira RM, Colli MHA, et al: Timed artificial insemination: current challenges and recent advances in reproductive efficiency in beef and dairy herds in Brazil. Anim Reprod 2017;14:558-571. 42. Crepaldi GA, Freitas BG, Mingoti RD, et al: Reproductive efficiency of Nelore cows submitted to three different reproductive strategies in a 64 days breeding season. Anim Reprod 2017;14:698 (abstract). 43. Penteado L, Rezende RG, Mingoti RD, et al: Pregnancy rate of Nelore cows submitted to resynchronization starting 14 or 22 days after prior FTAI. Anim Reprod 2016;13:450. (abstract). 44. Bó GA, Baruselli PS, Martinez MF. Pattern and manipulation of follicular development in Bos indicus cattle. Anim Reprod Sci 2003;78:307-326. 45. Baruselli PS, Ferreira , SA Filho M, et al: Review: Using artificial insemination v. natural service in beef herds. Animal 2018;12 (Suppl 1): S45-S52. 46. Bó GA, Baruselli PS: Synchronization of ovulation and fixed-time artificial insemination in beef cattle. Animal 2014;8 (Suppl 1):144-150. 47. Burtre C, Pérez Wallace S: Evaluation of FTAI, the synchronization and natural service in the reproductive and productive performance in beef cows in the north of patagonia. Proc XII International Symposium on Animal Reproduction, IRAC, Cordoba, Argentina 2017; p. 366 (abstract). 263 Clinical Theriogenology • Volume 11, Number 3 • September 2019 264Clinical Theriogenology • Volume 11, Number 3 • September 2019 264Clinical Theriogenology • Volume 11, Number 3 • September 2019 Introduction Synchronization of ovulation and fixed time artificial insemination protocols in beef cattle OMNIBLANK: