Treatments for the synchronisation of bovine recipients for fixed-time embryo transfer and improvement of pregnancy rates

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1 CSIRO PUBLISHING Reproduction, Fertility and Development, 2012, 24, Treatments for the synchronisation of bovine recipients for fixed-time embryo transfer and improvement of pregnancy rates Gabriel A. Bó A,B,E, Lucas Coelho Peres A, Lucas E. Cutaia A, Danilo Pincinato A, Pietro S. Baruselli C and R. J. Mapletoft D A Instituto de Reproducción Animal Córdoba (IRAC), Zona Rural General Paz, 5145, Córdoba, Argentina. B Instituto de Ciencias Básicas, Carrera de Medicina Veterinaria, Universidad Nacional de Villa María, Córdoba, Argentina. C Departamento de Reprodução Animal, FMVZ-USP, CEP São Paulo, Brazil. D Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, SK, Canada. E Corresponding author. gabrielbo@iracbiogen.com.ar Abstract. Although embryo transfer technology has been used commercially in cattle for many years, the inefficiency of oestrus detection, especially in recipients, has limited the widespread application of this technology. The most useful alternative to increase the number of recipients utilised in an embryo transfer program is the use of protocols that allow for embryo transfer without the need for oestrus detection, usually called fixed-time embryo transfer (FTET). Most current FTET protocols are based on progestin-releasing devices combined with oestradiol or GnRH, which control and synchronise follicular wave dynamics and ovulation. Conception rates to a single FTET have been reported to be similar to those after detection of oestrus, but pregnancy rates are higher because these treatments have increased the proportion of recipients that receive an embryo. Recent changes to treatments for FTET, such as the administration of ecg, have resulted in increased pregnancy rates and provide opportunities to make these treatments easier to perform on farm. Additional keywords: ecg, estradiol, fixed-time embryo transfer, progesterone, GnRH. Introduction Embryo transfer has been used widely to reproduce the most valuable females in the herd. However, factors such as nutrition, management and oestrus detection efficiency affect the widespread use of this technology in beef cattle operations. The most useful alternative to increase the number of recipients utilised in embryo transfer is to apply protocols that allow for embryo transfer without the need for oestrus detection, usually referred to as fixed-time embryo transfer (FTET). The objective of this manuscript is to review currently available protocols that synchronise ovulation in recipients, and discuss how these and other factors related to bovine embryo transfer impact on the effectiveness and application of commercial embryo transfer in beef cattle. Synchronisation of oestrus in recipients Treatment with prostaglandin F 2a (PGF) Prostaglandin F 2a has been the most commonly used treatment for synchronisation of oestrus in cattle (reviewed in Odde 1990). Treatments used for synchronisation of recipients generally Journal compilation Ó IETS 2012 involve the administration of PGF twice at 11- to 14-day intervals (Broadbent et al. 1991). If all the recipients are cycling,,80% should show oestrous behaviour within 5 days of the second treatment. However, due to inefficiency of oestrus detection and the variability in the interval from treatment to oestrus and ovulation,,50% of the treated recipients have been reported to receive an embryo 7 days after oestrus (reviewed in Bó et al. 2002). This situation may be even worse if the recipients have Bos indicus influence. Under these circumstances, the numbers of recipients that receive an embryo over those treated with PGF may be as low as 30%, due largely to the number of recipients seen in oestrus, and/or with a CL at the time of embryo transfer (reviewed in Bó et al. 2004). Treatment with progestins and PGF Studies evaluating different progestin protocols have shown that progestin treatments that are long enough to allow normal regression of the CL (i.e. $14 days), induce synchronous oestrus (Odde 1990), but result in the development of oversised (persistent) dominant follicles (Kinder et al. 1996) and low

2 Bovine recipient synchronisation protocols Reproduction, Fertility and Development 273 Table 1. Effect of treatments that induce ovulation of persistent follicles in Bos indicus-influenced recipients on progesterone concentrations and conception rates Mantovani et al. (2005) Moura et al. (2001) Control Persistent follicle Control Persistent follicle Number of recipients Diameter of the ovulatoryfollicle (mm) b a b a Diameter (cm) of the CL at embryo transfer b a b a Plasma progesterone (ng ml 1 ) b a b a Recipients transferred/treated (%) 36/70 (51.4) b 55/71 (77.5) a 38/66 (57.6) b 54/71 (76.1) a Conception rate (%) 21/36 (58.3) b 21/55 (38.2) a 17/38 (44.7) 19/54 (35.2) Pregnancy rate (%) 21/70 (30.0) 21/71 (29.6) 17/66 ( /71 (26.8) ab Within studies, different superscripts in a row denote significant differences (P, 0.05). fertility following AI (Savio et al. 1993). Persistent follicles develop because the low progesterone environment induced by the treatment in the absence of a CL (Savio et al. 1993). When the treatment results in progestin levels equivalent to sub-luteal concentrations of circulating progesterone (1 to 2 ng ml 1 in plasma), increased LH pulse frequency occurs, leading to prolonged growth and maintenance of the dominant follicle and elevated plasma concentrations of oestradiol-17b (Kinder et al. 1996). The development of a persistent ovarian follicle has a detrimental effect on conception after AI, because it leads to an altered oviducal environment (Binelli et al. 1999) or premature maturation of the oocyte (Revah and Butler 1996), or both. Since oocyte quality is not important to achieve pregnancy in an embryo transfer recipient, fertility may not be compromised if embryos are transferred to recipients following ovulation of a persistent follicle. In one study, ovulation of persistent follicles resulted in pregnancy rates following embryo transfer that did not differ from ovulation of non-persistent follicles (Wehrman et al. 1997), but the overall pregnancy rate in this study was low (,30%). Two experiments evaluated the effects of ovulation of persistent follicles on pregnancy rates in embryo recipients. In one, crossbred Bos indicus Bos taurus heifers were treated with progestin-releasing vaginal devices (1.9 g of progesterone, CIDR-B, Pfizer Animal Health, Sao Paulo, SP, Brazil) for 14 days, with PGF on Day 0 and Day 5, to ensure luteal regression (Mantovani et al. 2005). In the other, persistent follicles were induced by treating Bos indicus heifers with norgestomet implants (3 mg norgestomet, Crestar, Schering- Intervet, Sao Paulo, SP, Brazil) for 9 days, and PGF at insertion and removal (Moura et al. 2001). In both experiments, ovulation of persistent follicles resulted in larger CL and higher circulating progesterone concentrations at the time of embryo transfer, but lower conception rates than in controls (Table 1). Reduced progesterone concentrations during the development of the ovulatory follicle has been shown to affect uterine function in the late luteal phase of the subsequent oestrous cycle by prematurely increasing oestrogen receptor-a abundance and exacerbating PGF release (Cerri et al. 2011). Therefore, caution should be exercised in inducing ovulation of persistent follicles to produce a large CL with the idea of improving pregnancy rates in recipients. Treatments that control follicular wave emergence and synchronise ovulation GnRH-based treatments The treatment protocol that utilises GnRH and PGF for fixedtime AI in cattle is often referred to as Ovsynch (Pursley et al. 1995). The Ovsynch protocol currently used in dairy cattle consists of an injection of GnRH followed by PGF 7 days later, a second injection of GnRH 56 h after PGF treatment with fixedtime AI 16 h later (Brusveen et al. 2008). The rationale for this protocol is that the first GnRH will induce LH release and ovulation of a dominant follicle and emergence of a new follicular wave within 2 days. The administration of PGF 7 days later will induce luteolysis, and the second GnRH will induce LH release synchronising ovulation of the new dominant follicle (Pursley et al. 1995). GnRH-based protocols have also been used to synchronise ovulation in recipients that received in vivo- (Baruselli et al. 2000; Hinshaw 1999) or in vitro- (Ambrose et al. 1999) derived embryos. In these studies, more recipients received embryos because the GnRH-based protocol was not dependent on oestrus detection; although conception rates may be lower than controls, pregnancy rates have been higher. Recent studies have shown that the first GnRH resulted in ovulation in 44 to 54% of dairy cows (Bello et al. 2006; Colazo et al. 2009), 56% of beef heifers (Martinez et al. 1999) and 60% of beef cows (Small et al. 2009), and the emergence of a new follicular wave was synchronised only when treatment caused ovulation (Martinez et al. 1999). If the first GnRH does not synchronise follicular wave emergence, ovulation following the second GnRH may be poorly synchronised (Martinez et al. 2002), and recipients may be asynchronous to the stage of embryo development at the time of transfer. Prevention of the early ovulations by addition of a progestin-releasing device to a 7-day GnRH-based protocol has improved pregnancy rates in heifers after fixed-time AI (Martinez et al. 2002), and the addition of a norgestomet implant to a GnRH-based protocol for FTET resulted in similar pregnancy rates to those after observed oestrus (Hinshaw 1999). The same investigators treated 1637 recipients with GnRH plus a norgestomet implant or progestin-releasing vaginal device without oestrus detection; overall pregnancy rate was 59.9%. In summary, results indicate that acceptable pregnancy rates can

3 274 Reproduction, Fertility and Development G. A. Bó et al. be achieved when embryos are transferred to recipients that have been treated with GnRH and progestin device protocol to synchronise ovulation, without the necessity of oestrus detection. Another approach to increase the number of cows responding to a GnRH-based protocol is to pre-synchronise oestrus with two PGF treatments 14 days apart, with the last PGF given 10 to 12 days before the first GnRH (Moreira et al. 2001). This protocol has been named Pre-Synch Ovsynch and has been shown to improve pregnancy rates in GnRH-based FTAI protocols and could be used to synchronise ovulation in cycling recipients. Treatments with progestins and oestradiol Oestradiol treatments are the most commonly used treatment to synchronise follicle wave emergence and ovulation in beef and dairy recipients in South America (Baruselli et al. 2010). The protocol consists of insertion of a progestin-releasing device and the administration of 2 mg oestradiol benzoate (EB) on Day 0 (to synchronise follicular wave emergence), and PGF either 5 days later, or at the time of insertion and removal of the progestin device (to ensure luteolysis). The progestin device is usually removed on Day 8 and ovulation is induced by the administration of 0.5 or 1 mg of oestradiol cypionate at progestin device removal, 1 mg of EB 24 h after progestin removal, or GnRH 48 to 54 h after progestin removal (reviewed in Bó et al. 2002; Baruselli et al. 2010, 2011). As oestrus detection is usually not preformed, Day 9 is considered to be the day of oestrus. All cows with an apparently functional CL on Day 17 receive an embryo and conception rates are comparable to those obtained with embryo transfer 7 days after oestrus (Bó et al. 2002). Use of equine chorionic gonadotropin (ecg) to improve pregnancy rates Use of ecg in oestradiol/progestin-based protocols The most common strategy used to increase pregnancy rates in pasture-managed beef cattle in South America is the addition of 400 IU of ecg on either Day 5 or Day 8 of the oestradiol/ progestin treatment protocol. Overall, 75 to 85% of the recipients treated with this protocol receive an embryo (compared with 50% or less with PGF synchronisation), progesterone concentrations at the time of embryo transfer are high, and conception rates usually exceed 50% (reviewed in Bó et al. 2002; Baruselli et al. 2010, 2011). The FTET treatment protocol utilising EB, progestin and ecg has been evaluated in different parts of the world. In a commercial embryo transfer program in Argentina, 1309 (84.9%) of 1542 recipients were suitable for embryo transfer and 692 (44.9%) became pregnant following Direct Transfer (Bó et al. 2004). In a commercial embryo transfer program in Brazil, Nasser (pers.comm.) obtained 5801 (46.1%) pregnancies at 30 days and 5271 (41.7%) at 60 days of gestation following the transfer of in vitro-produced fresh embryos. In another study involving 988 recipients of in vitro-produced, frozen thawed embryos in China (Remillard et al. 2006), overall pregnancy rates were significantly higher in ecg-treated animals because of the higher percentage of recipients receiving embryos following treatment with ecg. In a study in Mexico with 949 Brahman-influenced recipients, treatment with ecg increased the number of recipients receiving an embryo, with similar conception rates, resulting in higher pregnancy rates (Looney et al. 2010). In summary, results indicate that the administration of ecg in an oestradiol/progestin-based synchronisation protocol for FTET resulted in increased pregnancy rates, especially in recipients that were nutritionally stressed or with Bos indicus influence. Considering that feeding recipients until they become pregnant is one of the most costly items in an embryo transfer program (Hinshaw 1999), a protocol that increases the number of pregnant recipients per synchronisation treatment seems cost-effective, especially considering that this treatment also avoids the necessity of oestrus detection (Looney et al. 2010). Use of ecg in GnRH-based protocols As oestradiol is not available in many countries, GnRH has been used to synchronise follicle wave emergence and ovulation in fixed-time protocols. In a Canadian study designed to evaluate the potential use of ecg in beef cattle recipients synchronised with GnRH/progestin for FTET (Small et al. 2007), recipient selection rates did not differ whether cows did or did not receive a progestin device (93.4% vs 85.5%) or ecg (91.0 vs 87.8%). In addition, pregnancy rates did not differ whether cows did or did not receive a progestin device (32.3 vs 32.4%) or ecg (35.2 and 29.2%). On the other hand, ecg significantly increased pregnancy rates in a Colombian study (Mayor et al. 2008). Bos indicus cross heifers were randomly allocated into one of three treatment groups. Heifers in the control group received a progestin device (1 g of progesterone, DIB, Syntex, Argentina) and 2 mg of EB on Day 0 and PGF plus 400 IU ecg on Day 5. Progestin devices were removed on Day 8 and 1 mg of EB was administered on Day 9. Heifers in the GnRH treatment group received a progestin device and GnRH on Day 0, PGF at progestin removal on Day 7 and GnRH on Day 9. Heifers in the GnRH þ ecg group were treated similarly except that they also received 400 IU ecg on Day 3. All heifers with a CL.16 mm in diameter received a frozen thawed embryo by Direct Transfer 7 days after GnRH or 8 days after EB. The number of recipients selected/treated was higher in the EB þ ecg (70.0%, 28/40) and the GnRH þ ecg (72.5%, 29/40) groups than in the GnRH group (47.5%, 19/40). Conception and pregnancy rates were also significantly higher in recipients in the EB þ ecg (16/28, 57.1% and 16/40, 40%) and GnRH þ ecg (16/26, 61.5% and 16/40, 40%) groups than in the GnRH group (9/19, 47% and 9/40, 22.5%). In summary, the addition of ecg to oestradiol- or GnRH-based protocols which included the use of progestin devices resulted in increased pregnancy rates depending on the type and body condition of the recipients. However, treatment with ecg may not improve pregnancy rates in Bos taurus beef recipients managed under near optimal conditions. Long-term effects of ecg treatments in cattle Since ecg is a complex glycoprotein with a high molecular weight that is produced by the pregnant mare (Murphy and Martinuk 1991), a potential immunological reaction against

4 Bovine recipient synchronisation protocols Reproduction, Fertility and Development 275 repeated use of ecg in cattle is a concern (Drion et al. 2001). A PhD thesis (University of Sao Paulo) evaluated the potential adverse effects of the repeated use of ecg in cattle (Mantovani 2010). A first experiment was designed to determine anti-ecg antibody production in response to 400 or 2000 IU of ecg, given once, twice or three times at 30-day intervals in Bos taurus and Bos indicus heifers. Animals were then submitted to weekly blood sampling for 63 days, and then at 30 to 60 day intervals for a total of 300 days. Antibody production was not affected by the number of ecg treatments; however, antibody production was higher in Bos taurus than in Bos indicus heifers. Higher antibodies levels were also observed in heifers receiving 2000 than 400 IU ecg. A second experiment conducted one year later focussed on the evaluation of the cellular and humoral immunological memory of the Bos taurus heifers previously treated with 400 or 2000 IU of ecg. Humoral immunological memory response was not observed in animals previously treated with 400 or 2000 IU of ecg, regardless of the number of previous treatments. However, cellular immunological memory response was observed to be higher in animals subjected to increased numbers of previous treatments; but no evidence of adverse biological effects (i.e. number of follicles stimulated with ecg) were observed. Results suggest that ecg, as used in synchronisation protocols, is unlikely to have adverse effects following subsequent treatments. Other treatments designed to increase pregnancy rates in recipients There have been several studies investigating the relationship between circulating progesterone concentrations and pregnancy rates in recipients (reviewed in Baruselli et al. 2010). However, the use of supplementary progesterone has resulted in inconsistent effects on pregnancy rates. An alternative strategy to increase circulating progesterone concentrations in recipients is to induce an accessory CL by induction of ovulation of the first wave dominant follicle around the time of embryo transfer (reviewed in Thatcher et al. 2001). Again, results have not been entirely clear. In Bos indicus recipients, treatment with human chorionic gonadotropin (hcg) on Day 7 increased progesterone concentrations (Marques et al. 2002) and treatment with GnRH, hcg, plh or a progestin device at the time of embryo transferresulted in increased pregnancy rates (Marques et al. 2003). However, pregnancy rate in non-treated (control) recipients was lower than normally expected. This result was confirmed by another experiment (Rodrigues et al. 2003) involving the induction of an accessory CL with GnRH at the time of embryo transfer in Bos indicus cross recipients. However, in another study involving Bos indicus cross recipients synchronised with progestin/oestradiol plus ecg protocol (Tribulo et al. 2005), pregnancy rates were not affected by treatment with hcg or GnRH at the time of FTET. Small et al. (2004) were also unable to improve pregnancy rates in Bos taurus recipients treated with GnRH or plh on Days 5 or 7 after oestrus. In a very recent study (Wallace et al. 2011), 719 beef recipients alternately received 1000 IU hcg or saline (control) at the time of embryo transfer. Serum progesterone concentrations at pregnancy diagnoses in pregnant cows were higher after hcg treatment than in controls, and conception rates were 61.8 and 53.9% for hcg and control groups, respectively. The authors concluded that giving hcg at embryo transfer increased the incidence of accessory CL, serum progesterone in pregnant recipients and pregnancy rates. With the exception of the last report, the beneficial effects of increasing circulating concentrations of progesterone seem to be evident when pregnancy rates in control (not treated) recipients were lower than expected. Factors affecting pregnancy rates following FTET with frozen]thawed embryos We have recently done logistic regression analysis to evaluate factors (other than the synchronisation treatment) that affect pregnancy rates following Direct Transfer in a FTET program (Peres 2011). Data were collected following transfer of 1333 embryos on the same farm in Argentina during the spring and summer (ambient temperature 20 to 408C, six replicates). All recipients were non-lactating, cycling, multiparous Bos indicus cross cows with body condition score between 3 and 4 (1 to 5 scale) and synchronised using the progestin/oestradiol plus ecg protocol for FTET, described earlier. All cows with a luteal area.76 mm 2 (by calculating the area of the CL minus the area of the cavity;,14 mm in diameter) the day before embryo transfer received frozen/thawed embryos by Direct Transfer one day later. Most embryos were IETS Grade 1, frozen in 1.5 M ethylene glycol by the same laboratory, thawed in a 308C water bath for 30 s and transferred by one of two technicians. Pregnancy rates were determined by ultrasonography 35 days after FTET. Of the various variables analysed, reproductive history (open in a previous FTET vs no history), embryo stage and embryo quality affected results (Table 2). The difference in pregnancy rates obtained between recipients that were open following a previous FTET compared with those that were not previously used confirm results reported by Looney et al. (2006), and questions the feasibility of re-using recipients when high valued embryos are transferred. However, it was surprising that the other factors such as body condition score and CL area did not affect pregnancy rates. Although site of placement of the embryo was not significant, very few embryos were transferred near the bifurcation of the uterus. Another factor that was critically evaluated was the effect of time from thawing to Direct Transfer on pregnancy rates (Bó et al. 2007). The thawing procedures and loading of the embryo transfer gun before transfer were done in an air conditioned room (20 to 258C). The time intervals to Direct Transfers were classified as,3 min, between 3 and 6 min and.6 min (between 6 and 16 min). Pregnancy rates did not differ among the three thawing to transfer intervals (Table 2), suggesting that there need not be a concern about time between thawing and transfer of embryos frozen in ethylene glycol (at least up to 16 min) into Bos indicus Bos taurus recipients synchronised for FTET. Summary and conclusions The inefficiency of oestrus detection in beef cattle operations has greatly limited the widespread application of embryo transfer,

5 276 Reproduction, Fertility and Development G. A. Bó et al. Table 2. Factors affecting pregnancy rates in recipients that received embryos by Direct Transfer following synchronisation for FTET (adapted from Peres 2011) Variables Pregnant/ transferred (%) P-value Oestrus detection (n ¼ 1333) Seen in oestrus 517/924 (55.9%) 0.36 Not seen in oestrus 219/409 (53.5%) CL diameter (1 day before FTET, n ¼ 1333) mm 128/228 (56.1%) mm 208/369 (56.4%).18 mm 400/736 (54.3%) CL numbers (n ¼ 1333) 1 673/1220 (55.2%) /99 (53.5%) 3 10/14 (71.4%) Embryo placement (n ¼ 1333) Proximal to the ovary 246/442 (55.7%) 0.23 Curvature 482/872 (55.3%) Bifurcation 8/22 (38.1%) Embryo stage (n ¼ 1333) Morula (Code 4) 259/468 (55.4%) ab Early blastocyst (Code 5) 275/463 (59.4%) a Blastocyst (Code 6) 179/344 (52.0%) bc Expanded blastocyst (Code 7) 23/58 (39.7%) c Embryo quality (n ¼ 1333) 1 693/1235 (56.1%) a /65 (49.2%) ab 3 11/33 (33.3%) b Technician (n ¼ 1333) A 313/542 (57.8%) 0.33 B 423/791 (53.5%) Reproductive history (n ¼ 837) Open in a previous FTET 116/231 (50.2%) 0.02 No history 347/606 (57.3%) Interval from thawing to transfer (n ¼ 1122) #3 min 251/385 (55.8%) min 372/655 (56.8%) 6 16 min 42/82 (51.2%) abc Denotes significant differences in pregnancy rates between embryos of different stages of development or quality (P, 0.01). especially in extensively managed herds. The incorporation of techniques designed to control follicular wave dynamics and ovulation reduce the need for oestrus detection and provide opportunities for the application of large scale genetic improvement programs using embryo transfer technology. Several treatment protocols originally developed for fixed-time AI provide for the planning of embryo transfer without the necessity of oestrus detection and without compromising results. Furthermore, recent changes incorporated into treatment protocols for FTET, such as the administration of ecg, have resulted in increased pregnancy rates. Finally, other factors have to be taken into consideration in large scale embryo transfer programs to achieve high pregnancy rates. The selection of the optimal program will depend on the availability and cost of treatments, the breed, age, physiological and nutritional status of the recipients, and other management factors such as the availability of qualified personnel. 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