影响马胚胎移植成功率的关键因素分析

旨在探讨影响马胚胎移植效率的几种关键因素。本研究统计了国内北京马场、河北马场和山东马场2013-2018年胚胎移植数据,3个马场供体马数量分别为15、21和25匹,受体母马数量分别为56、50和75匹。所有母马年龄为3～12岁。统计供体马冲胚时间对胚胎回收率的影响;胚胎日龄对移植后受体马妊娠率的影响;供、受体母马排卵同期化程度对移植后妊娠率的影响;受体母马居住移植基地时间对移植后妊娠率的影响。结果显示,母马在配种季节注射前列腺素(PG)+GnRH类似物或PG+hCG诱发排卵,发情周期分别为(14.5±0.8)和(14.3±1.1)d,显著低于对照组的((20.5±2.6)d,P0.05);排卵后第8天冲洗子宫的胚胎回收率均高于第7天,但差异不显著;8日龄胚胎移植后受体马的妊娠率均高于7日龄,差异不显著;供体母马排卵比受体母马早1d时,胚胎移植后的妊娠率最高;受体母马在移植基地居住时间大于1年时,移植后妊娠率高于居住时间小于0.5年的受体马。根据以上结果,本研究得出如下结论,PG与hCG或GnRH类似物联合使用可缩短母马发情周期,母马排卵后第8天的胚胎回收率和移植后妊娠率较高,胚胎移植时选择居住时间大于1年且排卵时间比供体晚1d的母马作受体。     

马胚胎移植现状

马的胚胎移植自七十年代首次移植成功以来有了很大的进展．目前，胚胎回收率达５５－８０％，移植妊娠率为７０％。而低生殖力的母马在其胚胎回收率．受体妊娠率及维持妊娠方面均较低．手术移植与非手术移植的妊娠率稍有不同。受体母马排卵时间在供体排卵前１－２天至排卵后２－３天的妊娠率比较高．卵巢摘除的母马用孕酮处理与供体同步排卵的前提下，也可以成功的作为胚胎受体．利用垂体制剂、促性腺素释放激素等类药品超排并不是很有效．每周期平均只有３个卵泡排卵．通过胚胎切割可以产生双驹，但成功率低；桑椹期较囊胚期胚胎冷冻效果好．胚胎在４℃保存２４小时其活力并未丧失。将体外受精的卵母细胞移植到受休进一步研究。     

和牛超数排卵及胚胎移植效果观察

为了研究和牛超数排卵和胚胎移植的效果,试验以和牛作供体,荷斯坦青年母牛作受体,采用PG+FSH方案对7头和牛供体进行超数排卵处理,用非手术法取胚并进行鲜胚移植。结果表明：采用PG+FSH方案头均获得胚胎数为(11.29±4.86)枚,可用胚胎(5.86±3.27)枚(A级和B级);鲜胚移植(A级和B级)妊娠率为51.22%。说明采用该方案对和牛供体进行超数排卵,并进行鲜胚移植的效果较为理想。     

猪胚胎移植技术研究

利用外源激素PMSG+HCG处理母猪58头,同期发情有效率和超排成功率分别为89.3％(25/28)和100％(30/30)。14头供体用手采集胚胎,平均每头排卵23.6±6.2个,共获得胚胎218枚,其中可利用胚胎206枚,胚胎可利用率为94.5％。将143枚胚胎移植给10头 受体,平均每头移植14.3±3.6枚,8头受体妊娠并产仔52头,移植妊娠率为80％(8/10),平均每头产仔6.5头,妊娠母猪胚胎存活产仔率为45.6％(总产仔数/胚胎移植总数)。仔猪初生平均体重为1.25±0.28公斤。其中12头供体手术冲卵后,第1个情期发情配种受胎率为91.8％(11/12)。     

非手术采集和移植骡胚的研究

选择已经配种的供体母马18匹,非手术冲洗子宫20次,其中排卵后4～5天的母马,冲洗5次,冲洗液平均回收率为94.80％,但未采到卵;排卵后5(1/2)～7(1/2)天的,冲洗15次,冲洗液平均回收率为85.66％,采到受精卵(骡胚)14个,采卵率为93.00％。结果表明,在排卵5(1/2)天之前,用非手术子宫采卵法未采到母马受精卵,而排卵5(1/2)天之后,非手术子宫采集马受精卵是有把握的。此外,另选没有配种而确切排卵的母马12匹,于排卵后5(1/2)～9天,回收马的未受精卵,虽冲洗液的平均回收率为89.30％,但也未采到未受精卵。显然,未受精的马卵没有进入子宫角,仍停留在输卵管中。经非手术采卵和二次重复采卵的母马都能按期发情,正常排卵。将14个骡胚中的7个以非手术法移植给7匹受体母马,结果一匹妊娠,妊娠率为14％。妊娠的受体母马生下一匹母骡驹,借腹怀胎350天。排卵同步差为-1(1/2)天。     

奶牛超数排卵及胚胎移植的试验研究

本试验采用FSH＋PG的方法对22头供体荷斯坦牛分两批进行了超数排卵处理,140头受体南阳黄牛和供体牛采用2次PG法分批进行了同期发情处理。结果如下：（1）共有20头供体牛、104头受体牛在第二次注射PG后96h内发情,同期发情率分别为90.90%和74.29%;（2）20头供体牛回收胚胎176枚,可用胚胎140枚/头,平均回收胚胎8.8枚/头,平均可用胚胎7.0枚/头,可用胚胎比率79.55%;（3）104头南阳黄牛受体均移植单胚,51头妊娠,妊娠率为49.04%。     

蒙古绒山羊胚胎移植效果研究

通过评价双胚胎移植方法和单胚胎移植方法对蒙古绒山羊妊娠率及产羔率的影响,为优质种羊的快速扩繁提供技术支持。选取5只供体母羊进行同期发情与超数排卵处理,供体母羊发情后进行人工授精,收集胚胎;利用收集的可用胚胎对选取的39只受体母羊进行胚胎移植,其中10只受体母羊做双胚移植,29只受体母羊做单胚移植;比较双胚移植和单胚移植受体母羊的妊娠率及产羔率。结果表明:供体母羊经CIDR处理,发情率达100%,超数排卵、人工授精后共收集胚胎64枚,其中可用胚胎49枚,可用胚胎率为76.56%;双胚移植受体母羊的妊娠率为90.00%(9/10),产羔率为180.00%(18/10),单胚移植受体母羊的妊娠率为68.97%(20/29),产羔率为62.07%(18/29)。双胚移植方法比单胚移植方法可获得更高的妊娠率及产羔率,表明双胚移植方法适用性良好,具有极大的推广价值。     

供受体羊发情同期化程度对胚胎移植效果的影响

将2008枚鲜胚移植给1146只受体羊,移植单胚和双胚的受体总妊娠率为63.1r3/1146,移植胚胎成羔率为52.861/2008。结果表明供、受体发情同步差在 1～ 0.5d、0d和-0.5～-1d时,移植单胚的受体妊娠率和胚胎成羔率没有明显的差异P>0.05;移植双胚的受体差异显著P<0.05,同步差为0d时的胚胎移植效果最好。移植双胚的受体中,奶山羊受体与供体同步差为-0.5～-1d和0d时,胚胎成羔率显著高于同步差为 1～ 0.5d的受体P<0.05;成都麻羊与供体同步差在 1～ 0.5d、0d和-0.5～-1d时,其胚胎成羔率均有显著差异P<0.05;南江黄羊和大耳羊做受体时,供、受体同步差在 1～ 0.5d、0d和-0.5～-1d时进行胚胎移植,胚胎成羔率无统计学差异。     

受体母猪排卵状况对体细胞克隆胚胎移植效率的影响

为了确定猪体细胞克隆胚胎的受体母猪最佳发情阶段,本研究比较了供受体同期化方案对猪体细胞克隆胚胎输卵管内移植后30 d受胎率和妊娠足月率的影响。将体外培养2 d的猪体细胞克隆胚胎,移植到发情第1~2天的受体母猪。根据受体母猪卵泡发育与排卵状况将受体猪群分为2个类别:卵泡发育处于即将排卵或正在排卵阶段(组一);卵泡发育处于排卵前阶段或完全排卵阶段(组二)。结果发现:(1)组一6头受体母猪胚胎移植后30d受胎率为100%;而组二10头受体母猪受胎率为40%,二者差异显著(P0.05)。(2)组一6头受体母猪全部产仔,妊娠足月率为100%;组二10头受胎母猪2头产仔,妊娠足月率为20%,二者差异极显著(P0.01)。以上结果表明:体外培养2 d的猪体细胞克隆胚胎发育阶段与即将排卵或正在排卵的受体母猪的子宫环境同期性最好,得到了理想的胚胎移植效率。     

波尔山羊供体和受体异地饲养鲜胚移植研究

为了进一步扩大良种波尔山羊的推广,提高胚胎移植效率,该文就饲养在不同牧场间的供体和受体实现鲜胚移植进行了系统研究,得到了以下结果:①洪体运输1h、2～3 h、＞4h、0h(对照)收集胚胎,分别得到可用胚胎数为15.8枚±4.3枚、13.0枚±5.7枚、13.8枚±6.6枚和14.2枚±6.3枚,不同运输时间回收可用胚胎数之间无显著性差异(P＞0.05).回收可用胚胎移植后,受体妊娠率分别为71.6％、67.6％、76.7％和80.7％,胚胎成羔率分别为74.1％、67.2％、69.9％和71.5％,供体不同运输时间回收胚胎移植妊娠率和胚胎成羔率无显著差异(P＞0.05).②胚胎在20℃,36.5℃和38.5℃运输2h后移植,受体妊娠率分别为42.0％、64.5％和66.7％,胚胎成羔率分别为41.7％、63.6％和66.7％.20℃运输胚胎受体妊娠率和胚胎成羔率显著低于36.5℃和38.5℃下运输胚胎(P＜0.01);胚胎在36.5℃分别运输0.5 h、1h、2h后移植,受体妊娠率分别为72.5 ％68.7％和64.9％,胚胎成羔率分别为69.1％、67.8％和63.0％,受体妊娠率和胚胎成羔率之间无显著性差异(P＞0.05).③受体在手术前2～4h通过卡车运输到另一畜牧场,运输时间分别为0.5 h、1h、≥2h,胚胎移植后受体妊娠率分别为61.2％、49.6％和42.0％.随着受体运输时间的延长,受体妊娠率呈下降趋势,运输时间超过2h的妊娠率显著低于运输0.5 h的妊娠率(P＜0.05).胚胎成羔率分别为60.9％、45.6％和32.0％,随着受体运输时间的延长,胚胎成羔率显著下降(P＜0.05或P＜0.01).从以上研究得出以下结论:山羊供体和受体饲养在不同饲养场,欲进行鲜胚移植,选择供体运输是最好的方法,其次为胚胎运输后移植.受体运输对胚胎移植效果(即受体妊娠率及胚胎成羔率)均有不利影响,不宜在生产中采用.     

Practical Experience with the Treatment of Recipient Mares with a Non‐Steroidal Anti‐Inflammatory Drug in an Equine Embryo Transfer Programme

As part of a commercial embryo transfer programme, 20 embryos were transferred to spontaneously synchronous or synchronized recipient mares. In 14 cases, embryo recipients were treated with non‐steroidal anti‐inflammatory drugs (NSAID), receiving flunixin meglumine i.v. at the time of transfer and vedaprofen orally twice a day on the 3 days after embryo transfer, while six embryos were transferred to untreated mares that served as controls. Out of the 14 recipient mares treated with NSAID, 11 (79%) were pregnant at 6–8 days after transfer and in 10 mares, the pregnancy was continued. From the six untreated recipients, only one became pregnant but underwent early embryonic death between day 14 and 35 after ovulation. In conclusion, pregnancy rate in NSAID‐treated recipients is higher than that in untreated recipients and above reported average values, indicating that treatment of recipient mares with NSAID helps to increase pregnancy rates after transcervical transfer and can be recommended for equine embryo transfer.     

Some Factors Affecting the Rate of Pregnancy after Embryo Transfer Derived from the Brazilian Jumper Horse Breed

This study aimed to evaluate the effects of certain embryo transfer parameters on the pregnancy rate after equine embryo transfer of the Brazilian Jumper Horse breed. The size, embryonic development stage, embryo quality, and synchronization of ovulation between the donor (n = 120) and recipient (n = 420) were evaluated in 396 embryos. Embryo recovery was performed on Day 6-9 after ovulation (Day 0 = day of ovulation). The recipient mares were chosen on the day of embryo recovery, and the transfers were performed that same day. The embryo size (diameter including envelopes; n = 396) ranged from 150 to 3000 μm; 67.1% measured between 400 and 1199 μm. The embryo size (400-1199 μm vs. ≤399 μm); stage of development (n = 396; blastocyst and expanded blastocyst versus compact morula and early blastocyst); quality (n = 396; grade 1 [excellent]), 2 [good], or 3 [poor]); and synchronization of ovulation between the donor and recipient (0, 1, 2, 3, and 4 days versus −1, 5, and 6 days, respectively) all affected pregnancy rate (P < .05). The pregnancy rate did not differ significantly among transfers performed on Days 0, 1, 2, 3, and 4. In conclusion, embryos measuring 400-1199 μm produced higher pregnancy rates in recipients than embryos measuring 150-399 μm, and blastocysts and expanded blastocysts produced pregnancy more efficiently than morulae and early blastocysts. The embryo quality also affected the pregnancy rate. Synchronization of donor and recipient ovulation to Days 0-4 improved the efficiency of embryo transplant.     

Strategies for Increasing Reproductive Efficiency in a Commercial Embryo Transfer Program With High Performance Donor Mares Under Training

Exercise stress has a negative impact on embryo transfer efficiency (ET). For example, a 34% embryo recovery rate, 43% incidence of poor quality embryos, and a 29% pregnancy rate after transfer have been reported. Administration of nonsteroidal anti-inflammatory drugs (NSAIDs) may reduce the inflammatory response produced after nonsurgical embryo transfer. In addition, progesterone supplementation is commonly administered to some recipient mares to improve uterine conditions before the transfer and to ensure adequate progestational support compatible with pregnancy. The aim of the study was to evaluate embryo recovery rates using BioRelease deslorelin versus hCG and to increase posttransfer pregnancy rates by jointly administering BioRelease progesterone and a NSAID (flunixin or meloxicam) to recipient mares. Seventeen upper-level showjumping mares stabled and in daily training were used as embryo donors. To induce ovulation, 1-mg IM BioRelease deslorelin (BioRelease Technologies, Lexington, KY) was injected in treated cycles (n = 66), or 2500-IU hCG IV (Ovusyn, Syntex, Buenos Aires, Argentina) was given in control cycles (n = 79) when a ≥35 mm follicle was present. Artificial insemination with extended fresh semen (at least 500 × 10⁶ progressively motile sperm) was carried out in both groups immediately after injecting the ovulation induction agent. Day 8 embryos were recovered and nonsurgically transferred using a speculum and a cervical traction forceps. Recipient mares (n = 73) were randomly assigned to one of three groups: Group A received a single injection of 1.5-g IM BioRelease progesterone (Progesterone LA 300, BioRelease Technologies) and 3 IV injections of 0.5 g of flunixin meglumine (Flunix Deltavet, Argentina), one injection administered the day of the transfer and one on each of the next two successive days. Group B received 1.5-g IM BioRelease progesterone and a single dose of 1.5-g IM BioRelease meloxicam (Meloxicam LA, BioRelease Technologies) at the moment of embryo transfer. Group C did not receive any treatment. Pregnancy diagnosis was carried out 7 days after transfer. Results were analyzed using comparisons of proportions. More embryos were recovered per cycle (13% increase) when donor mares in training were induced to ovulate with BioRelease deslorelin (60.6%; 40/66) than with hCG (46.8%; 37 of 79; P < .05). Although both recipient groups given NSAIDs in combination with BioRelease progesterone numerically had higher pregnancy rates (A: 70.8%; 17/24 and B: 75%; 15/20) compared with nontreated control recipients (47.1%; 33/70), pregnancy rates were significantly higher only in recipients given LA meloxicam treatment at the time of transfer (P < .05). The LA meloxicam is released over a 72-hour period making it more practical to use as it requires a single IM injection versus the 3 IV flunixin meglumine injections. Thus, to minimize the effects of exercise stress on ET efficiency, a combination of BioRelease deslorelin to induce ovulation in donors and BioRelease progesterone and LA meloxicam in recipients at the time of transfer may offer an interesting alternative for improving results in commercial ET programs.     

Japanese Society for Animal Reproduction: award for outstanding research 2002. The development and prevalence of the transfer technique for frozen-thawed embryos of Japanese black beef cattle in Tochigi Prefecture

The conditions of embryo transfer by the stepwise method, in which frozen-thawed embryos are transferred on day 7 (day 0=onset of estrus), were investigated with the aim of increasing pregnancy rates in frozen-thawed embryo transfer. The use of a vaginal speculum to prevent bacterial infection when passing an embryo transfer gun through the vagina yielded a pregnancy rate equal to or higher than that with application of a sheath cover to the transfer gun. Administration of a sedative, xylazine, to recipient cattle for preventing movement at the time of embryo transfer improved the pregnancy rate. The influence of the time from thawing of frozen embryos to transfer and of the transportation of the recipient by truck upon pregnancy rate was investigated. Embryo transfer within 60 minutes after aspiration into a straw or transportation of the bovine recipient, 1.5 hours each way before and after transfer, had no influence on pregnancy rate. Relations of the embryonic developmental stage and morphological quality after thawing of frozen embryos to pregnancy rate were investigated in recipients of nulliparous Holstein heifers. The pregnancy rate increased as the embryonic developmental stage advanced from compacted morula, early blastocyst, and blastocyst in that order. The pregnancy rate obtained with blastocyst stage embryos was significantly (P<0.05) higher than that with compacted morula stage embryos, and there was no significant difference in pregnancy rates between excellent morphological quality and good morphological quality for compacted morula stage embryos. When correlation of luteal function and pregnancy rate was investigated in bovine recipients, pregnancy rate showed a tendency to increase with increasing blood progesterone (P) concentration on the day before (on day 6 after estrus) and the day of embryo transfer. The pregnancy rate in bovine recipients, which showed a blood P concentration of > or =2.5 ng/ml on the day before embryo transfer, was significantly (P<0.05) higher than that in those with a blood P concentration of <2.5 ng/ml. Pregnancy rate showed a tendency to increase with decreasing blood estradiol-17beta (E2) concentration on the day of embryo transfer. Activation of luteal function by administration of human chorionic gonadotropin (hCG) in cycling cattle was investigated for its effect on increasing pregnancy rate in bovine recipients. A follicle coexisting with cyclic CL ovulated and induced CL formed after injection of hCG 1,500 IU 5 days after ovulation. The blood P concentration was significantly (P<0.05) higher in the administration group than in the control group, and the blood E2 concentration rapidly decreased, showing a lower concentration than in the control group. These results suggest the possibility that the pregnancy rate could be improved by administration of hCG. Pregnancy rate following intramuscular injection hCG 1,500 IU was comparatively investigated in parous Japanese Black beef cattle receiving frozen-thawed embryos 7 days after estrus. Pregnancy rate was 67.5% in the group in which hCG was administered on day 6 after estrus, and was significantly (P<0.05) higher than that in the control group (45.0%) and the group in which hCG was administered on day 1 after estrus (42.5%), revealing that hCG administration facilitated pregnancy. Transfer of frozen-thawed embryos in the blastocyst stage within 60 minutes after the aspiration into a straw, with a vaginal speculum after administration of xylazine is suggested as a way of improving pregnancy rate in bovine recipients with favorable luteal function and in those with luteal function activated by administration of hCG on the day before embryo transfer.     

Folliculogenesis, embryo parameters and post-transfer recipient pregnancy rate following equine follicle-stimulating hormone (eFSH) treatment in cycling donor mares

Background Induction of multiple ovulations, or superovulation, may potentially increase the efficiency of equine embryo transfer programs. Our objective was to investigate the effects of equine follicle‐stimulating hormone (eFSH) treatment on the success rate of embryo transfer programs in mares. Methods In the research facility of the University of Saskatchewan, Canada, we studied 12 donor mares and 37 recipient mares during the physiological breeding season. Donor mares were used in two consecutive oestrous cycles: the first served as the control cycle and in the second an eFSH regimen was applied (eFSH cycle). In the control cycle, mares were administered human chorionic gonadotropin (hCG) to induce ovulation when a follicle ≥35 mm in diameter was detected by transrectal ultrasonographic examination. In the second oestrous cycle, twice‐daily eFSH treatment was initiated when a follicle ≥25 mm was detected and treatment ceased when a follicle ≥35 mm was present, at which time hCG was administered. All donor mares were artificially inseminated while in oestrus using fresh semen collected from a stallion of proven fertility. At 8 days post‐ovulation, embryos were recovered transcervically and transferred individually to the uterus of a synchronised recipient mare. Results The eFSH treatment stimulated the ovary and resulted in greater numbers of ovulations and recovered embryos; however the recovered embryos tended to have a lower morphological grade than the control embryos, and the recipient pregnancy rate per transferred embryo was lower than anticipated. Conclusion The numbers of recipient pregnancies and foals born that resulted from eFSH treatment were not different from the control.     

Transcervical embryo transfer in performance mares

Pregnancy was established by transcervical transfer of embryos from performance mares into recipient mares. Estrus was synchronized between donor (n = 17) and recipient (n = 43) mares. After a greater than or equal to 25-mm follicle was detected, donor mares were bred artificially daily until ovulation. Day of ovulation was recorded. Uterine flushes (n = 111) were performed on donor mares 7 days after ovulation, and recovered embryos were transferred transcervically to recipient mares within 2 hours. Embryos were recovered from 40.5% of uterine flushes. Of transferred single embryos, 65.7% resulted in pregnancy, detectable by ultrasonographic examination 23 days after transfer. Only 35.3% of twin embryos resulted in pregnancy. Results over a 4-year period were as follows: uteri were flushed on 14, 44, 31 and 22 occasions, and 8, 21, 15, and 11 embryos were recovered (1 embryo was not transferred), with 6, 11, 4, and 6 resulting in 30-day pregnancy in years 1 to 4, respectively.     

Estradiol and progesterone concentrations resulting from the administration of an exogenous hormone regimen in diestrous embryo transfer recipients

Estradiol and progesterone concentrations were evaluated from diestrous embryo transfer recipient mares (5 to 14 days post-ovulation) which were treated with an exogenous hormone regimen. Upon detection of the donor mare's ovulation (0 hours), 10 mg PGF_2α was given to the recipient mare; at 12, 24 and 36 hours 20 mg estradiol cypionate; at 48 hours, 500 mg progesterone in oil and then 22 mg altrenogest at 60, 72 and 96 hours. Altrenogest (22 mg/day) was continued until end of the trial (detection of a fetal heart beat). Embryos were transferred non-surgically 6 or 7 days after the start of treatment.Plasma samples were evaluated over three periods; period 1-between recipient mare ovulation and prior to PGF_2α period 2-between PGF and embryo transfer and period 3-post-transfer. During periods 2 and 3, estradiol was higher (P<.05) for mares which were 10 to 14 days post-ovulation (late diestrous) as compared to mares which were 5 to 9 days post ovulation (mid-diestrous) when treatment began. Progesterone concentrations were higher (P<.05) for the mid-diestrous mares in the same periods. The pregnancy rate was higher for the late diestrous mares than the mid-diestrous mares (58% (7/12) vs 10% (1/10)). However, no difference (P>.05) was detected in estradiol or progesterone in the late diestrous mares which were pregnant or open. During period 2, estradiol was higher (P<.05) in the pregnant than open mares. Whereas, during period 3, progesterone was higher (P<.05) in the open mares.These data suggest that estradiol is important for the establishment of pregnancy in the mare. Furthermore, hormone treatment developed in this study appears to have some potential in synchronization of diestrus mares to be used as embryo recipients.     

Historical Aspects of Equine Embryo Transfer

Early embryo transfer in equids was undertaken simultaneously in the early 1970s in Cambridge, England, and Kyoto, Japan. Both groups achieved limited success when flushing the uterine horn ipsilateral to the side of ovulation but the rates improved markedly when the whole uterus was flushed on realization of the continued movement of the embryo throughout the uterine lumen after day 6. Initial transfers of embryos to recipient mares were carried out surgically, but nonsurgical transfer via the cervix has been used subsequently with increasing success, culminating in pregnancy rates of 75%–90% today. Experimental use of embryo transfer in horses and donkeys demonstrated the unique ability of equids to carry to term a full range of interspecies hybrid conceptuses and extraspecies pregnancies created by embryo transfer. Furthermore, splitting of day 4–8 cell embryos and day 6 compact morulae allowed the creation of genetically identical twin foals. But despite these and other significant advances over the past 45 years, a persisting limitation is the relatively low embryo recovery rates from donor mares treated with exogenous gonadotropins in attempts to induce them to superovulate. This is due to the toughness of the ovarian tunica albuginea which forces ovulation through the ventrally situated ovulation fossa where multiple follicles compete with each other and luteinize before they can ovulate properly.     

Use of a Low Dose of Equine Purified FSH to Induce Multiple Ovulations in Mares

The effects of a low dose of equine purified FSH (eFSH) on incidence of multiple ovulations and embryo recovery rate in mares were studied. During the physiological breeding season in Brazil (19°45′45′S), 14 Mangalarga Marchador donor mares were used in a crossover study and another 25 mares of the same breed, between 3 years and 12 years of age were used as recipients for the embryo transfers. Donors were monitored during two consecutive oestrus cycles, an untreated control cycle followed by a treated cycle, when eFSH was administered. In both cycles, after an embryo collection attempt on day 8 post‐ovulation all mares received 7.5 mg dinoprost and had their two largest follicles tracked daily by ultrasonography until the period of ovulation. Mares were inseminated every 48 h with extended fresh semen from a single stallion after the identification of a 35‐mm follicle until the period of ovulation. Ovulations were induced by intravenous administration of 2.500 IU of human chorionic gonadotropin, upon detection of a 35‐ to 40‐mm follicle. In the treated cycle, 5 mg eFSH was given intramuscularly once a day, from day 8 post previous ovulation until at least one follicle reached 35 mm in diameter. Embryo flushes were performed on day 8 of dioestrus (day 0 = ovulation). Treatment with eFSH resulted in higher (p < 0.05) ovulation rate and incidence of multiple ovulations compared to the control (1.6 vs 1.0 and 50% vs 0%, respectively – one mare had triple ovulation). However, embryo recovery rates in the control and treated cycles were similar (0.8 and 1.0, respectively; p > 0.05). Pregnancy rates in the recipient mares following embryo transfer were similar for the control and eFSH cycles (11/11 and 10/14, respectively). Additional studies are necessary in order to develop a low‐dose protocol for the use of eFSH that brings a more consistent contribution to the efficiency of commercial equine embryo transfer programs.     

Superovulation in cycling mares using equine follicle stimulating hormone (eFSH)

Superovulation would potentially increase the efficiency and decrease the cost of embryo transfer by increasing embryo collection rates. Other potential clinical applications include improving pregnancy rates from frozen semen, treatment of subfertility in stallions and mares, and induction of ovulation in transitional mares. The objective of this study was to evaluate the efficacy of purified equine follicle stimulating hormone (eFSH; Bioniche Animal Health USA, Inc., Athens, GA) in inducing superovulation in cycling mares. In the first experiment, 49 normal, cycling mares were used in a study at Colorado State University. Mares were assigned to 1 of 3 groups: group 1, controls (n = 29) and groups 2 and 3, eFSH-treated (n = 10/group). Treated mares were administered 25 mg of eFSH twice daily beginning 5 or 6 days after ovulation (group 2). Mares received 250 (of cloprostenol on the second day of eFSH treatment. Administration of eFSH continued until the majority of follicles reached a diameter of 35 mm, at which time a deslorelin implant was administered. Group 3 mares (n = 10) received 12 mg of eFSH twice daily starting on day 5 or 6. The treatment regimen was identical to that of group 2. Mares in all 3 groups were bred with semen from 1 of 4 stallions. Pregnancy status was determined at 14 to 16 days after ovulation.In experiment 2, 16 light-horse mares were used during the physiologic breeding season in Brazil. On the first cycle, mares served as controls, and on the second cycle, mares were administered 12 mg of eFSH twice daily until a majority of follicles were 35 mm in diameter, at which time human chorionic gonadotropin (hCG) was administered. Mares were inseminated on both cycles, and embryo collection attempts were performed 7 or 8 days after ovulation.Mares treated with 25 mg of eFSH developed a greater number of follicles (35 mm) and ovulated a greater number of follicles than control mares. However, the number of pregnancies obtained per mare was not different between control mares and those receiving 25 mg of eFSH twice daily. Mares treated with 12 mg of eFSH and administered either hCG or deslorelin also developed more follicles than untreated controls. Mares receiving eFSH followed by hCG ovulated a greater number of follicles than control mares, whereas the number of ovulations from mares receiving eFSH followed by deslorelin was similar to that of control mares. Pregnancy rate for mares induced to ovulate with hCG was higher than that of control mares, whereas the pregnancy rate for eFSH-treated mares induced to ovulate with deslorelin did not differ from that of the controls. Overall, 80% of mares administered eFSH had multiple ovulations compared with 10.3% of the control mares.In experiment 2, the number of large follicles was greater in the eFSH-treated cycle than the previous untreated cycle. In addition, the number of ovulations during the cycle in which mares were treated with eFSH was greater (3.6) than for the control cycle (1.0). The average number of embryos recovered per mare for the eFSH cycle (1.9 ± 0.3) was greater than the embryo recovery rate for the control cycle (0.5 ± 0.3).In summary, the highest ovulation and the highest pregnancy and embryo recovery rates were obtained after administration of 12 mg of eFSH twice daily followed by 2500 IU of hCG. Superovulation with eFSH increased pregnancy rate and embryo recovery rate and, thus, the efficiency of the embryo transfer program.

Introduction

Induction of multiple ovulations or superovulation has been an elusive goal in the mare. Superovulation would potentially increase the efficiency and decrease the cost of embryo transfer by increasing embryo collection rates.[1 and 2] Superovulation also has been suggested as a critical requirement for other types of assisted reproductive technology in the horse, including oocyte transfer and gamete intrafallopian transfer. [2 and 3] Unfortunately, techniques used successfully to superovulate ruminants, such as administration of porcine follicle stimulating hormone and equine chorionic gonadotropin have little effect in the mare. [4 and 5]The most consistent therapy used to induce multiple ovulations in mares has been administration of purified equine pituitary gonadotropins. Equine pituitary extract (EPE) is a purified gonadotropin preparation containing approximately 6% to 10% LH and 2% to 4% FSH.[6] EPE has been used for many years to induce multiple ovulations in mares [7, 8 and 9] and increase the embryo recovery rate from embryo transfer donor mares. [10] Recently, a highly purified equine FSH product has become available commercially.The objectives of this study were to evaluate the efficacy of purified eFSH in inducing superovulation in cycling mares and to determine the relationship between ovulation rate and pregnancy rate or embryo collection rate in superovulated mares.

Materials and methods

Experiment 1

Forty-nine normally cycling mares, ranging in age from 3 to 12 years, were used in a study at Colorado State University. Group 1 (control) mares (n = 29) were examined daily when in estrus by transrectal ultrasonography. Mares were administered an implant containing 2.1 mg deslorelin (Ovuplant, Ft. Dodge Animal Health, Ft. Dodge, IA) subcutaneously in the vulva when a follicle 35 mm in diameter was detected. Mares were bred with frozen semen (800 million spermatozoa; minimum of 30% progressive motility) from 1 of 4 stallions 33 and 48 hours after deslorelin administration. The deslorelin implants were removed after detection of ovulation.[11] Pregnancy status was determined at 14 and 16 days after ovulation.Group 2 mares (n = 10) were administered 25 mg of eFSH (Bioniche Animal Health USA, Inc., Athens, GA) intramuscularly twice daily beginning 5 or 6 days after ovulation was detected. Mares received 250 g cloprostenol (Estrumate, Schering-Plough Animal Health, Omaha, NE) intramuscularly on the second day of eFSH treatment. Administration of eFSH continued until a majority of follicles reached a diameter of 35 mm, at which time a deslorelin implant was administered. Mares were subsequently bred with the same frozen semen used for control mares, and pregnancy examinations were performed as described above.Group 3 mares (n = 10) received 12 mg of eFSH twice daily starting 5 or 6 days after ovulation and were administered 250 μg cloprostenol on the second day of treatment. Mares were randomly selected to receive either a deslorelin implant (n = 5) or 2500 IU of human chorionic gonadotropin (hCG) intravenously (n = 5) to induce ovulation when a majority of follicles reached a diameter of 35 mm. Mares were bred with frozen semen and examined for pregnancy as described above.

Experiment 2

Sixteen cycling light-horse mares were used during the physiologic breeding season in Brazil. Reproductive activity was monitored by transrectal palpation and ultrasonography every 3 days during diestrus and daily during estrus. On the first cycle, mares were administered 2500 IU hCG intravenously once a follicle 35 mm was detected. Mares were subsequently inseminated with pooled fresh semen from 2 stallions (1 billion motile sperm) daily until ovulation was detected. An embryo collection procedure was performed 7 days after ovulation. Mares were subsequently administered cloprostenol, and eFSH treatment was initiated. Mares received 12 mg eFSH twice daily until a majority of follicles were 35 mm in diameter, at which time hCG was administered. Mares were inseminated and embryo collection attempts were performed as described previously.

Statistical analysis

In experiment 1, 1-way analysis of variance with F protected LSD was used to analyze quantitative data. Pregnancies per ovulation were analyzed by x² analysis. In experiment 2, number of large follicles, ovulation rate, and embryo recovery rate were compared by Student,'s t-test. Data are presented as the mean S.E.M. Differences were considered to be statistically significant at p < .05, unless otherwise indicated.

Results

In experiment 1, mares treated with 25 mg eFSH twice daily developed a greater number of follicles 35 mm in diameter (p = .001) and ovulated a greater number of follicles (p = .003) than control mares (Table 1). However, the number of pregnancies obtained per mare was not significantly different between the control group and the group receiving 25 mg eFSH (p = .9518). Mares treated with 12 mg eFSH and administered either hCG or deslorelin to induce ovulation also developed more follicles 35 mm (p = .0016 and .0003, respectively) than untreated controls. Mares receiving eFSH followed by hCG ovulated a greater number of follicles (p = .003) than control mares, whereas the number of ovulations for mares receiving eFSH followed by deslorelin was similar to that of control mares (p = .3463). Pregnancy rate for mares induced to ovulate with hCG was higher (p = .0119) than that of control mares, whereas the pregnancy rate for eFSH-treated mares induced to ovulate with deslorelin did not differ from that of controls (p = .692). Pregnancy rate per ovulation was not significantly different between control mares (54.5%) and mares treated with eFSH followed by hCG (52.9%). The lowest pregnancy rate per ovulation was for mares stimulated with 25 mg eFSH and induced to ovulate with deslorelin. The mean number of days mares were treated with 25 mg or 12 mg of eFSH was 7.8 ± 0.4 and 7.5 ± 0.5 days, respectively. Overall, 80.0% of mares administered eFSH had multiple ovulations compared with 10.3% of control mares.