绵羊胚胎移植生殖内分泌调控及其效果研究

初产供体母羊（14月龄）德国美利奴15只、波德代15只、无角陶赛特18只分别用孕酮(CIDR)+促性腺激素(PMSG)+促卵泡素(FSH)进行处理,受体小尾寒羊母羊（2胎）124只用CIDR+PMSG进行处理,处理期测定供、受体外周血液中生殖激素孕酮（p4）、雌二醇（E2）、促卵泡素（FSH）和促黄体素（LH）的动态变化。结果显示：3个供体品种和受体各自发情同期化水平和效果达到非常理想的状态,但供、受体发情同步差相距1 d。黄体数德国美利奴（7.71）显著高于陶赛特（5.19）（P<0.05）;冲卵数德国美利奴（5.50）显著高于陶赛特（3.44）（P<0.05）;可用胚胎率德国美利奴、波德代、陶赛特分别为70.13%、55.74%、78.18%,陶赛特显著高于波德代（P<0.05）;德国美利奴、波德代、陶赛特胚胎移植受胎率分别为55.26%、54.17%、42.31%,德国美利奴显著高于陶赛特（P<0.05）。处理期供体和受体生殖激素动态变化的幅度大,第13天供、受体血清中p4达到峰值,小尾寒羊分别高于德国美利奴（5.37 ng/mL）、波德代（6.14 ng/mL）和陶赛特（5.37 ng/mL）;供体血清中E2于第14天开始发情达到峰值,小尾寒羊于第15天开始发情达到峰值;供体血清中FSH于第13天达到峰值,小尾寒羊第14天达到峰值;供体血清中LH于第14天开始发情达到峰值,小尾寒羊血清中LH于第15天开始发情达到峰值。     

Effect of estrus synchronization treatment after luteolysis on Holstein heifers as embryo transfer recipients

This study was designed to evaluate the effect of estrus synchronization treatments on recipient heifers for embryo transfer (ET). Holstein heifers were separated into the following three groups: (i) an administration of 50 µg GnRH (gonadotropin‐releasing hormone) analog was given to heifers at a random stage of the estrus cycle, followed 7 days later by two administrations of 7.5 mg prostaglandin F2 alfa analog (PG) as control; (ii) another administration of 100 µg GnRH was given to the control group at 48 h after the administration of PG as the second GnRH group; and (iii) an administration of 0.75 mg estradiol benzoate (E2) was given to the control group at 24 h after the administration of PG as the E2 group. Each method caused estrus synchronization. Fresh embryos were nonsurgically transferred into the suitable recipients that had a functional corpora lutea (CL) 7 days after estrus. The E2 group showed a significantly higher (P < 0.01) rate of estrus synchronization (98.9%) at 1–3 days after PG administration and the final pregnancy rate of the E2 group (50.6%) was also significantly higher than the other groups (37.1%, P < 0.05 and 30.9%, P < 0.01, respectively). These findings demonstrate that E2 administration 24 h after PG protocol is effective for estrus synchronization of Holstein heifers, thus improving the productivity of ET.     

Evaluation of the factors that affect the pregnancy rates during embryo transfer in beef heifers

The aim of this study was to evaluate the effects of the transfer side, transfer location, cervix transfer score, type and diameter of corpus luteum (CL) during embryo transfer on pregnancy rates in beef heifers. Progesterone-based synchronization and superovulation protocol were applied to Simmental cows used as donors (n = 168). Uterine flushings were performed on day 7 following artificial insemination. Obtained Code I (excellent or good) and II (fair) quality embryos were transferred to recipient beef heifers (n = 561). During embryo transfer, side of transfer (right or left), transfer location (the cranial or middle third of uterine horn), cervix transfer score (easy, moderate or difficult) and type (CLa, CLb and CLc) and diameter of CL were determined. Pregnancy rates following the transfer of Code I and II embryos were 44.66% and 33.07%, respectively (p < .05). The rates of pregnancy after transfers to the right and left uterine horn were 37% and 42.2%, respectively (p > .05). The pregnancy rates were 41.2%, 34.9% and 30.3% for cervix transfer scores as easy, moderate and difficult, respectively (p > .05). Pregnancy rates after transfer to the cranial third and middle third were 41.06% and 29.67%, respectively (p < .05). According to types of CL, pregnancy rates were 31.7%, 40.4% and 45.3% for CLa, CLb and CLc, respectively (p < .05). Moreover, it was found that as the CL diameter increased, the pregnancy rates increased. As a result, it was concluded that there was no effect of side of transfer and cervix transfer score, but embryo quality, transfer location, type and diameter of CL had significant effects on the pregnancy rate during embryo transfer in beef heifers.     

Effect of season and superstimulatory treatment on in vivo and in vitro embryo production in wood bison (Bison bison athabascae)

Two experiments were done using a two-by-two design to determine the effects of season and superstimulatory protocol on embryo production in wood bison. In Experiment 1 (in vivo-derived embryos), ovarian superstimulation was induced in female bison during the ovulatory and anovulatory seasons with either two or three doses of FSH given every-other-day (FSH × 2 vs. FSH × 3, respectively). Bison were given hCG to induce ovulation, inseminated 12 and 24 hr after hCG, and embryos were collected 8 days after hCG (n = 10 bison/group). In Experiment 2 (in vitro embryo production), ovarian superstimulation was induced in female bison during the ovulatory and anovulatory seasons with two doses of FSH, and in vivo maturation of the cumulus–oocyte complexes (COC) was induced with hCG at either 48 or 72 hr after the last dose of FSH. COC were collected 34 hr after hCG, and expanded COC were used for in vitro fertilization and culture. In Experiment 1, the number of follicles ≥9 mm, the proportion of follicles that ovulated, the number of CL, and the total number of ova/embryos collected did not differ between seasons or treatment groups, but the number of transferable embryos was greater (p < .05) in the ovulatory season. In Experiment 2, no differences were detected between seasons or treatment groups for any end point. The number of transferable embryos produced per bison was greatest (p < .05) using in vitro fertilization and was unaffected by season (1.5 ± 0.2 and 1.1 ± 0.3 during anovulatory and ovulatory seasons, respectively), in contrast to in vivo embryo production which was affected by season (0.1 ± 0.01 and 0.7 ± 0.2 during anovulatory and ovulatory seasons, respectively). Results demonstrate that transferable embryos can be produced throughout the year in wood bison by both in vivo and in vitro techniques, but the efficiency of embryo production of in vivo-derived embryos is significantly lower during the anovulatory season.     

受体因素对波尔山羊胚胎移植效果的影响

选择波杂一代羊、徐淮白山羊、长江三角洲白山羊作为受体，研究受体与供体发情同步化程度、受体的黄体状况、品种、营养状况等对受体妊娠率的影响。结果表明：受体发情同期化对其妊娠率有显著影响（P〈0．05），与供体同步发情的受体移植成功率最高（76．67％），受体黄体数及小卵泡的有无对其妊娠率没有显著影响（P〉0．05）。受体品种对其妊娠率有显著的影响（P〈0．05）；渡杂一代羊和徐淮白山羊比长江三角洲白山羊更适合作受体。     

胚胎质量和发育阶段对奶牛胚胎移植妊娠率的影响

[目的]为了评估胚胎质量和发育阶段对奶牛胚胎移植妊娠率的影响。[方法]使用63头青年奶牛作为供体进行超数排卵,评估回收胚胎质量和发育阶段。选择334头青年奶牛作为受体鲜胚移植不同质量和发育阶段胚胎。对胚胎质量分布、发育阶段分布、不同质量胚胎和不同发育阶段胚胎移植30 d妊娠率进行统计分析。[结果]可用胚胎中A级胚胎比例(60.78%)显著高于B级和C级胚胎比例(36.70%和2.52%)(P<0.05);致密桑椹胚比例(54.36%)显著高于早期囊胚,囊胚和扩张囊胚比例(18.35%,25.0%和2.29%)(P<0.05)。A级和B级胚胎移植30 d妊娠率(63.55%和64.35%)显著高于C级胚胎移植30 d妊娠率(44.44%)(P<0.05);致密桑椹胚、早期囊胚、囊胚和扩张囊胚移植30 d妊娠率差异不显著(P<0.05),早期囊胚、囊胚移植30 d妊娠率高于致密桑椹胚、扩张囊胚移植30 d妊娠率(P<0.05)。[结论]选择不同发育阶段的A级和B级胚胎能够获得较高胚胎移植妊娠率,增加早期囊胚和囊胚阶段胚胎移植数量能够提高胚胎移植妊娠率。     

胚胎移植试验羊群发情规律的研究

为了有效提高绵羊体细胞克隆和转基因等研究工作的效率,缩短胚胎移植试验羊群中空怀个体的利用周期间隔,试验对采用不同移植方法的胚胎移植受体及供体绵羊进行了移植前后的发情规律研究。结果表明:采用自制孕激素海绵栓和进口阴道孕激素释放装置(CIDR)对绵羊进行同期发情处理时发情率均接近90%,且92%以上的个体发情起始时间均集中在撤栓后24～48 h。在胚胎移植试验结束后,未参加胚胎移植个体和子宫角法未移植受体第2情期发情起始时间主要集中在上次发情后的14～20 d,而输卵管法未移植受体和输卵管法冲卵供体的发情时间则比较分散,呈现明显延后状态。未参加胚胎移植个体和子宫角法未移植受体人工授精后的受胎率接近往年羊场的平均水平,输卵管法未移植受体的受胎率略有下降,且输卵管法冲卵供体的受胎率明显降低。     

不同来源的山羊供核细胞对核移植重构胚发育的影响

以山羊的胚胎细胞、卵丘细胞、体细胞克隆胚胎细胞为核供体,比较了以这3种细胞为核供体得到的重构胚的体外发育能力.结果显示,以体内受精所得桑椹胚为核供体的重构胚发育能力最高,卵裂率和囊胚率分别为81.25%和25.64%以卵丘细胞为核供体的重构胚发育能力次之(79.68%,21.57%),再克隆胚胎发育能力最低(64%,12.5%),但是各组间差异不显著(P>0.05).     

不同胚胎移植方法对天祝白牦牛受胎率的影响

为提高胚胎移植技术在牦牛上的运用效果,选用51头甘肃省天祝县健康黑牦牛作为受体,以纯种天祝白牦牛作为供体生产胚胎,分别对同期发情处理和自然发情的受体牛进行鲜胚和冻胚移植试验。结果,同期发情处理的受体牛鲜胚移植的受胎率显著高于冻胚移植的受胎率(P0.05),分别为52%和38.5%;在自然发情受体牛的胚胎移植中也得到了相似结果,鲜胚和冻胚的移植受胎率分别为60%和50%,同期发情处理牛的平均妊娠率则低于自然发情受体牛的平均妊娠率(P0.05),二者分别为47.5%和54.5%。结论,牦牛鲜胚移植的受胎率明显高于冻胚移植的受胎率,而且自然发情受体牛的受胎率高于同期发情处理牛。     

Effect of mate selection on fuzzy selective mating criteria in closed dairy multiple ovulation and embryo transfer nucleus programs

In order to control rates of response and inbreeding, mate selection using fuzzy selective mating criteria (FMC) was investigated in adult multiple ovulation and embryo transfer nucleus schemes for dairy cattle. Stochastic simulation was used to model the closed nucleus scheme. This mate selection was examined in four alternative mating and male selection schemes: (i) a hierarchical scheme; (ii) a hierarchical sibship scheme (two males per sibship); (iii) a factorial scheme (two sires per dam); and (iv) a factorial sibship scheme (two males per sibship and two sires per dam). Genetic response and inbreeding rate tended to be reduced by increasing the trade-off parameter of FMC between the expected breeding value and inbreeding of progeny. Inbreeding rates in all schemes were reduced by reducing the variance of family size through selection and the average coancestry of mating pairs through mate allocation.     

Genomic selection strategies in dairy cattle breeding programmes: Sexed semen cannot replace multiple ovulation and embryo transfer as superior reproductive technology

The aim of this study was to test whether the use of X-semen in a dairy cattle population using genomic selection (GS) and multiple ovulation and embryo transfer (MOET) increases the selection intensity on cow dams and thereby the genetic gain in the entire population. Also, the dynamics of using different types of sexed semen (X, Y or conventional) in the nucleus were investigated. The stochastic simulation study partly supported the hypothesis as the genetic gain in the entire population was elevated when X-semen was used in the production population as GS exploited the higher selection intensity among heifers with great accuracy. However, when MOET was applied, the effect was considerably diminished as was the exchange of females between the nucleus and the production population, thus causing modest genetic profit from using X-sorted semen in the production population. In addition, the effect of using sexed semen on the genetic gain was very small compared with the effect of MOET and highly dependent on whether cow dams or bull dams were inseminated with sexed semen and on what type of semen that was used for the bull dams. The rate of inbreeding was seldom affected by the use of sexed semen. However, when all young bull candidates were born following MOET, the results showed that the use of Y-semen in the breeding nucleus tended to decrease the rate of inbreeding as it enabled GS to increase within-family selection. This implies that the benefit from using sexed semen in a modern dairy cattle breeding scheme applying both GS and MOET may be found in its beneficial effect on the rate of inbreeding.     

Use of oxytocin to attain cervical dilation for transcervical embryo transfer in sheep

The aim of this work was to determine whether a cervical dilation protocol (CDP) composed of only oxytocin can be used to perform transcervical (non-surgical) embryo transfer in sheep (NSET) without affecting the viability of the corpus luteum (CL). Likewise, we evaluated whether a cervical transposing test with a Hegar dilator (CT Hegar test), performed at oestrous time, could be used to screen ewes for NSET (greater or lower chances to transpose the cervix). For that, oestrous and ovulation synchronization was performed in 25 Santa Inês ewes to induce the dioestrous condition. Animals went through the following CDP in a crossover design: E + OX, oestradiol benzoate (100 µg intravenously [IV]) and oxytocin (100 IU IV); OX, oxytocin (100 IU IV); and SAL, saline solution (IV). Using a Hegar dilator, cervical transposing attempts were performed at oestrous (D0) and dioestrous time (D8). The viability of the CL (morphology, luteal blood flow and progesterone values) was evaluated by ultrasonography (colour Doppler and B-mode) and by serum progesterone measurement from D7 to D13. The cervical transposing rate was lower for the SAL group (64%; 16/25; p < .05) and did not differ between the E + OX (88%; 22/25, p > .05) and OX (84%; 21/25, p > .05) groups. No treatment affected the CL viability. The CT Hegar test showed a high sensitivity (85.7%–93.3%), satisfactory accuracy (72%–84%), low false-negative rate (6.7%–14.6%), but high false-positive rate (46%–66.7%). In conclusion, a CDP protocol composed exclusively of oxytocin can lead to good cervical transposing rates and does not affect the viability of the CL. In addition, a screening test (CT Hegar) performed at oestrus can identify ewes for which cervical transposing will likely not occur at NSET.     

受体因素对绵羊胚胎移植效果的影响

试验对绵羊胚胎移植过程中,受体不同发情方式、受体不同卵巢状态对妊娠率的影响进行了研究.结果表明,在受体不同发情方式,同期发情组鲜胚妊娠率为59.46%,自然发情组鲜胚为50.00%,在妊娠率上两组无显著差异(P＞0.05);在冻胚移植中,发情组妊娠率为46.22%,未发情组为16.00%,在妊娠率上两组差异极显著(P＜0.01);其妊娠率发情组为46.22%,二情期返情组为25.88%,在妊娠率上两组差异极显著(P＜0.01).有一个黄体受体羊,妊娠率38.37%,有多个黄体受体羊,妊娠率37.93%,两组妊娠率差异不显著(P＞0.05);有卵泡受体羊妊娠率29.55%,无卵泡受体羊妊娠率40.51%,两组妊娠率差异不显著(P＞0.05).     

Achievements and future perspectives of embryo transfer technology in pigs

Commercial embryo transfer (ET) has unprecedented productive and economic implications for the pig sector. However, pig ET has been considered utopian for decades mainly because of the requirements of surgical techniques for embryo collection and embryo deposition into recipients, alongside challenges to preserve embryos. This situation has drastically changed in the last decade since the current technology allows non‐surgical ET and short‐ and long‐term embryo preservation. Here, we provide a brief review of the improvements in porcine ET achieved by our laboratory in the past 20 years. This review includes several aspects of non‐surgical ET technology and different issues affecting ET programmes and embryo preservation systems. The future perspectives of ET technology are also considered. We will refer only to embryos produced in vivo since they are the only type of embryos with possible short‐term use in pig production.     

Effect of flunixin meglumine treatment during and after embryo transfer on the pregnancy rate in cattle

This study aimed to determine the effect of flunixin meglumine treatment during and after the transfer of in vivo produced embryos to Angus (cows) and Holstein (cows and heifers) breeds of cattle on pregnancy rate. Holstein cows were used as donors in the study. A double dose of prostaglandin F2α was administered to the recipient animals for synchronization. Uterine flushing was performed in donors on day 7 after artificial insemination. A total of 295 transferable embryos were obtained. These embryos were transferred to Angus cows (n = 85), Holstein heifers (n = 80) and Holstein cows (n = 130). After the transfer, these animals were divided into three subgroups. The first subgroup (TI) was administered flunixin meglumine during embryo transfer, and the second subgroup (TII) was administered flunixin meglumine both during embryo transfer and on days 8 and 9 after the transfer. The third subgroup (TIII) was not administered anything and it was considered the control group. Pregnancy examination of the recipients was performed on days 30–35 after the transfer using real-time ultrasonography. The pregnancy rates after embryo transfer were found to be 43.52% in Angus cows, 42.5% in Holstein heifers, and 24.61% in Holstein cows (p < .05). When the animals were not classified according to breed, the pregnancy rates in subgroups TI, TII and TIII were found to be 29.29%, 45.10% and 29.79%, respectively (p < .05). In addition, the pregnancy rates were higher in TII and TIII subgroups of Angus cows and Holstein heifers compared to that of Holstein cows (p < .05). As a result, the pregnancy rates obtained after embryo transfer in Angus cows and Holstein heifers were found to be higher than that in Holstein cows. In addition, it was concluded that the administration of flunixin meglumine during and during/after embryo transfer has a positive effect on pregnancy rates in Angus cows and Holstein heifers.     

非繁殖季节胚胎移植受体绵羊妊娠效果影响因素的研究

在非繁殖季节应用CIDR法和Progestagen Sponge法对受体绵羊进行同期发情处理后,进行冷冻胚胎移植,研究不同同期发情处理方法、卵巢状况、胚龄、移植方法和移植时间对冷冻胚胎移植妊娠率的影响,以期为当地实现绵羊胚胎移植产业化提供理论依据和技术参考。结果表明:经同期发情处理后,绵羊在去栓后24~72 h集中发情,在去栓后36~48 h发情最为集中;CIDR法和Progestagen Sponge法的同期发情率分别为91.11%和87.78%(P>0.05),同期发情绵羊移植利用率分别为82.93%和78.48%(P>0.05);单侧卵巢上含有1个和2个黄体其妊娠率分别为46.07%和48.78%(P>0.05);桑椹胚和囊胚的妊娠率分别为49.25%和42.11%(P>0.05);腹腔镜法和手术法的妊娠率分别为45.16%和48.52%(P>0.05);11月和12月进行冷冻胚胎移植的妊娠率分别为50.76%和43.08%(P>0.05),该试验的绵羊冻胚移植妊娠率为46.92%,基本接近国内冻胚移植水平,但低于目前国内羊鲜胚移植妊娠率(50%~60%)。由此可以得出,不同同期发情处理方法、卵巢状况、胚龄、移植方法和移植时间对胚胎移植妊娠率的影响均无显著性差异(P>0.05),大规模群体羊同期发情时,可选价格便宜的Progestagen Sponge法;只要受体绵羊黄体质量好即可移植胚胎;为了减轻受体绵羊的应激反应,降低术后子宫粘连的发病率,可广泛应用腹腔镜法进行胚胎移植;该试验的绵羊冻胚移植妊娠率低于目前国内羊鲜胚移植妊娠率,通过筛选高效低毒的抗冻保护剂,以及提高基层技术人员操作水平,则有望进一步提高绵羊冷冻胚胎移植妊娠率。     

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。     

家兔超数排卵和胚胎移植妊娠率的影响因素分析

应用FSH和HCG相结合的不同方法 (递减量与恒量法),对不同兔龄和是否经产的中国家兔进行了超数排卵处理。其中,递减量法超排处理:每间隔12 h依次肌肉注射15、15、10、10、5及5 U/只共60 U的FSH,6次之后间隔12 h,经耳缘静脉注射HCG 100 U/只,合笼;恒量法超排处理:每间隔12 h肌肉注射10 U/只共60 U的FSH,6次之后间隔12 h,经耳缘静脉注射HCG 100 U/只,合笼。17 h后,取卵经显微注射后移植到排卵点分布不同的家兔输卵管中,比较分析了超数排卵及胚胎移植妊娠率的影响因素,旨在优化兔超数排卵和胚胎移植方法。结果显示,①总量为60 U的FSH递减量肌肉注射法超排效果略优于恒量法(688枚、671枚),但差异不显著(P>0.05);②9～11月龄的母兔超排效果明显优于6～8月龄的母兔(794枚、565枚),差异显著(P<0.05);③经产母兔的超排效果明显优于未经产的母兔(887枚、472枚),差异显著(P<0.05);④经显微注射重组DNA的胚胎移植受体时,两侧卵巢均有排卵点的胚胎移植受体妊娠率明显高于一侧卵巢有排卵点和两侧卵巢均无排卵点的受体(81.25%、38.46%、37.50%),差异显著(P<0.05)。试验证明,通过肌肉注射递减总量为60 U的FSH对9～11月龄经产母兔的超排效果最佳,且两侧卵巢均有排卵点的受体胚胎移植妊娠率最高,为胚胎研究工作者提供了一定的参考依据。