牛活体采卵技术及其在胚胎体外生产中的应用

自第1头体外生产胚胎(IVP)牛诞生和非手术活体采卵(OPU)技术在牛上应用以来,为了改进IVP和OPU技术,许多研究者致力于提高牛IVP和OPU技术的效率.时至今日,OPU和IVP技术均已成为成熟的技术.阐述了过去十几年中OPU和IVP技术的主要研究成果和研究进展,并总结了该技术的应用历史、应用现状和应用前景.     

牛OPU卵子体外受精和早期胚胎培养的方法研究

为了提高牛OPU卵子来源的胚胎生产效率,本试验对牛活体采卵获得的卵子分别用不同系列的培养液培养,用不同精子离心方法及不同的脱颗粒细胞方法进行操作。通过卵裂率和桑囊率的结果,以确定培养液及各种操作方法的可行性。结果表明,进口成品培养液的桑囊率虽高于自配溶液(34.29±9.20%vs 26.45±20.01%),但差异不显著,不同的精子离心方法和扒卵方法也没有显著差异。通过本研究可以为源于牛OPU卵子的体外胚胎生产程序的优化提供参考。     

通过活体采卵(OPU)的方法来进行牛体外胚胎生产的研究

近年来，体外胚胎生产的技术日益提高，尤其是在牛上应用比较广泛，在1988年由体外生产的胚胎最后可移植到受体的数目达到30000枚，靠超声波引导从活体上采集卵母细胞（OPU）进行体外胚胎的生产的方法现在被广泛采用，这种方法可以使活体持续性的生产胚胎，大大地提高了繁殖效率。且活体采卵是一项非常灵活、方便的技术，通常1周可以采集2次，连续许多周，都不影响供体的繁殖潜力，通常依靠活全采卵的方法（OPU）所获得的平均每8～10个卵母细胞中就可生产出2枚可移植的胚胎，从而提高了胚胎的生产效率，缩短了世代间隔，而且用活体采卵的方法（OPU）所生产的胚胎质量也要好于屠宰的卵巢上所获得的卵母细胞生产的胚胎。目前，这种方法除在牛上应用外、也在其它动物如水牛，马上采用。     

全日制放牧BMY牛的活体采卵-体外胚胎生产-胚胎移植研究

为建立BMY牛的活体采卵(OPU)-体外胚胎生产(IVP)-胚胎移植(ET)快速扩繁技术体系,从人工草地全放牧牛群中选择健康的经产空怀BMY牛作为供体(n=14)集中OPU一次;卵母细胞经TCM-199体外成熟培养(IVM)、Fert-TALP液体外受精(IVF),受精卵用合成输卵管液(SOF)体外培养(IVC)生产胚胎;BMY牛受体(n=81)用CUE-MATETM孕酮阴道栓+EB+FSH+PG方法同期发情处理,对黄体合格的移植OPU-IVP来源鲜胚。研究结果表明:①OPU头均获卵母细胞23.50枚,获10枚以上卵母细胞的供体占85.7%;IVP的平均囊胚数和可用胚数分别为5.21和4.00枚;OPU前8～9d卵巢上有黄体(CL)的供体,头均获卵母细胞数及IVP的囊胚数、可移植胚数、囊胚率和可移植胚率分别比对照组高7.28枚(P<0.05)、7.29枚(P<0.05)、6.00枚(P<0.05)、27.3%(P<0.01)和23.2%(P<0.01);②受体黄体合格率69.1%(56/81),胚胎移植妊娠率37.5%(21/56),产犊率35.7%(20/56);③受体在胚胎移植时注射GnRH,其妊娠率比对照组高16.2%(P>0.05)。结果显示,BMY母牛具有良好的OPU潜力,但个体间差异较大;可通过严格供体选择及改善IVP技术等措施,实现OPU-IVP效率最大化。CUE-MATETM+EB+FSH+PG同期发情处理方法适于在牛规模化胚胎移植(ET)和人工授精(AI)中应用。综合应用OPU、IVP、同期发情和ET技术是加快优良BMY牛扩繁的重要途径之一。     

B型超声波导引采集牛卵母细胞

在牛胚胎移植技术的应用中由于母牛对外源激素超排反应的不稳定性,特别是连续进行超排处理对供体牛产生不良影响,限制了从一些优良母牛来获取多个胚胎。随着牛体外受精技术的成熟,可在体外生产胚胎,所需的卵母细胞主要从屠宰后的牛卵巢上获得,但对有价值的牛用屠宰取卵巢的方法显然是不可行的。近几年发展起来的活体采卵技术(OPU)使这一问题得到很好的解决。本试验用B型超声波导引采集卵母细胞取得较好的效果。1材料与方法1.1试验牛的选择试验在北京锦绣大地公司胚胎中心进行。从牛场选取皮埃蒙特杂交一代母牛3头,3岁,体重450kg左右,健康…     

奶牛活体采卵与屠宰场卵巢卵子体外生产胚胎的效果比较

先利用屠宰场卵巢获得的卵母细胞进行体外培养试验.在建立了培养体系获得较稳定的囊胚率的基础上,再利用具有优良乳用遗传性状的老龄奶牛进行活体采卵(OPU),获得的卵子进行体外胚胎生产,对奶牛活体采卵与屠宰场卵巢卵子体外生产胚胎的效果进行了比较.同时对奶牛重复OPU的效果及OPU卵子的培养天数对妊娠率的影响因素等进行探讨,试图为研究和生产建立一个高效的活体采卵体外胚胎生产方法.结果表明:(1)活体采卵获得的卵母细胞从形态以及后期的发育情况均明显差于屠宰场卵巢获得的卵子;OPU法的卵裂率、囊胚率(70.5%、18.3%)明显低于从屠宰场卵巢获得的卵子的卵裂率、囊胚率(75.1%、34.8%)(P＜0.01).(2)不同个体采集卵子数量差异明显(5.92枚/头与1.4枚/头)(P＜0.05).(3)同一头奶牛每次采卵数之间无显著差异,但有波动性变化.(4)奶牛OPU卵子经培养在第6 d或第7 d发育至囊胚的胚胎移植妊娠率高于第8 d发育至囊胚的胚胎.     

OPU卵母细胞体外成熟体系的优化研究进展

近年来，体外胚胎生产(in vitro embryo production, IVP)技术取得了很大进步，尤其是卵母细胞活体采卵(ovum pick-up, OPU)技术得到广泛应用。OPU是20世纪90年代发展起来的一项新型的能获得优秀种母牛卵母细胞的技术，该技术在动物胚胎生产和发育机理研究及人类辅助生育技术临床实践中有着广阔的应用前景。可以提高种母畜和珍贵野生动物的资源保护效率，提高优良母畜的遗传潜力，为奶牛生物技术发展提供必要的支撑技术。卵母细胞的质量是IVP系统取得成功的关键，卵母细胞的体外成熟(in vitro maturation, IVM)是成功受精和胚胎发育必不可少的步骤。卵母细胞IVM完成了卵母细胞的细胞质和核成熟，准备了发育至胚胎基因组激活所必需的所有必要成分。但体外成熟的卵母细胞发育能力低于体内成熟卵母细胞，卵母细胞减数分裂和细胞核质成熟不同步，氧化应激损伤等对卵母细胞成功受精和胚胎发育非常重要，因此，需要进一步提高卵母细胞体外成熟能力，以改善牛体外胚胎生产系统。本文从卵母细胞体外成熟面临的核质成熟不同步，氧化应激等问题进行概述，同时，叙述C型钠肽、褪黑素、FLI...     

牛体外胚胎高效生产技术优化研究

为了探讨小卵泡液、放线菌酮(CHX)对卵母细胞预成熟、囊胚滋养层细胞囊泡(TVS来源于体外受精培养14 d的滋养层细胞)、维生素对牛体外胚胎质量的影响。在B超仪下进行牛活体取卵(OPU),从牛活体卵巢采集卵母细胞,进行体外成熟、体外受精、早期胚胎体外培养。结果表明:10%小卵泡液及8%CHX的卵母细胞预成熟4 h组显著优于对照组;在体外受精及早期胚胎培养液CR1aa中添加10μg/m L维生素C组与TVS共培养组的卵裂率和囊胚发育率均高于其他试验组(P0.05);体外胚胎与TVS共移植受胎率显著高于对照组(P0.05)。说明活体采集的卵母细胞经体外预成熟处理,可以达到核质同期化的目的,早期胚胎的培育过程中加入TVS、抗氧化剂等可以克服早期胚胎的发育阻滞,提高体外胚胎的囊胚发育率。     

浅析牛体内胚胎生产的影响因素

体内胚胎生产是胚胎移植的重要组成部分。最近几年,体内胚胎生产结合胚胎移植技术在良种牛快速扩繁领域里发挥了重要作用,但在生产中,效果仍不稳定。国内外很多学者对提高牛体内胚胎生产的稳定性做了大量研究,本文就国内外专家学者对影响牛体内胚胎生产的外在和内在因素进行了分析总结。     

牛胚胎生产的全程质量控制

本文介绍了供体牛的准备、供体牛同期发情处理、供体牛的超排和配种以及胚胎回收、胚胎质量评定、胚胎冷冻等牛胚胎生产的各个环节中应采取的全程质量控制措施。     

Maturation competence of swamp buffalo oocytes obtained by ovum pick-up and from slaughterhouse ovaries

This study was designed with the final goal of improving in vitro embryo production in the Thai swamp buffalo (Bubalus bubalis carabensis). Oocytes were collected by ovum pick-up (OPU) from six non-lactating multiparous swamp buffalo twice per week for 10 consecutive sessions followed by once-weekly collection for 10 consecutive sessions without hormone stimulation. In addition, oocytes were collected from slaughterhouse ovaries that were classified as follows: ovaries from non-pregnant cows with a visible corpus luteum (NPCL); pregnant cows with a corpus luteum (P); and non-pregnant cows without a corpus luteum (NP). Follicles in each group of ovaries were categorized as small (2-4 mm), medium-sized (5-8 mm) or large follicles (≥ 9 mm). The quality of the oocytes was assessed by their capacity to undergo in vitro maturation. The total number of observed follicles per session (all sizes combined) was larger in the once-weekly OPU group compared with the twice-weekly OPU group. In particular, the numbers of small and large follicles were higher in the once-weekly OPU group (5.2 ± 0.7 and 0.9 ± 0.2, respectively) than in the twice-weekly OPU group (3.9 ± 0.5 and 0.5 ± 0.1). The number of medium-sized follicles did not differ between the groups. The percentages of oocytes with an abnormal spindle morphology were not different between oocytes from the twice-weekly (30.0%) and the once-weekly (28.6%) OPU groups. A higher percentage of oocytes obtained in vitro (49.5%) exhibited nuclear abnormalities compared with those obtained in vivo (≤34.8%) after in vitro maturation. In conclusion, oocytes can be successfully collected by OPU in the swamp buffalo, without hormonal pretreatment, and per week more good-quality oocytes can be collected by twice-weekly OPU. In addition, oocytes collected from slaughterhouse ovaries can be used with the reproductive status of the cow having no influence on the maturation competence of oocytes.     

Effect of Days Post-Partum, Breed and Ovum Pick-Up Scheme on Bovine Oocyte Recovery and Embryo Development

The objective of this study was to investigate (i) the effect of two different ovum pick-up (OPU) schemes (once vs twice weekly aspirations) on oocyte recovery rate, quality and subsequent in vitro embryo development, (ii) the influence of days post-partum on oocyte recovery and (iii) possible differences in OPU results from two different herds. In group A, OPU was performed twice weekly in two Holstein Friesian (HF) and three Danish Red and White (DRW) cows from a private herd. In the research herd, two groups of eight HF cows were investigated: group B (OPU once weekly) and group C (OPU twice weekly). The collected oocytes were subsequently submitted to in vitro embryo production. More oocytes were recovered from the private herd when compared with the research herd. In the research herd, the twice weekly scheme aspirated more oocytes than the once weekly scheme. The quality of the retrieved oocytes was significantly different between groups B and C but not between groups A and C, and HF cows yielded higher quality oocytes than DRW cows (p = 0.029). Oocytes from group C showed higher level of embryonic development than group B oocytes. No differences in blastocyst rates were observed between groups A and C. Session affected the number of retrieved oocytes and subsequent developmental rates, with these being lower in the first compared with the last sessions. Finally, there was no significant effect of days post-partum in the number and quality of the retrieved oocytes, likely because of the small group size and high variation between sessions.     

奶牛活体采卵-体外受精效率的影响因素研究

为建立稳定高效的活体采卵-体外受精技术体系,提高体外胚胎生产效率,本研究先利用屠宰场采集的新鲜卵巢卵母细胞进行体外受精,通过胚胎发育潜力来筛选最佳的体外胚胎培养液;再进一步研究不同种公牛精液和供卵母牛对活体采卵-体外受精效率的影响。结果显示,CR1aa培养液和mCR1aa培养液卵裂率差异不显著（P>0.05）,但mCR1aa组的囊胚发育率显著提高（28.1% vs 20.6%,P<0.05）;选取的3头荷斯坦种公牛精液的活体采卵-体外受精胚胎的卵裂率差异不显著（P>0.05）,但1号种公牛精液体外受精后囊胚率（38.7%）显著高于2号和3号（23.8%&22.9%）（P<0.05）;随机选择的3头活体采卵供体母牛（H1、H2、H3）获得的头均可用卵母细胞数无显著差异,但H1和H2供体母牛体外受精胚胎的卵裂率和囊胚率均显著高于H3供体牛（P<0.05）,且H1供体牛体外受精囊胚率显著高于H2供体牛（P<0.05）。结果表明,mCR1aa培养液能显著提高体外受精囊胚发育率,适用于体外胚胎生产;种公牛精液和供体母牛个体差异会直接影响活体采卵-体外受精胚胎的生产效率,为奶牛活体采卵-体外受精生产技术体系的优化提供参考。     

Effect of suckling on embryo production by repeated ovum pick-up before and after timed artificial insemination in early postpartum Japanese black cows

We investigated whether suckling would affect embryo production of cows bred by timed artificial insemination (TAI) following an ovulation synchronization protocol combined with ovum pick-up and progesterone releasing intravaginal device (OPU-PRID-TAI protocol). The number of oocytes and transferable embryos collected by repeated OPU, performed before and after TAI, were recorded. A total of 14 Japanese Black cows were divided into weaned (n=7) and suckled groups (n=7). All 14 cows were treated with OPU on day 0 (the first day of treatment) and then with a PRID for 9 days. Prostaglandin F(2alpha) analog was administered on day 7, GnRH analog was administered on day 10 (36 h after removal of the PRID) and TAI was performed 12 h later. Ovulation was confirmed by palpation per rectum the following day. After TAI, additional OPU sessions were performed on days 18, 25 and 32. The synchronized ovulation rates of the weaned and suckled groups were 100 and 85.7%, and the conception rates were 71.4 and 42.9%, respectively. Immature oocytes were fertilized and cultured in vitro. The numbers of oocytes collected and blastocysts generated were similar between the individual OPU sessions in both groups. However, the total numbers of oocytes collected, cultured oocytes, cleavage embryos and blastocysts as well as the proportions of cleavage embryos and blastocysts to cultured oocytes were all significantly (P<0.05) greater in the weaned group compared with the suckled group. These results suggest that the OPU-PRID-TAI protocol has the potential to produce a significant number of good-quality embryos in vitro after repeated OPU in early postpartum weaned Japanese Black cows. To collect more oocytes and produce more embryos, we suggest that calves be removed from cows scheduled for treatment using this protocol.     

水牛卵母细胞的体外受精及其胚胎发育的研究

本研究比较了不同来源的水牛卵母细胞的体外受精及其胚胎发育。对10头摩拉母水牛连续6周进行活体采卵,共采集292枚卵母细胞,头均回收卵母细胞4.87枚,头均A级卵数为3.07枚,从屠宰场收集74头水牛卵巢共采集559枚卵母细胞,头均回收卵母细胞和A级卵母细胞分别为7.55枚和5.20枚,均显著高于活体采集的水牛卵母细胞(P<0.01)。将收集的AB级水牛卵母细胞在相同的条件下进行体外成熟、体外受精和体外培养至囊胚。结果表明,活体采卵组和屠宰水牛卵母细胞组受精分裂率差异不显著,分别为54.81%和57.73%。屠宰水牛卵母细胞组的囊胚率则高于活体采卵组(28.78%vs21.34%,P<0.05)。利用两组的胚胎进行常规法冷冻保存,冻胚解冻后的存活率差异不显著(P>0.05)。     

Bovine OPU‐Derived Oocytes can be Matured In Vitro for 16–28 h with Similar Developmental Capacity

The aim of this study was to determine the optimal maturation culture period of ovum pick up (OPU)‐derived cumulus oocytes complexes (COCs) in relation to their developmental capacity. Embryo production, embryo cryotolerance, post‐transfer embryonic survival and calf characteristics such as gestation length, birthweight and sex ratio were investigated. This retrospective study covers the analyses of ovum pick up –in vitro production and calving results from a commercial programme that took place between March 1994 and September 2004. Donors were both heifers (of which approximately 90% pregnant) and cows (of which approximately 10% pregnant). Embryo production analyses were based on 7800 OPU sessions conducted from January 1995 until January 1999. Analyses of calving rate were based on 13 468 embryo transfers performed during January 1995 until May 2002. Analyses on calf characteristics were based on 2162 calves born between March 1994 and September 2004. The in vitro maturation culture period ranged from 16 to 28 h. The mean production rate of transferable embryos was 16.5% (1.2 embryos per OPU session). Length of maturation culture period did not affect the production of transferable embryos. Mean calving rate was 40.9% and 38.7% for fresh and frozen/thawed embryos, respectively. Calving rate was not affected by the maturation culture period. Mean birthweight, gestation length and proportion of male calves were 46 kg, 281.9 days and 52.8%, respectively. Maturation culture period did not affect these variables. In conclusion, this study shows that the in vitro maturation culture period within the range of 16–28 h does not affect in vitro embryo production, embryo cryotolerance, post‐transfer embryonic survival and calf characteristics, suggesting that all COC batches collected by OPU on the same day, can be fertilized in one IVF session without a significant loss in the production from oocyte to calf.     

牛活体采卵技术研究进展

应用活体采卵技术可以从活体母牛反复采集大量系谱明确的卵母细胞,用于胚胎的体外化生产。本文将对牛活体采卵技术的研究进展和操作方法进行综述,分析影响活体采卵效果的因素和存在问题,并论述活体采卵在家畜育种和胚胎生物技术等方面的应用前景。     

水牛分离精子与不同来源卵母细胞体外受精的效果研究

本研究的目的是探讨水牛分离精子与不同来源(活体采卵或屠宰场卵巢采卵)卵母细胞体外受精的效果。活体采卵是选用20头空怀河流型母水牛(其中摩拉母牛12头,尼里-拉菲母水牛8头)每间隔3 d采卵1次,连续采卵5~6周,活体采集卵母细胞;屠宰场卵巢采卵是收集屠宰场水牛卵巢,用10 mL注射器连接18 G针头吸取水牛卵巢上可视的卵泡来收集卵母细胞。将收集的AB级水牛卵母细胞在相同的条件下进行体外成熟、然后用分离或未分离精子进行体外受精以及体外培养至囊胚。结果发现:活体采卵组和屠宰场收集的水牛卵母细胞组用分离精子受精分裂率和囊胚率没有差异(P>0.05);分离精子和未分离精子的体外受精分裂率和囊胚率也没有差异(P>0.05)。由此说明,水牛分离精子可以用于体外生产性控胚胎。     

Anti-Muellerian hormone levels in plasma of Holstein-Friesian heifers as a predictive parameter for ovum pick-up and embryo production outcomes

The aim of this study was to investigate whether plasma anti-Muellerian hormone (AMH) levels of Holstein-Friesian heifers could be used to predict ovum pick-up (OPU) and embryo production outcomes. Plasma samples and data were collected from 64 heifers, which underwent repeated OPU with subsequent in vitro embryo production followed by embryo flushing after superovulation. AMH levels were significantly positively correlated with the number of follicles aspirated per OPU session (r = 0.45), recovered oocytes per OPU (r =0.43) and in vitro produced embryos per OPU (r = 0.28). No significant correlations between AMH and in vivo produced embryos were ascertained. Our results suggest that correlations between AMH and outcomes of an OPU-IVF program are too low to use AMH as a precise predictive parameter for the success of a particular OPU procedure in Holstein-Friesian heifers. However, AMH can help to identify groups of very good or very poor oocyte donors.