中国荷斯坦牛胚胎冷冻与移植受胎技术研究

牛冷冻胚胎解冻成活率达８４．４％（３８／４５）;冷冻胚胎移植总受胎率达到４６．７％（５７／１２２）,其中１９９６年达到５５．６％（２０／３６）,Ａ级胚胎达到７７．８％（２１／２７）。并在我国首次获得中国荷斯坦牛冷冻胚胎二分胚的同卵孪生牛犊,且达到４２．９％（３／７）的移植受胎率。     

牛胚胎冷冻与移植受胎技术研究

进行了牛胚胎冷冻技术研究，重点研究了提高超排效果和冷冻胚胎移植受胎率的方法。头均获可用胚胎数达到７．０－７．４枚，牛冷冻胚胎移植受胎率达到４６．７％（５７／１２２），其中１９９６年达到５５．６％（２０／３６），Ａ级胚胎达到７７．８％（２１／２７）。在我国首次获得中国荷斯坦牛冷冻胚胎二分胚的同卵孪生牛犊，二分胚移植受胎率达到４２．９％（３／７）。     

奶牛胚胎移植实用技术的研究

笔者于１９９５￣１９９７年进行了为期３年的奶牛胚胎移植实用技术的研究。３年共移植奶牛胚胎１３７枚，受胎率为３５．７７％（４９／１３７）。移植Ａ级胚胎的受胎率为７０％（７／１０），移植Ｂ级胚胎的受胎率为４３．０２％（３７／８６），移植Ｃ级胚胎的受胎率为１２．１９％（５／４１）。移植Ａ级胚胎的受胎率比移植Ｂ级胚胎的受胎率高２６．９８％（Ｐ〉０．０５）；移植Ａ级胚胎和Ｂ级胚胎的受胎率分别比移植Ｃ级胚胎的     

本地黄牛冷冻胚胎移植技术及其受胎率影响因素分析

笔者选用97头山东省平度市当地黄牛作为受体，采用玻璃化解冻进行加拿大进口北美性控高产荷斯坦奶牛冷冻胚胎移植。试验分别从冷冻胚胎级别、受体牛黄体直径、移植者技术水平、胚胎解冻后的停留时间，对冷冻胚胎移植受胎率进行了分析比较。结果表明：A级胚胎（46．3％）比B级胚胎（31．6％）、C级胚胎（0）受胎率高；受体牛黄体直径在1．0～1．5cm（49．1％）之间较2cm（28．1％）、2．5cm时受胎率要高；移植者技术水平的高（42．3％）低（21．1％）其受胎率相差较大；胚胎解冻后停留的时间10～20min（37．8％）、1～2h（46．9％）、4～6h（25％）对受胎率也影响较大。说明影响当地黄牛移植高产荷斯坦奶牛冷冻胚胎受胎率的因素主要有冷冻胚胎级别、受体牛黄体直径、移植者技术水平、胚胎解冻后停留的时间。     

奶牛胚胎体外保存时间和温度对移植受胎率的影响

试验对胚胎在体外保存时间的长短及温度与移植妊娠率的关系进行统计分析，胚胎收集后分别在0-3h,3-6h和6h后冷冻，解冻后移植受胎率分别为46.07%,43.33%,30.56%,0-3h和3-6h冷冻的胚胎，移植受胎率没有明显差异（P〉0。05），6h后的冻胚与6h以前的冻胚移植受胎率差异显著（P〈0．05）。胚胎分别在室温和37℃保存5h后冷冻，解冻后移植受胎率分别为39．74％、42．24％（P〉0．05）。     

切割取样后胚胎的冷冻保存技术的研究

研究了切割取样后小鼠胚胎和牛胚胎冷冻-解冻的技术方法。结果表明：（1）分别以10％甘油、10％乙二醇作为冷冻液以及在以上两种冷冻液中分别添加10％葡聚糖＋0．1mol／L蔗糖作为冷冻液常规方法冷冻切割后的小鼠胚胎，冷冻-解冻后体外培养72h总发育率分别为56．9％（259／455）、81．8％（604／738）、84．8％（540／637）和59．5％（308／518）。（2）取样后胚胎体外短暂培养（0-4h）并不能提高切割取样后小鼠胚胎冷冻-解冻体外培养发育率。（3）切割取样小鼠胚胎在25％甘油＋0．25mol／L蔗糖＋20％的血清为冷冻液，在-100～-110℃左右熏蒸2min快速冷冻后体外培养发育率为73．3％（33／45），切割取样牛胚胎快速冷冻-解冻移植妊娠率为57．1％（4／7）。     

进口奶牛冻胚一步解冻移植效果的研究

冷冻胚胎移植效果不仅能检验胚胎冷冻保存技术的可靠性,且直接影响牛胚移技术的普及应用与商业利益。与传统的胚胎冷冻一解冻处理技术相比较,牛冷冻胚胎解冻后立即移植给受体牛更适于现场操作,简化了移植前胚胎的分步脱甘油处理,具有省时、不需专门仪器设备等优越性。如果直接移植效果接近或超过冷冻精液人工授精技术水平（情期受胎率平均55％）,则对提高冷冻胚胎的利用率,增进国际问的引种,以及形成胚胎产业化、推动胚胎移植技术商业化具有重要意义。     

牛胚胎移植技术发展概况

家畜胚胎移植是家畜胚胎工程的重要组成部分,目前已成为一项比较成熟的生物技术。我国目前在牛的超数排卵、同期发情、胚胎移植、胚胎冷冻、胚胎分割、性别鉴定等技术已与国外基本同步。在正常情况下,平均每头供体牛获可用胚5．6枚,鲜胚移植受胎率为55％～70％,冻胚移植受胎率为45％～60%．每年移植受体牛在5000头以上。     

体外玻璃化冻胚在奶牛改良中的应用前景

本试验用2种不同的方法制作了体外玻璃化冷冻胚胎，通过解冻移植到受体母牛之后，它们的移植受胎率分别达到41.79％和57.78％。两者差异显著（P〈0．05）。     

青海高原地区奶牛胚胎移植效果探讨

在青海高原地区利用加拿大进口的荷斯坦供体牛群中制作的冷冻胚胎.对当地193头杂种牛、黄牛、犏牛、牦牛进行非手术法移植，经妊娠检查，受胎率达42.2％，与国内冻胚移植受胎率相比略偏低，其影响受胎率的因素有受体牛营养、环境等。     

影响绵羊冻胚移植妊娠率的因素分析

本研究对胚胎质量、胚胎移植数量、胚胎发育阶段、冷冻方法、移植方法及饲养管理水平等因素对移植产羔率的影响进行研究,以提高绵羊胚胎移植效率。结果表明:采用1.5mol/L乙二醇做冷冻保护剂对胚胎进行常规冷冻,冻前A级胚胎冷冻/解冻后可用胚率、存活率、降级率均高于B级,差异极显著(P<0.01)。移植1枚冷冻解冻后A级、B级或2枚(A+B)胚胎的受体移植产羔率分别为44.48%、46.84%和44.81%。经χ2检验分析,三者之间无显著差异(P>0.05)。移植双胚的受体双羔率为22(%53/241),以胚胎为单位计算,产羔率仅为33.40%。A级囊胚和桑椹胚的产羔率之间无显著差异(P>0.05)。1.5 mol/L乙二醇常规冷冻保存的体内胚胎移植产羔率高于EFS40玻璃化冷冻保存,但经统计学分析,二者无显著差异(P>0.05)。子宫手术移植和腹腔内窥镜法子宫移植之间产羔率不具统计学差异(P>0.05)。在牧场条件下大规模移植的平均产羔率为43.95%,范围在20%￣70%之间,受体饲养管理水平对绵羊移植产羔率有较大影响。     

The transfer of fresh and frozen embryos in an elite swamp buffalo herd

To investigate the development of fresh and frozen swamp buffalo embryos after transfer to synchronized recipients, 14 fresh embryos and 28 frozen embryos, collected from Thai swamp buffalo cows of an elite herd at the Surin Breeding Center, were transferred nonsurgically to 31 synchronized recipients buffalo cows. One fresh embryo was transferred to each of 14 recipients. Twenty eight frozen embryos were transferred to 17 recipients of which 7 cows received I embryo, 9 cows received 2 embryos and 1 cow received 3 embryos. Pregnancy was diagnosed by real time B-mode ultrasonography one month after transfer and confirmed by rectal palpation one and two months later. The pregnant cows were kept under observation until calving. The results of fresh embryo transfer showed that 5/14 (35.7%) were pregnant after 30 days, 4/14 (28.6%) remained pregnant until the 3rd month and 2/14 (14.3%) calved. With the frozen embryos, only one cow which received three embryos became pregnant and remained so for 3 months although the embryo did not survive to full term. The overall pregnancy rate using frozen embryos was 5.9% (1/17). The study demonstrated the possibility of performing embryo transfer in elite buffalo herds for genetic improvement, however the use of frozen embryos needed further investigation.     

Sex and PRNP genotype determination in preimplantation caprine embryos

The objective of this study was to test the accuracy of genotype diagnosis after whole amplification of DNA extracted from biopsies obtained by trimming goat embryos and to evaluate the viability of biopsied embryos after vitrification/warming and transfer. Whole genome amplification (WGA) was performed using Multiple Displacement Amplification (MDA). Sex and prion protein (PRNP) genotypes were determined. Sex diagnosis was carried out by PCR amplification of ZFX/ZFY and Y chromosome-specific sequences. Prion protein genotype determination was performed on codons 142, 154, 211, 222 and 240. Embryos were collected at day 7 after oestrus and biopsied either immediately after collection (blastocysts and expanded blastocysts) or after 24 h of in vitro culture (compacted morulae). Biopsied embryos were frozen by vitrification. Vitrified whole embryos were kept as control. DNA of biopsies was extracted and amplified using MDA. Sex diagnosis was efficient for 97.4% of biopsies and PRNP genotyping was determined in 78.7% of biopsies. After embryo transfer, no significant difference was observed in kidding rate between biopsied and vitrified control embryos, whereas embryo survival rate was different between biopsied and whole vitrified embryos (p = 0.032). At birth, 100% of diagnosed sex and 98.2% of predetermined codons were correct. Offspring PRNP profiles were in agreement with parental genotype. Whole genome amplification with MDA kit coupled with sex diagnosis and PRNP genotype predetermination are very accurate techniques to genotype goat embryos before transfer. These novel results allow us to plan selection of scrapie-resistant genotypes and kid sex before transfer of cryopreserved embryo.     

小鼠胚胎的玻璃化冷冻研究

用不同冷冻载体(玻璃管、塑料管和0.25 mL细管)及不同冷冻方法(程序化冷冻和玻璃化冷冻)对小鼠3.5 d~4 d桑椹胚和囊胚进行冷冻保存,并与不做任何冷冻保存处理直接培养进行对比。结果表明,使用玻璃管、塑料管和0.25 mL细管作为胚胎的承载材料进行玻璃化冷冻,效果差异不显著;采用程序化冷冻与OPS玻璃化冷冻法,对小鼠胚胎进行冷冻保存可以取得较好的结果。从而得出,用不同材质的冷冻载体进行玻璃化冷冻,可以获得与程序化冷冻相同的良好效果。     

Cryosurvival and pregnancy rates: One‐step protocol for freezing–thawing Shangri‐la Yak (Bos grunniens) Embryos

The yak is one of the most important and economically useful animals for highlanders. The decline in the yak population requires effective measures for the conservation and multiplication of elite germplasm. A standardized protocol will simplify the freezing and warming of yak embryos in straw and facilitate embryo transfer. In this work, we investigated a one‐step protocol that uses a stable basal medium, which comprised a warming medium (1.08 M sucrose) and a freezing medium (EFS40). We also assessed the effects of the new transfer method on embryo survival. A total of 145 yak frozen embryos were thawed in a standard medium system. The one‐step protocol led to a high recovery percentage (84.93) of yak embryos that survived vitrification and warming. The in vitro survival rates of these embryos significantly different from those of embryos frozen–thawed via the conventional method. The 95 embryos frozen–thawed via our one‐step protocol were then implanted in selected recipients. Thirty‐six singleton pregnancies were established. In conclusion, the proposed one‐step method is a simple, safe, and standardized freezing–thawing protocol that ensures embryo survival and quality under field conditions. This study establishes new possibilities for the widespread use of embryo transfer in yaks.     

白绒山羊性控胚胎生产及移植应用研究初探

应用X性控冷冻精液对超排供体白绒山羊进行人工授精,生产性控胚胎并进行鲜胚移植。结果发现,①用性控冻精和鲜精输精的供体母羊,平均卵子回收数分别为13.3和12.5枚,其中性控冻精组受精卵比例为29.6%（55/186）,极显著低于鲜精组94.0%（281/299）的比例（P<0.01）;②性控胚胎和普通胚胎移植受体母羊产羔率分别为42.2% 和58.1% ,二者差异极显著（P<0.01）;③性控胚胎移植受体母羊所产羔羊雌性所占百分数为100%,极显著高于普通胚胎移植（P<0.01）。表明通过白绒山羊X、Y精子分离、人工授精生产性控胚胎,同时结合胚胎移植技术,可以达到按照生产实际中的意愿得到性别控制子代白绒山羊的目的。     

绵羊玻璃化冷冻胚胎直接移植试验研究

应用EFS40玻璃化液对6.5～7日龄的绵羊胚胎进行玻璃化冷冻及解冻后直接移植试验.结果:桑椹胚、囊胚冷冻解冻后移植的妊娠率分别为37.50%(3/8)和54.55%(6/11),胚胎存活率分别为33.33%(3/9)和50.00%(6/12),差异均不显著(P＞0.05);胚胎解冻后用0.5 mol/L蔗糖脱防冻剂与直接用胚胎存放液脱除防冻剂的妊娠率分别为44.44%(4/9)和50.00%(5/10),胚胎存活率分别为40.00%(4/10)、45.45%(5/11)差异不显著(P＞0.05);10枚解冻后的胚胎细管内脱防冻剂后,直接装管移植给8只受体,妊娠率为50.00%(4/8),胚胎成活率为40.00%(4/10),与同期常规冷冻解冻组相比无显著差异(P＞0.05).     

Effect of semen and donor factors on multiple ovulation and embryo transfer (MOET) in sheep

Multiple ovulation and embryo transfer (MOET) is an important tool in the sheep industry for increasing numbers of genetically superior individuals. The objective of this study was to evaluate the effect of semen source (frozen or fresh), the number of embryo collection procedures for each donor (NECP), the season in which embryo transfer and collection was performed, and the age and breed of the donor, on the number of recovered embryos and pregnancy rates after embryo transfer. The Alamos Genetics’ flushing station database was used. This consisted of 140 embryo collection procedures, from 53 Dorper and White Dorper sheep donors, aged between one and eight years, totalling 1,200 collected embryos. Neither the number of retrieved embryos nor the pregnancy rate was affected by the semen preservation method (fresh or frozen), NECP or the age and breed of donor. The season did not affect the number of collected embryos but had a significant effect (p < 0.05) on the recipient pregnancy rate, with higher pregnancy rates reported in the winter (65.57% ± 25.33%) compared with spring (37.11% ± 33.27%), summer (29.95% ± 28.33%) or autumn (35.03% ± 31.66%). There is an estimated increase of 98.4% and 71.5% of embryos recovered in the spring and summer seasons, respectively, when winter is used as reference. The survival of embryos is significantly higher when implanted during the breeding season, more specifically in winter. Embryo collection can be carried out throughout the year in sheep, but there may be a marginal advantage in the use of superovulation and fresh embryo transfer programmes in the autumn and winter.     

湖羊胚胎和精液超低温保存效果的评价

【目的】 检验长期保存在国家家畜基因库的湖羊冷冻胚胎和冷冻精液的质量,评价超低温冷冻保存技术保种的效果。【方法】 对保存于国家家畜基因库的冷冻胚胎（保存20年）和冷冻精液（保存20和30年）进行复苏,进行相应鉴定后分别进行胚胎移植和人工授精,同时以0年冷冻精液和新鲜精液为对照,测定其受胎率和后代的羔皮性能、生长性能及繁殖性能,检验保存效果。【结果】 本试验共解冻胚胎36枚,其中,A级胚胎22枚,B级胚胎7枚,C级胚胎5枚,D级胚胎2枚,冷冻胚胎复苏利用率达到94.44%（34/36）,A级胚胎率达到61.11%（22/36）,移植34枚,产羔17只,冷冻胚胎复苏移植产羔率达50.00%;保存30年的冷冻精液复苏后平均精子活力达35%,受胎率为58.57%,平均产羔1.90只;保存20年冷冻精液复苏后平均精子活力达31%,受胎率为53.66%,平均产羔1.95只;胚胎复苏移植羊、30和20年冷冻精液后代体型外貌鉴定均符合纯种湖羊特征,没有畸形个体;羔羊一级羔皮率分别是31.25%、42.03%和23.81%,保持了当年湖羊的羔皮特性;生长发育及繁殖性能与现有湖羊群体性能基本保持一致。【结论】 利用超低温冷冻技术长期保存湖羊冷冻胚胎和冷冻精液的方法是可行的,对地方品种保种具有重要意义。     

高寒牧区无角陶赛特羊胚胎移植试验及其后裔的生长发育指标测定

在环青海湖地区的共和县引进澳大利亚无角陶赛特羊胚胎,以当地青海半细毛羊为受体,进行胚胎移植,结果移植冻胚60枚,产羔28只,移植妊娠率为46.67%,羔羊成活率为100%。羔羊生长发育指标测定结果表明,胚胎移植羔羊平均出生重3.25kg,4月龄前生长发育最为迅速,平均日增重达205.96g,6月龄平均体重32.47kg。