不同冷冻方法对水牛体外生产胚胎冷冻效果的研究

本研究采用不同来源的体外生产胚胎(屠宰场卵巢IVF胚胎,OPU-IVF胚胎,SCNT胚胎),系统研究了不同冷冻方法对水牛体外生产胚胎冷冻效果的影响,以完善水牛体外生产胚胎冷冻方法,进一步提高胚胎冷冻效果。试验选用6～7日龄囊胚分别用不同的冷冻液和不同冷冻方法进行胚胎冷冻。玻璃化冷冻液分别为40%EG、25%EG+25%DMSO和20%EG+20%DMSO+0.5 mol/L蔗糖;程序化冷冻液分别为10%甘油和0.05 mol/L海藻糖+1.8 mol/L EG+0.4%BSA。结果表明:(1)在玻璃化冷冻中,无论何种胚胎不同冷冻液的冷冻效果有明显的差异,但均以20%EG+20%DMSO+0.5 mol/L蔗糖作为冷冻液的冷冻效果最好,并且高于程序化冷冻的存活率;而对于程序化冷冻,用10%甘油作为冷冻液,屠宰场卵巢IVF胚胎的冷冻后存活率略高于用0.05 mol/L海藻糖+1.8 mol/L EG+0.4%BSA的冷冻后存活率,但差异不显著(P>0.05)。(2)VF胚胎利用程序化冷冻胚胎解冻后,在0～24 h内有76.5%胚胎复活,高于玻璃化冷冻的复苏率(48.9%)(P<0.05);而与此相反,在24～48 h内,玻璃化冷冻胚胎的复苏率(42.6%)则高于程序化冷冻(23.5%)(P<0.05)。综上所述,各种来源的水牛体外生产胚胎均可进行冷冻保存,应用玻璃化冷冻的效果好于程序化冷冻,且以20%EG+20%DMSO+0.5 mol/L蔗糖作为冷冻液进行玻璃化冷冻效果最好,但程序化冷冻后的胚胎复苏速度明显快于玻璃化冷冻的速度。     

ε-聚赖氨酸对小鼠胚胎玻璃化冷冻的影响

为研究ε-聚赖氨酸(ε-PL)对小鼠胚胎玻璃化冷冻效果的影响,探讨冷冻保护剂ε-PL适宜的浓度和平衡时间,试验采用了不同质量浓度的ε-PL(20,30,40 g/L)及不同的平衡时间(0.5,1.0,2.0 min)对小鼠囊胚进行玻璃化冷冻,统计胚胎解冻后存活率及孵化率。结果表明:在冷冻液中添加30 g/L的ε-PL,平衡时间为1.0 min时,冷冻效果最好,囊胚胚胎的存活率达91.2%,孵化率达89.7%。因此,ε-PL可以作为一种高分子非渗透性保护剂对胚胎玻璃化冷冻起保护作用。     

牛胚胎玻璃化超快速冷冻一步法移植试验

应用 30 %乙二醇 0 .3mol/ L 蔗糖 - m- PBS液 (VS1)、30 %乙二醇 0 .3m ol/ L 蔗糖 5 %葡聚糖 (T- 5 0 0 ) - m-PBS液 (VS2 )、30 %乙二醇 0 .3mol/ L蔗糖 10 %葡聚糖 (T- 5 0 0 ) - m- PBS液 (VS3)玻璃化超快速冷冻奶牛胚胎 ,一步法移植。结果显示 ,VS1、VS2、VS3组玻璃化超快速冷冻奶牛胚胎解冻后的形态正常率分别为 10 0 % (2 0 / 2 0 )、95 %(19/ 2 0 )和 5 5 .6 % (5 / 9) ;培养存活率分别为 70 % (14 / 2 0 )、75 % (15 / 2 0 )和 2 2 .2 % (2 / 9) ;囊胚孵化率分别为 0、2 0 % (4/2 0 )和 0。VS1、VS2组玻璃化冷冻奶牛胚胎解冻后的形态正常率极显著地高于 VS3组 (P<0 .0 1) ;VS1、VS2组玻璃化冷冻奶牛胚胎解冻后的培养存活率分别显著 (P<0 .0 5 )和极显著 (P<0 .0 1)地高于 VS3组。 VS1组冷冻的胚胎经一步法移植了 5头 (1枚 /头 ) ,未获得妊娠 (0 / 5 ) ;VS2组冷冻的胚胎用一步法移植了 10头 (1枚 /头 ) ,结果获得 2头妊娠(2 / 10 ) ,并产下 2头正常犊牛。     

ε-聚赖氨酸在牛胚胎玻璃化冷冻中的保护作用

通过研究ε-聚赖氨酸(ε-PL)对牛胚胎玻璃化冷冻的影响,探讨ε-PL在牛胚胎玻璃化冷冻中的保护作用,为优化牛胚胎玻璃化冷冻技术提供依据。单独以ε-PL作为冷冻保护剂,其质量浓度为20、35、50g/L,分别进行胚胎冷冻,结果均不能取得成活胚胎,说明ε-PL不能单独作为冷冻保护剂使用。在传统玻璃化冷冻剂中分别添加质量浓度为10、20、30g/L的ε-PL进行胚胎冷冻,结果发现添加ε-PL之后的冷冻保护液具有更好的保护效果,且ε-PL质量浓度为20g/L时效果最好,囊胚解冻孵化率可达42.37%。进一步探索了用该冷冻保护剂,缓冲时间分别为5、7.5、10min进行胚胎冷冻的效果,结果发现缓冲时间为7.5 min取得最高孵化率,孵化率为46.58%。结果表明,ε-PL对牛胚胎玻璃化冷冻具有一定的保护作用,且适当延长缓冲时间可取得更好的冷冻效果。     

猪孤雌激活4-细胞胚胎玻璃化冷冻保存效果的研究

本实验旨在研究猪孤雌激活4-细胞胚胎的冷冻保存效果。胚胎采用Cryotop法进行玻璃化冷冻保存,解冻后分析其存活率、胞内活性氧(ROS)和谷胱甘肽(GSH)水平、囊胚发育率及囊胚质量。结果表明:冷冻胚胎体外恢复2 h的存活率与新鲜胚胎无明显差异(100%vs.96.8%,P0.05),但当其培养时间达到24 h时,存活率显著下降至88.3%(P0.05)。另外,玻璃化冷冻导致胚胎内ROS水平显著升高(P0.05),GSH水平显著下降(P0.05)。与新鲜胚胎相比,冷冻胚胎获得的囊胚发育率明显降低(59.8%vs.26.4%,P0.05),但囊胚的凋亡细胞率、内细胞团数、滋养层细胞数及总细胞数均无明显差异(P0.05)。结果显示,玻璃化冷冻猪孤雌激活4-细胞胚胎导致其存活、氧化还原能力和囊胚发育下降,但仍能获得较高质量的囊胚。     

2-氨基乙基联苯基硼酸酯对玻璃化冷冻牛成熟卵母细胞发育能力的影响

【目的】研究内质网IP3受体(IP3R)抑制剂2-氨基乙基联苯基硼酸酯(2-APB)对玻璃化冷冻-解冻牛MⅡ期卵母细胞发育能力的影响。【方法】将体外成熟24 h的卵丘-卵母细胞复合体(COCs)用0.1%透明质酸酶消化去除卵丘颗粒细胞,获得的卵母细胞分为对照组和2-APB组,采用OPS法进行玻璃化冷冻,对照组直接进行玻璃化冷冻,2-APB组在玻璃化冷冻前先用10μmol/L 2-APB预处理10 min。2 d后解冻卵母细胞,统计解冻后(0 h)及恢复培养2 h时卵母细胞的存活率,用FITC-PNA荧光探针检测恢复培养2 h时卵母细胞皮质颗粒(CG)的分布,用2′,7′-DCHFDA和Cell Tracker Blue CMF2HC分别检测胞内活性氧(ROS)和谷胱甘肽(GSH)含量。将成熟培养24 h未冷冻卵母细胞(Fresh组),与解冻后恢复2 h的对照组和2-APB组卵母细胞同时进行孤雌激活,于培养的第2、7天分别统计孤雌胚胎的卵裂率和囊胚率。【结果】与对照组相比,2-APB组冷冻-解冻卵母细胞0和2 h的存活率均显著增加(P<0.05);2-APB组皮质颗粒皮质区分布比例...     

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).     

不同冷冻和解冻方法对小鼠桑椹胚发育的影响

本试验以2种程序化冷冻液和2种玻璃化冷冻液对昆明白系小鼠的桑椹胚进行细管法冷冻保存,比较程序化冷冻-管外解冻和玻璃化冷冻-管内解冻对胚胎体内、外发育的影响。胚胎体外培养结果表明:玻璃化冷冻组及程序化冷冻组胚胎发育率(95.3%￣95.8%,98.9%)无显著(P>0.05)差异。将程序化冷冻、EFS30玻璃化冷冻以及新鲜的胚胎各168枚移植给假孕受体鼠,妊娠受体产活仔率各组间相比(50.8%,58.3%,54.9%)无显著性(P>0.05)差异。结果证明,玻璃化冷冻保存的胚胎管内解冻效果好,为生产中家畜的胚胎移植提供了理论和技术参考。     

水牛徒手克隆胚胎培养及冷冻条件的优化

本试验利用微滴、微穴和平板培养系统对徒手克隆(hand-made clone,HMC)重组胚进行体外培养;采用了40% EG(ethylene glycol,EG)、25% EG＋25% DMSO(dimethylsulphoxide,DMSO) 和20% EG＋20% DMSO＋0.5 mol/L蔗糖作为玻璃化冷冻液对HMC囊胚进行了超低温冷冻;并且比较了HMC与传统核移植的胚胎生产效率及囊胚冷冻存活率。结果表明,微穴系统的卵裂率要显著高于平板系统(P<0.05),极显著高于微滴系统(P<0.01);且微穴系统的囊胚率(40.0%)极显著高于平板(19.8%)和微滴系统(8.3%)(P<0.01)。采用20% EG＋20% DMSO＋0.5 mol/L蔗糖作为冷冻保护剂时HMC囊胚存活率极显著高于40% EG(P<0.01);HMC重组胚的融合率和囊胚率均高于传统核移植法(P<0.05;P<0.01),而HMC囊胚的冷冻存活率与传统核移植生产的囊胚没有显著差异。以上结果说明水牛HMC可以替代传统核移植法生产克隆胚胎,微穴体系最适合水牛HMC胚胎的体外培养,且采用20% EG＋20% DMSO＋0.5 mol/L蔗糖对HMC囊胚进行玻璃化冷冻可以取得良好的冷冻效果。     

影响山羊胚胎冷冻效果因素的研究

分别用浓度为1.5 mol/L的乙二醇(EG),1.5 mol/L 1,2-丙二醇(PROH)和1.5 mol/L甘油为冷冻保护液对山羊胚胎进行常规冷冻保存,结果三者对山羊胚胎的冷冻保护效果无显著差异,其中以1.5 mol/L EG的冷冻保护效果为佳。以EFS40为玻璃化液对山羊胚胎进行细管法和OPS法玻璃化冷冻,其结果与常规冷冻间差异不显著,表明常规冷冻法、玻璃化细管法和OPS法均可用于山羊胚胎的冷冻保存。采用25℃和37℃水浴对常规冷冻和玻璃化冷冻后的山羊胚胎进行解冻,从解冻后的发育效果看,二者间无显著差异,但37℃水浴解冻后的胚胎发育效果略好于25℃。还比较了玻璃化液EFS40中添加FCS和BSA后与不添加其他成分的EFS40对胚胎冷冻保护效果的影响,结果表明添加BSA的EFS40的冷冻保护效果显著地高于不添加其他成分的EFS40,但与添加FCS的EFS40间不存在统计学上的差异。     

Conventional freezing of in vitro-produced and biopsied bovine blastocysts in the presence of a low concentration of glycerol and sucrose

The purpose of this study was to develop a practical cryopreservation method for in vitro-produced (IVP) and sex-predetermined bovine blastocysts that will be applicable to direct transfer of the post-thaw embryos. Blastocysts were harvested 7 days after IVF and allocated to either an intact or biopsy group. The cryoprotective solution contained 0.7 M glycerol and 0, 0.05 or 0.1 M sucrose. Slow cooling at a rate of -0.5 C/min was terminated at -25, -30, or -35 C, and rapid cooling in liquid nitrogen was followed. After one-step thawing and dilution, the IVP blastocysts were cultured for 3 days to assess their survival. The post-thaw survival rate of intact blastocysts after termination of slow cooling at -30 C in 0.7 M glycerol plus 0.1 M sucrose (96.2%) was significantly higher than that at -25 C in 0.7 M glycerol alone (44.4%). The post-thaw survival rate of biopsied bovine blastocysts after termination of slow cooling at -25 C in 0.7 M glycerol alone (53.8%) tended to be lower than that at -25 C in 0.7 M glycerol plus 0.05 M sucrose (91.3%) or -30 C in 0.7 M glycerol plus 0.1 M sucrose (92.3%). Thus, addition of a small amount of sucrose to 0.7 M glycerol cryoprotective solution shortened the process of slow cooling for both the intact and biopsied bovine embryos. Judged from the survival levels in vitro after thawing and one-step dilution of embryos (>80%), this is an improved method of cryopreservation for subsequent direct transfer of IVP and biopsied bovine blastocysts.     

Cryopreservation and sexing of in vivo- and in vitro-produced bovine embryos for their practical use

My research awarded includes contributions to cryopreservation and sexing of bovine embryos produced in vitro and in vivo, as follows; (1) In vivo-derived morulae and blastocysts were cryopreserved in the presence of 10% glycerol, and the embryos were transferred into recipients after two-step dilution of glycerol in straw, with a practically acceptable pregnancy rate. (2) The survival rate of 16-cell stage embryos frozen in the medium with ethylene glycol was higher than that with DMSO or 1,2-propanediol. Addition of linoleic acid-albumin to culture medium enhanced the survival rate of post-thaw bovine 16-cell stage in vitro-produced (IVP) embryos. (3) Polarization of cytoplasmic lipid droplets by centrifugation of 2-cell stage embryos was found effective to increase freezing tolerance in 16-cell stage embryos developed from the centrifuged embryos, because blastomeres of 16-cell stage embryos were mostly lipid-free. (4) The usefulness of gel-loading tip (GL-Tip) as a container for ultra-rapid vitrification was demonstrated in IVP embryos from 2-cell to blastocyst stages, with a higher in vitro survival than the conventional two-step freezing. (5) PCR analysis for sexing of in vivo-derived Day-7 embryos indicated that male embryos developed faster and graded higher than female embryos. But such correlation between genetic sex and embryonic development was not found in IVP embryos obtained from individual cows. (6) Addition of 0.1-1.0% deproteinized hemodialysate product from calf blood to culture medium increased the producing efficiency of demi-embryos with good quality. Female embryos rather than male embryos required a longer time to repair after bisection. (7) In vivo-derived bovine embryos after biopsy for sexing by PCR analysis and subsequent vitrification using GL-Tips are available to practical use in the field. (8) Introduction of primer extension preamplification-PCR and purification of DNA product before standard sexing PCR of biopsy samples from Day 3-4 in vitro-derived embryos allowed accurate sex determination, and Day-7 blastocysts developed from Day 3-4 embryos were cryopreserved by GL-Tip vitrification without a loss of their viability. Thus the field application of bovine embryo transfer is in part supported by improvements of technologies in embryo cryopreservation and sex pre-determination.     

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。     

牛胚胎冷冻与移植受胎技术研究

本文进行了牛胚胎冷冻与移植受胎技术研究。摸索出了提高牛冷冻胚胎成活率和移植受胎率的综合配套技术。牛冷冻胚胎解冻成活率达８４．４％（５７／１２２）。其中１９９６年移植受胎率达到５５．６％（２０／３６）。Ａ级胚胎达以７７．８％（２１／２７），并在我国首次获得中国荷斯坦牛冷冻胚胎的二分胚的同孵孪生牛犊。且达到４２．９％（３／７）移植受胎率。     

Effects of resveratrol treatment on mitochondria and subsequent embryonic development of bovine blastocysts cryopreserved by slow freezing

This study evaluated the effects of cryopreservation by slow freezing on the mitochondrial function, DNA integrity, and developmental ability of bovine embryos and examined whether resveratrol treatment of the frozen‐thawed blastocysts improved embryonic viability. In vitro produced bovine embryos were subjected to slow freezing. After thawing, the ATP content and mitochondrial DNA integrity (mtDNA), determined by real‐time PCR targeting short and long mitochondrial sequences, was found to be lower in frozen‐thawed embryos than in fresh embryos, and mtDNA copy number was significantly reduced during the 24‐hr incubation post warming. Furthermore, immunostaining against double‐strand DNA revealed DNA damage in frozen‐thawed embryos. When frozen‐thawed embryos were incubated in the medium containing 0.5 µM resveratrol, SIRT1 expression, and survival rate of the embryos significantly improved compared with the vehicle‐treated embryos. In addition, cell‐free mtDNA content in medium was higher in case of resveratrol‐treated embryos than of vehicle‐treated embryos. In conclusion, slow freezing affects mitochondrial integrity and function in the blastocysts. In the frozen‐thawed embryos, mitochondria were removed during post‐thawing incubation and resveratrol enhanced the process, resulting in improved survivability of the embryos.     

Recent progress in bovine in vitro-derived embryo cryotolerance: Impact of in vitro culture systems,advances in cryopreservation and future considerations

Cryopreservation of in vitro-derived bovine embryos is a crucial step for the widespread reproduction and conservation of valuable high-merit animals. Given the current popularity of bovine in vitro embryo production (IVP), there is a demand for a highly efficient ultra-low temperature storage method in order to maximize donor ovum pickup (OPU) turn-over, recipient availability/utilization and domestic/overseas commercial trading opportunities. However, IVP bovine embryos are still very sensitive to chilling and cryopreservation, and despite recent progress, a convenient (simple and robust) protocol has not yet been developed. At the moment, there are two methods for bovine IVP embryo cryopreservation: slow programmable freezing and vitrification. Both of the aforementioned techniques have pros and cons. While controlled-rate slow cooling can easily be adapted for direct transfer (DT), ice crystal formation remains an issue. On the other hand, vitrification solved this problem but the possibility of successful DT commercial incorporation remains to be determined. Moreover, simplification of the vitrification protocol (including warming) through the use of an in-straw dilution without the use of a microscope is a prerequisite for its use under farm conditions. This review summarizes the bovine IVP embryo cryopreservation achievements, strengths and limitations of both freezing systems and prospective improvements to enhance cryosurvival, as well as perspectives on future directions of this assisted reproductive technology.     

Production of Lambs After the Transfer of Fresh and Cryopreserved in vitro Produced Embryos from Prepubertal Lamb Oocytes and Unsorted and Sex-sorted Frozen-thawed Spermatozoa

Cumulus-oocyte complexes from hormone-stimulated 3-4-week-old (n=43) and 6-7-week-old (n=12) prepubertal lambs were matured in vitro and incubated with unsorted, or X- or Y-spermatozoa separated with a high-speed cell sorter (SX MoFlo)frozen-thawed. Presumptive zygotes were then cultured to the blastocyst stage, and transferred to recipients fresh or after cryopreservation (frozen). Oocyte cleavage was higher (p <0.05) with unsorted (515/926, 55.6%) than X- or Y-spermatozoa (261/672, 38.8% and 229/651, 35.2%, respectively) and blastocyst formation (% zygotes) by Day 9 of in vitro culture was lower (p <0.05) for X- (102/261, 39.1%) than unsorted spermatozoa (249/515, 48.3%), but did not differ between Y-spermatozoa (103/229, 45.0%) and unsorted spermatozoa, or between X- and Y-spermatozoa (p >0.05). For fresh embryos, survival to term was 50.0% (3/6) for unsorted, 0.0% (0/6) for X- and 16.7% (1/6) for Y-spermatozoa-derived embryos (p >0.05), and for frozen embryos was 4.0% (2/50) for unsorted, 9.1% (2/22) for X- and 2.9% (1/34) Y-spermatozoa-derived embryos (p >0.05). Of the two lambs born from X-spermatozoa-derived embryos, one was female (50%), and from the two Y-spermatozoa-derived lambs, both were male (100%), demonstrating that lambs can be produced after the transfer of fresh and cryopreserved IVP embryos derived from prepubertal lamb oocytes and frozen-thawed sex-sorted sperm.     

In vitro development of IVF-derived bovine embryos following cytoplasmic microinjection for the episomal expression of the IGF2 gene

Important genomic imprinting changes usually occur following the in vitro production (IVP) of bovine embryos, especially in the imprinting pattern of components of the IGF system. This study aimed to evaluate the effects of a transient episomal overexpression of the IGF2 gene in bovine IVP embryos following embryo cytoplasmic microinjection (CMI) at the 1-cell stage on embryo survival, early and late developmental kinetics and morphological quality up to Day 7 of development. Selected cumulus–oocyte complexes (COCs) were matured and fertilized in vitro and subsequently segregated into six experimental groups: non-CMI control group and five CMI groups at increasing doses (0, 10, 20, 40 and 80 ng/μl) of a GFP vector built for the episomal expression of bovine IGF2. Zygote CMI was effective in delivering the expression vector into the ooplasm, irrespective of the groups, with 58% of positive GFP fluorescence in Day 7 blastocysts. Considering developmental rates and late embryo kinetics, the 10-ng/μl CMI vector dose promoted a lower blastocyst rate (10.4%), but for blastocysts at more advanced stages of development (93.0% blastocysts and expanded blastocysts), and higher number of cells (116.0 ± 3.0) than non-CMI controls (23.3%, 75.0% and 75.0 ± 6.8 were obtained, respectively). In conclusion, CMI at the 1-cell stage did not compromise subsequent in vitro development of surviving embryos, with the 10-ng/μl group demonstrating a possible growth-promoting effect of the IGF2 gene on embryo development, from the 1-cell to the blastocyst stage.     

Technical note: Vitrification of goat embryos by the open pulled-straw method

The suitability of the open pulled-straw (OPS) method for vitrifying bovine embryos was tested for goat embryos. Of 14 does receiving OPS-vitrified embryos, all became pregnant and 13 (93%) kidded. The corresponding values for an established conventional freezing program were 58% pregnant and 50% (6/12) kidding. Overall embryo survival amounted to 64% (18/28) for OPS-vitrified and 42% (10/24) for conventionally frozen embryos. All differences were statistically significant. It is concluded that OPS vitrification is a suitable method for cryopreserving caprine d-7 blastocysts.     

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。