山羊胚胎冷冻保存技术研究

本研究探讨了不同抗冻保护剂、不同冷冻方法、玻璃化液(EFS40)中添加FCS和BSA,以及冷冻前细胞松驰素B处理对山羊胚胎冷冻保存效果的影响。结果表明,山羊胚胎常规冷冻时以1.5 mol/L EG为抗冻保护剂的保护效果最好,解冻后胚胎发育率为70.59%,孵化率为58.82%;玻璃化冷冻细管法和OPS法以EFS40为保护液的冷冻效果较好,其解冻后胚胎的发育率分别为67.57%和52.94%;EFS40中添加BSA的冷冻保护效果显著地高于不添加其他成分的EFS40;山羊胚胎冷冻前用细胞松驰素B处理,能提高冷冻保存的效果。     

冷冻方法和冷冻保护液对山羊卵母细胞发育的影响

比较了不同冷冻方法和冷冻保护液对山羊卵母细胞冷冻-解冻后体外受精和激活后的发育效果.结果表明,开放式拉长塑料细管(OPS)法的效果好于玻璃化细管法和常规冷冻法;在OPS法中,20%EG 20%DMSO对卵母细胞的冷冻保护效果好于EDFS40,而在玻璃化细管法冷冻中,则是EDFS40好于20%EG 20%DMSO.对于常规冷冻法而言,则是1.5mol/L EG的冷冻效果好于1.5mol/L PROH.     

澳洲波尔山羊胚胎3种冷冻方法对其胚胎移植效果的影响

在25℃室温下,采用细管法(一步法、二步法)和OPS法,以不同浓度的EFS、EDFS为玻璃化冷冻液,对澳洲波尔山羊致密桑椹胚和囊胚进行玻璃化冷冻保存。同时利用1.5mol/LEG为抗冻保护剂对胚胎进行常规法冷冻保存。分别将上述3种方法冷冻解冻后的胚胎移植于同期发情后6~7d的云南黑山羊受体。结果表明,细管法胚胎玻璃化冷冻保存效果均以EFS40组为佳,解冻后胚胎移植产羔率分别为40.54%(15/37;一步法)和51.35%(19/37;二步法)。与新鲜胚胎移植产羔率(52.50%,21/40)和常规法冷冻保存的胚胎移植产羔率(45.16%,14/31)相比无显著性差异(P>0.05)。另外,用EDFS30玻璃化溶液,OPS法冷冻解冻后的胚胎移植产羔率高达51.43%(18/35),为整个玻璃化冷冻试验的最佳值。玻璃化冷冻方法简便、迅速,无论是细管法还是OPS法均获得了比较理想的胚胎移植效果。     

小鼠4-细胞胚胎OPS法冷冻保存技术

在25℃室温和37℃恒温台条件下，利用玻璃化冷冻溶液EFS30、EFS40、EDFS30或EDFS40，对小鼠4-细胞胚胎进行玻璃化冷冻保存．以解冻后培养72h的囊胚发育率为其体外发育能力的考核指标，同时对解冻后培养1～3h的胚胎进行移植以判定其体内发育潜力。开放式拉长塑料细管（OPS）二步法冷冻保存，即胚胎首先移入预处理液（10％EG或10％EG＋10％DMSO）中平衡30s，再移入玻璃化溶液中洗涤后吸入OPS管中，分别经35、30或25s后直接投入液氮中冷冻保存。一步法冷冻保存则无需预处理液处理。结果表明，小鼠胚胎4-细胞一步法和二步法冷冻后囊胚最高发育率分别为87．7％和88.6％，与对照组（93.0％）差异不显著（P〉0．05）。利用最佳冷冻组获得的143枚胚胎移植于12只假妊娠50～60h的受体母鼠，结果有4只妊娠产仔17只，妊娠产仔率为42．5％（17／40），与对照组59．4％（19／32）差并不显著（P〉0.05）。     

小鼠2-细胞胚胎细管法和OPS法玻璃化冷冻保存技术的研究

本试验在室温 (2 0℃和 2 5℃ )条件下 ,利用不同浓度的玻璃化溶液 (EFS和EDFS) ,对小鼠 2 细胞胚胎进行细管法和OPS法玻璃化冷冻保存。在 2 0℃室温条件下 ,用EFS4 0平衡 1min细管一步法冷冻 ,解冻后囊胚发育率仅为35 .0 % ,和新鲜 2 细胞体外培养的对照组 (6 5 .0 % )的差异极显著 (P <0 .0 1)。当 2 细胞胚胎在 10 %EG +10 %D溶液中预处理 5min ,再移入EDFS中平衡 30s二步法冷冻保存 ,解冻后囊胚发育率达 4 7.8%～ 4 8.8% ;当室温升至2 5℃时 ,二步法冷冻保存后 2 细胞的囊胚发育率达到 5 2 .2 % ,与对照组无显著差异 (P >0 .0 5 )。改用OPS二步法EFS30冷冻组保存后的 2 细胞胚胎的囊胚发育率高达 6 2 .2 % ,为试验中的最佳组。用最佳细管法和OPS法冷冻组解冻后培养至囊胚移植给受体母鼠均获得产仔     

昆明小鼠成熟卵母细胞冷冻保存方案的优化与改进

5-6周龄雌性昆明小鼠的成熟卵母细胞,在不同前处理液(10% EG或10% EG 10% DMSO)中平衡5 min,然后在冷冻溶液(EFS30、EFS40、EDFS30或EDFS40)中平衡30 s后进行开放式拉长麦管法(OPS,open pulled straw)和固体表面冷冻法(SSV,solid-surface vitrification)玻璃化冷冻保存研究.试验结果表明:玻璃化冷冻中,多种抗冻保护剂组合使用的效果优于使用单一抗冻保护剂,而且EDFS冷冻液冷冻保存小鼠卵母细胞的效果好于EFS,尤其是EDFS30;SSV法冷冻保存小鼠成熟卵母细胞效果优于OPS法.     

哺乳动物胚胎玻璃化冷冻保存的研究进展

自1985年胚胎玻璃化冷冻(v itrification)保存技术发明以来,玻璃化法先后在小鼠、兔、绵羊、牛、猪、山羊等动物胚胎上获得成功,近年来有关哺乳动物胚胎玻璃化冷冻保存的研究主要集中在冷冻和解冻方法上,相继发明了一些新的玻璃化冷冻方法:冷环玻璃化法(cryo loop)和开放式细管法(open pu lled straw,OPS),并且对解冻后细管内直接脱除防冻剂进行了广泛深入的研究,使得冷冻胚胎移植更易于在生产上推广应用。现将胚胎玻璃化冷冻的原理、冷冻保护剂、冷冻方法、解冻后保护剂脱除方法的最新研究进展作一综述。     

牛GV期卵母细胞冷冻保存后发育潜力的研究

二甲基亚砜(DMSO)、丙二醇(PROH)、乙二醇(EG)和甘油(GL)4种冷冻保护剂程序化冷冻牛GV期卵母细胞的结果表明,EG和PROH的保护效果比GL和DMSO好。4种不同冷冻方法冷冻保存牛GV期卵母细胞,比较解冻后卵母细胞的体外成熟率、受精后卵裂率。结果表明,在程序化冷冻法与细管玻璃化法(Straw)之间的差异不显著(P>0.05),在开放式拉管法(OPS)与毛细玻管法(GMP)之间的差异不显著(P>0.05);但OPS和GMP与程序化冷冻法和Straw之间的差异极显著(P<0.01)。玻璃化冷冻效果优于程序化冷冻。说明GMP和OPS玻璃化冷冻优于Straw玻璃化冷冻。说明可以采用GMP方法冷冻保存牛GV期卵母细胞。     

水牛卵母细胞冷冻保存初步研究

①用EFS30、EFS40、EDFS30、EDFS40四种玻璃化冷冻液对MⅡ期水牛卵母细胞进行毒性试验,结果表明:试验组卵母细胞形态正常率与对照组均无显著性差异(P>0.05);对卵母细胞孤雌激活后EDFS30、EDFS40组的卵裂率与对照组(75.28%)及EFS30、EFS40组差异显著(P<0.05);利用4种冷冻保护剂采用OPS法冷冻保存MⅡ期水牛卵母细胞,其中以EDFS40作为冷冻液时,卵母细胞冷冻解冻后孤雌激活卵裂率最高,达31.60%;以EDFS40作为冷冻液,比较了GMP法和OPS法的冷冻效果,结果表明GMP法冷冻效果好于OPS法。②采用不同预处理时间和平衡时间使用细管法常规冷冻G V期卵母细胞,结果表明预处理5 min、平衡15min组的形态正常率和极体排出率相对较高,分别为72.73%、27.27%。     

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。     

Vitrification of Boer Goat Morulae and Early Blastocysts by Straw and Open-Pulled Straw Method

The aim of this study was to investigate the effects of different vitrification solutions [EFS30 or EFS40 contains 30% (v/v) ethylene glycol (EG), 40% (v/v) EG; EDFS30 or EDFS40 contains 15% (v/v) EG and 15% (v/v) dimethyl sulfoxide (DMSO), 20% (v/v) EG and 20% (v/v) DMSO], equilibrium time during vitrification (0.5-2.5 min) and vitrification protocols [one-step straw, two-step straw and open-pulled straw (OPS)] on in vivo development of vitrified Boer goat morulae and blastocysts after embryo transfer. In the one-step straw method, the lambing rates of vitrified embryos in EFS30 (37.5%), EFS40 (40.5%) or EDFS30 (38.2%) group were similar to that of fresh embryos (57.5%) and conventional freezing method (46.7%) when the equilibrium time was 2 min. In the two-step straw method, the highest lambing rate was obtained when embryos were pretreated with 10% EG for 5 min and then exposed to EFS40 for 2 min (51.4%), showing similar lambing rates compared with fresh embryos (56.1%) or the embryos cryopreserved by conventional freezing method (45.2%). In the OPS method, the lambing rate in EFS40, EDFS30 or EDFS40 groups were similar to that (57.1%) of fresh embryos, or to that (46.0%) of embryos cryopreserved by conventional freezing method. The highest lambing rate (51.4%) of the group of OPS was obtained when the embryos were vitrified with EDFS30. In conclusion, either the two-step straw method in which embryos were pretreated in 10% EG for 5 min and then exposed to EFS40 for 2 min, or the OPS method in which embryos were pretreated in 10% EG + 10% DMSO for 30 s and then exposed to EDFS30 for 25 s was a simple and efficient method for the vitrification of Boer goat morulae and blastocysts.     

Survival Rate and Ultrastructure of Vitrified Bovine in vitro and in vivo Developed Embryos

The capacity of different vitrification media and methods was tested onto in vivo and in vitro produced bovine morula/blastocysts and their ultrastructure and survival studied post-thawing. Two vitrification solutions were finally selected, named 40 ES (40% ethylene glycol in PBS containing 0.5 M sucrose) and 35 EFS (composed of 35% (v/v) ethylene glycol in PBS containing 0.5 M/l sucrose and 30% (w/v) Ficoll 70). The straws were either precooled or not precooled in nitrogen vapour, plunged and stored in LN₂ for 10–25 days, and then thawed in a 20° C waterbath. The content of the straws was rediluted in 1M sucrose solution in PBS and later cocultured with BOEC for 48 h. The overall survival rates for in vitro and in vivo embryos were 36% (12 of 33) and 20% (3 of 15) after 24 h and 21% (7 of 33) and 33% (5 of 15 ) after 48 h. The survival rates for precooled embryos were significantly higher than for not precooled (48% vs 13% after 24 h and 44% vs 4% after 48 h) when tested across vitrification media. The in vitro-produced embryos presented an ultrastructure similar to the pre-freeze state, irrespective of the vitrification media used. The in vivo developed embryos showed a rather modified post-thaw ultrastructure, with clear signs of osmotic changes at both the trophoblastic and embryonic cells. The results indicated that in vitro and in vivo developed bovine embryos can survive vitrification using ethylene glycol as a cryoprotectant.     

牛IVP胚胎玻璃化冷冻技术的研究

作者比较了乙二醇传统冷冻和OPS玻璃化冷冻牛IVP胚胎的效果,目的是改善其冷冻效果,提高IVP胚胎的移植妊娠率。结果表明,玻璃化冷冻后的存活率(D7-71)明显高于D7-71L(P<0.01),D7-72组与D7-71L组(传统冷冻方法)的存活率基本一致(P>0.05)。     

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。     

Stepwise in-straw dilution and direct transfer using open pulled straws (OPS) in the mouse: a potential model for field manipulation of vitrified embryos

In the present study, mouse blastocysts were employed to investigate the feasibility and efficiency of stepwise in-straw dilution and direct transfer using the open pulled straw (OPS) method. In experiment I, the effects of various vitrification solutions (VS) on embryo survival were examined. After thawing, the expanded blastocyst rates (97.59 and 95.05%) and hatching rates (80.48 and 78.95%) achieved in the EDFS30 [15% ethylene glycol (EG), 15% dimethyl sulfoxide (DMSO), Ficoll, and sucrose] and EFS40 [40% EG, Ficoll, and sucrose] groups were no different from those (96.15% and 83.33%) of the control group. However, the rates in the EFS30 [30% EG, Ficoll, and sucrose] (87.80 and 55.43%) and EDFS40 [20% EG, 20% DMSO, Ficoll, and sucrose] (95.69 and 70.97%) groups were significantly lower than those (96.15 and 83.33%) of the control group (P<0.05). In the experiment II, the effects of the volume of VS in the OPS on the survival of embryos after in-straw thawing were investigated. When the length of the VS in the column was less than 1 cm, the in vitro viability of embryos thawed by stepwise in-straw dilution was no different among the experimental and control groups. The embryos could be successfully thawed by immersing the OPS in 0.5 M sucrose for 3 min and then 0.25 M sucrose for 2 min. In experiment III, the effect of immersion time of the OPS in diluent (PBS) on the viability of vitrified embryos was investigated. After in-straw thawing, OPSs were immersed immediately in 1 ml PBS for 0 to 30 min. When the immersion time of the OPSs in PBS was less than 12 min, in vitro development of the in-straw thawed embryos was no different from that of the controls. In experiment IV, in-straw thawed blastocysts were directly transferred to pseudopregnant mice to examine their in vivo developmental viability. The pregnancy (91.67%) and birth rates (42.42%) of embryos in-straw thawed and directly transferred were no different from those of the unvitrified controls (90.90 and 40%) and embryos thawed by the conventional method (84.61 and 46.94%). These results demonstrate that mouse embryos vitrified with OPS could be successfully thawed by stepwise in-straw dilution and transferred directly to a recipient and that this method might be a model for field manipulation of vitrified embryos in farm animals.     

Vitrification of In Vitro-produced Bovine Embryos by Addition of Ethylene Glycol in One-step

The objective of this study was to simplify two-step addition of cryoprotectant for vitrification of bovine embryos by developing a one-step procedure. Survival was calculated as a percentage of non-vitrified controls developed from the same batch of oocytes. In experiment 1, bovine blastocysts were vitrified following one- or two-step addition of cryoprotectant. Exposure of embryos to cryoprotectant in one-step resulted in survival rates not significantly lower (p > 0.1) than those obtained by two-step addition (85% vs 98%, respectively). Based on these results, experiments 2-4 were designed to test one-step addition of cryoprotectant more rigorously. Experiment 2 exposed day 7 blastocysts to 6, 7 or 8 M ethylene glycol for 2.5 or 3.5 min. At 24 h post-vitrification, survival of embryos was similar, irrespective of ethylene glycol concentration or exposure time (6 M 38%, 7 M 51%, 8 M 59%; 2.5 min 54%, 3.5 min 45%). In experiment 3, blastocysts were exposed to 7 M ethylene glycol for shorter times (30 or 60 s); 30 s exposure resulted in decreased survival (8% vs 31%, p < 0.05). Experiment 4 concerned one-step addition of cryoprotectant to day 6 bovine morulae, exposed to 7 M ethylene glycol for 1 or 1.5 min. There was no difference in survival between exposure times of 1 or 1.5 min (28% vs 45%, respectively; p > 0.1). It is unclear why many embryos survive vitrification with one-step addition of cryoprotectant, but others do not. Although, one-step addition of cryoprotectant simplifies the vitrification procedure, survival rates were inadequate for routine cryopreservation of in vitro-produced bovine embryos.