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%。     

玻璃化冷冻对猪MⅡ卵母细胞皮质颗粒分布及其激活后胚胎发育能力的影响

本实验旨在探讨玻璃化冷冻保存对猪MⅡ期卵母细胞皮质颗粒分布和孤雌激活后早期胚胎发育能力的影响。实验将卵母细胞随机分为对照组、毒性实验组和冷冻组。采用EFS40和EDFS40两种玻璃化冷冻液处理,卵母细胞经恢复后对其进行染色,观察皮质颗粒的分布;并对另一部分卵母细胞实施孤雌激活,观察早期胚胎的发育。结果表明:毒性实验组和冷冻组卵母细胞皮质颗粒部分释放、完全释放的比例无显著差异,但均显著高于对照组(P<0.05)。不同毒性处理组和不同冷冻组间对皮质颗粒的分布无显著差异。与毒性实验组相比,冷冻处理组显著降低皮质颗粒在皮质区分布的比例(P<0.05)。EFS40毒性实验组孤雌激活后的存活率、卵裂率、囊胚发育率均显著高于EDFS40毒性实验组(86.6%vs.75.0%)、(61.8%vs.40.7%)、(30.2%vs.23.5%)(P<0.05)。EFS40冷冻组存活率显著高于EDFS40冷冻组,但均显著低于对照组。结果显示,抗冻保护剂处理和玻璃化冷冻均导致猪卵母细胞皮质颗粒释放,与EDFS40相比采用EFS40较适合猪MⅡ期卵母细胞冷冻保存。     

绵羊体外成熟卵母细胞OPS法玻璃化冷冻保存试验

研究以EDFS30为玻璃化冷冻液,以卵母细胞解冻后孤雌激活和体外受精后的卵裂率、囊胚发育率作为评价指标,探讨了以OPS法玻璃化冷冻保存体外成熟绵羊卵母细胞的效果。结果表明:卵母细胞孤雌激活后的卵裂率,冷冻组(64.2%)显著(P<0.05)低于毒性组(76.7%)和对照组(79.1%),而毒性组和对照组无显著(P>0.05)差异;卵母细胞孤雌激活后的囊胚发育率,冷冻组(4.2%)和毒性组(5.8%)均显著(P<0.05)低于对照组(20.2%),毒性组和冷冻组无显著(P>0.05)差异;冷冻组和毒性试验组卵母细胞体外受精后的卵裂率和囊胚发育率(67.6%和7.1%;62.3%和9.1%)均显著低于对照组(78.4%和28.4%)(P<0.05),而毒性组和冷冻组无显著(P>0.05)差异。可见以EDFS30为玻璃化冷冻液,采用OPS法冷冻保存绵羊体外成熟卵母细胞会在一定程度上降低其受精能力和胚胎发育能力。     

小鼠卵母细胞SSV法冷冻保存技术的研究

试验用2种前处理液(10%EG和10%EG 10%DMSO)和4种玻璃化冷冻液(EFS30、EFS40、EDFS30和EDFS40)对小鼠卵母细胞进行玻璃化(SSV)法冷冻保存,研究小鼠卵母细胞冷冻后的发育潜力。结果表明:小鼠未成熟卵母细胞形态正常率最高可达92.3%,成熟率达57.2%;成熟卵母细胞形态正常率最高可达91.5%。     

纽胞松弛素B(CB)对水牛卵母细胞玻璃化冷冻保存的影响

研究主要探讨冷冻前细胞松弛素B(CB)预处理水牛M Ⅱ期卵母细胞对其冷冻效果的影响.以水牛M Ⅱ期的卵母细胞为材料,通过玻璃毛细管(GMP)和玻璃化冷冻液EDS33(16.5%EG 16.5%DMSO Sucrose)对水牛M Ⅱ期的卵母细胞进行两步法玻璃化冷冻保存,即卵母细胞首先放入预平衡液(7.5%EG 7.5%DMSO Sucrose)中平衡3 min,然后再移入玻璃化冷冻液中30 s后装管直接投入液氮.冷冻前卵母细胞随机分为2组,一组用7.5μg/mL CB预处理30 min,另一组未用CB预处理作对照组,然后都冷冻保存.解冻是在蔗糖浓度逐渐降低的解冻液中进行的.解冻后存活的卵母细胞进行孤雌激活,以分裂率和囊胚发育率作为评定卵母细胞冷冻效果的指标.结果发现,GMP组和GMP CB组卵母细胞解冻后的存活率(88.89%、94.20%)和培养后发育到囊胚的比率(8.82%、14.55%)差异均不显著(P＞0.05),但GMP组和GMP CB组存活卵母细胞激活后的分裂率差异显著(32.69%、55.56%,P＜0.05),所以细胞松弛素B预处理可以提高GMP冷冻卵母细胞的发育能力.     

玻璃化冷冻对猪MⅡ期卵母细胞及其DNA的影响

研究旨在筛选最适合猪MⅡ期卵母细胞玻璃化冷冻的冷冻液,并探究玻璃化冷冻对猪MⅡ期卵母细胞DNA的影响。选取目前应用最多的7种冷冻液（分别为1、2、3、4、5、6、7组）,将MⅡ期卵母细胞随机分为8组,其中对照组直接进行孤雌激活,其余7组分别进行7种冷冻液处理后不经液氮冷冻直接于解冻液中解冻,解冻后进行孤雌激活,通过卵裂率、囊胚率和囊胚细胞数的统计结果筛选出最适冷冻液;应用筛选的3种冷冻液,进行猪MⅡ期卵母细胞玻璃化冷冻,解冻后恢复2 h,统计卵母细胞形态正常率,孤雌激活44~48 h统计卵裂率;应用透射电子显微镜观察正常MⅡ期卵母细胞与玻璃化冷冻-复苏后的MⅡ期卵母细胞超微结构的变化;将猪MⅡ期卵母细胞随机分成对照组、冷冻液处理组和冷冻组,应用彗星电泳技术检测玻璃化冷冻对卵母细胞DNA的损伤。结果发现,与对照组相比,除5组卵裂率、1组囊胚率显著降低（P<0.05）外,其余各组卵裂率、囊胚率均差异不显著（P>0.05）;各组间囊胚细胞数均低于对照组,但差异均不显著（P>0.05）,3、6、7组卵裂率和囊胚率较高;玻璃化冷冻-解冻后,7组卵母细胞的形态正常率、卵裂率均显著低于3、6组（P<0.05）,6组卵裂率高于3组;MⅡ期卵母细胞移入预处理液中后可见明显的皱缩,移入冷冻液中迅速脱水,解冻后可见卵母细胞透明带断裂,胞质皱缩、分布不均;透射电子显微镜下,冷冻后猪MⅡ期卵母细胞透明带及细胞膜损伤,微绒毛严重损伤甚至消失,皮质颗粒排列在质膜下且数量减少,脂滴形态破坏、形成空泡,内质网与脂滴的联系损坏,线粒体肿胀、嵴不明显;彗星电泳发现,与对照组相比,冷冻液处理组头部DNA、尾部DNA和Olive尾矩值均差异不显著（P>0.05）,有彗星拖尾现象;冷冻组头部DNA损伤、尾部DNA损伤与Olive尾矩值均显著高于冷冻液处理组和对照组（P<0.05）,有明显彗星拖尾现象。结果表明,以二甲基亚砜（DMSO）和乙二醇（EG）为主要成分的冷冻液适于猪MⅡ期卵母细胞玻璃化冷冻;玻璃化冷冻对猪MⅡ期卵母细胞超微结构及其DNA存在一定损伤作用,其损伤机制有待进一步研究。     

云岭山羊卵母细胞玻璃化冷冻的研究

试验以屠宰场云岭黑山羊卵巢卵母细胞为材料,研究其玻璃化冷冻的效果。试验中选用20% EG+20% DMSO为冷冻液、冷冻环为载体,以20 s、40 s玻璃化时间冷冻GV和MⅡ期的卵母细胞。结果表明,GV期卵母细胞的形态正常率、成熟率和卵裂率都很低,且解冻成熟培养后冷冻组的成熟率和卵裂率极显著低于对照组(P<0.01)。而MⅡ期卵母细胞冷冻效果较好,毒性试验组和冷冻组形态正常率分别为91.1%和83.3%,明显高于GV期;孤雌激活后毒性组卵裂率与对照组无显著性差异(P>0.05),冷冻组的卵裂率显著低于对照组(P<0.05)。用20 s、40 s玻璃化时间冷冻的卵母细胞解冻后GV和MⅡ期各组均无显著差异。根据试验结果得出在冷冻保存中最好冷冻MⅡ期的卵母细胞,以便提高后期的卵裂率和囊胚率;卵母细胞玻璃化时间在40 s内均不影响卵母细胞的活力和发育潜力。     

玻璃化冷冻牛卵母细胞单精子注射后体外发育能力的研究

实验研究了不同成熟培养时间的牛卵母细胞玻璃化冷冻及胞质内单精子注射(ICSI)后的受精效果。结果表明:成熟后的新鲜牛卵母细胞按照ICSI注射方法穿刺而不注射精子组与未经穿刺的对照组相比,孤雌激活后的卵裂率、囊胚发育率及囊胚细胞数无显著差异(P>0.05);成熟培养16h(MⅠ)和23h(MⅡ)卵母细胞冷冻解冻后形态正常率均显著低于新鲜对照组(76.66%、87.33%vs100.0%)(P<0.05),冷冻解冻后二者分别成熟培养至24h,ICSI后胚胎的囊胚发育率(5.29%、14.41%)显著低于新鲜对照组(24.40%)(P<0.05);成熟培养23h与成熟培养16h的卵母细胞冷冻解冻后形态正常率及ICSI后囊胚发育率(14.41%vs5.29%)均有显著性差异(P<0.05)。实验证明,ICSI操作不会影响卵母细胞发育潜力;玻璃化冷冻影响卵母细胞解冻后形态正常率以及ICSI后胚胎的发育能力;成熟培养23h比16h的卵母细胞冷冻保存后经ICSI的胚胎发育潜力高。     

水牛卵母细胞玻璃化冷冻保存

以水牛MII期的卵母细胞为材料,利用玻璃化冷冻液EDS33(16.5%EG+16.5%DMSO+sucrose)对水牛MII期的卵母细胞进行两步法(玻璃毛细管(GMP)和拉细的开口塑料细管(OPS))玻璃化冷冻保存,即卵母细胞首先放入预平衡液(7.5%EG+7.5%DMSO+sucrose)中平衡3 min,再移入玻璃化冷冻液中30 s后装管直接投入液氮.解冻是在蔗糖浓度逐渐降低的解冻液中进行的.解冻后存活的卵母细胞孤雌激活,通过囊胚发育率作为评定卵母细胞冷冻效果的指标.结果发现,GMP和OPS法冷冻保存的水牛卵母细胞解冻后的存活率(分别为96.80%和97.41%)与对照组卵母细胞的存活率(100%)3者之间差异均不显著(P>0.05).GMP法和OPS法冷冻的水牛卵母细胞激活后的胚胎分裂率和囊胚发育率2者均明显低于对照组(分别为30.58%和28.32% vs50.94%,10.81%和9.38% vs 29.63%,P<0.05),而这2种方法冷冻的水牛卵母细胞激活后的分裂率和囊胚发育率差异均不显著(P>0.05).这表明GMP和OPS玻璃化冷冻方法可以用于水牛卵母细胞的冷冻,并且玻璃化冷冻的卵母细胞能继续分裂并发育到囊胚.     

细胞松驰素B（CB）对水牛卵母细胞玻璃化冷冻保存的影响

研究主要探讨冷冻前细胞松弛素B（CB）预处理水牛MⅡ期卵母细胞对其冷冻效果的影响。以水牛MⅡ期的卵母细胞为材料，通过玻璃毛细管（GMP）和玻璃化冷冻液EDS33（16．5％EG＋16．5％DMSO＋Sucrose）对水牛MⅡ期的卵母细胞进行两步法玻璃化冷冻保存，即卵母细胞首先放入预平衡液（7．5％EG＋7．5％DMSO＋Sucrose）中平衡3min，然后再移入玻璃化冷冻液中30s后装管直接投入液氮。冷冻前卵母细胞随机分为2组，一组用7．5μg／mLCB预处理30min，另一组未用CB预处理作对照组。然后都冷冻保存。解冻是在蔗糖浓度逐渐降低的解冻液中进行的。解冻后存活的卵母细胞进行孤雌激活，以分裂率和囊胚发育率作为评定卵母细胞冷冻效果的指标。结果发现，GMP组和GMP＋CB组卵母细胞解冻后的存活率（88．89％、94．20％）和培养后发育到囊胚的比率（8．82％、14．55％）差异均不显著（P〉0．05），但GMP组和GMP＋CB组存活卵母细胞激活后的分裂率差异显著（32．69％、55．56％，P〈0．05），所以细胞松弛素B预处理可以提高GMP冷冻卵母细胞的发育能力。．     

奶牛GV期卵母细胞OPS法和GMP法超低温冷冻效果

采用OPS管和GMP管对GV期的牛的卵母细胞进行玻璃化冷冻.在不同的前处理液中平衡5 min,然后在冷冻液(EFS30,EFS40,EDFS30或EDFS40)中平衡30 s,进行OPS法和GMP法玻璃化冷冻保存.结果显示,OPS法用EFS40液和EDFS40液冷冻后形态正常卵率为69.6%和76.1%,2组差异显著(P<0.05),成熟率最高达19.2%和33.3%,2组差异显著(P<0.05);GMP法用EFS40液和EDFS40液冷冻后形态正常卵率最高达75.6%和80.8%,2组差异显著(P<0.05),成熟率最高达15.6%和34.9%,2组差异显著(P<0.05).而采用EDFS40液,OPS法和GMP法对GV期卵母细胞体外发育的影响差异均不显著,但GMP法的冷冻效率较高.表明采用EDFS40液GMP法对GV期卵母细胞的冷冻效率优于OPS法.     

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.     

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.     

冷冻后小鼠卵母细胞的透明带和质膜对体外受精的影响

以冷冻保存后的小鼠卵母细胞为实验材料,研究其膜对体外受精效果的影响。在室温(25±0.5)℃条件下,以10%乙二醇(EG)+10%二甲基亚砜(DMSO)为预处理液,EDFS30为玻璃化溶液。将卵母细胞于10%EG+10%DMSO溶液中预处理30s,然后再移入EDFS30中处理25s,以开放式拉长细管(OPS)为承载器投入液氮中,即两步法玻璃化冷冻保存。结果表明:冷冻卵母细胞受精后的卵裂率(46.67%)显著低于新鲜组的(86.06%)(P<0.05)。冷冻卵母细胞去透明带后质膜上的平均精子结合数(10.70)与对照组(10.81)无显著性差异(P>0.05),形成雌雄原核数也无显著性差异(2.49vs.2.59)(P>0.05)。冷冻卵母细胞透明带打孔后,其体外受精后卵裂率与新鲜组差异不显著(84.73%vs.91.19%)(P>0.05)。因此玻璃化冷冻保存小鼠卵母细胞透明带的变化是影响体外受精效果的主要因素之一。     

小尾寒羊羔羊超数排卵及卵母细胞玻璃化冷冻保存效果研究

以我国地方品种小尾寒羊作为供体,对不同年龄羔羊(6～8周龄和12～14周龄)超数排卵效果以及卵母细胞冷冻保存对体外受精、体外胚胎发育、胚胎移植产羔的影响进行研究。结果表明:6～8周龄组羔羊只均超排处理获卵数和可用卵数(60.8枚和58.2枚)显著高于12～14周龄组(27.3枚和26.0枚)(P<0.05);经玻璃化冷冻—解冻后的卵母细胞体外受精,冷冻组的卵裂率和桑椹胚发育率(67.8%和35.6%)均显著降低于对照组(79.2%和53.8E)(P<0.05);玻璃化冷冻保存体外生产的胚胎,经移植后成功产下4只健康羔羊(4/52)。     

小鼠桑椹胚简易玻璃化冷冻技术再探讨

本试验继小鼠扩张囊胚玻璃化冷冻保存成功后，在室温（２５℃）下利用不同浓度的ＥＦＳ玻璃化溶液，对小鼠的桑椹胚简易玻璃化冷冻技术进行再探讨。结果是胚胎在１０％ＥＧ溶液中预先处理５分钟，再移入事先配置好含有ＥＦＳ３０的０．２５ｍｌ塑料细管中１分钟平衡后直接投入液氮中冷冻，解冻后获得的发育率最高（９４％）。冻胚移植后妊娠率和产仔率分别为５６％（９／１６）及４２％（４９／１１６）。与对照组相比差异不显著（Ｐ＞０．０５）     

High survival rate of bovine oocytes matured in vitro following vitrification

Improving pregnancy rates associated with the use of cryopreserved human oocytes would be an important advance in human assisted reproductive technology (ART). Vitrification allows glasslike solidification of a solution without ice crystal formation in the living cells. We have attempted to improve the survival rates of oocytes by a vitrification technique using bovine models. In vitro matured oocytes with or without cumulus cells were vitrified with either 15.0% (v/v) ethylene glycol (EG) + 15% (v/v) dimethylsulfoxide (DMSO) + 0.5 M sucrose or 15% (v/v) EG + 15% (v/v) 1,2-propanediol (PROH) + 0.5 M sucrose, using 'Cryotop' or 'thin plastic sticker', respectively. The oocyte survival rates after vitrifying-warming, and the capacity for fertilization and embryonic development were examined in vitro. The rate of embryonic development to blastocyst was significantly higher (P<0.05) in the oocytes vitrified with 15% (v/v) EG + 15% (v/v) PROH + 0.5 M sucrose than in the oocytes vitrified with 15% (v/v) EG + 15% (v/v) DMSO + 0.5 M sucrose (7.4% +/- 4.1 vs. 1.7% +/- 3.0, respectively). Oocytes vitrified without cumulus cells had a higher survival rate after thawing and a superior embryonic developmental capacity compared with oocytes vitrified with cumulus cells. Prolonged pre-incubation time after thawing adversely affected the rates of embryonic cleavage and development. These results indicate that in vitro matured bovine oocytes can be vitrified successfully with the mixture of the cryoprotectants, EG + PROH, the absence of cumulus cells for vitrification does not affect oocyte survival rate after warming, and vitrified and warmed oocytes do not require pre-incubation before in vitro fertilization.