OPS法玻璃化冷冻山羊卵母细胞的研究

本实验采用两种不同的冷冻保护液VSⅠ(EG DMSO)和VSⅡ(PROH DMSO),用OPS法对不同期的卵母细胞进行冷冻。结果表明两种冷冻液VSⅠ和VSⅡ的冷冻效果差异不显著(P>0.05),但从数据看VSⅠ要优于VSⅡ;而不同期的卵母细胞对于冷冻后的发育能力有明显的影响,无论是VSⅠ还是VSⅡ,GV期和培养10h卵母细胞的形态正常率、成熟率、受精率差异不显著(P>0.05),但GV期和培养10h卵母细胞受精率均低于IVM期卵母细胞,它们之间差异显著(P<0.05)。     

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.     

OPS法玻璃化冷冻山羊卵母细胞的研究

采用两种不同的冷冻保护液VS Ⅰ(EG DMSO)和VSⅡ(PROH DMSO),用OPS法对不同期的卵母细胞进行冷冻.结果表明,两种冷冻液的冷冻效果差异不显著(P＞0.05),但从数据看,VS Ⅰ要优于VSⅡ;而不同期的卵母细胞对于冷冻后的发育能力有明显的影响,无论是VS Ⅰ还是VSⅡ,GV期和培养10h卵母细胞的形态正常率、成熟率、受精率差异不显著(P＞0.05),但GV期和培养10 h卵母细胞受精率均低于ⅣM期卵母细胞,它们之间差异显著(P＜0.05).     

山羊卵母细胞冷冻保存及其对发育效果的影响

在程序冷冻条件下,冷冻保护剂种类对山羊卵母细胞发育效果有显著影响。对于山羊GV期卵母细胞,解冻后的形态正常率,PROH(83 1%)和DMSO(81 7%)均高于甘油(70 7%)(P<0 05);而体外成熟率则是PROH(20 3%)高于DMSO(14 2%)和甘油(9 8%),DMSO又高于甘油(P<0 05)。对于山羊IVM卵母细胞,解冻后的形态正常率,PROH(84 5%)和DMSO(86 4%)均高于甘油(74 2%)(P<0 05);而受精率则是PROH(23 2%)高于DMSO(17 5%)和甘油(13 1%),DMSO又高于甘油(P<0 05)。冷冻方法和卵母细胞发育阶段对冷冻效果有显著影响。从冷冻方法看,程序冷冻和OPS玻璃化冷冻,GV期卵母细胞的成熟率分别为19 7%、27 6%,受精率为3 3%、8 6%;培养9h卵母细胞的成熟率分别为20 6%、30 9%,受精率为4 4%、10 3%;IVM卵母细胞的受精率分别为20 9%、29 4%,2 细胞率为4 4%、8 8%,均是OPS玻璃化高于程序冷冻,差异显著(P<0 05);从卵母细胞发育阶段看,不论是程序冷冻还是OPS玻璃化冷冻,GV期和培养9h卵母细胞的成熟率、受精率差异均不显著(P>0 05),但受精率均显著低于IVM卵母细胞(P<0 05)。     

小鼠卵母细胞冷冻技术研究

采用PBS作为冷冻基础液,分别用甘油和二甲基亚砜(DMSO)作为冷冻保护液,在程序化冷冻保存和玻璃化冷冻保存条件下,研究小鼠生发泡期(GV期)卵母细胞的抗冻能力。结果表明,2种冷冻方法对小鼠GV期卵母细胞解冻后形态正常率和存活率无显著影响(P>0.05)。冷冻保护剂种类对小鼠GV期卵母细胞解冻后形态正常率无显著影响(P>0.05);但对存活率有显著影响,玻璃化冷冻采用二甲基亚砜作为冷冻保护液效果极显著优于甘油(P<0.01)。以冷冻效果较好的二甲基亚砜作为冷冻保护液,采用玻璃化冷冻不同发育阶段(GV期和MⅡ期)的小鼠卵母细胞,解冻后形态正常率无显著差异(P>0.05),但存活率GV期要显著优于MⅡ期卵母细胞(P<0.05)。     

OPS法玻璃化冷冻山羊卵母细胞的研究

采用两种不同的冷冻保护液VSⅠ（EG＋DMSO）和VSⅡ（PROH＋DMSO），用OPS法对不同期的卵母细胞进行冷冻。结果表明：两种冷冻液VSⅠ和VSⅡ的冷冻效果差异不显著（P〉0．05），但从数据看。VSⅠ要优于VSⅡ；而不同期的卵母细胞对冷冻后的发育能力有明显的影响，无论是VSⅠ还是VSⅡ，GV期和培养10小时卵母细胞的形态正常率、成熟率、受精率差异不显著（P〉0．05），但GV期和培养10小时卵母细胞受精率均低于IVM期卵母细胞，它们之间差异显著（P〈0．05）。     

3种因素对牛卵母细胞玻璃毛细管玻璃化冷冻效果的影响研究

室温下 ,采用GMP(glassmicropipette)玻璃化法对牛卵母细胞进行冷冻保存。结果表明 :不同发育阶段的牛卵母细胞GMP冷冻、解冻后体外受精卵裂率在 36 .6 7%～ 52 .38%之间 ,其中体外培养 2 2h牛卵母细胞的受精卵裂率最高 ,为 52 .38%,与对照组 (6 8.42 %)差异不显著 (P >0 .0 5) ;采用不同的预处理溶液处理冻前牛GV期卵母细胞 ,其中以 3%的EG稀溶液预处理可获得最高的体外受精卵裂率。结果表明 :牛体外成熟卵母细胞冷冻效果最好 ,采用3%EG稀溶液处理玻璃化前卵母细胞可以提高冷冻效果。     

玻璃化冷冻对小鼠GV期卵母细胞发育能力的影响

试验首次采用OPS法玻璃化冷冻小鼠GV期卵母细胞(不带卵丘细胞,下同),同时尝试用EDFS30对小鼠卵巢进行细管法玻璃化冷冻,以研究GV期卵母细胞冷冻后的发育潜力。首先,利用MEM培养和MEM-腔前卵泡培养新鲜GV期卵母细胞,并把较好的培养方式用于冷冻后培养试验。2种培养方式培养24h后新鲜GV期卵母细胞成熟率无显著性差异;OPS法冷冻的GV期卵母细胞解冻后成熟率及体外受精后卵裂率与对照组差异不显著(P>0.05)。细管法冷冻卵巢组织的GV期卵母细胞成熟率极显著低于对照组(P<0.01),其受精后未获得受精卵。结果表明:OPS法可有效地冷冻保存小鼠GV期卵母细胞,而细管法冷冻小鼠卵巢对GV期卵母细胞损伤较大。     

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.     

不同冷冻和解冻方法对绵羊卵母细胞发育效果的影响

卵母细胞冷冻保存具有广泛而潜在的应用价值.试验主要分析了不同冷冻及解冻方法对绵羊GV期和MII卵母细胞发育效果的影响.GV期卵母细胞程序化冷冻解冻后形态正常率(54.8%)以及体外培养成熟率(14.7%)均显著低于细管玻璃化(71.8%,29.5%;P＜0.05)和OPS玻璃化(78.3%、35.4%;P＜0.05),卵裂率OPS玻璃化高于细管玻璃化,但差异不显著(26.1%,16.7%;P＞0.05);MII期卵母细胞形态正常率程序化冷冻显著低于细管玻璃化和OPS玻璃化冷冻(67.2%,77.6%,84.8%;P＜0.05),卵裂率OPS玻璃化显著高于程序化冷冻法(31.3%,10.3%;P＜0.05);3种不同方法冷冻不同发育时期卵母细胞成熟率和卵裂率与对照组相比较均差异极显著(P＜0.01).解冻后卵母细胞形态正常率三步与五步解冻法极显著高于一步解冻法(85.9%,82.7%,63.1%; P＜0.01);成熟率为三步法和五步法显著高于一步法(10.8%,26.9%,25.3%;P＜0.05);卵裂率三步法和五步法高于一步法,但没有差异显著性(14.3%,23.9%,21.1%; P＞0.05).     

Effect of Different Combinations of Cryoprotectants on In Vitro Maturation of Immature Buffalo (Bubalus bubalis) Oocytes Vitrified by Straw and Open‐Pulled Straw Methods

This study was designed to evaluate effects of different combinations of cryoprotectants on the ability of vitrified immature buffalo oocytes to undergo in vitro maturation. Straw and open‐pulled straw (OPS) methods for vitrification of oocytes at the germinal vesicle stage also were compared. The immature oocytes were harvested from ovaries of slaughtered animals and were divided into three groups: (i) untreated (control); (ii) exposed to cryoprotectant agents (CPAs); or (iii) cryopreserved by straw and OPS vitrification methods. The vitrification solution (VS) consisted of 6 m ethylene glycol (EG) as the standard, control vitrification treatment, and this was compared with 3 m EG + 3 m dimethyl sulfoxide (DMSO), 3 m EG + 3 m glycerol, and 3 m DMSO + 3 m glycerol. Cryoprotectants were added in two steps, with the first step concentration half that of the second (and final) step concentration. After warming, oocyte samples were matured by standard methods and then fixed and stained for nuclear evaluation. Rates of MII oocytes exposed to CPAs without vitrification were lower (54.3 ± 1.9% in EG, 47.5 ± 3.4% in EG + DMSO, 36.8 ± 1.2% in EG + glycerol and 29.9 ± 1.0% in DMSO + glycerol; p < 0.05) than for the control group (79.8 ± 1.3%). For all treatments in each vitrification experiment, results were nearly identical for straws and OPS, so all results presented are the average of these two containers. The percentages of oocytes reaching telophase‐I or metaphase‐II stages were lower in oocytes cryopreserved using all treatments when compared with control. However, among the vitrified oocytes, the highest maturation rate was seen in oocytes vitrified in EG + DMSO (41.5 ± 0.6%). Oocytes cryopreserved in all groups with glycerol had an overall low maturation rate 19.0 ± 0.6% for EG + glycerol and 17.0 ± 1.1% for DMSO + glycerol. We conclude that the function of oocytes was severely affected by both vitrification and exposure to cryoprotectants without vitrification; the best combination of cryoprotectants was EG + DMSO for vitrification of immature buffalo oocytes using either straw or OPS methods.     

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。     

小鼠卵母细胞和胚胎不同冷冻方法的比较研究

探讨程序化冷冻与玻璃化冷冻对小鼠GV期卵母细胞及二细胞期胚胎的复苏率及其发育潜能的影响。通过小鼠的卵母细胞与早期胚胎的不同冷冻方法的比较,为后续阿旺绵羊的胚胎冷冻保存提供参考。采用程序化冷冻与玻璃化冷冻技术,分别冷冻小鼠GV期卵母细胞及二细胞期胚胎,复苏后培养,比较不同冷冻处理后的复苏率、成熟率与囊胚率。小鼠GV期卵母细胞程序化冷冻复苏率(48.00%±5.29%)显著低于玻璃化冷冻复苏率(65.00%±5.00%),有统计学差异(P=0.0147<0.05);而程序化冷冻后复苏卵母细胞的发育成熟率略高于玻璃化冷冻组,但无统计学意义。小鼠二细胞期胚胎程序化冷冻组复苏率(76.00%±2.00%)显著高于玻璃化冷冻组复苏率(70.00%±2.00%),有统计学差异(P=0.0213<0.05);冷冻后复苏胚胎发育的囊胚率程序化冷冻略低于玻璃化冷冻及对照组,但无统计学意义。     

不同冷冻方法对GV期绵羊卵母细胞发育效果的影响

卵母细胞冷冻保存具有广泛而潜在的应用价值。文章主要分析了不同冷冻方法对绵羊GV期卵母细胞发育效果的影响。GV期卵母细胞程序化冷冻解冻后形态正常率（54.8%）和体外培养成熟率（14.7%）均显著或极显著低于细管玻璃化冷冻（71.8%,29.5%;P〈0.05）和OPS玻璃化冷冻（78.3%,P〈0.01;35.4%,P〈0.05）;3种不同方法冷冻GV期卵母细胞成熟率与对照组相比均差异极显著（P〈0.01）。     

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.     

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%。     

Studies on MⅡ Stage Porcine Oocytes Vitrification

The study was designed to select right cryoprotectants and carrier for porcine matured oocytes to improve oocyte viability after vitrification and lay a good foundation for porcine oocytes continue development. Using 40% EG, 15% EG+15% DMSO+20% FBS, 15% EG+15% DMSO+20% SPS as cryoprotectants and taking GMP and cryotop as carrier to do porcine oocytes vitrification. The results showed that VS3 including SPS made better active of FDA (59.40%±4.93%) than VS1 （42.12%±4.08%） and VS2 (37.57%±1.21%）（P<0.05);VS3 as cryoprotectants and cryotop as carrier made better active of FDA (83.33%±3.33%) than GMP (59.40%±4.93%)(P<0.05). The data demonstrated that use SPS to instead of FBS and cryotop as carrier could significantly improve active of oocyte after vitrification.     

Comparative Study between Slow Freezing and Vitrification of Mouse Embryos Using Different Cryoprotectants

The objective of this study was to evaluate the effects of different cryoprotectants and different cryopreservation protocols on the development of mouse eight-cell embryos. Mouse eight-cell embryos were cryopreserved by using propylene glycerol (PROH), ethylene glycerol (EG), dimethyl sulfoxide (DMSO) or glycerol (G) as cryoprotectant with slow-freezing or Vit-Master vitrification protocol. After thawing, the survival rate, blastocyst formation rate and blastocyst hatching rate of the embryos were compared. When the mouse eight-cell embryos were cryopreserved by the slow-freezing, the survival rate, the blastocyst formation rate and the blastocyst hatching rate of the embryos with PROH were significantly higher than those of DMSO and G (p < 0.05, respectively), but not significantly different among those of DMSO, G and EG (p > 0.05, respectively), and not significantly different between those of PROH and EG (p > 0.05, respectively). When the mouse eight-cell embryos were cryopreserved by Vit-Master vitrification, the survival rate, the blastocyst formation rate and the blastocyst hatching rate of the embryos with EG were significantly higher than those of PROH, DMSO and G (p < 0.05, respectively). Yet, there were no significant differences among those of PROH, DMSO and G (p > 0.05, respectively). In conclusion, PROH was the optimal cryoprotectant for the cryopreservation of mouse eight-cell embryos by slow-freezing protocol. EG was the optimal cryoprotectant for the cryopresevation of mouse eight-cell embryos by Vit-Master vitrification protocol, which may be commonly used in clinical and laboratory practice.