玻璃化冷冻对猪卵母细胞体外发育能力的影响

采用猪未成熟(GV期)和体外成熟(MⅡ期)卵母细胞分组进行冷冻保护剂毒性试验,试验组经冷冻液处理,对照组不用冷冻液处理。结果表明,GV期与MⅡ期卵母细胞相比,两者经冷冻保护剂处理后,其存活率、体外受精胚卵裂率均无显著差异(P>0.05);试验组GV期卵母细胞存活率、体外成熟率及体外受精胚卵裂率虽略低于对照组(85.9%,72.4%和50.9%对91.3%,73.9%和53.3%),但差异不显著(P>0.05);试验组MⅡ期卵母细胞存活率虽显著低于对照组,但卵裂率与对照组无显著差异(P>0.05)。表明所用冷冻液及其处理程序,对卵母细胞无明显毒性。分3组对猪卵母细胞进行冷冻保存试验:I组为对照组;II组为GV期卵母细胞冷冻组;III组为MⅡ期卵母细胞冷冻组。结果表明,经开放式拉管(Open pulled straw,OPS)法玻璃化冷冻后,GV期和MⅡ期卵母细胞均获得较高的形态正常率,但GV期卵母细胞冷冻后存活率要显著高于MⅡ期卵母细胞(P<0.05)。经玻璃化冷冻后,GV期卵母细胞的体外成熟率和卵裂率均明显低于新鲜卵母细胞(42.6%和7.79%对73.9%和53.3%,P<0.05),MⅡ期卵母细胞冷冻-解冻后未获得卵裂。在GV期卵母细胞冷冻组,154枚卵母细胞冷冻后经体外成熟、体外受精及体外培养,共有12枚发生卵裂,其中6枚发育至8-细胞,3枚发育至16-细胞,3枚发育至桑椹胚。本研究表明,所用冷冻方案更适合于猪GV期卵母细胞的冷冻保存。

Influence of vitrification on viability of porcine oocytes in vitro

Porcine GV stage and M II stage oocytes were divided into test groups and control groups in order to investigate the toxicity of cryoprotectants.The oocytes of test groups were treated with freezing fluids but no such a treatment for the control.The results showed that there were not significant differences in the survival rate and cleavage rate between treated GV stage and M II stage oocytes(P>0.05).The survival rate,in vitro maturation rate and cleavage rate of treated GV stage oocytes were slightly lower than those of the control(85.9%,72.4% and 50.9% versus 91.3%,73.9% and 53.3%),but their differences between both were not significant(P>0.05).The survival rate of treated M II stage oocytes was significantly lower than that of the control but the difference in the cleavage rate between both was not significant,which indicated that the freezing fluids and operational procedures used in this experiment were not toxic to the porcine oocytes.Porcine oocytes were divided into three groups in cryopreservation experiment.Group I was the control.Group II and III were the vitrification groups respectively for GV stage oocytes and for M II stage oocytes(P>0.05).The results showed that after vitrification with OPS method a relatively high normal rate could be obtained from either GV stage or M II stage oocytes,while the pos-thaw survival rate of GV stage oocytes was significantly higher than that of M II stage oocytes(P<0.05).The in vitro maturation rate and cleavage rate of vitrified GV stage oocytes were significantly lower than those of fresh oocytes(42.6% and 7.79% versus 73.9% and 53.3%,P<0.05).No cleavage was found in the M II stage oocytes after freezing-thaw.In group II,12 of 154 vitrified GV stage oocytes produced cleavage through in vitro maturation,in vitro fertilization and in vitro culture.Six of them developed into 8-cell stage,three of them into 16-cell stage and the rest into morula stage.The study showed that the vitrification protocol used was more suitable to the cryopreservation of porcine GV stage oocytes.