水牛卵母细胞冷冻保存初步研究

Morphology and Developmental Competence of Buffalo(Bubalus bubalis) Oocytes:Effect of Vitrification and Slow Freezing

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%。

This study was first employed to investigate the developmental potential of in vitro matured buffalo oocytes vitrified by open-pulled straw（OPS） and glass micropipette（GMP） methods.The oocytes were first pretreated in 10 % EG for 0.5 min and then 25 sec equilibration in EFS30（group 1） or EFS40（group 2）,or in 10 %EG＋10 %DMSO for 0.5 min and then 25 sec equilibration in EDFS30（group 3） or EDFS40（group 4） before they were subsequently diluted in 0.25 mol/L sucrose for 1 min and 0.15 mol/L sucrose for 5 min.After dilution,the morphologically normal rates of the oocytes in the 4 groups were similar to that of the control.After parthenogenetic activation,the cleavage rate of oocytes in group 3 and group 4 were significantly lower（P0.05） than that in group 1 and group 2,which were similar to that of control（75.28%）.After OPS vitrification and warming,the oocytes in group 4 showed the highest cleavage rate（31.60%）.However,when the oocytes in group 4 were cryopreserved by GMP method,the cleavage rate were 45.63%.Secondly,the G V oocytes were cryopresered by slow freezing.After thawing,the morphologically normal rate and the polar body extrusion rate of the oocytes were significantly lower when compared with those of control.