【摘要】 采用毛细玻璃管法对牛卵母细胞进行玻璃化冷冻保存,解冻后再进行体外受精(IVF)和早期胚胎的体外培养(IVC)。在此技术的基础上,分别对冷冻前平衡时间、解冻处理、卵丘细胞层数以及卵母细胞所处的减数分裂阶段等影响卵母细胞冷冻保存的因素进行研究,以期筛选出适合牛卵母细胞冷冻保存的方法。结果发现,处于MⅡ期卵母细胞在10%二甲基亚砜(DMSO)+10%乙二醇(EG)液(VS1)中平衡1~3min,然后进行玻璃化冷冻保存。解冻时将卵母细胞先移入VS1液中处理15s,然后移入蔗糖稀释液中。另外发现,冷冻保存时部分卵丘细胞对卵母细胞有保护作用。而减数分裂阶段不影响解冻后卵母细胞形态正常率,但对胚胎发育率有严重影响。更多还原

【Abstract】 The objective of this study was to provide an effective vitrification protocol to cryopreserve bovine oocytes for research and practice of in vitro fertilization and nuclear transfer. The effect of equilibration time,thawing procedure,number of cumulus cell layers and meiotic stages of bovine oocytes vitrified by glass capillaries were compared. The results showed as follows:bovine oocytes matured in vitro in 10%ethylene glycol (EG) and 10%dimethylsulfoxide (DMSO) in TCM199 plus 20%fetal bovine serum (FBS)1 to 3 min,the blastocyst development rates of vitrified oocytes following in vitro fertilization were higher than those of other treated groups (P<0.05). Thawing conducted by immersing the end of the capillaries directly into 10%EG and 10%DMSO in TCM199 plus 20% FBS for 15s and then placing oocytes stepwise in 0.3M sucrose in holding medium for 1min,0.15M sucrose in holding medium 5min each,the rates of morphological normality and development to 2-cell,4-cell and 8-cell stages were not different from those of non-treated groups (directly into 0.3M sucrose in holding medium),but the blastocyst rate was higher than that of non-treated group (P<0.05). Oocytes matured in vitro partially stripped of cumulus cells by a short exposure to 0.1% hyaluronidase followed by gentle pipetting,the morphological normal rate was higher than those of non-treated and naked oocytes (P<0.05). Oocytes matured in vitro for 0,4,8,15 and 22h showed similar morphological normal rates,however the rates of development to 2-cell,4-cell,8-cell and blastocyst were the highest in 22h group (P<0.05).更多还原