水牛卵母细胞玻璃化冷冻后孤雌激活方法的优化

Optimization of parthenogenetic activation protocol of in vitro matured buffalo oocytes following vitrification

【目的】了解玻璃化冷冻—解冻及不同孤雌激活条件对水牛卵母细胞发育潜能的影响,为构建完善的水牛卵母细胞孤雌激活培养体系奠定基础。【方法】利用玻璃化冷冻法对水牛体外成熟卵母细胞进行冷冻保存1-2 d,解冻复苏后进行孤雌激活,以新鲜的体外成熟卵母细胞为对照,探讨离子霉素浓度、6-二甲氨基嘌呤（6-DMAP）浓度和处理时间对玻璃化冷冻复苏后水牛卵母细胞孤雌激活效果的影响。【结果】与新鲜的体外成熟卵母细胞相比,玻璃化冷冻—解冻复苏后水牛体外成熟卵母细胞孤雌激活的卵裂率、4-细胞率、8-细胞率和囊胚发育率均极显著降低（P〈0.01）。水牛体外成熟卵母细胞经玻璃化冷冻—解冻复苏后,以3.5μmol/L离子霉素激活5 min联合2 mmol/L 6-DMAP培养2 h的孤雌激活效果最佳,其卵裂率、4-细胞率、8-细胞率和囊胚发育率分别为60.6%、45.1%、33.7%和14.8%。【结论】水牛体外成熟卵母细胞经玻璃化冷冻—解冻复苏后,其激活阈值发生改变,因此不宜采用与新鲜体外成熟卵母细胞一致的激活程序进行孤雌激活。

[Objective]The effect of vitrification and different parthenogenetic activations on the potential development of vitrified-warmed buffalo oocytes was studied to provide references for establishment of a specific activation pro-tocol. 【 Method 】 The matured buffalo oocytes were cryopreserved for 1- 2 days by vitrification method. Oocytes were treated by parthenogenetic activation after vitrification and thawing, and untreated fresh matured oocytes were used as the control. Following parthenogenetic activation, the effects of ionomycin concentration, concentration and processing time of 6-DMAP on vitrified- warmed oocytes were evaluated. 【 Result 】 The cleavage, 4- cell, 8- cell and blastocyst development rates of vitrified-thawed oocytes were all significantly lower than the control group（P〈0.01）. The vitrifiedthawed matured buffalo oocytes were cultured in 6-DMAP at 2 mmol/L for 2 hours after treated with 3.5 μmol/L ionomycin for 5 minutes, and the rates of cleavage, 4-cell, 8-cell and blastocyst were 60.6%, 45.1%, 33.7% and14.8%, respectively. 【Conclusion】The threshold of vitrified-thawed oocytes after activation might have changed, therefore, vitrified matured buffalo oocytes need a specific parthenogenetic activation protocol different from the unvitrified fresh oocytes.