PDE3与亮甲酚蓝在绵羊卵母细胞体外培养中的应用研究

Application of PDE3 and Brilliant Cresyl Blue in in-vitro Culture of Sheep Oocyte

[目的]探讨磷酸二酯酶3(PDE3)特异性抑制剂Milrinone与亮甲酚蓝染色液(BCB)相结合在绵羊卵母细胞体外培养中的应用。[方法]比较了BCB对绵羊卵母细胞的筛选与传统的形态分级之间的差异,以及对胚胎后期发育的影响;研究了Milrinone对绵羊卵母细胞的最佳抑制时间,并用于BCB-卵母细胞体外培养。[结果]A、B级卵母细胞中BCB+卵母细胞的比例为64.42%,极显著高于C级卵母细胞的17.00%;BCB+卵母细胞的成熟率(86.16%)、卵裂率(85.29%)、囊胚率(34.40%)分别极显著高于BCB-卵母细胞(50.94%、36.19%和6.73%);6 h是Milrinone对绵羊卵母细胞的最佳抑制时间,该试验组体外培养效率显著高于其他组;Milrinone对BCB-卵母细胞抑制培养6 h,极显著提高了体外胚胎发育率。[结论]为提高绵羊卵母细胞体外培养效率提供了依据。

[Objective] The aim of this study was to increase the viability of sheep oocytes in vitro by using phosphodiesterase type 3（PDE 3） inhibitors milrinone and brilliant cresyl blue（BCB） staining.[Method] The quality between BCB tested and morphologically selected oocytes,as well as the embryo development was compared;and then suitable inhibitive time of milrinone to sheep oocytes in vitro was studied and used in BCB-oocytes for in vitro embryo production（IVEP）.[Result] The BCB＋ oocytes percentage in A-and B-level sheep oocytes was 64.42%,which was extremely significantly higher than that in C-level（17.0%）.The maturing rate,cleavage rate and blastocyst rate of BCB＋ oocytes（86.16%,85.29% and 34.40%） of was significantly higher than those of BCB-oocytes（50.94%,36.19% and 6.73%）.The best time for PDE 3 inhibitor delaying the sheep oocyte mature in vitro was 6 h.In addition,the rate of embryo development in vitro could be significantly increased by inhibiting the BCB-oocytes for 6 h with Milrinone.[Conclusion] The study will provide reference for improving the efficiency of sheep oocytes culture in vitro.