不同化学激活方法及卵丘细胞层数对牛体外成熟卵母细胞孤雌发育的影响

为探索适宜的牛体外成熟卵母细胞孤雌激活方式,提高核移植效率及研究胚胎孤雌发育。研究比较了不同浓度的乙醇( EH)、离子霉素(ionomycin)、钙离子载体A23187与蛋白激酶抑制剂6-二甲氨基嘌呤(6-DMAP)、细胞松驰素(cytochalasin B, CCB)对牛卵母细胞激活发育的影响,并在相同条件下,比较了不同卵丘细胞层数对卵母细胞激活后胚胎发育能力的影响。结果表明: (1) 5 μmol·L-1 Ionomycin，10 μmol·L-1 A23187，7% EH分别联合2 mmol·L-1 6-DMAP，CCB均可以有效地激活牛孤雌胚,其中以Ionomycin＋6-DMAP+CCB组囊胚发育率极显著高于其他各组(P<0.01),并且激活液中CCB的存在对牛孤雌胚胎的发育有利；(2) 卵母细胞包被的卵丘细胞层数不同对卵母细胞成熟激活有显著的影响,卵丘细胞层数3～5层和多于6层的卵丘—卵母细胞复合体(COCs)孤雌激活的分裂率和囊胚率分别为69.09%,31.58%和75.14%,38.85%,这2组之间差异不显著(P>0.05), 但其显著高于1～2层组与混合组(P< 0.05), 因此,这2组细胞是最佳的试验研究材料。

Effects of different chemical activation methods and number of cumulus cell layers on in vitro parthenogenetic development of matured bovine oocytes

To explore the suitable activation methods for in vitro parthenogenetic development of matured bovine oocytes and increase the efficiency of nuclear transfer for embryonic parthenogenesis, we have studied the effects of activation by ethanol (EH), ionomycin (Ion), A23187, 6-dimethyl-aminopurine (6\|DMAP: 2 mmol·L-1) or cytochalasin B (CCB) on the development of bovine oocytes. Meanwhile, we have analyzed the effects of different numbers of cumulus cell layers on parthenogenetic development of bovine oocytes in the same condition. The results showed that (1) Ion(5 μmol·L-1), A23187(10 μmol·L-1) or ethanol(7%) applied together with 6-DMAP(2 mmol·L-1) and cytochalasin B (CCB) significantly activated the growth of bovine parthenogenetic embryo, with treatment of Ion＋6-DMAP＋CCB showing the strongest effects as compared to other groups(P<0. 01). Thus, CCB is beneficial to the development of bovine parthenogenetic embryos.(2) The number of cumulus cell layers coating oocytes had significant effects on parthenogenesis development after parthenogenetic activation. The rate of cleavage and blastocysts for bovine cumulus oocyte complexes (COCs) containing 3-5 or above 6 cumulus cell layers were 69.09%, 31.58% and 75.14%, 38.85%, respectively. These two groups had no significant difference(P>0. 05), but were significantly higher than the group containing 1-2 lays and the mixed group(P<0.05). Therefore, cells of these two groups were the optimal research material for experiment of parthenogenetic activation of in vitro embryo development in bovine．