不同激活方法对猪体外成熟卵母细胞孤雌发育的影响

Comparison of different treatments for parthenogenetic activation of porcine oocytes matured in vitro

通过系统探讨不同孤雌激活方法对猪卵母细胞体外激活后发育效果的影响,比较了乙醇（Ethanol,EH）、离子霉素（Ionomycin,Ion：5μmol/L）、氯化锶（Strontium chloride hexahydrate,Sr^2＋：10 mmol/L）、6-二甲氨基嘌呤（6-dimethylaminopurine,6-DMAP：2 mmol/L）和放线菌酮（cycloheximide,CHX：10 mg/L）对猪卵母细胞激活发育的效果。结果表明：（1）9%EH激活处理10 min效果好于15 min;（2）使用9%EH激活处理10 min,再结合CHX、6-DMAP、Sr^2＋、CHX＋Sr^2＋、Sr^2＋＋6-DMAP、CHX＋6-DMAP或CHX＋6-DMAP＋Sr^2＋组处理3～4 h,以EH＋6-DMAP组效果最好,分裂率及囊胚率分别达到82.86%和22.86%;（3）在使用化学激活（Ion＋6-DMAP组和9%乙醇10 min＋6-DMAP组）和电激活（50 V/mm,50μs,2t）的方法中,Ion法激活猪卵母细胞效果较好,囊胚率达到37.50%;（4）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4～6层和多于6层的卵丘-卵母细胞复合体孤雌激活的分裂率和囊胚率分别为（68.99%,32.56%）和（75.36%,37.68%）,2组之间差异不显著（P〉0.05）;但其显著高于其他组（P〈0.05）,这2组细胞在猪孤雌激活发育研究中是最佳的实验研究材料。

The systematical analysis on the effect of parthenogenetic activation protocols in vitro maturation of porcine oocytes was carried out in the present study. Comparison of parthenogenetic development of porcine oocytes treated by ethanol（ EH）, ionomycin（ Ion ： 5μmol/L）, strontium chloride hexahydrate（ Sr^2＋ ： 10 mmol/L）, 6-dimethylaminopurine（6-DMAP：2 mmol/L）and cycloheximide（CHX： 10 mg/L） activation treatments. The results showed that（1）The treatment of concentrations of 9%EH in the NCSU-23 medium and exposure times of 10 min for the in vitro matured porcine oocytes was better than 9%EH in the NCSU-23 medium and exposure times of 15 min. （2）Oocytes were activated with the NCSU-23 medium containing 9% ethanol for 10 min. After that,Oocytes were incubated in the NCSU-23 medium supplemented with CHX, 6-DMAP, Sr^2＋ , CHX＋ Sr^2＋ , Sr^2＋ ＋6-DMAP, CHX＋ 6- DMAP and CHX＋6-DMAP＋Sr^2＋ for 3-4 h respectively,the EH＋6-DMAP treatment was better,the rate of cleavage and the rate of blastocyst formation were 82.86% and 22.86% respectively. （3）Chemical activation （Ion＋ 6- DMAP and 9GEH for 10 min＋6-DMAP）and electric activation（50 V/mm, 50 μs, 2 t）of porcine oocytes were explored in this study, the development effect of the porcine parthenogenetic embryos after ionomycin activation was better and the rate of blastocyst of Ion＋6-DMAP treatment was 37. 50%. （4）The effect of different quality of porcine cumulus oocyte complexes（COCs） was studied on parthenogenesis development after parthenogenetic activation. The rate of cleavage and blastocysts in the group of 4-6 layers of cumulus cells and the group of 〉6 layers of cumulus cells were （68.99% ,32.56%） and （75.36% ,37.68%） respectively and they were not significantly（P〉0.05） ,but these two groups were significantly higher（P〈0.05） and were more efficient than other groups. Therefore,cells of the two groups were the optimal research material of experiment for parthenogenetic activation in vitro embryo development in porcine.