猪卵母细胞激活方法的研究

Study on electrical activation of porcine oocytes

【目的】探讨猪卵母细胞体外胚胎生产的最佳激活条件。【方法】以不同电场强度(100,150,200,250V/mm)、脉冲时间(2,30,40,60,80μs)、电脉冲次数(1,2,3次)的电激活方法及电激活(电场强度为200 V/mm,脉冲时间为60μs,激活次数为1次)联合化学处理(NCSU-23+CHX、NCSU-23+6-DMAP和NCSU-23+CHX+6-DMAP)方法对猪卵母细胞进行激活,研究不同激活方法对猪卵母细胞孤雌活化的影响。【结果】电激活猪卵母细胞时,在200 V/mm、60μs、1次激活的条件下其卵裂率和囊胚率均最高。电激活联合NCSU-23+CHX、NCSU-23+6-DMAP、NCSU-23+CHX+6-DMAP化学处理时,在孤雌激活后期的卵裂率分别为69.17%,82.31%和79.67%,囊胚率分别为24.17%,39.46%和39.84%,在统计学上各处理间无显著差异。【结论】猪卵母细胞孤雌激活的最佳条件,是在电场强度为200 V/mm,脉冲时间为60μs,激活次数为1次的电激活后,联合NCSU-23+6-DMAP处理4 h,再移入胚胎培养液NCSU-23+4 g/L BSA中继续孵育(40±4)h。

[Objective] The objective of this study was to determine the best method of activation to produce porcine embryo in vitro. [Method] The porcine oocytes were activated by different electric field strengths （100,150,200 and 250 V/mm）, different time （20,30,40,60 and 80 μs）, different electric pulsing numbers （1,2 and 3 times）. The porcine oocytes were also activated by electrical pulses（200 V/mm,60 μs,1 time） combined with chemical activation（NCSU-23 ＋ CHX, NCSU-23 ＋ 6-DMAP and NCSU-23 ＋ CHX＋ 6-DMAP）. [Result] 200 V/mm of electric field strength and 60 μs of electric field duration one electric pulsing can get the higher rate of oocyte activation,cleavage and parthenogenetic embryo. There were no significant differences in the cleavage rates（69. 17%,82.31% and 79.67%） and blastocyst rates（24. 17%,39.46% and 39.84%） after parthenogenetic activation using NCSU-23＋CHX, NCSU-23＋6-DMAP and NCSU-23＋CHX＋6-DMAP. [Conclusion] The better method of activation is the activation of electrical pulses（200 V/mm,60 μs） combined with NCSU-23＋6-DMAP in 4 h,then the occytes are cultured in the embryo media NCSU-23＋4 g/L BSA in （40＋4） h,which can get a high cleavage rate and blastocyst rate.