不同化学激活剂对牛卵丘细胞重构胚发育的影响

The Effect of Different Activation Methods on Bovine Reconstructed Embryos Produced by Cumulus Cells

本文主要研究了①离子霉素(Ion)+6-二甲基氨基嘌呤(6-DMAP)、②Ion+放线菌酮(CHX)、③体积分数7%乙醇+6-DMAP、④体积分数7%乙醇+CHX四种不同化学激活方法对牛胞质内卵丘细胞全细胞注射法所获得重构胚的激活及前期发育的影响。Ion和7%乙醇的处理时间为5min,6-DMAP的处理时间为4h,CHX的处理时间为5h。结果表明,重构胚与颗粒细胞单层细胞共培养2d后,各组卵裂率分别为52.7%、51.5%、53.2%和54.0%,差异不显著(P>0.05);培养8d后,①组激活的囊胚发育率(20.0%)极显著高于其他组(P<0.01),其他各组之间差异不显著,分别为8.5%、10.2%和6.1%;培养10d后,①组激活的囊胚孵化率(10.7%)极显著高于其他组(P<0.01),其他各组之间差异不显著,分别为2.3%、3.0%和1.8%。表明,4种激活方法均可以有效地激活重构胚,但Ion+6-DMAP法激活重构胚的囊胚率和囊胚孵化率均极显著高于其他3组,说明Ion+6-DMAP激活法有利于牛重构胚的发育,可以获得较理想的囊胚率,是胞质内全细胞注射法克隆的理想激活方法。

The objective of this article was to investigate the effect of 4 different chenmical activation reagents on the rate of development of bovine reconstucted embryos produced by intracytoplasmic direct injection. The reconstucted embryos were randomly divided into 4 groups to be activated： （1） Ionomycin＋6-DMAP; （2） Ionomycin＋Cycloheximide （CHX）; （3） 7% ethanol＋6-DMAP; （4） 7% ethanol＋CHX. The reconstructed embryos were activated for 5min in Ionomycin and 7% ethanol;6-DMAP, 4h; CHX, 5h. The reconstructed embryos were co-cultured with cumulus cell monolayer. The rate of cleavage of the 4 groups were 52.7% ,51.5% ,53.2% and 54.0%, respectively. There was no significant difference among the 4 groups in terms of the rate of cleavage. The rate of blastocyst at day 8 were 20.0% ,8.5%, 10.2% and 6.1% for group （1）,（2）, （3）and （4）, respectively. The rate of blastocyst of group （1） was significantly higher than that of three other groups. The rate of hatching blastocyst of group（1） showed very significant difference with that of group （2）, （3）and （4） （10.7%, 2.3%, 3.0% and 1.8%, respectively, P〈0.01）. The results showed that all of the d kinds of activation reagents could successfully activated the reconstructed bovine embryos. The group （1） gained the highest rate of blastocyst and hatching blastocyst, which demonstrated that Ionomycine＋6-DMAP benefited the development of bovine reconstructed embryos and could obtain an ideal rate of blastocyst. Ionomycine＋6-DMAP were ideal activation reagents for bovine reconstructed embryos produced by intracytoplasmic direct injection.