Ma YuFang, Li Yan, Wei HengXi, Li QiuYan, Fang Rui, Zhao Rui, Zhang Kun, Xue Kai, Lou YanKun, Dai YunPing, Lian LinSheng, Li Ning
State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing, 100193, China.
Sci China C Life Sci. 2009 Jul;52(7):657-64. doi: 10.1007/s11427-009-0087-3. Epub 2009 Jul 30.
The effects of chemical activation on birth efficiency of cloned pigs were studied by investigating the developmental process from porcine oocyte activation to birth of cloned pigs. Three different activation methods were used: (i) Electroporation (Ele); (ii) Ele followed by incubation with 6-dimethylaminopurine (6-DMAP); and (iii) Ele followed by a treatment with cycloheximide (CHX). In experiment 1, the rates of cleavage, developmental rates and cell number of porcine parthenogenetic (PA) embryos were investigated in the three treatment groups. In experiment 2, NT embryos produced by the three different activation treatments were compared for the rates of cleavage, development and cell number. Finally, the effects of Ele and Ele+CHX activation methods on birth efficiency of cloned pigs were compared. The activated oocytes treated by combination activation generally showed a higher (P<0.05) blastocyst rate and produced more expanded blastocysts than oocytes activated with Ele. The rates of cleavage and total cell number of parthenotes were not significantly different. Parthenogenetic embryos activated with 6-DMAP developed into blastocyst and expanded blastocyst stages at a significantly (P<0.05) higher rate than those treated with Ele, but the developmental capability was dramatically decreased in NT embryos. With the CHX activation method, the NT embryo blastocyst rate was substantially (P<0.05) increased although the production of expanded blastocysts was not significantly different from that by the other two methods. The birth rate of cloned pigs increased in the CHX group, though the rate was not significantly different from Ele. The effects of season on developmental rate of the porcine PA embryos and birth rate of cloned pigs were also examined in our study. Porcine oocytes collected in the spring had higher developmental capabilities than those collected in the winter. However, no difference in birth rate of the cloned pigs was found between the oocytes collected in the two seasons. The results obtained from PA and NT embryos, following different activation methods, were inconsistent, suggesting that activation mechanisms are dissimilar in PA and NT embryos. Although the chemical activation in our study leads to an elevation of the blastocyst rate, it does not improve the oocyte's molecular programming and so does not significantly improve the efficiency of producing cloned pig births.
通过研究从猪卵母细胞激活到克隆猪出生的发育过程，探讨了化学激活对克隆猪出生效率的影响。采用了三种不同的激活方法：（i）电穿孔（Ele）；（ii）电穿孔后用6-二甲基氨基嘌呤（6-DMAP）孵育；（iii）电穿孔后用环己酰亚胺（CHX）处理。在实验1中，研究了三个处理组中猪孤雌生殖（PA）胚胎的卵裂率、发育率和细胞数量。在实验2中，比较了三种不同激活处理产生的核移植（NT）胚胎的卵裂率、发育率和细胞数量。最后，比较了电穿孔和电穿孔+CHX激活方法对克隆猪出生效率的影响。联合激活处理的激活卵母细胞通常比用电穿孔激活的卵母细胞具有更高的（P<0.05）囊胚率，产生更多扩张囊胚。孤雌生殖胚胎的卵裂率和总细胞数量没有显著差异。用6-DMAP激活的孤雌生殖胚胎发育到囊胚和扩张囊胚阶段的比率显著（P<0.05）高于用电穿孔处理的胚胎，但在核移植胚胎中发育能力显著下降。采用CHX激活方法，核移植胚胎的囊胚率显著（P<0.05）提高，尽管扩张囊胚的产生与其他两种方法没有显著差异。克隆猪的出生率在CHX组有所提高，尽管该比率与电穿孔组没有显著差异。本研究还考察了季节对猪孤雌生殖胚胎发育率和克隆猪出生率的影响。春季采集的猪卵母细胞比冬季采集的具有更高的发育能力。然而，两个季节采集的卵母细胞在克隆猪出生率上没有差异。不同激活方法处理后的孤雌生殖和核移植胚胎的结果不一致，表明孤雌生殖和核移植胚胎的激活机制不同。尽管本研究中的化学激活导致囊胚率升高，但它并没有改善卵母细胞的分子编程，因此没有显著提高克隆猪出生的效率。
