Effect of donor cell and trichostatin A(TSA) on development of cloned dairy cattle embryos | 供体细胞和曲古抑菌素(TSA)对奶牛克隆胚胎发育的影响

Chinese. 本研究探讨供核细胞处理策略(新鲜消化、-196℃冻存组(复苏后)和曲古抑菌素(TSA)处理组 )和TSA-CR1aa胚胎培养液处理时间对克隆胚发育的影响。利用体细胞核移植技术生产奶牛克隆胚胎，将部分克隆胚移植给自然发情同步的受体，检验克隆胚的体内发育能力。结果显示：TSA处理体细胞组的克隆胚卵裂率和囊胚率与新鲜消化和-196℃冻存组相比差异显著(P＜0.05)；-196℃冻存组与新鲜消化组差异不显著；用TSA-CR1aa分别处理新鲜消化细胞构建的克隆胚0、24、48和60 h，其中，60 h处理组的囊胚率最高(36.11±1.78%)与其他组对比差异显著(P＜0.05)； 用TSA-CR1aa分别处理TSA处理组构建的重构胚24、48和60 h，结果发现，60 h TSA-CR1aa处理组囊胚率(37.39±1.78%)最高，显著高于24 h(25.48±1.34%)TSA-CR1aa处理组，且差异显著(P＜0.05)。将所获得的克隆胚胎分别移植给40头自然发情的受体母牛，并观察移植25，45，65和90 d后的返情情况，结果显示，各组受孕率无统计学差异，但经TSA处理细胞和胚胎组移植的受体中，获得一头存活的体细胞克隆奶牛。说明优化的处理方法处理胚胎可以完成体内发育。

English. The objective of present study was to investigate the effects of treatments to donor cells with fresh digestion (FD), cryopreservation/thawning (CT), trichostatin A (TSA) and durations of culture using TSA-CR1aa medium on in vitro development of dairy cow cloned embryos. In addition, some somatic cell cloned embryos were transferred to surrogates in heat to evaluate the in vivo developmental competence. The results showed that pretreatment of donor cells using TSA could significantly increase both cleavage and blastocyst rates of embyos (P＜0.05) compared with FD and CT group，while no significant difference was found between FD and CT group. When cloned embryos were subjected to TSA treatment in CR1aa for different times (0, 24 , 48 and 60 h), the results showed that the blastocyst rate in the 60 h group was the highest (36.11±1.78%) compared with the other groups (P＜0.05). While the reconstructed embryos derived from donor cells treated with TSA for 24 h were continually cultured in TSA for different times (24, 48 and 60 h), the results showed that the blastocyst rate (37.39±1.78%) in the 60 h group was significantly higher than that of 24 h (25.48±1.34%) group (P＜0.05). Finally, when the cloned embryos from different groups were respectively transferred to 40 natural estrus recipients, no significant difference in terms of pregnancy rate among groups was found, however, a viable cloned calf was successfully obtained from TSA-treated donor cells and cloned embryo. Therefore, cloned embryos treated with optimized methods can development to term.