小型猪克隆胚胎体外培养阶段的优化

Optimization of Miniature Porcine Cloning Embryos in vitro Stage

本试验从卵母细胞卵丘多少和初次卵裂时间早晚2个方面进行研究，旨在改善小型猪克隆方案的体外培养环节，提高克隆效率。将卵母细胞按卵丘多少分成3组，比较卵母细胞的成熟效果，取成熟效果好的卵母细胞进行后续试验；PA和SCNT试验均按初次卵裂时间早晚分成3组，在体外培养条件下，比较胚胎的卵裂率和囊胚率。结果表明，卵丘多的卯母细胞体外成熟39～40 h和卵丘少的卵母细胞体外成熟41～42 h，比不区分卵丘多少的卵母细胞体外成熟39～42 h的成熟效果要好；初次卵裂时间发生在26 h以前的胚胎比26 h之后的胚胎在数量和质量上都明显优越，前者胚胎的卯裂率和囊胚率显著高于后者，此结果在孤雌激活（parthenogenetic，PA）胚胎和体细胞核移植（somatic cell nucleartransfer，SCNT）胚胎的体外试验中均得到验证。本研究完善了小型猪克隆方案的体外培养环节，为器官异种移植提供相关技术参考。

To improve the in vitro maturation of pig oocytes and to modify the protocols of cloning, the size of cumulus-oo-cyte and timing of the first zygotic cleavage were mainly studied in the present study. The oocytes were divided into threegroups by the number of cumulus cells, subsequently the maturation efficiency was compared, and the matured oocytes wereused for the following experiments. According to timing of the first zygotic cleavage, PA and SCNT embryos were divided intothree groups to compare the rates of cleavage and blastocysts in the in vitro culturing. Our results showed that a better abilityof maturation was observed in the oocytes which had more cumulus cells （39 to 40 h） and few cumulus cells （41 to 42 h） thanthose oocytes of which the number of cumulus ceils （39 to 42 h） didn＇t being distinguish. Both of PA and SCNT, the embryoscleaved in 26 h had a better developmental competence compared to the embryos cleaved after 26 h, which was evaluated by therates of cleavage and blastocysts. Therefore, in the present study, not only the miniature porcine cloning programs was im-proved, but also that it provided technical reference for organ xenotransplantation.