Cloned Embryo Reconstruction In Sheep Somatic And The Research Of The Embryo Development

This study reconstructed the embryos with adult sheep skin fibroblasts - sheep oocytes and fetal sheep skin Fibroblasts -sheep oocytes, and compared the rate of fusion, cleavage, morula of the two kinds of reconstructed embryos.The condition of 37℃,100% humidity and 5% CO2, DMEM-F12 supplemented with 10% FBS was used to support the sheep skin fibroblasts growth in vitro. The method of explanted tissue culture is better than the method of enzyme digestion for the primary cells. The sheep skin fibroblasts could be separated and purified by enzyme digestion and repeatitial attachments for two or three times of subcultures during the course of subcultures. The trypsin dissolved in D-Hanks was feasible for digestion of sheep skin fibroblasts; The adult sheep fibroblasts grew more slowly than fetal cells, the time of group multiplication of the fibroblasts were 48 hours and 24 hours, respectively; Sheep skin fibroblast had 80.0% normal chromosome content after at least ten passages (2n=54). Matured sheep oocytes were efficiently activated by Ionomycin together with 6-DMAP and 30.6 rate of blastocyst were achieved. It was higher than the rate of blastocyst achieved by the method of cytochalasin B. The condition of 1500v/cm voltage, 40μsec impulse time was suitable for the fusion of reconstructed embryos. It was optimal for them to mature in vitro for 18 to 21 hours with blind absorption in the guarantee of some maturation rate of sheep oocytes.The rate of fusion, cleavage, morula of the two kinds of embryos, which were reconstructed by adult sheep skin fibroblasts - sheep oocytes and sheep fetal skin Fibroblasts -sheep oocytes 76.0%,76.2%,26.9%,75.5%,79.5%,28.3%, respectively. There were no obvious differences between the rate of fusion, cleavage, morula of reconstructed embryos, which used adult and fetal sheep skin fibroblasts as donor cells (P>0.05).