Heterogeneous Embryos Reconstructed By Horse Somatic Cell And Bovine And Ovine Oocytes And Their Developmental Potential

In this study, horse skin fibroblasts were cultured in vitro and were used as donor of nuclear to reconstruct cloned embryos with enucleated bovine and ovine oocytes respectively, then the early developmental potential of the two different reconstructed embryos were observed.Horse skin tissue fibroblasts were dissociated and cultured in vitro. In DMEM-F12 supplemented with 10% FBS at 37℃,100% humidity and 5% CO2. The method of explant tissue culture was easier to obtain horse skin fibroblasts than the method of enzyme digestion. The horse skin fibroblasts were dissociated and purified after four times of subcultures, and had 76.3% normal chromosome content after at least ten passages(2n=64).Matured ovine oocytes were efficiently activated by 5mM Ionomycin together with 6-DMAP and the oocentage of cleavage and blastocyst were 81.68% and 34.43%,respectively. But in electroactivation of ovine oocytes, under the condition of 1300v/cm voltage, 10μs duration, 2 pulses and 0.5 seconds internal, the cleavage and blastocyst rates were 74.22% and 29.69%. So chemokinesis was better than electroactivation for the parthenogenetic activation of ovine oocytes.The reconstructed embryos were obtained by sub zona pellucida injection associated with electrofusion after blindfold enucleation. The result indicated that when the voltage at 1580v/cm, the fusion and cleavage rates of the two kinds of reconstructed embryos achieved the highest. The fusion and cleavage rates of horse - bovine reconstructed embryos were 49.01% and 55.65% respectively, and the horse - bovine reconstructed embryos could develop to early morula. The fusion and cleavage rates of horse - ovine reconstructed embryos were 55.36% and 58.06% respectively, and the horse - ovine reconstructed embryos could develop to hatched blastocyst. The number and morpha of chromosome in horse - ovine blastocyst were same to that of the horse fibroblasts, which suggested that ovine oocytes could support the horse nucleus to develop.