Study On Somatic Cell Nuclear Transfer Of Horse

The technique of somatic cell nuclear transfer provides a new method and opportunities for fast breeding and genome preservation in domestic animals. In China, this technology has been applied in some domestic animals, such as cattle and sheep. However, there are fewer researches for horse reproduction on this area. In this study, we reconstruct somatic cell nuclear transfer embryo by fibroblast of thoroughbred horse and equine oocytes, and also reconstruct interspecies somatic cell nuclear transfer embryo by fibroblast of thoroughbred horse and donkey oocytes. We have established in vitro maturation system for equine and donkey oocytes. We also do some work for embryo in vitro culture and transplanted early heterogeneous cloned embryos into recipient oviducts by surgical method.1. The establishment of equine somatic cell linesIn the study, the suitable culture and preservation system for the horse fibroblasts was set up during the study of culturing the horse leg skin cells in vitro, freezing and thawing the cells.2. Reconstruction somatic nuclear tranfer embryo of horseBoth of the M199 and DMEM/F12 were used for in vitro maturation and the result show that there have no significant difference (p>0.05).After 24-28h and 30-36h maturation for (Ex) COCs and (Cp) COCs,gained 65% and 43% maturation rate ,respectively. M?stage oocyte wether gained from (Ex) COCs or (Cp) COCs have same developmental competence. In the study for equine nuclear transfer ,wo found that the highest fusion rate wound gained(72.5%) when the fusion parameter were 140 V,20 Us, 2 pulse?The cleavage rate of reconstructed embryo is 41.9%.3. Reconstruction of horse-donkey interspecies somatic nuclear tranfer embryosThree medium that M199?MEM/F12 and equal parts (v/v) of M199 and DMEM/F12 were evaluated for their ability to support in vitro maturation of donkey oocytes and their development after parthenogenetic activation. There were no significant differences among the three maturation media for oocyte maturation and their development after parthenogenetic activation (p> 0.05). As equine COCs,there also have two types[(Ex) COCs and (Cp) COCs]for donkey COCs. After 24-28h and 30-36h maturation for (Ex) COCs and (Cp) COCs, gained approximate 70% and 50% maturation rate, respectively. Transport donkey ovaries within 6 h were not affect the maturation rate of donkey oocytes. There were no effect for maturation rate of donkey oocytes when them were treated with EH medium for 12h at room temperature. Wether transport donkey ovary or oocyte for a long time(20-22h),or maturation of the oocytes occurs during shipment using a portable incubator were effectless for in vitro maturation of donkey oocytes. Both the SOFaa and DMEM/F12 can used for in vitro culture of donkey embryo, there have no significant difference(p>0.05).DMEM/F12 supplement with high level of glucose (10mM) wound be better for in vitro culture of donkey parthenogenetic embryo, but there have no significant difference . In the study of produced horse - donkey heterogeneous embryo use equine fibroblasts and anucleated donkey oocytes by somatic cell nuclear transfer, we found that the high fusion rate(60-70%) were gained when the fusion parameter were 130-150 V,20 Us, 2 pulse. The cleavage rate of reconstructed embryo is 48.4%. We transplanted 46 early horse - donkey heterogeneous embryos into oviducts of five recipient horses, no pregant were detected after 90 days...