Studies On The Interspecies Somatic Cell Nuclear Transfer In Boer Goat And Cattle

The present study was undertaken to establish an optimized system of the interspecies somatic cell nuclear transfer(iSCNT)in Boer goat and cattle,and in this study Boer goat somatic cells cultured in vitro were used as nuclear donors while bovine enucleated oocytes matured in vitro were uesd as recipients in order to optimize key procedures involved in iSCNT.Six experiments were performed in this study.Experiment 1 was designed to investigate the effects of preservation temperature and transportation time of bovine ovaries during transportation on nucleus maturation of bovine oocytes maturated in vitro, and the result that when transportation time ranged between 10-12 h,the nucleus maturation rate of bovine oocytes from ovaries preserved at 26-30℃was significantly higher than that from bovine ovaries preserved at 21-25℃and 16-20℃(78.94±4.50 vs. 72.08±5.99 vs.68.70±3.36,P＜0.05),and while the ovaries were preserved at 20-25℃, there was no significant difference between the nucleus maturation rates of bovine oocytes from ovaries transported for 4-6 h and 10-12 h(P＞0.05).Experiment 2 was designed to evaluate the influence of cumulus cells on maturation and parthenogenetic development in vitro of bovine oocytes,and the results showed that the rates of maturation and blastocyst formation in intact cumulus oocytes complexes(COCs)group were higher than those in denuded oocytes group(71.49±5.52 vs.39.12±10.36,P＜0.05;35.24±5.61 vs.22.60±2.74, P＜0.05),evaluated effects of investment of different layers of cumulus cells and attached or not together with follicular shell pieces(FSP)on the ability of maturation and parthenogenetic development in vitro of COCs,and the result indicates that there was no difference in maturation rate between COCs invested by different layers of cumulus cells(P＞0.05),but the cleavage rate of oocytes enclosed with more layers of cumulus cells was higher significantly than that of COCs attached with FSP(79.90±16.70 vs. 66.94±11.47,P＜0.05).However,the blastocyst rate of oocytes enclosed with more layers of cumulus cell and attached together with FSP was higher than oocytes enclosed with fewer layers of cumulus cells(27.60±5.10 vs.30.06±4.05 vs.20.36±4.33,P＜0.05). Experiment 3 was designed to investigate the effects of fusion voltage on the fusion rate and death rate of interspecies reconstructed oocytes,and the results showed that the fusion rate of reconstructed oocytes,when fusion parameter was at 230v/mm 10μs,was higher significantly than that at 200v/mm 10μs and 250v/mm 10μs(53.16±5.94 vs.26.97±3.86 vs. 33.07±8.15,P＜0.05).Furthermore,the death rate of reconstructed ooyctes at 230v/10μs was lower.Experiment 4 was designed to compared the reconstruction rate and development ability of reconstructed embryos derived from the sub-zonal injection method and whole cell intra-cytoplasmic injection method,and the results showed that there was no difference between two methods(P＞0.05).Experiment 5 was designed to used cumulus cells of different passages as feeder cells to culture reconstructed embryos, and the results showed that the blastocyst rate of two co-culture group was higher than that of control group(24.66±5.18 vs.24.21±4.99 vs.17.64±1.47,P＜0.05),and that there was no difference between the blastocyst rates of co-culture group of the original cumulus cells and 3 passages cumulus cells(P＞0.05).Experiment 6 was designed to investigate the effect of 50 nM trichostatin A treatment on the development of interspecies reconstructed embryos,and the results showed that blastocyst rate of reconstructed embryos treated with TSA for 48 h was higher significantly than that for 24 h and 0 h(33.85±2.88 vs. 19.07±3.70 vs.23.82±3.23,P＜0.05).Taken together,in the current study we established a modified and effective technical system for iSCNT between goat somatic cells and bovine oocytes as following:firstly, bovine ovaries were preserved at 26～30℃and transported to laboratory,and we selected oocytes,attached 5 layers of cumulus cells and FSP,and subjected them to in vitro maturation and utilized matured oocytes as recipients;secondly,we used goat somatic cells as nucleus donors,and we made reconstructed oocytes by sub-zonal injections or whole cell intra-cytoplasmic injections to enucleated matured ooctyes;finally,the reconstructed oocytes were activated by chemicals,and we cultured cloned embryos together with feeder cells or treated them with 50 nM trichostatin A for 48 h,and successfully obtain cloned blastocysts.