Study On Somatic Unclear Transfer Of Goats

In order to optimize the protocols in somatic nuclear transfer, this thesis investigatedthree main factors affecting somatic nuclear transfer on goats.(1)Effects of culture system onthe development of goat parthenogenetic embryos；(2)Effects of cytoplasm enucleating andcytochalasin B(CB) on embryos from parthenogenesis and somatic nuclear transfer；(3)Influence of donor cell types, reconstruct means and donor refrigeration on embryos ofsomatic nuclear transfer. Goat parthenogenetic embryos were cultured in vitro in SOFaa,CR1aa and mCR1aamedium supplemented with different serum concentrations. The results of this study showedthat 10 percent is better supplemented concentration of NGS in all these three media thancontrol groups for development of goat parthenogenetic embryos, the ratio of blastocyst canreach 62.79%(81/129), 53.52%(38/71),13.64%(12/88)respectively； For the blastocystrate and blastocyst cell number in all treatment groups, mCR1aa group is lower significantlythan SOFaa and CR1aa group, SOFaa group is better than CR1aa group though there is nosignificant difference. Oocytes after in vitro maturation(IVM)were treated with only CB(group I), onlycytoplasm enucleating(group II),CB plus cytoplasm enucleating(group III)and with nothing(control).Developmental potential of parthenogenetic or nuclear transfer embryos wereobserved after in vitro culture. The results showed that experiment group I, II, III andcontrol group all had high cleavage rate(72.35%,71.55%,64.87%,86.15%)and blastocystrate(55.50%,37.43%,39.78%,59.82%).However, abnormal cleavage form in group Iand group II is higher significantly than group III and control group. Nuclear transfer was carried out with galactophore cells , fetal fibroblasts andcumulus-granulosa cells by means of intracytoplast injection and electro-fusion respectively.The donor cells were treated with or without refrigeration and synchronized at G0/G1 stage byserum deprivation before cell transfer. Development of reconstructive embryos was recorded.The results showed capacity of cloned embryos from galactophore cells(blastocyst rate,7.14%) is low significantly than fetal fibroblasts (16.19%)and cumulus-granulosa cells(19.01%)(P<0.05)；Influence of cell refrigeration on development of cloned embryos hasno significant difference between three kinds of cell(P>0.05)； The successful rate ofreconstruction by intracytoplast injection(59.20%)in galactophore cell is lower significantlythan electro-fusion(69.52%)(P<0.01),in fetal fibroblasts have on significant differencebetween two means(P>0.05),however, in cumulus-granulosa cells intracytoplast injection(82.17%) mean is higher significantly than electro-fusion mean(50.99%)(P<0.05). 2山羊体细胞核移植的研究 Conclusion 1 SOFaa medium plus 10%NGS is better combination of in vitro culture inparthenogenetic goat embryos, the ratio of blastocyst is high to 62.79%. 2 CB treatment has no effect on parthenogenetic embryos of goat. The proportion ofcytoplasm enucleated under 1/3 has no significant effect on development of parthenogeneticembryos. CB combined with cytoplasm enucleation can improve the development capacityand reduce the abnormal cleavage. 3 In the three kinds of donor cells mentioned above , fetal fibroblasts orcumulus-granulosa cells is superior to galactophore cells in sustaining development capacityof cloned embryos. Cell refrigeration has no influence on that. In reconstructive mean,intracytoplasm injection mean is superior to electro-fusion to galactophore cells, but isinferior to electro-fusion to cumulus-granulosa cells. Both means are fit to fetal fibroblasts.