Study On Somatic Interspecies Cloning Technology Of Capra Ibex

Shi Qiao-ting (Grassland Science)Surpervised by Professor Guo Zhi-qin This paper researched in the interspecies somatic nuclear transfer of Capra ibex and goat. For this aim, we do the follow experiments: A, IVM goat oocytes treated with different concentrations of FSH and LH were compared. And we compared the effect of different transported temperature of goat ovaries and different IVM time of goat oocytes on the maturation of goat. B, the effects of different activation methods on parthenogenetic development of goat oocytes was discussed. C, We compared with different primary culture methods of fibroblasts of goat and Capra ibex, and tested the karyotype analysis and growth curve of cell line of goat and Capra ibex. D, the effects of the fresh and culture- frozen-thawed granulosa cells on nuclear transfer of goat also were tested. And we compared the effects of serum starvation with confluent cells of granulosa and fibroblasts on nuclear transfer of goat, and of different electric pulse parameter on fusing rate and development of reconstructed embryos. And we also compared the effect of different treated methods, different electric strength and CB on the development of interspecies cloning embryos of Capra ibex. E, we tested the cell cycle phase of Capra ibex and goat ear fibroblasts under different growth conditions. F, we transferred the interspecies reconstructed embryos of Capra ibex to surrogate goat.The main results were followed:1,The appropriate temperature of goat ovaries during transportation was 20~35℃; FSH 8μg/ml,LH0.6IU/ml was proper concentration for goat oocyte IVM. With the IVM time extending, the maturation rate was increased but the enucleate rate was declined.2,When the oocytes were stimulated primely with electric pulse then activated by chemical methods, the activation rates were significantly higher than that stimulated with electric pulse or chemical active alone. The cleavage rate was significant high when CB contained in activated media; Goat oocytes were treated by 1.2kv/cm~1.6kv/cm in 1~2 pulses had higher activation rate. And goat IVM oocytes could activate by 7% ethanol for 7min. 3,The primary culture was better used method of minced tissue than enzyme digestion for cell culture. The number of the chromosomes 2n＝60 in goat and Capra ibex, All of the chromosomes were acrocentric chromosomes. The cell lines of goat and Capra ibex were normal cells because they grown slowly at beginning and then had contact inhibition. The fibroblast cell growth of Capra ibex was slower than that of goat. The population doubling time was 4.46d,2.51d respectively.4,Analysis of cell cycle distribution by fluorescence-activated cell sorting (FACS) showed that 79.9, 7.5, 11.6% of serum starvation cycling cells were at G1/G0, S and G2+M phase, respectively, and confluent cells for 84.9, 4.5, 9.4% respectively. As donor, the rate of fuse of fresh cells was significantly higher than that of culture-frozen-thawed cells(p<0.05), but cleavage rate and murulae/blastocyst rate had no significant difference, and fibroblasts had significantly lower than granulosa cells in fuse and cleavage rate(p<0.05), but the murulae/ blastocyst rate had no significant difference. The confluent and serum starvation cells as donor, the embryo had no significant difference in fuse, cleavage and murulae/blastocyst rate. And the reconstructed embryos were treated with 1.2~1.6kv/cm, 15~20 μs and 2 pulses electric stimulation had high activation rate. The reconstructed embryos of interspecies of Capra ibex were treated with 1.6kv/cm had high fusing rate; CB was good at the activation of reconstructed embryos of interspecies.5,The cell cycles stages were different when cells grew to 50%~60%, 70~85% and complete confluent for the ninth passage cells of Capra ibex and the sixth passage cells of goat. The synchronization ability of serum deprivation or keeping confluency was similar. It was suggested that we could obtain cells at G0/G1 stage by culturing to confluency instead of serum starvation in nuclear tran...