The Research On The Establishment Of Bovine Fibroblast Cell Line And In Vitro Fertilization And Nuclear Transfer Of Bovine

In this research, bovine fibroblast cell culture technology system, somatic cell nuclear transfer technology system and bovine in vitro fertilization technology system are established in an newly-built embryology laboratory. This research provided the foundation for preservation of genetic resource of superiority bull, production of bovine cheap test-tube embryo and study of transgenic bovine. The results as following shown :1. Adopting adherent cell culture method, bull fibroblast cell lines were established. It is best time for freezing bull fibroblast cell when it is passed over the 3rd or 4th generation with the density of cell over 1×106/ml. In our study, cell livability after revival have no difference, both keep more than 90%, and the growth curve line of cow's fibroblast is S-shaped. The results of Microorganism experiment are negative, the cells are not contaminated by bacterium and mycoplasma.2. Establishing and ripening bovine in vitro fertilization technology system. 547 oocytes were collected and the growth rate and growth speed of the parthenogenetic embryo and fertilized embryo were compared. The result showed that in vitro fertilization embryo's cleavage rate (69.8%),8-cell rate (47.3%) and blastocyst rate (26.1%) were lower than the parthenogenetic embryo's cleavage rate (73.0%),8 cell rate (49.2%) and blastocyst rate (32.6%). In both two groups, blastocysts came out earliest on 6d. Moreover, in the experiment, cumulus cell of Cumulus-Oocyte Complex can significantly improve in vitro fertilization blastocyst rate from 10.2% to 27.5%. And Vero cells as feeder can also significantly improve in vitro fertilization blastocyst rate from 12.4% to 27.5%.3. The establishment of bovine somatic cell nuclear transfer technology system. The cleavage rate (72.4%),8cell rate (38.8%) and blastocyst rate (11.2%) of bovine somatic cell nuclear transfer embryo are lower than the cleavage rate (78.8%),8cell rate (55.3%) and blastocyst rate (34.9%) of parthenogenetic embryo. In both two groups the cleavage rate had no difference, but 8 cell rate and blastocyst rate significantly different. As in vitro fertilization showed, vero cells as feeder could significantly improve somatic cell nuclear transfer blastocyst rate from 0% to 11.2%.