Study On The Optimization Of In Vitro Culture System Of Embryos And Isolation And Generated Culture Technology Of Embryo Stem Cells In Goats

This study was to optimize the technologies for production of embryo in vitro, including in vitro maturation (IVM ) of oocytes, in vitro fertilization (IVF) and in vitro culture (IVC) of embryos in goats to establish a perfect system for in vitro embryo production (IVP) and to make these technologies going out of laboratory to be used in the fields of embryonic engineering. At the same time, the isolation and generative culture of embryonic stem (ES) cells in goats were studied to provide the theoretic bases for post–embryonic engineering technologies such as nuclear transfer and transgenic technology etc. Experiment one studied the IVM methods to increase the maturation rate of oocytes. Droplet method was used to culture oocytes to study of collection of oocytes, modification to maturation medium, supplementation of different hormones and antioxidants on the IVM of oocytes. Oocytes of grade A with uniform and compact cytoplasm and parceled by at least three layers of cumulus cells were chosen. The maturation rate increased by adding 1 μg E2, 5 μM β–merocaptoethanol (β–ME), which was a kind of antioxdant, and culturing 27h. Imported FSH and LH could be completely replaced by domestic chorionic gonadotrophin (the main gredients were FSH and LH) and reduced the experiment cost under the same maturation effects. Experiment two studied the optimization of IVF conditions to increase the quality and quantity of in vitro matured oocytes. The effects of three capacitation materials of heparin,caffeine and Ca2+ vector IA23187 and different capacitation methods of Percoll and swim–up methods were compared. The results showed that only the heparin group was better and had higher percentages of fertilization, cleavage and blastocyst. The cleavage rate of Percoll method was significantly higher than that of swim–up method. But there was no significant difference for blastocyst rate between the two methods. The motility rate of sperm by Percoll method was higher than that by swim–up method. However, the overall fertilization and development rates were still lower. Experiment three aimed to establish a suitable culture system for IVC of goat embryos by optimizing culture procedure and screening culture medium. Studies by comparison among different co–culture cells, replacement procedures and basal culture media showed that, if only co–culture cells without considering the type of them, the embryos could overcome the 8~16–cell inhibition stage. The best procedure of using fetal calf serum (FCS) and BSA was adding BSA in the former stage of 48h and replacing it by FCS hereafter. All three kinds of medium, TCM199, CR1 and SOF, could be used as embryo culture media and SOF could be prepared simply and thus recommended to be used as basal medium.