| In order to study whether closed pulled straw(CPS) can be used to refrigerate bovine oocytes, we researched separately the effect of epidermal growth factor(EGF) on in vitro maturation of bovine oocytes, the effect of co-culture methods on embry development, the best choice of vitrification solution for bovine oocytes, the effect of different developmental stages on vitrification for bovine oocytes and the utilization of CPS for freezing bovine oocytes. The results are as followed:1 Bovine oocytes were cultured in different concentrations of EGF to mature, and the optimal concentration of EGF was analyzed based on maturing rate finally. Fertilized embryos were cultured by different co-culture methods, and then according to the embryo developmental rates, the effects of two co-culture systems on embryonic development were studied. The results showed that: the developmental rates of the two groups that concentrations of EGF were 15μg/ml and 20μg/ml were higher significantly than other groups (p<0.05); The embryo developmental rates of the two co-culture systems had no obvious difference(p>0.05). The conclusions: the maturing rates would be best on condition that the concentration of EGF was 15-20μg/ml; The effects of two co-culture methods that using granular cells and fibroblasts on embryonic development had no obvious difference. Consequently, according to actual condition, we can choose different kinds of cells to be co-cultured.2 Maturing bovine oocytes were frozed with four kinds of refrigerant, and then used in vitro fertilization after thawing, finally the best refrigerant was definited according to the embryo developmental rates. The results proved that the embryo developmental rate of the group with a refrigerant concentration of 20%EG and 20%DMSO was higher obviously than others(p<0.05). The conclusion: the refrigerating effect would be the best when 20%EG and 20%DMSO were mixed-used as a kind of cryoprotectant added into vitrification solution.3 Bovine oocytes were froze after being cultured to mature for different time, and then were cultured to mature and used in vitro fertilization after thawing, the best freezing period for bovine oocytes was settled based on the embryo developmental rates. The maturing rate, the cleavage rate, and the blastocyst rate of the bovine oocytes cultured to mature for 22 hours were 77.2%, 56.5% and 16.9% after vitrificated, unfreezed and in vitro fertilization, which are higher significantly than other oocytes in different maturing stages. The conclusion: bovine oocytes grown to maturity basically when cultured in vitro for 22 hours, and they need to be cultured in maturing solution for another 2 hours, which contributed to wipe the cryoprotectant out absolutely, and made the vitrification effect be the best.4 Bovine oocytes in GV stage were frozed with microtubules, OPS and CPS, cultured and fertilized in vitro. Compared to the embryo development rates between the three groups, we made a conclusion that: the normal morphology rates of group microtubules, group OPS and group CPS were 67.1%, 88.1% and 85.8%, the maturing rates were separately 49.1%, 66.9% and 64.4%, the cleavage rates were separately 19.6%, 45.8% and 43.4%, the blastocyst rates were separately 0, 6.6% and 6.0%. There were no obvious difference between group OPS and group CPS on these index mentioned above(p>0.05), but both of them are higher significantly than group microtubules(p<0.01). The conclusion: not only can CPS cool fastly as well as OPS, but also it can avoid the direct contact between oocytes and liquid nitrogen which can reduce the danger that the oocytes were polluted, consequently. The method of CPS can be used in refrigeration of bovine oocytes.
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