| In order to raise the effect of the development and cryopreserved of the oosperms in vitro, oocytes which were extracted from the follicles (2-6mm) of the bovine ovaries in a slaughterhouse, were cultured and fertilized in vitro in routine method. Then the following experiment have been done on oosperm in vitro.1.The oosperm and cumulus cells were co-cultured in basic development culture fluid (TCM-199+10%CS). During the culture period, the experiment group exchanged the culture fluid, the control one did not so. The cleavage rates (66.9%/60.4%) and blastula development rates(14. 2%/13. 4%) were no significant difference between them.2.The oosperm co-cultured with cumulus cells in basic development culture fluid(TCM-199+10%CS) with ingredient of taurine, which concentration was 0 mM (control group), 5 mM (group I), 10 mM (group II), 20 mM (group III),25(group IV), 50 mM (group V) in different group respectively. The blastula development rate was 20.9%, 28.0%, 34.7%, 25. 7% , 28. 6%and 22. 6%. The group II was different from the control group significantly(P<0. 01) and there was no significant difference between the other experiment groups and control one(P>0.05). Among the experiment groups, the group II was better than group III(P<0.05) and group V significantly(P<0. 01). This result indicated that: on this study designed conditions, taurine can promote the development of embryo, and the concentration of the group 3 was better than others.3. In order to study the best stage of adding tautine to the culture fluid during the in vitro maturation and development culture, 5 group experiments were designed.Group IVM: lOmM taurine was added to oocytes' maturation culture fluid during in vitro development maturation only.Group IVC(0~48h): lOmM taurine was added to oosperms' culture fluid during the first 48h development culture after in vitrofertilization only.Group IVC(48h ~ ) : Before adding 10mM taurine to oosperms' development culture fluid, they were cultured 48h in vitro.Group IVM+IVC: lOmM taurine was added to culture fluid during in vitro maturation and development culture from beginning to end.The control group: In vitro maturation and development culture followed the routine methods.The blastula development rates was 32.2%, 36.1%, 41.9%, 44.0% and 31.7%, respectively. Group IVC (48h~)and IVM+IVC were different from the control group and Group IVM(P<0. 05) , but Group IVM and IVC were not different from the control group (P>0. 05) . There was no difference among the group IVC(0~48h), IVC(48h~) and IVM+IVC(P>0.05). This result indicated that: taurine can promote blastula development rates during the later development or maturation culture period and all the development culture period on conditions that the oosperm and cumulus cells were co-cultured in TCM-199 basic culture fluid. The taurine can promote the oocytes' maturating and developing of oosperm.4. To study the effect of direct freezing method of oosperms' cryopreserved, 4 experiments were designed. The experiment design and result were as the following table.The result shown that there was significant difference between D3 and the control group (P<0. 01) only. And there was no difference among the experiment groups. So we could concluded that: D, was not different from control group in the rate of normal embryo after thawed, development after 48h' s cultivation and hatch after 72h' s cultivation. In thebovine blastula' s rate of development , Direct freezing method (D2)was lower than the control group, but there was no difference in hatching rate.The study believed that there was no need to exchange culture fluid during the culturing of bovine oosperm in vitro. Taurine(10mM) can promote the development of embryo, especially during the in vitro maturation and development culture from beginning to end or the later stage of the development culture. There was no significant difference between direct freezing method (D1)using 1. 5M PG as cryopreserved and 0. 2M SU as diluent and one step method. Direct freezing... |
