| For the sake of enhancing the effectiveness of mammalian nuclear transplantation, it is necessary to research on how to improve the quality of oocyte after in vitro maturation(IVM), which is useful for explaining the mechanism of reprogram induced by factors in oocyte cytoplasm; and it is also necessary to study the method that could make the donor chromosome sensitive to the reprogram factors. Based on Bore Goat Fetal Fibroblast(BGFF) as the nuclear transfer donor cell, this paper researched influence of treatments on the donor cells to the cloned embryo developmental competency in early stage.Fetal fibroblasts were obtained either by two basic culture media DMEM and M199 or were digested by a mixture of 0.2% collagenasell and 0.25% trypsin at the ratio of 2/3. The cells could be stored both at -196℃ for a long time and at -70 ℃ for ashort time under the condition that 5% DMSO was added into freezing media.The influencing factors concerning goat oocyte IVM in culture medium were studied through 4 methods. That is: the origination and concentration of serum( both fetal bovine serum(FBS) and estrogenic goat serum(EGS)) and gonadotropins(FSH/ LH and human chorionic gonadotropin(HMG)), the concentrtion of epithelial growth factor(EGF) and the origination of water. The result showed that the ratio of goat oocyte IVM could be as high as 85.7% if they were cultured in the medium of TCM199+10% EGS+0.075IU/mL HMG+1 n g/mLE2. It showed that EGS was better than FBS in cuturing goat oocytes and the efficiency was improved alone with the rising concentration of EGS; HMG was slightly superior to FSH/LH; EOF could not inhance the maturating efficiency at a concentration of 30 ng/mL but was useful for early embryo development; it is advisable to use fresh water dissolve media, if purchase, a small package is recommended it should be used up as soon as possible.The efficency of ethanol and ionomycin united with 6-dimethyladenine (6-DMAP) in stimulating goat IVM oocytes were studied. The result showed that ethanol was not fit for parthenogenetic development of goat oocytes while 5u.mol/L ionomycin did. The ratio of blastocysts between them were greatly different (P<0.01), which was 23.5% to 0% respectitively.The influence of the cell cycle stage of receptor, Mil and Til, on goat nuclear transfer were studied. The result showed that Til stage method could enhance the enucleation efficiency significantlly (P<0.05) , however the blastocysts that obtained from both methods had no significant difference (P>0.05) .Four methods were introduced to capture G0 /G1 stage fetal fibroblasts and the efficiency were examined by immuno-flourescent. The result demonstrated that 79.5% and 84.5% cells were at GO/G1 stage that were induced by serum deprivation and by Roscovitine, a inhibitor of G0/G1, respectively. Both of them had significantly higher effectiveness than control group(60.3%). High confluncy method had a inducing ratio of GO/G1 cells of 70.1%, which was also higher than control group but the difference was not significant (P>0.05) between them.The influence of cell synchronization and heat denaturaton of donor cells on the early development of nuclear transfer embryo were evaluated. Heat denaturation donor cells were cloned by introcytoplasmic injection and the blastocysts developmental ratio was 11.8%. GO/G1 induced cells were also cloned and the blastocysts developmental tatio were 15.9%(serum deprivation), 11.9%(Roscovitine treatment), 7.5%(high confluency treatment)and 0%(control group). The result suggested that both heat denaturing method and GO/G1 stage incucement could be applied to goat cloning and there were no significant differences in blastocyst development (P>0.05) among them. |
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