| 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 factor 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 Bovine Fetal Fibroblasts (BFF) as the nuclear transfer donor cell,by modifying the nuclear transfer process of donor cells,receptor oocytes and culture environment of cloned embryos, in an angle of apoptosis, this paper researched influence of VE on bovine nuclear transfer embryo developmental competency in early stage. Bovine fetal fibroblasts were obtained either by two basic culture media DMEM and M199 or were digested by a mixture of 0.2 % collagenase Ⅳand 0.25 % trypsin at the rate of 2/3.The cell could be stored both at -196℃for a long time and at -70 ℃for a short time under the condition that 10 % DMSO was added into freezing media. Serum deprivation method was introduced to capture G0/G1 stage fetal fibroblasts and the efficiency was examined by Flow cytometry. the result demonstrated that,contrast to the control, 82.5 % cells were at G0/G1 stage that were induced by serum deprivation, the induced effectiveness is significantly higher than control group(61.2 %)(p<0.05)。In the same time, we examined the apoptosis rate of bovine fetal fibroblasts when added VE to the media after serum deprivation with Hoechest33342 fluorescence staining, DNA agarose gel and Flow cytometry.the result showed that serum starvation made the apoptosis rate of bovine fetal fibroblasts rised significantly, the reduced apoptosis rate of VE was significantly distinct (p<0.01), Thus the optimum inhibitor concentration of VE was 100 μmol/L. The efficiency of Ionomycine united with 6-dimethyladenine(6-DMAP) in stimulating bovine IVM oocytes and the affect of Vitmine E and V_C on bovine parthenogenetic embryo development in vitro. the result shows that 5μmol/L Ionomycine can activate the IVM oocytes effectively,and the parthenogenetic embryo early blast rate is 39.3 %, expanded blast rate is 24.6 %,hatched blast rate is 10.7 %。compare to the control, that when added 100μmol/L V_E to the culture medium, the development rate of early blast(43.5 %), expanded blast(30.4 %) and hatched blast(17.4 %) rised distinctly(p<0.05). when added VE and VC to the medium, the number of early blast, expanded blast, hatched blast are higher than the control, but the difference with VE treatment alone was not significant(p>0.05). By Hoechst33342 fluorescence staining and morphological observing, we studied the influence of donor cells treated by VE on early development and apoptosis of cloned embryo. The result shows that the apoptosis appeared when oocytes are in the course of development and maturing, the blastomeres undergone apoptosis in the development of cloned blasts. The blastomeres apoptosis rate of blasts that cloned by 100 μmol/L VE treated fibroblasts reduced significantly(p<0.05), so the development rate of early embryos rised in vitro; long-term cultured donor cells(>25 PD) used for SCNT did not affect the developmental competence of bovine cloned embryos and the total number of blasts, but increased the incidence of apoptotic blastomeres. The influence of donor cells,receptor oocytes and cloned embryos cultured with VE on the early development of nuclear transfer embryo were evaluated. The result suggested that the blastocyst rate of cloned embryo treated with VE is 23.9 %,contrast to the control (18.8%),the difference between them is significant(p<0.01). |
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