Studie On The Effect Of β-ME Treat On Donor Cell For Somatic Cell Nuclear Transfer And Embryo Development

Somatic cell nuclear transfer (SCNT) is useful not only for shortening the breeding time and increasing the availability of good species, but also for the study of fertilization mechanism. There are many steps in the procedure of mammalian SCNT from donor cell preparation to embryo transfer to foetus birth, and every step can have influence on the efficiency of somatic cell nuclear transfer. Donor cell preparation and reconstructed embryo culture are the most important steps. The aim of this study was to improve the efficiency of nuclear transfer at these two steps. We studied on the choices and treatments of donor cell, and the influence of beta-ME on in vitro reconstruction of embryos. Salient results of the present study were as follows:1. Establishment of donor cell lines for SCNT and MTT tested cell activity to select the best donor cells.We chose three kinds of cells, Bovine mammary epithelial cells (BMec), fibroblast cells from a cross between the Holstein and the Jersey (HJF), and yellow cattle fibroblast cells (YCF) to culture, and used MTT to test the cells of passage 2,6,13, and 20. It showed that the cell viability and proliferation of the second and sixth passage are significantly better than passage 13 and 20 (P<0.05), especially the passage 6 cells (P<0.01).2. The variation tendency of MTT-OD value of the three kinds of cells by Beta-ME treatment showed us the best concentration.We used beta-ME of concentration gradient (0,50,100,150μmol/L) to treat the different source of cells, then cell proliferation was examined with MTT. The results showed that Beta-ME enhances cell proliferation rate at 100μmol/L significantly (P<0.05).3. Antioxidant capacity of the three kinds of cells which were treated by Beta-ME.In this part, we took the pre-treatment in the same way as former part, after 0,50, 100,150μmol/L beta-ME added to culture solution for the cells, we tested five oxidation indicators (GSH-Px, SOD, CAT, MDA, T-AOC), the results showed that:when supplemented with (3-ME 100μmol//L,The GSH-Px, SOD, CAT, T-AOC concentrations were significantly (P<0.05) higher than the other groups and MDA value was significantly (P<0.05) lower, it demonstrated thatβ-ME enhances cell oxidation resistance, and 100μmol//Lis optimal concentration. 4. The cleavage and blastocyst rate of nuclear transferWe induced the 6th passage cells into GO/G1 stage with drop serum concentration to 0.5%, and then we designed three experiments:group one, blank control, injected the cell into enucleated mature oocytes, group two, base on group one,we cultured reconstructed embryos with Beta-ME (100μmol//L), group three, cultured donor cells with Beta-ME (100μmol//L) 24h, then reconstructed embryos, and cultured reconstructed embryos with Beta-ME(100μmol//L). The results showed that:the rate of cleavage and blastocyst of group two and three significantly (P<0.05) increased when compared with group one, however, there was no significant (P>0.05) difference between group two and three. Moreover, the data showed that the cleavage and blastocyst rate of group three are highest.In conclusion, the cells oxidation resistance and the rate of cleavage and blastocyst are improved afterβ-ME treatment.