Study On Bovine Somatic Cell Nuclear Transfer

This study focused on bovine somatic cell nuclear transfer. For obtaining a simple and effective method for bovine somatic cloning, the different protocols and experiments were adopted to reach an optimized procedure.1. Recover, in vitro mature and activation of bovine oocytes(1) Two different methods, follicle incision and aspiration, were used for recovering usable cumulus-oocyte complexes (COCs) from follicles of slaughtered bovine ovaries. The result indicates that the method of follicle incision collects more (9.22ova/ovary) COCs than aspiration(6.58ova/ovary) , but consumes more time (117.57s/ovary VS 52.13s/ovary) , COCs recovering methods does not affect oocyte maturation.(2) The maturation rates in droplets containing 100ul medium, 4-well plates containing 400μl medium overlaid with mineral oil and 35mm plates containing 3ml medium were not significantly different. The result indicates that culture medium volume has not effect on oocyte maturation.(3) Bovine oocytes were treated with ionomycin combined with 6-DMAP or ethanol combined with 6-DMAP, the percentage of cleavage 8-cell rate and blastocyst rate in these two methods were not significantly different, but the parthenogenesis development following treatment with ionomycin combined with 6-DMAP treatment was a bit higher than the treatment with ethanol combined with 6-DMAP.2. Separation and culture of bovine somatic cells(1) Bovine granulose cells were cultured in three different ways. The result indicates that, culturing granulose cells obtained from in vitro matured COCs is a convenient, safe and efficient method.(2) Proliferation-promoting rule of bovine granulose cells and ear fibroblast cells were studied by protracting the growth curve. The result indicates that, proliferation-promoting rule of bovine granulose cells is similar with ear fibroblast cells.(3) Bovine granulose cells and ear fibroblast cells were cryo-preserved with DMSO as cryoprotectants in manual way. After freeze-thaw cell viability was 68.13% or 72.43% in granulosecell or ear fibrobiast cells, respectively. The result indicates that, cryo-preservation with DMSO as cryoprotectants in manual way is an effective way for cryo-preserving bovine granulose cell or ear fibrobiast cells.3. Bovine somatic cell nuclear transfer(1) Bovine reconstructed embryos were treated with ethanol combined with 6-DMAP and ionomycin combined with 6-DMAP. The result indicates that, the treatment with ethanol combined with 6-DMAP is a better way to activate bovine reconstructed embryos, however, the development rates in two group was not significantly different.(2) Bovine reconstructed oocytes were treated or not treated with PHA before fusion, The fusion percentage of the group treated with PHA (68.01%) is significantly higher than that of the not-treated group (49.65%), but cleavage re^ 8-ceIl rate and blastocyst rate were not significantly different between two methods, however, the blastocyst rate in the group not-treated (40.36% ) is far higher than that of treated (21.31%) . The result indicates that, PHA can increase the fusion rate of bovine reconstructed oocyte, but it may have negative effect on later development of the embryos.(3) Bovine reconstructed embryos were cultured in atmosphere consists of 5%CC>2 -AIR or 5%CO2-5%O2-90%N2. The development rates were not significantly different between two methods-However, all development rates in atmosphere containing 5%CO2-5%O2-90%N2 are a bit higher than another one. The result indicates that, atmosphere of 5%CO2-5%O2-90%N2 may have active effect on later development of bovine reconstructed embryos.(4) Bovine reconstructed embryos were obtained from serum-starved and not starved donor cells. The development rates were not significantly different between two methods. The result indicates that, serum-starved or not can not significantly affect bovine nuclear transfer, but the fusion rate is a bit higher in not starved group.(5) Bovine reconstructed embryos were obtained from two different types of donor cells, bovine granulose cells and ear fibrobiast cells. The result indicates that, within granulose cells and ear fibrobiast cells, donor cell type can not significantly affect the result of bovine nuclear transfer.(6) Bovine reconstructed embryos were obtained from donor granulose cells of two different origins (donor), donor B or donor E. The result indicates that, within these two granulose cells, donor cell origin can not significantly affect the result of bovine nuclear transfer, however, fusion rate in donor B is higher than that in donor E.(7) Bovine reconstructed embryos were obtained from 1-5 passages of donor cells, the blastocystrate (8.47%) of reconstructed embryos obtained from first passage is significantly lower than that of passage 2 or 3 (28.36% or 30.33%) , there has not significant difference among other groups. The result indicates that, first passage of donor cells has negative effect on the development of bovine reconstructed embryos.(8) Cloned blastocysts were cryo-preserved using OPS vitrification method. The embryo viability is 72.09%. A total of 70 blastocysts were nonsurgically transferred into 31 surrogate cows, 9.68% (3/31) females became pregnant, and one of them aborted at 4th month after blastocyst implantation. Expected dates of birth of the remaining two cows are June and July, 2005.