Study On Bovine Somatic Cell Nuclear Transfer

The success of nuclear transfer of somatic cells not only indicates that the differentiation of animal somatic cells is not irreversible theoretically,but also provided a new method of preservation and amplification of endangered animals.The production of animals by SCNT involves multiple steps,The donor nuclei gained is one of the most important step.Currently,use intracytoplasmic nuclear injection to take out nuclear transfer,the membrane of donor cells must be fully lysed.Therefore,a simple and convenient method for accomplish this purpose is required. This study focused on to prepare abbit anti-bovine-spleen serum which could be following utilized in the processing of lysising the membrane of the donor cells,and then applicated in the bovine oocyte intracytoplasmic nuclear injection,which would provided a new method of acquiring donor nuclei in nuclear transfer,accordingly.1.In vitro mature and activation of bovine oocytes(1) Comprision the doses of hormones that affecting maturation of bovine oocytes,thus find a optimal environment for the oocytes maturing,the results indicated that the demands of maturation of bovine oocytes in vitro would be satisfied fully by hormones made in China,and when the concentration of follicle-stimulating hormone(FSH) and luteinizing hormone(LH) was 10μg/mL and 20μg/mL respectively,the maxium rate of the matured was 82.1%;so the optimal doses of hormones was FSH:10μg/mL,LH:20μg/mL.(2) In order to comparision the effects of BFF on the maturation of oocytes,additional 10% BFF was added in the maturation medium,the results indicated that the maturing rates had no significant difference between BFF added or not(83.2%versus 82.1%).(3) Different methods of parthenogenetic bovine oocytes were studied,the results indicated, when the oocytes were activated by ionomycin+6-DMAP±CB respectively,the cleavage rates and the blastocyst rates of oocytes did not have significantly difference(83.3%versus 79.0%,37.3% versus 35.9%,respectively);on the contrast,the cleavage rates and the blastocyst rates between the two groups of ethanol+6-DMAP±CB had significantly difference(82.1%versus 67.5%,36.2% versus 25.0%,respectively).Furthermore,the cleavage rates and the blastocyst rates between ionomycin+6-DMAP+CB and ethanol+6-DMAP+CB had also no significant difference(83.3% versus 82.1%,37.3%versus 36.2%,respectively).This indicate ionomycin+6-DMAP±CB and ethanol+6-DMAP±CB can effectively activate the bovine oocytes both.(4) Bovine parthenogenetic embryos cultured in SOFaa continued,comparision the two cultured methods,with medium replaced and without medium replaced,the results showed that the blastocyst rates had not significantly difference(39.7%versus 33.9%).This indicate the bovine parthenogenetic embryos cultured in the large volume of the developmental medium,the blastocyst rates did not influnced by medium replaced or not.2.Separation and culture of bovine somatic cells(1) Culturing granular cells obtained from in vitro matured COCs is a convenient,safe and efficient method.And the time of the primary culturing is short.(2) Bovine ear fibroblast cells were cryo-preserved with liquid nitrogen,and to thaw with the method of quickly thaw(38℃,1～2 min),the adherence condition of the cells was good.(3) Bovine granular cells and ear fibroblast cells were cryo-preserved with DMSO as cryoprotectants in manual way is an effective way for the bovine granular cells or ear fibroblast cells.(4) The bovine granular cells in 10^th passage were observed by the karyotype analyzed.The results indicated that 75.56%of the cells maintained diploid karyotype in 10^th Passages,and can be used for the constructed of the nuclear transferred embryos.3.Preparation of abbit anti-bovine-spleen serum (1) Rabbit anti-bovine-spleen serum was produced in rabbits which was intravenous injection of bovine spleen cells.Analysis of potency of serum antibody,and its titer was achieve the expecting aim:when the serum was diluted into 1/64,red blood cells still have the dissolution reaction.(2) Reaction of the anti-bovine-spleen polyclonal antibody serum with the donor cells,the results showed,the antibody serum can lysising the membrane of the donor cells and islation the donor nuclei.This indicated it is feasible to prepare the donor nuclei by immunosurgery.4.Bovine somatic cell nuclear transfer(1) Bovine reconstructed embryos were obtained from two different types of donor cells, bovine granular cells and ear fibroblast cells.When investigated and compared the effects of different donor cells in the development of the reconstructed embryos,the cleavage rates of granular cells and ear fibroblasts were 62.62%and 67.89%,the blastocyst rates were 23.88%and 18.92%,respectively,these results had no significant difference(P＞0.05).(2) When compared the effects of the different donor cells types treated by low tempreture(4℃) preservation or not in the effects of the nuclear transferred,The results indicated:the cleavage rates of granular cells and ear fibroblasts were 63.34%and 62.03%,70.76%and 66.67%,respectively; moreover,the blastocyst rates were 26.67%and 24.45%,21.74%and 19.56%,respectively,there was no obvious difference(P＞0.05) respectively.(3) Compared the effects of two different delaying activation time(1～3 h and 3～5 h) after nuclear injection in the cleavage and the development of the reconstructed embryos.The results indicated:the delaying time was 3～5 h has the higher cleavage rates compared with 1～3 h,and had significant difference(73.91%versus 56.52%,P＜0.05),but the blastocyst rates had no significant difference(23.53%,versus 15.38%,P＞0.05).