Study On Improving The Key Steps Of Sexing Bovine Embryos With Multiplex Polymerase Chain Reaction

In the study, four key steps of sexing bovine embryo with PCR were improved so this method would become more perfect and more useful.The first, a rapid, sensitive multiple PCR was developed for sex determination. The stl/st2 and bovine 1.715 satellite DNA sequences were selected for the determination by amplifying male cattle DNA and bovine-specific DNA respectively. But the unequal number of copies of the two repetitive sequences required some modification of the multiplex PCR method. In the multiplex PCR, the first 10 PCR cycles were done with male-specific primers followed by additional 20 cycles with bovine-specific primers. The sex of embryos at the preimplantation stage has determined by the multiplex polymerase chain reaction. 30 sectioned embryos were detected. Of these 50 embryos, 21 were fresh ones and 9 were frozen ones. The results showed that the rate of accuracy is 100% IN the twice experiments of sexing identification.The second, three pairs of primers, stl/st2 and BOV97M and K49057/k49058 were chosen as the sexing candidate primers. Finally, primers stl/st2 was selected for sexing identification, because they only showed specific for the Y-chromosome of bovine but not for that of mice, human beings, and other mammals.The third, the blastocysts were ordinarily divided into five grades and four development stages. The correctly recognizing of embryo grades and stages was benefit to evaluate embryo quality, select the biopsied embryo stage and position for sexing. In the experiment, 30 A- and B- grade morula and blastula were selected for sexing. The PCR method was applied successfully using 5-10 cells dissociated from bovine fetal fibroblast cell line.Furthermore, we resolved some of the problems associated with single-cell PCR and overcame them with a highly efficient PCR system.