Study On The Bovine Embryo Sexing By PCR Method

The PCR is a new way of early bovine embryo sexing. By this method, the bovine embryo sexing can be done quickly, exactly and easily. So it is the best way to sex early bovine embryo at present. In this study, we aimed to improve the sensitivity and try to reduce the time that it will take, and then establish a suitable and applicable PCR system that can be used to sex the early bovine embryo. In order to do these, we have done lots of things and also reach the aim that we designed before. Bellow are some of them:1. Using bovine gene for testis determining factor SRY as male specific gene and bovine alpha-s1 casein gene as marker gene, two pairs of primers were designed respectively and according to each gene sequence, we designed to pairs of nest primers, if the multiplex PCR system was not suitable for the bovine embryo sexing then we would try the nest PCR. The length of inner and outer primer of these two genes were 357bp,232bp,528bp and 367bp respectively. The result showed that the multiplex PCR system was stable and exact but its sensitivity was low, so it was not suitable for sexing bovine embryo.2. So we established the nest PCR system on the base of the multiplex PCR system. After trying time by time, by this system, using just one cell from embryo, we can sex the bovine early embryo.3. Because the Sry just has one copy in a genomic, the system that established by this gene can not be very sensitive. So we choose another sequence on Y chromosome of bovine as the male bovine specific gene. It is a repeated sequence, some of them repeat thousands of times. When the number of cell from embryo was determined, these sequences would have more copies in a PCR system than Sry gene. The bovine tumor necrosis factor alpha (TNFa) gene was chose as the marker of bovine. Just as above, the lengths of primers were 534bp, 480bp, 357bp and 272bp. The result showed that the sexing can be done just using three cells.4. Although the multiplex PCR system established using sequence on Y chromosome of bovine can be used to sex bovine embryo, the result of PCR was very weak when the number of cell was small and the system become unstable. So we must use the nest PCR system. Just like above, the bovine embryo sexing can be done using one cell from embryo by this nest PCR.