Sex Determination Of Rabbit Early Embryos By PCR

In this study, two pairs of primers were designed according to the sex determining region of the Y (Sry) gene of rabbit as male specific primers, and three pairs of primers were designed according to the amyloid precursor protein gene (APP) of rabbit euchromosome as control primers. These primers were combined by twos in order to establish the multiplex PCR and nest PCR system used to sex rabbit embryo, and then, the system were chosen and optimized. The results of nest PCR were also compared using electrophoresis and non-electrophoresis. At the same time, the sex determining region of the Y (Sry) gene of rabbit was compared with the gene of human, bovine, sheep and mouse, and using the nest PCR system established in the study, the gene of human, bovine, sheep and mouse were amplified in order to detect the specific bands and the possible pollution during the sexing embryo.The genomic DNA from blood and liver of twenty-four male rabbits and nine female rabbits were used to multiplex PCR and nest PCR.The result shows that there are two specific bands—the 282bp of SRY gene and 173bp of APP gene when the male rabbits DNA were amplified using multiplex PCR, but there is only one specific band—173bp of APP gene when the female rabbits genomic DNA were amplified. The products of nest PCR were detected after reaction using electrophoresis and non-electrophoresis, the result shows that there are specific band about 282bp when the genomic DNA from male rabbits were used after electrophoresis and there is bright fluorescence in the PCR tubes when they were put in the ultraviolet radiation analyzer and the sensitivity is 10pg, but nothing can be seen when the genomic DNA of female were used. The sensitivity of multiplex PCR is obviously low than nest PCR, but the results of embryo sexing of these two kinds of PCR are consistent with practical sex and the accuracy was 100%.Twelve rabbits were supervulated using imported FSH+hCG(8) and PMSG+hCG(4),and the result shows that the ovulation points and embryos of FSH+hCG are 72.4±13.6 and 52.6±14.4 respectively.It's remarkably better than PMSG+hCG—32±6 and 21.8±4.2 respectively. A part of embryos were transfered into four rabbits that were synchronizated, as a result, two rabbits gestated and one of them aborted after twenty days,the other layed eight small rabbits.The rest were splitted by BIO-RAID Micromanipulator or hand. The result is using hand cut the embryo is more flexible and simple, and it's also easy to be used on the field, but the disadvantage is the number of cells that get from embryo aren't exact.