Sex Identification Of Embryos In Different Developmental Stages By Using Pregnant Dairy Cattle Blood

As a complementarity of embryos sex identification in early pregnant stages for dairy cattle, It has important significence to improve the female rate of dairy cattle and economic effect. The traditional method of sex identification for dairy cattle's early embryos are mainly jugular vein-puncturing or sampling from chorion, it is harmful for matrix and fetus to some extent. It supplied the theoretic bases for dairy fetus sex identifying in early stage when pregnant dairy cattle perpheral blood has free fetal DNA. The harmfulness can be avoided and alleviated for embryos in different developmental stages by using pregnant dairy cattle's blood.The test mainly aims at the pregnant dairy cattle, which were fertilized to identify sex of early embryos. The fetal DNA was extracted from plasma, which was separated from jugular vein blood, which sampled from pregnant dairy cattle. Specific primers were designed based on SRY gene (sex-determining region of Y chromosome) sequences. Nested PCR was applied to amplify specific gene fragments and the specific products of amplified fragments ofβ-actin primers as the internal control simultaneously and to explore a new and non-invasive method of embryo sex identification for pregnant dairy cattle.The amelogenin (AML) gene primers were designed and compared with specific SRY primers during the test. The amplified results were compared of two primers and showed that the protocols of nested PCR to amplify SRY gene primers are complex and may take long time, but the effect of amplification is well; and the protocols of normal PCR to amplify AML gene primers is simple and it takes only short time, but it's effect of amplifying is bad. The effect of amplified AML primers is better for gDNA and that of N-PCR for scale free DNA in plasma. In this test, 32 embryos in different developmental stages were identified by using the method of N-PCR.The result showed that the average accurate rate of sex identification in different pregnant stages is 80%, and the stage in 1~2 months, 2~3 months, 4~8 months are 60%, 85.7% and 92.3%, respectively.