睾丸特异蛋白基因对奶牛早期胚胎性别的鉴定

为方便性别鉴定方法在现场应用，利用睾丸特异蛋白基因（TSPY）建立奶牛早期胚胎的非电泳性别鉴定方法。本实验首先设计并合成TSPY雄性特异引物和雌、雄共有引物并利用已知性别的奶牛血液DNA检测了利用TSPY基因鉴定胚胎性别的可能性。结果显示：雄性特异引物和共有引物对在10pg～60pg范围内性别符合率均为100%；采用非电泳的方法检测TSPY基因鉴定了49枚胚胎的性别，同时，用LAMP法对其中的6枚胚胎的性别鉴定结果进行验证，结果二者完全一致；对其中鉴定为雌性的21枚进行移植，结果9头出生的犊牛均为母牛。结果表明，TSPY基因是一个很好的雄性特异标记，非电泳检测TSPY基因对奶牛早期胚胎性别的鉴定结果准确可靠。

Use of Testis Specific Protein Gene for Sex determination in Preimplantation Bovine Embryos

The objective of this study was to examine the possibility of using a non-electrophoretic method to detect the TSPY gene for sexing bovine early embryos, which made bovine embryo sexing under farm condition more feasible. Primers were designed to amplify a portion of the TSPY gene and a common gene as an internal control primer. PCR optimization was carried out using a DNA template from bovine whole blood. Furthermore, six embryo samples were diagnosed by this method and the sexing results were contrasted with those of the Loop-Mediated Isothermal Amplification (LAMP) method. The result showed that TSPY was as reliable a sexing method as LAMP. Forty-three morula and blastocyst embryos collected from superovulated donor dairy cattle were sexed by this method, and twenty-one embryos judged to be female embryos were transferred non-surgically to recipients 6 to 8 days after natural estrus. Out of 21 recipients, 9 were pregnant and all delivered female calves. The results showed that the sex predicted accuracy of this protocol was 100%. In conclusion, TSPY gene was a good male specific marker and indicated that a non-electrophoretic method was feasible and accurate to detect TSPY gene for sexing preimplantation bovine embryos.