性控奶山羊精液X和Y精子分离比例检测方法的建立

旨在建立可以定量计算奶山羊精液中X、Y精子数量的双重TaqMan荧光定量PCR方法，用以检测经过分离的奶山羊精液X和Y精子的数量和比例，为性控技术的开发和生产应用提供技术支撑。本研究选择X、Y染色体中特异基因F9及ZFY片段设计引物，建立标准曲线，优化荧光定量PCR反应体系和条件。通过对阳性标准品梯度稀释以及对60支已知纯度的性控精液进行测定（3次重复）来检验方法的敏感性和可靠性。结果显示，所建立的双重TaqMan荧光定量PCR方法特异性和重复性好，X和Y精子检测灵敏性分别为47和51 copies·μL^-1；利用该方法对商品化的奶山羊性控冷冻精液中X和Y精子的数量和比例进行计算，其结果与销售公司提供的X和Y精子的纯度无显著差异（P>0.05），表明该方法结果可靠。本研究建立的计算奶山羊X、Y精子数量的双重TaqMan荧光定量PCR方法特异性和重复性好，灵敏度高，结果可靠，为计算奶山羊精液分离后X、Y精子数量及比例提供了快速可靠的方法。

Establishment of Assay for Detecting the Ratio of X and Y Sperm Separation in Gender Controlled Dairy Goat

The aim of the study was to establish a dual TaqMan Real-time PCR assay that could quantitatively calculate the number of X and Y sperm in dairy goat semen, detect the number and ratio of separated dairy goat X and Y sperm and provide technical support for development and application of gender control technology. The dual real-time PCR assay was established through designing primers targeted to the specific gene F9 and ZFY fragments in the X and Y sperm, a standard curve was established, and TaqMan real-time PCR reaction conditions and system were optimized. The sensitivity and reliability of the method were verified by serial dilution of positive standards and determination of 60 gender controlled semen (3 repetitions) of known purity. The results showed that the dual real-time PCR assay had good specificity and repeatability, and the sensitivities of X and Y sperm detection were 47 and 51 copies·μL^-1, respectively. The assay was used to calculate the numbers and ratios of X and Y sperm of dairy goat gender controlled semen, and the results were not significantly different from the purity of the X and Y sperm provided by the sales company (P>0.05), which indicated that this assay was reliable. The dual real-time PCR assay for calculating the number of X and Y sperm of dairy goats established in this study has good specificity and reproducibility, high sensitivity and reliable results, which provides a fast and reliable assay for calculating the number and ratio of X and Y sperm after semen separation in dairy goat semen.