绵羊精液中X、Y 精子比率快速评价方法的建立  

Establishment of Rapid Method for Evaluating the Ratio of X and Y Spermatozoa in Sheep Semen

作　　者：古丽孜巴·艾力 李伟建 艾力亚木·土尔衮 张慧 王旭光[1] Guliziba·Aili;LI Weijian;Ailiyamu·Tuergun;ZHANG Hui;WANG Xuguang(College of Animal Science,Xinjiang Agricultural University,Urumqi 830000,China)

机构地区：[1]新疆农业大学动物科学学院,乌鲁木齐830000

出　　处：《中国草食动物科学》2024年第3期33-38,共6页China Herbivore Science

基　　金：新疆维吾尔自治区自然科学基金项目(2020D01A56)。

摘　　要：研究旨在建立以TaqMan荧光PCR为基础的快速检测绵羊X、Y精子比率的方法,为绵羊X、Y精子分离技术的发展与应用提供技术支撑。试验分别针对X、Y染色体特异性基因F9和SRY设计引物与探针,以绵羊基因组DNA为模板进行片段扩增、质粒连接、扩增提纯后按不同比率混合构建标准曲线,计算拟合方程。并通过对3份商用X、Y性控精液及2份鲜精进行测定来检验方法的准确性、稳定性及可靠性。结果表明,商用X性控精液中X精子的比率分别为(89.46±3.37)%、(89.86±2.85)%、(90.02±2.19)%,商用Y性控精液中Y精子的比率分别为(89.02±2.68)%、(88.21±2.63)%、(88.72±3.93)%,检测结果与质控报告所标注的X、Y精子比率无显著性差异(P<0.05)。鲜精的检测结果表明,X、Y精子比率分别为(49.86±2.90)%、(49.30±2.10)%,与理论值50%无显著性差异(P<0.05)。综上,研究建立的以TaqMan探针荧光定量PCR为基础的快速检测绵羊X、Y精子比率的方法速度快、检测所需精液体积小,准确性、稳定性及可靠性高,能够为绵羊X、Y精子分离检测提供技术支撑。The aim of this experiment was to establish a TaqMan fluorescence PCR-based method for rapid calculation of X and Y sperm ratio in sheep,and to provide technical support for the development and application of X and Y sperm separation technology in sheep.In this study,specific primers and probes were designed for the X and Y chromosome-specific genes F9 and SRY,respectively,and sheep genomic DNA was used as the template for fragment amplification,plasmid ligation,and standard curve was constructed by mixing at different ratios after amplification and purification,and the fitting equations were calculated.The accuracy,stability and reliability of the method were tested by measuring commercial X-sex-controlled semen(n=3),Y-sex-controlled semen(n=3)and fresh semen(n=2).The results showed that the ratios of X spermatozoa in commercial X-sex-controlled semen were(89.46±3.37)%,(89.86±2.85)%,(90.02±2.19)%,respectively;the ratios of Y spermatozoa in commercial Y-sex-controlled semen were(89.02±2.68)%,(88.21±2.63)%,(88.72±3.93)%;and there was no significant difference between the results of the assay and the ratios of X and Y spermatozoa as labelled in the quality control report(P<0.05).The assay results of fresh sperm showed that the X and Y sperm ratios were(49.86±2.90)%and(49.30±2.10)%,respectively,which were not significantly different from the theoretical value of 50%(P<0.05).In summary,the method established in this study based on TaqMan probe fluorescence quantitative PCR for rapid calculation of X and Y sperm ratio in sheep is fast,the volume of semen required for the test is small, and the accuracy, stability and reliability are high, which can provide technicalsupport for the detection of X and Y sperm separation in sheep.

关 键 词：绵羊 性控精液 X、Y精子 TAQMAN探针 荧光定量PCR 

分 类 号：S826.3[农业科学—畜牧学]