Establishment of Rapid Method for Evaluating the Ratio of X and Y Spermatozoa in Sheep Semen
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 signifi-cantly 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 technical support for the detection of X and Y sperm separation in sheep.
sheepsexually controlled semenX and Y spermatozoaTaqMan probefluorescence quantitative PCR
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