At present, the only proven method for producing sexed semen in mammals is the cell sorting by flow cytometry, based on DNA difference. Cell sorting by flow cytometer provides a powerful tool for artificial insemination and production of predefined sexed embryos. Due to high costs and decrease in fertility rate, the extensive use of sexed semen in livestock depends extremely on sorting purity of sperm cells. Validating the accuracy of sperm sexing requires reliable procedures, therefore real time PCR assay can determinate sex ratio as reliable assay. In this study a SYBR Green real time PCR assay was used to determinate sex ratio in bovine sperm. Two primers were designed on specific X- and Y- chromosome genes. The Y-specific primers pair were designed on a conserved region of the bovine Y- chromosome-linked SRY gene that is responsible for male sex determination. the Yproduct amplification length was 120 bp (GenBank accession no. HQ908797). Oligonucleotide X-specific primers were designed to amplify a 149 bp DNA fragment on the bovine proteolipid protein gene (PLP) (GenBank accession no.HQ875721). Two certified standard curves were obtained using of two plasmids containing the X- and Y- amplicons. The method was validated by a series of accuracy, repeatability, and reproducibility and by testing two sets of sorted and unsorted samples for X- and Y-chromosomes. The evolution of X-chromosome bearing sperm content in unsorted samples showed an average of 50.3 ::!: 0.97% for ejaculates and 51.60 ::!:0.18% for the commercial semen. Accuracy (95.2%), repeatability (CV = 3.19%) and reproducibility ,(CV = 2.24%) was shown. This new method for quantification of the sexual chromosome content in spermatozoa might be reliable approach and providing a valid support to the sperm sexing technologies with low costs.

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