Comparative Evaluation of Candidate Genes for Efficient Sex Determination in Bovine Sorted Semen and Embryos

Sex-sorted sperm and preimplantation sex determination are indispensable tools for improving reproductive efficiency and herd management in livestock production. The present study aimed to identify the most effective 'X' and 'Y' specific genes for sexing sorted bovine sperm and embryos. We selected five Y-specific (SRY, HSFY, TSPY, ZFY and OFD1Y) and four X-specific (PLCXD1, SHROOM 2, PLP and MAOA) genes and evaluated their specificity and sensitivity by PCR and qPCR analyses in X-sorted sperm, Y-sorted sperm, mixed sperm and female blood. HSFY and TSPY showed more pronounced and specific amplification in Y-sorted sperm DNA, making them robust Y-specific markers, while MAOA and SHROOM 2 were more specific to X-sorted sperm DNA and identified as effective X-specific markers. These results were further validated in buffalo IVF embryos, confirming their effectiveness in embryo sexing. The findings of this study can be applied in duplex or multiplex PCR assays, which minimise the risk of amplification failure and false negatives, providing a rapid and reliable toolkit for bovine sperm and embryo sexing. Implementing such a molecular approach can significantly benefit livestock management by improving breeding outcomes, reducing the costs and inefficiencies associated with undesired sex ratios.