Development of qPCR Assay for Determination of Sperm Sex Ratio in Indian Cow Bull

Abstract

Background: Currently, the sex selection of cattle offspring holds great promise for genetic advancement and meeting consumer demand. Sperm sorting by flow cytometry provides a reliable tool for artificial insemination and the creation of embryos with predetermined sexes. A precise evaluation of the yield of sperm separation is still needed for a field application of this method or for the advancement and validation of other related semen sexing technologies. The current study was conducted to develop a qPCR-based method for the determination of sperm sex ratio in cow bull semen. Methods: The SYBR Green-based Real-Time PCR chemistry targeting PLP (X chromosome-specific gene) and SRY (Y chromosome-specific gene) genes were used for the development of the assay. To determine the efficiency of PCR amplification, standard curves were generated. Result: The developed assay revealed a negligible variation in the semen sex ratio (51.7±0.465% X and 48.23±0.465% Y) of unsorted semen. The repeatability and reproducibility of this approach were evaluated. For PLP and SRY, the standards produced a linear relationship with regression coefficients of 0.994 and 0.997, respectively. The low mean values of CV obtained in repeatability and reproducibility trials demonstrated the high dependability of this novel method for assessing the sexual chromosomal content in semen samples.