Sex Determination of X and Y Separated Fresh Bull Sperm Using SRY Gene Copy Number Analysis.

The present study employed absolute quantitative PCR to determine the sex of sperm fractions after subjecting fresh bull semen to motility-based separation using resiquimod, an agonist of Toll-like receptors 7 and 8, which is supposed to selectively impair the motility of sperm carrying the X chromosome. The upper and lower fractions obtained from fresh bull sperm following the swim-up procedure, in both the control (untreated) and resiquimod-treated groups, were subjected to sperm DNA extraction and absolute quantification by real-time PCR to determine the number of Y chromosome-bearing sperm. Ten different dilutions were prepared from the cloned plasmid with SRY sequence. Isolated sperms from the upper and lower layers in the resiquimod-treated group and the fresh non-treated sperm were also used for in vitro embryo production. The SRY gene copy number was significantly higher in the upper than the lower layer in the resiquimod-treated group (p < 0.05). The sex of the embryos produced with sperm harvested from the upper and lower layers of the treatment group confirmed the results of the PCR, because the number of male embryos produced from the upper layer sperm was significantly higher than the lower layer (73.33% vs. 28.57%) (p < 0.05). Considering that this sexing method is low-cost and can be done with little facilities, it can be used in the future for the isolation of X and Y sperms in bulls.