Molecular evidence of Resiquimod-induced sperm sexing in bulls and its effect on semen cryosurvival

Demand for female calves in dairy herds has generated considerable interest in developing simple and cost-effective alternatives to conventional sperm sex-sorting techniques. The present study evaluated a Toll-like receptor (TLR7/8)-mediated sperm enrichment approach using Resiquimod (R848) during routine semen processing in Frieswal bulls. A total of 24 ejaculates collected from three clinically healthy bulls were incubated with Resiquimod at concentrations of 0.2, 0.4, and 0.6 µM, separated into upper and lower sperm fractions, cryopreserved, and subsequently evaluated for semen quality, cryosurvivability, and X/Y sperm enrichment. Semen quality parameters, including individual progressive motility, viability, plasma membrane integrity and acrosome integrity were assessed before and after cryopreservation. Conventional PCR and qPCR targeting MAOA (X-linked) and SRY (Y-linked) markers demonstrated fraction-specific enrichment patterns. Molecular analysis revealed enrichment of X-bearing spermatozoa in the lower fraction and Y-bearing spermatozoa in the upper fraction following Resiquimod treatment. The 0.4 µM group showed the most pronounced enrichment, with significantly lower MAOA CT values in the lower fraction and lower SRY CT values in the upper fraction compared with the control. Following cryopreservation, semen quality declined in all groups; however, post-thaw functional parameters at 0.2 and 0.4 µM remained comparable to the control, whereas 0.6 µM significantly impaired sperm quality ( p < 0.05). Overall, the findings indicated that Resiquimod, particularly at 0.4 µM, may provide a practical, economical and less invasive approach for partial enrichment of X- and Y-bearing spermatozoa under routine semen processing conditions without markedly compromising cryosurvivability.