Application of R848 Ligand to Improve the Efficiency of Sex Separation in Ram Spermatozoa

The present study aims to evaluate the application of the TLR7/8 agonist – Resiquimod (R848) to improve the efficiency of sex separation of ram spermatozoa. Semen samples collected from five clinically healthy rams (Ovis aries) of the Lacon breed were treated with the R848 ligand to separate spermatozoa carrying X and Y chromosomes into separate fractions (RX and RY). Kinematic parameters were analyzed using a computer-assisted sperm analyzer (CASA), and sperm vitality was assessed by staining with eosin-nigrosin. Real-time PCR tracked the expression of genes specific to X and Y chromosomes – TLR7 and SRY. The results showed successful separation of ram spermatozoa by sex, with expression of TLR7 and SRY in the RX or RY fraction, respectively (p < 0.05). An enrichment trend was recorded for X-carrying (84.9%) and Y-carrying (86.5%) fractions. In conclusion, the use of ligand R848 in this study showed possible application in reproductive medicine of small ruminants as well as introducing a novel approach to molecular sex determination based on gene expression analysis. This highlights the need for future studies and the application of sorted semen fractions in in vivo experiments.