Effects of TLR7/8 Activator on Sperm Quality, Energy Metabolism and the Separation Efficiency of X- and Y-Chromosome-bearing Sperm in Frozen-thawed Bull Semen

Background: This study investigated the effects of Requimod (R848), a TLR7/8 activator on sperm quality, energy metabolism and the separation efficiency of X- and Y-chromosome-bearing sperm in frozen-thawed bull semen. Methods: Bull semen samples were divided into six groups: A control group and five experimental groups treated with different concentrations of R848 (0.05, 0.5, 1, 2 and 4 μmol/L). Following incubation at 37oC for 60 min by upstream methods, sperm quality parameters were assessed for upper- and lower-layer sperm. Further evaluations included acrosomal integrity, plasma membrane integrity, mitochondrial activity and ATP content. Result: The results demonstrated that treatment with 0.5 μmol/L R848 significantly reduced sperm quality parameters in lower-layer X-sperm. This concentration also inhibited mitochondrial activity and ATP content in X-sperm. However, no significant differences in acrosomal or plasma membrane integrity were observed between the control and experimental groups. Additionally, the R848 treatment had no significant effects on sperm quality parameters, acrosomal integrity, or plasma membrane integrity of the upper-layer Y-sperm compared to the control group. Flow cytometric analysis of separated sperm indicated that 76.13% of the sperm in the upper layer were Y-sperm, while 61.78% of the sperm in the lower layer were X-sperm. These findings suggest that 0.5 μmol/LR 848 effectively enhances the separation of X- and Y-sperm in frozen-thawed bull semen.