Expression of TLR7/8 in canine sperm and evaluation of the effect of ligand R848 on the sorting of canine X/Y sperm

The aim of this study was to analyze the expression pattern of Toll receptor 7/8 (TLR7/8) in canine sperm, and explore the feasibility of using TLR7/8 ligand resiquimod(R848)to separate canine X and Y sperm. In this study, cellular immunofluorescence was used to analyze the expression of TLR7/8 in canine sperm, real-time fluorescence quantitative PCR was used to calculate the proportion of X sperm in the lower layer of the incubation solution with R848 to evaluate the sorting effect of R848 on canine X/Y sperm, and sperm quality detection system was used to analyze the effect of R848 on the motility of canine sperm. The mechanism of effect of R848 on canine sperm motility was analyzed by Western blot. The results showed that TLR8 was not expressed in all canine sperm, while TLR7 was expressed in all canine sperm and was localized in the head and tail of sperm. When 0.4 μM R848 was incubated with canine sperm for 1 h, the total motility, average path velocity (VAP), average straight-line velocity (VSL), and average curved-line velocity (VCL) of canine sperm were significantly decreased(P < 0.05). There was no significant difference between the lower and upper layers of the R848 treatment group and the control group(P > 0.05), and the proportion of X sperm was nearly half. The levels of NF-κB and GSK3α/β phosphorylation of sperm in R848 treatment group were significantly increased compared with control group(P < 0.05). The above results showed that TLR7/8 was not differentially expressed in canine X and Y sperm. R848 could decrease the motility of canine spermatozoa and inhibit sperm motility by the GSK3α/β-hexokinase pathway through the phosphorylation of NFκB and GSK3α/β, while could not separate X and Y spermatozoa. The method of sorting X/Y sperm based on TLR7/8 is not feasible for dogs.