Effects of Post-Thaw Toll-Like Receptor 1/2 Agonist and Antagonist Exposure on Motility Subpopulations and Functional Characteristics of Bovine Spermatozoa.

Toll-like receptor 2 (TLR2) has been implicated in fertilization-related functions of bovine spermatozoa, but post-thaw responses to pharmacological TLR1/2 modulation across kinematic and membrane-related characteristics remain less defined. One ejaculate from each of four Holstein bulls was used and semen was exposed after thawing to a TLR1/2 agonist, a TLR1/2 antagonist or no additive. Five independent experimental repetitions per bull were performed using separate straws from the same ejaculate; therefore, the biological donor sample size was four bulls. Computer-assisted sperm analysis and k-means clustering identified six kinematically defined motility subpopulations and flow cytometry assessed plasma membrane and acrosomal integrity, membrane fluidity and mitochondrial superoxide-related fluorescence using MitoSOX Red. At 3 h, antagonist exposure reduced total motility compared with control and agonist treatments (30.1% ± 6.3% vs. 40.4% ± 5.6% and 37.7% ± 5.8%, respectively; adjusted p = 0.001). Conversely, antagonist-treated samples contained more spermatozoa with intact plasma membrane and acrosome than agonist-treated samples (52.4% ± 3.4% vs. 48.4% ± 3.1%; adjusted p = 0.001) and more viable spermatozoa with low MitoSOX fluorescence (31.1% ± 6.1% vs. 22.4% ± 4.7%; adjusted p = 0.001). Agonist exposure increased the proportion of viable spermatozoa with high membrane fluidity and was associated with higher MitoSOX fluorescence than antagonist exposure. These findings describe distinct post-thaw functional profiles following pharmacological TLR1/2 agonist and antagonist exposure; they do not establish TLR2-specific causality or effects on fertilizing competence.