119 Effect of deacetylase inhibitors on kinematic parameters of stallion sperm

One of the major obstacles to achieve IVF in horses is the inability to reliably induce in vitro sperm capacitation. Recent studies in humans and mice have shown that induction of a hyperacetylated state in the sperm can induce capacitation-like modifications. The aim of the present study was to evaluate the presence of acetylation and hyperactivated motility in stallion sperm by evaluation of kinematic parameters after treatment with two deacetylase inhibitors: trichostatin A (TSA) and nicotinamide (NAM). Two stallions (A and B) of known fertility were collected, and the semen was centrifuged and diluted at 10 × 10⁶ spermatozoa mL⁻¹ in modified Whitten’s media (MW). Experimental groups were defined as follows: Zero hours (ZH; MW only) evaluated at 0 h and Non-capacitating (NCAP; MW only), CAP (MW + 0.7% bovine serum albumin + 25 mM NaHCO₃) and iDAC (MW + 5 µM TSA + 0.5 mM NAM), evaluated at 3 h. Sperm samples of all groups were fixed and stained by immunofluorescence using an antibody against lysine acetylated proteins. Images were analysed with the Image J software (National Institutes of Health) for relative acetylation. Computer-assisted sperm analysis (CASA) parameters were evaluated. Sperm was considered hyperactivated if a decrease in linearity (LIN) and straightness (STR), along with an increase in the amplitude of lateral head displacement (ALH) and curvilinear velocity (VCL), were recorded. Data were analysed by one-way ANOVA and differences considered significant when P < 0.05. After 4 replicates, there was no difference in relative protein acetylation among the groups. Total sperm motility decreased over time but was not different from before incubation for both stallions. Progressive sperm motility, however, decreased in the CAP group for stallion A (ZH: 46.1 ± 3.7%; CAP: 17.6 ± 2.4%, NCAP: 28.3 ± 6.6%, and iDAC: 31.9 ± 7.2%), whereas it was the same among groups for stallion B. VCL decreased in the CAP group only when compared to the ZH group in stallion A (163 ± 9.7 vs. 272.2 ± 10.9 µm s⁻¹, respectively), whereas it decreased in the NCAP and iDAC groups compared to ZH (213.7 ± 1.7 and 203.7 ± 5.6 vs. 272.2 ± 10.9 µm s⁻¹, respectively) for stallion B. ALH significantly decreased in stallion B for the NCAP and iDAC groups compared with the ZH group (8.6 ± 0.3 and 8.5 ± 0.2 vs. 10.4 ± 0.4 µm s⁻¹). In stallion A, LIN decreased in the CAP compared to ZH group (37.7 ± 1. vs. 46.1 ± 2.6%, respectively), whereas there were no differences in STR among groups. Based on a recent literature search, we have shown the presence of acetylation in equine sperm for the first time; however, the incubation time and iDAC doses used in this experiment did not increase relative acetylation and induce sperm hyperactivation.