94 Effect of glycine and creatine on the in vitro capacitation-related events in frozen/thawed equine sperm

Commercial IVF remains unsuccessful in the horse. It is known that the post-ovulatory oviducal environment promotes sperm capacitation and fertilisation. Recent studies in equine species have reported that glycine and creatine concentrations increase in post-ovulatory oviducal fluid compared to preovulatory oviducal fluid. Specifically, creatine has been proven to increase protein tyrosine phosphorylation in mouse sperm, and glycine has been proven to be involved in the acrosome reaction. Yet no information exists on the effect of these molecules on in vitro equine sperm capacitation. Therefore, the objective of this study was to evaluate the effects of glycine and creatine on capacitation-related parameters of protein tyrosine phosphorylation and acrosome reaction. Semen was collected from three stallions (three replicates per stallion) with an artificial vagina and frozen through a standard protocol utilising a commercial freezing extender containing glycerol and an amide (E-Z Freezin® Cryomax LE, Animal Reproduction Systems Inc.). Semen was thawed at 37°C and rinsed twice in Whitten’s medium (WM) without NaHCO₃ and BSA (WM−) or WM with 25 mM NaHCO₃ and 7 mg mL⁻¹ BSA (WM++) at 600 × g for 10 min, and then resuspended in one of the treatments. There were eight treatments in this experiment, which utilised either WM++ or WM−. The WM++ variations consisted of 500 or 1000 µM creatine, 1 mM glycine, and 500 µM creatine with 1 mM glycine. The WM− variations included 1 mM glycine and 500 µM creatine with 1 mM glycine. All of these concentrations were decided based on previous papers’ results. Additionally, WM− and WM++ without added glycine or creatine were used as controls. The semen was incubated for 2 h at 37°C in air atmosphere. After incubation, acrosome status was evaluated using fluorescein isothiocyanate-conjugated peanut agglutinin (FITC-PNA) and protein tyrosine phosphorylation using an anti-phosphotyrosine (clone 4G10®) mouse monoclonal primary antibody and a goat anti-mouse (Alexa Fluor 488)-conjugated secondary antibody. Images were captured utilising a fluorescence deconvolution microscope. Percentage data was arcsine transformed and was analysed by the Type III test of fixed effects and Tukey media separation utilising the Proc Glimmix of SAS 9.4 (P < 0.05). No difference in acrosome reaction or in protein tyrosine phosphorylation was found between treatments. Results of the experiment showed that glycine and creatine did not increase the acrosome reaction rate or induce protein tyrosine phosphorylation during a 2-h incubation period in frozen/thawed equine sperm. A longer incubation time may increase protein tyrosine phosphorylation and/or acrosome reaction, but it may result in a decline in sperm survival.