143 The effect of media and incubation temperature on in vitro capacitation of frozen-thawed equine sperm

In vitro fertilisation (IVF) has been optimised to work on many different species, although this tool has not yet proven to be a viable option for equine species. Previous findings suggest that equine sperm is unable to successfully capacitate under the in vitro conditions studied so far, highlighting the need to determine the optimal incubation environment for the induction of capacitation. Importantly, low sperm viability often results from incubation under capacitation conditions, especially of frozen-thawed equine sperm. Capacitation can be characterised by acrosomal exocytosis and increased protein tyrosine phosphorylation and is often associated with hyperactivated sperm motility. In this study, it was hypothesised that lowering the post-thawing incubation temperature will result in greater motility and sperm viability after 4 h of incubation in capacitation conditions. Therefore, the objective of this study was to determine the optimal medium and incubation temperature for in vitro capacitation of frozen-thawed equine semen. Frozen-thawed equine sperm (n = 4 stallion, three repetitions per stallion) was incubated in human tubal fluid (HTF) or Whitten’s medium (WM) under non-capacitation (NC) and capacitation (CAP) (WM: 2.4 mM Ca lactate, 7 mg/mL BSA, 25 mM NaHCO₃; HTF: 7 mg/mL BSA, 25 mM NaHCO₃) conditions for 4 h at both room temperature (RT) and at 37°C, resulting in eight treatments. Computer-assisted sperm analysis (CASA) was used to assess motility-related parameters of at least 400 million sperm at 0 and 4 h of incubation. Sperm viability and capacitation-like parameters of tyrosine phosphorylation and acrosome reaction were assessed (at least 200 sperm analysed per parameter) by immunofluorescence after 4 h of incubation. Results were evaluated using the Glimmix procedure (P ≤ 0.05) of the Statistical Analysis System (SAS 9.4), where percentages were square root arcsine transformed. CASA results revealed that sperm incubated at RT in NC conditions, regardless of the medium, had the highest progressive motility and curvilinear velocity (VCL) after 4 h of incubation. Sperm incubated for 4 h at RT tended to show higher levels of tyrosine phosphorylation, whereas semen incubated at 37°C tended to have a higher proportion of sperm with an exposed acrosomal region. The interaction of capacitation conditions and incubation temperature had an effect on viability, with incubation at RT in NC conditions yielding the highest viability and incubation at 37°C in media supplemented with capacitation-inducing agents showing the lowest survivability. In conclusion, the results indicate that, under our experimental conditions, the supplementation of media with capacitation-inducing agents did not have an effect on equine sperm capacitation events. However, incubation at room temperature in non-capacitation conditions yielded higher progressive motility, VCL, and sperm viability after 4 h of incubation compared to sperm incubated at 37°C.