156 Effect of human tubal fluid medium and hyperactivation inducers on stallion sperm capacitation and hyperactivation

Abstract

Conventional IVF has not yet been implemented in the equine species. One of the main reasons has been the inability to develop a culture medium and incubation conditions supporting high levels of stallion sperm capacitation and hyperactivation in vitro. Although different culture media have been used for this purpose, human tubal fluid (HTF) medium, widely used in the manipulation of human and mice gametes, has not been reported so far in stallion sperm culture. Thus, the first part of this study aimed to compare HTF (Summers and Biggers, 2003 Hum. Reprod. Update9, 557-582, DOI: 10.1093/humupd/dmg039) and Whitten’s (McPartlin et al. 2009 Biol. Reprod.81, 199-206, DOI: 10.1095/biolreprod.108.074880) media on different stallion sperm quality and capacitation variables. Additionally, the effect of procaine, aminopyridine and caffeine on sperm motility parameters was evaluated in both media at different incubation times. Fresh semen from 3 Chilote stallions was collected, diluted to 10×106 sperm mL−1 in capacitating (7mg mL−1 BSA and 25mM NaHCO3) and non-capacitating (without BSA and NaHCO3) HTF and Whitten’s media and incubated for 30 and 120min at 38°C in air atmosphere. Integrity and destabilisation of the plasma membrane were evaluated by merocyanine 540/SYTOX Green (MC540), mitochondrial membrane potential (ΔΨm) using tetramethylrhodamine methyl ester perchlorate, acrosome membrane integrity by peanut agglutinin/fluorescein isothiocyanate and tyrosine phosphorylation by P-tyrosine mouse mAb conjugated to Alexa Fluor® in a FACSCanto II flow cytometer (Becton, Dickinson and Co., Franklin Lakes, NJ, USA). A total of 10,000 sperm events were acquired from each measurement (n=3 replicates for each stallion). Motility parameters were evaluated using the integrated semen analysis system (ISAS®, Selinion Medical, Brussels, Belgium). Percentage data were arcsine transformed and subjected to a 2-way ANOVA with Bonferroni’s post hoc test using Prism 7 software (GraphPad Software, La Jolla, CA, USA). We found no differences between Whitten’s and HTF media in terms of sperm viability, uninduced acrosome membrane damage or mitochondrial membrane potential at 30 and 120min of incubation. Membrane fluidity (MC540) increased in both media at 30 and 120min of incubation compared with non-capacitating conditions. Similarly, tyrosine phosphorylation increased in both media in capacitating conditions at 2 and 4h of incubation compared with non-capacitating conditions, without differences between media. Although procaine showed the best result in terms of sperm hyperactivated motility in both media, aminopyridine also showed parameters consistent with hyperactivation including an increase in curvilinear velocity and decrease in straightness. In conclusion, HTF medium and aminopyridine equally support capacitation-related parameters in stallion sperm. Funding support was received from FONDECYT 1160467 CONICYT, Chile.