116 Effect of different energy sources on motility and calcium ionophore-induced acrosome reaction in equine sperm

Abstract

Equine sperm appears to differ from sperm of other species, preferring oxidative phosphorylation over glycolysis for energy production. However, there is little information about the effects of different energy sources on capacitation-related events in equine sperm. We evaluated stallion sperm incubated in the presence of different energy sources at a pH (~8.3) known to support some aspects of capacitation. Fresh stallion semen (n = 9 ejaculates) was washed, then diluted to 30 × 10⁶ sperm mL⁻¹ with modified Whitten's medium containing 25 mM bicarbonate, 7 mg mL⁻¹ bovine serum albumin (BSA), and only one energy source (5 mM glucose, 10 mM lactate, or 10 mM pyruvate). The diluted sperm were incubated in air for 0.5, 2, or 4 h, then exposed to 5 µM calcium ionophore A23187 (CaI) or vehicle (V) for 10 min. Sperm were washed and incubated in the assigned medium for 30 min, then evaluated by computer-assisted sperm motility analysis and by live/dead staining (Fixable Live/Dead Red, Molecular Probes) followed by fixation (2% v/v paraformaldehyde) and staining for acrosome reaction, using Pisum sativum agglutinin (FITC-PSA, Sigma Aldrich). Stained sperm were evaluated via flow cytometry to detect live acrosome-reacted sperm. Data were analysed using ANOVA with Tukey post hoc comparison (SAS 9.4, SAS Institute). Medium pH at 4 h was 8.37 to 8.48. There were no significant differences between CaI and V treatments for any experimental endpoint (P > 0.05). At 4 h, pyruvate values for total motile sperm (46 ± 5%) and curvilinear velocity (113 ± 8 µm s⁻¹) were significantly higher than those for lactate (32 ± 5% and 86 ± 5 µm s⁻¹) or glucose (27 ± 4% and 90 ± 5 µm s⁻¹). The percentage of live sperm that were acrosome-reacted (%L-AR) did not increase significantly until 4 h; at this time, %L-AR was higher for lactate (46 ± 3%) than for pyruvate (21 ± 3%) or glucose (18 ± 3%; P < 0.05). To the best of our knowledge, this is the first critical evaluation of the effect of different energy sources on motility and acrosome reactivity of equine sperm. The higher motility in pyruvate supports the concept that stallion sperm utilises oxidative phosphorylation more effectively than glycolysis. The apparent lack of effect of CaI on %L-AR may be related to the 1- to 2-h delay typically seen after its use in equine sperm; the present data show that incubation even for 4 h in potentially capacitating conditions does not prepare equine sperm to respond quickly to CaI. Surprisingly, a high rate of spontaneous acrosome reaction was achieved in the lactate treatment. The notably higher %L-AR with lactate than with pyruvate suggests that this effect is not due to utilisation of substrate for oxidative phosphorylation but may be associated with conversion of lactate to pyruvate via lactate dehydrogenase, or possibly with effects of lactate through other pathways. Although pyruvate was associated with greater motility, this could be related to higher oxidative stress, which could have influenced the %L-AR. Further work is needed to determine whether the acrosomal changes observed are related to functional capacitation.