139 Effect of protein and calcium ionophore A23187 concentration on hyperactivated motility and acrosome status of stallion sperm

Abstract

In vitro fertilization does not occur readily in the horse. Fertilization can be achieved using sperm treated with the calcium ionophore A23187 (CaI), but rates are low and variable. In order to fertilize, it is thought that the sperm must show hyperactivated motility and undergo the acrosome reaction. The presence of protein in the media is thought to suppress the effect of CaI, but protein is needed for maintenance of sperm motility. Therefore, the objective of this study was to assess the effect of CaI in the presence or absence of protein on the acrosome and on hyperactivated motility of equine sperm. For this purpose, sperm from 4 stallions were exposed for 10 min at 37°C to vehicle or to 1 (C1), 5 (C5) or 10 (C10) μM CaI, with (BSA) or without (N) 7 mg mL⁻¹ BSA. The sperm were then washed and incubated at 37°C for 2 h. Total and hyperactivated motility were measured by computer-assisted semen analysis. Sperm were considered hyperactivated if curvilinear velocity was >180 μm s⁻¹, amplitude of lateral head displacement was >12 μm, linearity was <30% and fractal dimension value was >1.3. The percentage of live acrosome-reacted sperm was measured by flow cytometry after staining with propidium iodide and Pisum sativum agglutinin. Data were analysed by repeated-measures 2-way ANOVA. Results were expressed as mean ± standard error. Total motility in C5 and C10 treatments was significantly decreased in relation to control (BSA-vehicle) starting at 30 min of incubation (35.42 ± 13.57 to 28.20 ± 13.10% v. 71.72 ± 9.21%, respectively; P < 0.05). Hyperactivated motility was significantly lower in C10, C5 and N-C1 than in control after 2 h of incubation (1.46 ± 0.64 v. 3.10 ± 0.58%, respectively). Live acrosome-reacted sperm were significantly higher (P < 0.05) for BSA-C5 (14.04 ± 1.99%) and BSA-C10 (14.85 ± 2.52%) than for control (7.50 ± 1.62%) after 2 h of incubation. The exposure to sperm of concentrations ≥5 μM CaI was associated with loss of motility from 30 min of incubation on. However, 2 h of incubation after ≥5 μM CaI in the presence of BSA were needed to increase the percentage of live acrosome-reacted sperm. This mismatch between motility and acrosome response helps to clarify the reasons for the variable effect of sperm CaI treatment on equine IVF. Further studies measuring calcium influx and assessing the effect of sperm pre-incubation on CaI response are needed to explore mechanisms for equine in vitro sperm capacitation.