Differential Modulation of Capacitation-Associated Responses in Boar Spermatozoa by In Vitro Capacitation Media: A Kinetic Study

In vitro capacitation of boar spermatozoa remains highly variable among laboratories due to differences in media composition, incubation conditions, and previous semen handling. Since capacitation is a dynamic process, identifying how different media modulate specific sperm responses over time is crucial to improving the standardization of porcine in vitro fertilization protocols. This study evaluated the temporal dynamics of membrane lipid disorder, acrosomal integrity, mitochondrial activity, and motility patterns in response to different media. Commercial boar semen doses were centrifuged and resuspended in a non-capacitating control solution or in three capacitation media differing mainly in bicarbonate, caffeine, and energy substrate composition, and evaluated every 15 min using flow cytometry and computer-assisted semen analysis at 38 °C for up to 2 h. Media containing higher bicarbonate and caffeine concentrations induced rapid membrane lipid destabilization and increased acrosomal exocytosis during the initial incubation period. Subsequently, these changes were accompanied by a more rapid decline in cell viability over time. In contrast, the medium containing lower bicarbonate together with lactate and pyruvate preserved higher viability, mitochondrial activity, and a more sustained, linear movement pattern (higher linearity and straightness). Interestingly, training-associated responses were detected immediately at time 0, suggesting that semen storage conditions may prime spermatozoa before incubation. Overall, medium composition and pre-treatment handling conditions should both be considered when standardizing porcine reproductive technologies.