Seminal extracellular vesicles influence porcine spermatozoa physiology by modulating key functional parameters

Seminal plasma (SP) contains a heterogeneous population of extracellular vesicles (EVs) recognized as key modulators of sperm function. However, the specific functional roles of each seminal EV (sEV) subset remain poorly understood. This study aimed to evaluate the interaction of two sized sEV subsets (small [S-sEVs] and large [L-sEVs]) with pig liquid-stored spermatozoa under different pH conditions and their effect on specific sperm functional parameters. Seminal EV subsets were isolated from SP samples using size exclusion chromatography and characterized following the MISEV2023 guidelines. Semen samples were incubated with each sEV subset or without sEVs (control) for 6 h at 37 ºC, 100 % humidity and 5 % CO₂ under different pH conditions (6.5, 7.0, or 7.5). Sperm functional parameters were assessed by flow cytometry (Cytoflex®S and LX, Beckman Coulter), under capacitating and non-capacitating conditions. Confocal microscopy revealed that both sEV subsets bound to and were internalized by spermatozoa as early as 30 min after incubation, regardless of pH. Flow cytometry revealed that both sEVs decreased reactive oxygen species production (P ≤ 0.0001), mitochondrial membrane potential (P ≤ 0.0001) and mitochondrial O₂^•⁻ levels (P ≤ 0.01) and increased apoptosis (active caspase-3) in viable spermatozoa (P ≤ 0.0001). However, the influence of sEV on acrosome integrity in viable sperm was time- and condition-dependent (P ≤ 0.05). This study showed that both S- and L-sEVs interact with porcine spermatozoa across a range of physiological pH conditions. This interaction is reflected by decreased oxidative stress and mitochondrial activity, as well as increased apoptosis in spermatozoa.