Amniotic mesenchymal stromal cell-derived extracellular vesicles improve sperm motility in aged stallions

Introduction: Age-related testicular degeneration is a major cause of subfertility in stallions and is commonly associated with impaired sperm motility. Extracellular vesicles (EVs) derived from mesenchymal stromal/stem cells (MSCs) have recently emerged as promising regenerative mediators due to their ability to transfer bioactive molecules to target cells. This pilot study evaluated the effects of equine amniotic MSC-derived EVs (AMSC-EVs) on sperm motility and functional integrity in aged stallions. Methods: Semen samples collected from four aged stallions were analyzed by conventional spermiogram and computer-assisted sperm analysis (CASA). Amniotic MSC-EVs were isolated and characterized by nanoparticle tracking analysis, transmission electron microscopy, and Western blot analysis of EV markers. Following a dose–response assay, spermatozoa were co-incubated with PKH26-labelled AMSC-EVs, and sperm kinetic parameters, EV interaction, mitochondrial membrane potential, viability, and acrosomal status were assessed. Results: Co-incubation with AMSC-EVs at 400 × 10⁶ EVs/mL significantly modulated sperm kinetic parameters, attenuating the decline in progressive motility and maintaining higher linearity (LIN) and straightness (STR) values than untreated controls, while dynamically modifying the amplitude of lateral head displacement (ALH) (p < 0.05). Progressive motility was preserved up to 4 h post collection in EV-treated samples (16.00 ± 0.20%) compared with untreated controls (0.90 ± 0.10%). Fluorescence analyses by confocal microscopy confirmed EV localization predominantly at the sperm midpiece. EV-treated spermatozoa showed dynamic modulation of movement patterns without evidence of premature capacitation or acrosome reaction in viable spermatozoa. Discussion: These results suggest that AMSC-EVs may represent a promising cell-free strategy for modulating sperm functionality in aged stallions while preserving sperm physiological integrity. Considering the limited number of stallions included, the findings should be interpreted as preliminary proof-of-concept observations.