Effect of conditioned medium from equine Wharton's jelly mesenchymal stem/stromal cells on cool-stored stallion semen

Artificial insemination in horses largely relies on cooled transported semen, but stallion spermatozoa are highly sensitive to cold stress, limiting storage duration. Mesenchymal stem/stromal cell-derived conditioned medium (CM) has shown protective effects on sperm in other species, yet its impact on equine semen remains unexplored. This study investigated the effects of equine Wharton's jelly-derived MSC-CM (eWJ-MSC-CM), on the quality of cooled stallion semen. Semen from five stallions of proven fertility was extended in INRA96 supplemented with 0, 5, or 15% eWJ-MSC-CM at 5 °C for 0, 24 and 48 h; motility (CASA), viability, acrosome integrity, mitochondrial activity, and intracellular reactive oxygen species (ROS) levels (flow cytometry) were evaluated at the same time points. Storage time significantly reduced motility and viability, and increased ROS levels (P < 0.05), whereas eWJ-MSC-CM supplementation did not influence any parameter. A marked stallion effect was detected for several traits, consistently with well-known variability within stallions in semen quality and cold sensitivity; however, some treatment × stallion interactions were detected for specific kinematic parameters and acrosome integrity, indicating that individual stallions responded differently to eWJ-MSC-CM supplementation. No adverse effects were found at any concentration, indicating that eWJ-MSC-CM is safe for direct sperm exposure. In conclusion, eWJ-MSC-CM maintains sperm quality during cooled storage and could be a promising candidate for in vivo applications in equine reproduction, where its anti-inflammatory properties may support female fertility.