The secretome from human amniotic membrane-derived mesenchymal stem cells alleviate oxidative damage in bovine sperm

Bovine spermatozoa are highly vulnerable to oxidative stress (OS), which can impair motility, viability, membrane integrity, and DNA integrity. Human amniotic membrane-derived mesenchymal stem cells (AM-MSCs) secrete paracrine factors with antioxidant properties, making their secretome (AM-MSC-Sec) a potential tool for protecting sperm function. This study evaluated the protective effects of AM-MSC-Sec on bovine sperm under OS. Frozen Holstein bull semen was thawed, purified using a two-layer density gradient, and divided into four groups: control (no treatment), H₂O₂ (10 μM), H₂O₂ + 20 % AM-MSC-Sec, and H₂O₂ + 20 % DMEM/F12. After 2 h at 38.5°C and 5 % CO₂, sperm quality parameters-including motility, progressive motility, viability, plasma membrane integrity, acrosome reaction, DNA fragmentation, malondialdehyde (MDA), and total antioxidant capacity (TAC)-were assessed. AM-MSC-Sec significantly improved the percentage of total and progressive motility, enhanced viability and membrane integrity, and decreased DNA fragmentation compared with control and DMEM groups. The percentage of acrosome-reacted sperm increased, and TAC levels were elevated, indicating improved antioxidant defense. DMEM/F12 alone did not show a protective effect. MDA levels increased non-significantly under stress in the control group; however, the difference was not statistically significant. These findings suggest that AM-MSC-Sec could mitigate oxidative damage and preserve multiple functional parameters in bovine sperm. AM-MSC-Sec may serve as a promising therapeutic agent for maintaining sperm quality, with potential applications in bovine reproductive technologies.