Seminal extracellular vesicles improve cryosurvival and fertility potential of kundhi buffalo sperm.

Buffaloes are important contributors to agricultural production in many developing countries, yet poor semen cryosurvival remains a major challenge for efficient artificial insemination and genetic improvement. In recent years, small extracellular vesicles (sEVs) have emerged as important mediators of intercellular communication and reproductive function; however, their potential as supplements for improving buffalo semen cryosurvival remains poorly understood. Therefore, this study evaluated the influence of adding a seminal plasma-derived small extracellular vesicle-enriched preparation to a Tris extender using post-thaw sperm traits, oxidative status, DNA integrity, HSP70 and PRM1 transcript abundance, and fertility of Kundhi buffalo bull semen. Seminal plasma-derived particles were isolated and characterized by nanoparticle tracking analysis, showing a mean diameter of 117.8 ± 2.47 nm, modal diameter of 89.4 ± 1.41 nm, and concentration of (3.21 ± 0.05) × 1011 particles/mL. Thirty-two ejaculates from four Kundhi buffalo bulls were pooled and extended with a Tris extender supplemented with 0, 25, 50, 100, or 200 μg/mL sEV-enriched preparation before cryopreservation. Post-thaw sperm quality, early apoptosis, DNA fragmentation, lipid peroxidation, antioxidant enzymes, and relative mRNA abundance of HSP70 and PRM1 were assessed. All evaluated post-thaw variables differed (P < 0.05) after supplementation, whereas 50 μg/mL revealed the strongest response. The EV-50 group showed higher total motility, progressive motility, viability, and HOST-positive spermatozoa, however reduced early apoptotic sperm, DNA fragmentation, and MDA concentration than the control group. EV-50 also enhanced SOD, catalase, total glutathione, HSP70 and PRM1 expression (1.91 ± 0.07-fold and 1.69 ± 0.05-fold, respectively). In fertility validation, pregnancy rate increased from 41.7% in the control group to 56.7% in the EV-50-treated semen group, with higher odds of pregnancy (OR = 1.83; P = 0.028). In conclusion, supplementation with 50 μg/mL of the sEV-enriched preparation improved post-thaw sperm quality, redox homeostasis, DNA integrity, HSP70 and PRM1 transcript abundance, and field fertility, supporting its potential as a cryopreservation supplement for enhancing the reproductive performance of Kundhi buffalo semen.