Effect of seminal plasma extracellular vesicles in post-thaw functional parameters of cryopreserved ram sperm

Cryopreservation of ram semen induces structural and functional alterations that compromise sperm function. Given that seminal plasma contributes to sperm physiology regulation, increasing attention has focused on extracellular vesicles (EVs), which carry proteins involved in membrane organization and capacitation. These properties suggest that EVs may contribute to maintaining sperm stability during cryopreservation. Therefore, this study evaluated the effect of seminal plasma-derived EVs on post-thaw functional parameters of ram sperm. Semen was cryopreserved in the presence or absence of EVs isolated by ultracentrifugation previously characterized by nanoparticle tracking analysis, TEM, FTIR and Western blotting. Post-thaw sperm quality was assessed through viability, membrane lipid disorder, reactive oxygen species production, protein phosphorylation, acrosome status, intracellular calcium levels, and motility. Sperm cryopreserved with EV-supplemented extender showed reduced membrane lipid disorder and lower intracellular calcium levels compared to controls (p < 0.05). CASA analysis revealed no significant differences in total or progressive motility; however, EV supplementation was associated with a modified kinematic profile, characterized by increased velocity and trajectory-related parameters (p < 0.001), suggesting a potential effect on sperm movement patterns. No significant differences were observed in viability, ROS levels, protein phosphorylation (tyrosine residues or PKA), or acrosome status. These findings provide proof-of-concept evidence that supplementation with seminal plasma-derived EVs during ram semen cryopreservation is associated with changes in membrane lipid organization, intracellular calcium homeostasis, and selected sperm kinematic parameters. Further studies are required to determine whether these changes translate into improved fertilizing ability.