Estrous Sheep Serum-Derived Extracellular Vesicles Deliver Proteins That Modulate Sperm Motility in Sheep.

Sperm capacitation requires signals from the female reproductive environment, although the molecular mechanisms governing this process remain poorly understood. Estrous sheep serum (ESS) is widely used in in vitro fertilization of small ruminants, contributing to mimic the female reproductive fluids. Among ESS components, extracellular vesicles (ESS-EVs) may mediate the transfer of molecular signals to spermatozoa and support the modifications observed when sperm are incubated with ESS. To explore this possibility, ESS-EVs were isolated and validated according to MISEV2023 guidelines, and their proteome was characterized and compared with the protein signature of sperm incubated with ESS. In addition, sperm were co-incubated with ESS-EVs, and sperm motility and mitochondrial activity were assessed. Notably, 27.1% of proteins previously reported to be upregulated in capacitated ram sperm were identified in ESS-EVs, representing a significant enrichment of capacitation-related factors. Functionally, ESS-EVs modulated sperm kinematics, producing a motility profile comparable to that observed under capacitation conditions, while mitochondrial activity remained unchanged. Together, these findings suggest that ESS-EVs may act as molecular conveyors delivering proteins that reshape sperm motility.