Ovine follicular fluid extracellular vesicles improve oocyte maturation and exhibit ribosome-related cargo signatures

Oocyte developmental competence is critical for efficient assisted reproduction in livestock. In vitro maturation (IVM) is often the first step in in vitro embryo production, making improvements in oocyte quality an important goal. We supplemented IVM medium with ovine follicular fluid-derived extracellular vesicles (oFF-EVs), evaluated oocyte maturation and subsequent embryonic development, and characterized vesicle cargo. The isolated oFF-EVs exhibited typical small extracellular vesicle features. Among the concentrations examined, 30 μg/mL yielded the highest mean maturation rate and greatest cumulus expansion. PKH67 tracing revealed time-dependent uptake by cumulus cells. Supplementation reduced reactive oxygen species levels, increased glutathione content, and maintained mitochondrial membrane potential. It also improved cortical F-actin organization, cortical granule distribution, chromosome distribution, and spindle morphology, accompanied by increased total AKT abundance. Following in vitro fertilization, the cleavage rate and blastocyst total and inner cell mass cell numbers increased, whereas the blastocyst rate was not significantly altered. Small RNA sequencing identified 84 microRNAs dominated by the let-7 family, while proteomics identified 4,730 proteins enriched in RNA processing, ribonucleoprotein complexes, intracellular transport, and protein homeostasis. Integrated analyses revealed a ribosomal protein module centered on FAU ubiquitin-like and ribosomal protein S30 fusion (FAU), suggesting ribosome-related cargo signatures. These findings support oFF-EV supplementation as a strategy to improve sheep oocyte maturation and subsequent developmental competence and identify cargo features for further functional investigation.