The use of in vitro reproduction techniques is increasing worldwide in human and domestic species. However, the oocyte to embryo turnover in vitro remains marginal leading to the conclusion that the response could be in the in vivo events. Extracellular vesicles naturally present in follicular fluid (ffEVS) play a role in cell-to-cell communication and can participate in follicular development and oocyte maturation. However, there is limited understanding regarding the impact of the isolation methods on ffEVs contents and their use as a supplement during in vitro maturation (IVM). Here, we provide evidence that ffEVs isolated by ultracentrifugation (ffEVs-UC) and size exclusion chromatography (ffEVs-SEC) were different in terms of proteins, microRNAs (miRNAs), transcripts, and lipids contents. Proteins such as NPR2, and EGFR were detected in ffEVS-SEC at higher levels in ffEVS-UC. The bioinformatics analysis of miRNA and proteins indicates that these ffEVs contents play a role in modulating pathways associated with the regulation of transzonal projections (TZPs), such as the axon guidance. To evaluate the effects of ffEVS supplementation during oocyte IVM, ffEVs isolated by ffEVs-UC or ffEVs-SEC were added or not (Control group) in bovine cumulus-oocyte-complex (COCs) during IVM. COCs supplemented with ffEVs-SEC had lower number of TZPs, and an increased maturation rate than the Control group (P < 0.05). The analysis of transcriptional variants in cumulus cells supplemented for 9 h during IVM revealed that PDE5A-203 was upregulated in ffEVs-SEC group compared to the Control and ffEVs-UC group. PDE5A can decrease the levels of cGMP in cumulus cells in addition to increased levels of EGFR transferred by ffEVs-SEC contributing to meiosis progression and consequently TZP numbers reduction during maturation. Together, these findings suggest that EVs’ isolation method can recover EVs with different contents that, upon supplementation, can possibly transfer molecules (such as protein, lipids, miRNAs) and induce changes in transcripts as well as intracellular pathways involved in meiosis progression and TZPs-mediated communication in COCs during IVM.