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Extracellular vesicles (EVs) have been identified as one of the communication mechanisms amongst embryos. They are secreted into the embryo culture medium and, as such, represent a source of novel biomarkers for identifying the quality of cells and embryos. However, only small amounts of embryo-conditioned medium are available, which represents a challenge for EV enrichment. Our aim is to assess the suitability of different EV separation methods to retrieve EVs with high specificity and sufficient efficiency. Bovine embryo-conditioned medium was subjected to differential ultracentrifugation (DU), OptiPrep TM density gradient (ODG) centrifugation, and size exclusion chromatography. Separated EVs were characterized by complementary characterization methods, including Western blot, electron microscopy, and nanoparticle tracking analysis, to assess the efficiency and specificity. OptiPrep TM density gradient centrifugation outperformed DU and SEC in terms of specificity by substantial removal of contaminating proteins such as ribonucleoprotein complexes (Argonaute-2 (AGO-2)) and lipoproteins (ApoA-I) from bovine embryo-derived EVs (density: 1.02-1.04, 1.20-1.23 g/mL, respectively). In conclusion, ODG centrifugation is the preferred method for identifying EV-enriched components and for improving our understanding of EV function in embryo quality and development.

The Separation and Characterization of Extracellular Vesicles from Medium Conditioned by Bovine Embryos