Figure 1.

Schematic overview of three methods to separate EVs from bovine embryo-conditioned medium. A total of 1500 presumed zygotes were cultured in groups of 25 in 50 µL droplets of ultracentrifuged synthetic oviductal fluid enriched with non-essential and essential amino acids (SOFaa), ITS (5 µg/mL insulin; 5 µg/mL transferrin; 5 ng/mL selenium) and medium droplets were covered with mineral oil and incubated at 38 °C in 5% CO₂, 5% O₂, and 90% N₂. After 8 days of post insemination, the bovine embryo-conditioned medium was pooled and equally divided over three separation methods: differential ultracentrifugation (DU), OptiPrep^TM density gradient centrifugation (ODG), and size exclusion chromatography (SEC).