Fig. 3

EV content, size, and PRDX5/PRDX6 expression in SEC fractions from bovine seminal plasma. In bovine seminal plasma SEC fractions 4–7, 72% of the detected particles were membranous (Cell Mask Green positive, CMG +). Of these, 61% expressed at least one EV-related surface marker (CD9 or CD63). The population marked in purple represents CMG + and CD63 + particles, the green population represents CMG + and CD9 + particles, and the yellow population represents CMG + , CD63 + , and CD9 + particles (A). EVs from SEC fractions 4–7 were sized between 100 and 200 nm (B). Western blot analysis of proteins separated by non-reducing SDS-PAGE, using anti-PRDX5 (C) and anti-PRDX6 (D) antibodies, demonstrated the presence of PRDX5 in the exosomal vesicle (EV) fraction, with no detectable signal in the exosome-depleted seminal plasma (SP). Arrows indicate 8 reactive bands for PRDX5 with wide MW range for PRDX5 in the EV fraction. In contrast, PRDX6 was detected in both the EV fraction (EVs) and the exosome-depleted seminal plasma (SP), with arrows indicating 3 reactive bands for PRDX6 with high MW for PRDX6, although the signal was weaker in both