68 Characterization of Bovine Extracellular Vesicles Isolated from Colostrum

Abstract Bovine colostrum is composed of a variety of biologically active compounds including microRNAs (miRNA), which are found within extracellular vesicles (EVs). Newborn calves are agammaglobulinemic. Hence, the ability of the bovine neonate to fight off pathogens is therefore almost entirely dependent upon colostrum ingestion. Extracellular vesicle-derived miRNAs may have a crucial role in the regulation of the immune response. Thus, we aimed to characterize the EVs morphology from bovine colostrum using stimulated emission depletion (STED) super resolution microscopy. Bovine colostrum samples (approx. 3-4 h after parturition) were collected from primiparous Holstein dairy cows (n = 10) at a commercial dairy farm. Colostrum samples (15 mL) were subjected to a series of centrifugation steps to achieve defatted supernatant and to label EV pellets with EV specific markers. The EV pellets were resuspended in 100µL of 1 x PBS for EV staining methods or resuspended in 1 mL of trizol reagent for further miRNA isolation. The EVs suspensions were incubated with a primary polyclonal antibody anti-CD63 and with a secondary antibody (Alexa Fluor 594) for approximately 90 minutes each. Labeled EV-antibody complex were analyzed using confocal and STED microscopy. Based on the confocal and super resolution STED imaging capability obtained from the EVs-derived from bovine colostrum (Figure 1), labeled EVs with anti-CD63 and the enhanced resolution of STED over conventional confocal imaging, allowed to image the EVs at their actual size (30-200 nm). Further in-depth view on EV morphology, marker proteins expressed on the membrane, and size will contribute to our understanding of EVs-derived from colostrum and their functions. This will be crucial in studies to evaluate EVs uptake by intestinal cells for the correct identification of compartments involved in subcellular trafficking.