Different approaches to assess extracellular vesicles from bovine colostrum

<h2>Abstract</h2> Extracellular vesicles (EVs) are lipid-bilayer nanoparticles secreted by most cell types and implicated in intercellular communication, immune regulation, and disease processes. Milk-derived EVs, particularly those present in bovine colostrum (BCEVs), may play critical roles in neonatal calf development and immunity; however, their characterization remains limited due to methodological challenges in isolating pure vesicle populations. This study aimed to evaluate different methods to detect and characterize BCEVs. We isolated BCEVs from colostrum of multiparous dairy cows using differential ultracentrifugation and employed complementary analytical techniques to evaluate their morphology, size distribution, and EV-associated markers. Dynamic light scattering (DLS) indicated an average BCEV diameter of 127.3 ± 9.5 nm, whereas single particle interferometric reflectance imaging sensing (SP-IRIS) revealed a smaller population of 40–45 nm vesicles expressing CD9 and CD63. Transmission electron microscopy (TEM) further confirmed spherical vesicles averaging 38.7 ± 2 nm, while cryogenic electron microscopy (cryo-EM) visualized intact lipid bilayer structures. These findings demonstrate that BCEVs comprise heterogeneous populations detectable by multiple complementary approaches and underscore the importance of combined methodologies for their accurate characterization.