Milk-Derived EVs from Different Animal Sources: An Overview on Their Detection, Isolation and Pleiotropic Exerted Effects

Milk is a primary source of vital nutrients and bioactive components fundamental for the growth and development of both newborn animals and humans. Produced by economi-cally significant livestock species (including cattle, buffaloes, goats, sheep and camels) milk is a complex matrix rich in caseins, vitamins, fats and proteins. In addition to its nu-tritional profile, milk serves as a vehicle for milk-derived extracellular vesicles (mEVs), a specialized class of food-derived EVs (fEVs) that exert pleiotropic effects aligned with the One Health concept, relating animal health, human nutrition, and ecosystem stability. mEVs offer unique advantages, such as high biocompatibility and gastrointestinal stabil-ity, rendering them also potential therapeutic tools, as drug delivery systems. However, challenges remain regarding the standardization of mEVs and the variability of their mo-lecular cargo. This review provides a comparative analysis of mEVs across diverse spe-cies, including bovines, water buffaloes, yaks, camels, goats, pigs, horses, donkeys, and humans, with a focus on their unique functional profiles. Indeed, a critical issue in mEVs research is the isolation process: recommendations to minimize contamination from milk fat globules and casein micelles (which can cover EV signals) are given. Finally, current detection methods and instrumentation, with a specific focus on advancing Flow Cytom-etry (FC) approaches are discussed. Key insights include the use of Conventional FC (with fluorescence triggering, the necessity of rigorous controls and calibration, and the utility of Bead-Based Assays to overcome resolution limits) and of Imaging Flow Cytometry (IFC). In both technical approaches, the application of different EVs generic fluorescent markers and the strategic selection of tetraspanins (i.e. CD9, CD63, CD81), is mandatory, empha-sizing the importance of selecting appropriate antibody clones or considering cross-reactivity when targeting these antigens across different mammalian species.