Molecular profiling of extracellular vesicles in buffalo colostrum and milk across lactation by an integrated multi-omics approach

Milk-derived extracellular vesicles are bioactive nanostructures involved in intercellular communication and immune regulation through their cargo of proteins, lipids, metabolites and RNA, which contribute to their immunomodulatory potential. Although bovine milk has been extensively investigated, buffalo extracellular vesicles are still poorly characterized, particularly across lactation. Exploring species-specific differences is crucial to identify optimal sources for clinical and therapeutic applications. This study aimed to characterize the small RNA cargo and metabolites of colostrum EVs (ColosEVs) and milk EVs (mEVs) at two lactation stages (15 and 60 days post-parturition) by integrating transcriptomics (small RNA next-generation sequencing) and metabolomics (high-performance liquid chromatography-mass spectrometry analyses). Vesicle preparations were characterized by nanoparticle tracking analysis and scanning electron microscopy, confirming the expected size distribution and morphology. Mean particle size was about 175.6 ± 3.2 nm in colostrum-derived extracellular vesicles and 174.0 ± 1.5 nm in milk-derived extracellular vesicles, with concentrations in the order of 10^12 particles/mL. Flow cytometry, performed using both single-vesicle and bead-based approaches, showed expression of the canonical tetraspanins CD9, CD81 and CD63. Colostrum samples contained a higher abundance of extracellular vesicles than mature milk, whereas milk extracellular vesicles collected at both time points showed greater molecular diversity and shared the largest number of detected features. Small RNA sequencing identified 51 microRNAs enriched in colostrum-derived vesicles, 81 in milk vesicles at 15 days and 79 at 60 days. Metabolomic analysis revealed 379 molecules specifically enriched in colostrum-derived vesicles, while 619 and 764 were enriched in milk vesicles at 15 and 60 days, respectively. Preliminary integrative multi-omics analysis highlighted a clear separation among the three groups in principal component analysis and integrated heatmaps, together with strong microRNA-metabolite associations (r = 0.85), supporting a stage-dependent remodeling of vesicular cargo throughout lactation. Functional analysis of class-specific microRNA targets indicated enrichment of pathways related to immune and stress responses, nucleic acid metabolism, protein signaling and intracellular transport, whereas metabolite pathway analysis suggested dynamic changes in amino acid, lipid, carbohydrate and signaling metabolism. These findings provide a systems-level view of buffalo milk extracellular vesicles and support their potential role in neonatal development, immune modulation and future clinical applications.