Multi-omics characterization of small RNA and metabolite cargo in buffalo colostrum and milk extracellular vesicles across lactation

Buffalo colostrum and milk extracellular vesicles (EVs) are emerging mediators of mother-neonate communication, but their small RNA and metabolic cargo during lactation remains poorly characterized. This study aimed to define the molecular signatures of EVs isolated from buffalo colostrum, transitional milk and mature milk, and to identify coordinated small RNA-metabolite patterns involved in neonatal adaptation. EVs from colostrum collected within 6 h post-partum and milk at 15 and 60 days were isolated from seven subjects and analyzed by nanoparticle tracking analysis, scanning electron microscopy, flow cytometry, untargeted UPLC-MS/MS metabolomics, Illumina small RNA sequencing, differential analysis and multi-omics integration. Vesicle preparations showed the expected morphology and size distribution, with mean particle sizes of 175.6 ± 3.2 nm in colostrum-derived EVs and 174.0 ± 1.5 nm in milk-derived EVs, concentrations in the order of 10^12 particles/mL, and expression of CD9, CD81 and CD63. Untargeted metabolomics identified 4,323 metabolites, 3,449 with database-supported annotation, and clearly separated colostrum- from milk-derived EVs. Differential analysis revealed 1,993 metabolites differing between transitional milk EVs and colostrum EVs, 2,224 between mature milk EVs and colostrum EVs, and 201 between mature and transitional milk EVs. Colostrum EVs were enriched in amino acid derivatives, small peptides, glycoconjugates and lipid/coenzyme-related features; transitional milk EVs showed organic acids, carbohydrates and glycan-related pathways; mature milk EVs displayed a more restricted profile involving amino acid derivatives, heterocyclic compounds, lipid-related classes and coenzymatic metabolism. Small RNA sequencing identified 49, 75 and 14 differentially abundant miRNA/small RNA features in the same comparisons, respectively. bta-miR-432 and bta-miR-375 were enriched in milk EVs, whereas bta-miR-139, bta-miR-34a, bta-miR-206, bta-miR-182 and bta-miR-183 characterized colostrum. Multi-omics integration identified 327 highly correlated miRNA-metabolite pairs, suggesting coordinated cargo remodelling and highlighting lipid, glycan, nucleotide and coenzymatic pathways for functional validation in future in vitro assays.