Characterisation and Comparative Evaluation of miRNA Contained in Small Extracellular Vesicles Isolated From Bovine Milk and Whey

ABSTRACT Small extracellular vesicles (EVs) from bovine milk and whey are emerging as biologically active carriers of microRNA (miRNA), yet their composition and variability among different dairy sources remain incompletely understood. We conducted an integrated characterisation of EV‐associated miRNAs isolated from milk and whey collected from three dairy farms. EVs were purified and validated according to MISEV2023 criteria, and small RNA sequencing was performed on milk and whey samples collected on three different days at each farm (18 samples). After quality filtering and adaptor trimming, mapped reads were normalised to counts per million (CPM). To ensure robust detection, only miRNAs present in at least 67% of samples were retained, resulting in a high‐confidence set of 329 miRNAs for downstream analyses. Milk‐derived EVs showed a higher overall miRNA read count than whey‐derived EVs (9.45 ± 5.3 million vs. 5.03 ± 2.5 million reads; p < 0.10), with Farm Z displaying the greatest overall abundance. Stability assessment using Z ‐scored coefficients of variation demonstrated that miRNA reproducibility varied considerably between farms and collections, indicating that farm‐ and collection‐specific factors, rather than matrix origin alone, were major contributors to expression variability. Differential expression analysis (DESeq2) identified 32 miRNAs significantly modulated between milk and whey EVs, with most ( n = 29) enriched in whey. Functional enrichment of predicted targets indicated involvement in immune and inflammation‐related pathways, including NF‐κB signalling, cytokine–receptor interaction and Ras signalling. Pairwise comparison of farms revealed only a small number of differentially expressed miRNAs in both matrices, and KEGG analyses did not identify significant pathways after multiple‐testing correction. This study presents a comparative overview of bovine milk and whey EV‐associated miRNAs, highlighting higher miRNA abundance in milk, significant farm‐dependent variability, and enrichment of immune‐related pathways that distinguish the two matrices.