Comparative small RNA profiling of donkey, human and bovine milk-derived extracellular vesicles reveals conserved and distinct miRNA abundance patterns

Abstract

Milk-derived extracellular vesicles (EVs) carry small RNAs, but the EV-associated miRNA repertoire of donkey milk remains poorly characterized. Small-RNA sequencing was performed on independently prepared EV-enriched preparations from three donkeys, with representative EV characterization performed on one preparation. Public human and bovine milk EV datasets were reprocessed with the donkey data using a harmonized workflow, and sequence-identical mature miRNAs were compared descriptively. Across the three donkey libraries, 292 miRNAs were identified, including 244 known and 48 putative novel miRNAs. Donkey and human abundance rankings showed the strongest correlation (Spearman's ρ = 0.773), miR-143-3p and miR-122-5p showed higher mean normalized abundance in the donkey dataset than in both external comparator datasets. These findings support a conserved yet compositionally distinctive donkey milk EV-associated miRNA repertoire and provide candidates for future functional validation.