Donkey milk study: miRNAs compared with buffalo, goat, and sheep (Cendron 2024)

In donkey milk from 49 donkeys in Lazio, miRNAs accounted for 7.74% of the annotated small RNAs, compared to only 0.87–1.57% in buffalo, goat, and sheep milk. Only four miRNAs were found in all four types of milk.

Study at a Glance

• Original title: Analysis of miRNAs in milk of four livestock species
• Authors: Cendron F., Rosani U., Franzoi M. et al.
• Journal: BMC Genomics, 25, 859 (2024)
• Country: Italy · Study type: Laboratory study

Study Design

Individual milk samples from commercial farms in the Lazio region (Italy) were collected during routine machine milking: 49 donkeys of the Amiata (n = 37) and Ragusana (n = 12) breeds from two farms (1st to 7th lactation, 2nd to 7th month of lactation), as well as goats (n = 498), sheep (n = 443), and water buffalo (n = 648) from five farms each. The donkeys, goats, and sheep grazed and were supplemented with hay and grain. The samples were pooled into 20 donkey samples and then combined into one overall pool per animal species (50 ml).

Key Figures

• 49 – donkeys (37 Amiata, 12 Ragusana) from two farms in Lazio
• 20 – donkey milk samples, combined into one overall pool
• 70,549,149 – raw sequences (reads) from the donkey milk sample
• 9,385,067 – donkey milk reads after quality filtering
• 94.6% – proportion of mapped reads in donkey milk (highest value; sheep: 84.8%)
• 7.74% – proportion of miRNAs in the annotated RNA of donkey milk

Results

After quality filtering, 9,385,067 reads remained for donkey milk; 94.6% of these could be mapped to the reference genome, the highest proportion among the species (buffalo 92%, goat 89.3%, sheep 84.8%).

The proportion of miRNAs in the annotated RNA was 7.74% in donkey milk, significantly higher than in buffalo (0.87%), goat (1.57%), and sheep milk (1.12%). 130 miRNAs were identified in donkey milk, 132 in goat milk, 83 in buffalo milk, and 43 in sheep milk.

Only four miRNAs – miR-200a, miR-200b, miR-200c, and miR-23a – were found in all four types of milk. miR-200a accounted for 15% of all miRNA reads in donkey milk (sheep 11%, buffalo 6%, goat 5%); miR-200b and miR-200c were also proportionally most abundant in donkey milk, while miR-23a was most abundant in goat milk.

Study Limitations

• One pool per species: All donkey samples were combined into a single pool and sequenced once. There were therefore no biological replicates or statistical analysis of differences between the species; the effects of breed, stage of lactation, or farm cannot be separated. The authors themselves recommend studies on individual animals.
• Horse genome used as reference: The donkey reads were mapped to the horse genome, and the buffalo reads to the cow genome. Donkey-specific miRNAs may therefore have been overlooked or incorrectly assigned; the high mapping rate should be interpreted with this limitation in mind.
• Inconsistent group numbers: The combination of goat–donkey–buffalo is mentioned twice in the results section (with 10 and 11 miRNAs), while the discussion refers to 12. Furthermore, it mentions "10 miRNAs in all four species," although the Venn diagram shows that only four are common to all.
• Further inconsistencies: According to the results section, donkey milk had the most miRNAs, but the figures given show 132 for goat and 130 for donkey. miR-22-3p is described as absent from donkey milk, while elsewhere the presence of miR-22 in donkey milk is confirmed. Normalization is referred to as RPKM, but described as reads per million.
• Functions only predicted: Target genes and signaling pathways are based on computer predictions. The authors' statements regarding their importance for immune defense, growth, or development in young animals, as well as the intake of miRNAs through food, were not experimentally tested; effects in humans were not the subject of the study.

Level of evidence: Basic research (laboratory) – describes the properties of milk; no statement on effects in humans.

Our Assessment

The study describes which small RNA molecules can be detected in pooled raw milk from Italian donkeys compared to three ruminant species. The functional information is based on predictions; no conclusions about effects in humans or on individual commercial products can be drawn from it.

Source: Cendron F., Rosani U., Franzoi M., Boselli C., Maggi F., De Marchi M., Penasa M. (2024). Analysis of miRNAs in milk of four livestock species. BMC Genomics. DOI: 10.1186/s12864-024-10783-4 · Summarized by the DON DONKEY'S editorial team, as of October 2026.

End of study summary. The study was conducted independently by DON DONKEY'S® and does not refer to our products. Our donkey milk is a food product.