Pasture-based milk presents several advantages over milk from intensive industrial farming in terms of human health, the environment, animal welfare, and social aspects. This highlights the need for reliable methods to differentiate milk according to its origin on the market. Here, we explored whether miRNA profiles could serve as a marker of milk production systems. We compared levels of previously described miRNAs in milk from four production systems (altogether 112 milk samples): grazing, zero grazing, grass silage or corn silage. Total RNA was extracted from the fat phase, and miRNAs levels were quantified by real-time quantitative PCR. The levels of the miRNAs bta-miR-155 and bta-miR-103 were higher in the grazing system than in corn silage farms. The levels of bta-miR-532, bta-miR-103 and bta-miR-7863 showed differences between different farm managements. The miRNAs bta-miR-155 and bta-miR-103 were predicted to participate in common functions related to fat metabolism and fatty acid elongation. All four differentially expressed miRNAs were predicted to participate in transport, cell differentiation, and metabolism. These results suggest that the dairy production system influences the levels of some miRNAs in milk fat, and that bta-miR-155 and bta-miR-103 may be potential biomarkers to identify milk from pasture-managed systems.

Studying microRNA (miRNAs) in certain agri-food products is attractive because (1) they have potential as biomarkers that may allow traceability and authentication of such products; and (2) they may reveal insights into the products’ functional potential. The present study evaluated differences in miRNAs levels in fat and cellular fractions of tank milk collected from commercial farms which employ extensive or intensive dairy production systems. We first sequenced miRNAs in three milk samples from each production system, and then validated miRNAs whose levels in the cellular and fat fraction differed significantly between the two production systems. To accomplish this, we used quantitative PCR with both fractions of tank milk samples from another 20 commercial farms. Differences in miRNAs were identified in fat fractions: overall levels of miRNAs, and, specifically, the levels of bta-mir-215, were higher in intensive systems than in extensive systems. Bovine mRNA targets for bta-miR-215 and their pathway analysis were performed. While the causes of these miRNAs differences remain to be elucidated, our results suggest that the type of production system could affect miRNAs levels and potential functionality of agri-food products of animal origin.

Dairy production systems significantly impact environmental sustainability, animal welfare, and human health. Intensive farming maximizes output through high-input practices, raising concerns about environmental degradation, animal welfare, and health risks from antibiotic residues. Conversely, organic farming emphasizes sustainable practices, animal welfare, and minimal synthetic inputs, potentially enhancing biodiversity, soil health, and milk quality. MicroRNAs (miRNAs), non-coding RNAs regulating gene expression, are promising biomarkers due to their response to various conditions. In this study, miRNAs bta-miR-103 and bta-miR-155, which are abundant in milk from pasture-fed cows, were selected. Additionally, bta-miR-215, which is abundant in milk fat from intensive systems, was also studied, in order to differentiate dairy production systems. A novel, cost-effective gold nanoparticle (AuNP)-based sensor was developed for miRNA detection, leveraging the unique plasmonic properties of AuNPs for visual detection. The method involves functionalizing AuNPs with complementary RNA probes and detecting miRNA-induced aggregation through colorimetric changes. This rapid, results in 30 min, and sensitive, visual limit of detection of 200 nM, assay requires minimal instrumentation and can be easily interpreted, offering significant advantages for field implementation in characterizing dairy production systems. This study demonstrates the successful application of this sensor in detecting miRNAs in 350 nM miRNA spiked raw milk, highlighting its potential for in situ dairy industry applications.

Dairy farming practices significantly affect the nutritional and functional properties of milk. This study compares miRNAs in milk exosomes from extensive and intensive dairy systems and explores their potential implications for human consumers. Extensive systems are believed to produce milk of higher quality with better animal welfare compared to intensive systems. Milk samples from eight extensive and nine intensive dairy farms were analysed. Milk-derived extracellular vesicles were isolated through sequential ultracentrifugation and characterised through Dynamic Light Scattering and Nanosight to determine the size and the concentration of the extracellular vesicles, in addition to immunoblotting to ensure the presence of exosome-specific proteins in their membrane. miRNA levels were quantified using RT-qPCR, and metabolic pathways associated with miRNAs showing significant differences between farm groups were analysed. EVs from extensive farms had higher concentrations. Notably, bta-miR-451 levels were significantly higher in milk from extensive farms (p = 0.021). Like human miRNA hsa-miR-451, it is linked to pathways related to Parkinson’s disease and cancer. Our research suggests that milk production in extensive systems not only provides socioeconomic and environmental benefits but may also have positive effects on human health. Further research is warranted to explore the bioactive potential of these miRNAs and their implications for human health.