Signals in the milk: what melatonin and miRNAs reveal about mastitis in dairy cows

Bovine mastitis is one of the most common and economically significant diseases in the dairy industry, creating the need for novel, non-invasive diagnostic tools and alternative strategies to control udder health. This thesis investigated two aspects of the mammary gland immune response, with the main aim of identifying bioactive molecule mediators and molecular biomarkers that could enhance, respectively, mastitis prevention and monitoring. The first part of the study (experiment 1) focused on determining melatonin concentrations in bovine milk collected during daytime and nighttime milkings in winter, under natural photoperiod conditions. Milk samples were collected from 40 Holstein Friesian cows at 4:00 am and 4:00 pm and analysed for composition, melatonin content, somatic cell count (SCC), and differential somatic cell count (DSCC). Melatonin quantification was performed with an ELISA kit, while SCC and DSCC were determined with a Fossomatic™ 7DC analyser. Melatonin concentrations were significantly higher in nighttime milk samples (15.63 ± 1.9 pg/mL vs. Day group: 6.80 ± 0.75 pg/mL) (p < 0.001). Furthermore, Night milk samples showed a SCC reduction (129.70 ± 25.01 ×103 cells/mL vs. Day group: 196.39 ± 33.80 ×103 cells/mL) (p < 0.001), confirming the hypothesis that melatonin may play a protective and immunomodulatory role on udder health. DSCC showed no differences between Day and Night samples (p = 0.78), indicating that the proportion of immune cell subpopulations does not vary during the day and that melatonin may act by enhancing the local immune response rather than altering its composition. These results highlight melatonin potential as a natural bioactive molecule that contributes to mammary gland defence and can be modulated through improved livestock management. The second part of the study (experiment 2) evaluated the potential as biomarkers for mastitis early diagnosis of four circulating microRNAs (miR-26a-5p, miR-142-5p, miR-146a, and miR-223-3p) in milk, already associated with mammary gland inflammation. The study assessed the c-miRNAs quantification by qPCR both in vivo in milk samples grouped by inflammatory status (healthy, susceptible, chronic, and acute mastitis), and in vitro in the inflammatory-stimulated supernatants of bovine immune cells (polymorphonuclear leukocytes or PMNs, lymphocytes, monocytes). 72 milk samples from Italian Brown Swiss cows were collected and classified into four groups based on SCC and DSCC values, determined using a Fossomatic™ 7DC analyser. Immune cells were stimulated with PHA (lymphocytes) or LPS (PMNs and monocytes) to evaluate miRNA expression and nitric oxide production. miR-26a showed a downregulation in susceptible (0.7-fold) (p < 0.05), chronic (0.8-fold) (p < 0.01) and acute mastitis (0.8-fold) (p < 0.01) groups, suggesting its potential utility as an early inflammation biomarker. miR-223 was significantly upregulated only in the acute mastitis group (vs. control: 26.5-fold, vs. susceptible: 24.4-fold, vs. chronic: 25.7-fold) (p < 0.01). These expression profiles, together with DSCC values, were integrated into a diagnostic decision model that can discriminate between different mastitis stages. In immune-cell cultures, miR-26 exhibited reduced expression following lipopolysaccharide stimulation in neutrophils (0.4-fold) (p < 0.01), whereas miR-223 shows increased expression in lymphocytes activated with phytohemagglutinin (0.2-fold) (p < 0.05). In conclusion, this thesis contributes to a better understanding of the interaction mechanisms between the immune system and the mammary gland, identifying melatonin and c-miRNAs as promising and non-invasive indicators of udder health. Their use in combination with SCC and DSCC could improve the accuracy and timing of mastitis diagnosis and prevention, supporting farming approaches aimed at reducing antibiotic use and improving animal welfare.