Saliva and serum profiling by high-throughput TMT-based proteomics for the investigation of metritis in dairy cows.

Metritis affects up to 20 % of dairy cows after giving birth, causing significant losses in terms of animal welfare and dairy farm profitability. The aim of the present transversal study is to evaluate the possible changes in salivary and serum proteomes in bovine metritis using a high-throughput quantitative proteomic analysis to potentially discover biomarkers of this disease. For this, proteomes of paired saliva and serum samples from cows with metritis (n = 5) and healthy controls (n = 5) were analyzed using Tandem Mass Tags (TMT). In addition, an independent set of n = 33 salivary samples was employed to further evaluate calprotectin in saliva as a candidate biomarker of metritis. TMT proteomic approach identified 326 proteins in saliva and 275 in serum (with 2 unique peptides and 1 % FDR), with 15 and 22, respectively, showing significant changes in the metritis group in comparison to controls. These proteins with different abundance between the two groups were related to different pathways including the immune system, inflammation, lipid metabolism and homeostasis, oxidative stress, DNA repair, cellular signaling, and structural and mucosal functions. One protein, calprotectin (S100A8-A9), was further validated in bovine saliva for the first time, showing higher levels in cows with metritis (median = 0.44, IQR = 0.41-0.50 mg/L) than in controls (median = 0.17, IQR = 0.10-0.26 mg/L), consistent with the increase in S100A8 found in the proteomic results. Overall, this study indicates that saliva and serum proteomes can reflect physiopathological changes caused during metritis, being a potential source of potential biomarkers of the disease. SIMPLE SUMMARY: This study identifies distinct protein profiles in the saliva and serum of healthy cows compared to cows with metritis using a gel-free proteomic approach. Significant changes in protein abundance were observed in 15 salivary and 22 serum proteins, involved in inflammation, lipid metabolism, and oxidative stress. Since S100A8 protein was found at higher levels in cows with metritis, calprotectin (S100A8-A9) was further validated in saliva in an independent sample cohort. These findings offer potential biomarkers for metritis in dairy cows and could contribute to understanding disease-related changes, impacting animal health management and milk production.