Identification of possible new salivary biomarkers of stress in sheep using a high-resolution quantitative proteomic technique

The aim of this study was to identify biological pathways and proteins differentially expressed in the saliva proteome of sheep after the application of a model of stress, using high-resolution quantitative proteomics. In addition, one of the proteins differently expressed was verified and evaluated as a possible biomarker of stress in this species. Saliva paired samples from eight sheep before and after the application of a model of stress based on shearing were analysed using tandem mass tags (TMT). The TMT analysis allowed for the identification of new stress-related metabolic pathways and revealed 13 proteins, never described in saliva of sheep, that were differentially expressed between before and after the stress. Six of these proteins pertain to four major metabolic pathways affected, namely: canonical glycolysis, oxygen transport, neural nucleus development, and regulation of actin cytoskeleton reorganization. The rest of proteins were unmapped original proteins such as acyl-coenzyme-A-binding protein; complement C3; alpha-2-macroglobulin isoform-Xl; type-II small proline-rich protein; lactoferrin; secretoglobin family-1D-member; and keratin, type-II cytoskeletal 6. Of these proteins, based on its biological significance and specific immunoassay availability, lactoferrin was selected for further validation. The immunoassay intra- and inter-assay coefficients of variation were lower than 13%. The method showed good linearity under dilution and recovery, and the detection limit was low enough to detect salivary lactoferrin levels. A significant decrease (P < 0.01) in salivary lactoferrin concentration in the sheep following the application of the model of stress was observed, suggesting that this protein could be a potential salivary biomarker of stress situations in sheep.