Potential new biomarkers in pig saliva for differentiating between the effects of different conditions of mixing and density at pre-slaughter: a preliminary proteomics study

) production systems raises concerns regarding animal welfare, particularly during pre-slaughter conditions, a phase associated with significant stress. Saliva is increasingly recognised as a non-invasive matrix for detecting stress-related biomarkers in pigs. This preliminary study aimed to explore salivary protein changes in pigs subjected to two distinct pre-slaughter conditions at the slaughterhouse, improved (Group A) and stressful (Group B), by tandem mass tag (TMT)-based proteomics. Proteomic analysis of saliva from three pigs per group revealed 13 proteins with a statistically significant difference in relative abundance between the groups. Group B showed elevated levels of proteins linked to metabolic stress, inflammation, and coagulation, such as cystatin-C and fibrinogen chains, while proteins like vimentin and follistatin-related protein were decreased. Cystatin-C and vimentin were further validated by immunoassays in 12 additional pigs per group, confirming their differential abundance. These findings suggest that salivary cystatin-C and vimentin, along with the other 11 proteins that showed changes at proteomics, may serve as candidate biomarkers of acute stress at slaughter. While further validation is required, our results support the potential of salivary proteomics for welfare monitoring in livestock.