Investigation of salivary microRNAs as non-invasive biomarkers for weaning stress in piglets: A preliminary study

Weaning exposes piglets to abrupt nutritional and environmental changes that induce physiological stress. Saliva, unlike serum or tissue samples, enables repeated and stress-free sampling, making it a practical biological matrix for monitoring early-life adaptation. To evaluate whether salivary microRNAs (miRNAs) can serve as non-invasive indicators of these responses, we profiled miRNA expression in saliva collected from suckling and weaning piglets. Small RNA sequencing was conducted to detect salivary miRNAs and a total of 41 differentially expressed miRNAs (DEMs) were detected between the two groups, with 20 showing highly expression and 21 showing lowly expression in weaning piglets. Several of these DEMs have been previously associated with immune activity, inflammatory regulation, oxidative stress responses, and disease processes, highlighting their biological relevance during the weaning transition. Functional annotation of predicted target genes revealed enrichment in pathways such as TGF-beta, HIF-1, and AMPK signaling, as well as autophagy-related processes. These pathways are known to play key roles in stress adaptation, cellular metabolism, and immune modulation. These findings suggest that salivary miRNA profiles may reflect molecular changes associated with the weaning transition and may serve as candidate non-invasive indicators of physiological stress and health status in piglets. This approach may provide a basis for future development of saliva-based monitoring strategies after validation in larger cohorts and cost-effective targeted assays.