Investigating Targeted and Untargeted Approaches to Biomarker Discovery in Fearful, Adult Dogs

Chronic fear and anxiety experienced by domestic dogs pose significant welfare concerns, yet the molecular mechanisms related to these emotional states remain poorly understood. The objective of this study was to explore physiological correlates of fearfulness in dogs using targeted and untargeted metabolomic approaches to identify metabolic markers. One hundred privately owned adult dogs of various breeds (48 females; 52 males, 4.2 ± 2.1 years, and 25.7 ± 11.6 kg [mean ± SD]) were recruited for this study, spanning a range of owner-reported fearfulness categories/levels. Behavior was assessed using a previously published survey and the Canine Behavioral Assessment and Research Questionnaire (C-BARQ), resulting in ten behavioral outcome categories. Serum samples were collected for enzyme-linked immunosorbent assay (ELISA) analysis of serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA), alongside untargeted LC-MS metabolomics analysis. Ordinal logistic regression models assessed associations between metabolites and fear-related behavior categories, adjusting for age, sex, body weight, and dog as a random effect. Multiple testing for metabolomics data was controlled by Benjamini-Hochberg FDR (PFDR < 0.05). Pathway analysis identified tryptophan metabolism enriched for touch sensitivity (PFDR = 0.001) and pyrimidine metabolism enriched for dog-directed fear (PFDR = 0.041). At the metabolite level, formiminoglutamic acid, a marker of impaired one-carbon metabolism, was positively associated with combined fear (C-BARQ), while lauric acid, a medium-chain fatty acid, was positively associated with touch sensitivity (PFDR < 0.05). In targeted analyses, the 5-HIAA:5-HT ratio was inversely related to separation-related behavior (P = 0.03), and 5-HIAA was or tended to be weakly associated with combined fear (Temesi P = 0.04; C-BARQ P = 0.08), whereas 5-HT alone was not associated with any behavioral category. Metabolomics and ELISA rank estimates of 5-HT and 5-HIAA did not correlate, highlighting methodological differences and supporting the need to use both targeted and untargeted methods in biomarker discovery. Taken together, these exploratory results point to biologically plausible pathway-level patterns, particularly involving tryptophan, one-carbon/folate, and lipid metabolism. These may guide future research on nutritional and therapeutic strategies for fear-related behaviors in dogs.