311 Investigating metabolomic profiles associated with canine fearfulness

Abstract Chronic fear and anxiety experienced by domestic dogs pose significant welfare concerns, yet the molecular mechanisms related to these states remain poorly understood. Therefore, the objective of this study was to explore the relationships between type and level of fear shown by dogs and peripheral metabolites, aiming to identify metabolic markers that could inform future nutritional strategies for managing fearfulness. One hundred and one client-owned adult dogs of various breeds (48 females; 53 males, 4.2 ± 2.1 years, and 25.7 ± 11.6 kg [mean ± SD]) were recruited for this study. Included dogs ranged from those described by their owners as not generally fearful to very fearful and none were on medications or supplements. Owners completed a questionnaire, based on the Canine Behavioral Assessment and Research Questionnaire (C-BARQ), which includes scenario-based questions scored on a scale from 0 (no fear) to 4 (extreme fear). Responses were categorized into three fear subtypes: stranger-oriented (fear of unfamiliar people), dog-oriented (fear of other dogs), and non-social (fear of unfamiliar situations, objects, or noises) for analysis. To standardize dietary nutrient profiles, dogs were fed a commercial kibble (Acana, Classics Red Meat Recipe) for ≥ 4 weeks prior to blood collection. Serum samples underwent non-targeted liquid chromatography-mass spectrometry metabolite profiling. Logistic regression was used to examine the relationship between fear subtypes and individual metabolites using SAS Studio, with dog as a random effect. Multiple comparisons were corrected using the Benjamini-Hochberg false discovery rate (FDR) adjustment and data was considered significant at pFDR < 0.05. Two metabolites, L-homophenylalanine and indoleacetate, were associated with higher owner-reported non-social fear (pFDR < 0.04). No associations were found between metabolites and human- or dog-oriented fear (pFDR > 0.05). Indoleacetate is a microbial metabolite of tryptophan that acts as a signaling molecule in the gut-brain axis in other mammals, suggesting a potential link between gut-derived metabolites and some types of fear in dogs. While the role of L-homophenylalanine is not well understood in mammals, it is commonly detected in the canine fecal microbiome, further supporting a possible connection between gut microbial metabolism and pathways involved in fear responses. These findings contribute to the growing body of evidence highlighting the important role of gut-brain interactions in fear and anxiety, laying the groundwork for future research in this area. By identifying specific metabolites associated with canine fearfulness, this study provides a foundation for continued exploration of microbiome-metabolite links and the development of targeted nutritional strategies to support behavioral well-being in dogs.