PS6-19. Evaluation of Microbiome Composition Combined with Fecal Untargeted Metabolomics Profiling in Healthy Adult Dogs Consuming Diets with Increasing Levels of Red Sorghum Bran Inclusion.

Abstract The aim of this study was to evaluate the effects of up to 28% inclusion of red sorghum bran (RSB), a polyphenol-rich ingredient, on the fecal microbiome composition and untargeted metabolome in healthy adult dogs. A control (0% RSB) and three treatment diets containing 7% (RSB7), 14% (RSB14), and 28% (RSB28) RSB were formulated to have similar macronutrient compositions. Red sorghum bran was included at the expense of rice and chicken meal, while the inclusion of beet pulp and cellulose was adjusted to balance soluble and insoluble fiber across diets. Twelve adult Beagles (age = 3.97 ± 0.02 yr) were enrolled in a replicated 4 x 6 Latin Rectangle design. In each of the four 28-day periods, fresh fecal samples were collected during the last 5 days and subjected to microbiome (16S rRNA gene amplicon sequencing) and untargeted metabolome (gas chromatography-time of flight-mass spectrometry) analyses. Univariate analyses were performed using the GLIMMIX procedure of SAS 9.4 with diet as a fixed effect and dog and period as random effects. Partial least squares-discriminant analysis (PLS-DA) was performed in MetaboAnalyst 6.0 and Spearman correlation in RStudio. Dietary inclusion of RSB caused modest alterations in the fecal microbiome, resulting in greater (P < 0.05) alpha-diversity (observed features, Shannon Index, Fisher’s Alpha, and Simpson Index) in dogs fed RSB7 compared to control. In contrast, fecal metabolomics revealed more pronounced shifts; 28 of the 243 identified metabolites were altered (q < 0.05), generally demonstrating linear or quadratic responses to increasing dietary RSB. Control and RSB28 groups were clearly discriminated from each other in PLS-DA (R2=0.75; Q2=0.45; accuracy=0.58), and the major metabolites contributing to the discrimination were beta-sitosterol, 3,4-dihydroxycinnamic acid, pimelic acid, and 4-aminobenzoic acid. Dogs fed RSB28 had greater levels of 3,4-dihydroxycinnamic acid compared to control (P < 0.05), as well as greater (P < 0.05) fecal polyphenol-derived intermediates (catechol, 4-hydroxybenzoate, and hydroquinone), indicating enhanced microbial transformation of phenolic compounds in the gut. In addition, fecal samples from dogs fed RSB28 exhibited alterations in lipid metabolism, characterized by greater free fatty acids and monoacylglycerols and lower sterols (P < 0.05). These results align with the observed decrease in relative abundance of Collinsella and Faecalibacterium (P < 0.05), suggesting reduced microbial lipid metabolism and altered sterol biotransformation in the gut. Consistent with this, Spearman correlation analysis showed that Collinsella was moderately negatively correlated with linoleic acid (ρ=-0.45; P < 0.05) and 1-monoolein (ρ=-0.41; P < 0.05), while Faecalibacterium was moderately positively correlated with dihydroxycholesterol (ρ = 0.42; P < 0.05). Overall, the results indicate that the dietary inclusion of RSB in extruded dog food modulated the fecal microbiome and microbial metabolic activity of healthy adult dogs, potentially contributing to shifts in lipid- and polyphenol-related metabolites and supporting metabolic processes related to gut health.