|Abstract:||The pet population is growing at a fast pace and 91.7% of 2,668 American pet owners consider their dog as part of their family. Thus, the importance of longevity and health of companion animals has been a constant concern for pet parents, and diet plays an important role in that. The use of prebiotics and dietary fibers have gained renewed interest in the pet food industry as a strategy to modulate gut health. Dietary fibers are heterogeneous compounds that exert different physiological responses and health benefits depending on their physical and chemical characteristics. Different types of prebiotics can be incorporated in diets of dogs and their efficacy is related to their structure, form of supplementation, and dosage. Both prebiotics and dietary fibers are non-digestible ingredients that may confer benefits to the host by selectively stimulating beneficial intestinal bacteria and affecting rate of fermentation and concentrations of fermentative end-products. Therefore, the aim of this experiment was to evaluate the effects of a prebiotic and dietary fiber blend with or without a gut health promoter on outcomes pertaining to gastrointestinal health and nutrient digestibility by adult dogs. Four diets containing either 5% of cellulose (CT), 5% fiber and prebiotic blend (FP), 0.02% of a gut health promoter additive (GP), or 5% fiber and prebiotic blend plus 0.02% of gut health promoter (FG) were formulated to meet or exceed the AAFCO (2017) nutritional requirements of adult dogs. Eight adult female beagles (mean age 4.2 ± 1.1 yr; mean BW = 10.8 ± 1.4 kg; mean BCS = 5.8 ± 0.6) were randomly assigned to one of the four dietary treatments using a replicated 4 x 4 Latin square design. Each experimental period consisted of 14 days (10 days of diet adaptation + 4 days of total and fresh fecal and total urine collection). Food was offered twice daily and fed to maintain body weight. Food intake and total fecal and urine output were measured and sampled for macronutrient analyses and digestibility calculations. Blood samples were collected at the end of each period for serum chemistry analysis and complete blood count. A fresh fecal sample from each dog was collected within 15 minutes of defecation and analyzed for dry matter (DM), phenols and indoles, short-chain fatty acids (SCFA), branched-chain fatty acids (BCFA), and microbiota. The data were analyzed using The MIXED procedure using SAS, version 9.4. Total DNA from fresh fecal samples was extracted using Mo-Bio PowerSoil kits, sequencing was performed on a MiSeq and data were analyzed using QIIME 1.9.1. All animals remained healthy throughout the study, with serum metabolites being within reference ranges for adult dogs. All diets were well accepted by the dogs, resulting in similar (P > 0.05) daily food intakes among treatments. Likewise, fecal output and scores did not differ (P > 0.05) among dietary treatments, with the latter being within an ideal range (2.5-2.9). All diets were highly digested by dogs, and had a similar (P > 0.05) apparent total tract digestibility (ATTD) of dry matter (DM) (81.6-84.4%), organic matter (OM) (86.4-87.3%), and crude protein (CP) (86.6-87.3%). However, ATTD of total dietary fiber (TDF) was greater for dogs fed the FG diet (P < 0.05) in contrast with dogs fed the GP diet, while dogs fed the CT and FP diets had intermediate TDF digestibilities but not different from either FG or GP. Fecal acetate and propionate concentrations were greater (P < 0.05) for dogs fed FP and FG diets. The concentration of fecal butyrate did not differ among treatments (P > 0.05). Fecal concentrations of isobutyrate and isovalerate were greater for dogs fed CT (P < 0.05) compared to dogs fed the other three treatments. No shifts in fecal microbial richness and diversity were observed when dogs consumed diets containing the fiber and prebiotic blend and (or) the gut health promoter additive. Overall, the data suggest that dietary supplementation of fiber and prebiotic blend were well tolerated by dogs, did not cause detrimental effects on fecal quality or nutrient digestibility, and resulted in beneficial shifts in fecal fermentative end-products that may support gut health. They also suggest a potential synergistic effect between fiber and prebiotic blend wth gut heath promoter that warrants further investigation.