**Background:** Dietary fiber from whole grains, particularly rye, has been associated with beneficial effects on gut microbiota and host metabolism in humans, but few studies have examined these effects in dogs. The gut microbiota plays a critical role in fiber fermentation, production of short-chain fatty acids (SCFA), and immune function. *Prevotella* and *Ruminococcaceae* are key fiber-fermenting bacteria linked to metabolic health. This study aimed to evaluate how inclusion of whole grain rye, compared with refined wheat, influences fecal microbiota composition, SCFA profiles, and apparent total tract digestibility (ATTD) in dogs.
**Methods:** Six male Beagle dogs (mean age 4.6 years, SEM 0.95; mean body weight 14.6 kg, SEM 0.32) were fed three experimental diets in the same order (W, then RW, then R), each for 21 days including a 6-day acclimatization period, with 2–2.5 months washout between periods. Diets were formulated to be similar in energy and protein but differed in carbohydrate source: refined wheat flour (W), a 1:1 mixture of whole grain rye meal and refined wheat (RW), or whole grain rye meal alone (R), each comprising 50% of total dry matter. Titanium dioxide was added as an inert marker for ATTD determination. Fecal samples were collected 1–3 days before each diet period (baseline) and during the last three days of each period. Microbiota was analyzed by sequencing 16S rRNA gene amplicons (V3-V4 region) on the Illumina MiSeq platform. SCFA (acetate, propionate, butyrate) were measured by high-performance liquid chromatography. ATTD of crude protein, crude fat, neutral detergent fiber, and gross energy was calculated. Statistical analyses included principal coordinate analysis (PCoA), analysis of similarity (ANOSIM), similarity percentage tests, and linear mixed effects models with diet, time, and their interaction as fixed effects and dog as random effect.
**Key Results:** All dogs completed the W and RW periods; one dog was excluded from the R period due to weight loss exceeding 5% in one week. PCoA and ANOSIM revealed significant differences in fecal microbiota composition depending on diet (P = 0.002, R = 0.19). The R diet produced a significant shift from baseline (ANOSIM P = 0.014, R = 0.67), primarily driven by an increase in *Prevotella* (mean relative abundance 9.9% at baseline vs. 54.3% after R diet; P < 0.001). *Prevotella* abundance was also significantly higher after the R diet compared with the W diet (P < 0.007). *Romboutsia* and an unclassified *Peptostreptococcaceae* decreased significantly after the R diet (P < 0.02 for both). No significant differences in SCFA proportions were found overall, but there was a tendency for a diet-by-time interaction for acetic acid (P < 0.06), and post-hoc comparison showed higher molar proportions of acetic acid after the R diet (mean before 52.2%, SEM 1.4%; mean after 67.0%, SEM 6.3%; P = 0.005). ATTD was significantly lower for the R diet compared with W and RW for crude protein (79.6% vs. 89.3% and 87.0%; P < 0.001), crude fat (95.7% vs. 96.8% and 97.0%; P = 0.01), neutral detergent fiber (34.0% vs. 69.9% and 64.0%; P < 0.001), and gross energy (83.1% vs. 93.6% and 90.5%; P < 0.001). Fecal score and dry matter did not differ between diets.
**Clinical Implications:** This study provides evidence that high inclusion of whole grain rye (50% of DM) in dog food can significantly alter the gut microbiota, particularly by increasing *Prevotella* abundance, and may shift fermentation patterns toward greater acetate production. These changes have been associated with favorable metabolic effects in humans and warrant further investigation in dogs. However, the concurrent reduction in ATTD of all major nutrients suggests that a lower inclusion rate of rye may be advisable to avoid compromising digestibility. The small sample size (n=6), lack of randomization, and potential confounding from seasonal temperature variation and coprophagia are important limitations. Further studies with larger sample sizes and randomized designs are needed to confirm these findings and determine optimal rye inclusion levels for canine diets.