What our six-month feeding study found about fresh-frozen food, stool quality, and gut health in dogs, results of our AAFCO feeding trial

ABSTRACT

The pet food market now offers a wide range of diets, with fresh-frozen options gaining popularity for their high-quality ingredients and digestibility. While research on their long-term benefits is still emerging, they are widely valued for their freshness and nutritional quality. This ongoing study with 16 dogs is evaluating how a commercially available fresh-frozen diet (FF) compares to a commercial dry extruded kibble (KB) in terms of overall health, fecal parameters, and activity levels. Adult dogs were randomly assigned to either diet and fed at maintenance levels. Study endpoints include fresh fecal sampling at the beginning, middle, and end to measure moisture and pH. Fecal consistency scores and food intake were recorded daily, while body weight and body condition scores (BCS) were tracked weekly. Total activity levels were assessed monthly using accelerometer-equipped collars. Fecal microbiome profiles were assessed at baseline and after one month. Data collected through week 12 have been analyzed using a repeated measures mixed model. By day 91, CBC and chemistry values remained within normal reference ranges. 

Body weights and BCS did not differ by treatment, time, or treatment × time interaction (P ≥ 0.11). Fecal quality scores were significantly greater in the FF diet compared to KB (P < 0.01), but there were no interactions by week (P = 0.13). Analysis of the first 10 days of the study (diet transition period) revealed a significant treatment × time interaction (P < 0.01), with FF fecal scores surpassing KB by day 8. Fecal moisture and pH were not affected by treatment, time, or treatment × time interaction after one and 3 months, respectively (P ≥ 0.13). Daily activity (total, inside, or outside) was not affected by treatment or treatment × time interaction (P ≥ 0.31). Inside activity increased in the second month, as they were kept inside for the majority of the day due to inclement weather (P < 0.01). 

After one month, the microbiomes of both groups diverged, indicating a dietary impact, but no clear evidence favored one diet over the other yet. The fresh-frozen diet tested here generated rapid and sustained improvements in fecal consistency, a desirable trait for many pet owners.Fresh-frozen dog diets are becoming increasingly popular among pet owners, but little is known regarding the impact of this diet on canine gut health and the microbiome. This study evaluated the impacts of a fresh-frozen versus an extruded kibble diet on the fecal consistency, metabolites, and microbiome in 16 adult Labrador Retrievers (average age 4.2 ± 0.26 years; average weight: 25.7 kg ± 3.18 kg) over the course of 6 months. Dogs were randomly assigned to one of the two diets in a parallel design. Feces were collected at baseline, 3 months, and 6 months for short-chain fatty acid, ammonia, and microbiome analyses. An additional sample was collected at 1 month for microbiome analysis. Fecal microbiome was analyzed via 16S rRNA gene sequencing. Fecal consistency scores were recorded daily, and body weights and body condition scores were recorded weekly. Data were analyzed across time and treatments using a repeated measures ANOVA. Results showed that fecal consistency was improved in the dogs fed the fresh diet, with differential changes detected shortly after the transition phase onto the test diets (P < 0.01). Fecal butyrate concentrations were greater in the fresh group (P < 0.01), and fecal valerate concentrations fluctuated more in the kibble group while the fresh group remained more consistent over time (P = 0.05). No differences were observed in fecal moisture, pH, ammonia, or other fecal short-chain or branched-chain fatty acids. General dog health was not negatively impacted by either diet, as observed through weight maintenance and veterinary wellness exams. Diet type impacted fecal microbiome composition, as shown by differences in beta diversity between the fresh and kibble groups (P < 0.01). Additionally, microbiomes of dogs fed a fresh diet were enriched in short-chain fatty acid producers such as Turicibacter sanguinis, Ruminococcus, and Clostridium compared to dogs fed kibble. Overall, the fresh diet increased fecal consistency and fecal butyrate, though other fecal parameters were not different.

Two diets, six months, 16 dogs: How the study was designed

Sixteen adult Labrador Retrievers (8 male and 8 female, mean age 4.2 years, mean weight 25.7 kg) were randomly assigned to one of two diets for 184 days. Eight dogs were fed Ollie's fresh-frozen Chicken Dish with Carrots, and eight were fed Blue Buffalo Life Protection Chicken and Brown Rice Recipe, a widely available dry extruded kibble. Before the trial began, all dogs ate a standard kennel diet.

Both diets were fed at maintenance levels, and researchers monitored body weight, body condition, skin and coat quality, bloodwork, stool quality, and more. The study was conducted at Four Rivers Kennel in Missouri under AAFCO feeding trial protocols, the framework used to substantiate that a pet food is complete and balanced. The research team included veterinarians and PhD researchers from both Four Rivers and Ollie. To see what was happening inside the gut, researchers analyzed frozen stool samples using gas chromatography-mass spectrometry (GC-MS) to measure short-chain fatty acids, and full-length 16S rRNA gene sequencing (PacBio Sequel IIe) to identify gut bacteria. Microbiome differences were evaluated using PERMANOVA and LinDA differential abundance analysis.

The study results at a glance

• Stool consistency (mean score): 3.73 for fresh-frozen vs. 2.71 for kibble. This was significantly better for the fresh-frozen group (P < 0.01).

• Fecal butyrate, overall (µmol/g DM): 72.94 for fresh-frozen vs. 48.74 for kibble. This was significantly higher for the fresh-frozen group (P < 0.01).

• Fecal butyrate at month 6 (µmol/g DM): 92.30 for fresh-frozen vs. 49.30 for kibble. This was the highest level recorded in the fresh-frozen group.

• Fecal moisture, pH, and ammonia: Comparable between diets, with no significant difference.

• Acetate, propionate, and isovalerate: Comparable between diets, with no significant difference.

• Microbial diversity (alpha): Increased compared with the kennel diet in both groups, with no significant difference between the two diets.

• Microbial community (beta): Distinct for each diet, and the communities differed by diet from month 1 through month 6 (P < 0.01).

• Body weight and body condition: Maintained on both diets. Both diets supported healthy weight and condition.

How fresh-frozen food affects stool consistency

Dogs fed Ollie's fresh-frozen diet had significantly better stool consistency than dogs fed kibble, with a mean score of 3.73 compared to 2.71 (P < 0.01). Stools were scored on a 5-point scale, with higher scores reflecting firmer, better-formed stools that are easier to pick up. The difference showed up fast. Stool scores between the two groups began to separate within the first 10 days of the diet transition and held throughout the trial. Over the six months, the kibble group's scores were more variable, with more frequent loose stools. Stool consistency is one of the most practical, everyday signals of digestive health. Loose, inconsistent, or hard-to-pass stools can indicate that a diet isn't agreeing with a dog's digestion, while firm, consistent stools suggest the digestive system is handling food well. Researchers also measured fecal moisture, pH, and ammonia. These were comparable between the two groups, with no significant differences in moisture (P ≥ 0.35), pH (P ≥ 0.13), or ammonia (P ≥ 0.17).

Further findings from the gut, higher butyrate and steadier short-chain fatty acids

Stool quality is the most visible result of the study, but researchers also looked at what was happening inside the gut, measuring the fatty acids gut bacteria produce and sequencing the bacteria themselves.

Gut bacteria produce short-chain fatty acids (SCFAs) when they ferment parts of a dog's food. Butyrate, also called butyric acid, is one of the most important. It's the main energy source for the cells that line the colon, and it helps support the intestinal lining and regulate gut inflammation. Dogs on the fresh-frozen diet had significantly higher fecal butyrate overall than dogs on kibble (72.94 vs. 48.74 µmol/g dry matter, P < 0.01), and the gap grew over time. By month 6, butyrate in the fresh-frozen group reached 92.30 µmol/g, compared to 49.30 in the kibble group. Valerate, another fatty acid associated with gut balance, showed a significant diet-by-time interaction (P = 0.05). Levels stayed steady in the fresh-frozen group and fluctuated in the kibble group over the study. Other SCFAs and branched-chain fatty acids, including acetate, propionate, isobutyrate, and isovalerate, did not differ significantly between the two diets.

A distinct microbiome on each diet

Moving off the standard kennel diet increased overall gut microbial diversity (alpha diversity) in both groups (P < 0.05), and the fresh-frozen and kibble groups did not differ on this measure (P > 0.05). However, the makeup of each group's gut bacteria did differ. The overall microbial community (beta diversity) was significantly different between diets from month 1 through month 6 (P < 0.01), meaning each diet cultivated its own distinct microbial fingerprint.

Dogs fed the fresh-frozen diet had higher relative abundances of SCFA-producing and protein-fermenting (proteolytic) bacteria, including Turicibacter sanguinis, Clostridium dakarense, Clostridium perfringens, Romboutsia ilealis, and Escherichia coli. Dogs fed kibble had higher relative abundances of fiber-fermenting bacteria, particularly Prevotella copri, Catenibacterium mitsuokai, and Lactobacillus species.

What these findings mean for pup parents

• Firmer, more consistent stools. Dogs fed fresh-frozen food produced firmer, better-formed stools than dogs fed kibble, and the difference appeared within the first 10 days of switching.

• Higher butyrate. Fresh-frozen food significantly increased butyrate, a fatty acid that fuels colon cells and supports the intestinal lining.

• A different, not lesser, microbiome on each diet. Both diets increased gut diversity compared to a standard kennel diet, and each supported its own community of beneficial bacteria.

• Both diets maintained health. Dogs on both diets kept a healthy weight, body condition, and overall physical health for the full six months.

Makenzie C Melby, Claire L Timlin, Fiona Burmeister McCracken, Connie A Rojas, Catherine Carchedi, Brittany White, Lindsay Cassibry, Amanda Blake, Craig N Coon, Impacts of a fresh-frozen dog food on fecal consistency and microbiome in colony dogs, Journal of Animal Science, 2026; skag303, https://doi.org/10.1093/jas/skag303