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Journal of Animal Science logoLink to Journal of Animal Science
. 2022 Aug 15;100(10):skac265. doi: 10.1093/jas/skac265

Effects of a mildly cooked human-grade dog diet on gene expression, skin and coat health measures, and fecal microbiota of healthy adult dogs

Elizabeth L Geary 1, Patrícia M Oba 2, Catherine C Applegate 3,4,5, Lindsay V Clark 6, Christopher J Fields 7, Kelly S Swanson 8,9,10,
PMCID: PMC9527297  PMID: 35965387

Abstract

Purported benefits of human-grade pet foods include reduced inflammation, enhanced coat quality, and improved gut health, but research is scarce. Therefore, we compared gene expression, skin and coat health measures, and the fecal microbiome of dogs consuming a mildly cooked human-grade or extruded kibble diet. Twenty beagles (BW = 10.25 ± 0.82 kg; age = 3.85 ± 1.84 yr) were used in a completely randomized design. Test diets included: 1) chicken and brown rice recipe [feed-grade; extruded; blue buffalo (BB)]; and 2) chicken and white rice [human-grade; mildly cooked; Just Food for Dogs (JFFD)]. The study consisted of a 4-week baseline when all dogs ate BB, and a 12-week treatment phase when dogs were randomized to either diet (n = 10/group). After the baseline and treatment phases, fresh fecal samples were scored and collected for pH, dry matter (DM), and microbiome analysis; blood samples were collected for gene expression analysis; hair samples were microscopically imaged; and skin was analyzed for delayed-type hypersensitivity (DTH), sebum concentration, hydration status, and transepidermal water loss (TEWL). Data were analyzed as a change from baseline (CFB) using the Mixed Models procedure of SAS (version 9.4). At baseline, fecal pH was higher (P < 0.05) and hair surface score, superoxide dismutase (SOD) expression, and tumor necrosis factor-α (TNF-α) expression was lower (P < 0.05) in dogs allotted to JFFD. The decrease in CFB fecal pH and DM was greater (P < 0.05) in dogs fed JFFD, but fecal scores were not different. The increase in CFB hair surface score was higher (P < 0.05) in dogs fed JFFD. The decrease in CFB TEWL (back region) was greater (P < 0.05) in dogs fed JFFD, but TEWL (inguinal and ear regions), hydration status, and sebum concentrations in all regions were not different. Hair cortex scores and DTH responses were not affected by diet. The increase in CFB gene expression of SOD, COX-2, and TNF-α was greater (P < 0.05) in dogs fed JFFD. PCoA plots based on Bray–Curtis distances of bacterial genera and species showed small shifts over time in dogs fed BB, but dramatic shifts in those fed JFFD. JFFD increased (adj. P < 0.05) relative abundances of 4 bacterial genera, 11 bacterial species, 68 KEGG pathways, and 167 MetaCyc pathways, and decreased (adj. P < 0.05) 16 genera, 25 species, 98 KEGG pathways, and 87 MetaCyc pathways. In conclusion, the JFFD diet dramatically shifted the fecal microbiome but had minor effects on skin and coat measures and gene expression.

Keywords: canine nutrition, fecal microbiome, skin and coat health


The current study expanded the research on mildly cooked human-grade dog diets, demonstrating that they dramatically alter the fecal microbiome without changing the fecal scores of healthy adult dogs. While minor differences in blood gene expression and skin and coat health measures were observed after 12 weeks of feeding, our results suggest that improvements would require a longer period of study or testing in animals with lower health status.

Introduction

With many dog owners considering their pets to be a family member instead of just a companion, the pet industry continues to expand. Owners are focused on providing a high quality of care for their pets and keeping their pets as healthy as possible. Therefore, many owners are starting to critically think about what they are feeding their dogs. Following recent human trends, many pet owners are moving away from more processed foods, such as kibble, and are turning to fresh and mildly cooked alternatives. Human-grade pet foods are also on the rise because of the notion that their dogs should eat food that was made with the same standards as human food. AAFCO (2022) defines human-grade as whole products with every ingredient being processed, handled, stored, and transported in accordance with the regulations for current good manufacturing practices for human foods as dictated in 21 CFR part 117. The products must be labeled with the intended use and species using the same size or larger font than the human-grade label. Because the entire product must be human-grade, the term human-grade cannot be used in the ingredient list.

Human-grade mildly cooked pet foods have been shown to have a higher apparent total tract digestibility of dry matter, organic matter, energy, and acid-hydrolyzed fat than a kibble diet and other fresh diets (Do et al., 2021). Dogs fed human-grade pet foods also had higher apparent total tract digestibility of crude protein, required lower caloric intake (kcal/d), and had lower fecal dry matter (DM) and as-is fecal output than those fed a kibble diet. Because they were more digestible, that study showed that human-grade pet foods also require a lower daily DM food intake than kibble or fresh diets. Finally, dogs eating the human-grade diets had fecal microbial communities that shifted from those fed the extruded and fresh diets. In addition to the ones listed above, human-grade dog diets have many other purported, but unsubstantiated, benefits such as increased physical activity, stronger immunity, reduced inflammation, improved skin and coat health, and a longer lifespan.

Given the paucity of research on this category of dog food, the objective of this study was to determine whole blood gene expression, skin and coat health measures, and the fecal microbiome of healthy adult dogs consuming a human-grade or an extruded kibble diet. We hypothesized that dogs consuming the human-grade dog diet would have reduced whole blood expression of genes associated with inflammation and oxidative stress, improved skin and coat health, and beneficial shifts to the fecal microbiome compared with those consuming the extruded kibble diet because the human-grade pet food is less processed than the extruded diet.

Materials and Methods

All animal care procedures were approved by the University of Illinois Urbana-Champaign Institutional Animal Care and Use Committee prior to the start of the experiment (IACUC #20109).

Animals and housing

Twenty healthy (8 spayed; 12 intact) female adult beagles (BW = 10.25 ± 0.82 kg; age = 3.85 ± 1.84 yr) were used in a completely randomized design (sample size of n = 10 determined by previous lab research). Dogs were housed in two environmentally (temperature and light) controlled rooms in an animal facility at the University of Illinois at Urbana-Champaign. Dogs were housed individually in runs (approximately 1.2 m wide × 2.4 m long) for the duration of the study. A 12 h light:12 h dark schedule, with lights being on from 7 am to 7 pm, was used. Dogs had free access to fresh water at all times. They were fed twice a day (9 a.m. and 4 p.m.) to maintain BW. The amount of food offered was based on previous feeding records and the estimated caloric content of the diets given by the manufacturers. Daily food intake was recorded. Dogs had access to toys at all times and were socialized at least two times per week when they were given other toys, further enrichment, and socialization with each other and humans.

Bathing schedule

Because skin and coat measures were taken, dogs were maintained on a tightly controlled bathing schedule. Dogs were bathed every other week with Oster Oatmeal Essentials Extra Soothing shampoo (country apple variety) (Sunbeam Products, Boca Raton, FL). Dogs were first rinsed off so that their entire body except for their head and upper neck was wet. A tablespoon of shampoo was measured out for each dog and approximately 3/4 was applied to the neck, upper back, legs, and tail. The remaining shampoo was applied to the chest, belly, and inguinal area. The shampoo was rubbed in until it was bubbly. Dogs were then completely rinsed until no soap remained and dried with a towel. During bathing, dogs had their ears cleaned with Virbac Epiotic Advanced Ear Cleaner (Virbac AH, Fort Worth, TX). One squeeze of the ear cleaner was applied to each ear, massaged in, and cleaned out with cotton balls. During bathing, dogs also had their nails trimmed. Dogs were brushed three times a week on non-bathing weeks and twice per week on bathing weeks. They were brushed with an EPI slicker brush all over their back and sides until there was not an excess of loose fur coming off of their coats.

Experimental timeline and diets

The study consisted of three main phases, including a baseline phase (4 wk), a diet adaptation phase (1 wk), and a treatment phase (12 wk). During the baseline phase, all dogs consumed an extruded kibble diet (BB: Life Protection Formula Chicken and Brown Rice Recipe; Blue Buffalo, Wilton, CT). After the baseline phase, fresh fecal, blood, and hair samples were collected, and skin measurements were conducted. Dogs were then randomly allotted to one of the two test diets [BB or JFFD: Chicken and White Rice; Just Food for Dogs, Irvine, CA] (Table 1). To avoid gastrointestinal distress from the vastly different diet formats, dogs were transitioned to diets over 7 d (days 1–2: 75% kcal from prior dietary treatment + 25% kcal from new dietary treatment; d 3–4: 50% kcal from prior dietary treatment + 50% kcal from new dietary treatment; d 5–6: 25% kcal from prior dietary treatment + 75% kcal from new dietary treatment; d 7: 100% kcal from new dietary treatment). Both dietary treatments tested were commercial diets formulated to meet all AAFCO (2020) nutrient recommendations for adult dogs at maintenance (Table 1). After the transition phase, dogs were fed their diet for 12 wk. At the end of the treatment phase, fresh fecal, blood, and hair samples were collected, and skin measurements were conducted. Throughout the study, dogs were weighed and body condition scores (BCS) were assessed using a 9-point scale (Laflamme, 1997) once a week prior to the morning feeding.

Table 1.

Chemical analysis of experimental diets1 tested

Item BB4 JFFD5
Dry matter, % 91.45 22.62
------ DM basis ------
Crude protein, % 26.96 34.74
Acid-hydrolyzed fat, % 17.16 15.27
Total dietary fiber, % 12.43 11.42
 Insoluble fiber, % 8.23 5.64
 Soluble fiber, % 4.20 5.78
Ash, % 7.08 6.93
Nitrogen-free extract, % 36.37 31.64
Gross energy, kcal/kg 5152 5150
Metabolizable energy2, kcal/kg 4078 4029
Metabolizable energy3, kcal/kg 3675 3621

BB, Blue Buffalo (Wilton, CT); JFFD, Just Food for Dogs (Irvine, CA).

Metabolizable energy estimated using Atwater factors (4 kcal/g for protein and nitrogen-free extract; 9 kcal/g for fat).

Metabolizable energy estimated using modified Atwater factors (3.5 kcal/g for protein and nitrogen-free extract; 8.5 kcal/g for fat).

Extruded diet: deboned chicken, chicken meal, brown rice, barley, oatmeal, pea starch, flaxseed (source of omega 3 and 6 fatty acids), chicken fat (preserved with mixed tocopherols), dried tomato pomace, natural flavor, peas, pea protein, salt, potassium chloride, dehydrated alfalfa meal, potatoes, dried chicory root, pea fiber, alfalfa nutrient concentrate, calcium carbonate, choline chloride, dl-methionine, preserved with mixed tocopherols, dicalcium phosphate, sweet potatoes, carrots, garlic, zinc amino acid chelate, zinc sulfate, vegetable juice for color, ferrous sulfate, vitamin E supplement, iron amino acid chelate, blueberries, cranberries, barley grass, parsley, turmeric, dried kelp, Yucca Schidigera extract, niacin (vitamin B3), glucosamine hydrochloride, calcium pantothenate (vitamin B5), copper sulfate, biotin (vitamin B7), L-ascorbyl-2-polyphosphate (source of vitamin C), L-lysine, L-carnitine, vitamin A supplement, copper amino acid chelate, manganese sulfate, taurine, manganese amino acid chelate, thiamine mononitrate (Vitamin B1), riboflavin (vitamin B2), vitamin D3 supplement, vitamin B12 supplement, pyridoxine hydrochloride (vitamin B6), calcium iodate, dried yeast, dried Enterococcus faecium fermentation product, dried Lactobacillus acidophilus fermentation product, dried Aspergillus niger fermentation extract, dried Trichoderma longibrachiatum fermentation extract, dried Bacillus subtilis fermentation extract, folic acid (vitamin B9), sodium selenite, oil of rosemary.

Mildly cooked human-grade diet: chicken thigh, long grain white rice, spinach, carrots, apples, chicken gizzard, chicken liver, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), dicalcium phosphate dihydrate, calcium, sodium chloride, choline bitartrate, dried seaweed meal, zinc oxide, magnesium amino acid chelate, vitamin E (as a-tocopherol succinate), ferrous amino acid chelate, copper amino acid chelate, vitamin D3 (as cholecalciferol), vitamin B5 (as calcium d-pantothenate), riboflavin, vitamin B12 (as cyanocobalamin).

Fecal scoring, sample collection, and analysis

Fresh fecal samples were collected at the end of the baseline and treatment phases. All fecal samples collected were scored according to the following scale: 1: hard, dry pellets, small hard mass; 2: hard, formed, dry stool, remains firm and soft; 3: soft, formed, and moist stool, retains shape; 4: soft, unformed stool, assumes the shape of the container; and 5: watery, liquid that can be poured. Fresh feces (within 15 min of defecation) were collected for measurement of pH, DM content, and microbiome analysis. Fecal pH was measured immediately using an AP10 pH meter (Denver Instrument, Bohemia, NY) equipped with a Beckman Electrode (Beckman Instruments Inc., Fullerton, CA), and then aliquots were collected. One aliquot was used for fresh fecal DM determination (2 pre-weighed pans/dog; 1–2 g/pan). Fecal DM content was measured according to AOAC (AOAC, 2006) using a 105 °C oven. Four aliquots of fresh feces were collected for microbiome analysis. These samples were immediately transferred to sterile cryogenic vials (Nalgene, Rochester, NY), quickly frozen in dry ice, and stored at −80oC until they were analyzed.

Fecal microbiome analysis

Fecal bacterial DNA was extracted according to the manufacturer’s instructions using the MO BIO PowerSoil Kit (MO BIO Laboratories, Carlsbad, CA) with bead beating using a vortex adaptor, followed by quantification of extracted DNA using a Qubit® 3.0 Fluorometer (Life Technologies, Grand Island, NY). Shotgun sequencing analysis was conducted using an Illumina NovaSeq (Illumina Inc., San Diego, CA), generating sequences with a length of 150 base pairs. Microbiome analysis was conducted in collaboration with the HPCbio Bioinformatics Unit of the Roy J. Carver Biotechnology Center at the University of Illinois at Urbana-Champaign. First, host DNA was removed using kneadData. The remaining 150-base pair reads were classified using HUMAnN3 v3a5, with UniRef50 as the reference database. For MetaCyc community pathway (abundance of pathways across all taxa), genus, species, MetaCyc pathway × species, KEGG community pathway, and KEGG pathway × species, abundances were transformed to proportions, and the Bray–Curtis distance was calculated using the vegan R package. In addition, weighted and unweighted UniFrac distances were calculated for species using the rbiom package and the CHOCOPhlAn species tree. Taxa abundances were calculated by MetaPhlan, and pathway abundances (community and by species) were calculated by HUMAnN. Principal coordinates analysis (PCoA) was performed using the cmdscale function in R. MaAsLin2 was used to detect the differential abundance of pathways and taxa. Default parameters were used, including filtering features that were present in less than 10% of samples. The human_regroup_table command was used with the -groups uniref50_ko to quantify KEGG orthologs. Mappings between KEGG orthologs and KEGG pathways were obtained using the getGeneKEGGLinks function from the limma1 R package and exported to HUMAnN format. The humann_regroup_table was used a second time to quantify KEGG pathways from KEGG orthologs. Treatment × time interactions were tested and deemed significantly different when the FDR-corrected P-value (q-value) < 0.05.

Blood sample collection and analysis

At the end of the baseline and treatment phases, blood (approximately 11–15 mL) was collected via jugular or cephalic puncture using 20–22 gauge needles. Dogs were fasted for at least 12 h overnight prior to the blood collection but had free access to water. Blood was collected for serum metabolite concentrations, complete blood count (CBC), and gene expression analyses. Samples were immediately transferred to appropriate vacutainer tubes, with 0.5 mL going into #367841 BD Vacutainer Plus plastic whole blood tubes (Lavender with K2EDTA additive), 3–5 mL going into #367974 BD Vacutainer Plus plastic serum tubes (red/grey with clot activator and gel for serum separation; BD, Franklin Lakes, NJ), and 7.5 mL of blood going into PAXgene Blood Tubes (#762165; Qiagen, Valencia, CA). Blood tubes for serum isolation were centrifuged at 1,300 × g at 4 °C for 10 min (Beckman CS-6R centrifuge; Beckman Coulter Inc., Brea, CA). The serum was transported to the University of Illinois Veterinary Medicine Diagnostics Laboratory for serum chemistry analysis. K2EDTA tubes were cooled (but not frozen) and transported to the University of Illinois Veterinary Medicine Diagnostics Laboratory for CBC analyses.

Total RNA from blood cells were isolated using a PAXgene Blood RNA Kits (#762331; Qiagen, Valencia, CA, USA). Tubes were stored at −20 °C until RNA extraction. RNA concentrations were determined using an ND-1000 spectrophotometer (Nanodrop Technologies, Wilmington, DE). cDNA was synthesized using SuperScript IV reverse transcriptase (Invitrogen, Carlsbad, CA, USA). Gene expression was measured by real-time two-step RT-PCR using an Applied Biosystems 7900HT real-time PCR system (Applied Biosystems, Waltham, MA) and was carried out with SYBR Green chemistry (Bio-Rad Laboratories, Hercules, CA) in a QuantStudio 7 instrument (Thermo Fisher Scientific, Waltham, MA) using validated forward and reverse primers (Bio-Rad Laboratories, Hercules, CA). The genes of interest included glutathione peroxidase (GPX, UniqueAssayID: qCfaCED0030791), superoxide dismutase (SOD, UniqueAssayID: qCfaCED0038911), cyclooxygenase-2 (COX-2, UniqueAssayID: qCfaCED0024663), myeloperoxidase (MPO, UniqueAssayID: qCfaCID0034597), glutathione reductase (GSR, UniqueAssayID: qCfaCED0031064), catalase (CAT, UniqueAssayID: qCfaCED0028561), tumor necrosis factor-alpha (TNF-α, UniqueAssayID: qCfaCED0030703), chemokine C-C ligand-2 (CCL-2, UniqueAssayID: qCfaCED0031550), interferon-gamma (IFN-γ, UniqueAssayID: qCfaCID0023928), interleukin-1 beta (IL-1β, UniqueAssayID: qCfaCED0025591), and interleukin-6 (IL-6, UniqueAssayID: qCfaCID0020842). All gene expression data were analyzed using the 2−∆∆Ct method, represented as gene expression relative to the housekeeping gene ribosomal protein S5 (RPS5, UniqueAssayID: qCfaCED0028510) (Brinkhof et al., 2006).

Hair collection and analysis

At the end of the baseline and treatment phases, a 2″ square of hair, from a spot directly left of the spine and in between the ribs and hips, was clipped and placed into Whirl Pak bags (Nasco, Fort Atkinson, WI) and stored at −20 oC until analysis. Hair was imaged at the Beckman Institute for Advanced Science and Technology using a scanning electron microscope (SEM; FEI Quanta FEG 450 ESEM; FEI Company, Hillsboro, OR). At least five hairs per dog were cut at an approximately 45° angle and fixated by carbon tape to a SEM specimen stub on top of a 2.5 cm or 1.5 round metal SEM specimen mount disk. These disks received a gold and palladium sputter coating and were inserted into the microscope for photographic surveying. Hairs were scored by three researchers that were blinded to treatment using a grading system similar to that established by Kim et al. (2010) to assess damage to the hair through observation of the hair surface and cortex. The following scale was used to assess hair surface damage: 0: intact hair, regular overlay of the cuticle; 1: irregular overlay of the cuticle; 2: lift-up of the cuticle; 3: severe lift-up of the cuticle, or over half scale size gone; 4: partial presence of cuticle. The following scale was used to assess hair cortex damage: 0: intact, thick, and dense cortex, regular overlay of the cortex; 1: thick and dense cortex, with minor damage, presence of a crack; 2: thick cortex, with moderate damage, presence of a crack; 3: thick cortex, with severe damage, presence of a crack and hole; 4: thin cortex, with severe damage, presence of a crack, blurry separation between cortex and medulla; and 5: very thin cortex, with severe damage, presence of a crack, loss of distinction between cortex and medulla.

Skin and coat condition

At the end of the baseline and treatment phases, skin and coat were scored by three blinded researchers according to Rees et al. (2001). The scale for hair scoring was: 1: dull, coarse, dry; 2: poorly reflective, non-soft; 3: medium reflective, medium soft; 4: highly reflective, very soft; 5: greasy. The scale for skin scoring was: 1: dry; 2: slightly dry; 3: normal; 4: slightly greasy; and 5: greasy. After skin and coat scoring was done, dogs were sedated by an intramuscular injection of a combination of butorphanol (Torbugesic; 0.2 mg/kg BW) (Zoetis Manufacturing and Research, Girona, Spain) and dexmedetomidine (0.02 mg/kg BW) (manufactured by Orion Corporation, Espoo, Finland; distributed by Zoetis Inc., Kalamazoo, MI) so that transepidermal water loss [TEWL; Tewameter TM 300 MDD (Courage + Khazaka Electronic GmbH, Cologne, Germany)], hydration status [Corneometer CM 825 (Courage + Khazaka Electronic GmbH, Cologne, Germany)], and sebum concentrations [external Sebumeter SM 815 (Courage + Khazaka Electronic GmbH, Cologne, Germany)], and delayed-type hypersensitivity (DTH) response could be measured. All skin and coat measurements were conducted in the same environmentally controlled room for the baseline and treatment periods.

Delayed-type hypersensitivity (DTH)

DTH response was tested as described by Kim et al. (2000). Briefly, dogs were injected intradermally in the flank area with 100 μL of saline (8.5 mg/mL; functions as control), an attenuated vaccine (Duramune Max 5 Dog Vaccine; Elanco Animal Health, Greenfield, IN; functions as specific antigen), and phytohemagglutinin (PHA; 0.5 mg/mL) and concanavalin A (ConA; 0.5 mg/mL), which both function as non-specific antigens. Three replicates (located in the dorsal, ventral, or central side of the dog) of each of the four different antigens or saline were injected for a total of twelve injections per dog. The injection site was clipped and wiped with 70% ethyl alcohol before injections. After skin measurements and DTH injections were completed, an injection of the reversal agent for dexmedetomidine, atipamezole (0.2 mg/kg BW) (manufactured by Orion Corporation, Espoo, Finland; distributed by Zoetis Inc., Kalamazoo, MI), was administered intramuscularly. Skin inflammation was measured using a digital caliper and reported as the average wheal diameter [(horizontal diameter + vertical diameter)/2] of the inflamed skin surrounding the injection site. Measurements were taken at baseline, 15, 30, 45, and 60 min after injection, and 24, 48, and 72 h after injection.

Diet chemical analyses

The JFFD diet was first dried at 55 °C in a forced-air oven. Both diets were then ground in a Wiley mill (model 4, Thomas Scientific, Swedesboro, NJ) through a 2-mm screen and then analyzed for DM and ash according to AOAC (2006; methods 934.01 and 942.05), with OM being calculated. Crude protein was calculated from Leco (FP2000 and TruMac) total nitrogen values according to AOAC (2006; method 992.15). Total lipid content (acid-hydrolyzed fat) was determined according to the methods of the American Association of Cereal Chemists (AACC, 1983) and Budde (1952). Total dietary fiber was determined according to Prosky et al. (1992). Gross energy was measured using an oxygen bomb calorimeter (model 6200, Parr Instruments, Moline, IL).

Statistical analyses

Data were analyzed using the Mixed Models procedure of SAS (version 9.4, SAS Institute, Inc., Cary, NC). The fixed effect of treatment was tested and a dog was considered a random effect. Data were tested for normality using the UNIVARIATE procedure of SAS. Changes from baseline differences between treatments were determined using a Fisher-protected least significant difference with a Tukey adjustment to control for experiment-wise error. A probability of P < 0.05 was accepted as statistically significant. Reported pooled standard errors of the mean were determined according to the Mixed Models procedure of SAS.

Results

At baseline, food intake, BW, BCS, fecal scores, and fecal DM % were not different between groups (Table 2). Fecal pH, however, was higher (P < 0.05) in dogs allotted to JFFD than those allotted to BB at baseline. BW and BCS did not change over time, but change from baseline food intake on a DM basis was lower (P < 0.0001), while the change from baseline food intake on an as-is basis was higher (P < 0.0001) in dogs fed JFFD than those fed BB (Table 3). This resulted in a lower (P < 0.0001) food intake (g/d DM):body weight (kg) ratio in dogs fed JFFD vs. those fed BB. The dogs eating JFFD had a lower (P < 0.0001) change from baseline caloric intake than the dogs eating BB. Changes from baseline fecal scores were not different between diets, but changes from baseline fecal pH and fecal DM % were lower (P < 0.001) in dogs fed JFFD than in those fed BB.

Table 2.

Baseline food and caloric intake, body condition scores, body weight, and fecal characteristics of healthy adult dogs consuming a kibble diet1

Item BB baseline2 JFFD baseline2 P-value
Food intake, g/day [dry matter (DM)] 143.47 ± 3.77 159.45 ± 7.95 0.0860
Food intake, g/day (as-is) 154.40 ± 4.05 171.60 ± 8.56 0.0860
Body condition score3 5.43 ± 0.16 5.12 ± 0.17 0.1355
Body weight, kg 10.44 ± 0.22 10.05 ± 0.29 0.2961
Food intake (g/d DM)/body weight (kg) 13.80 ± 0.48 16.08 ± 1.11 0.0751
Caloric intake, kcal/day4 585.00 ± 15.36 650.17 ± 32.42 0.0860
Caloric intake, kcal/day5 527.27 ± 13.84 586.00 ± 29.22 0.0860
Caloric intake (kcal)/body weight (kg)4 56.34 ± 1.95 65.61 ± 4.50 0.0749
Caloric intake (kcal)/body weight (kg)5 50.78 ± 1.76 59.13 ± 4.05 0.0749
Fecal score6 2.70 ± 0.19 2.45 ± 0.14 0.2978
Fecal pH 5.77 ± 0.09b 6.08 ± 0.11a 0.0496
Fecal DM, % 31.61 ± 0.94 31.77 ± 0.68 0.8953

BB, Blue Buffalo (Wilton, CT); JFFD, Just Food for Dogs (Irvine, CA); all dogs consumed BB at baseline and then were allotted to BB or JFFD after baseline measurements.

Mean ± standard error.

9-point scale (Laflamme, 1997).

Metabolizable energy estimated with Atwater factors.

Metabolizable energy estimated with modified Atwater factors.

Fecal scores: 1 = hard, dry pellets, small hard mass; 2 = hard, formed, dry stool, remains firm and soft; 3 = soft, formed, and moist stool; retains shape; 4 = soft, unformed stool, assumed shape of container; 5 = watery, liquid that can be poured.

Table 3.

Change from baseline food intake, body condition scores, body weight, and fecal characteristics of healthy adult dogs consuming a kibble or mildly cooked human-grade diet1

Item ∆BB2 ∆JFFD2 P-value
Food intake, g/day [dry matter (DM)] 14.26 ± 3.69a −36.69 ± 3.52b <0.0001
Food intake, g/day (as-is) 15.35 ± 3.97b 371.10 ± 23.80a <0.0001
Body condition score3 0.04 ± 0.03 0.05 ± 0.04 0.6574
Body weight, kg −0.18 ± 0.05 −0.31 ± 0.12 0.3312
Food intake (g/d DM)/body weight (kg) 1.69 ± 0.44a −3.40 ± 0.53b <0.0001
Caloric intake, kcal/day4 58.16 ± 15.05a −155.54 ± 14.35b <0.0001
Caloric intake, kcal/day5 52.42 ± 13.57a −141.49 ± 12.94b <0.0001
Caloric intake (kcal)/body weight (kg)4 6.77 ± 1.76a −14.71 ± 2.16b <0.0001
Caloric intake (kcal)/body weight (kg)5 6.10 ± 1.59a −13.39 ± 1.95b <0.0001
Fecal score6 0.00 ± 0.27 0.55 ± 0.16 0.0822
Fecal pH 0.27 ± 0.19a −1.00 ± 0.18b 0.0001
Fecal DM, % 0.14 ± 1.22a −7.45 ± 0.91b 0.0004

BB, Blue Buffalo (Wilton, CT); JFFD, Just Food for Dogs (Irvine, CA).

Mean ± standard error.

9-point scale (Laflamme, 1997).

Metabolizable energy estimated with Atwater factors.

Metabolizable energy estimated with modified Atwater factors.

Fecal score: 1 = hard, dry pellets, small hard mass; 2 = hard, formed, dry stool, remains firm and soft; 3 = soft, formed, and moist stool; retains shape; 4 = soft, unformed stool, assumed shape of container; 5 = watery, liquid that can be poured.

None of the serum chemistry measures were different between groups at baseline and were within reference ranges, with the exception of globulin (slightly lower than reference ranges) and albumin:globulin ratio (slightly higher than reference ranges) (Supplementary Table S1). Changes from baseline serum total protein and globulin were higher (P < 0.001), while changes from baseline serum albumin:globulin ratio and phosphorus were lower (P < 0.01) in dogs fed JFFD than in those fed BB (Table 4). Change from baseline serum cholesterol was lower (P < 0.05) in dogs fed JFFD than those fed BB. All other serum chemistry measures were not different between groups. All baseline CBC were within reference ranges (Supplementary Table S2). Baseline basophil concentration and % were higher (P < 0.05) in dogs allotted to JFFD, but none of the other measures were different between groups. None of the CBC measures were affected by diet (Table 5).

Table 4.

Change from baseline serum chemistry of healthy adult dogs consuming a kibble or mildly cooked human-grade diet1

Item ∆BB2 ∆JFFD2 p-value
Creatinine, mg/dL -0.03 ± 0.03 -0.09 ± 0.04 0.2080
Blood urea nitrogen, mg/dL -1.30 ± 0.50 -1.60 ± 0.54 0.6876
Total protein, g/dL -0.16 ± 0.07b 0.31 ± 0.05a <0.0001
Albumin, g/dL -0.19 ± 0.04 -0.14 ± 0.05 0.4590
Globulin, g/dL 0.03 ± 0.06b 0.45 ± 0.07a 0.0002
Albumin: globulin ratio -0.09 ± 0.04a -0.26 ± 0.04b 0.0099
Ca, mg/dL -0.31 ± 0.09 -0.21 ± 0.07 0.3814
P, mg/dL -0.19 ± 0.13a -1.01 ± 0.17b 0.0013
Na, mmol/L -1.30 ± 0.47 -0.90 ± 0.43 0.5406
K, mmol/L -0.01 ± 0.07 0.05 ± 0.10 0.6255
Na: K ratio -0.20 ± 0.66 -0.50 ± 0.82 0.7793
Cl, mmol/L -1.00 ± 0.37 0.20 ± 0.61 0.1091
Glucose, mg/dL 0.60 ± 1.61 1.00 ± 2.48 0.8937
Alkaline phosphatase (ALP), U/L 7.80 ± 5.69 4.70 ± 3.17 0.9697
Corticosteroid-induced ALP, U/L 0.40 ± 1.51 -0.60 ± 0.22 0.5007
Alanine transaminase, U/L -1.50 ± 1.06 -2.50 ± 2.03 0.9090
Gamma glutamyltransferase, U/L 0.50 ± 0.22 0.80 ± 0.36 0.4872
Total bilirubin, mg/dL 0.00 ± 0.03 -0.01 ± 0.01 0.7545
Creatine phosphokinase, U/L -5.40 ± 9.00 -2.50 ± 14.12 0.8644
Cholesterol, mg/dL 21.00 ± 17.03a -34.80 ± 13.44b 0.0192
Triglycerides, mg/dL 6.40 ± 8.60 1.30 ± 3.48 0.4720
Bicarbonate, mmol/L 0.20 ± 0.33 -0.50 ± 0.64 0.3410
Anion gap -0.30 ± 0.26 -0.60 ± 0.58 0.6432

BB, Blue Buffalo (Wilton, CT); JFFD, Just Food for Dogs (Irvine, CA).

Mean ± standard error.

Table 5.

Change from baseline complete blood count of healthy adult dogs consuming a kibble or mildly cooked human-grade diet1

Item ∆BB2 ∆JFFD2 p-value
Red blood cells, 10^6/µL 0.27 ± 0.30 0.82 ± 0.26 0.1848
Reticulocyte count, % 0.06 ± 0.05 −0.06 ± 0.08 0.2057
A reticulocyte count,/µL 4522.22 ± 4100.43 −679.13 ± 8279.44 0.7003
Hemoglobin, g/dL 0.76 ± 0.63 1.69 ± 0.64 0.3126
Hematocrit, % 2.06 ± 1.71 4.87 ± 1.60 0.2450
Mean cell volume, fl 0.25 ± 0.49 −1.14 ± 0.45 0.0507
Mean corpuscular hemoglobin, pg 0.21 ± 0.15 −0.19 ± 0.20 0.1274
Mean corpuscular hemoglobin, g/dL 0.18 ± 0.15 0.25 ± 0.26 0.8168
Platelets, 10^3/µL 8.70 ± 16.45 40.90 ± 38.89 0.4557
Mean platelet volume, fl −0.40 ± 0.28 −0.64 ± 0.22 0.5117
White blood cell count, 10^3/µL −0.46 ± 0.21 0.05 ± 0.49 0.3445
Lymphocytes, % −4.66 ± 2.91 −1.06 ± 2.26 0.3414
Monocytes, % −0.18 ± 0.76 0.29 ± 0.53 0.6183
Eosinophils, % −0.76 ± 0.60 −0.94 ± 0.86 0.8498
Basophils, % 0.09 ± 0.06 −0.11 ± 0.10 0.1126
A Lymphocytes, 10^3/µL −0.41 ± 0.18 −0.14 ± 0.16 0.1413
A Monocytes, 10^3/µL −0.07 ± 0.06 0.03 ± 0.05 0.2600
A Eosinophils, 10^3/µL −0.07 ± 0.05 −0.06 ± 0.05 0.7329
A Basophils, 10^3/µL 0.00 ± 0.00 -0.01 ± 0.01 0.1066

BB, Blue Buffalo (Wilton, CT); JFFD, Just Food for Dogs (Irvine, CA).

Mean ± standard error.

Of the skin and coat measures, the baseline hair surface score was lower (p<0.05; less damaged) in dogs allotted to JFFD than those allotted to BB (Supplementary Table S3). None of the other skin and coat measures at baseline were different between groups. Change from baseline hair surface score was higher (P < 0.01; more damaged) in dogs fed JFFD than those fed BB (Table 6), which was likely due to the differences noted at baseline. Change from baseline TEWL in the back region was lower (P < 0.01; less water loss) in dogs fed JFFD than those fed BB. Change from baseline TEWL in other regions (inguinal; ear), hydration status, sebum concentrations, and skin and coat scores were not affected by diet. Baseline DTH response to PHA injection was lower (P < 0.01) at 48 h and 72 h in dogs allotted to JFFD than those allotted to BB (Supplementary Table S4). None of the other baseline DTH responses were different. Change from baseline DTH responses were not affected by diet (Table 7). During the final DTH measures, two of the dogs appeared to have allergic reactions to at least one of the DTH injections. Upon examination, a veterinarian administered diphenhydramine (Armas Pharmaceuticals, Freehold Township, NJ) and dexamethasone (Sparhawk Laboratories Inc., Lexena, KS). Because of this reaction, the data from these two dogs were excluded from all DTH analyses. Four other dogs had mild reactions (small red rashes on the belly, swollen faces), but were not given medicine and not excluded.

Table 6.

Change from baseline hair and skin scores, sebum concentrations, hydration status, and transepidermal water loss (TEWL) of healthy adult dogs consuming a kibble or mildly cooked human-grade diet1

Item ∆BB2 ∆JFFD2 P-value
Hair score3 0.05 ± 0.13 0.15 ± 0.12 0.6703
Skin score4 0.12 ± 0.11 0.08 ± 0.10 0.8576
Hair surface score5 −0.27 ± 0.15b 0.28 ± 0.14a 0.0048
Hair cortex score6 0.13 ± 0.20 1.30 ± 0.30 0.0701
Sebum concentration (back)7, arbitrary unit −0.33 ± 0.32 −0.07 ± 0.45 0.7371
Sebum concentration (inguinal)8, arbitrary unit −2.00 ± 0.80 −3.83 ± 1.35 0.7661
Sebum concentration (ear)9, arbitrary unit −36.40 ± 8.65 −20.77 ± 10.81 0.3650
Hydration (back)7, arbitrary unit −1.24 ± 0.61 −1.09 ± 0.82 0.7920
Hydration (inguinal)8, arbitrary unit −10.98 ± 2.65 −11.15 ± 2.33 0.9674
Hydration (ear)9, arbitrary unit −17.18 ± 3.26 −11.82 ± 3.72 0.2826
TEWL (back)7, g/h/m2 −2.88 ± 0.92a −6.98 ± 1.30b 0.0027
TEWL (inguinal)8, g/h/m2 −4.83 ± 1.06 −3.84 ± 1.03 0.5047
TEWL (ear)9, g/h/m2 −2.35 ± 0.64 −3.10 ± 0.96 0.6412

BB, Blue Buffalo (Wilton, CT); JFFD, Just Food for Dogs (Irvine, CA); all dogs consumed BB at baseline and then were allotted to BB or JFFD after baseline measurements.

Mean ± standard error.

Hair scores: 1 = dull, coarse, dry; 2 = poorly reflective, non-soft; 3 = medium reflective, medium soft; 4 = highly reflective, very soft; 5 = greasy.

Skin scores: 1 = dry; 2 = slightly dry; 3 = normal; 4 = slightly greasy; 5 = greasy.

Hair surface scores: 0 = intact hair, regular overlay of the cuticle; 1 = irregular overlay of the cuticle; 2 = lift-up of the cuticle; 3 = severe lift-up of the cuticle, or over half scale size gone; 4 = partial presence of cuticle.

Hair cortex scores: 0 = intact, thick, and dense cortex, regular overlay of the cortex; 1 = thick and dense cortex, with minor damage, presence of a crack; 2 = thick cortex, with moderate damage, presence of a crack; 3 = thick cortex, with severe damage, presence of a crack and hole; 4 = thin cortex, with severe damage, presence of a crack, blurry separation between cortex and medulla; 5 = very thin cortex, with severe damage, presence of a crack, loss of distinction between cortex and medulla.

Back measurements were taken on the right side along the spine between the rib and hip bone.

Inguinal measurements were taken on the left side.

Ear measurements were taken on the left side towards the inside of the ear.

Table 7.

Change from baseline delayed-type hypersensitivity measurements of healthy adult dogs consuming a kibble or mildly cooked human-grade diet1

Time Item ∆BB ∆JFFD p-value
15 min PHA3, mm 1.63 ± 1.22 1.36 ± 0.95 0.8671
ConA, mm 1.15 ± 0.75 2.41 ± 1.28 0.3837
Vaccine, mm 0.68 ± 0.64 1.46 ± 1.21 0.5567
30 min PHA, mm 3.05 ± 1.12 3.09 ± 1.28 0.9791
ConA, mm 0.68 ± 0.95 0.94 ± 1.04 0.8548
Vaccine, mm 0.11 ± 0.89 -0.61 ± 1.35 0.6560
45 min PHA, mm 5.01 ± 1.21 2.93 ± 1.51 0.2947
ConA, mm 2.87 ± 1.87 -0.04 ± 1.06 0.2481
Vaccine, mm 0.58 ± 1.25 -0.37 ± 1.89 0.6688
60 min PHA, mm 6.07 ± 2.29 4.11 ± 1.09 0.4866
ConA, mm 4.42 ± 2.95 -0.52 ± 1.26 0.1097
Vaccine, mm 1.16 ± 1.46 -0.84 ± 2.16 0.4411
24 h PHA, mm 5.75 ± 1.66 3.47 ± 1.89 0.3772
ConA, mm 6.74 ± 3.38 -0.74 ± 2.83 0.1202
Vaccine, mm 6.33 ± 2.55 5.73 ± 3.24 0.8861
48 h PHA, mm 0.62 ± 1.94 2.10 ± 1.13 0.5496
ConA, mm -9.38 ± 3.99 -9.15 ± 3.07 0.9652
Vaccine, mm -1.06 ± 3.28 1.80 ± 1.73 0.4841
72 h PHA, mm 1.05 ± 2.26 3.35 ± 0.99 0.4053
ConA, mm -6.48 ± 3.13 -7.24 ± 3.81 0.8789
Vaccine, mm -2.03 ± 2.95 1.88 ± 1.91 0.3085

BB, Blue Buffalo (Wilton, CT); JFFD, Just Food for Dogs (Irvine, CA).

Mean ± SE.

PHA, phytohaemagglutinin; ConA, concanavalin A; Vaccine, Duramune Max5 Dog Vaccine (Elanco Animal Health, Greenfield, IN).

Baseline blood gene expression of SOD and TNF-α were lower (P < 0.05) in dogs allotted to JFFD than those allotted to BB (Supplementary Table S5). The expression of the other genes was not different at baseline. All Ct values for IFN-γ were undetermined so the results are not reported. One dog was an extreme outlier for most of the genes at the baseline and treatment phases and was removed from all gene expression analyses. The removal did not affect any of the significant results. Change from baseline gene expression of SOD, COX-2, and TNF-α was higher (P < 0.05) in dogs fed JFFD than in those fed BB (Table 8). The change from baseline expression of all other genes was not affected by diet.

Table 8.

Change from baseline whole blood gene expression of healthy adult dogs consuming a kibble or mildly cooked human-grade diet1

Category Item ∆BB2 ∆JFFD2 p-value3
Oxidative Stress Glutathione peroxidase, 2−∆∆Ct 0.75 ± 0.66 0.14 ± 0.19 0.5242
Superoxide dismutase, 2−∆∆Ct 0.21 ± 0.24b 1.62 ± 0.61a 0.0013
Cyclooxygenase-2, 2−∆∆Ct 0.05 ± 0.13b 0.43 ± 0.13a 0.0198
Myeloperoxidase, 2−∆∆Ct 0.03 ± 0.42 0.44 ± 0.46 0.3987
Glutathione reductase, 2−∆∆Ct 0.58 ± 0.72 0.41 ± 0.41 0.8881
Catalase, 2−∆∆Ct 0.55 ± 0.40 0.59 ± 0.36 0.5340
Inflammation Tumor necrosis factor-α, 2−∆∆Ct 0.20 ± 0.14b 0.70 ± 0.13a 0.0156
Chemokine C–C ligand 2, 2−∆∆Ct 0.73 ± 0.78 0.58 ± 0.38 0.6700
Interleukin-6, 2−∆∆Ct 0.16 ± 0.13 0.07 ± 0.21 0.8511
Interleukin-1β, 2−∆∆Ct 0.03 ± 0.08 0.07 ± 0.20 0.7714

BB, Blue Buffalo (Wilton, CT); JFFD, Just Food for Dogs (Irvine, CA); all dogs consumed BB at baseline and then were allotted to BB or JFFD after baseline measurements.

Mean ± SE.

Statistics were conducted using ∆Ct values to generate P-values; data are reported as fold change in relation to a housekeeping gene (2−∆∆Ct ).

The fecal microbiome was dramatically altered by diet in this study. While microbiome profiles were similar in dogs consuming BB over time, dogs consuming JFFD for 12 wk had strong shifts. From a taxonomic view, this is clearly observed when viewing the top 15 bacterial families (Supplementary Figure S1), genera (Figure 1), and species (Supplementary Figure S2) in dogs at baseline when all were consuming BB and after 12 wk of consuming BB or JFFD. The PCoA plots based on Bray distances of bacterial genera (Figure 2) and bacterial species (Supplementary Figure S3) show some minor shifts over time in dogs fed BB, but much greater shifts in those fed JFFD. When examining specific taxonomic groups, JFFD increased the relative abundance of 4 bacterial genera and decreased the relative abundance of 16 bacterial genera (Table 9). The bacterial genera that decreased in dogs consuming JFFD included Adlercreutzia, Asaccharobacter, Enorma, Bacteroides, Prevotella, Blautia, Butyricicoccus, Catenibacterium, Dorea, Erysipelatoclostridium, Faecalibacterium, Firmicutes unclassified, Gemmiger, Holdemanella, Ileibacterium, and Peptostreptococcaceae unclassified. The genera that increased in dogs eating JFFD were Bifidobacterium, Escherichia, Helicobacter, and Lactobacillus (Table 9). Similarly, JFFD increased the relative abundance of 11 bacterial species and decreased the relative abundance of 25 bacterial species (Supplementary Table S6). Similar shifts were demonstrated when considering gene content. The PCoA plots based on Bray distance of KEGG pathways (Figure 3) and MetaCyc community pathways (Supplementary Figure S4) showed much larger shifts in dogs fed JFFD compared with those fed BB over time. When examining specific pathways, JFFD increased the relative abundance of 68 KEGG pathways and decreased the relative abundance of 98 KEGG pathways (Supplementary Table S7). Similarly, JFFD increased 167 MetaCyc pathways and decreased 87 MetaCyc pathways (Supplementary Table S8).

Figure 1.

Figure 1.

Relative abundance of the top 15 bacterial genera present in feces of dogs fed a kibble diet (BB; Blue Buffalo, Wilton, CT) at baseline (top panels) and then a kibble or mildly cooked human-grade (JFFD; Just Food For Dogs, Irvine, CA) diet after 12 wk (bottom panels).

Figure 2.

Figure 2.

Principal coordinates analysis (CoA) of fecal samples based on Bray distance of bacterial genera of dogs fed a kibble diet (BB; Blue Buffalo, Wilton, CT) at baseline (B_BB; B_JFFD) and then a kibble (T_BB) or mildly cooked human-grade (JFFD; Just Food For Dogs, Irvine, CA) diet after 12 wk (T_JFFD).

Table 9.

Fecal bacterial genera differences of healthy adult dogs consuming a mildly cooked human-grade diet as compared with a kibble diet1

Phylum Genus Coefficient SE Adj. P-value
Actinobacteria Adlercreutzia −0.5799 0.2178 0.0389
Asaccharobacter −0.5566 0.2113 0.0408
Bifidobacterium 0.5650 0.2116 0.0389
Enorma −0.5872 0.1785 0.0104
Bacteroidetes Bacteroides −0.6754 0.1743 0.0032
Prevotella −1.7187 0.2292 <0.0001
Firmicutes Blautia −0.7129 0.1310 0.0001
Butyricicoccus −1.2957 0.1794 <0.0001
Catenibacterium −1.3424 0.4043 0.0103
Dorea −0.9098 0.2407 0.0038
Erysipelatoclostridium −1.0154 0.1430 <0.0001
Faecalibacterium −0.4877 0.1644 0.0204
Firmicutes unclassified −1.5721 0.1844 <0.0001
Gemmiger −1.2685 0.1715 <0.0001
Holdemanella −0.6786 0.2098 0.0113
Ileibacterium -0.4177 0.1547 0.0389
Lactobacillus 1.7133 0.3638 0.0005
Peptostreptococcaceae unclassified −0.5487 0.1144 0.0005
Proteobacteria Escherichia 0.8439 0.2510 0.0092
Helicobacter 1.1166 0.2330 0.0005

BB, Blue Buffalo (Wilton, CT); JFFD, Just Food for Dogs (Irvine, CA); all dogs consumed BB at baseline and then were allotted to BB or JFFD after baseline measurements.

Figure 3.

Figure 3.

Principal coordinates analysis (CoA) of fecal samples based on Bray distance of KEGG pathways of dogs fed a kibble diet (BB; Blue Buffalo, Wilton, CT) at baseline (B_BB; B_JFFD) and then a kibble (T_BB) or mildly cooked human-grade (JFFD; Just Food For Dogs, Irvine, CA) diet after 12 wk (T_JFFD).

Discussion

Although there is little research on human-grade dog diets, there are several companies producing commercially available products in this category. For a variety of reasons, including mistrust of more traditional dog food formats, partly due to recalls (FDA, 2022), a desire to feed food that is more similar to human food (Nielson, 2016), and a notion that this type of diet has more health benefits than other diets, some owners are wanting to feed their dogs human-grade foods. Because of the growing popularity of this diet format, more research is warranted. The current study aimed to further the research done on human-grade pet foods (Do et al., 2021) by determining whole blood gene expression, skin and coat health measures and characterizing the fecal microbiome of healthy adult dogs consuming a human-grade dog diet.

The processing techniques, ingredient inclusion, and chemical analysis of the diets tested varied substantially. The feed-grade kibble diet was produced via extrusion. The human-grade diet was “mildly-cooked” in skillets according to hazard analysis and critical control points and US food and drug administration guidelines, with standard operating procedures for ingredient weights and cooking temperatures and times (target temperature between 76 and 82 °C for 20–40 min). From a “clean label” perspective, JFFD has a much shorter list of ingredients and contained no rendered ingredients. Although the diets had similar concentrations of fat, total dietary fiber, ash, and gross energy (on a DM basis), JFFD had higher concentrations of protein, a higher proportion of soluble fiber, and lower amounts of digestible carbohydrates. The diets had different fiber sources, with the majority of fiber in JFFD likely coming from spinach, carrots, and apples. On the other hand, the majority of fiber in BB came from brown rice, barley, oatmeal, flaxseed, tomato pomace, peas, alfalfa meal, and chicory root. Although they share the same protein source, BB contained a lot more grains (e.g., rice, barley, oatmeal), while JFFD contained fruits and vegetables (e.g., spinach, carrots, apples) and white rice. The feed-grade extruded diet also contained many live microorganisms and fermentation products (dried yeast, dried Enterococcus faecium fermentation product, dried Lactobacillus acidophilus fermentation product, dried Aspergillus niger fermentation extract, dried Trichoderma longibrachiatum fermentation extract, and dried Bacillus subtilis fermentation extract).

The different ingredient profiles and processing of diets tested likely had a large impact on nutrient digestibility, as previously reported (Do et al., 2021). These differences would have resulted in different amounts and types of substrates entering the colon, consequently affecting fecal characteristics and the microbiome. The higher digestibility of protein and energy would likely decrease the amount of protein and carbohydrates entering the colon and available for fermentation by microbes. The lower food intake (DM basis) and fecal DM% of dogs fed the human-grade diet were likely due to the higher digestibility of that diet (Do et al., 2021). Ingredient composition and lower processing likely contributed to the higher digestibility. Fecal pH is largely influenced by the amount of organic acids produced by intestinal microbiota. Higher dietary fiber inclusion levels and the proportion of soluble fiber are known to increase short-chain fatty acid (SCFA) production and lower fecal pH (Wakshlag et al., 2011). A lower pH can reduce the growth of pathogenic bacteria, decrease ammonia absorption, and decrease bile acid reabsorption, but increase mineral absorption (Hooda et al., 2012). The lower fecal pH observed in dogs fed JFFD in the current study was likely due to greater pectin concentrations with the inclusion of apples (Biagi et al., 2010). Although there was a large difference in fecal pH, stool quality was adequate for dogs fed both diets.

More hydrated skin has a higher dielectric constant and more hydrated skin is healthier and better maintains its barrier function (Fluhr et al., 1999). Generally, healthier skin has a high sebum content, but it is unhealthy to have too much sebum, causing oily skin. Transepidermal water loss is a measure of the evaporation of moisture from the skin. Although all skin has a certain level of water loss, skin with a damaged barrier has a higher water loss. Transepidermal water loss could be affected by the thickness and lipid content of the stratum corneum, the corneocyte formation, blood flow, and skin temperature (Lau-Gillard et al., 2010). The present study noted no differences in sebum concentrations or hydration levels between the two diets, but the change in TEWL was lower in the back region for dogs fed JFFD. The reduced loss in moisture could be due to the higher as-is water content of JFFD. If the dogs all drank a similar amount of water, although water intake was not measured, the dogs consuming JFFD might have consumed more total water than the dogs eating BB, which could cause the skin to reduce TEWL (Bentivegna et al., 2021). It could also be due to JFFD containing DHA and EPA, which contain omega-3 that has been shown to reduce TEWL (Parke et al., 2021).

Some prior studies have noted large day-to-day and site-to-site variation (Lau-Gillard et al., 2010) and large between-dog variability (Hester et al., 2004) with TEWL measurement, while another study had relatively low variation in the TEWL values, moderate for the hydration values, and high for sebum values (Jeong et al., 2017). A large variation in the measurement can make it challenging to detect differences due to other factors, such as diet. To attempt to reduce variability as much as possible, dogs in the current study were bathed consistently every other week, with a strict bathing protocol using the same amount of shampoo per dog and using the same shampoo throughout the entire study. Pilot testing was also performed before the current study to identify the areas of the body with the most consistent measurements. Ultimately, three areas of the body were selected (ear with no hair, inguinal with sparse hair shaved, and back with thick hair shaved) that typically gave consistent readings and also allowed for testing of three different hair statuses. Dogs were shaved 24 h before the measurements, the measurements were performed in a temperature-controlled room, and dogs were given 15–30 min to adjust to the room before measurements were taken. Despite the challenges with these measurements, skin and coat health is important to pet owners so continued efforts should be made to study this area using the methods and/or devices in this study or others that are developed and shown to be more informative.

The microbiome is influenced by many factors, including genetics, the environment, age, disease, and medical interventions, but diet is arguably the most important. Dietary factors that may influence the microbiome include the ingredients, nutrients, and processing methods (Barko et al., 2018). Diets contain distinct macronutrients, micronutrients, and phytochemicals that influence the microbiota (Compher, 2021). The addition of fiber to foods can increase the proportion of saccharolytic bacteria and decrease proteolytic bacteria, improving stool quality. The ratio of saccharolysis to proteolysis bacteria can be characterized by high levels of free saccharides and low levels of free amino acids in the feces. Proteolysis increases branched-chain SCFAs and can generate detrimental metabolites and cause catabolic putrefaction of hydrolyzed amino acids. Coriobacteriaceae unclassified, Dorea, Paraprevotellaceae unclassified, Slackia, Turicibacter, Catenibacterium, Streptomyces, Enterobacteriaceae unclassified, Parabacteroides, Ruminococcus, Methanococcus, Luteolibacter, Microthrixaceae unclassified, and Bifidobacterium all increased upon the addition of fiber to a hydrolyzed meat diet and are known to be saccharolytic (Jackson and Jewell, 2019). In that same study, 11 saccharolytic genera decreased with the addition of the fiber, including Bacteroides, Coprococcus, Dialister, Megamonas, Oscillospira, and Roseburia. Desulfovibrionaceae unclassified and Erysipelotrichaceae unclassified are known to be proteolytic and decreased with the addition of fiber to the hydrolyzed meat diet. Similarly, nine saccharolytic genera increased, including Blautia, Collinsella, Roseburia, Succinivibrio, and Brenneria, for the grain-rich food and four decreased, including Dialister. Three genera known to be proteolytic decreased with the addition of fiber, including Peptococcus and Peptostreptococcus. Fiber can also influence nutrient digestibility and transit rate, which reduces digestibility and increases the substrate load to the colon.

There were no statistical differences in microbial taxa or pathways at baseline but were greatly affected by diet. Although there was a large difference in the microbiome profile of the dogs consuming the mildly cooked human-grade diet versus the extruded diet (beta diversity), there was no statistical difference in alpha diversity as estimated using the Shannon, Simpson, and inverse Simpson indexes. In past research studying these two diets (Do et al., 2021), relative abundances of fecal Prevotella, Butyriciococcus, Holdemanella, and Catenibacterium were all higher in the dogs consuming the feed-grade extruded diet vs. the mildly cooked human-grade chicken diet, which is in agreement with the current study. In addition, the relative abundance of fecal Dialister was higher in dogs consuming the extruded diet. In the current study, there was an increase in relative abundances of fecal Escherichia, Helicobacter, Bifidobacterium, and Lactobacillus in dogs consuming the mildly cooked human-grade dog food, changes that were not noted in Do et al. (2021). There was a decrease in the relative abundances of fecal Adlercreutzia, Bacteroides, Blautia, Dorea, Erysipelatoclostridium, Faecalibacterium, which were also not noted in Do et al. (2021). Asaccharobacter, Enorma, Firmicutes unclassified, Gemmiger, Ileibacterium, and Peptostreptococcaceae unclassified all decreased in the present study, but these genera were not reported in Do et al. (2021).

With the exception of Bifidobacterium and Lactobacillus, most genera in the phyla Actinobacteria, Bacteroidetes, and Firmicutes decreased in the dogs fed JFFD in the current study. Many taxa within the Firmicutes, Bacteroidetes, and Actinobacteria phyla produce SCFA, strengthen epithelial tight junctions, and increase anti-inflammatory compound production (Mondo et al., 2019).

Adlercreutzia (including Adlercreutzia equolifaciens) decreased in dogs consuming JFFD. Although the same diets were tested in the current study and that reported by Do et al. (2021), the results may differ due to animal variation, the utilization of different databases, or other factors. Adlercreutzia equolifaciens metabolizes isoflavones to equol, which has antioxidant properties greater than its parent isoflavone compounds (Hwang et al., 2003; Maruo et al., 2008). The peas in the extruded diet might explain the higher prevalence of Adlercreutzia equolifaciens because peas are a legume and usually have moderate to high isoflavone concentrations, depending on the processing and portions of the peas used (Bhagwat et al., 2008). In addition, in a dog study, the food containing fiber sources such as citrus, carrot, tomato, and spinach, had a numerical but not significant increase in fecal equol concentration and had a significantly increased relative abundance of Adlercreutzia (Gebreselassie et al., 2018). Similarly, Asaccharobacter celatus, closely related to Adlercreutzia equolifaciens, can also convert the isoflavone, daidzein, to equol (Clavel et al., 2014).

The relative abundances of fecal Bifidobacterium and Lactobacillus were increased in dogs consuming the mildly cooked human-grade diet. Bifidobacterium and Lactobacillus are generally considered to be beneficial and are commonly used as probiotics or as targets for a prebiotic. Many Bifidobacterium strains have anti-microbial activity, inhibit pathogens, and stimulate the immune system (Liévin et al., 2000). Lactobacillus and Bifidobacterium also produce SCFA (LeBlanc et al., 2017). Some species of Lactobacillus break down pectin and so its increase may have been due to the potentially higher amounts of pectin present in apples (Vidhyasagar et al., 2013). In addition, Lactobacillus are acid-tolerant so their numbers may have increased due to the lower fecal pH in dogs fed the mildly cooked human-grade diet.

Some species in Bacteroidetes can be opportunistic pathogens, but they also can benefit the host by contributing to protein and complex sugar degradation (Rajilić-Stojanović and de Vos, 2014). The reason that Bacteroides might have been higher in dogs fed the kibble diet is the higher amount of carbohydrates present (calculated by nitrogen-free extract). In a canine study done by Bermingham et al. (2017), the family Bacteroidaceae was correlated with a higher dietary carbohydrate. Dogs eating a kibble diet had a greater relative abundance of fecal Bacteroides than dogs eating a red meat diet (Bermingham et al., 2017). In a dog study comparing a low-protein, high-carbohydrate diet to a high-protein, low-carbohydrate diet, Bacteroidetes was overrepresented in the low-protein, high-carbohydrate group (Li et al., 2017).

The relative abundance of fecal Catenibacterium decreased in the mildly cooked human-grade group in the current study. The decrease may be due to the kibble containing more carbohydrates because Catenibacterium can produce acetate, lactate, and isobutyrate from the hydrolysis and breakdown of carbohydrates (Kageyama and Benno, 2000). Furthermore, more KEGG pathways related to carbohydrate metabolism were decreased (9) than increased (3) in dogs consuming the mildly cooked human-grade diet in the current study. The MetaCyc community pathways supported this response, with 14 pathways decreasing (as opposed to 6 increasing) for carbohydrate biosynthesis and 6 decreasing (as opposed to 6 increasing) for carbohydrate degradation, suggesting that the difference in carbohydrate concentrations between the two diets had a substantial impact on the microbiota.

The relative abundance of fecal Prevotella and Prevotella copri, which were decreased in dogs fed JFFD in the current study, were also decreased in dogs changed from a kibble diet to a mildly cooked diet in previous studies (Do et al., 2021; Tanprasertsuk et al., 2021). The decrease in Prevotella could be due to the mildly cooked human-grade diet containing less carbohydrates, as in the study by Schauf et al. (2018) where dogs consuming a high-fat, low-starch diet had a lower relative abundance of Prevotella. In addition, Prevotella copri has been shown to increase in humans eating a non-Western diet containing greater fiber (Tett et al., 2019). Although both diets tested in the current study contained similar amounts of total fiber, the increase could be due to the fact that the extruded diet had more insoluble fiber.

The relative abundance of fecal Blautia decreased in dogs fed the mildly cooked human-grade diet in the current study. Fructooligosaccharides, likely present in the chicory root in the extruded diet, have been shown to increase the prevalence of Blautia (Bai et al., 2016; Mao et al., 2018). Furthermore, the increase in Blautia in dogs fed the extruded diet may have also been due to greater resistant starch or whole grain content (Martínez et al., 2013; Yang et al., 2013). The extruded kibble diet contained multiple fiber sources that have been shown to increase Blautia.

The relative abundances of fecal Butyricicoccus, Erysipelatoclostridium, Faecalibacterium, and Dorea were higher in dogs fed the extruded diet. The extruded diet contained peas and diets containing peas have been shown to increase these genera in past studies (Beloshapka et al., 2016; Reilly et al., 2021a, 2021b). Faecalibacterium is a known SCFA producer, particularly butyrate (LeBlanc et al., 2017). Faecalibacterium prausnitzii, the same species that decreased in dogs eating the mildly cooked human-grade diet in the current study, decreased in dogs switched from an extruded to mildly cooked diet previously (Tanprasertsuk et al., 2021).

The relative abundances of fecal Escherichia coli and Helicobacter increased in dogs consuming the mildly cooked human-grade diet in the current study. Helicobacter canis was reported to be one of the dominant Proteobacteria detected in the feces of healthy dogs (Chaban et al., 2012). Helicobacter is an acid-tolerant neutralophile that does best with a neutral pH, but can still survive and multiply in an acidic environment, which is perhaps why it increased in dogs fed the mildly cooked human-grade diet (Scott et al., 1998).

More KEGG pathways associated with amino acid metabolism decreased (12) in dogs consuming the mildly cooked human-grade diet than increased (6). This is also true for the MetaCyc community pathways, with 12 pathways decreasing for amino acid biosynthesis and 7 pathways decreasing for amino acid degradation. Ten pathways increased for amino acid biosynthesis and 1 pathway increased for amino acid degradation. In addition, all seven MetaCyc community pathways relating to amine and polyamine biosynthesis decreased. In terms of lipid metabolism, 5 KEGG pathways involving lipid metabolism increased in dogs consuming the mildly cooked human-grade diet, while 2 decreased. However, all 18 of the MetaCyc community pathways for fatty acid and lipid biosynthesis decreased and all four of the MetaCyc community pathways for fatty acid and lipid degradation increased.

In addition, more KEGG pathways decreased (7) for cofactor and vitamin metabolism than increased (3). The MetaCyc community pathways support this, with 31 pathways increasing for cofactor, carrier, and vitamin biosynthesis and only 10 decreasing. Interestingly, KEGG pathways for antimicrobial and antineoplastic drug resistance increased for all four pathways listed and MetaCyc community pathways for antibiotic resistance increased for all three pathways reported. Furthermore, all four KEGG pathways relating to the immune system decreased in dogs consuming the mildly cooked human-grade diet. Both of the KEGG pathways for nucleotide metabolism increased for dogs eating the mildly cooked human-grade diet. Nine of the MetaCyc community pathways for nucleoside and nucleotide biosynthesis increased while six decreased, but all nine of the pathways for nucleoside and nucleotide degradation decreased.

The current study noted an increase in the change from baseline gene expression of SOD, TNF-α, and COX-2 in the dogs consuming the mildly cooked human-grade diet. SOD converts superoxide radicals into hydrogen peroxide and oxygen, so a higher expression of SOD indicates more potential for protection against oxidative stress (Mruk et al., 2002). Hydrogen peroxide is then converted to water by CAT and GPX. However, SOD expression was higher in the dogs eating the extruded diet at baseline, so the increase from baseline in the mildly cooked human-grade group could have simply been a return to the mean. On the other hand, TNF-α is pro-inflammatory and activates NF-kappaB, which then leads to the expression of more inflammatory genes, such as COX-2 (Sethi et al., 2008). Although an increase in the expression of TNF-α would indicate more inflammation, TNF-α was also higher in the dogs consuming the extruded diet at baseline, so again the change from baseline in the mildly cooked human-grade group could be a return to the mean. COX-2 is also pro-inflammatory and produces prostanoids in acute and chronic inflammatory conditions, so an increase in COX-2 would indicate more inflammation (Minghetti, 2004). Despite the changes in gene expression, complete blood count and most serum chemistry measures were not different between diets, indicating that both diets were adequate and maintained the health of the dogs.

There were some limitations to the present study, with several factors that may have influenced the results of the skin devices. First, the relative humidity decreased drastically from the baseline collection (54–57%) to the wk-12 collection (24–25%), likely because the former was taken in the summer and the latter in the winter, which may have impacted the skin device results. In addition, hair was clipped 24 h prior to the skin measurement. In a prior study, clipping hair immediately before measurements had no effect on TEWL (Lau-Gillard et al., 2010). In another study, however, shaving the hair caused a 44% drop in the TEWL values (compared with parting the hair) (Watson et al., 2002). Additionally, in a study by Oh and Oh (2009), TEWL and hydration values were not different between conscious and anaesthetized dogs in the ear and inguinal region, but they were lower in the back of anesthetized dogs. However, an advantage of sedating the dogs in our study was that it kept them very still, which helps to decrease variation in measurements. A study done by Watson et al. (2002) demonstrated that TEWL is significantly affected by the movement of the dog during measurements. There was also considerable variation in the hair surface and hair cortex scoring. First, there were only approximately five different hairs fixed per dog. Second, only one hair per dog was imaged and analyzed, with the hair facing the camera at the best angle (to enable successful cortex scoring) being the one selected. Future studies should probably score more hairs per dog and take an average of the scores. Another limitation lies within the study design, which tested two diets that differed in numerous ways, including processing method, product grade, ingredients, and macronutrient composition. Many of the differences observed cannot be directly attributable to one element, due to the multiple factors present.

In conclusion, mildly cooked human-grade pet foods are an emerging popular diet format, but there is a lack of research in this area. Previous studies have shown that mildly cooked human-grade diets are highly digestible (Oba et al., 2020; Do et al., 2021) and lead to lower stool volume (Do et al. 2021) when fed to dogs. However, there has been no other research on mildly cooked human-grade foods. Therefore, this study aimed to assess skin and coat health measures, whole blood gene expression, fecal characteristics, and the fecal microbiome of mildly cooked human-grade pet foods. All dogs remained healthy during the study and there were minimal differences between skin and coat quality, although there was a decrease in TEWL in dogs fed the mildly cooked human-grade diet. There were also few differences in gene expression markers of inflammation and oxidative stress. However, the fecal microbiome was vastly different between the two diets, with the mildly cooked human-grade diet clustering away from the extruded kibble diet.

Supplementary Material

skac265_suppl_Supplementary_Material

Acknowledgments

The funding for this study was provided by Just Food For Dogs, Irvine, CA, USA.

Glossary

Abbreviations

BW

body weight

CFB

change from baseline

COX-2

cyclooxygenase-2

DM

dry matter

DTH

delayed-type hypersensitivity

GI

gastrointestinal

PCoA

principal coordinate analysis

qPCR

quantitative polymerase chain reaction

SCFA

short-chain fatty acids,

SOD

superoxide dismutase

TEWL

transepidermal water loss

TNF-α

tumor necrosis factor-α

Contributor Information

Elizabeth L Geary, Department of Animal Sciences, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.

Patrícia M Oba, Department of Animal Sciences, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.

Catherine C Applegate, Department of Animal Sciences, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA; Department of Bioengineering, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA; The Beckman Institute of Advanced Science and Technology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.

Lindsay V Clark, High Performance Computing in Biology, Roy J. Carver Biotechnology Center, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.

Christopher J Fields, High Performance Computing in Biology, Roy J. Carver Biotechnology Center, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.

Kelly S Swanson, Department of Animal Sciences, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA; Division of Nutritional Sciences, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA; Department of Veterinary Clinical Medicine, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.

Conflict of Interest Statement

The authors have no conflicts of interest.

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