Effects of diets supplemented with bioactive peptides on nutrient digestibility, immune cell responsiveness, and fecal characteristics, microbiota, and metabolites of adult cats.

Bioactive peptides (BP) are recognized for their ability to function as antioxidants and maintain lipid stability. They may have positive health effects, including antihypertensive, anti-inflammatory, antimicrobial, osteoprotective, gut health, and immunomodulatory properties, but are poorly tested in cats. Our primary objective was to determine the apparent total tract digestibility (ATTD) of BP-containing kibble diets and assess how the fecal characteristics, metabolites, and microbiota were affected in adult cats. Our secondary objective was to test whether BP could impact blood oxidative stress markers and cytokine concentrations following transport stress. Twelve adult cats (4.83 ± 0.37 yr; 4.76 ± 0.14 kg) were used in a replicated 4 × 4 Latin square design to test four extruded kibble diets: Control (no BP), Chicken (4% chicken BP), Marine1 (2% marine BP), and Marine2 (4% marine BP). Each experimental period lasted 28 d, with a 20-d adaptation phase, 5 d for fecal collection, 2 d for blood collection, and 1 d for transport stress testing (driven in vehicle in individual carriers for 45 min). Salivary cortisol and blood oxidative stress markers and cytokines were measured after transport. Fecal microbiota data were evaluated using 16S rRNA gene amplicon sequencing and QIIME2. All other data were analyzed using the Mixed Models procedure of SAS, with P < 0.05 being considered significant and P < 0.10 considered trends. No differences were observed in animal health outcomes, with all cats remaining healthy and serum metabolites remaining within reference ranges. Cats fed the Marine2 diet had higher (P < 0.05) ATTD of dry matter (84.5% vs. 80.9%) and organic matter (88.3% vs. 85.8%) than those fed the control diet. The ATTD of protein and energy tended to be higher (P < 0.10) for cats fed the Marine2 diet. Fecal characteristics, metabolites, and bacterial alpha and beta diversity measures were not affected by treatment. However, the relative abundances of six bacterial genera were different (P < 0.05) and two bacterial genera tended to be different (P < 0.10) across treatments. Treatment did not alter salivary cortisol, blood oxidative stress markers, or blood cytokines after transport stress. Our data suggest that BP inclusion may increase nutrient digestibility and modify fecal microbiota and immune measures. More testing is required, however, to determine whether BP may provide additional benefits to cats.

Dietary bioactive peptides (BP) may have positive health effects, but are poorly tested in cats. Our primary objective was to determine the apparent total tract digestibility of BP-containing kibble diets and assess how fecal characteristics, metabolites, and microbiota were affected in adult cats. Our secondary objective was to test whether BP could impact blood oxidative stress markers and cytokines following transport stress. Adult cats were used in a replicated 4 × 4 Latin square design to test four extruded kibble diets containing different BP concentrations. After diet adaptation, fecal and blood samples were collected and transport stress testing was done in each experimental period. All cats remained healthy and serum metabolites remained within reference ranges. Cats fed one of the BP diets had higher dry matter and organic matter digestibilities and tended to have higher protein and energy digestibilities. Fecal characteristics, metabolites, and microbiota diversity measures were not different, but the relative abundances of eight bacterial genera differed or tended to differ across treatments. Treatments did not alter oxidative stress markers after transport stress. Our data suggest that BP inclusion may increase nutrient digestibility and modify fecal microbiota. Further testing is required to determine whether BP provides additional benefits to cats.

Effects of a novel dental chew on oral health outcomes, halitosis, and microbiota of adult dogs.

Halitosis in dogs is an initial indication of periodontitis, highlighting its significance as a vital marker for underlying problems. Moreover, the oral microbial population has a significant influence on periodontal disease. Measuring the oral microbiota may be used in addition to breath odor, dental plaque, and gingivitis scoring to assess the impact of dental chews on oral health. In this study, we aimed to determine the differences in breath odor, oral health outcomes, and oral microbiota of adult dogs consuming a novel dental chew compared with control dogs consuming only a diet. Twelve healthy adult female beagle dogs were used in a crossover design study. Treatments (n = 12/group) included: diet only (control) or the diet + a novel dental chew. Each day, one chew was provided 4 h after mealtime. On days 1, 7, 14, 21, and 27, breath samples were analyzed for total volatile sulfur compound concentrations using a halimeter. On day 0 of each period, teeth were cleaned by a veterinary dentist blinded to treatments. Teeth were scored for plaque, calculus, and gingivitis by the same veterinary dentist on day 28 of each period. After scoring, subgingival and supragingival plaque samples were collected for microbiota analysis using Illumina MiSeq. All data were analyzed using SAS (version 9.4) using the Mixed Models procedure, with P < 0.05 being significant. Overall, the dental chews were well accepted. Dogs consuming the dental chews had lower calculus coverage, thickness, and scores, lower gingivitis scores, and less pocket bleeding than control dogs. Breath volatile sulfur compounds were lower in dogs consuming the dental chews. Bacterial alpha-diversity analysis demonstrated that control dogs had higher bacterial richness than dogs fed dental chews. Bacterial beta-diversity analysis demonstrated that samples clustered based on treatment. In subgingival and supragingival plaque, control dogs had higher relative abundances of potentially pathogenic bacteria (Pelistega, Desulfovibrio, Desulfomicrobium, Fretibacterium, Helcococcus, and Treponema) and lower relative abundances of genera associated with oral health (Neisseria, Actinomyces, and Corynebacterium). Our results suggest that the dental chew tested in this study may aid in reducing periodontal disease risk in dogs by beneficially shifting the microbiota population and inhabiting plaque buildup.

In this study, we aimed to determine the effects of a novel dental chew on the breath odor, oral health outcomes, and oral microbiota of dogs. Healthy adult dogs were used in a crossover design study to test a diet only (control) or the diet plus a novel dental chew. Each day, one chew was provided 4 h after mealtime. Breath samples were analyzed over time and teeth were scored for plaque, calculus, and gingivitis by a veterinary dentist on day 28 of each period. After scoring, subgingival and supragingival plaque samples were collected for microbiota analysis. Dogs consuming dental chews had lower calculus coverage, thickness, and scores, lower gingivitis scores, and less pocket bleeding than control dogs. Breath volatile sulfur compounds were lower in dogs consuming dental chews. Bacterial alpha-diversity was higher in control dogs than in dogs fed dental chews. Bacterial beta-diversity analysis demonstrated sample clustering based on treatment. Control dogs had higher relative abundances of potentially pathogenic bacteria and lower relative abundances of genera associated with oral health. Our results suggest that the dental chew tested may aid in reducing periodontal disease risk in dogs by beneficially shifting microbiota and inhabiting plaque buildup.

Green banana flour as a novel functional ingredient in retorted feline diets.

Green banana flour (GBF) is a novel ingredient that is high in resistant starch and could be a dietary fiber source in companion animal nutrition. In addition, with its light brown color and pectin content, GBF could potentially serve as a natural color additive and thickening agent in pet food manufacturing. The purpose of this research is to evaluate different sources of GBF, the effect of GBF on texture and color in canned foods, and its effect on apparent total tract digestibility (ATTD), fecal characteristics, and fecal fermentative end-products in healthy adult cats. Prior to the feline study, different sources of GBF were analyzed for chemical composition, manufacturing properties, true metabolizable energy, and fermentability. For the feline feeding trial, all treatment diets were formulated to meet or exceed the Association of American Feed Control Officials (Association of American Feed Control Officials (AAFCO) 2020. Official Publication. Champaign, IL.) guidelines for adult cat maintenance. There were five dietary treatments: rice control (4% rice flour), potato control (4% dehydrated potato flakes), 1% GBF (1% GBF and 3% rice flour), 2% GBF (2% GBF and 2% rice flour), and 4% GBF. All treatment diets were analyzed for texture and color. The animal study was conducted using a completely randomized design with 39 adult domestic cats. There was a 7-d diet adaptation period followed by a baseline fresh fecal collection to determine fecal score, pH, short-chain fatty acid, branched-chain fatty acid, phenol, indole, ammonia, and microbiota. The treatment period lasted for 21 d and a total fecal collection was performed during the last 4 d of this period to determine the ATTD. A fresh fecal sample was also collected during the total fecal collection to evaluate fecal score, pH, metabolites, and microbiota. The MIXED model procedures of SAS version 9.4 were used for statistical analysis. Treatment diets containing GBF had a lower hardness from the texture profile analysis (P < 0.05). For color analysis, the 4% GBF diet was darker in color compared with the rice diet (P < 0.05). There was no difference in food intake, fecal output, or ATTD of macronutrients among the treatment groups (P > 0.05). There was no interaction of treatment and time or main effects shown in fecal score, pH, metabolites, or microbiota diversity (P > 0.05). In conclusion, adding GBF to canned diets may affect the texture and color of the product, but GBF was comparable to traditional carbohydrate sources, rice, and potato, from a nutritional aspect.

Green banana flour (GBF) is a novel ingredient in the pet food industry but has been gaining popularity in human nutrition. Not only can GBF be a source of dietary fiber in pet foods, but the natural brown color and hygroscopic properties also show the potential in contributing to physical characteristics. With its soluble fiber content, green banana flour has fewer calories than a digestible starch and is partially fermentable. The current study aimed to examine the effect of green banana flour on canned cat foods in comparison to traditional starch sources. Canned diets were made with predominately chicken and the test carbohydrate sources of rice flour, dehydrated potato flakes, and/or green banana flour. Canned food with a high inclusion rate of green banana flour showed differences in texture and color when compared with traditional diets; the finding indicated that green banana flour could be utilized to obtain desirable wet food characteristics, including color and texture. As a carbohydrate source in the canned diet, green banana flour had comparable effects on digestibility and gut microbiota to traditional starches when fed to cats. In conclusion, green banana flour can be used as an alternative carbohydrate source in canned diets and contribute to product texture and color.

Dietary Inclusion of Yellow Mealworms (T. molitor) and Lesser Mealworms (A. diaperinus) Modifies Intestinal Microbiota Populations of Diet-Induced Obesity Mice.

BACKGROUND: Insect-based proteins are high-quality alternatives to support the shift toward more sustainable and healthy diets. Additionally, insects contain chitin and have unique fatty acid profiles. Studies have shown that mealworms may beneficially affect metabolism, but limited information is known regarding their effects on gut microbiota. OBJECTIVES: We determined the effects of defatted yellow mealworm (Tenebrio molitor) and whole lesser mealworm (Alphitobius diaperinus) meals on the intestinal microbiota of diet-induced obesity mice. METHODS: Male C57BL/6J mice were fed a high-fat diet (HFD; 46% kcal) to induce obesity. Obese mice were then randomly assigned to treatments (n = 10/group) and fed for 8 wk: HFD, HFD with casein protein; B50, HFD with 50% protein from whole lesser mealworm; B100, HFD with 100% protein from whole lesser mealworm; Y50, HFD with 50% protein from defatted yellow mealworm; Y100, HFD with 100% protein from defatted yellow mealworm. Lean mice (n = 10) fed a low-fat-diet (10% kcal) were included. Fresh feces were collected at baseline and every 2 wk, with cecal digesta collected at kill. Fecal and cecal DNA was analyzed for microbiota using 16S rRNA MiSeq Illumina sequencing. RESULTS: In feces and cecal digesta, mice fed mealworms had greater (P < 0.05) bacterial alpha diversity, with changes occurring in a time-dependent manner (P < 0.05). Beta diversity analyses of cecal samples showed a clear separation of treatments, with a time-based separation shown in fecal samples. Widespread microbial differences were observed, with over 45 genera altered (P < 0.05) by diet in cecal digesta. In feces, over 50 genera and 40 genera were altered (P < 0.05) by diet and time, respectively. CONCLUSION: Mealworm consumption changes the intestinal microbiota of obese mice, increasing alpha diversity measures and shifting bacterial taxa. More investigation is required to determine what mealworm components are responsible and how they may be linked with the metabolic benefits observed in mealworm-fed mice.

Effects of commercial and traditional kefir supplementation on apparent total tract macronutrient digestibility and the fecal characteristics, metabolites, and microbiota of healthy adult dogs.

Kefir is a fermented dairy beverage that has been consumed by humans for centuries, but poorly studied in pets. The objective of this study was to determine the effects of commercial or traditional kefir supplementation on apparent total tract macronutrient digestibility (ATTD) and fecal characteristics, microbiota populations, and metabolite and immunoglobulin (Ig) A concentrations of healthy adult dogs. Twelve healthy adult dogs (5.67 ±â€…1.72 yr, 7.27 ±â€…1.15 kg) were used in a replicated 3 × 3 Latin square design (n = 12/group). All dogs were fed a commercial diet and allotted to 1 of 3 treatments (60 mL/d): 2% reduced-fat milk treated with lactase [CNTL; 4.57E + 03 lactic acid bacteria (LAB) colony-forming units (CFU)/mL], commercial kefir (C-Kefir; 6.95E + 04 LAB CFU/mL), or traditional kefir brewed daily from 2% reduced-fat milk and kefir grains (T-Kefir; 1.79E + 09 LAB CFU/mL). The experiment was composed of three 28-d periods, with each consisting of a 22-d transition phase, a 5-d fecal collection phase, and 1 d for blood collection. Fecal samples were collected for determination of ATTD and fecal pH, dry matter, microbiota, and metabolite, and IgA concentrations. Data were analyzed using the Mixed Models procedure of SAS 9.4. The main effects of treatment were tested, with significance set at P ≤ 0.05 and trends set at P ≤ 0.10. Kefir products differed in microbial density and profile, but fecal microbiota populations were weakly impacted. Bacterial alpha diversity tended to be greater (P = 0.10) in dogs fed T-Kefir than those fed CNTL. Bacterial beta diversity analysis identified a difference (P < 0.0004) between dogs-fed CNTL and those fed C-Kefir. Dogs-fed C-Kefir tended to have a greater (P = 0.06) relative abundance of Fusobacteriota than those fed CNTL or T-Kefir. Dogs-fed T-Kefir had a greater (P < 0.0001) relative abundance of Lactococcus than those fed CNTL or C-Kefir. Dogs-fed T-Kefir also tended to have a lower (P = 0.09) relative abundance of Escherichia Shigella and greater (P = 0.09) relative abundance of Candidatus stoquefichus than dogs-fed CNTL or C-Kefir. Dogs-fed C-Kefir tended to have lower (P = 0.08) fecal valerate concentrations than those fed CNTL or T-Kefir. All other measures were unaffected by kefir treatments. Our results suggest that kefir supplementation had minor effects on the fecal microbiota populations and fecal metabolite concentrations of healthy adult dogs without impacting ATTD, fecal characteristics, or fecal IgA concentrations.

Kefir is a fermented dairy beverage that has been consumed by humans for centuries, but poorly studied in pets. Our objective was to determine the effects of commercial or traditional kefir supplementation on apparent total tract macronutrient digestibility and fecal characteristics, microbiota populations, and metabolite and immunoglobulin A concentrations of healthy adult dogs. Using a replicated Latin square design, milk (control; CNTL), commercial kefir (C-Kefir), and traditional kefir (T-Kefir) treatments were tested. Kefir products differed in microbial density and profile, but fecal microbiota populations were weakly impacted. Bacterial alpha diversity tended to be greater in T-Kefir than CNTL. Bacterial beta diversity analysis identified differences between CNTL and C-Kefir. Dogs-fed C-Kefir tended to have greater relative abundance of Fusobacteriota than those fed CNTL or T-Kefir. Dogs-fed T-Kefir had a greater relative abundance of Lactococcus than those fed CNTL or C-Kefir. Dogs-fed T-Kefir tended to have a lower relative abundance of Escherichia-Shigella and greater relative abundance of Candidatus stoquefichus than dogs-fed CNTL or C-Kefir. Dogs-fed C-Kefir tended to have lower fecal valerate than those fed CNTL or T-Kefir. Our results suggest that kefir supplementation had minor effects on the fecal microbiota and metabolites of healthy adult dogs.

Standardized amino acid digestibility and nitrogen-corrected true metabolizable energy of frozen and freeze-dried raw dog foods using precision-fed cecectomized and conventional rooster assays.

Commercial raw or minimally-processed diets, often referred to holistically as raw meat-based diets (RMBD) represent a small portion of the pet food market, but the growth of this sector has been significant in recent years. While traditionally, high-moisture, frozen options were the standard format of commercially available raw diets, freeze-dried raw diets have become more prevalent as of late. Despite the increasing popularity of these commercial raw diet formats, there is a dearth of literature describing their nutritional properties, particularly regarding freeze-dried diets. Therefore, the objective of this experiment was to determine and compare the standardized amino acid (AA) digestibilities and nitrogen-corrected true metabolizable energy (TMEn) of raw frozen and freeze-dried dog foods using precision-fed cecectomized and conventional rooster assays. Three formats of frozen or freeze-dried raw diets provided by Primal Pet Foods (Fairfield, CA, USA) were tested: traditional freeze-dried nuggets (T-FDN), hybrid freeze-dried nuggets (H-FDN), and frozen nuggets (FZN). Diets were fed to cecectomized roosters (4 roosters/diet) to determine AA digestibilities, while conventional roosters (4 roosters/diet) were used to determine TMEn. In both cases, after 26 h of feed withdrawal, roosters were tube-fed 12 to 13 g of test diets and 12 to 13 g of corn. Following crop intubation, excreta were collected for 48 h. Endogenous corrections for AA were made using five additional cecectomized roosters. All data were analyzed using the Mixed Models procedure of SAS version 9.4. There were no significant differences in standardized AA digestibilities among diets, with digestibilities being high for all diets tested. For most of the indispensable AA, digestibilities were greater than or equal to 90% for all diets. Histidine and lysine were the exceptions, with digestibilities ranging from 82% to 87% and 87% to 92%, respectively. Moreover, the reactive lysine:total lysine ratio, a measure of heat damage, ranged from 0.91 to 0.95. TMEn values were higher (P = 0.0127) in T-FDN (6.1 kcal/g) and FZN (5.9 kcal/g) than H-FDN (5.3 kcal/g) and were most similar to those estimated by Atwater factors. In general, all diets tested had high AA digestibilities and had TMEn values that were most similar to Atwater factors.

Commercial raw or minimally-processed diets represent a small portion of the pet food market, but the growth of this sector has been significant in recent years. Despite the increasing popularity of commercial frozen and freeze-dried raw diet formats, there is a dearth of literature describing their nutritional properties. The objective of this experiment was to determine the standardized amino acid (AA) digestibilities and nitrogen-corrected true metabolizable energy (TMEn) of raw frozen and freeze-dried dog foods using precision-fed cecectomized and conventional rooster assays. Diets tested included traditional freeze-dried nuggets (T-FDN), frozen nuggets (FZN), and hybrid freeze-dried nuggets (H-FDN). Diets were fed to cecectomized roosters to determine AA digestibilities, while conventional roosters were used to determine TMEn. In both cases, fasted roosters were tube-fed test diets, and excreta was collected. Standardized AA digestibilities were high for all AA (>90% for most indispensable AA) and were not different among diets. The reactive lysine: total lysine ratio, a measure of heat damage, ranged from 0.91 to 0.95. TMEn values were higher in T-FDN (6.1 kcal/g) and FZN (5.9 kcal/g) than H-FDN (5.3 kcal/g). In general, all diets tested had high AA digestibilities and had acceptable reactive lysine:total lysine ratios.

Effects of overfeeding on the digestive efficiency, voluntary physical activity levels, and fecal characteristics and microbiota of adult cats.

The incidence of feline obesity continues to rise despite it being a preventable disease. There are many risks and health perturbations associated with obesity, with several of those impacting a pet's quality of life, wellness, and longevity. Feline obesity is commonly studied, but most research has been focused on weight loss rather than weight gain. To our knowledge, feline studies have not examined the implications of overfeeding and weight gain on gastrointestinal transit time (GTT) nor the association it has with the fecal microbiota. Therefore, the objective of this study was to determine the effects of overfeeding and weight gain on apparent total tract digestibility (ATTD), GTT, blood hormones, serum metabolites, hematology, fecal microbiota populations, and voluntary physical activity of cats. Eleven lean adult spayed female cats [body weight (BW) = 4.11 ±â€…0.43 kg; body condition score = 5.41 ±â€…0.3; age = 5.22 ±â€…0.03 y] were used in a longitudinal weight gain study. After a 2-wk baseline phase, cats were allowed to overeat for 18 wk. A commercially available complete and balanced diet was fed during the baseline phase to identify the intake needed to maintain BW. Cats were then fed the same diet ad libitum to induce weight gain. Fecal samples, blood samples, and voluntary physical activity data were collected at baseline (week 0) and 6, 12, and 18 wk after weight gain. Fecal samples were collected for microbiota analysis, determination of ATTD, and GTT measurement while blood samples were collected for serum chemistry, hematology, and insulin and leptin measurements. Microbiota data were evaluated using QIIME2. All other measures were evaluated statistically using the mixed models procedure of SAS using repeated measures analysis, with time effects being the focus. A P < 0.05 was considered significant. The ATTD of dry matter (P = 0.0061), organic matter (P = 0.0130), crude protein (P < 0.0001), fat (P = 0.0002), and gross energy (P = 0.0002), and GTT (P = 0.0418) decreased with overfeeding and weight gain. Fecal bacterial alpha diversity measures were unchanged, but fecal bacterial beta diversity was impacted (P < 0.05) with overfeeding and weight gain. The relative abundances of 16 bacterial genera, including Bifidobacterium, Collinsella, Erysipelatoclostridium were affected (P < 0.05) by overfeeding and weight gain. In conclusion, overfeeding and subsequent weight gain reduced ATTD, reduced GTT, and caused changes to the fecal microbial community of adult cats.

Feline obesity continues to rise, impacting the wellness, quality of life, and longevity of cats. Understanding the metabolic and gastrointestinal changes that companion animals face with the onset of weight gain and obesity may help with future prevention and treatment plans. The implications of overfeeding and weight gain on gastrointestinal transit time (GTT) and its association with fecal microbiota populations have not been studied. Therefore, the objective of this study was to determine the effects of overfeeding and weight gain on apparent total tract digestibility, GTT, blood hormones, serum metabolites, hematology, fecal microbiota populations, and voluntary physical activity of cats. After a 2-week baseline phase, adult cats were allowed to overeat for 18 weeks. Fecal and blood samples were collected, and voluntary physical activity was measured using accelerometers over time. Dry matter, organic matter, protein, fat, and energy digestibilities and GTT were decreased with overfeeding and weight gain. Fecal bacterial beta diversity was impacted by overfeeding and weight gain, impacting the relative abundances of 1 bacterial phylum and 16 bacterial genera. In conclusion, overfeeding and subsequent weight gain reduced nutrient digestibility, reduced GTT and caused changes to the fecal microbial community of adult cats.

Effects of weight loss and feeding specially formulated diets on the body composition, blood metabolite profiles, voluntary physical activity, and fecal metabolites and microbiota of overweight cats.

Feline obesity is a common and preventable disease, posing a myriad of health risks and detriments. Specially formulated diets and restricted feeding may serve as an intervention strategy to promote weight loss and improve feline health. In this study, our objective was to determine the effects of restricted feeding and weight loss on body composition, voluntary physical activity, blood hormones and metabolites, and fecal microbiota of overweight cats. Twenty-two overweight adult spayed female and neutered male cats [body weight (BW) = 5.70 ± 1.0 kg; body condition score (BCS) = 7.68 ± 0.6; age = 4 ± 0.4 yr] were used in a weight loss study. A control diet (OR) was fed during a 4-wk baseline to identify intake needed to maintain BW. After baseline (week 0), cats were allotted to OR or a test diet (FT) and fed to lose ~1.0% BW/wk for 24 wk. At baseline and 6, 12, 18, and 24 wk after weight loss, dual-energy x-ray absorptiometry scans were performed and blood samples were collected. Voluntary physical activity was measured at weeks 0, 8, 16, and 24. Fecal samples were collected at weeks 0, 4, 8, 12, 16, 20, and 24. Change from baseline data were analyzed statistically using the Mixed Models procedure of SAS, with P < 0.05 considered significant. Restricted feeding of both diets led to weight and fat mass loss, lower BCS, and lower blood triglyceride and leptin concentrations. Cats fed the FT diet had a greater reduction in blood triglycerides and cholesterol than cats fed the OR diet. Restricted feeding and weight loss reduced fecal short-chain fatty acid, branched-chain fatty acid, phenol, and indole concentrations. Fecal valerate concentrations were affected by diet, with cats fed the OR diet having a greater reduction than those fed the FT diet. Fecal bacterial alpha diversity was not affected, but fecal bacterial beta diversity analysis showed clustering by diet. Restricted feeding and weight loss affected relative abundances of 7 fecal bacterial genera, while dietary intervention affected change from baseline relative abundances of 2 fecal bacterial phyla and 20 fecal bacterial genera. Our data demonstrate that restricted feeding promoted controlled and safe weight and fat loss, reduced blood lipids and leptin concentrations, and shifted fecal metabolites and microbiota. Some changes were also impacted by diet, highlighting the importance of ingredient and nutrient composition in weight loss diets.

The objective of this study was to determine the effects of diet, restricted feeding and weight loss on body composition, voluntary physical activity, blood hormones and metabolites, and fecal metabolites and microbiota of overweight cats. Overweight cats were allotted to a control diet (OR) or weight loss diet (FT) and fed to lose ~1.0% body weight/week for 24 wk. Body weight, body composition, and voluntary physical activity were measured, while fecal and blood samples were collected over time. Restricted feeding led to weight and fat mass loss, and lower blood triglyceride and leptin concentrations. Cats fed FT had a greater reduction in blood triglycerides and cholesterol than cats fed OR. Restricted feeding reduced fecal metabolite concentrations and affected relative abundances of 7 fecal bacterial genera. Fecal bacterial beta diversity analysis showed clustering by diet. Dietary intervention affected change from baseline relative abundances of 2 fecal bacterial phyla and 20 fecal bacterial genera. Our data demonstrate that restricted feeding promoted controlled and safe weight and fat loss, reduced blood lipids and leptin concentrations, and shifted fecal metabolites and microbiota. Some dietary differences were noted, highlighting the importance of ingredient and nutrient composition in weight loss diets.

Comparing the standardized amino acid digestibility of an alternative protein source with commercially available protein-based ingredients using the precision-fed cecectomized rooster assay.

Using single-cell-based proteins in pet foods is of interest, but little testing has been done. Therefore, our objective was to determine the amino acid (AA) digestibilities, assess protein quality of a novel microbial protein (MP) (FeedKind), and compare it with other protein-based ingredients using the precision-fed cecectomized rooster assay. Test ingredients included: MP, chicken meal (CM), corn gluten meal (CGM), pea protein (PP), and black soldier fly larvae. Thirty cecectomized roosters (n = 6/ingredient) were randomly assigned to test ingredients. After 24 h of feed withdrawal, roosters were tube-fed 15 g test ingredient and 15 g corn, and then excreta were collected for 48 h. Endogenous AA corrections were made using additional roosters. Digestible indispensable AA score (DIAAS)-like values were calculated to determine protein quality according to Association of American Feed Control Officials (AAFCO), The European Pet Food Industry Federation, and National Research Council reference values for growing and adult dogs and cats. Data were analyzed using the Mixed Models procedure of SAS 9.4, with P ≤ 0.05 being significant. All reactive lysine:total lysine ratios, an indicator of heat damage, were higher than 0.9, except for CM (0.86). Digestibility of indispensable and dispensable AA were >85% and >80% for MP, respectively, with indispensable AA digestibilities being >80% for all other ingredients. In general, CGM had the highest, while CM had the lowest AA digestibilities. Two exceptions were lysine and tryptophan. Lysine digestibility for MP was higher than that of all other ingredients, while tryptophan digestibility for MP was higher than that of CM, CGM, and PP. Threonine digestibility was highest for CGM and MP. Valine digestibility was highest for CGM, PP, and MP. DIAAS-like calculations identified limiting AA of each ingredient and depended on the reference used and life stage and species of animal. Using AAFCO guidelines, all DIAAS-like values for MP were >100 suggesting that it could be used as the sole source of protein in adult dog and cat diets; only methionine had DIAAS-like values <100 for growing kittens. For dogs, limiting AA was most commonly methionine, threonine, and tryptophan in the other protein sources. For cats, limiting AA was most commonly lysine and methionine. Lysine was severely limited in CGM across all life stages considered. Further research in dogs and cats is necessary, but our data suggest that the MP tested has high AA digestibilities and is a high-quality protein source that may be useful in pet foods.

Single-cell-based proteins are of interest for use in pet foods, but little testing has been done. The objective of this experiment was to compare the amino acid (AA) digestibilities and protein quality of a novel microbial protein (MP) (FeedKind) with chicken meal (CM), corn gluten meal (CGM), pea protein (PP), and black soldier fly larvae ingredients using the precision-fed cecectomized rooster assay. Cecectomized roosters were tube-fed the test ingredients and excreta were collected. All reactive lysine:total lysine ratios, an indicator of heat damage, were higher than 0.9, except for CM. Digestibility of indispensable and dispensable AA were >85% and >80% for MP, respectively, with indispensable AA digestibilities being >80% for all other ingredients. In general, CGM had the highest, while CM had the lowest AA digestibilities. Lysine and tryptophan were exceptions, being highest for MP. Threonine and valine digestibilities were also high for MP. Digestible indispensable AA score-like values identified limiting AA of each ingredient. Limiting AA was most commonly methionine, threonine, and tryptophan for dogs and lysine and methionine for cats. Our data suggest that the MP tested has high AA digestibilities and is a high-quality protein source that may be useful in pet foods.

Yellow Mealworm (Tenebrio molitor) and Lesser Mealworm (Alphitobius diaperinus) Proteins Slowed Weight Gain and Improved Metabolism of Diet-Induced Obesity Mice.

BACKGROUND: High-protein diets not only meet amino acid needs but also modulate satiety and energy metabolism. Insect-based proteins are sustainable, high-quality proteins. Mealworms have been studied, but limited information is known about their ability to impact metabolism and obesity. OBJECTIVE: We determined the effects of defatted yellow mealworm (Tenebrio molitor)- and whole lesser mealworm (Alphitobius diaperinus)-based proteins on the body weight (BW), serum metabolites, and liver and adipose tissue (AT) histology and gene expression of diet-induced obesity mice. METHODS: Male C57BL/6J mice were fed a high-fat diet (HFD; 46% kcal) to induce obesity and metabolic syndrome. Obese mice were then assigned to treatments (n = 10/group) and fed for 8 wk: HFD: HFD with casein protein; B50: HFD with 50% protein from whole lesser mealworm; B100: HFD with 100% protein from whole lesser mealworm; Y50: HFD with 50% protein from defatted yellow mealworm; Y100: HFD with 100% protein from defatted yellow mealworm. Lean mice (n = 10) fed a low-fat-diet (LFD; 10% kcal) were included. Longitudinal food intake, BW, body composition, and glucose response were measured. At time of killing, serum metabolites, tissue histopathology and gene expression, and hepatic triglycerides were analyzed. RESULTS: After 8 wk, HFD, B50, and B100 had greater (P < 0.05) weight gain than LFD, whereas Y50 and Y100 did not. Y50, B100, and Y100 had a lower (P < 0.05) BW change rate than HFD. Mealworm-based diets led to increased (P < 0.05) serum high-density lipoprotein (HDL) and reduced (P < 0.05) serum low-density lipoprotein (LDL) concentrations and reduced (P<0.05) LDL/HDL ratio. Mealworm-based diets led to increased (P < 0.05) hepatic expression of genes related to energy balance, immune response, and antioxidants and reduced (P < 0.05) AT expression of genes associated with inflammation and apoptosis. Mealworm-based diets altered (P < 0.05) hepatic and AT expression of glucose and lipid metabolism genes. CONCLUSIONS: In addition to serving as an alternative protein source, mealworms may confer health benefits to obese patients.

Effects of a Saccharomyces cerevisiae fermentation product on fecal characteristics, metabolite concentrations, and microbiota populations of dogs undergoing transport stress.

Previously, a Saccharomyces cerevisiae fermentation product (SCFP) positively altered fecal microbiota, fecal metabolites, and immune cell function of adult dogs. Our objective was to determine the fecal characteristics, microbiota, and metabolites of SCFP-supplemented dogs subjected to transport stress. All procedures were approved by the Four Rivers Kennel IACUC prior to experimentation. Thirty-six adult dogs (18 male, 18 female; age: 7.1 ± 0.77 yr; body weight: 28.97 ± 3.67 kg) were randomly assigned to be controls or receive SCFP supplementation (250 mg/dog/d) (N = 18/group) for 11 wk. At that time, fresh fecal samples were collected before and after transport in a hunting dog trailer with individual kennels. The trailer was driven 40 miles round trip for about 45 min. Fecal microbiota data were evaluated using Quantitative Insights Into Microbial Ecology 2, while all other data were analyzed using the Mixed Models procedure of Statistical Analysis System. Effects of treatment, transport, and treatment × transport were tested, with P < 0.05 being considered significant. Transport stress increased fecal indole concentrations and relative abundances of fecal Actinobacteria, Collinsella, Slackia, Ruminococcus, and Eubacterium. In contrast, relative abundances of fecal Fusobacteria, Streptococcus, and Fusobacterium were reduced by transport. Fecal characteristics, metabolites, and bacterial alpha and beta diversity measures were not affected by diet alone. Several diet × transport interactions were significant, however. Following transport, relative abundance of fecal Turicibacter increased in SCFP-supplemented dogs, but decreased in controls. Following transport, relative abundances of fecal Proteobacteria, Bacteroidetes, Prevotella, and Sutterella increased in controls, but not in SCFP-supplemented dogs. In contrast, relative abundances of fecal Firmicutes, Clostridium, Faecalibacterium, and Allobaculum increased and fecal Parabacteroides and Phascolarctobacterium decreased after transport stress in SCFP-supplemented dogs, but not in controls. Our data demonstrate that both transport stress and SCFP alter fecal microbiota in dogs, with transport being the primary cause for shifts. SCFP supplementation may provide benefits to dogs undergoing transport stress, but more research is necessary to determine proper dosages. More research is also necessary to determine if and how transport stress impacts gastrointestinal microbiota and other indicators of health.

The objective of this study was to determine the fecal characteristics, microbiota, and metabolites of dogs supplemented with a Saccharomyces cerevisiae fermentation product (SCFP) and subjected to transport stress. Thirty-six adult dogs were randomly assigned to a control diet or an SCFP-supplemented diet (N = 18 per group) and fed for 11 wk. At that time, a transport stress challenge was conducted. Fresh fecal samples were collected for measurement of general characteristics, microbiota, and metabolites before and after transport stress. Transport stress increased fecal indoles and Actinobacteria, Collinsella, Slackia, Ruminococcus, and Eubacterium populations and decreased fecal Fusobacteria, Streptococcus, and Fusobacterium populations. Fecal characteristics, metabolites, and bacterial alpha and beta diversity measures were not affected by diet alone, but several diet × transport interactions were significant. Following transport, fecal Turicibacter increased in SCFP-supplemented dogs, but decreased in controls. Following transport, fecal Proteobacteria, Bacteroidetes, Prevotella, and Sutterella increased in controls, but not in SCFP-supplemented dogs. Fecal Firmicutes, Clostridium, Faecalibacterium, and Allobaculum increased and fecal Parabacteroides and Phascolarctobacterium decreased after transport stress in SCFP-supplemented dogs, but not in controls. Our data demonstrate that both transport stress and SCFP alter fecal microbiota in dogs. SCFP supplementation may provide benefits to dogs undergoing stress, but proper dosages need to be determined.

Effects of a Lactobacillus fermentation product on the fecal characteristics, fecal microbial populations, immune function, and stress markers of adult dogs.

The objective of this study was to measure the effects of a Lactobacillus fermentation product (LBFP) on fecal characteristics and microbiota, blood biomarkers, immune function, and serum oxidative stress markers of adult dogs. Thirty adult beagle dogs [23 M, 7 F; mean age = 8.47 ± 2.65 yr old; mean BW = 15.43 ± 4.17 kg] were used in a completely randomized design study. All dogs were fed a basal diet to maintain BW for 5 wk, followed by baseline blood and fecal sample collections. Dogs remained on the same diet, but then were randomly assigned to a placebo (dextrose) or LBFP supplement (Limosilactobacillus fermentum and Lactobacillus delbrueckii). Both treatments were dosed at 4 mg/kg BW via gelatin capsule for 5 wk (n = 15/treatment). Fecal and blood samples were collected at that time. Change from baseline data were analyzed using the Mixed Models procedure of SAS 9.4, with P < 0.05 being significant and P < 0.10 being trends. Most circulating metabolites and immunoglobulins (Ig) were unaltered by treatment, but LBFP-supplemented dogs had lower changes in serum corticosteroid isoenzyme of alkaline phosphatase (P < 0.05), alanine aminotransferase (P < 0.10), and IgM (P < 0.10) than controls. The change in fecal scores tended to be lower (P = 0.068) in LBFP-supplemented dogs than controls, signifying firmer feces in LBFP-supplemented dogs. Regarding the fecal microbiota, alpha diversity indicators tended to be higher (P = 0.087) in LBFP-supplemented dogs than controls. One fecal bacterial phylum (Actinobacteriota) was altered by treatments, with its relative abundance tending to have a greater (P < 0.10) increase in controls than LBFP-supplemented dogs. Fifteen bacterial genera were altered (P < 0.05 or P < 0.10) by treatments, including relative abundances of fecal Peptoclostridium, Sarcina, and Faecalitalea that had a greater (P < 0.05) increase in controls than LBFP-supplemented dogs. In contrast, relative abundances of fecal Faecalibaculum, Bifidobacterium, and uncultured Butyricicoccaceae had a greater (P ≤ 0.05) increase in LBFP-supplemented dogs than controls. After week 5, dogs underwent transport stress (45-min vehicle ride) to assess oxidative stress markers. The change in serum superoxide dismutase after transport had a greater (P < 0.0001) increase in LBFP-supplemented dogs than controls. Our data suggest that LBFP may provide benefits to dogs by stabilizing stool quality, beneficially shifting fecal microbiota, and protecting against oxidative damage when subjected to stress.

Our objective was to measure the effects of a Lactobacillus fermentation product (LBFP) on fecal characteristics and microbiota, immune function, and oxidative stress markers of dogs. Thirty adult dogs were used in a completely randomized design study. All dogs were fed a basal diet to maintain body weight for 5 wk and then randomly assigned to a placebo or LBFP supplement for five more weeks. Fecal and blood samples were collected after baseline and treatment phases. Change from baseline data were analyzed statistically. Most blood markers were unaltered by treatment, but LBFP-supplemented dogs had lower changes in liver enzymes and IgM than controls. Change in fecal scores tended to be lower in LBFP-supplemented dogs than controls, signifying firmer feces. Fecal bacterial alpha diversity tended to be higher in LBFP-supplemented dogs than controls. One fecal bacterial phylum and 15 bacterial genera were altered by treatments. After 5 wk, dogs underwent transport stress (45-min vehicle ride) to assess oxidative stress markers. The increase in serum superoxide dismutase after transport was greater in LBFP-supplemented dogs than controls. Our data suggest that LBFP may provide benefits to dogs by stabilizing stool quality, beneficially shifting fecal microbiota, and protecting against oxidative damage when undergoing stress.

Effects of weight loss and feeding specially formulated diets on the body composition, blood metabolite profiles, voluntary physical activity, and fecal metabolites and microbiota of obese dogs.

Canine obesity negatively influences health and well-being, but can be managed by altering diet composition and caloric intake. Restricted feeding, dietary intervention, and consequent weight loss may be used to improve health and modify gastrointestinal microbiota. In this study, we aimed to determine the effects of restricted feeding of specially formulated foods on weight loss, body composition, voluntary physical activity, serum hormones and oxidative stress markers, and fecal metabolites and microbiota populations of obese dogs. Twenty-four obese dogs [body weight (BW) = 15.2 ±â€…1.7 kg; body condition score (BCS) = 8.7 ±â€…0.4; muscle condition score (MCS) = 3.5 ±â€…0.3; age = 7.2 ±â€…1.6 yr] were used in a 24-wk study. A control (OR) food was fed during a 4-wk baseline to identify intake needed to maintain BW. After baseline, dogs were allotted to one of two diets: OR or test (FT), and then fed to lose 1.5% BW/wk. Food intake, BW, BCS, and MCS were measured, blood and fecal samples were collected, DEXA scans were performed, and voluntary physical activity was measured over time. Microbiota data were evaluated using QIIME2 and change from baseline data from other measures were evaluated using the Mixed Models procedure of SAS, with P < 0.05 being significant. Restricted feeding led to reduced BW, BCS, fat mass, and blood cholesterol, triglyceride, glucose, and leptin concentrations, and increased MCS and lean body mass percentage. Blood cholesterol reduction was greater in dogs fed FT vs. OR. Fecal metabolites and bacterial alpha-diversity were affected by diet and weight loss. Dogs fed FT had greater reductions in fecal short-chain fatty acid, branched-chain fatty acid, and ammonia concentrations than those fed OR. Dogs fed OR had a higher alpha-diversity than those fed FT. Weight loss increased alpha-diversity (weeks 16, 20, and 24 > weeks 0 and 4). Beta-diversity showed separation between dietary groups and between week 0 and all other time points after week 8. Weight loss increased fecal Allobaculum and Ruminococcus torques. Weight loss also increased fecal Bifidobacterium, Faecalibaculum, and Parasutterella, but were greater in dogs fed OR. Weight loss decreased fecal Collinsella, Turicibacter, Blautia, Ruminococcus gnavus, Faecalibacterium, and Peptoclostridium, but were greater in dogs fed OR. In summary, restricted feeding promoted safe weight and fat loss, reduced blood lipid and leptin concentrations, and altered fecal microbiota of obese dogs.

In this study, we aimed to determine the effects of restricted feeding of specially formulated foods on weight loss, body composition, voluntary physical activity, serum hormones and oxidative stress markers, and fecal metabolites and microbiota populations of obese dogs. A control (OR) food was fed during a 4-wk baseline to identify intake needed to maintain the body weight (BW). After baseline, dogs were allotted to one of two diets: OR or test (FT) and then fed to lose 1.5% BW per week for 24 wk. Restricted feeding and weight loss led to reduced BW, body condition score, fat mass, and blood cholesterol, triglyceride, glucose and leptin concentrations and increased muscle condition score and lean body mass percentage. The reduction in blood cholesterol was greater in dogs fed FT vs. OR. Fecal metabolites and bacterial alpha-diversity were affected by diet and weight loss, with dogs fed with OR having a higher alpha-diversity than those fed with FT. Restricted feeding and weight loss increased alpha-diversity, affected beta-diversity, and impacted the relative abundances of nearly 20 bacterial genera. In summary, restricted feeding with high-protein, low-starch kibble diets promoted safe weight and fat loss, reduced blood lipid and leptin concentrations, and altered fecal microbiota of obese dogs.

Amino acid digestibility and nitrogen-corrected true metabolizable energy of mildly cooked human-grade vegan dog foods using the precision-fed cecectomized and conventional rooster assays.

The pet food market is constantly changing and adapting to meet the needs and desires of pets and their owners. One trend that has been growing in popularity lately is the feeding of fresh, human-grade foods. Human-grade pet foods contain ingredients that have all been stored, handled, processed, and transported in a manner that complies with regulations set for human food production. While most human-grade pet foods are based on animal-derived ingredients, vegan options also exist. To our knowledge, no in vivo studies have been conducted to analyze the performance of human-grade vegan diets. Therefore, the objective of this study was to investigate the amino acid (AA) digestibility and nitrogen-corrected true metabolizable energy (TME n ) of mildly cooked human-grade vegan dog foods using precision-fed cecectomized rooster and conventional rooster assays. Three commercial dog foods were tested. Two were mildly cooked human-grade vegan dog diets (Bramble Cowbell diet (BC); Bramble roost diet (BR)), while the third was a chicken-based extruded dog diet (chicken and brown rice recipe diet (CT)). Prior to the rooster assays, both mildly cooked diets were lyophilized, and then all three diets were ground. Diets were fed to cecectomized roosters to determine AA digestibility, while conventional roosters were used to determine TME n . All data were analyzed using the mixed models procedure of SAS (version 9.4). The majority of indispensable and dispensable AA across all diets had digestibilities higher than 80%, with a few exceptions (BC: histidine, lysine, threonine, and valine; BR: histidine). The only difference in indispensable AA digestibility among diets was observed with tryptophan, with its digestibility being higher (P = 0.0163) in CT than in BC. TME n values were higher (P = 0.006) in BC and BR (4.55 and 4.66 kcal/g dry matter, respectively) than that in CT (3.99 kcal/g dry matter). The TME n /GE was also higher (P = 0.0193) in BR than in CT. Metabolizable energy (ME) estimates using Atwater factors accurately estimated the energy content of CT, but modified Atwater factors and the predictive equations for ME recommended by the National Research Council underestimated energy content. All calculations underestimated the measured TME n values of BC and BR, with Atwater factors being the closest. Although testing in dogs is required, these data suggest that mildly cooked human-grade vegan dog diets are well-digested. Moreover, TME n data suggest that existing methods and equations underestimate the ME of the mildly cooked human-grade vegan foods tested.

Apparent total tract macronutrient digestibility of mildly cooked human-grade vegan dog foods and their effects on the blood metabolites and fecal characteristics, microbiota, and metabolites of adult dogs.

Vegan, mildly cooked, and human-grade dog foods are becoming more popular, as beliefs and views of pet owners change. To our knowledge, however, dog studies have not examined the digestibility of commercial vegan diets. Therefore, the objective of this study was to determine the apparent total tract digestibility (ATTD) of mildly cooked human-grade vegan dog foods and their effects on blood metabolites and fecal microbiota, characteristics, and metabolites of adult dogs consuming them. Three commercial dog foods were tested. Two were mildly cooked human-grade vegan dog diets, while the third was a chicken-based extruded dog diet. Twelve healthy adult female beagles (7.81 ± 0.65 kg; 7.73 ± 1.65 yr) were used in a replicated 3 × 3 Latin Square design. The study consisted of three experimental periods, with each composed of a 7 d diet adaptation phase, 15 d of consuming 100% of the diet, a 5 d phase for fecal collection for ATTD measurement, and 1 d for blood collection for serum chemistry and hematology. During the fecal collection period, a fresh sample was collected for fecal scoring and dry matter, pH, metabolite, and microbiota measurements. All data were analyzed using the Mixed Models procedure of SAS (version 9.4). All three diets were shown to be highly digestible, with all macronutrients having digestibility values above 80%. The vegan diets had higher (P < 0.001) ATTD of fat, but lower (P < 0.05) ATTD of organic matter than the extruded diet. Dogs consuming the vegan diets had lower circulating cholesterol (P < 0.001), triglyceride (P < 0.001), and platelet (P < 0.009) concentrations and lower (P < 0.010) blood neutrophil percentages than dogs consuming the extruded diet. Dogs consuming vegan diets had lower (P < 0.001) fecal dry matter percentages, lower (P < 0.001) fecal phenol and indole concentrations, and higher (P = 0.05) fecal short-chain fatty acid concentrations than those consuming the extruded diet. Fecal bacterial alpha and beta diversities were not different (P > 0.05) among diets, but dogs consuming vegan diets had altered (P < 0.05) relative abundances of nearly 20 bacterial genera when compared with those consuming the extruded diet. In conclusion, the mildly cooked human-grade vegan dog foods tested in this study performed well, resulting in desirable fecal characteristics, ATTD, and serum chemistries. The vegan diets tested also led to positive changes to serum lipids and fecal metabolites, and interesting changes to the fecal microbial community.

Vegan, mildly cooked, and human-grade dog foods are increasing in popularity, but few studies have been performed to examine their performance. Our objective was to determine the apparent total tract digestibility (ATTD) of mildly cooked human-grade vegan dog foods and their effects on blood metabolites and fecal microbiota, characteristics, and metabolites of dogs. Two mildly cooked human-grade vegan dog diets and a chicken-based extruded dog diet were tested using 12 healthy adult dogs in a replicated 3 × 3 Latin Square design. All diets were highly digestible, with all macronutrients having digestibility values >80%. Vegan diets had higher ATTD of fat, but lower ATTD of organic matter than the extruded diet. Dogs consuming vegan diets had lower circulating cholesterol, triglycerides, platelets, and neutrophils than dogs consuming the extruded diet. Dogs consuming vegan diets had lower fecal dry matter percentages and phenol and indole concentrations, and higher fecal short-chain fatty acid concentrations than those consuming the extruded diet. Finally, ~20 bacterial genera were altered between dogs consuming vegan and extruded diets. In conclusion, the mildly cooked human-grade vegan dog foods tested performed well, resulting in desirable fecal characteristics, high ATTD, adequate serum chemistries, positive changes to serum lipids and fecal metabolites, and interesting changes to fecal microbiota.

Evaluation of high-protein diets differing in protein source in healthy adult dogs.

Given the dynamic market for protein-based ingredients in the pet food industry, demand continues to increase for both plant- and animal-based options. Protein sources contain different amino acid (AA) profiles and vary in digestibility, affecting protein quality. The objective of this study was to evaluate the apparent total tract digestibility (ATTD) of canine diets differing in protein source and test their effects on serum metabolites and fecal characteristics, metabolites, and microbiota of healthy adult dogs consuming them. Four extruded diets were formulated to be isonitrogenous and meet the nutrient needs for adult dogs at maintenance, with the primary difference being protein source: 1) fresh deboned, dried, and spray-dried chicken (DC), 2) chicken by-product meal (CBPM), 3) wheat gluten meal (WGM), and 4) corn gluten meal (CGM). Twelve adult spayed female beagles (body weight [BW] = 9.9 ± 1.0 kg; age = 6.3 ± 1.1 yr) were used in a replicated 4 × 4 Latin square design (n = 12/treatment). Each period consisted of a 22-d adaptation phase, 5 d for fecal collection, and 1 d for blood collection. Fecal microbiota data were analyzed using QIIME 2.2020.8. All other data were analyzed using the Mixed Models procedure of SAS version 9.4. Fecal scores were higher (P < 0.05; looser stools) in dogs fed DC or CBPM than those fed WGM or CGM, but all remained within an appropriate range. Dry matter ATTD was lower (P < 0.05) in dogs fed CBPM or CGM than those fed DC or WGM. Crude protein ATTD was lower (P < 0.05) in dogs fed DC or CGM than those fed WGM. Dogs fed CBPM had lower (P < 0.05) organic matter, crude protein, and energy ATTD than those fed the other diets. Fecal indole was higher (P < 0.05) in dogs fed CBPM than those fed WGM. Fecal short-chain fatty acids were higher (P < 0.05) in dogs fed DC than those fed CGM. Fecal branched-chain fatty acids were higher (P < 0.05) in dogs fed DC or CBPM than those fed WGM. Fecal ammonia was higher (P < 0.05) in dogs fed DC or CBPM than those fed WGM or CGM. The relative abundances of three bacterial phyla and nine bacterial genera were shifted among treatment groups (P < 0.05). Considering AA profiles and digestibility data, the DC diet protein sources provided the highest quality protein without additional AA supplementation, but the animal-based protein diets resulted in higher fecal proteolytic metabolites. Further studies evaluating moderate dietary protein concentrations are needed to better compare plant- and animal-based protein sources.

Pet food trends are constantly changing. Because consumers are often focused on dietary proteins, with ingredient sources, dietary inclusion levels, and processing methods being important, they are a popular research topic. Protein sources contain different amino acid (AA) profiles and vary in digestibility, affecting protein quality. Our objective was to evaluate the apparent total tract digestibility of canine diets differing in protein source and test their effects on serum metabolites and fecal characteristics, metabolites, and microbiota of healthy adult dogs. Test diets were formulated to be similar nutritionally, but differed in protein source: fresh deboned, dried, and spray-dried chicken (DC), chicken by-product meal (CBPM), wheat gluten meal (WGM), and corn gluten meal (CGM). Fecal scores were higher in dogs fed chicken-based diets, but remained within an appropriate range. Dogs fed CBPM had lower nutrient and energy digestibilities than those fed the other diets, with protein digestibility also being lower in dogs fed DC or CGM than those fed WGM. Fecal metabolites and microbiota were shifted among diets, with animal-based protein diets increasing fecal protein metabolites. All diets were complete and balanced and performed well. When considering AA profiles and digestibility, however, the DC diet provided the highest protein quality.

Amino acid digestibility and protein quality of mealworm-based ingredients using the precision-fed cecectomized rooster assay.

Mealworms may serve as an alternative protein source for pet foods because of their high protein content and low environmental footprint. The amino acid (AA) content and protein quality of mealworm-based ingredients may vary depending on their composition and processing, however, so testing is required. Our objective was to measure the AA composition, AA digestibility, and protein quality of mealworm-based ingredients using the precision-fed cecectomized rooster assay. The University of Illinois Institutional Animal Care and Use Committee approved all animal procedures prior to experimentation. Sixteen cecectomized roosters (4 roosters per substrate) were randomly allotted to one of four test substrates: 1) whole lesser mealworm (A. diaperinus) meal (ADw); 2) defatted lesser mealworm (A. diaperinus) meal (ADd); 3) defatted yellow mealworm (T. molitor) meal (TMd); and 4) hydrolyzed T. molitor protein meal (TMh). Ingredients were provided by Ynsect, France. After 26 h of feed withdrawal, roosters were tube-fed test substrates. Following crop intubation, excreta samples were collected for 48 h. Endogenous loss corrections for AA were made by using five additional cecectomized roosters. All data were analyzed using SAS version 9.4. All substrates had high AA digestibilities, with all indispensable AA digestibilities being >90% with the exception of histidine (87.9% to 91.1%) and valine (77.9% to 79.7%). Amino acid digestibilities were not different among substrates (P > 0.05). Digestible indispensable AA score (DIAAS)-like values were calculated to determine protein quality according to Association of American Feed Control Officials (AAFCO) nutrient profiles, The European Pet Food Industry Nutritional Guidelines (FEDIAF) nutritional guidelines, National Research Council (NRC) recommended allowances for adult dogs, adult cats, growing puppies, and growing kittens, and NRC minimal requirements for growing puppies and growing kittens. In general, TMh had the highest and TMd had the lowest DIAAS-like values for most indispensable AA. Methionine (TMh; TMd; ADw) and phenylalanine (ADd) were the first-limiting AA. Our results demonstrate that mealworm-based ingredients are high-quality protein sources. Further research in dogs and cats is necessary to confirm sufficient palatability and digestibility, but these data suggest that they are valuable sources of protein for pet foods.

Mealworms may serve as an alternative protein source for pet foods because of their high protein content and low environmental footprint. The amino acid (AA) content and protein quality of mealworm-based ingredients may vary depending on their composition and processing, however, so testing is required. Our objective was to measure the AA composition, AA digestibility, and protein quality of the following ingredients using the precision-fed cecectomized rooster assay: 1) defatted yellow mealworm (Tenebrio molitor) flour (TMd); 2) hydrolyzed T. molitor proteins (TMh); 3) whole lesser mealworm (Alphitobius diaperinus) flour (ADw); and 4) defatted lesser mealworm (A. diaperinus) flour (ADd). All ingredients had high AA digestibilities, with all indispensable AA digestibilities being >90% with the exception of histidine and valine. Digestible indispensable AA score (DIAAS)-like values were calculated to determine protein quality for adult dogs, adult cats, growing puppies, and growing kittens. In general, TMh had the highest and TMd had the lowest DIAAS-like values for most indispensable AA. Methionine (TMh; TMd; ADw) and phenylalanine (ADd) were the first-limiting AA. Our results demonstrate that mealworm-based ingredients are high-quality protein sources. Dog and cat research is necessary, but these data suggest that they are valuable sources of protein for pet foods.

Effects of a milk oligosaccharide biosimilar on fecal characteristics, microbiota, and bile acid, calprotectin, and immunoglobulin concentrations of healthy adult dogs treated with metronidazole.

In recent dog and cat experiments, a novel milk oligosaccharide biosimilar (GNU100) positively modulated fecal microbiota and metabolite profiles, suggesting benefits to gastrointestinal health. The objective of this study was to investigate the effects of GNU100 on the fecal characteristics, microbiota, and bile acid (BA) concentrations of healthy adult dogs treated with antibiotics. Twelve healthy adult female dogs (mean age: 3.74 ± 2.4 yr) were used in an 8-wk crossover design study (dogs underwent both treatments). All dogs were fed a control diet during a 2-wk baseline, then randomly allotted to 1 of 2 treatments (diet only or diet + 1% GNU100) for another 6 wk. From weeks 2 to 4, dogs were orally administered metronidazole (20 mg/kg BW) twice daily. Fecal scores were recorded daily and fresh fecal samples were collected at weeks 2, 4, 5, 6, and 8 for measurement of pH, dry matter, microbiota populations, and BA, immunoglobulin A, and calprotectin concentrations. On weeks 0, 4, and 8, blood samples were collected for serum chemistry and hematology analysis. All data were analyzed as repeated measures using the Mixed Models procedure of SAS version 9.4, with significance considered P < 0.05. Metronidazole increased (P < 0.0001) fecal scores (looser stools) and modified (P < 0.05) fecal microbiota and BA profiles. Using qPCR, metronidazole reduced fecal Blautia, Fusobacterium, Turicibacter, Clostridium hiranonis, and Faecalibacterium abundances, and increased fecal Streptococcus and Escherichia coli abundances. DNA sequencing analysis demonstrated that metronidazole reduced microbial alpha diversity and influenced the relative abundance of 20 bacterial genera and families. Metronidazole also increased primary BA and reduced secondary BA concentrations. Most antibiotic-induced changes returned to baseline by week 8. Fecal scores were more stable (P = 0.01) in GNU100-fed dogs than controls after antibiotic administration. GNU100 also influenced fecal microbiota and BA profiles, reducing (P < 0.05) the influence of metronidazole on microbial alpha diversity and returning some fecal microbiota and secondary BA to baseline levels at a quicker (P < 0.05) rate than controls. In conclusion, our results suggest that GNU100 supplementation provides benefits to dogs treated with antibiotics, providing more stable fecal scores, maintaining microbial diversity, and allowing for quicker recovery of microbiota and secondary BA profiles which play an essential role in gut health.

Our objective was to test the effects of a novel milk oligosaccharide biosimilar (GNU100) on the fecal characteristics, microbiota, and bile acid (BA) concentrations of healthy adult dogs treated with antibiotics. Dogs were fed a control diet during a 2-wk baseline, then randomly allotted to 1 of 2 treatments (diet only or diet + 1% GNU100) for another 6 wk. From weeks 2 to 4, dogs were given an oral antibiotic. Fecal scores were recorded and fresh fecal samples were collected over time to assess fecal characteristics, microbiota populations, and BA concentrations. The antibiotic was shown to increase fecal scores (looser stools) and modify fecal microbiota populations (altered diversity and ~20 bacterial genera and families) and BA profiles (increased primary and reduced secondary BA). Most antibiotic-induced changes returned to baseline by week 8. In dogs fed GNU100, fecal scores were more stable and changes to microbial diversity were lower than controls after antibiotic administration. Fecal microbiota and secondary BA of GNU100-fed dogs also returned to baseline levels at a quicker rate than controls. These results suggest that GNU100 provides benefits to dogs given antibiotics, providing more stable fecal scores, maintaining microbial diversity, and allowing for quicker recovery of microbiota and BA profiles.

Effects of a Saccharomyces cerevisiae fermentation product-supplemented diet on fecal characteristics, oxidative stress, and blood gene expression of adult dogs undergoing transport stress.

Previously, a Saccharomyces cerevisiae fermentation product (SCFP) was shown to positively alter fecal microbiota, fecal metabolites, oxidative stress, and circulating immune cell function of adult dogs. The objective of this study was to measure the effects of SCFP on fecal characteristics, serum oxidative stress biomarkers, and whole blood gene expression of dogs undergoing transport stress. Sixteen adult pointer dogs [8M, 8F; mean age = 6.7 ± 2.1 yr; mean body weight (BW) = 25.5 ± 3.9 kg] were used in a randomized crossover design study. All dogs were fed a control diet for 4 wk, then randomly assigned to a control or SCFP-supplemented diet (formulated to include approximately 0.13% of the active SCFP ingredient) and fed to maintain BW for 11 wk. A 6-wk washout preceded the second 11-wk experimental period with dogs receiving opposite treatments. After 11 wk, fresh fecal and blood samples were collected before and after transport in a van for 45 min. Change from baseline data (i.e., before and after transport) were analyzed using the Mixed Models procedure of SAS 9.4, with P < 0.05 being significant and P < 0.10 being trends. Change in serum malondialdehyde concentrations increased (P < 0.05) and serum 8-isoprostane concentrations tended to increase (P < 0.10) in dogs fed SCFP, but decreased (P < 0.05) in control dogs after transport. Other serum markers were unaffected by diet during transport stress. Fecal dry matter percentage tended to be affected (P < 0.10) by diet during transport stress, being reduced in control dogs, but stable in dogs fed SCFP. Other fecal characteristics were unaffected by diet during transport stress. Genes associated with activation of innate immunity were impacted by diet in response to transport stress, with blood cyclooxygenase-2 and malondialdehyde mRNA expression being increased (P < 0.05) in control dogs, but stable or decreased in dogs fed SCFP. Expression of other genes was unaffected by diet during transport stress. These data suggest that the benefits of feeding a SCFP during transport stress may be mediated through suppression of innate immune cell activation.

Saccharomyces cerevisiae fermentation product (SCFP) is a yeast product containing bioactive fermentation metabolites, residual yeast cells, and yeast cell wall fragments. In this study, SCFP was investigated for its impacts on fecal characteristics and oxidative stress of dogs undergoing transport stress. Using a randomized crossover study design, 16 adult pointer dogs were used to compare changes in fecal characteristics, oxidative stress marker concentrations, and gene expression when fed a SCFP-supplemented diet or control diet. After transport, change in serum malondialdehyde concentrations increased and serum 8-isoprostane concentrations tended to increase in dogs fed SCFP, but decreased in control dogs. Fecal moisture percentage tended to be affected by diet during transport stress, being reduced in control dogs, but stable in dogs fed SCFP. Blood cyclooxygenase-2 and myeloperoxidase mRNA gene expression was affected by diet during transport stress, being increased in control dogs, but stable or decreased in dogs fed SCFP. In conclusion, these data suggest that the benefits of feeding a SCFP during transport stress may be mitigated through suppression of innate immune cell activation rather than through suppressing oxidative damage to lipids.

Effects of a Saccharomyces cerevisiae fermentation product on fecal characteristics, metabolite concentrations, and microbiota populations of dogs subjected to exercise challenge.

The objective of this study was to determine the fecal characteristics, microbiota, and metabolites of dogs fed a Saccharomyces cerevisiae fermentation product (SCFP) and subjected to exercise challenge in untrained and trained states. Thirty-six adult dogs (18 male, 18 female; mean age: 7.1 yr; mean body weight: 29.0 kg) were randomly assigned to control or SCFP-supplemented (250 mg/dog/d) diets and fed for 10 wk. After 3 wk, dogs were given an exercise challenge (6.5 km run), with fresh fecal samples collected pre- and post-challenge. Dogs were then trained by a series of distance-defined running exercise regimens over 7 wk (two 6.4 km runs/wk for 2 wk; two 9.7 km runs/wk for 2 wk; two 12.9 km runs/wk for 2 wk; two 3.2 km runs/wk). Dogs were then given exercise challenge (16 km run) in the trained state, with fresh fecal samples collected pre- and post-challenge. Fecal microbiota data were evaluated using QIIME2, while all other data were analyzed using the Mixed Models procedure of SAS. Effects of diet, exercise, and diet*exercise were tested with P < 0.05 considered significant. Exercise challenge reduced fecal pH and ammonia in both treatments, and in untrained and trained dogs. After the exercise challenge in untrained dogs, fecal indole, isobutyrate, and isovalerate were reduced, while acetate and propionate were increased. Following the exercise challenge in trained dogs, fecal scores and butyrate decreased, while isobutyrate and isovalerate increased. SCFP did not affect fecal scores, pH, dry matter, or metabolites, but fecal Clostridium was higher in controls than in SCFP-fed dogs over time. SCFP and exercise challenge had no effect on alpha or beta diversity in untrained dogs. However, the weighted principal coordinate analysis plot revealed clustering of dogs before and after exercise in trained dogs. After exercise challenge, fecal Collinsella, Slackia, Blautia, Ruminococcus, and Catenibacterium were higher and Bacteroides, Parabacteroides, Prevotella, Phascolarctobacterium, Fusobacterium, and Sutterella were lower in both untrained and trained dogs. Using qPCR, SCFP increased fecal Turicibacter, and tended to increase fecal Lactobacillus vs. controls. Exercise challenge increased fecal Turicibacter and Blautia in both untrained and trained dogs. Our findings show that exercise and SCFP may affect the fecal microbiota of dogs. Exercise was the primary cause of the shifts, however, with trained dogs having more profound changes than untrained dogs.

The objective of this study was to determine the fecal characteristics, microbiota, and metabolites of dogs fed a Saccharomyces cerevisiae fermentation product (SCFP) and subjected to exercise challenge in untrained and trained states. Thirty-six adult dogs were randomly assigned to control or SCFP-supplemented (250 mg/d) diets and fed for 10 wk. An exercise challenge was administered while dogs were in an untrained state and a trained state (after 7 wk of an exercise regimen), with fresh fecal samples collected pre- and post-challenge. Exercise challenge reduced fecal pH and ammonia in all dogs. After the exercise challenge in untrained dogs, fecal indole, isobutyrate, and isovalerate concentrations were reduced, while acetate and propionate concentrations were increased. Following exercise challenge in trained dogs, fecal scores and butyrate concentrations decreased, while isobutyrate and isovalerate increased. SCFP reduced fecal Clostridium over time vs. controls. Beta diversity analysis revealed clustering of dogs before and after exercise in trained dogs. After exercise challenge, over 10 bacterial genera were altered in untrained and trained dogs. Our findings show that exercise and SCFP may affect the fecal microbiota of dogs, but exercise was the primary cause of the shifts and trained dogs had more profound changes than untrained dogs.