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Evaluation of a novel animal milk oligosaccharide biosimilar: macronutrient digestibility and gastrointestinal tolerance, fecal metabolites, and fecal microbiota of healthy adult dogs and in vitro genotoxicity assays.

ABSTRACT: Milk oligosaccharides (MO) are bioactive compounds in mammalian milk that provide health benefits to neonates beyond essential nutrients. GNU100, a novel animal MO biosimilar, was recently tested in vitro, with results showing beneficial shifts in microbiota and increased short-chain fatty acid (SCFA) production, but other effects of GNU100 were unknown. Three studies were conducted to evaluate the safety, palatability, and gastrointestinal (GI) tolerance of GNU100. In study 1, the mutagenic potential of GNU100 was tested using a bacterial reverse mutation assay and a mammalian cell micronucleus test. In study 2, palatability was assessed by comparing diets containing 0% vs. 1% GNU100 in 20 adult dogs. In study 3, 32 adult dogs were used in a completely randomized design to assess the safety and GI tolerance of GNU100 and explore utility. Following a 2-wk baseline, dogs were assigned to one of four treatments and fed for 26 wk: 0%, 0.5%, 1%, and 1.5% GNU100. On weeks 2, 4, and 26, fresh fecal samples were collected to measure stool quality, immunoglobulin A, and calprotectin, and blood samples were collected to measure serum chemistry, inflammatory markers, and hematology. On weeks 2 and 4, fresh fecal samples were collected to measure metabolites and microbiota. On week 4, total feces were collected to assess apparent total tract macronutrient digestibility. Although revertant numbers were greater compared with the solvent control in tester strain WP2uvrA(pKM101) in the presence of metabolic activation (S9) in the initial experiment, they remained below the threshold for a positive mutagenic response in follow-up confirmatory tests, supporting that GNU100 is not mutagenic. Similarly, no cytotoxicity or chromosome damage was observed in the cell micronucleus test. The palatability test showed that 1% GNU100 was strongly preferred (P < 0.05; 3.6:1 consumption ratio) over the control. In study 3, all dogs were healthy and had no signs of GI intolerance or illness. All diets were well accepted, and food intake, fecal characteristics, metabolite concentrations, and macronutrient digestibilities were not altered. GNU100 modulated fecal microbiota, increasing evenness and Catenibacterium, Megamonas, and Prevotella (SCFA producers) and reducing Collinsella. Overall, the results suggest that GNU100 is palatable and well-tolerated, causes no genotoxicity or adverse effects on health, and beneficially shifts the fecal microbiota, supporting the safety of GNU100 for the inclusion in canine diets.

SUBMITTER: Lee AH

PROVIDER: S-EPMC7851890 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:GNU100 is a novel animal milk oligosaccharide (AMO) biosimilar. In a recent in vitro fermentation study, GNU100 was shown to be fermentable by feline gastrointestinal microbiota and lead to increased short-chain fatty acid production. Our objectives herein were to evaluate the palatability, safety, and gastrointestinal tolerance of GNU100 in healthy adult cats. Exploratory end-points were measured to assess utility. In study 1, 20 adult cats were used to test the palatability of diets containing 0% or 1% GNU100. In study 2, 32 (mean age = 1.9 yr; mean body weight = 4.6 kg) male (n = 12) and female (n = 20) adult cats were used in a completely randomized design. After a 2-wk baseline, cats were assigned to one of the following treatment groups and fed for 26 wk: control (CT, no GNU100), low dose (LD, 0.5% GNU100), medium dose (MD, 1.0% GNU100), and high dose (HD, 1.5% GNU100). On weeks 2, 4, and 26, fresh fecal samples were collected for the measurement of stool quality and immune and inflammatory markers and on weeks 2 and 4 for microbiota and metabolites. On week 4, total feces were collected to measure apparent total tract macronutrient digestibility. On weeks 2, 4, and 26, blood samples were collected for serum chemistry, hematology, and inflammatory marker measurement. The palatability test showed that 1% GNU100 was strongly preferred (P < 0.05), with GNU100 having a 17.6:1 consumption ratio compared with control. In the long-term study, all cats remained healthy, without any signs of gastrointestinal intolerance or illness. All diets were well accepted, resulting in similar (P > 0.05) food intake, fecal characteristics, immunoglobulin A, and calprotectin, and dry matter, organic matter, fat, and crude protein digestibilities. Fecal butyrate was greater (P = 0.02) in cats fed HD than cats fed LD or MD. Fecal indole was lower (P = 0.02) in cats fed HD than cats fed LD. Cats fed CT had a higher (P = 0.003) relative abundance of Actinobacteria than cats fed LD. The relative abundance of Peptococcus was impacted by diet and time. At 4 wk, Campylobacter was lower in fecal samples of cats fed HD. Overall, the data suggest that dietary GNU100 supplementation was highly palatable, well tolerated, did not cause detrimental effects on fecal quality or nutrient digestibility, increased fecal butyrate concentrations, and reduced fecal indole concentrations, supporting the safety of GNU100 for inclusion in feline diets and suggesting potential benefits on gastrointestinal health of cats.

Project description: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.

Project description:Obesity and estrogen reduction are known to affect the gut microbiota and gut microbial-derived metabolites in some species, but limited information is available in dogs. The aim of this study was to determine the effects of dietary macronutrient profile on apparent total tract macronutrient digestibility, fecal microbiota, and fecal metabolites of adult female dogs after spay surgery. Twenty-eight adult intact female beagles (age: 3.02 ± 0.71 yr, BW: 10.28 ± 0.77 kg; BCS: 4.98 ± 0.57) were used. After a 5-wk baseline phase (week 0), 24 dogs were spayed and randomly allotted to one of three experimental diets (n = 8 per group): 1) control (CO) containing moderate protein and fiber (COSP), 2) high-protein, high-fiber (HPHF), or 3) high-protein, high-fiber plus omega-3 and medium-chain fatty acids (HPHFO). Four dogs were sham-operated and fed CO (COSH). All dogs were fed to maintain BW for 12 wk after spay and then allowed to consume twice that amount for 12 wk. Fecal samples were collected at weeks 0, 12, and 24 for digestibility, microbiota, and metabolite analysis. All data were analyzed using repeated measures and linear mixed models procedure of SAS 9.4, with results reported as a change from baseline. Apparent organic matter and energy digestibilities had greater decreases in HPHF and HPHFO than COSH and COSP. Increases in fecal acetate, total short-chain fatty acids, and secondary bile acids were greater and decreases in primary bile acids were greater in HPHF and HPHFO. Principal coordinates analysis of weighted UniFrac distances revealed that HPHF and HPHFO clustered together and separated from COSH and COSP at weeks 12 and 24, with relative abundances of Faecalibacterium, Romboutsia, and Fusobacterium increasing to a greater extent and Catenibacterium, Bifidobacterium, Prevotella 9, Eubacterium, and Megamonas decreasing to a greater extent in HPHF or HPHFO. Our results suggest that high-protein, high-fiber diets alter nutrient and energy digestibilities, fecal metabolite concentrations, and fecal gut microbiota, but spay surgery had minor effects. Future research is needed to investigate how food intake, nutrient profile, and changes in hormone production influence gut microbiota and metabolites of dogs individually and how this knowledge may be used to manage spayed pets.

Project description:Resistant starch (RS) is fermentable by gut microbiota and effectively modulates fecal short-chain fatty acid concentrations in pigs, mice, and humans. RS may have similar beneficial effects on the canine gut but has not been well studied. The objective of this study was to evaluate the effects of 0%, 1%, 2%, 3%, and 4% dietary RS (Hi-maize 260) on apparent total tract macronutrient digestibility, fecal characteristics, fermentative end-product concentrations, and microbiota of healthy adult dogs. An incomplete 5 × 5 Latin square design with seven dogs and five experimental periods was used, with each treatment period lasting 21 d (days 0 to 17 adaptation; days 18 to 21 fresh and total fecal collection) and each dog serving as its own control. Seven dogs (mean age = 5.3 yr; mean body weight = 20 kg) were randomly allotted to one of five treatments formulated to be iso-energetic and consisting of graded amounts of 100% amylopectin cornstarch, RS, and cellulose and fed as a top dressing on the food each day. All dogs were fed the same amount of a basal diet throughout the study, and fresh water was offered ad libitum. The basal diet contained 6.25% RS (dry matter [DM] basis), contributing approximately 18.3 g of RS/d based on their daily food intake (292.5 g DM/d). Data were evaluated for linear and quadratic effects using SAS. The treatments included 0%, 1%, 2%, 3%, and 4% of an additional RS source. Because Hi-maize 260 is approximately 40% digestible and 60% indigestible starch, the dogs received the following amounts of RS daily: 0% = 18.3 g (18.3 + 0 g), 1% = 20.1 g (18.3 + 1.8 g), 2% = 21.9 g (18.3 + 3.6 g), 3% = 23.7 g (18.3 + 5.4 g), and 4% = 25.5 g (18.3 + 7.2 g). Apparent total tract DM, organic matter, crude protein, fat, and gross energy digestibilities and fecal pH were linearly decreased (P < 0.05) with increased RS consumption. Fecal output was linearly increased (P < 0.05) with increased RS consumption. Fecal scores and fecal fermentative end-product concentrations were not affected by RS consumption. Although most of the fecal microbial taxa were not altered, Faecalibacterium were increased (P < 0.05) with increased RS consumption. The decrease in fecal pH and increase in fecal Faecalibacterium would be viewed as being beneficial to gastrointestinal health. Although our results seem to indicate that RS is poorly and/or slowly fermentable in dogs, the lack of observed change may have been due to the rather high level of RS contained in the basal diet.

Project description:Milk oligosaccharides (MO) confer multiple potential physiological benefits, such as the selective growth promotion of beneficial microbiota, inhibition of enteric pathogen growth and adhesion to enterocytes, maturation of the gut mucosal barrier, and modulation of the gastrointestinal immune system. This study was conducted to determine the fermentation potential of GNU100, an animal MO biosimilar, in an in vitro system using healthy canine and feline fecal inocula. Single feline and single canine fecal samples were used to inoculate a batch fermentation system. Tubes containing a blank control (BNC), GNU100 at 0.5% (5 g/L; GNU1), or GNU100 at 1.0% (10 g/L; GNU2) were incubated for 48 h. Gas pressure, pH, lactate, short-chain fatty acids (SCFA; acetate, propionate, and butyrate), and branched-chain fatty acids (BCFA; isobutyrate, isovalerate, and valerate) were measured after 6, 24, and 48 h. Ammonium and microbiota (total bacteria by flow cytometry and Pet-16Seq; Lactobacillus and Bifidobacterium by quantitative polymerase chain reaction ) were measured after 24 and 48 h. Data were analyzed using the Mixed Models procedure of SAS. Substrates were considered to be a fixed effect and replicates considered to be a random effect. Tukey's multiple comparison analysis was used to compare least squares means, with differences considered significant with P < 0.05. In feline and canine incubations, SCFA increases were greater (P < 0.0001) in GNU100 compared with BNC, with acetate making up the largest SCFA proportion (P < 0.0001). GNU100 cultures led to greater increases (P < 0.0001) in lactate and ammonium than BNC in the feline incubations. GNU100 cultures led to greater increases (P < 0.0001) in ammonium than BNC in canine incubations and greater increases (P < 0.0001) in BCFA than BNC in feline incubations. Pet-16Seq microbial profiles from the feline and canine fecal incubations exhibited a modulation after GNU100 fermentation, with a reduction of the genera Escherichia/Shigella and Salmonella. In feline incubations, Bifidobacterium populations had greater increases (P < 0.0001) in GNU100 than BNC. In feline incubations, Lactobacillus populations had greater increases (P = 0.01) in GNU100 than BNC, with GNU1 leading to greater increases (P = 0.02) in Lactobacillus than BNC tubes in canine incubations. Overall, this study demonstrated that GNU100 was fermented in an in vitro fermentation system inoculated with canine and feline microbiota, resulting in the growth of beneficial bacteria and the production of SCFA, BCFA, and ammonium.

Project description:Human-grade (HG) pet foods are commercially available, but they have not been well studied. Our objective was to determine the apparent total tract digestibility (ATTD) of HG pet foods and evaluate their effects on fecal characteristics, microbiota, and metabolites, serum metabolites, and hematology of dogs. Twelve dogs (mean age = 5.5 ± 1.0; BW = 11.6 ± 1.6 kg) were used in a replicated 4 × 4 Latin square design (n = 12/treatment). The diets included 1) Chicken and Brown Rice Recipe (extruded; Blue Buffalo); 2) Roasted Meals Tender Chicken Recipe (fresh; Freshpet); 3) Beef and Russet Potato Recipe (HG beef; JustFoodForDogs); and 4) Chicken and White Rice Recipe (HG chicken; JustFoodForDogs). Each period consisted of 28 d, with a 6-d diet transition phase, 16 d of consuming 100% of the diet, a 5-d phase for fecal collection, and 1 d for blood collection. All data were analyzed using the Mixed Models procedure of SAS 9.4. Dogs fed the extruded diet required a higher (P < 0.05) daily food intake (dry matter basis, DMB) to maintain BW. The ATTD of dry matter (DM), organic matter (OM), energy, and acid-hydrolyzed fat (AHF) were greater (P < 0.05) in dogs fed the HG diets than those fed the fresh diet, and greater (P < 0.05) in dogs fed the fresh diet than those fed the extruded diet. Crude protein ATTD was lower (P < 0.05) for dogs fed the extruded diet than those fed all other diets. Dogs fed the extruded diet had greater (P < 0.05) fecal output (as-is; DMB) than dogs fed fresh (1.5-1.7 times greater) or HG foods (2.0-2.9 times greater). There were no differences in fecal pH, scores, and metabolites, but microbiota were affected by diet. Dogs fed HG beef had higher (P < 0.05) relative abundance of Bacteroidetes and lower (P < 0.05) relative abundance of Firmicutes than dogs fed the fresh or HG chicken diets. The Actinobacteria, Fusobacteria, Proteobacteria, and Spirochaetes phyla were unchanged (P > 0.05), but diet modified the relative abundance of nearly 20 bacterial genera. Similar to previous reports, these data demonstrate that the fecal microbiota of dogs fed HG or fresh diets is markedly different than those consuming extruded diets, likely due to ingredient, nutrient, and processing differences. Serum metabolites and hematology were not greatly affected by diet. In conclusion, the HG pet foods tested resulted in significantly reduced fecal output, were highly digestible, maintained fecal characteristics, serum chemistry, and hematology, and modified the fecal microbiota of dogs.

Dataset Information

Evaluation of a novel animal milk oligosaccharide biosimilar: macronutrient digestibility and gastrointestinal tolerance, fecal metabolites, and fecal microbiota of healthy adult dogs and in vitro genotoxicity assays.

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