Usefulness of muscle condition score and ultrasonographic measurements for assessment of muscle mass in cats with cachexia and sarcopenia.

OBJECTIVE: To compare muscle condition scores (MCSs) and muscle ultrasonographic measurements in cats with and without muscle loss and to evaluate repeatability and reproducibility of MCS assessment. ANIMALS: 40 cats of various ages, body condition scores (BCSs), and MCSs. PROCEDURES: A prospective cross-sectional study was conducted. Body weight, BCS, MCS, epaxial muscle height (EMH), vertebral epaxial muscle score (VEMS), and forelimb epaxial muscle score (FLEMS) were assessed in each cat. The MCS for each cat was assessed 3 separate times by each of 5 raters. RESULTS: The MCS was significantly correlated with EMH (r = 0.59), VEMS (r = 0.66), and FLEMS (r = 0.41). For MCS, the overall value of the κ coefficient for interrater agreement (reproducibility) was 0.43 and the overall value of the κ coefficient for intrarater agreement (repeatability) ranged from 0.49 to 0.76. CONCLUSIONS AND CLINICAL RELEVANCE: Ultrasonographic measurements of muscle may be useful for assessing muscle loss in individual cats over time. However, for the cats of this study, no advantage was observed for assessment of VEMS or FLEMS over EMH. Substantial repeatability and moderate reproducibility were shown when MCS was used for assessment of muscle mass in cats. Prospective ultrasonographic studies are warranted to evaluate the usefulness of MCS and EMH assessment for evaluation of changes in muscle mass of cats over time.

Evaluation of the use of muscle condition score and ultrasonographic measurements for assessment of muscle mass in dogs.

OBJECTIVE: To evaluate repeatability and reproducibility of muscle condition score (MCS) in dogs with various degrees of muscle loss; to compare MCS, muscle ultrasonographic measurements, and quantitative magnetic resonance (QMR) measurements; and to identify cutoff values for ultrasonographic measurements of muscle that can be used to identify dogs with cachexia and sarcopenia. ANIMALS: 40 dogs of various age, body condition score (BCS), and MCS. PROCEDURES: A prospective cross-sectional study was conducted. Body weight, BCS, QMR measurements, thoracic radiographic measurements, and muscle ultrasonographic measurements were assessed once in each dog. The MCS for each dog was assessed 3 separate times by 4 separate raters. RESULTS: For the MCS, overall κ for interrater agreement was 0.50 and overall κ for intrarater agreement ranged from 0.59 to 0.77. For both interrater and intrarater agreement, κ coefficients were higher for dogs with normal muscle mass and severe muscle loss and lower for dogs with mild and moderate muscle loss. The MCS was significantly correlated with age (r = -0.62), vertebral epaxial muscle score (VEMS; r = 0.71), forelimb epaxial muscle score (FLEMS; r = 0.58), and BCS (r = 0.73), and VEMS was significantly correlated (r = 0.84) with FLEMS. Cutoff values for identification of mild muscle loss determined by use of VEMS and FLEMS were 1.124 and 1.666, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: MCS had substantial repeatability and moderate reproducibility for assessment of muscle mass in dogs. Prospective studies of MCS, VEMS, and FLEMS for assessment of muscle mass in dogs are warranted.

Fecal metabolomics of healthy breast-fed versus formula-fed infants before and during in vitro batch culture fermentation.

Nontargeted metabolomics analyses were used (1) to compare fecal metabolite profiles of healthy breast-fed (BF) and formula-fed (FF) infants before and during in vitro fermentation in batch culture and (2) to evaluate fecal metabolomics in assessing infant diet. Samples from healthy BF (n = 4) or FF (n = 4) infants were individually incubated at 37( °)C in anaerobic media containing 1% (wt/vol) galactooligosaccharides, 6'-sialyllactose, 2'-fucosyllactose, lacto-N-neotetraose, inulin, and gum arabic for up to 6 h, and supernatants were analyzed using GC/MS and LC/MS/MS to assess changes in various compounds. Comparison of over 250 metabolites prior to incubation showed that BF samples contained higher relative concentrations (P ≤ 0.05) of 14 compounds including human milk oligosaccharides and other metabolites presumably transferred through breast feeding (linoelaidate, myo-inositol) (P ≤ 0.05). Conversely, feces from FF infants contained 41 identified metabolites at higher levels (P ≤ 0.05) with many indicative of carbon limitation and protein fermentation. Our data are consistent with the notion that carbon-limited cultures catabolize protein and amino acids to obtain energy, whereas the provision of fermentable carbohydrate creates anabolic conditions relying on amino acids for bacterial growth. Results also suggest that fecal metabolomics can be a useful tool for studying interactions among diet, microbes, and host.

Subcutaneous and gonadal adipose tissue transcriptome differences in lean and obese female dogs.

Canine obesity leads to shortened life span and increased disease incidence. Adipose tissue depots are known to have unique metabolic and gene expression profiles in rodents and humans, but few comparisons of depot gene expression have been performed in the dog. Using microarray technology, our objective was to identify differentially expressed genes and enriched functional pathways between subcutaneous and gonadal adipose of lean and obese dogs to better understand the pathogenesis of obesity in the dog. Because no depot × body weight status interactions were identified in the microarray data, depot differences were the primary focus. A total of 946 and 703 transcripts were differentially expressed (FDR P < 0.05) between gonadal and subcutaneous adipose tissue in obese and lean dogs respectively. Of the adipose depot-specific differences in gene expression, 162 were present in both lean and obese dogs, with the majority (85%) expressed in the same direction. Both lean and obese dog gene lists had enrichment of the complement and coagulation cascade and systemic lupus erythematosus pathways. Obese dogs had enrichment of lysosome, extracellular matrix-receptor interaction, renin-angiotensin system and hematopoietic cell lineage pathways. Lean dogs had enrichment of glutathione metabolism and synthesis and degradation of ketone bodies. We have identified a core set of genes differentially expressed between subcutaneous and gonadal adipose tissue in dogs regardless of body weight. These genes contribute to depot-specific differences in immune function, extracellular matrix remodeling and lysosomal function and may contribute to the physiological differences noted between depots.

In vitro fermentation characteristics of select nondigestible oligosaccharides by infant fecal inocula.

This study sought to determine the fermentation potential of human milk oligosaccharides by mixed cultures of fecal microbiota from breast-fed (BF; n = 4) and formula-fed (FF; n = 4) infants. Infant fecal inocula were incubated with galactooligosaccharide (GOS), gum arabic (GA), HP inulin (HP), 2'-fucosyllactose (2'FL), 6'-sialyllactose (6'SL), and lacto-N-neotetraose (LNnt). GOS, 2'FL, and LNnT had a lower pH than other substrates after 3 h (P < 0.05). Total short chain fatty acids were greater in FF compared to BF infants at 6 h (P = 0.03) and 12 h (P = 0.01). GOS, 2'FL, and LNnT led to more lactate than 6'SL, HP, and GA (P < 0.05). Bifidobacteria populations were greater (P = 0.02) in FF at 6 and 12 h. Overall, GOS, 2'FL, and LNnT were rapidly fermented by infant fecal inocula, 6'SL and HP had intermediate fermentability, while GA had little fermentation. Inocula from FF infants fermented substrates more rapidly than inocula from BF infants, which should be accounted for when evaluating substrate fermentability. These data will aid in future infant formulas to promote optimal gut health in FF infants.

Dietary fibre fermentability but not viscosity elicited the 'second-meal effect' in healthy adult dogs.

The present study evaluated the effects of fibre fermentability and viscosity in a morning meal on glucose, insulin and glucagon-like peptide-1 (GLP-1) responses to a glucose challenge later in the day in six healthy female dogs. For this purpose, two Latin square design experiments were performed. In Expt 1, dogs were fed a low-fibre (LF; 1 % Solka-Floc (International Fiber Corporation) and 1% soya hulls) diet, a low-fermentable fibre (LFF; 5% Solka-Floc (International Fiber Corporation) and 3% soya hulls) diet or a high-fermentable fibre (HFF; 5% pectin and 3% short-chain fructo-oligosaccharides) diet. In Expt 2, dogs were fed a low-viscosity fibre (5% Solka-Floc (International Fiber Corporation) and 3% soya hulls) diet, a moderate-viscosity fibre (MVF; 2% Solka-Floc (International Fiber Corporation), 2% soya hulls, 2% psyllium and 2% pectin) diet or a high-viscosity fibre (HVF; 4% psyllium and 4% pectin) diet. Dogs were fed at 08.00, 12.00 and 16.00 hours on days 1-6 of each period. On day 7, dogs were fed at 08.00 hours and then dosed with maltodextrin at 12.00 hours. Data were analysed to identify baseline and incremental AUC (IAUC) changes among the treatments. In Expt 1, glucose IAUC(0-180) min was lower (P< 0.05) in dogs fed the HFF v. LF and LFF diets. Insulin and GLP-1 IAUC(0-180 min) were not affected. In Expt 2, baseline GLP-1 was greater (P< 0.005) and baseline insulin was lower (P< 0.05) in dogs fed the HVF v. MVF diet, but glucose, insulin and GLP-1 IAUC(0-180) min were not affected. In summary, HFF in a morning meal has the potential to decrease blood glucose response in a consequent meal.

The gut microbiome of kittens is affected by dietary protein:carbohydrate ratio and associated with blood metabolite and hormone concentrations.

High-protein, low-carbohydrate (HPLC) diets are common in cats, but their effect on the gut microbiome has been ignored. The present study was conducted to test the effects of dietary protein:carbohydrate ratio on the gut microbiota of growing kittens. Male domestic shorthair kittens were raised by mothers fed moderate-protein, moderate-carbohydrate (MPMC; n 7) or HPLC (n 7) diets, and then weaned at 8 weeks onto the same diet. Fresh faeces were collected at 8, 12 and 16 weeks; DNA was extracted, followed by amplification of the V4­V6 region of the 16S rRNA gene using 454 pyrosequencing. A total of 384 588 sequences (average of 9374 per sample) were generated. Dual hierarchical clustering indicated distinct clustering based on the protein:carbohydrate ratio regardless of age. The protein:carbohydrate ratio affected faecal bacteria. Faecal Actinobacteria were greater (P< 0·05) and Fusobacteria were lower (P< 0·05) in MPMC-fed kittens. Faecal Clostridium, Faecalibacterium, Ruminococcus, Blautia and Eubacterium were greater (P< 0·05) in HPLC-fed kittens, while Dialister, Acidaminococcus, Bifidobacterium, Megasphaera and Mitsuokella were greater (P< 0·05) in MPMC-fed kittens. Principal component analysis of faecal bacteria and blood metabolites and hormones resulted in distinct clusters. Of particular interest was the clustering of blood TAG with faecal Clostridiaceae, Eubacteriaceae, Ruminococcaceae, Fusobacteriaceae and Lachnospiraceae; blood ghrelin with faecal Coriobacteriaceae, Bifidobacteriaceae and Veillonellaceae; and blood glucose, cholesterol and leptin with faecal Lactobacillaceae. The present results demonstrate that the protein:carbohydrate ratio affects the faecal microbiome, and highlight the associations between faecal microbes and circulating hormones and metabolites that may be important in terms of satiety and host metabolism.

Effects of dietary fiber on the feline gastrointestinal metagenome.

Four healthy adult cats were used in a crossover design to determine phylogeny and metabolic functional capacity of the cat's gastrointestinal microbiota using a metagenomic approach. Healthy adult cats (1.7 years old) were fed diets containing 4% cellulose, fructooligosaccharides (FOS), or pectin for 30 d, at which time fresh fecal samples were collected. Fecal DNA samples from each cat consuming each diet were subjected to 454 pyrosequencing. Dominant phyla determined using two independent databases (MG-RAST and IMG/M) included Firmicutes (mean=36.3 and 49.8%, respectively), Bacteroidetes (mean=36.1 and 24.1%, respectively), and Proteobacteria (mean=12.4 and 11.1%, respectively). Primary functional categories as determined by KEGG were associated with carbohydrates, clustering-based subsystems, protein metabolism, and amino acids and derivatives. Primary functional categories as determined by COG were associated with amino acid metabolism and transport, general function prediction only, and carbohydrate transport and metabolism. Analysis of carbohydrate-active enzymes revealed modifications in several glycoside hydrolases, glycosyl transferases, and carbohydrate-binding molecules with FOS and pectin consumption. While the cat is an obligate carnivore, its gut microbiome is similar regarding microbial phylogeny and gene content to omnivores.

Effects of inulin or yeast cell-wall extract on nutrient digestibility, fecal fermentative end-product concentrations, and blood metabolite concentrations in adult dogs fed raw meat-based diets.

OBJECTIVE: To determine the effects of raw meat-based diets with and without inulin or yeast cell-wall (YCW) extract on macronutrient digestibility, blood cell counts, serum metabolite concentrations, and fecal fermentative end-product concentrations in healthy adult dogs. ANIMALS: 6 healthy adult spayed female dogs (mean ± SD age, 5.5 ± 0.5 years; mean body weight, 8.5 ± 0.5 kg). PROCEDURES: Dogs were fed each of the following 6 diets for 21 days, the order of which was randomly assigned in a Latin square design: beef control, beef and 1.4% inulin, beef and 1.4% YCW extract, chicken control, chicken and 1.4% inulin, and chicken and 1.4% YCW extract. Each diet trial consisted of a phase for diet adaptation (days 0 to 14) and a phase for measurement of urine and fecal output and content (days 15 to 20). On day 21, food was withheld for blood sample collection. Afterward, the next diet trial began immediately. RESULTS: All dogs maintained desirable fecal quality characteristics and produced low fecal volume. All diets were highly digestible (protein digestibility > 88%; fat digestibility > 97%). Differences in fermentative end-product concentrations among all diets were minor, but a significant increase in fecal short-chain fatty acid concentrations was evident when dogs were fed beef-based diets with inulin and YCW extract. Fecal spermine concentrations were higher with diets containing inulin and YCW extract than with control diets. Blood cell counts and serum metabolite values were within reference limits after each trial. All diets resulted in maintenance of nitrogen balance. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested the raw meat-based diets evaluated were highly digestible in dogs. The increase in fecal short-chain fatty acid concentrations achieved when inulin and YCW extract were included may be beneficial to canine health.

Acute satiety response of mammalian, avian and fish proteins in dogs.

Fish proteins have been reported to be more satiating than meat proteins. The objective was to determine the effect of different animal protein pre-meals on satiety. A total of ten intact female hounds were fed pork loin, beef loin, chicken breast, salmon fillet or pollock fillet. Each pre-meal was fed to contain 100 g protein. Blood was collected at 0, 5, 15, 30, 60, 90 and 120 min postprandially and analysed for glucose, insulin, total ghrelin, active glucagon-like peptide-1 (GLP-1) and plasma amino acids (AA). Dogs were fed 2 ×  metabolisable energy, 3 h following the pre-meal, and intake was determined 30, 60, 180 and 1440 min after food presentation. Glucose decreased over time (P < 0·001), but was lowest (P = 0·01) when dogs consumed pollock or chicken. Insulin increased (P < 0·0001) over time, and was greater (P = 0·09) when dogs consumed salmon. GLP-1 increased (P < 0·001) over time, and was greatest (P = 0·04) when dogs consumed beef. Ghrelin decreased (P < 0·0001) over time for all pre-meals. The tryptophan:large neutral AA ratio tended to be greater (P = 0·08) when dogs consumed pork, salmon and pollock. Different protein sources may influence blood markers in dogs, but it does not appear that fish substrates have different satiating abilities than mammalian or avian sources.

Adipose tissue transcriptome changes during obesity development in female dogs.

During the development of obesity, adipose tissue undergoes major expansion and remodeling, but the biological processes involved in this transition are not well understood. The objective of this study was to analyze global gene expression profiles of adipose tissue in dogs, fed a high-fat diet, during the transition from a lean to obese phenotype. Nine female beagles (4.09 ± 0.64 yr; 8.48 ± 0.35 kg) were randomized to ad libitum feeding or body weight maintenance. Subcutaneous adipose tissue biopsy, blood, and dual x-ray absorptiometry measurements were collected at 0, 4, 8, 12, and 24 wk of feeding. Serum was analyzed for glucose, insulin, fructosamine, triglycerides, free fatty acids, adiponectin, and leptin. Formalin-fixed adipose tissue was used for determination of adipocyte size. Adipose RNA samples were hybridized to Affymetrix Canine 2.0 microarrays. Statistical analysis, using repeated-measures ANOVA, showed ad libitum feeding increased (P < 0.05) body weight (0 wk, 8.36 ± 0.34 kg; 24 wk, 14.64 ± 0.34 kg), body fat mass (0 wk, 1.36 ± 0.24 kg; 24 wk, 6.52 ± 0.24 kg), adipocyte size (0 wk, 114.66 ± 17.38 µm(2); 24 wk, 320.97 ± 0.18.17 µm(2)), and leptin (0 wk, 0.8 ± 1.0 ng/ml; 24 wk, 12.9 ± 1.0 ng/ml). Microarrays displayed 1,665 differentially expressed genes in adipose tissue as weight increased. Alterations were seen in adipose tissue homeostatic processes including metabolism, oxidative stress, mitochondrial homeostasis, and extracellular matrix. Adipose transcriptome changes highlight the dynamic and adaptive response to ad libitum feeding and obesity development.

Age and diet affect gene expression profiles in canine liver tissue.

BACKGROUND: The liver plays a central role in nutrient and xenobiotic metabolism, but its functionality declines with age. Senior dogs suffer from many of the chronic hepatic diseases as elderly humans, with age-related alterations in liver function influenced by diet. However, a large-scale molecular analysis of the liver tissue as affected by age and diet has not been reported in dogs. METHODOLOGY/PRINCIPAL FINDINGS: Liver tissue samples were collected from six senior (12-year old) and six young adult (1-year old) female beagles fed an animal protein-based diet (APB) or a plant protein-based diet (PPB) for 12 months. Total RNA in the liver tissue was extracted and hybridized to Affymetrix GeneChip® Canine Genome Arrays. Using a 2.0-fold cutoff and false discovery rate <0.10, our results indicated that expression of 234 genes was altered by age, while 137 genes were differentially expressed by diet. Based on functional classification, genes affected by age and/or diet were involved in cellular development, nutrient metabolism, and signal transduction. In general, gene expression suggested that senior dogs had an increased risk of the progression of liver disease and dysfunction, as observed in aged humans and rodents. In particular for aged liver, genes related to inflammation, oxidative stress, and glycolysis were up-regulated, whereas genes related to regeneration, xenobiotic metabolism, and cholesterol trafficking were down-regulated. Diet-associated changes in gene expression were more common in young adult dogs (33 genes) as compared to senior dogs (3 genes). CONCLUSION: Our results provide molecular insight pertaining to the aged canine liver and its predisposition to disease and abnormalities. Therefore, our data may aid in future research pertaining to age-associated alterations in hepatic function or identification of potential targets for nutritional management as a means to decrease incidence of age-dependent liver dysfunction.

Gene expression profiles of colonic mucosa in healthy young adult and senior dogs.

BACKGROUND: We have previously reported the effects of age and diet on nutrient digestibility, intestinal morphology, and large intestinal fermentation patterns in healthy young adult and senior dogs. However, a genome-wide molecular analysis of colonic mucosa as a function of age and diet has not yet been performed in dogs. METHODOLOGY/PRINCIPAL FINDINGS: Colonic mucosa samples were collected from six senior (12-year old) and six young adult (1-year old) female beagles fed one of two diets (animal protein-based vs. plant protein-based) for 12 months. Total RNA in colonic mucosa was extracted and hybridized to Affymetrix GeneChip® Canine Genome Arrays. Results indicated that the majority of gene expression changes were due to age (212 genes) rather than diet (66 genes). In particular, the colonic mucosa of senior dogs had increased expression of genes associated with cell proliferation, inflammation, stress response, and cellular metabolism, whereas the expression of genes associated with apoptosis and defensive mechanisms were decreased in senior vs. young adult dogs. No consistent diet-induced alterations in gene expression existed in both age groups, with the effects of diet being more pronounced in senior dogs than in young adult dogs. CONCLUSION: Our results provide molecular insight pertaining to the aged canine colon and its predisposition to dysfunction and disease. Therefore, our data may aid in future research pertaining to age-associated gastrointestinal physiological changes and highlight potential targets for dietary intervention to limit their progression.

Phylogenetic characterization of fecal microbial communities of dogs fed diets with or without supplemental dietary fiber using 454 pyrosequencing.

BACKGROUND: Dogs suffer from many of the same maladies as humans that may be affected by the gut microbiome, but knowledge of the canine microbiome is incomplete. This work aimed to use 16S rDNA tag pyrosequencing to phylogenetically characterize hindgut microbiome in dogs and determine how consumption of dietary fiber affects community structure. PRINCIPAL FINDINGS: Six healthy adult dogs were used in a crossover design. A control diet without supplemental fiber and a beet pulp-supplemented (7.5%) diet were fed. Fecal DNA was extracted and the V3 hypervariable region of the microbial 16S rDNA gene amplified using primers suitable for 454-pyrosequencing. Microbial diversity was assessed on random 2000-sequence subsamples of individual and pooled DNA samples by diet. Our dataset comprised 77,771 reads with an average length of 141 nt. Individual samples contained approximately 129 OTU, with Fusobacteria (23-40% of reads), Firmicutes (14-28% of reads) and Bacteroidetes (31-34% of reads) being co-dominant phyla. Feeding dietary fiber generally decreased Fusobacteria and increased Firmicutes, but these changes were not equally apparent in all dogs. UniFrac analysis revealed that structure of the gut microbiome was affected by diet and Firmicutes appeared to play a strong role in by-diet clustering. CONCLUSIONS: Our data suggest three co-dominant bacterial phyla in the canine hindgut. Furthermore, a relatively small amount of dietary fiber changed the structure of the gut microbiome detectably. Our data are among the first to characterize the healthy canine gut microbiome using pyrosequencing and provide a basis for studies focused on devising dietary interventions for microbiome-associated diseases.

Carbohydrates blended with polydextrose lower gas production and short-chain fatty acid production in an in vitro system.

Maximizing health benefits of prebiotics, while limiting negative side effects, is of importance to the food industry. This study examined several oligosaccharides and their blends in an in vitro fermentation model. Substrates included medium- and long-chain fructooligosaccharides (FOS), oligofructose-enriched inulin, galactooligosaccharide, polydextrose (POL), and 50:50 substrate blends. Substrates and blends were fermented in vitro using human fecal inoculum, and fermentation characteristics were quantified at 0, 4, 8, and 12 hours. We hypothesized that mixtures of short- and long-chain oligosaccharides would generate less gas than do short-chain oligosaccharides and modulate gut microflora to a greater extent than do long-chain oligosaccharides. Carbohydrates blended with POL had decreased (P < .01) total gas volume and H(2) produced after 4, 8, and 12 hours of fermentation compared with individual carbohydrates. Mixing of 2 oligofructose-enriched inulin products led to less (P < .05) gas produced and a slower (P < .05) rate of production. When mixed with POL, all carbohydrates tested in the present study produced less total short-chain fatty acids (P < .04) and butyrate (P < .0001) after 12 hours of in vitro fermentation, compared with individual carbohydrates. The bifidogenic effect of medium-chain FOS and oligofructose-enriched inulin after 12 hours of in vitro fermentation was lower (P < .05) when mixed with POL. Mixing the pure carbohydrates with galactooligosaccharide increased (P < .05) bifidobacteria counts measured after 12 hours of in vitro fermentation, except when mixed with medium-chain FOS. In general, when mixed with POL, all carbohydrates had lower gas production, gas production rates, butyrate and total short-chain fatty acid production, and bifidobacteria counts than when fermented alone for 12 hours.