Effects of the Dietary Protein and Carbohydrate Ratio on Gut Microbiomes in Dogs of Different Body Conditions.

Obesity has become a health epidemic in both humans and pets. A dysbiotic gut microbiota has been associated with obesity and other metabolic disorders. High-protein, low-carbohydrate (HPLC) diets have been recommended for body weight loss, but little is known about their effects on the canine gut microbiome. Sixty-three obese and lean Labrador retrievers and Beagles (mean age, 5.72 years) were fed a common baseline diet for 4 weeks in phase 1, followed by 4 weeks of a treatment diet, specifically, the HPLC diet (49.4% protein, 10.9% carbohydrate) or a low-protein, high-carbohydrate (LPHC) diet (25.5% protein, 38.8% carbohydrate) in phase 2. 16S rRNA gene profiling revealed that dietary protein and carbohydrate ratios have significant impacts on gut microbial compositions. This effect appeared to be more evident in obese dogs than in lean dogs but was independent of breed. Consumption of either diet increased the bacterial evenness, but not the richness, of the gut compared to that after consumption of the baseline diet. Macronutrient composition affected taxon abundances, mainly within the predominant phyla, Firmicutes and Bacteroidetes The LPHC diet appeared to favor the growth of Bacteroides uniformis and Clostridium butyricum, while the HPLC diet increased the abundances of Clostridium hiranonis, Clostridium perfringens, and Ruminococcus gnavus and enriched microbial gene networks associated with weight maintenance. In addition, we observed a decrease in the Bacteroidetes to Firmicutes ratio and an increase in the Bacteroides to Prevotella ratio in the HPLC diet-fed dogs compared to these ratios in dogs fed other diets. Finally, analysis of the effect of diet on the predicted microbial gene network was performed using phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt). IMPORTANCE: More than 50% of dogs are either overweight or obese in the United States. A dysbiotic gut microbiota is associated with obesity and other metabolic problems in humans. HPLC diets have been promoted as an effective weight loss strategy for many years, and potential effects were reported for both humans and dogs. In this study, we explored the influence of the protein and carbohydrate ratio on the gut microbiome in dogs with different body conditions. We demonstrated significant dietary effects on the gut microbiome, with greater changes in obese dogs than in lean dogs. The HPLC diet-fed dogs showed greater abundances of Firmicutes but fewer numbers of Bacteroidetes than other dogs. This knowledge will enable us to use prebiotics, probiotics, and other nutritional interventions to modulate the gut microbiota and to provide an alternative therapy for canine obesity.

A High-Resolution SNP Array-Based Linkage Map Anchors a New Domestic Cat Draft Genome Assembly and Provides Detailed Patterns of Recombination.

High-resolution genetic and physical maps are invaluable tools for building accurate genome assemblies, and interpreting results of genome-wide association studies (GWAS). Previous genetic and physical maps anchored good quality draft assemblies of the domestic cat genome, enabling the discovery of numerous genes underlying hereditary disease and phenotypes of interest to the biomedical science and breeding communities. However, these maps lacked sufficient marker density to order thousands of shorter scaffolds in earlier assemblies, which instead relied heavily on comparative mapping with related species. A high-resolution map would aid in validating and ordering chromosome scaffolds from existing and new genome assemblies. Here, we describe a high-resolution genetic linkage map of the domestic cat genome based on genotyping 453 domestic cats from several multi-generational pedigrees on the Illumina 63K SNP array. The final maps include 58,055 SNP markers placed relative to 6637 markers with unique positions, distributed across all autosomes and the X chromosome. Our final sex-averaged maps span a total autosomal length of 4464 cM, the longest described linkage map for any mammal, confirming length estimates from a previous microsatellite-based map. The linkage map was used to order and orient the scaffolds from a substantially more contiguous domestic cat genome assembly (Felis catus v8.0), which incorporated ∼20 × coverage of Illumina fragment reads. The new genome assembly shows substantial improvements in contiguity, with a nearly fourfold increase in N50 scaffold size to 18 Mb. We use this map to report probable structural errors in previous maps and assemblies, and to describe features of the recombination landscape, including a massive (∼50 Mb) recombination desert (of virtually zero recombination) on the X chromosome that parallels a similar desert on the porcine X chromosome in both size and physical location.

Veterinary Medicine and Multi-Omics Research for Future Nutrition Targets: Metabolomics and Transcriptomics of the Common Degenerative Mitral Valve Disease in Dogs.

Canine degenerative mitral valve disease (DMVD) is the most common form of heart disease in dogs. The objective of this study was to identify cellular and metabolic pathways that play a role in DMVD by performing metabolomics and transcriptomics analyses on serum and tissue (mitral valve and left ventricle) samples previously collected from dogs with DMVD or healthy hearts. Gas or liquid chromatography followed by mass spectrophotometry were used to identify metabolites in serum. Transcriptomics analysis of tissue samples was completed using RNA-seq, and selected targets were confirmed by RT-qPCR. Random Forest analysis was used to classify the metabolites that best predicted the presence of DMVD. Results identified 41 known and 13 unknown serum metabolites that were significantly different between healthy and DMVD dogs, representing alterations in fat and glucose energy metabolism, oxidative stress, and other pathways. The three metabolites with the greatest single effect in the Random Forest analysis were γ-glutamylmethionine, oxidized glutathione, and asymmetric dimethylarginine. Transcriptomics analysis identified 812 differentially expressed transcripts in left ventricle samples and 263 in mitral valve samples, representing changes in energy metabolism, antioxidant function, nitric oxide signaling, and extracellular matrix homeostasis pathways. Many of the identified alterations may benefit from nutritional or medical management. Our study provides evidence of the growing importance of integrative approaches in multi-omics research in veterinary and nutritional sciences.

Endogenous retrovirus insertion in the KIT oncogene determines white and white spotting in domestic cats.

The Dominant White locus (W) in the domestic cat demonstrates pleiotropic effects exhibiting complete penetrance for absence of coat pigmentation and incomplete penetrance for deafness and iris hypopigmentation. We performed linkage analysis using a pedigree segregating White to identify KIT (Chr. B1) as the feline W locus. Segregation and sequence analysis of the KIT gene in two pedigrees (P1 and P2) revealed the remarkable retrotransposition and evolution of a feline endogenous retrovirus (FERV1) as responsible for two distinct phenotypes of the W locus, Dominant White, and white spotting. A full-length (7125 bp) FERV1 element is associated with white spotting, whereas a FERV1 long terminal repeat (LTR) is associated with all Dominant White individuals. For purposes of statistical analysis, the alternatives of wild-type sequence, FERV1 element, and LTR-only define a triallelic marker. Taking into account pedigree relationships, deafness is genetically linked and associated with this marker; estimated P values for association are in the range of 0.007 to 0.10. The retrotransposition interrupts a DNAase I hypersensitive site in KIT intron 1 that is highly conserved across mammals and was previously demonstrated to regulate temporal and tissue-specific expression of KIT in murine hematopoietic and melanocytic cells. A large-population genetic survey of cats (n = 270), representing 30 cat breeds, supports our findings and demonstrates statistical significance of the FERV1 LTR and full-length element with Dominant White/blue iris (P < 0.0001) and white spotting (P < 0.0001), respectively.

Discrepancy between use of lean body mass or nitrogen balance to determine protein requirements for adult cats.

This study was undertaken to contrast the minimum protein intake needed to maintain nitrogen balance or lean body mass (LBM) in adult cats using a prospective evaluation of 24 adult, neutered male cats fed one to three different diets. Following a 1-month baseline period during which all cats consumed a 34% protein diet, cats were fed a 20% (LO), 26% (MOD) or 34% (HI) protein diet for 2 months. During the baseline period and following the 2-month feeding period, nitrogen balance was assessed using a 96-h complete collection of urine and feces, and LBM was assessed using dual energy X-ray absorptiometry. Weight loss increased in a linear manner with decreasing protein intake (P <0.01), despite no significant difference in calorie intake. Linear regression of the data indicated that approximately 1.5 g protein/kg (2.1 g/kg(0.75)) body weight is needed to maintain nitrogen balance, while 5.2 g protein/kg (7.8 g/kg(0.75)) body weight is needed to maintain LBM. This study provides evidence that nitrogen balance studies are inadequate for determining optimum protein requirements. Animals, including cats, can adapt to low protein intake and maintain nitrogen balance while depleting LBM. Loss of LBM and an associated reduction in protein turnover can result in compromised immune function and increased morbidity. Current Association of American Feed Control Officials (AAFCO) and National Research Council (NRC) standards for protein adequacy may not provide adequate protein to support LBM. The minimum daily protein requirement for adult cats appears to be at least 5.2 g/kg (7.8 g/kg(0.75)) body weight, well in excess of current AAFCO and NRC recommendations. Further research is needed to determine the effect, if any, of body condition, age and gender on protein requirements.

Wnt/ß-catenin signaling is downregulated but restored by nutrition interventions in the aged heart in mice.

Aging hallmarks include decreased progenitor cell functions. The Wnt/ß-catenin signaling pathway has emerged as a key player in cellular aging in recent years. Wnt activity changes in aged tissues including skin, serum, muscle and artery. In heart, it is hypothesized that Wnt signaling increases with aging and this signaling drives cardiac progenitor cells into fibrogenic lineage. However, experimental evidence supporting this hypothesis has not been established. Here we take a bioinformatics data mining approach, utilizing pre-existing data, to study cardiac aging gene expression data in mice. Contrary to the hypothesis, our study shows that the Wnt/ß-catenin signaling is down-regulated in aged heart in mice. Nutrition treatment, with calorie restriction and Resveratrol supplementation, known to retard aging, opposes heart aging by restoring Wnt/ß-catenin signaling level in the old heart. In addition, the expression of ß-catenin gene, a key regulator of the Wnt/ß-catenin signaling pathway, decreases up to 3-fold in aged heart, but is restored to levels found in young heart with methods of nutrition intervention. Combined with database search, our study suggests that some of bioflavonoids may have potential therapeutic benefits to heart aging.

Plasma phospholipid fatty acid and ex vivo neutrophil responses are differentially altered in dogs fed fish- and linseed-oil containing diets at the same n-6:n-3 fatty acid ratio.

The effect of diets containing either 18-carbon n-3 fatty acids (FA) or 20/22-carbon n-3 FA on canine plasma and neutrophil membrane fatty acid composition, superoxide and leukotriene B4 and B5 production when fed at the same n-6:n-3 fatty acid ratio was investigated. Four groups of ten dogs each were fed a low fat basal diet supplemented with safflower oil (SFO), beef tallow (BTO), linseed oil (LSO), or Menhaden fish oil (MHO) for 28 days. Dietary fat provided 40.8% of energy and the n-6:n-3 of the diets were ~100:1, 9.7:1, 0.38:1, and 0.34:1 for the SFO, BTO, LSO and MHO groups, respectively. The MHO and LSO groups had increased incorporation of EPA and DPA in both the plasma and neutrophil membranes compared to the BTO and SFO groups. DHA was observed in the MHO but not in the LSO group. Neutrophils from the MHO diet fed dogs had less LTB4 and greater LTB5 than the other three groups. The LSO group also showed a reduction in LTB4 and greater LTB5 production compared to the SFO and BTO groups. Both LSO and MHO groups had lower superoxide production compared to the SFO and BTO groups. Diets containing 18 or 20/22 carbon n-3 FA fed at the same n-6:n-3 resulted in differential incorporation of long chain n-3 FA into neutrophil membranes. Thus, fatty acid type and chain length individually affect neutrophil membrane structure and function and these effects exist independent of dietary total n-6:total n-3 FA ratios.

Defining and mapping mammalian coat pattern genes: multiple genomic regions implicated in domestic cat stripes and spots.

Mammalian coat patterns (e.g., spots, stripes) are hypothesized to play important roles in camouflage and other relevant processes, yet the genetic and developmental bases for these phenotypes are completely unknown. The domestic cat, with its diversity of coat patterns, is an excellent model organism to investigate these phenomena. We have established three independent pedigrees to map the four recognized pattern variants classically considered to be specified by a single locus, Tabby; in order of dominance, these are the unpatterned agouti form called "Abyssinian" or "ticked" (T(a)), followed by Spotted (T(s)), Mackerel (T(M)), and Blotched (t(b)). We demonstrate that at least three different loci control the coat markings of the domestic cat. One locus, responsible for the Abyssinian form (herein termed the Ticked locus), maps to an approximately 3.8-Mb region on cat chromosome B1. A second locus controls the Tabby alleles T(M) and t(b), and maps to an approximately 5-Mb genomic region on cat chromosome A1. One or more additional loci act as modifiers and create a spotted coat by altering mackerel stripes. On the basis of our results and associated observations, we hypothesize that mammalian patterned coats are formed by two distinct processes: a spatially oriented developmental mechanism that lays down a species-specific pattern of skin cell differentiation and a pigmentation-oriented mechanism that uses information from the preestablished pattern to regulate the synthesis of melanin profiles.

A domestic cat X chromosome linkage map and the sex-linked orange locus: mapping of orange, multiple origins and epistasis over nonagouti.

A comprehensive genetic linkage map of the domestic cat X chromosome was generated with the goal of localizing the genomic position of the classic X-linked orange (O) locus. Microsatellite markers with an average spacing of 3 Mb were selected from sequence traces of the cat 1.9x whole genome sequence (WGS), including the pseudoautosomal region 1 (PAR1). Extreme variation in recombination rates (centimorgans per megabase) was observed along the X chromosome, ranging from a virtual absence of recombination events in a region estimated to be >30 Mb to recombination frequencies of 15.7 cM/Mb in a segment estimated to be <0.3 Mb. This detailed linkage map was applied to position the X-linked orange gene, placing this locus on the q arm of the X chromosome, as opposed to a previously reported location on the p arm. Fine mapping placed the locus between markers at positions 106 and 116.8 Mb in the current 1.9x-coverage sequence assembly of the cat genome. Haplotype analysis revealed potential recombination events that could reduce the size of the candidate region to 3.5 Mb and suggested multiple origins for the orange phenotype in the domestic cat. Furthermore, epistasis of orange over nonagouti was demonstrated at the genetic level.

An autosomal genetic linkage map of the domestic cat, Felis silvestris catus.

We report on the completion of an autosomal genetic linkage (GL) map of the domestic cat (Felis silvestris catus). Unlike two previous linkage maps of the cat constructed with a hybrid pedigree between the domestic cat and the Asian leopard cat, this map was generated entirely with domestic cats, using a large multi-generational pedigree (n=256) maintained by the Nestlé Purina PetCare Company. Four hundred eighty-three simple tandem repeat (STR) loci have been assigned to linkage groups on the cat's 18 autosomes. A single linkage group spans each autosome. The length of the cat map, estimated at 4370 cM, is long relative to most reported mammalian maps. A high degree of concordance in marker order was observed between the third-generation map and the 1.5 Mb-resolution radiation hybrid (RH) map of the cat. Using the cat 1.9x whole-genome sequence, we identified map coordinates for 85% of the loci in the cat assembly, with high concordance observed in marker order between the linkage map and the cat sequence assembly. The present version represents a marked improvement over previous cat linkage maps as it (i) nearly doubles the number of markers that were present in the second-generation linkage map in the cat, (ii) provides a linkage map generated in a domestic cat pedigree which will more accurately reflect recombination distances than previous maps generated in a hybrid pedigree, and (iii) provides single linkage groups spanning each autosome. Marker order was largely consistent between this and the previous maps, though the use of a hybrid pedigree in the earlier versions appears to have contributed to some suppression of recombination. The improved linkage map will provide an added resource for the mapping of phenotypic variation in the domestic cat and the use of this species as a model system for biological research.

Four independent mutations in the feline fibroblast growth factor 5 gene determine the long-haired phenotype in domestic cats.

To determine the genetic regulation of "hair length" in the domestic cat, a whole-genome scan was performed in a multigenerational pedigree in which the "long-haired" phenotype was segregating. The 2 markers that demonstrated the greatest linkage to the long-haired trait (log of the odds > or = 6) flanked an estimated 10-Mb region on cat chromosome B1 containing the Fibroblast Growth Factor 5 (FGF5) gene, a candidate gene implicated in regulating hair follicle growth cycle in other species. Sequence analyses of FGF5 in 26 cat breeds and 2 pedigrees of nonbreed cats revealed 4 separate mutations predicted to disrupt the biological activity of the FGF5 protein. Pedigree analyses demonstrated that different combinations of paired mutant FGF5 alleles segregated with the long-haired phenotype in an autosomal recessive manner. Association analyses of more than 380 genotyped breed and nonbreed cats were consistent with mutations in the FGF5 gene causing the long-haired phenotype in an autosomal recessive manner. In combination, these genomic approaches demonstrated that FGF5 is the major genetic determinant of hair length in the domestic cat.

A homozygous single-base deletion in MLPH causes the dilute coat color phenotype in the domestic cat.

Three proteins have been described in humans and mice as being essential for even distribution, transport, and translocation of pigment granules, with defects in these molecules giving rise to lighter skin/coat color. The dilute phenotype in domestic cats affects both eumelanin and phaeomelanin pigment pathways; for example, black pigmentation combined with dilute appears gray and orange pigments appear cream. The dilute pigmentation segregates as a fully penetrant, autosomal recessive trait. We conducted classical linkage mapping with microsatellites in a large multigeneration pedigree of domestic cats and detected tight linkage for dilute on cat chromosome C1 (theta=0.08, LOD=10.81). Fine-mapping identified a genomic region exhibiting conserved synteny to human chromosome 2, which included one of the three dilute candidate genes, melanophilin (MLPH). Sequence analysis in dilute cats identified a single base pair deletion in exon 2 of MLPH transcripts that introduces a stop codon 11 amino acids downstream, resulting in the truncation of the bulk of the MLPH protein. The occurrence of this homozygous variant in 97 unrelated dilute cats representing 26 cat breeds and random-bred cats, along with 89 unrelated wild-type cats representing 29 breeds and random-bred cats, supports the finding that dilute is caused by this single mutation in MLPH (p<0.00001). Single-nucleotide polymorphism analyses in dilute individuals identified a single haplotype in dilute cats, suggesting that a single mutation event in MLPH gave rise to dilute in domestic cats.

Correlations of fatty acid supplementation, aeroallergens, shampoo, and ear cleanser with multiple parameters in pruritic dogs.

Seventy-two pruritic dogs were fed one of four diets controlled for n-6:n-3 fatty acid ratios and total dietary intake of fatty acids. Multiple parameters were evaluated, including clinical and cytological findings, aeroallergen testing, microbial sampling techniques, and effects of an anti-fungal/antibacterial shampoo and ear cleanser. Significant correlations were observed between many clinical parameters, anatomical sampling sites, and microbial counts when data from the diet groups was combined. There were no statistically significant differences between individual diets for any of the clinical parameters. The importance of total clinical management in the control of pruritus was demonstrated.

Diet modulates proteinuria in heterozygous female dogs with X-linked hereditary nephropathy.

Young adult heterozygous (carrier) female dogs with X-linked hereditary nephropathy (XLHN) have glomerular proteinuria but are otherwise healthy. Because data regarding dietary influences on the magnitude of proteinuria in dogs with spontaneous glomerular disease are not available, 12 such dogs were studied in a double crossover experiment intended to determine effects of altering dietary protein intake for up to 6 weeks. Dogs were blocked by urine protein : creatinine ratio (UPC) and randomly assigned to receive 2 diets: high protein (34.6% dry matter [DM], HP) or low protein (14.1% DM, LP) fed in HP-LP-HP or LP-HP-LP sequence. Food intake was measured daily, body weight (BW) was measured twice weekly, and UPC, plasma creatinine, blood urea nitrogen, phosphorus, albumin, and protein concentrations were measured at 2-week intervals. Nutrient digestibility was measured during the third treatment period. Diet had a significant effect (P < .0001) on all measured variables except plasma phosphorus (P > .5), but unintended differences in digestibility of protein and energy (P < or = .01) prevented assignment of the diet effect exclusively to protein. Proteinuria was greater (UPC 4.7 +/- 2.2 versus 1.8 +/- 1.1, P < .0001) when the HP diet was fed, but the LP diet did not maintain starting BW or plasma albumin concentration within the normal reference range. Diet greatly affects the magnitude of proteinuria in XLHN carrier females. Dietary protein restriction can reduce proteinuria in dogs with glomerular disease, but BW and blood protein concentrations may not be maintained if the restriction is too severe.

Effect of n-3 fatty acid ratio and dose on clinical manifestations, plasma fatty acids and inflammatory mediators in dogs with pruritus.

The use of n-3 fatty acids is often recommended to manage pruritus. The purpose of this study was to determine the effect of various doses of n-3 fatty acids at different n-6:n-3 ratios on plasma fatty acids, clinical response and inflammatory mediators in pruritic dogs. After baseline assessment, dogs were randomly assigned to receive diets varying in both total n-3 and n-6 fatty acid dose and n-6:n-3 ratio. The total clinical score decreased significantly in all four diet groups after 8 weeks with no difference between groups. Plasma fatty acid changes generally mirrored the fatty acid content of the test diets, although alterations appeared to depend on both the dose of n-3 fatty acids and the n-6:n-3 ratio. In this clinical trial, which controlled dietary intake of fatty acids, n-3 fatty acid supplementation did not appear to have an added benefit on clinical signs over thorough clinical management.

Molecular genetics and evolution of melanism in the cat family.

Melanistic coat coloration occurs as a common polymorphism in 11 of 37 felid species and reaches high population frequency in some cases but never achieves complete fixation. To investigate the genetic basis, adaptive significance, and evolutionary history of melanistic variants in the Felidae, we mapped, cloned, and sequenced the cat homologs of two putative candidate genes for melanism (ASIP [agouti] and MC1R) and identified three independent deletions associated with dark coloration in three different felid species. Association and transmission analyses revealed that a 2 bp deletion in the ASIP gene specifies black coloration in domestic cats, and two different "in-frame" deletions in the MC1R gene are implicated in melanism in jaguars and jaguarundis. Melanistic individuals from five other felid species did not carry any of these mutations, implying that there are at least four independent genetic origins for melanism in the cat family. The inferred multiple origins and independent historical elevation in population frequency of felid melanistic mutations suggest the occurrence of adaptive evolution of this visible phenotype in a group of related free-ranging species.