Stratification of Companion Animal Life Stages from Electronic Medical Record Diagnosis Data.

Aging is a complex, multifactorial process, where different life stages reflect changes in metabolic processes, immune capacities, and genetic/epigenetic repertoires. With accumulating exposure to environmental stresses and deterioration of physiological functions, body systems become more prone to low-grade chronic inflammation and an increasing range of pathologies. We hypothesized that differential susceptibility to diseases across life span reflects phased changes in an organism's physiological capacity that may highlight when interventions may be appropriately used. Furthermore, the number of life stages may vary between species and be impacted by signalment such as breed. We tested this hypothesis using disease diagnoses data from veterinary electronic medical records containing almost 2 million cats and over 4 million dogs. Bi-clustering (on rates of disease diagnoses) and adaptive branch pruning were used to identify age clusters that could be used to define adult life stages. Clustering among diagnoses were then interpreted within the context of each defined life stage. The analyses identified 5 age clusters in cats and 4 age clusters within each of the 4 canine breed size categories used. This study, using population scale data for two species, one with differential size and life expectancies, is the first to our knowledge to use disease diagnosis data to define adult life stages. The life stages presented here are a result of a data-driven approach to age and disease stratification and are intended to support conversations between clinicians and clients about appropriate health care recommendations.

Suitability of Dried Blood Spots for Accelerating Veterinary Biobank Collections and Identifying Metabolomics Biomarkers With Minimal Resources.

Biomarker discovery using biobank samples collected from veterinary clinics would deliver insights into the diverse population of pets and accelerate diagnostic development. The acquisition, preparation, processing, and storage of biofluid samples in sufficient volumes and at a quality suitable for later analysis with most suitable discovery methods remain challenging. Metabolomics analysis is a valuable approach to detect health/disease phenotypes. Pre-processing changes during preparation of plasma/serum samples may induce variability that may be overcome using dried blood spots (DBSs). We report a proof of principle study by metabolite fingerprinting applying UHPLC-MS of plasma and DBSs acquired from healthy adult dogs and cats (age range 1-9 years), representing each of 4 dog breeds (Labrador retriever, Beagle, Petit Basset Griffon Vendeen, and Norfolk terrier) and the British domestic shorthair cat (n = 10 per group). Blood samples (20 and 40 µL) for DBSs were loaded onto filter paper, air-dried at room temperature (3 h), and sealed and stored (4°C for ~72 h) prior to storage at -80°C. Plasma from the same blood draw (250 µL) was prepared and stored at -80°C within 1 h of sampling. Metabolite fingerprinting of the DBSs and plasma produced similar numbers of metabolite features that had similar abilities to discriminate between biological classes and correctly assign blinded samples. These provide evidence that DBSs, sampled in a manner amenable to application in in-clinic/in-field processing, are a suitable sample for biomarker discovery using UHPLC-MS metabolomics. Further, given appropriate owner consent, the volumes tested (20-40 µL) make the acquisition of remnant blood from blood samples drawn for other reasons available for biobanking and other research activities. Together, this makes possible large-scale biobanking of veterinary samples, gaining sufficient material sooner and enabling quicker identification of biomarkers of interest.

Responses in randomised groups of healthy, adult Labrador retrievers fed grain-free diets with high legume inclusion for 30 days display commonalities with dogs with suspected dilated cardiomyopathy.

Early responses in healthy adult dogs fed grain-free diets with high inclusion of split peas (20%) and lentils (40%) that may lead to canine diet-induced dilated cardiomyopathy (DCM) were investigated. To help understand the clinical relevance of the findings, a survey of electronic health records (EHR) was conducted of dogs with and without suspected DCM for comparison. Control and Test diets were fed to Labrador retriever dogs for 30 days (n = 5 and 6, respectively). Blood and urine samples collected at baseline and days 3, 14 and 28/30 were analyzed for hematology, clinical biochemistry and taurine concentrations. The EHRs of dogs at Banfield® Pet Hospitals in the 2-year period 2018-2019 were surveyed, revealing 420 dogs diagnosed with DCM, which were compared with 420 breed, gender and age-matched healthy control dogs. Compared to baseline values, feeding the Test diet for 28 days caused progressive, significant (p < 0.001) decreases in red blood cell counts (RBC), hematocrit and total hemoglobin by 7.7, 8.3 and 6.3%, respectively, and a 41.8% increase in plasma inorganic phosphate. Commonalities in these parameters were observed in clinical DCM cases. Regarding taurine status, Test dogs transiently increased whole-blood (23.4%) and plasma (47.7%) concentrations on day 14, while taurine:creatinine ratio in fresh urine and taurine in pooled urine were reduced by 77 and 78%, respectively, on day 28/30. Thus grain-free, legume-rich Test diets caused reduced RBC and hyperphosphatemia, findings also indicated in dogs with suspected DCM. Changes in taurine metabolism were indicated. The data will aid in generating hypotheses for future studies.

Adaptations Supporting Plasma Methionine on a Limited-Methionine, High-Cystine Diet Alter the Canine Plasma Metabolome Consistent with Interventions that Extend Life Span in Other Species.

BACKGROUND: Using indicator amino acid oxidation methodology, the mean dietary requirement of adult dogs for methionine (Met) was estimated to be ∼66% of the current recommended allowance. Dogs fed a diet formulated to provide the estimated mean Met requirement for 32 wk maintained plasma Met, seemingly supported by betaine oxidation. OBJECTIVE: To gain a better understanding of the metabolic changes that were associated with supporting plasma Met when dogs were fed a limited Met diet over 32 wk, we analyzed plasma samples taken from that study using a data-driven metabolomics approach. METHODS: Labrador retrievers (20 females/13 males; mean age: 4.9 y; range: 2.0-7.9 y) were fed semi-purified, nutritionally complete diets. After 4 wk of feeding a control diet (DL-Met; 1.37 g/1000 kcal), 17 dogs remained on this diet and 16 were transitioned to a test diet formulated to the estimated mean Met requirement (0.55 g/1000 kcal), with dietary total sulfur amino acid maintained with additional l-cystine (Cys). Dogs were individually fed diets to maintain a stable body weight at an ideal body condition score for 32 wk. Plasma samples from fasted blood collected at baseline and 8 and 32 wk were analyzed using untargeted metabolic profiling. RESULTS: Analysis of metabolites (n = 593) confirmed our primary findings (increased Met, betaine, and dimethylglycine). Metabolite changes consistent with repartitioning choline to support Met cycling included reduced pools of lipids derived via phosphatidylethanolamine N-methyltransferase and enhanced fatty acid oxidation. Some changes were consistent with metabolomics studies reported in other species that used interventions known to extend life span (caloric- and Met-restricted diets or feeding strategy). CONCLUSIONS: Changes in the plasma metabolome were consistent with reported adaptations to support Met-dependent activities. We propose that feeding a limited-Met, high-Cys diet using the estimated mean Met requirement in adult Labrador retrievers alters regulation of the Met cycle, thereby altering metabolism, similar to interventions that extend life span.

No Observed Adverse Effects on Health Were Detected in Adult Beagle Dogs When Fed a High-Calcium Diet for 40 Weeks.

The implications of long-term high calcium (Ca) intake are well documented in growing dogs and in adult dogs of large breed size, however, the consequences on other breeds and breed sizes are yet to be determined. Eighteen neutered adult beagles, nine males and nine females aged 1.4-4.4 years, were randomized to control or test diets providing in g∙4184 kJ-1 (1000 kcal-1): 1.44 and 7.19 total Ca balanced with 1.05 and 4.25 total phosphorus, respectively, for 40 weeks. Health parameters, ultrasound scans, radiographs, glomerular filtration rate, and mineral balance were measured at eight-week intervals. All dogs remained healthy with no measured evidence of orthopedic, urinary, or renal disease. The test diet resulted in a 5.2 fold increase in fecal Ca excretion. Apparent Ca digestibility (%) and Ca balance (g/d) did not significantly (p > 0.05) change from baseline in the test diet group, although dogs displayed a positive Ca balance (maximum at week 8, 1.11 g/d with 95% CI (0.41, 1.80)) before a neutral Ca balance was restored at week 32. Despite an initial positive Ca balance, we can conclude that no measurable adverse health effects were observed as a result of the test diet fed in this study in beagles over a period of 40 weeks.

Clusterin secretion is attenuated by the proinflammatory cytokines interleukin-1ß and tumor necrosis factor-α in models of cartilage degradation.

The protein clusterin has been implicated in the molecular alterations that occur in articular cartilage during osteoarthritis (OA). Clusterin exists in two isoforms with opposing functions, and their roles in cartilage have not been explored. The secreted form of clusterin (sCLU) is a cytoprotective extracellular chaperone that prevents protein aggregation, enhances cell proliferation and promotes viability, whereas nuclear clusterin acts as a pro-death signal. Therefore, these two clusterin isoforms may be putative molecular markers of repair and catabolic responses in cartilage and the ratio between them may be important. In this study, we focused on sCLU and used established, pathophysiologically relevant, in vitro models to understand its role in cytokine-stimulated cartilage degradation. The secretome of equine cartilage explants, osteochondral biopsies and isolated unpassaged chondrocytes was analyzed by western blotting for released sCLU, cartilage oligomeric protein (COMP) and matrix metalloproteinases (MMP) 3 and 13, following treatment with the proinflammatory cytokines interleukin-1ß (IL-1ß) and tumor necrosis factor-α. Release of sulfated glycosaminoglycans (sGAG) was determined using the dimethylmethylene blue assay. Clusterin messenger RNA (mRNA) expression was quantified by quantitative real-time polymerase chain reaction. MMP-3, MMP-13, COMP, and sGAG release from explants and osteochondral biopsies was elevated with cytokine treatment, confirming cartilage degradation in these models. sCLU release was attenuated with cytokine treatment in all models, potentially limiting its cytoprotective function. Clusterin mRNA expression was down-regulated 7-days post cytokine stimulation. These observations implicate sCLU in catabolic responses of chondrocytes, but further studies are required to evaluate its role in OA and its potential as an investigative biomarker.

Rapid Reconstitution of the Fecal Microbiome after Extended Diet-Induced Changes Indicates a Stable Gut Microbiome in Healthy Adult Dogs.

The gut microbiome has an important role in health, and diet represents a key lever for shaping the gut microbiome across all stages of life. Maternal milk consumption in neonates leads to long-term health effects, indicating that pliability in the infant gut microbiome in response to diet can drive enduring change. The ability of diet to drive lasting changes in the adult gut microbiome is less understood. We studied the effect of an extreme dietary shift on the fecal microbiome of 46 Labrador retriever dogs (mean age, 4.6 years) over 11 months. Dogs were fed a nutritionally complete, commercially available complex diet (CD) for a minimum of 5 weeks, followed by highly purified diets (PDs) for 36 weeks, and the initial CD for at least a further 4 weeks. Fecal samples were collected at regular intervals for DNA extraction. By analyzing 16S rRNA genes and the metagenomes, we observed minor effects on microbial diversity but significant changes in bacterial taxa and genetic potential when a PD was fed. Specifically, metagenomics identified an enrichment of quinone- and GABA-related pathways on PD, providing insights into dietary effects on cross-feeding strategies impacting community structure. When dogs returned to the CD, no significant differences were found with the initial time point. These findings are consistent with the gut microbiome being rapidly adaptable but capable of being reconstituted when provided with similar diets. These data highlight that long-term changes in the adult dog gut microbiome may only be achieved through long-term maintenance on a specified diet, rather than through feeding a transitionary diet.IMPORTANCE Diet can influence the adult gut microbiome (the community of bacteria) and health outcomes, but the ability to make changes persisting beyond feeding of a particular diet is poorly understood. We investigated whether feeding highly purified diets to adult dogs for 36 weeks would alter bacterial populations sufficiently to result in a persistent change following the dogs' return to a commercial diet. As expected, the microbiome changed when the purified diet was fed, but the original microbiome was reconstituted within weeks of the dogs returning to the commercial diet. The significance of these findings is in identifying an intrinsic stability of the host microbiome in healthy dogs, suggesting that dietary changes to support adult dog health through modifying the gut microbiome may be achieved only through maintenance on a specified diet, rather than through feeding transitionary diets.

The Significance of Environmental Attributes as Indicators of the Life Cycle Environmental Impacts of Packaging and Food Service Ware.

The environmental impacts of packaging and food service ware (FSW) are increasingly the subject of government policy, public discourse, and industry commitments. While some consideration is given to reducing the impacts of packaging across its entire life cycle, most of the focus is on packaging waste or feedstock substitution. Efforts typically focus on specific packaging characteristics, or material attributes, commonly perceived to be environmentally preferable. This article summarizes an extensive meta-review of existing published literature that was performed to determine whether the material attributes recyclability, recycled content, compostability, and biobased, commonly considered to be environmentally beneficial, correlate with lower net environmental impacts across the full life cycle of the packaging and FSW. Seventy-one unique life cycle assessment (LCA) studies that quantify the environmental impacts throughout the entire life cycle of packaging and FSW were analyzed. These studies included over 5000 comparisons for 13 impact categories commonly analyzed in LCA studies. The results from the meta-review identified a number of instances where material attributes do not correlate with environmental benefits for packaging and FSW. Rather, other characteristics such as material choice or mass of the packaging/FSW products can have higher influence in determining life cycle impacts.

Short-term determination and long-term evaluation of the dietary methionine requirement in adult dogs.

Methionine, an essential sulphur-containing amino acid (SAA), plays an integral role in many metabolic processes. Evidence for the methionine requirements of adult dogs is limited, and we employed the indicator amino acid oxidation (IAAO) method to estimate dietary methionine requirements in Labrador retrievers (n 21). Using semi-purified diets, the mean requirement was 0·55 (95 % CI 0·41, 0·71) g/4184 kJ. In a subsequent parallel design study, three groups of adult Labrador retrievers (n 52) were fed semi-purified diets with 0·55 g/4184 kJ (test diet 1), 0·71 g/4184 kJ (test diet 2) or 1·37 g/4184 kJ (control diet) of methionine for 32 weeks to assess the long-term consequences of feeding. The total SAA content (2·68 g/4184 kJ) was maintained through dietary supplementation of cystine. Plasma methionine did not decrease in test group and increased significantly on test diet 1 in weeks 8 and 16 compared with control. Reducing dietary methionine did not have a significant effect on whole blood, plasma or urinary taurine or plasma N-terminal pro B-type natriuretic peptide. Significant effects in both test diets were observed for cholesterol, betaine and dimethylglycine. In conclusion, feeding methionine at the IAAO-estimated mean was sufficient to maintain plasma methionine over 32 weeks when total SAA was maintained. However, choline oxidation may have increased to support plasma methionine and have additional consequences for lipid metabolism. While the IAAO can be employed to assess essential amino acid requirements, such as methionine in the dog using semi-purified diets, further work is required to establish safe levels for commercial diet formats.

Metabolomic profiling to identify effects of dietary calcium reveal the influence of the individual and postprandial dynamics on the canine plasma metabolome.

Short-term feeding studies have highlighted a phenomenon in Ca regulation that raises concerns around Ca absorption in dogs that may make an impact on commercial diets near to the maximum recommended level. A recent study to determine responses in dogs fed one of two diets differing in dietary Ca over 40 weeks found no evidence to suggest a concern across a range of biological parameters hypothesised to be affected by Ca. Unforeseen consequences of dietary Ca could have occurred and metabolic profiling was deemed a suitable data-driven approach to identify effects of dietary Ca. The objectives were to compare the fasted plasma metabolome (sampled at 8-week intervals over 40 weeks) of dogs fed one of two diets, near to the minimum and maximum recommended levels of dietary Ca. Comparisons with the control diet were also investigated across the postprandial time course (1-4 h) following acute (1 d) and long-term (24 weeks) feeding of the test diet. Comparing fasted plasma samples at each time point, no significant effect (adjusted P < 0·05) of diet on metabolites was observed. In the postprandial state, only phosphate was consistently different between diets and was explained by additional dietary P to maintain Ca:P. Metabolic profiling analysis supports the view that the dietary Ca upper limit is safe. Additionally, the canine plasma metabolome was characterised, providing insights into the stability of individual profiles across 40 weeks, the response to consumption of a nutritionally complete meal over a 4 h postprandial time course and different kinetic categories of postprandial absorption.

Impact of dietary macronutrient profile on feline body weight is not consistent with the protein leverage hypothesis.

The protein leverage hypothesis proposes that the need to prioritise protein intake drives excess energy intake (EI) when the dietary ratio of protein to fat and carbohydrate is reduced. We hypothesised that cats may become prone to overconsuming energy content when moderate protein diets were offered, and considered the potential influence of fat and carbohydrate on intake. To determine the effect of dietary protein and macronutrient profile (MNP) on EI, weight and body composition, cats (1-4 years) were offered food in excess of energy requirements (ER). A total of six diets were formulated, containing moderate (approximately 7 % w/w; approximately 22 % metabolisable energy (ME)) or high (approximately 10 % w/w; approximately 46 % ME) protein and varying levels of carbohydrate and fat. For 4 weeks, 120 cats were offered 100 % of their individual ER of a diet at the MNP selected by adult cats (50:40:10 protein energy ratio:fat energy ratio:carbohydrate energy ratio). EI, body weight (BW), body composition, activity and palatability were measured. Subsequently, cats were offered one of the six diets at 200 % of their individual ER for 4 weeks when measurements were repeated. Cats offered excess high protein diets had higher EI (kJ/kg) throughout, but at 4 weeks BW was not significantly different to baseline. Cats offered excess moderate protein diets reduced EI and gradually lost weight (average loss of 0·358 (99 % CI 0·388, 0·328) kg), irrespective of fat:carbohydrate and initial palatability. The data do not support the protein leverage hypothesis. Furthermore, cats were able to adapt intake of a wet diet with high protein in an overfeeding environment within 28 d.

Linoleate-enriched diet increases both linoleic acid esterified to omega hydroxy very long chain fatty acids and free ceramides of canine stratum corneum without effect on protein-bound ceramides and skin barrier function.

Few studies have investigated the influence of increased amounts of dietary linoleic acid on the epidermal lipid biochemistry and TEWL in healthy subject. The influence of dietary linoleic acid on canine stratum corneum (SC) lipids was studied by feeding two groups of five dogs differential amounts of linoleic acid (LA) for three months. SC was harvested by tape stripping and lipids were analyzed by thin-layer chromatography and mass spectrometry. The dogs that were fed the higher concentration of LA showed high increases in the contents of both linoleic acid and free ceramides in the SC, whereas the protein-bound ceramide content was unchanged. Acylacids that represent the esterified form of linoleic acid in omega hydroxy very long chain fatty acids (ω-OH VLCFA) accounted for most of the elevation of LA, whereas the concentration of the free form was not significantly changed. Corroborating the absence of change in the protein-bound ceramides content of healthy dogs SC, TEWL was nearly unaffected by the linoleic acid-enriched diet.

Adult dogs are capable of regulating calcium balance, with no adverse effects on health, when fed a high-calcium diet.

Although the implications of long-term high Ca intakes have been well documented in growing dogs, the health consequences of Ca excess in adult dogs remain to be established. To evaluate the impact of feeding a diet containing 7·1 g/4184 kJ (1000 kcal) Ca for 40 weeks on Ca balance and health parameters in adult dogs, eighteen neutered adult Labrador Retrievers, (nine males and nine females) aged 2·5-7·4 years were randomised to one of two customised diets for 40 weeks. The diets were manufactured according to similar nutritional specifications, with the exception of Ca and P levels. The diets provided 1·7 and 7·1 g/4184 kJ (1000 kcal) (200(SD26) and 881(SD145) mg/kg body weight0·75 per d, respectively) Ca, respectively, with a Ca:P ratio of 1·6. Clinical examinations, ultrasound scans, radiographs, health parameters, metabolic effects and mineral balance were recorded at baseline and at 8-week intervals throughout the study. Dogs in both groups were healthy throughout the trial without evidence of urinary, renal or orthopaedic disease. In addition, there were no clinically relevant changes in any of the measures made in either group (all P>0·05). The high-Ca diet resulted in a 3·3-fold increase in faecal Ca excretion (P0·05). Ca intakes of up to 7·1 g/4184 kJ (1000 kcal) are well tolerated over a period of 40 weeks, with no adverse effects that could be attributed to the diet or to a high mineral intake.

The impact of time of neutering on weight gain and energy intake in female kittens.

Neutering is a risk factor for obesity in companion animals. In a study to determine the total energy requirements of kittens (15-52 weeks) the impact of neutering and age when neutered on intake and body weight (BW) was investigated. Females (n 14), neutered when 19 (early neuter; EN) or 31 (conventional neuter; CN) weeks old (n 7/group), were individually fed to maintain an ideal body condition score (BCS). EN kittens gained weight gradually whilst CN kittens' BW gain slowed from week 24, weighing less than EN kittens from week 30 with a reduced energy intake (kcal/kg BW0·67) in weeks 24-32 (P < 0·05). Following neutering, CN cats' BW and energy intake increased rapidly (energy intake CN > EN in weeks 36-40). Although EN required earlier diet restriction, acute hyperphagia and increased rate of BW gain following neutering were not observed. Earlier neutering may aid healthy weight management through growth when regulating intake to maintain an ideal BCS.

Ultra high performance liquid chromatography-high resolution mass spectrometry plasma lipidomics can distinguish between canine breeds despite uncontrolled environmental variability and non-standardized diets.

INTRODUCTION AND OBJECTIVES: The purpose of this study was to use high accurate mass metabolomic profiling to investigate differences within a phenotypically diverse canine population, with breed-related morphological, physiological and behavioural differences. Previously, using a broad metabolite fingerprinting approach, lipids appear to dominate inter- and intra- breed discrimination. The purpose here was to use Ultra High Performance Liquid Chromatography-High Resolution Mass Spectrometry (UHPLC-HRMS) to identify in more detail, inter-breed signatures in plasma lipidomic profiles of home-based, client-owned dogs maintained on different diets and fed according to their owners' feeding regimens. METHODS: Nine dog breeds were recruited in this study (Beagle, Chihuahua, Cocker Spaniel, Dachshund, Golden Retriever, Greyhound, German Shepherd, Labrador Retriever and Maltese: 7-12 dogs per breed). Metabolite profiling on a MTBE lipid extract of fasted plasma was performed using UHPLC-HRMS. RESULTS: Multivariate modelling and classification indicated that the main source of lipidome variance was between the three breeds Chihuahua, Dachshund and Greyhound and the other six breeds, however some intra-breed variance was evident in Labrador Retrievers. Metabolites associated with dietary intake impacted on breed-associated variance and following filtering of these signals out of the data-set unique inter-breed lipidome differences for Chihuahua, Golden Retriever and Greyhound were identified. CONCLUSION: By using a phenotypically diverse home-based canine population, we were able to show that high accurate mass lipidomics can enable identification of metabolites in the first pass plasma profile, capturing distinct metabolomic variability associated with genetic differences, despite environmental and dietary variability.

Metabolic Profiling Reveals Effects of Age, Sexual Development and Neutering in Plasma of Young Male Cats.

Neutering is a significant risk factor for obesity in cats. The mechanisms that promote neuter-associated weight gain are not well understood but following neutering, acute changes in energy expenditure and energy consumption have been observed. Metabolic profiling (GC-MS and UHPLC-MS-MS) was used in a longitudinal study to identify changes associated with age, sexual development and neutering in male cats fed a nutritionally-complete dry diet to maintain an ideal body condition score. At eight time points, between 19 and 52 weeks of age, fasted blood samples were taken from kittens neutered at either 19 weeks of age (Early Neuter (EN), n = 8) or at 31 weeks of age (Conventional Neuter (CN), n = 7). Univariate and multivariate analyses were used to compare plasma metabolites (n = 370) from EN and CN cats. Age was the primary driver of variance in the plasma metabolome, including a developmental change independent of neuter group between 19 and 21 weeks in lysolipids and fatty acid amides. Changes associated with sexual development and its subsequent loss were also observed, with differences at some time points observed between EN and CN cats for 45 metabolites (FDR p<0.05). Pathway Enrichment Analysis also identified significant effects in 20 pathways, dominated by amino acid, sterol and fatty acid metabolism. Most changes were interpretable within the context of male sexual development, and changed following neutering in the CN group. Felinine metabolism in CN cats was the most significantly altered pathway, increasing during sexual development and decreasing acutely following neutering. Felinine is a testosterone-regulated, felid-specific glutathione derivative secreted in urine. Alterations in tryptophan, histidine and tocopherol metabolism observed in peripubertal cats may be to support physiological functions of glutathione following diversion of S-amino acids for urinary felinine secretion.

Characterisation of the main drivers of intra- and inter- breed variability in the plasma metabolome of dogs.

INTRODUCTION: Dog breeds are a consequence of artificial selection for specific attributes. These closed genetic populations have metabolic and physiological characteristics that may be revealed by metabolomic analysis. OBJECTIVES: To identify and characterise the drivers of metabolic differences in the fasted plasma metabolome and then determine metabolites differentiating breeds. METHODS: Fasted plasma samples were collected from dogs maintained under two environmental conditions (controlled and client-owned at home). The former (n = 33) consisted of three breeds (Labrador Retriever, Cocker Spaniel and Miniature Schnauzer) fed a single diet batch, the latter (n = 96), client-owned dogs consisted of 9 breeds (Beagle, Chihuahua, Cocker Spaniel, Dachshund, Golden Retriever, Greyhound, German Shepherd, Labrador Retriever and Maltese) consuming various diets under differing feeding regimens. Triplicate samples were taken from Beagle (n = 10) and Labrador Retriever (n = 9) over 3 months. Non-targeted metabolite fingerprinting was performed using flow infusion electrospray-ionization mass spectrometry which was coupled with multivariate data analysis. Metadata factors including age, gender, sexual status, weight, diet and breed were investigated. RESULTS: Breed differences were identified in the plasma metabolome of dogs housed in a controlled environment. Triplicate samples from two breeds identified intra-individual variability, yet breed separation was still observed. The main drivers of variance in dogs maintained in the home environment were associated with breed and gender. Furthermore, metabolite signals were identified that discriminated between Labrador Retriever and Cocker Spaniels in both environments. CONCLUSION: Metabolite fingerprinting of plasma samples can be used to investigate breed differences in client-owned dogs, despite added variance of diet, sexual status and environment.

A Longitudinal Study of the Feline Faecal Microbiome Identifies Changes into Early Adulthood Irrespective of Sexual Development.

Companion animals provide an excellent model for studies of the gut microbiome because potential confounders such as diet and environment can be more readily controlled for than in humans. Additionally, domestic cats and dogs are typically neutered early in life, enabling an investigation into the potential effect of sex hormones on the microbiome. In a longitudinal study to investigate the potential effects of neutering, neutering age and gender on the gut microbiome during growth, the faeces of kittens (16 male, 14 female) were sampled at 18, 30 and 42 weeks of age. DNA was shotgun sequenced on the Illumina platform and sequence reads were annotated for taxonomy and function by comparison to a database of protein coding genes. In a statistical analysis of diversity, taxonomy and functional potential of the microbiomes, age was identified as the only factor with significant associations. No significant effects were detected for gender, neutering, or age when neutered (19 or 31 weeks). At 18 weeks of age the microbiome was dominated by the genera Lactobacillus and Bifidobacterium (35% and 20% average abundance). Structural and functional diversity was significantly increased by week 30 but there was no further significant increase. At 42 weeks of age the most abundant genera were Bacteroides (16%), Prevotella (14%) and Megasphaera (8%). Significant differences in functional potential included an enrichment for genes in energy metabolism (carbon metabolism and oxidative phosphorylation) and depletion in cell motility (flagella and chemotaxis). We conclude that the feline faecal microbiome is predominantly determined by age when diet and environment are controlled for. We suggest this finding may also be informative for studies of the human microbiome, where control over such factors is usually limited.