Glycine supplementation can partially restore oxidative stress-associated glutathione deficiency in ageing cats.

Intracellular levels of glutathione, the major mammalian antioxidant, are reported to decline with age in several species. To understand whether ageing affects circulating glutathione levels in cats, blood was sampled from two age groups, < 3 years and > 9 years. Further, to determine whether dietary supplementation with glutathione precursor glycine (GLY) affects glutathione concentrations in senior cats (> 8 years), a series of free GLY inclusion level dry diets were fed. Subsequently, a 16-week GLY feeding study was conducted in senior cats (> 7 years), measuring glutathione, and markers of oxidative stress. Whole blood and erythrocyte total, oxidised and reduced glutathione levels were significantly decreased in senior cats, compared with their younger counterparts (P ≤ 0·02). The inclusion level study identified 1·5 % free GLY for the subsequent dry diet feeding study. Significant increases in erythrocyte total and reduced glutathione were observed between senior cats fed supplemented and control diets at 4 weeks (P ≤ 0·03; maximum difference of 1·23 µM). Oxidative stress markers were also significantly different between groups at 8 (P = 0·004; difference of 0·68 nG/ml in 8-hydroxy-2'-deoxyguanosine) and 12 weeks (P ≤ 0·049; maximum difference of 0·62 nG/mG Cr in F2-isoprostane PGF2α). Senior cats have lower circulating glutathione levels compared with younger cats. Feeding senior cats a complete and balanced dry diet supplemented with 1·5 % free GLY for 12 weeks elevated initial erythrocyte glutathione and altered markers of oxidative stress. Dietary supplementation with free GLY provides a potential opportunity to restore age-associated reduction in glutathione in cats.

The MARS PETCARE BIOBANK protocol: establishing a longitudinal study of health and disease in dogs and cats.

BACKGROUND: The veterinary care of cats and dogs is increasingly embracing innovations first applied to human health, including an increased emphasis on preventative care and precision medicine. Large scale human population biobanks have advanced research in these areas; however, few have been established in veterinary medicine. The MARS PETCARE BIOBANK™ (MPB) is a prospective study that aims to build a longitudinal bank of biological samples, with paired medical and lifestyle data, from 20,000 initially healthy cats and dogs (10,000 / species), recruited through veterinary hospitals over a ten-year period. Here, we describe the MPB protocol and discuss its potential as a platform to increase understanding of why and how diseases develop and how to advance personalised veterinary healthcare. METHODS: At regular intervals, extensive diet, health and lifestyle information, electronic medical records, clinicopathology and activity data are collected, genotypes, whole genome sequences and faecal metagenomes analysed, and blood, plasma, serum, and faecal samples stored for future research. DISCUSSION: Proposed areas for research include the early detection and progression of age-related disease, risk factors for common conditions, the influence of the microbiome on health and disease and, through genome wide association studies, the identification of candidate loci for disease associated genetic variants. Genomic data will be open access and research proposals for access to data and samples will be considered. Over the coming years, the MPB will provide the longitudinal data and systematically collected biological samples required to generate important insights into companion animal health, identifying biomarkers of disease, supporting earlier identification of risk, and enabling individually tailored interventions to manage disease.

Dietary calcium to phosphorus ratio affects postprandial phosphorus concentrations in feline plasma.

The impact of dietary phosphorus on chronic renal disease in cats, humans and other species is receiving increasing attention. As Ca and P metabolism are linked, the ratio of Ca:P is an important factor for consideration when formulating diets for cats and other animals. Here, we describe a fully randomised crossover study including twenty-four healthy, neutered adult cats, investigating postprandial responses in plasma P, ionised Ca and parathyroid hormone (PTH) following one meal (50 % of individual metabolic energy requirement) of each of six experimental diets. Diets were formulated to provide P at either 0·75 or 1·5 g/1000 kcal (4184 kJ) from the soluble phosphorus salt sodium tripolyphosphate (STPP, Na5P3O10), variable levels of organic Ca and P sources, and an intended total Ca:P of about 1·0, 1·5 or 2·0. For each experimental diet, baseline fasted blood samples were collected prior to the meal, and serial blood samples collected hourly for 6 h thereafter. For all diets, a significant increase from baseline was observed at 120 min in plasma PTH (P < 0·001). The diet containing the highest STPP inclusion level and lowest Ca:P induced the highest peaks in postprandial plasma P and PTH levels (1·8 mmol/l and 27·2 pg/ml, respectively), and the longest duration of concentrations raised above baseline were observed at 3 h for P and 6 h for PTH. Data indicate that Ca:P modulates postprandial plasma P and PTH. Therefore, when formulating diets containing soluble P salts for cats, increasing the Ca:P ratio should be considered.

Towards establishing no observed adverse effect levels (NOAEL) for different sources of dietary phosphorus in feline adult diets: results from a 7-month feeding study.

High dietary phosphorus (P), particularly soluble salts, may contribute to chronic kidney disease development in cats. The aim of the present study was to assess the safety of P supplied at 1 g/1000 kcal (4184kJ) from a highly soluble P salt in P-rich dry format feline diets. Seventy-five healthy adult cats (n 25/group) were fed either a low P control (1·4 g/1000 kcal [4184kJ]; Ca:P ratio 0·97) or one of two test diets with 4 g/1000 kcal (4184 kJ); Ca:P 1·04 or 5 g/1000 kcal (4184kJ); Ca:P 1·27, both incorporating 1 g/1000 kcal (4184 kJ) sodium tripolyphosphate (STPP) - for a period of 30 weeks in a randomised parallel-group study. Health markers in blood and urine, glomerular filtration rate, renal ultrasound and bone density were assessed at baseline and at regular time points. At the end of the test period, responses following transition to a commercial diet (total P - 2·34 g/1000 kcal [4184kJ], Ca:P 1·3) for a 4-week washout period were also assessed. No adverse effects on general, kidney or bone (skeletal) function and health were observed. P and Ca balance, some serum biochemistry parameters and regulatory hormones were increased in cats fed test diets from week 2 onwards (P ≤ 0·05). Data from the washout period suggest that increased serum creatinine and urea values observed in the two test diet groups were influenced by dietary differences during the test period, and not indicative of changes in renal function. The present data suggest no observed adverse effect level for feline diets containing 1 g P/1000 kcal (4184 kJ) from STPP and total P level of up to 5 g/1000 kcal (4184 kJ) when fed for 30 weeks.

Development of an enzyme-linked immunosorbent assay for the diagnosis of feline tuberculosis.

Cases of feline tuberculosis (TB) can be challenging to diagnose. Currently, this is achieved through a combination of mycobacterial culture, polymerase chain reaction (PCR), or interferon-gamma release assay (IGRA); however, these each have limitations. There is limited data regarding the use of humoral immunodiagnostics for TB in cats. Therefore, we sought to develop an enzyme-linked immunosorbent assay (ELISA) to further facilitate the diagnosis of feline TB. A comparative PPD (purified protein derivative) antibody ELISA was optimised for use on serum and plasma, and was tested against samples from 14 cats with culture-confirmed TB and 24 uninfected controls. Selection of an appropriate positive cut-off value based on receiver-operator characteristic curve analysis gave test sensitivity of 64.3 % and specificity of 100 %. When tested on further samples from cats with strongly suspected mycobacteriosis, 32.9 % (23/70) were antibody positive. Notably, positive results were recorded in cats that failed to respond to the IGRA, and in one PCR and IGRA negative cat. No positive responses were identified in cats with non-tuberculous mycobacterial infections, or with non-mycobacterial diseases (n = 12). Therefore, antibody-based diagnostics may be useful adjunctive tests for cases of TB missed by the IGRA, helping protect both feline and, in turn, human health.

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.

Understanding How Dogs Age: Longitudinal Analysis of Markers of Inflammation, Immune Function, and Oxidative Stress.

As in human populations, advances in nutrition and veterinary care have led to an increase in the lifespan of companion animals. Detrimental physiological changes occurring later in life must be understood before interventions can be made to slow or reduce them. One important aspect of human aging is upregulation of the inflammatory response and increase in oxidative damage resulting in pathologies linked to chronic inflammation. To determine whether similar processes occur in the aging dog, changes in markers of inflammation and oxidative stress were investigated in 80 Labrador retrievers from adulthood to the end of life. Serum levels of immunoglobulin M (p < .001) and 8-hydroxy-2-deoxyguanosine (p < .001) increased with age, whereas no effect of age was detected for immunoglobulin G or C-reactive protein unless the last year of life was included in the analysis (p = .002). Baseline levels of heat shock protein 70 decreased with age (p < .001) while those after exposure to heat stress were maintained (p = .018). However, when excluding final year of life data, a decline in the heat shock protein 70 response after heat stress was observed (p = .004). These findings indicate that aging dogs undergo changes similar to human inflammaging and offer the possibility of nutritional or pharmacological intervention to delay or reduce these effects.

The effect of reducing energy density, via the addition of water to dry diet, on body weight and activity in dogs.

Approximately 40 % of pet dogs are estimated to be overweight and this is associated with health conditions significantly reducing life span and quality. In cats, dietary energy dilution has been reported to increase activity levels and aid healthy body-weight maintenance. Our aim was to investigate this in dogs. For 28 d, a complete and balanced standard dry diet, hydrated to a total moisture content (TMC) of 72 %, was offered to forty-six dogs at individual maintenance energy requirements (MER). Intake, body weight and activity were measured. For the following 28 d, the dry diet was offered at 200 % of MER with or without hydration (7·6 or 72 % TMC) and measurements repeated. When offered diets in excess, body weight increased significantly faster (19·3 g/d) with the hydrated diet (P = 0·001), but activity levels did not change from baseline (P = 0·392), while activity reduced significantly with the dry diet (about 15 %; P < 0·001). Dogs completely compensated for the reduction of dietary energy content, indicating that this is not a useful strategy for maintaining body weight when offered excess food.

Energy requirements for growth in the Yorkshire terrier.

The 2006 National Research Council (NRC) equation calculating puppy energy requirements does not account for reported breed differences in growth pattern. Energy requirements of toy breed puppies are unknown and it is unclear whether feeding guidelines should differ between breeds. Energy requirements of Yorkshire terrier (YT) puppies were observed over their first year of life and compared with those predicted by the NRC and those previously observed in large (Labrador retriever) and medium (miniature Schnauzer; MS) breed puppies. Twenty-two puppies (from eight litters) were offered complete and balanced diets to maintain ideal body condition score (BCS). Energy intake, body weight and BCS were recorded from 10 to 52 weeks of age. Every 12 weeks, health was monitored by veterinary examination, routine haematology and plasma biochemistry. Puppies remained clinically healthy with normal skeletal development throughout. After analysis by linear mixed models it was observed that the NRC equation overestimates YT energy requirements between 10 and 20 weeks of age by up to 324·3 (95 % CI 390·4, 258·2) kJ/kg0·75. Energy intake was lower (P < 0·05) in YT than Labradors until 29 weeks by up to 376·6 (95 % CI 477·4, 275·3) kJ/kg0·75 and lower than MS between 16 and 25 weeks by up to 216·3 (95 % CI 313·0, 119·7) kJ/kg0·75 (P < 0·05). Data indicate differences in toy, medium and large breed energy requirements for growth. The NRC equation for puppy energy requirements overestimated the requirements of this YT population, suggesting the need for breed-specific feeding guides for growth to avoid overfeeding.

The effect of reducing dietary energy density via the addition of water to a dry diet, on body weight, energy intake and physical activity in adult neutered cats.

Increasingly domestic cats live in an overfeeding and underexercising environment where obesity is a major health concern. One strategy to aid healthy body weight maintenance is dietary energy dilution. Published data indicate that increasing dietary moisture content leads to a reduction in energy intake and increased activity. However, a number of different methodologies were employed in these studies and associated changes in physical activity have only been measured once. The aim of the present study was to determine the effect of diets of three different moisture contents offered in excess of energy requirements, on body weight, energy intake and physical activity in adult neutered cats. Sixty-nine adult cats randomised into three groups, received 100 % of their daily individual maintenance energy requirements (IMER) of dry diet or dry diet hydrated to 40 or 80 % total moisture content (tmc). Baseline activity, intake, body weight and body composition were measured. Following this baseline phase, the cats received the same diets at 200 % of daily IMER and the measurements repeated over the next 28 d. When offered the diets at 200 % IMER, cats fed the dry diet significantly increased body weight and percentage of body fat (P < 0·01), while those offered the hydrated diets did not (P > 0·01). The levels of physical activity in cats offered the hydrated 80 % tmc diet were significantly (P < 0·01) higher than those offered the dry or 40 % tmc diet. We suggest that dietary energy dilution by addition of water may be a useful strategy for healthy body weight maintenance in overfed cats.