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While a large set of in vitro models are available to study the effects of specific food ingredients (e.g. pre- and probiotics) on the human gut microbiome, the availability of such models for companion animals is limited. Since improving gut health of such animals is an emerging research field, the Simulator of the Canine Intestinal Microbial Ecosystem (SCIME™) was recently developed and validated with in vivo data. The current study presents a further improvement of this model by using an alternative method for feed preparation, i.e. by administering digestive enzymes to mimic upper gastro-intestinal digestion followed by a dialysis approach to mimic small intestinal absorption. As opposed to the previously implemented method, this resulted in a more optimal simulation of protein digestion and absorption. Further, upon entrance in the colon, increased production of the health-promoting butyrate and lower levels of Lactobacillus spp. and Bifidobacterium spp. were observed, which corresponded better with obtained in vivo data. A second model improvement consisted of the implementation of a mucosal environment to not only simulate luminal but also mucosal microbiota. In consistency with the human model for which this technology was previously validated, it was found that for all canine microbiota mucin beads were enriched with members of the Lachnospiraceae (~ Clostridium cluster XIVa), a family containing multiple well-known butyrate producers. The SCIME™ was thus upgraded to a so-called Mucosal SCIME™ (M-SCIME™). In conclusion, the current study presents improvements of the SCIME™, further increasing the relevance of obtained data with this in vitro model for dogs., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Many horses are fed differently than their wild ancestors. They often have limited access to pasture and are fed conserved forage and concentrates rich in starch and sugars, in only 2 meals per day. Feeding practices in contrast to natural feeding behavior can lead to gastrointestinal issues. Standard nutritional evaluation is warranted because of its important role in prevention and in treatment and management of diseases. When medical and nutritional treatments are combined, success rates are higher. New techniques to characterize equine microbiota have been used, allowing for microbiota manipulation to prevent and treat intestinal diseases., Competing Interests: Disclosure The authors have nothing to disclose., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Inappropriate nutrition is a priority welfare challenge. Nutritional mistakes are common and can lead to adverse events, such as poor growth and performance, colic, laminitis, and obesity. A detailed nutritional assessment involving evaluating the equine patient, current diet/ration, and management is essential in creating an effective nutritional plan. Goal(s) should be established and used to inform the plan. Management or resource barriers should be considered. Effective communication and a team-based approach versus an authoritarian one are likely to enhance the success of the nutritional plan. Developing a plan should be an interactive process, adjusting as needed after intentional monitoring., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Symmetric dimethylarginine (SDMA) is a promising renal marker that correlates well with the glomerular filtration rate and could allow earlier detection of impaired renal function. The main objectives of this study were to assess the long-term variability of SDMA in healthy dogs and examine the influence of an increased body fat percentage on the level of SDMA. Sixteen lean Beagles were randomly assigned to the control group or weight-change group in age- and gender-matched pairs. The energy intake of the control group (n = 8) was strictly regulated to maintain an ideal body weight for 83 weeks, while the weight-change group (n = 8) was fed to induce weight gain (week 0-47), to maintain stable excessive body weight (week 47-56) and to lose weight (week 56-83), consecutively. At 8 specified time points, the body condition score, body composition, glomerular filtration rate, serum concentration of SDMA and creatinine were analyzed. In the control group, the within-subject coefficient of variation, between-subject coefficient of variation, reference change value (type I error = 5%) and index of individuality were 0.16, 0.22, 0.43 and 0.73, respectively. The control group and weight-change group did not differ significantly in SDMA concentration. SDMA showed a significant negative association (coefficient = -0.07) with body fat percentage (p<0.01) in the weight-change group and a significant positive association (coefficient = 7.79) with serum creatinine (p<0.01) in the entire study population. In conclusion, SDMA concentration has high long-term stability in healthy adult dogs. For the evaluation of SDMA concentrations, subject-specific reference values are preferred over a population-based reference value seen their higher sensitivity. Moreover, an increased body fat percentage does seem to affect the serum SDMA concentration of otherwise healthy dogs, but its clinical relevance has to be clarified in further research., Competing Interests: The authors declare the following competing financial interests: SDMA analyses were financially supported and performed by IDEXX Laboratories, Inc.. The diet used in this study and blood and urine analyses were financially supported by Virbac. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

In order to investigate the effect and appropriate dose of prebiotics, this study evaluated the effect of two levels of xylooligosaccharides (XOS) in cats. Twenty-four healthy adult cats were divided into three groups: no-XOS control diet with 1% cellulose; low XOS supplementation (LXOS) with 0.04% XOS and 0.96% cellulose; and high XOS supplementation (HXOS) with 0.40% XOS and 0.60% cellulose. Both XOS groups increased blood 3-hydroxybutyryl carnitine levels and decreased hexadecanedioyl carnitine levels. Both XOS treatments displayed an increased bacterial abundance of Blautia , Clostridium XI, and Collinsella and a decreased abundance of Megasphaera and Bifidobacterium . LXOS groups increased fecal pH and bacterial abundance of Streptococcus and Lactobacillus , decreased blood glutaryl carnitine concentration, and Catenibacterium abundance. HXOS group showed a more distinct microbiome profile and higher species richness, and an increased bacterial abundance of Subdoligranulum , Ruminococcaceae genus (unassigned genus), Erysipelotrichaceae genus, and Lachnospiraceae . Correlations between bacterial abundances and blood and fecal parameters were also observed. In conclusion, XOS could benefit feline gut health by altering microbiota; its effects dependant on the dose. The higher-dose XOS increased bacterial populations that possibly promoted intestinal fermentation, while the lower dose altered populations of carbohydrate-metabolic microbiota and possibly modulated host metabolism. Low-dose prebiotics may become a trend in future studies.

Current laboratory tests are unable to distinguish healthy from allergic dogs. Unlike serum antibody responses, circulating antibody-secreting cells (ASC) are temporarily induced after each contact with the antigen. These ASC can be identified using ELISPOT and the observation of allergen-specific ASC might correlate with the causative allergens in dogs with an allergic dermatitis. In this study, blood was sampled from six privately-owned allergic dogs and six non-allergic laboratory beagles to determine the frequency of circulating allergen-specific ASC for common allergens. Blood IgE+, IgA + and IgG + cells were magnetically isolated to determine the number of allergen-specific ASC with ELISPOT for Dermatophagoides farinae, Dermatophagoides pteronyssinus, Alternaria alternata, birch, timothy grass, wheat, cow's milk, bovine, chicken and lamb meat. For IgA and IgG, allergen-specific spots were observed, however for IgE, no spots were detected for any of the allergens. ELISPOT could not differentiate allergic from non-allergic dogs. When the responses to the different allergens were compared, more IgA ASC for D. pteronyssinus were observed compared to some of the other allergens which was statistically significant for the non-allergic dogs and approached significance in the allergic dogs. These findings indicate that ELISPOT can be used to identify circulating allergen-specific IgA- and IgG-secreting cells. The technique did however not detect allergen-specific IgE ASC and was unable to distinguish allergic from non-allergic dogs. Only a small number of studies have studied allergen-specific IgA in dogs. The finding that dogs have higher numbers of D. pteronyssinus-specific IgA ASC points out that apart from IgE and IgG, it might be interesting to include IgA measurements for certain allergens to analyse the complete spectrum of both the protective and pro-allergic antibody responses., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The relationship between microbial community and host has profound effects on the health of animals. A balanced gastrointestinal (GI) microbial population provides nutritional and metabolic benefits to its host, regulates the immune system and various signaling molecules, protects the intestine from pathogen invasion, and promotes a healthy intestinal structure and an optimal intestinal function. With the fast development of next-generation sequencing, molecular techniques have become standard tools for microbiota research, having been used to demonstrate the complex intestinal ecosystem. Similarly to other mammals, the vast majority of GI microbiota in cats (over 99%) is composed of the predominant bacterial phyla Firmicutes , Bacteroidetes , Actinobacteria , and Proteobacteria . Many nutritional and clinical studies have shown that cats' microbiota can be affected by several different factors including body condition, age, diet, and inflammatory diseases. All these factors have different size effects, and some of these may be very minor, and it is currently unknown how important these are. Further research is needed to determine the functional variations in the microbiome in disease states and in response to environmental and/or dietary modulations. Additionally, further studies are also needed to explain the intricate relationship between GI microbiota and the genetics and immunity of its host. This review summarizes past and present knowledge of the feline GI microbiota and looks into the future possibilities and challenges of the field., (Copyright © 2020 Lyu, Su, Verbrugghe, Van de Wiele, Martos Martinez-Caja and Hesta.)

Early detection of obesity-related glomerulopathy in humans is challenging as it might not be detected by routine biomarkers of kidney function. This study's aim was to use novel kidney biomarkers and contrast-enhanced ultrasound (CEUS) to evaluate the effect of obesity development and weight-loss on kidney function, perfusion, and injury in dogs. Sixteen healthy lean adult beagles were assigned randomly but age-matched to a control group (CG) (n = 8) fed to maintain a lean body weight (BW) for 83 weeks; or to a weight-change group (WCG) (n = 8) fed the same diet to induce obesity (week 0-47), to maintain stable obese weight (week 47-56) and to lose BW (week 56-83). At 8 time points, values of systolic blood pressure (sBP); serum creatinine (sCr); blood urea nitrogen (BUN); serum cystatin C (sCysC); urine protein-to-creatinine ratio (UPC); and urinary biomarkers of glomerular and tubular injury were measured. Glomerular filtration rate (GFR) and renal perfusion using CEUS were assayed (except for week 68). For CEUS, intensity- and time-related parameters representing blood volume and velocity were derived from imaging data, respectively. At 12-22% weight-gain, cortical time-to-peak, representing blood velocity, was shorter in the WCG vs. the CG. After 37% weight-gain, sCysC, UPC, glomerular and tubular biomarkers of injury, urinary immunoglobulin G and urinary neutrophil gelatinase-associated lipocalin, respectively, were higher in the WCG. sBP, sCr, BUN and GFR were not significantly different. After 23% weight-loss, all alterations were attenuated. Early weight-gain in dogs induced renal perfusion changes measured with CEUS, without hyperfiltration, preceding increased urinary protein excretion with potential glomerular and tubular injury. The combined use of routine biomarkers of kidney function, CEUS and site-specific urinary biomarkers might be valuable in assessing kidney health of individuals at risk for obesity-related glomerulopathy in a non-invasive manner., Competing Interests: The authors have declared that no competing interests exist.

Background: Research in humans and mice suggests that obesity influences the abundance and diversity of gastrointestinal (GI) microbiota, and that an "obese microbiome" influences energy metabolism and fat storage in the host. Microbiota membership and composition have been previously assessed in healthy cats. However, research investigating the effects of obesity and weight loss on the cat's fecal microbiota is limited. Therefore, this study's objective was to evaluate differences in fecal microbial abundance and biodiversity, as well as serum cobalamin and folate concentrations in obese cats, before and after weight loss, and compare to lean cats. Fourteen lean and 17 obese healthy client-owned cats were fed a veterinary therapeutic weight loss food at maintenance energy requirement for 4 weeks. At the end of week 4, lean cats finished the study, whereas obese cats continued with a 10-week weight loss period on the same food, fed at individually-tailored weight loss energy requirements. Body weight and body condition score were recorded every 2 weeks throughout the study. At the end of each period, a fecal sample and food-consumption records were obtained from the owners, and serum cobalamin and folate concentrations were analysed. DNA was extracted from fecal samples, polymerase chain reaction (PCR) was performed, and products were sequenced using next-generation sequencing (Illumina MiSeq)., Results: No significant differences in the relative abundance of taxa and in biodiversity indices were observed between cats in either group (P > 0.05 for all tests). Nevertheless, some significantly enriched taxa, mainly belonging to Firmicutes, were noted in linear discriminant analysis effect size test in obese cats before weight loss compared to lean cats. Serum cobalamin concentrations were significantly higher in lean compared to obese cats both before and after weight loss. Serum folate concentrations were higher in obese cats before weight loss compared to after., Conclusions: The association between feline obesity and the fecal bacterial microbiota was demonstrated in enriched taxa in obese cats compared to lean cats, which may be related to enhanced efficiency of energy-harvesting. However, in obese cats, the fecal microbial abundance and biodiversity were only minimally affected during the early phase of a standardized weight loss plan.

The in vitro gas production technique (IVGPT) has been a valuable tool in ruminant nutrition research for decades and has more recently been used in horse nutrition studies to investigate fermentation activities of the equine hindgut though primarily using feces as inoculum. This study was conducted to evaluate the use of equine rectal content in the IVGPT system as a viable inoculum that can be considered representative of the activities throughout the equine hindgut. Additionally, the study was conducted to measure the effects on fermentation kinetics and end-product production using inoculum from horses fed supplemental levels of coated sodium butyrate in an IVGPT system. Eight warmblood horses were fed a diet consisting of haylage (1% DM intake based on ideal body weight [BW]) and a mash concentrate formulated to provide 2.5 g nonstructural carbohydrate (NSC)/kg BW per meal. The diet was intended to create a NSC challenge to the microbial populations of the hindgut. The horses were randomly assigned to treatment or control group and after a 1-wk diet-adaptation period, the treatment group received 0.4 g/kg BW per day of a coated sodium butyrate supplement, while the control group received a placebo (coating only). After a 3-wk treatment period, the animals were sacrificed and digesta from the cecum, left ventral colon, right dorsal colon, and the rectum were collected within 30 min postmortem and used as inocula for the IVGPT trial. Haylage and concentrates fed to the test animals were also used as substrates in vitro. Sodium butyrate supplementation was not significant for gas production parameters or VFA measured suggesting no effect of sodium butyrate supplementation on the extent or kinetics of gas production or microbial end-product production (P ≥ 0.073). Differences in inocula were significant for organic matter corrected cumulative gas production (P = 0.0001), asymptotic gas production of the second phase (A2) (P < 0.0001); and maximal rate of OM degradation of the second phase (Rmax2) (P = 0.002). Inocula had a significant effect on total VFA (P = 0.0002), butyrate (Bu) (P = 0.015), branched chain fatty acids (P < 0.0001), pH (P < 0.0001), and ammonia (NH3) (P = 0.0024). In conclusion, based on observed results from this study, total tract digestibility may be overestimated if using rectal content inoculum to evaluate forage-based feeds in an IVGPT system., (© The Author(s) 2020. Published by Oxford University Press on behalf of the American Society of Animal Science.All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Whereas a wide variety of in vitro models have been developed and validated to assess the effect of specific food ingredients on the human gut microbiome, such models have only been developed and applied to a limited extent for companion animals. Since the use of pre- and probiotics to improve gut health is an emerging research topic in the field of companion animals and as dogs are often used as laboratory animals in developing and testing of pharmaceuticals, the current study aimed to establish an adequate canine in vitro model. This consisted of a four-stage reactor composed of a stomach and small intestinal compartment followed by a proximal and distal colon. This semi-continuous gastrointestinal tract model allowed a long-term, region-dependent, and pH-controlled simulation of the colon-associated microbial community of dogs. Upon reaching a functional steady state, the simulated canine microbial community composition proved to be representative of the in vivo situation. Indeed, the predominant bacterial phyla present in the in vitro proximal and distal colon corresponded with the main bacterial phyla detected in the fecal material of the dogs, resulting in an average community composition along the simulated canine gastrointestinal tract of 50.5% Firmicutes, 34.5% Bacteroidetes, 7.4% Fusobacteria, 4.9% Actinobacteria, and 2.7% Proteobacteria. A parallel in vivo-in vitro comparison assessing the effects of fructooligosaccharides (FOS) on the canine microbial community composition showed a consistent stimulation of Lactobacillus concentrations in the in vivo fecal samples as well as in the in vitro canine gut model. Furthermore, the in vitro platform provided additional insights about the prebiotic effect of FOS supplementation of dogs, such as a reduced abundance of Megamonas spp. which are only present in very low abundance in in vivo fecal samples, indicating an interesting application potential of the developed canine in vitro model in research related to gastrointestinal health of dogs., (© The Author(s) 2019. Published by Oxford University Press on behalf of the American Society of Animal Science. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Contrast-enhanced ultrasound may be helpful for detecting early renal microvascular damage and dysfunction in dogs. However, before this noninvasive imaging method can be tested as an early-stage screening tool in clinical patients, an improved understanding of long-term variation in healthy animals is needed. In this prospective, secondary, longitudinal, serial measurements study, variability of contrast-enhanced ultrasound renal perfusion parameters was described for eight healthy dogs, using seven time points and a period of 83 weeks. Dogs were sedated with butorphanol (0.4 mg/kg), and contrast-enhanced ultrasound of each kidney was performed after an intravenous bolus injection of a microbubble contrast agent (0.04 mL/kg). Time-intensity curves were created from regions-of-interest drawn in the renal cortex and medulla. Intensity-related parameters representing blood volume and time-related parameters representing blood velocity were determined. A random-effects model using restricted maximum likelihood was used to estimate variance components. Within-dog coefficient of variation was defined as the ratio of the standard deviation over the mean. Time-related parameters such as time-to-peak, rise and fall time had lowest within-dog variability. Intensity-related parameters such as peak enhancement, wash-in and wash-out area under the curve, total area under the curve, and wash-in and washout rates had high within-dog variability (coefficient of variation > 45%). Authors therefore recommend the use of time-related parameters for future studies of renal perfusion. Within-dog variability for bilateral kidney measurements was extremely low, therefore contrast-enhanced ultrasound may be particularly useful for detecting unilateral changes in renal perfusion. Future studies are needed to compare contrast-enhanced ultrasound findings in healthy dogs versus dogs with renal disease., (© 2018 American College of Veterinary Radiology.)

Sentinel lymph node (SLN) mapping is a valuable and crucial diagnostic procedure in staging malignancies. We compared two non-invasive techniques, near-infrared (NIR) fluorescence imaging and contrast-enhanced ultrasound (CEUS), to identify the SLNs in three superficial anatomical regions in an animal model. Six healthy laboratory dogs were included in a proof-of-concept trial. A NIR fluorescent dye (Indocyanine Green) and microbubbles (Sonovue) were consecutively injected subdermally in the Inguinal, axillary and popliteal region to map the SLNs. Transcutaneous NIR fluorescence imaging identified SLNs in 17 out of a total of 18 occasions. CEUS identified SLNs in all regions (18/18). Whereas NIR fluorescence imaging performed better in the visualization of the afferent lymphatic tract, CEUS demonstrated different filling patterns of the SLNs, a feature potentially critical for the concept of SLN mapping in cancer patients. Both NIR fluorescence imaging and CEUS are safe, non-invasive, practical and accurate methods to perform real-time transcutaneous SLN mapping with potential in a clinical setting., (© 2018 John Wiley & Sons Ltd.)

Pronounced variations in faecal consistency have been described anecdotally for some carnivore species fed a structure-rich diet. Typically two faecal consistencies are distinguished, namely hard and firm versus liquid and viscous faeces. It is possible that a separation mechanism is operating in the carnivore digestive tract, as in many herbivore species. Six beagle dogs were fed two experimental diets in a cross-over design of 7 days. Test diets consisted of chunked day old chicks differing only in particle size (fine = 7.8 mm vs coarse = 13 mm) in order to vary dietary structure. Digestive retention time was measured using titanium oxide (TiO2) as marker. The total faecal output was scored for consistency and faecal fermentation profiles were evaluated through faecal short-chain fatty acid (SCFA) and ammonia (NH3) analyses. A total of 181 faecal samples were collected. Dietary particle size did not affect faecal consistency, fermentative end products nor mean retention time (MRT). However, a faecal consistency dichotomy was observed with firm faeces (score 2-2.5) and soft faeces (score 4-4.5) being the most frequently occurring consistencies in an almost alternating pattern in every single dog. Firm and soft faeces differed distinctively in fermentative profiles. Although the structure difference between diets did not affect the faecal dichotomy, feeding whole prey provoked the occurrence of the latter which raises suspicion of a digestive separation mechanism in the canine digestive tract. Further faecal characterisation is however required in order to unravel the underlying mechanism.

Background: High protein diets shift the faecal microbiota into a more unfavourable composition in obese humans. In lean dogs, higher protein consumption is accompanied with increased production of putrefactive fermentation products, whereas obese dogs have a different gut microbiota compared to lean dogs. Still, the impact of high dietary protein on gut microbiota in obese dogs remains unclear. The aim of this study was to investigate faecal microbial changes in lean and obese dogs in response to two different levels of dietary protein. Six healthy lean and six obese Beagles were fed a high protein diet (HP) and a low protein diet (LP) for 28 days each in a crossover design. Denaturing gradient gel electrophoresis and quantitative PCR were performed on faecal samples for microbial profiling. Plasma acylcarnitine and fermentation metabolites were measured., Results: Dogs fed HP had higher concentrations of protein fermentation metabolites including faecal ammonia, isovalerate, isobutyrate, phenol, indole, serum indoxyl sulphate and plasma 3-OH isovalerylcarnitine compared to dogs fed LP, whereas no changes in faecal concentrations of acetate and butyrate were observed. The abundances of clostridial clusters IV and XIVa, covering the majority of butyrate-producing bacteria, and of the butyrate kinase gene, one of the terminal genes of the butyrate synthesis pathway were higher in dogs on HP compared to LP. Significant interactions between diet and body condition were found for the abundance of Firmicutes, Lactobacillus and clostridial cluster I. The similarity coefficient of faecal microbiota between the two diets was smaller in obese dogs than in lean dogs., Conclusions: High protein diet increased the abundance and activity of butyrate-producing bacteria in Beagles independent of the body condition. In addition, increasing dietary protein content had a greater overall impact on faecal microbiota in obese compared to lean dogs.

Although scintigraphy is an important tool for the assessment of thyroid function in cats, time variation of commonly used thyroid variables has not been investigated to this day. The aim of this study was to investigate whether a week-to-week variation of scintigraphic variables exists in healthy cats of a magnitude that is clinically relevant, as this could lead to misinterpretation of results. Fourteen adult, healthy, experimental cats were included in the study. At 3 time points, with 7-day intervals, the cats underwent a thyroid pertechnetate scan and blood samples were collected. The scintigraphic variables calculated were the thyroid to salivary gland ratio (T/S), thyroid to background ratio (T/B), percentage technetium uptake in the thyroid glands (%TcU T ) and additionally percentage technetium uptake in the salivary glands (%TcU SG ). Two thyroid hormones, total thyroxine (TT 4 ) and thyroid stimulating hormone (TSH), were included in the analysis. All scintigraphic variables, with the exception of the %TcU T and T/B neck ratio, were within the normal reference ranges reported in literature. No clinically relevant week-to-week variation was observed for any of the variables included in this study., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The domestic cat's wild ancestors are obligate carnivores that consume prey containing only minimal amounts of carbohydrates. Evolutionary events adapted the cat's metabolism and physiology to this diet strictly composed of animal tissues and led to unique digestive and metabolic peculiarities of carbohydrate metabolism. The domestic cat still closely resembles its wild ancestor. Although the carnivore connection of domestic cats is well recognised, little is known about the precise nutrient profile to which the digestive physiology and metabolism of the cat have adapted throughout evolution. Moreover, studies show that domestic cats balance macronutrient intake by selecting low-carbohydrate foods. The fact that cats evolved consuming low-carbohydrate prey has led to speculations that high-carbohydrate diets could be detrimental for a cat's health. More specifically, it has been suggested that excess carbohydrates could lead to feline obesity and diabetes mellitus. Additionally, the chances for remission of diabetes mellitus are higher in cats that consume a low-carbohydrate diet. This literature review will summarise current carbohydrate knowledge pertaining to digestion, absorption and metabolism of carbohydrates, food selection and macronutrient balancing in healthy, obese and diabetic cats, as well as the role of carbohydrates in prevention and treatment of obesity and diabetes mellitus., Competing Interests: The authors declare no conflict of interest. Adronie Verbrugghe is the Royal Canin Veterinary Diets Endowed Chair in Canine and Feline Clinical Nutrition at the Ontario Veterinary College.

The trace element selenium is essential to both dogs and cats. Dry diets are formulated with a large range of ingredients, which may vary in selenium concentration and accessibility. This paper reports equations to predict the average in vitro selenium accessibility from dry pet foods based on essential dietary nutrient concentrations, including crude protein, amino acids and crude fat. Predictive equations were made using stepwise linear regression for extruded and pelleted diets. The equations can be used to aid diet formulation to optimize selenium accessibility within the diet and to prevent selenium deficiency or toxicity., (Journal of Animal Physiology and Animal Nutrition © 2016 Blackwell Verlag GmbH.)

Contrast-enhanced ultrasound is a valuable and safe technique for the evaluation of organ perfusion. Repeated injections of ultrasound contrast agent are often administered during the same imaging session. However, it remains unclear if quantitative differences are present between the consecutive microbubble injections. Therefore, the first and second injection of contrast agent for the left renal cortex, renal medulla and the splenic parenchyma in healthy cats were compared. A lower peak intensity and area under the curve were observed for the first injection of contrast agent in the feline kidney, both for the renal cortex and medulla, and spleen. Moreover, for the renal cortex, the time-intensity curve was steeper after the second injection. Findings from the present study demonstrate that a second injection of contrast agent provides stronger enhancement. The exact mechanism behind our findings remains unclear; however, saturation of the lung macrophages is believed to play an important role., (Copyright © 2016 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.)

Contrast-enhanced ultrasound (CEUS) is an emerging technique to evaluate tissue perfusion. Promising results have been obtained in the evaluation of renal perfusion in health and disease, both in human and veterinary medicine. Renal scintigraphy using 99mTc-Mercaptoacetyltriglycine (MAG3) is another non-invasive technique that can be used to evaluate renal perfusion. However, no data are available on the ability of CEUS or 99mTc- MAG3 scintigraphy to detect small changes in renal perfusion in cats. Therefore, both techniques were applied in a normal feline population to evaluate detection possibilities of perfusion changes by angiotensin II (AT II). Contrast-enhanced ultrasound using a bolus injection of commercially available contrast agent and renal scintigraphy using 99mTc-MAG3 were performed in 11 healthy cats after infusion of 0,9% NaCl (control) and AT II. Angiotensin II induced changes were noticed on several CEUS parameters. Mean peak enhancement, wash-in perfusion index and wash-out rate for the entire kidney decreased significantly after AT II infusion. Moreover, a tendency towards a lower wash-in area-under-the curve was present. Renal scintigraphy could not detect perfusion changes induced by AT II. This study shows that CEUS is able to detect changes in feline renal perfusion induced by AT II infusion., Competing Interests: The authors have declared that no competing interests exist.

Glutathione peroxidase activity is used as a biomarker of selenium status in dogs. Freshly collected blood samples are usually measured, due to the lack of knowledge on the effect of storing the samples. This study investigated if the analysis of glutathione peroxidase activity in whole blood collected from dogs was affected by storage of between 5 and 164 days. Results indicated that glutathione peroxidase activity was more variable in the freshly analysed samples compared to the stored samples. Although the mean differences between fresh and stored samples were not always equal to zero, this is thought to be caused by the variability of reagent preparation rather than by storage, as no consistent increase or decrease in glutathione peroxidase activity was found. Therefore, it can be concluded that heparinised dog blood samples can be successfully stored up to 164 days before analysis of glutathione peroxidase activity.

Background: Research in cats has shown that increased fermentation-derived propionic acid and its metabolites can be used as alternative substrates for gluconeogenesis, thus sparing amino acids for other purposes. This amino acid sparing effect could be of particular interest in patients with kidney or liver disease, where this could reduce the kidneys'/liver's burden of N-waste removal. Since dogs are known to have a different metabolism than the obligatory carnivorous cat, the main objective of this study was to assess the possibility of altering amino acid metabolism through intestinal fermentation in healthy dogs. This was studied by supplementing a low-protein diet with fermentable fibres, hereby providing an initial model for future studies in dogs suffering from renal/liver disease., Results: Eight healthy dogs were randomly assigned to one of two treatment groups: sugar beet pulp and guar gum mix (SF: soluble fibre, estimated to mainly stimulate propionic acid production) or cellulose (IF: insoluble fibre). Treatments were incorporated into a low-protein (17 %) extruded dry diet in amounts to obtain similar total dietary fibre (TDF) contents for both diets (9.4 % and 8.2 % for the SF and IF diet, respectively) and were tested in a 4-week crossover feeding trial. Apparent faecal nitrogen digestibility and post-prandial fermentation metabolites in faeces and plasma were evaluated. Dogs fed the SF diet showed significantly higher faecal excretion of acetic and propionic acid, resulting in a higher total SCFA excretion compared to IF. SF affected the three to six-hour postprandial plasma acylcarnitine profile by significantly increasing AUC of acetyl-, propionyl-, butyryl- + isobutyryl-, 3-OH-butyryl-, 3-OH-isovaleryl- and malonyl-L-carnitine. Moreover, the amino acid plasma profile at that time was modified as leucine + isoleucine concentrations were significantly increased by SF, and a similar trend for phenylalanine and tyrosine's AUC was found., Conclusion: These results indicate that guar gum and sugar beet pulp supplementation diminishes postprandial use of amino acids favoring instead the use of short-chain fatty acids as substrate for the tricarboxylic acid (TCA) cycle. Further research is warranted to investigate the amino acid sparing effect of fermentable fibres in dogs with kidney/liver disease.

Background: Inflammatory bowel disease (IBD) refers to a diverse group of chronic gastrointestinal diseases, and gut microbial dysbiosis has been proposed as a modulating factor in its pathogenesis. Several studies have investigated the gut microbial ecology of dogs with IBD but it is yet unclear if this microbial profile can alter the nutrient metabolism of the host. The aim of the present study was to characterize the faecal bacterial profile and functionality as well as to determine host metabolic changes in IBD dogs. Twenty-three dogs diagnosed with IBD and ten healthy control dogs were included. Dogs with IBD were given a clinical score using the canine chronic enteropathy clinical activity index (CCECAI). Faecal short-chain fatty acids (SCFA) and ammonia concentrations were measured and quantitative PCR was performed. The concentration of plasma amino acids, acylcarnitines, serum folate, cobalamin, and indoxyl sulfate was determined., Results: No significant differences in the abundance of a selection of bacterial groups and fermentation metabolites were observed between the IBD and control groups. However, significant negative correlations were found between CCECAI and the faecal proportion of Lactobacillus as well as between CCECAI and total SCFA concentration. Serum folate and plasma citrulline were decreased and plasma valine was increased in IBD compared to control dogs. Increased plasma free carnitine and total acylcarnitines were observed in IBD compared with control dogs, whereas short-chain acylcarnitines (butyrylcarnitine + isobutyrylcarnitine and, methylmalonylcarnitine) to free carnitine ratios decreased. Dogs with IBD had a higher 3-hydroxyisovalerylcarnitine + isovalerylcarnitine to leucine ratio compared to control dogs., Conclusions: Canine IBD induced a wide range of changes in metabolic profile, especially for the plasma concentrations of short-chain acylcarnitines and amino acids, which could have evolved from tissue damage and alteration in host metabolism. In addition, dogs with more severe IBD were characterised by a decrease in faecal proportion of Lactobacillus.

There is a growing concern for the long-term health effects of selenium (Se) over- or underfeeding. The efficiency of utilization of dietary Se is subject to many factors. Our study in dogs evaluated the effect of diet type (canned versus kibble) and dietary protein concentration on Se digestibility and bioactivity. Canned and kibble diets are commonly used formats of dog food, widely ranging in protein concentration. Twenty-four Labrador retrievers were used and four canned and four kibble diets were selected with crude protein concentrations ranging from 10.1 to 27.5 g/MJ. Crude protein concentration had no influence on the digestibility of Se in either canned or kibble diets, but a lower Se digestibility was observed in canned compared to kibble diets. However, the biological activity of Se, as measured by whole blood glutathione peroxidase, was higher in dogs fed the canned diets than in dogs fed the kibble diets and decreased with increasing crude protein intake. These results indicate that selenium recommendations in dog foods need to take diet type into account.

Background: Inadequate dietary selenium (Se) intake in humans and animals can lead to long term health problems, such as cancer. In view of the owner's desire for healthy longevity of companion animals, the impact of dietary Se provision on long term health effects warrants investigation. Little is currently known regards biomarkers, and rate of change of such biomarkers in relation to dietary selenium intake in dogs. In this study, selected biomarkers were assessed for their suitability to detect changes in dietary Se in adult dogs within eight weeks., Results: Twenty-four dogs were fed a semi-purified diet with an adequate amount of Se (46.1 μg/MJ) over an 8 week period. They were then divided into two groups. The first group remained on the adequate Se diet, the second were offered a semi-purified diet with a low Se concentration (6.5 μg/MJ; 31% of the FEDIAF minimum) for 8 weeks. Weekly urine and blood was collected and hair growth measurements were performed. The urinary Se to creatinine ratio and serum Se concentration were significantly lower in dogs consuming the low Se diet from week 1 onwards, by 84% (adequate 25.3, low 4.1) and 7% (adequate 257 μg/L, low 238 μg/L) respectively. Serum and whole blood glutathione peroxidase were also significantly lower in dogs consuming the low Se diet from weeks 6 and 8 respectively. None of the other biomarkers (mRNA expression and serum copper, creatine kinase, triiodothyronine:thyroxine ratio and hair growth) responded significantly to the low Se diet over the 8 week period., Conclusions: This study demonstrated that urinary Se to creatinine ratio, serum Se and serum and whole blood glutathione peroxidase can be used as biomarkers of selenium status in dogs. Urinary Se to creatinine ratio and serum Se concentrations responded faster to decreased dietary Se than the other parameters. This makes these biomarkers candidates for early screening of long term effects of dietary Se provision on canine health.

Reasons for Performing Study: Commercial immunoglobulin E (IgE)-based tests are available for diagnosis of food allergies and are commonly used in equine practice. However, these tests have been proven unreliable as a screening method in man and other species, but not critically evaluated in equids. Therefore, a commercially available IgE-based test for horses was evaluated., Objectives: To evaluate the consistency of the results obtained with a commercially available IgE-based test for food allergy diagnosis in ponies (Phase I) and to subject ponies to a provocation trial with the presumed allergens (Phase II)., Study Design: Allergen screening followed by experimental food provocation trials in healthy ponies., Methods: Blood samples of 17 healthy Shetland ponies were taken at 2 different time points, sent blinded to a commercial laboratory for screening of common food allergens and the results were evaluated for consistency (Phase I). Ponies that were positive for food allergens were consecutively challenged orally with each allergen separately for 14 days (Phase II). A washout period of one week was applied in ponies with multiple positive results. Clinical parameters and serum amyloid A were monitored during the provocation trial., Results: Only 7/17 ponies were negative on the IgE-based test at the 2 time points, 3 had positive results twice but only one tested positive twice for the same food allergen. No abnormalities were noted during the provocation trials., Conclusions: This study demonstrated that this IgE-based test is not a reliable screening tool for food allergy in healthy equids., (© 2015 EVJ Ltd.)

Since the first description of feline hyperthyroidism (HT) in 1979, several studies have been undertaken to define the etiology of the disease. Epidemiologic studies, after investigating non-food- and food-associated factors, suggest a multifactorial etiology. However, in the absence of prospective cohort studies that can confirm a cause-and-effect relationship between HT and associated risk factors, no causative factor for HT has been identified to date. Feline HT resembles toxic nodular goiter in humans, with autonomously functioning upregulated iodide uptake systems. Contribution of the diet to HT development remains controversial. The purpose of this paper is to review critically the reported food-associated risk factors for HT., (© ISFM and AAFP 2014.)

Se bioavailability in commercial pet foods has been shown to be highly variable. The aim of the present study was to identify dietary factors associated with in vitro accessibility of Se (Se Aiv) in pet foods. Se Aiv is defined as the percentage of Se from the diet that is potentially available for absorption after in vitro digestion. Sixty-two diets (dog, n 52; cat, n 10) were in vitro enzymatically digested: fifty-four of them were commercially available (kibble, n 20; pellet, n 8; canned, n 17; raw meat, n 6; steamed meat, n 3) and eight were unprocessed (kibble, n 4; canned, n 4) from the same batch as the corresponding processed diets. The present investigation examined if Se Aiv was affected by diet type, dietary protein, methionine, cysteine, lysine and Se content, DM, organic matter and crude protein (CP) digestibility. Se Aiv differed significantly among diet types (P< 0·001). Canned and steamed meat diets had a lower Se Aiv than pelleted and raw meat diets. Se Aiv correlated positively with CP digestibility in extruded diets (kibbles, n 19; r 0·540, P =0·017) and negatively in canned diets (n 16; r - 0·611, P =0·012). Moreover, the canning process (n 4) decreased Se Aiv (P =0·001), whereas extrusion (n 4) revealed no effect on Se Aiv (P =0·297). These differences in Se Aiv between diet types warrant quantification of diet type effects on in vivo Se bioavailability.

Understanding the symbiotic relationship between gut microbes and their animal host requires characterization of the core microbiota across populations and in time. Especially in captive populations of endangered wildlife species such as the cheetah (Acinonyx jubatus), this knowledge is a key element to enhance feeding strategies and reduce gastrointestinal disorders. In order to investigate the temporal stability of the intestinal microbiota in cheetahs under human care, we conducted a longitudinal study over a 3-year period with bimonthly faecal sampling of 5 cheetahs housed in two European zoos. For this purpose, an integrated 16S rRNA DGGE-clone library approach was used in combination with a series of real-time PCR assays. Our findings disclosed a stable faecal microbiota, beyond intestinal community variations that were detected between zoo sample sets or between animals. The core of this microbiota was dominated by members of Clostridium clusters I, XI and XIVa, with mean concentrations ranging from 7.5-9.2 log10 CFU/g faeces and with significant positive correlations between these clusters (P<0.05), and by Lactobacillaceae. Moving window analysis of DGGE profiles revealed 23.3-25.6% change between consecutive samples for four of the cheetahs. The fifth animal in the study suffered from intermediate episodes of vomiting and diarrhea during the monitoring period and exhibited remarkably more change (39.4%). This observation may reflect the temporary impact of perturbations such as the animal's compromised health, antibiotic administration or a combination thereof, which temporarily altered the relative proportions of Clostridium clusters I and XIVa. In conclusion, this first long-term monitoring study of the faecal microbiota in feline strict carnivores not only reveals a remarkable compositional stability of this ecosystem, but also shows a qualitative and quantitative similarity in a defined set of faecal bacterial lineages across the five animals under study that may typify the core phylogenetic microbiome of cheetahs.

Microbubbles have shown potential as intralymphatic ultrasound contrast agents while nanoparticle-loaded microbubbles are increasingly investigated for ultrasound-triggered drug and gene delivery. To explore whether mRNA-nanoparticle loaded microbubbles could serve as theranostics for detection of and mRNA transfer to the lymph nodes, we investigate the behavior of unloaded and mRNA-loaded microbubbles using contrast-enhanced ultrasound imaging after subcutaneous injection in dogs. Our results indicate that both types of microbubbles are equally capable of rapidly entering the lymph vessels and nodes upon injection, and novel, valuable and detailed information on the lymphatic structure in the animals could be obtained. Furthermore, additional observations were made regarding the dynamics of microbubble lymph node uptake. Importantly, neither the microbubble migration distance within the lymphatics, nor the observed contrast signal intensity was influenced by mRNA-loading. Although further optimization of acoustic parameters will be needed, this could represent a first step towards ultrasound-guided, ultrasound-triggered intranodal mRNA delivery using these theranostic microbubbles.

Domestic cats are obligate carnivores and in this light hindgut fermentation has been considered unimportant in this species. However, a diverse microbiota has been found in the small and large intestines of domestic cats. Additionally, in vitro and in vivo studies support the hypothesis that microbial fermentation is significant in felines with potential benefits to the host. Results on microbiota composition and microbial counts in different regions of the feline gastrointestinal tract are compiled, including a description of modulating host and technical factors. Additionally, the effects of dietary fibre supplementation on the microbiota composition are described. In a second section, in vitro studies, using inocula from fresh feline faeces and focusing on the fermentation characteristics of diverse plant substrates, are described. In vivo studies have investigated the effects of dietary fibre on a broad range of physiological outcomes. Results of this research, together with studies on effects of plant fibre on colonic morphology and function, protein and carbohydrate metabolism, and the effects of plant fibre on disease conditions that require a decrease in dietary protein intake, are shown in a third section of the present review. Conclusively, for fructans and beet pulp, for example, diverse beneficial effects have been demonstrated in the domestic cat. Both dietary fibre sources are regularly used in the pet food industry. More research is warranted to reveal the potential benefits of other fibre sources that can be used on a large scale in feline diets for healthy and diseased cats.

Contrast-enhanced ultrasonography of the left kidney was performed using a commercial contrast agent in six healthy adult purpose-bred cats. A cross-over design was used to compare three protocols: (1) awake, (2) butorphanol (0.4 mg/kg IM), and (3) propofol (3.5-7.7 mg/kg IV boluses to effect). Time-intensity curves were created from two regions-of-interest drawn in the renal cortex. The curves were analyzed for blood flow parameters representing blood volume (base intensity, peak intensity, area-under-curve) and blood velocity (arrival time, time-to-peak, wash-in/out). There was no difference in the subjective enhancement pattern between the three protocols. No significant effect of butorphanol was observed in any of the perfusion parameters (P > 0.05). Propofol did not influence the most important perfusion parameter, area-under-the-curve, and is adequate for use in contrast-enhanced ultrasound studies., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The present study evaluated the effect of different levels of energy restriction on metabolic parameters in obese ponies. Relative weight changes, markers of lipid metabolism and oxidant/antioxidant balance were monitored. A total of eighteen obese (body condition score ≥ 7/9) Shetland ponies were studied over a 23·5-week trial, which was divided into three periods. The first period involved a 4-week adaptation period in which each animal was fed 100% of their maintenance energy requirements needed to maintain a stable obese body weight (MERob). This was followed by a 16·5-week weight-loss period in which ponies were assigned to receive either 100% (control group, CONTROL), 80% (slow weight-loss (SLOW) group) or 60% (rapid weight-loss (RAPID) group) of their MERob. During the 3-week end-phase period, all ponies were again fed 100% of their MERob. Relative weight loss was higher in the RAPID group (P< 0·001) compared with the SLOW group. No linear relationship was found as a doubling of the percentage of energy restriction was accompanied by a tripling of the percentage of weight loss. Relative weight gain afterwards in the end-phase period was higher in the RAPID group (P< 0·001) compared with the SLOW and CONTROL groups. During the weight-loss period, TAG and NEFA concentrations were highest in the RAPID group, as were α-tocopherol and ferric-reducing ability of plasma concentrations. After 8 weeks of weight loss, the concentrations of advanced oxidation protein products were higher in the RAPID group compared with the SLOW and CONTROL groups (P< 0·001). In conclusion, the level of energy restriction influences the extent of changes in oxidant/antioxidant balance. Practically, more severe energy restriction regimens may be associated with a greater regain of weight after the restriction period.

Renal and gastrointestinal pathologies are widespread in the captive cheetah (Acinonyx jubatus) population but are often diagnosed at a late stage, because diagnostic tools are limited to the evaluation of clinical signs or general blood examination. Presently, no data are available on serum proteins and acute-phase proteins in cheetahs during health or disease, although they might be important to improve health monitoring. This study aimed to quantify serum proteins by capillary electrophoresis in 80 serum samples from captive cheetahs, categorized according to health status and disease type. Moreover, serum amyloid A concentrations were measured via a turbidimetric immunoassay validated in domestic cats, whereas haptoglobin and C-reactive protein were determined by non-species-specific functional tests. Cheetahs classified as healthy had serum protein and acute phase protein concentrations within reference ranges for healthy domestic cats. In contrast, unhealthy cheetahs had higher (P < 0.001) serum amyloid A, alpha2-globulin, and haptoglobin concentrations compared with the healthy subgroup. Moreover, serum amyloid A (P = 0.020), alpha2-globulin (P < 0.001) and haptoglobin (P = 0.001) concentrations in cheetahs suffering from chronic kidney disease were significantly greater compared to the reportedly healthy cheetahs. Our study indicates that serum proteins in the cheetah can be analyzed by routine capillary electrophoresis, whereas acute-phase proteins can be measured using available immunoassays or non-species-specific techniques, which are also likely to be applicable in other exotic felids. Moreover, results suggest that serum amyloid A and haptoglobin are important acute-phase proteins in the diseased cheetah and highlight the need to evaluate their role as early-onset markers for disease.

In strict carnivorous domestic cats, a metabolic competition arises between the need to use amino acids for gluconeogenesis and for protein synthesis both in health and disease. The present study investigated the amino acid-sparing potential of propionic acid in cats using dietary propionylated starch (HAMSP) supplementation. A total of thirty cats were fed a homemade diet, supplemented with either HAMSP, acetylated starch (HAMSA) or celite (Control) for three adaptation weeks. Propionylated starch was hypothesised to provide propionic acid as an alternative gluconeogenic substrate to amino acids, whereas acetic acid from HAMSA would not provide any gluconeogenic benefit. Post-adaptation, a 5-d total faecal collection was carried out to calculate apparent protein digestibility coefficients. Fresh faecal and blood samples were collected to analyse fermentation endproducts and metabolites. The apparent protein digestibility coefficients did not differ between supplements (P = 0·372) and were not affected by the protein intake level (P = 0·808). Faecal propionic acid concentrations were higher in HAMSP than in HAMSA (P = 0·018) and Control (P = 0·003) groups, whereas concentrations of ammonia (P = 0·007) were higher in HAMSA than in HAMSP cats. Tendencies for or higher propionylcarnitine concentrations were observed in HAMSP compared with HAMSA (P = 0·090) and Control (P = 0·037) groups, and for tiglyl- + 3-methylcrotonylcarnitine concentrations in HAMSP as compared with Control (P = 0·028) cats. Methylmalonylcarnitine concentrations did not differ between groups (P = 0·740), but were negatively correlated with the protein intake level (r -0·459, P = 0·016). These results suggest that HAMSP cats showed more saccharolytic fermentation patterns than those supplemented with HAMSA, as well as signs of sparing of valine in cats with a sufficient protein intake.

Background: Oxidative stress and inflammation can be altered by dietary factors in various species. However, little data are available in true carnivorous species such as domestic cats. As numerous anti-inflammatory and anti-oxidative additives become available and might be of use in cats with chronic low-grade inflammatory diseases, the current study aimed to develop a model of diet-induced inflammation by use of two opposite diets. It was hypothesized that a high fat diet enhanced in n-6 PUFA and with lower concentrations of antioxidants would evoke inflammation and oxidative stress in domestic cats., Results: Sixteen healthy adult cats were allocated to two groups. One group received a moderate fat diet, containing pork lard and salmon oil (AA:(EPA + DHA) ratio 0.19) (MFn-3), while the other group was fed a high fat diet, containing pork lard and chicken fat (AA:(EPA + DHA) ratio 2.06) (HFn-6) for 12 weeks. Prior to and 2, 4, 6, 8, 10 and 12 weeks after starting the testing period, blood samples were collected. Erythrocytic fatty acid profile showed clear alterations in accordance to the dietary fatty acid profile. Serum thiobarbituric acid reactive substances was higher when fed MFn-3 compared to the HFn-6, suggesting augmented oxidative stress. This was associated with a reduced serum vitamin E status, as serum α-tocopherol concentrations were lower with MFn-3, even with higher dietary levels of vitamin E. Serum cytokine and serum amyloid A concentrations were not influenced by diet., Conclusion: These results point towards a resistance of cats to develop dietary fat-induced inflammation, but also suggest a high susceptibility to oxidative stress when fed a fish oil-supplemented diet even with moderate fat level and additional vitamin E.

Background: Imbalanced feeding regimes may initiate gastrointestinal and metabolic diseases in endangered felids kept in captivity such as cheetahs. Given the crucial role of the host's intestinal microbiota in feed fermentation and health maintenance, a better understanding of the cheetah's intestinal ecosystem is essential for improvement of current feeding strategies. We determined the phylogenetic diversity of the faecal microbiota of the only two cheetahs housed in an EAZA associated zoo in Flanders, Belgium, to gain first insights in the relative distribution, identity and potential role of the major community members., Results: Taxonomic analysis of 16S rRNA gene clone libraries (702 clones) revealed a microbiota dominated by Firmicutes (94.7%), followed by a minority of Actinobacteria (4.3%), Proteobacteria (0.4%) and Fusobacteria (0.6%). In the Firmicutes, the majority of the phylotypes within the Clostridiales were assigned to Clostridium clusters XIVa (43%), XI (38%) and I (13%). Members of the Bacteroidetes phylum and Bifidobacteriaceae, two groups that can positively contribute in maintaining intestinal homeostasis, were absent in the clone libraries and detected in only marginal to low levels in real-time PCR analyses., Conclusions: This marked underrepresentation is in contrast to data previously reported in domestic cats where Bacteroidetes and Bifidobacteriaceae are common residents of the faecal microbiota. Next to methodological differences, these findings may also reflect the apparent differences in dietary habits of both felid species. Thus, our results question the role of the domestic cat as the best available model for nutritional intervention studies in endangered exotic felids.

Background: In humans, adipose tissue (AT) originating from different depots shows varying gene expression profiles. In horses, the risk of certain metabolic disorders may also be influenced by the impact of specific AT depots. Macrophage infiltration in human and rat AT is considered to be a source of inflammatory changes. In horses, this relationship has not been extensively studied yet. Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR), a useful method to evaluate differences in mRNA expression across different tissues, can be used to evaluate differences between equine AT depots. For a correct interpretation of the RT-qPCR results, expression data have to be normalized by the use of validated reference genes. The main objectives of this study were to compare mRNA expression of inflammation-related genes, as well as adipocyte morphology and number between different equine AT depots; and in addition, to investigate the presence of antigen presenting cells in equine AT and any potential relationship with adipokine mRNA expression., Results: In this study, the mRNA expression of inflammation-related genes (leptin, chemokine ligand 5, interleukin 1β, interleukin 6, interleukin 10, adiponectin, matrix metalloproteinase 2, and superoxide dismutase 2) and candidate reference gene stability was investigated in 8 different AT depots collected from the nuchal, abdominal (mesenteric, retroperitoneal, and peri-renal) and subcutaneous (tail head and loin) AT region. By using GeNorm analysis, HPRT1, RPL32, and GAPDH were found to be the most stable genes in equine AT. The mRNA expression of leptin, chemokine ligand 5, interleukin 10, interleukin 1β, adiponectin, and matrix metalloproteinase 2 significantly differed across AT depots (P < 0.05). No significant AT depot effect was found for interleukin 6 and superoxide dismutase 2 (P > 0.05). Adipocyte area and number of antigen presenting cells per adipocyte significantly differed between AT depots (P < 0.05)., Conclusions: Adipose tissue location was associated with differences in mRNA expression of inflammation-related genes. This depot-specific difference in mRNA expression suggests that the overall inflammatory status of horses could be partially determined by the relative proportion of the different AT depots.

Across species obesity is associated with several disorders but in companion animals little information is available on the impact of chronic obesity on immune competence. The aim of the present study was to investigate whether weight gain and stable obese bodyweight affects the immune cell response. Obesity was induced in eight adult healthy beagle dogs (weight gain group; WGG) by a weight gain period (WGP) of 47 weeks, which was immediately followed by a period (stable period: SP) of stable obesity of 26 weeks. Eight adult healthy beagle dogs were included as a control group (CG) and remained at their ideal bodyweight throughout the entire study. Body composition was measured at five intervening time-points. Concentration of serum leptin and inflammatory cytokines, functionality of lymphocytes and phagocytic activity of neutrophils and monocytes were evaluated at ten intervening time-points. Serum leptin concentration was rising during the WGP in the WGG but went to lower concentrations during the SP. At the end of long-term weight gain, a decreased mitogen-induced proliferation of T-lymphocytes was noted but this alteration seemed to be transient after stabilization of bodyweight. This finding may imply an altered immune response for dogs with different energy balances. However, no systemic low grade inflammation or alteration in other immune cell functions was observed. Consequently it is suggested that the change in energy balance during the onset of obesity (becoming obese versus being obese), evokes an additional obesity-related disorder in dogs, i.e. impaired T-lymphocyte immune function., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The present study evaluated the potential of affecting amino acid metabolism through intestinal fermentation in domestic cats, using dietary guar gum as a model. Apparent protein digestibility, plasma fermentation metabolites, faecal fermentation end products and fermentation kinetics (exhaled breath hydrogen concentrations) were evaluated. Ten cats were randomly assigned to either guar gum- or cellulose-supplemented diets, that were fed in two periods of 5 weeks in a crossover design. No treatment effect was seen on fermentation kinetics. The apparent protein digestibility (P= 0.07) tended to be lower in guar gum-supplemented cats. As a consequence of impaired small-intestinal protein digestion and amino acid absorption, fermentation of these molecules in the large intestine was stimulated. Amino acid fermentation has been shown to produce high concentrations of acetic and butyric acids. Therefore, no treatment effect on faecal propionic acid or plasma propionylcarnitine was observed in the present study. The ratio of faecal butyric acid:total SCFA tended to be higher in guar gum-supplemented cats (P= 0.05). The majority of large-intestinal butyric acid is absorbed by colonocytes and metabolised to 3-hydroxy-butyrylcoenzyme A, which is then absorbed into the bloodstream. This metabolite was analysed in plasma as 3-hydroxy-butyrylcarnitine, which was higher (P= 0.02) in guar gum-supplemented cats. In all probability, the high viscosity of the guar gum supplement was responsible for the impaired protein digestion and amino acid absorption. Further research is warranted to investigate whether partially hydrolysed guar gum is useful to potentiate the desirable in vivo effects of this fibre supplement.

With the use of perfusion tracers, in vivo examination of the regional cerebral blood flow in cats can be performed with single photon emission computed tomography (SPECT). Reliable perfusion data of normal, healthy cats are necessary for future clinical studies or other research use. Therefore, this dataset of the regional perfusion pattern of the normal feline brain was created. Twelve cats were used in this study. Technetium-99m-ethyl cysteinate dimer ((99m)Tc-ECD) was injected intravenously and the acquisition, using a triple head gamma camera equipped with three multi-pinhole collimators (pinhole SPECT), was started 40 mins after tracer administration under general anaesthesia. Nineteen regions of interest were defined using 7T magnetic resonance images of the feline brain and a topographical atlas. Regional counts were normalised to the counts of two reference regions: the total brain and the cerebellum. The highest tracer uptake was noticed in the subcortical structures, and the lowest in the frontal cortex and the cerebellum. Also left-right asymmetry in the temporal cortex and a rostrocaudal gradient of 5% were observed.

This study aimed to evaluate correlations between fermentation characteristics and end products of selected fermentable fibres (three types of fructans, citrus pectin, guar gum), incubated with faecal inocula from donor cats fed two diets, differing in fibre and protein sources and concentrations. Cumulative gas production was measured over 72 h, fermentation end products were analysed at 4, 8, 12, 24, 48 and 72 h post-incubation, and quantification of lactobacilli, bifidobacteria and bacteroides in fermentation liquids were performed at 4 and 48 h of incubation. Partial Pearson correlations, corrected for inoculum, were calculated to assess the interdependency of the fermentation characteristics of the soluble fibre substrates. Butyric and valeric acid concentrations increased with higher fermentation rates, whereas acetic acid declined. Concentrations of butyric acid (highest in fructans) and propionic acid were inversely correlated with protein fermentation end products at several time points, whereas concentrations of acetic acid (highest in citrus pectin) were positively correlated with these products at most time points. Remarkably, a lack of clear relationship between the counts of bacterial groups and their typically associated products after 4 h of incubation was observed. Data from this experiment suggest that differences in fibre fermentation rate in feline faecal inocula coincide with typical changes in the profile of bacterial fermentation products. The observed higher concentrations of propionic and butyric acid as a result of fibre fermentation could possibly have beneficial effects on intestinal health, and may be confounded with a concurrent decrease in the production of putrefactive compounds. In conclusion, supplementing guar gum or fructans to a feline diet might be more advantageous compared with citrus pectin. However, in vivo research is warranted to confirm these conclusions in domestic cats.

In six normal-weight and six obese cats, the metabolic effect of propionate absorbed from the colon was assessed. Two colonic infusions were tested in a crossover design with intervals of 4 weeks. The test solution contained 4 mmol sodium propionate per kg ideal body weight in a 0.2% NaCl solution. Normal saline was given as control solution. Solutions were infused into the hindgut over 30 min. Blood samples were obtained prior to and at various time points after starting the infusion. As body condition did not affect evaluated parameters, all data were pooled. Plasma glucose concentrations showed differences neither over time nor during or after infusion with propionate or control. Plasma amino acid concentrations rose over time (p < 0.001), but were similar for both infusions. Plasma propionylcarnitine rose markedly towards the end of the propionate infusion and decreased afterwards (p < 0.001), whereas 3-hydroxy-3-methylglutarylcarnitine was lower 30 (p = 0.005) and 60 min (p = 0.032) after ending propionate infusions and acetylcarnitine tended to fall at the same time points (p = 0.079; p = 0.080), suggesting inhibition of gluconeogenesis from pyruvate and amino acids, but initiation of propionate-induced gluconeogenesis. In conclusion, propionate absorbed from the colon is hypothesized to act as gluconeogenic substrate, regardless of the cat's body condition., (© 2011 Blackwell Verlag GmbH.)

The natural diet of felids contains highly digestible animal tissues but also fractions resistant to small intestinal digestion, which enter the large intestine where they may be fermented by the resident microbial population. Little information exists on the microbial degradability of animal tissues in the large intestine of felids consuming a natural diet. This study aimed to rank animal substrates in their microbial degradability by means of an in vitro study using captive cheetahs fed a strict carnivorous diet as fecal donors. Fresh cheetah fecal samples were collected, pooled, and incubated with various raw animal substrates (chicken cartilage, collagen, glucosamine-chondroitin, glucosamine, rabbit bone, rabbit hair, and rabbit skin; 4 replicates per substrate) for cumulative gas production measurement in a batch culture technique. Negative (cellulose) and positive (casein and fructo-oligosaccharides; FOS) controls were incorporated in the study. Additionally, after 72 h of incubation, short-chain fatty acids (SCFA), including branched-chain fatty acids (BCFA), and ammonia concentrations were determined for each substrate. Glucosamine and glucosamine-chondroitin yielded the greatest organic matter cumulative gas volume (OMCV) among animal substrates (P < 0.05), whereas total SCFA production was greatest for collagen (P < 0.05). Collagen induced an acetate production comparable with FOS and a markedly high acetate-to-propionate ratio (8.41:1) compared with all other substrates (1.67:1 to 2.97:1). Chicken cartilage was rapidly fermentable, indicated by a greater maximal rate of gas production (R(max)) compared with all other substrates (P < 0.05). In general, animal substrates showed an earlier occurrence for maximal gas production rate compared with FOS. Rabbit hair, skin, and bone were poorly fermentable substrates, indicated by the least amount of OMCV and total SCFA among animal substrates (P < 0.05). The greatest amount of ammonia production among animal substrates was measured after incubation of collagen and rabbit bone (P < 0.05). This study provides the first insight into the potential of animal tissues to influence large intestinal fermentation in a strict carnivore, and indicates that animal tissues have potentially similar functions as soluble or insoluble plant fibers in vitro. Further research is warranted to assess the impact of fermentation of each type of animal tissue on gastro-intestinal function and health in the cheetah and other felid species.

Objective: To evaluate the impact of modulation of the membrane-bound efflux pump P-glycoprotein (P-gp) on plasma concentrations of orally administered prednisolone in dogs., Animals: 7 healthy adult Beagles., Procedures: Each dog received 3 treatments (control [no treatment], rifampicin [100 mg/d, PO, for 21 days, as an inducer of P-gp], and ketoconazole [100 mg/d, PO, for 21 days, as an inhibitor of P-gp]). A single dose of prednisolone (1 mg/kg, PO) was administered on day 8 of each treatment period. There was a 7-day washout period between subsequent treatments. Plasma concentrations of prednisolone were determined by use of a validated liquid chromatography-tandem mass spectrometry method. Duodenum and colon biopsy specimens were obtained endoscopically from anesthetized dogs and assessed for P-gp protein labeling via immunohistochemical analysis and mRNA quantification via real-time PCR assay. Total fecal collection was performed for evaluation of effects of P-gp modulation on digestion of nutrients., Results: Rifampicin treatment upregulated duodenal P-gp in dogs and significantly reduced the area under the plasma concentration-time curve of prednisolone. Ketoconazole typically downregulated expression of duodenal P-gp, with a subsequent increase in the area under the plasma concentration-time curve of prednisolone. There was a noticeable interindividual difference in response. Digestion of nutrients was not affected., Conclusions and Clinical Relevance: Modulation of P-gp expression influenced plasma concentrations of prednisolone after oral administration in dogs. Thus, treatment response to prednisolone may be influenced by coadministration of P-gp-modulating medications or feed ingredients.

Overweight in dogs is, as in other companion animals, a major risk factor for several metabolic disorders. However, it is not yet known whether immunity is challenged by increased body weight in dogs. The aim of this study was to investigate the effect of a short-term increase in body weight on immunological variables in adult healthy beagle dogs. Sixteen dogs, divided into a control group (CG) and weight gain group (WGG), were included. During a period of 13 weeks, the CG was fed at maintenance energy requirement (MER), whereas the WGG received a double amount of food. After 13 weeks, blood samples were taken for immunological and biochemical analyses. Weight gain and increased body condition score in the WGG were accompanied by a significant higher leptin concentration. Weight gain increased the number of lymphocytes and immunoglobulins A and M and was responsible for a higher proliferation of peripheral blood mononuclear cells (PBMC). Short-term increase of body weight thus seems to trigger immunological variables in dogs., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Cats are strict carnivores that rely on nutrients in animal tissues to meet their specific and unique nutritional requirements. In their natural habitat, cats consume prey high in protein with moderate amounts of fat and minimal carbohydrates in contrast to commercial diets, which are sometimes moderate to high in carbohydrates. This change in diet has been accompanied by a shift from an outdoor environment to an indoor lifestyle and decreased physical activity, because cats no longer need to hunt to obtain food. This transformation of the lifestyle of cats is thought to be responsible for the recent increase in incidence of obesity, insulin resistance, and diabetes mellitus in domestic cats. At first, an overview of the evolutionary physiological adaptations of carbohydrate digestion in the feline digestive tract and of the hepatic carbohydrate and protein metabolism reflecting the true carnivorous nature of cats is given. Secondly, this literature review deals with nutritional modulation of insulin sensitivity, focusing on dietary macronutrients, carbohydrate sources, and dietary fiber for prevention and treatment of insulin resistance.

Inflammatory bowel disease (IBD) is an idiopathic chronic inflammatory disease of the stomach, the small intestine and/or the large intestine. Loss of integrity of the intestinal barrier may be an important factor in the pathogenesis of IBD. In dogs, lymphoplasmacytic enteritis (LPE) is one of the recognized forms of IBD. P-glycoprotein (P-gp) is a membrane-bound efflux pump constituting an important component of the intestinal barrier. Changes in P-gp expression at the level of the intestinal barrier may be important in the pathogenesis of canine LPE, as this may lead to variable protection against xenobiotics and bacterial products in the intestine. The aim of the present study was to evaluate the expression of epithelial P-gp in the intestine in dogs with LPE compared with disease-free animals. Formalin-fixed intestinal biopsy samples from 57 dogs with histopathological evidence of LPE were immunolabelled with anti-P-gp antibodies (C494 and C219). Endoscopic biopsy samples of the duodenum and colon from 16 healthy beagles were used as controls. None of the control dogs had P-gp expression in the apical membrane of duodenal enterocytes, but all had P-gp labelling at the colonic epithelial surface. Twenty out of 57 dogs with LPE had P-gp expression at the apical surface membrane of villus epithelial cells in the duodenum, jejunum and/or ileum. Six out of 16 colonic samples from dogs with LPE had decreased P-gp expression at the epithelial surface compared with controls. It is unclear whether these changes in P-gp expression in dogs with LPE are a cause or a consequence of the inflammation. The observed changes could affect bioavailability of therapeutic drugs used in LPE., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The effect of dietary oligofructose and inulin supplementation on glucose metabolism in obese and non-obese cats was assessed. Two diets were tested in a crossover design; a control diet high in protein (46 % on DM basis), moderate in fat (15 %), low in carbohydrates (27 %), but no soluble fibres added; and a prebiotic diet, with 2.5 % of a mixture of oligofructose and inulin added to the control diet. Eight non-obese and eight obese cats were allotted to each of two diets in random order at intervals of 4 weeks. At the end of each testing period, intravenous glucose tolerance tests were performed. Area under the glucose curve (AUCgluc) was increased (P = 0.022) and the second insulin peak was delayed (P = 0.009) in obese compared to non-obese cats. Diets did not affect fasting plasma glucose concentrations, blood glucose response at each glucose time-point after glucose administration, AUCgluc, fasting serum insulin concentrations, area under the insulin curve, and height and appearance time of insulin response. Yet, analysis of acylcarnitines revealed higher propionylcarnitine concentrations (P = 0.03) when fed the prebiotic diet, suggesting colonic fermentation and propionate absorption. Prebiotic supplementation reduced methylmalonylcarnitine (P = 0.072) and aspartate aminotransferase concentrations (P = 0.025), both indicating reduced gluconeogenesis from amino acids. This trial evidenced impaired glucose tolerance and altered insulin response to glucose administration in obese compared to non-obese cats, regardless of dietary intervention; yet modulation of glucose metabolism by enhancing gluconeogenesis from propionate and inhibition of amino acid catabolism can be suggested.

Depending on type and inclusion level, dietary fibre may increase and maintain satiety and postpone the onset of hunger. This 7-week study evaluated the effect of fibre fermentability on physiological satiety-related metabolites and voluntary food intake (VFI) in dogs. Sixteen healthy adult dogs were fed a low-fermentable fibre (LFF) diet containing 8.5 % cellulose or a high-fermentable fibre (HFF) diet containing 8.5 % sugarbeet pulp and 2 % inulin. Large intestinal fibre degradation was evaluated by apparent faecal digestibility of nutrients and faecal SCFA and NH3 concentrations. Postprandial blood samples were obtained to determine postprandial plasma glucose, insulin, total peptide tyrosine-tyrosine (PYY), total glucagon-like peptide-1 (GLP-1) and total ghrelin concentrations. At the end of the study, the dogs were given a single meal of a dry dog food to determine VFI. Dogs fed the HFF diet had a significantly higher large intestinal fibre degradation and production of SCFA compared with the dogs fed the LFF diet. The HFF-fed dogs tended (P = 0.058) to show a lower VFI at the end of the study. No treatment effects were found for postprandial plasma glucose, PYY, GLP-1 and ghrelin responses. The concentrations of these metabolites could not be related to the observed difference in VFI. The inclusion of fermentable fibre in canine diets may contribute to the prevention or mitigation of obesity through its effects on satiety. The underlying mechanisms require further investigation.