High starch intake favours body weight control in neutered and spayed cats living in homes fed ad libitum.

Different starch-to-protein ratios were compared among neutered and spayed domiciled cats. Male and female obese and non-obese cats were fed kibble diets ad libitum for 4 months high in starch (HS (38 % crude protein (CP)): starch 32 %, protein 38 %; DM basis) or high in protein (HP (55 % CP): starch 19 %, protein 55 %) but similar in energy and fat in a crossover design. Physical activity was evaluated using an accelerometer, and body composition (BC), energy expenditure (EE) and water turnover (WT) using the doubly labelled water method. Results were compared in a 2 diet × 2 sex × 2 body condition factorial arrangement. Cats fed the HS (38 % CP) diet maintained a constant body weight, but lean mass (LM) tended to be reduced in female obese but to be increased in male non-obese (P < 0·08) and increased in female non-obese cats (P = 0·01). The HP (55 % CP) diet induced an increase in cat body weight and LM (P < 0·05) without altering BC proportion. EE tended to be higher in males (351 (se 8) kJ/kg0·67/d) than females (330 (se 8) kJ/kg0·67/d; P = 0·06), was unaffected by diet or BC, decreased as age increased (R 2 0·44; P < 0·01) and increased as physical activity increased (R 2 0·58; P < 0·01). WT was higher for the HP (55 % CP) diet (P < 0·01) and increased with EE (R 2 0·65; P < 0·01). The HS (38 % CP) diet favoured body weight control during 4 months of ad libitum feeding. Caution is necessary to balance protein in diets of female obese cats over 5 years, as they may have low energy and food intake, with LM loss.

The Impact of Fiber Source on Digestive Function, Fecal Microbiota, and Immune Response in Adult Dogs.

This study evaluated the impact of different fiber sources on intestinal function, fecal microbiota, and overall health in dogs. Twelve dogs were used in a crossover design, involving three periods of 6 weeks and three diets: a low-fiber diet (CTR), a cereal-fiber and beet-pulp-supplemented diet (BRA), and a fruit-fiber-supplemented diet (FRU). Each period included a digestibility trial and fecal and blood sampling in the last week. Short-chain fatty acids (SCFAs) and microbiota taxonomy (16S rRNA Illumina-MiSeq) and functionality (Shotgun-NovaSeq 6000) were determined in the feces. General biochemistry, complete blood cells, and lymphocyte subsets were also analyzed. The fiber-supplemented diets showed lower digestibility without significant changes in the fecal consistency. The BRA diet showed higher total SCFA concentrations (p = 0.056), with increases in alpha diversity and particular beneficial genera, such as Lachnospira, Bifidobacterium, and Faecalibacterium. The BRA microbiota was also associated with an overabundance of genes related to carbohydrate and amino acid metabolism. The FRU diet had a distinct impact on the microbiota composition and functionality, leading to higher levels of CD8 lymphocytes. These findings emphasize the importance of selecting the right fiber source when formulating dog diets, as it can have a differential impact on gut microbiota and animal health.

Age-associated changes in intestinal health biomarkers in dogs.

The gut microbiome is critical for maintaining host health. In healthy humans, the aging process is one of the main factors modulating the changes in the intestinal microbiota. However, little is known about the relationship between gut health, microbiota, and the aging process in dogs. The present study aims to explore the differences in the intestinal microbiota and intestinal health based on fecal biomarkers in a population of dogs of different ages. The study involved 106 dogs of different breeds aged between 0.2 and 15 years categorized as senior (>7 years; n = 40), adult (2-7 years; n = 50), and junior (< 2 years; n = 16). Fecal samples were collected during the same period at the same facilities. The analysis included the following gut health indicators: 16S rRNA gene sequencing to investigate the differences in the fecal microbiota; qPCR to determine the dysbiosis index; fecal short-chain fatty acid concentrations; fecal calprotectin; and immunoglobulin A. Beta diversity analysis revealed a significant difference with a small effect size (p = 0.003; R = 0.087) among age categories based on the unweighted UniFrac metric, but no significance was observed based on the weighted UniFrac metric or Bray-Curtis distances. There were no significant differences in the alpha diversity measures or the fecal dysbiosis index among age categories. Senior dogs had significantly higher relative abundance proportions in phyla Bacteroidota and Pseudomonadota and the genus Faecalibacterium, but not on qPCR analysis. At the family level, Ruminococcaceae, Uncl. Clostridiales.1, Veillonellaceae, Prevotellaceae, Succinivibrionaceae, and Bacteroidaceae abundances were higher in the senior category than in the adult and/or junior categories. Relative proportions, but not concentrations of fecal acetate, were higher in the senior category, while butyrate, isovaleric acid, and valeric acid were lower. The valeric acid concentration was significantly lower in the senior category than in the adult category. Calprotectin and immunoglobulin A levels did not differ significantly across groups. In conclusion, this study observed multiple minor changes in the fecal microbiota composition and the relative amount of short-chain fatty acids in dogs among different age groups, but studies in larger populations representative of all ages are warranted to refine the present results.

Adherence of Uropathogenic Escherichia Coli in Dog Urine After Consumption of Food Supplemented with Cranberry (Vaccinium Macrocarpon).

Introduction: Escherichia coli is the most common pathogen isolated from the urine of dogs with urinary tract infections (UTIs). While there are many studies in humans investigating the potential for the prevention of UTIs by dietary consumption of cranberry, few analogous studies have been carried out in dogs. Material and Methods: Eight dogs, four male and four female, were successively fed two diets, first a control without cranberry, and then the second diet containing cranberry extracts. Naturally excreted urine was collected on the tenth day after the start of each diet for 24 h and used for bacterial growth. Madin-Darby canine kidney cell adherence by the uropathogenic E. coli G1473 strain expressing type 1 pili and positive for P pili and haemolysin gene markers was quantified after growth in urine samples. Results: Significant reductions in bacterial adherence to MDCK cells (from -16.5 to -73.4%, P < 0.05) were observed in the four females but not in the males after consumption of the cranberry extracts compared to the same animals consuming the control diet. Conclusion: Dietary supplementation with cranberry may provide some degree of protection to female dogs against adhesion of uropathogenic E. coli to urinary epithelial cells.

Evaluation of Efficacy of a Dental Chew to Reduce Gingivitis, Dental Plaque, Calculus, and Halitosis in Toy Breed Dogs.

The aim of this study was to determine the clinical efficacy of a dental chew with mechanical and chemical properties in 2 toy dog breeds. Eight Yorkshire terriers and 9 Chihuahuas participated in a crossover design trial. Gingivitis, dental plaque, calculus, and volatile sulfur compounds (VSC) in the breath were assessed after 4 weeks and 9 weeks, respectively. When dogs were fed 1 dental chew per day they had significantly reduced gingivitis (-20%, P < .001), accumulation of plaque (-15%, P < .001), calculus (-35%, P = .001), and VSC concentration (-19%, P < .001) compared to when receiving no chew. A significant breed effect was observed on all the average dental indices and VSC concentration.

Effect of dietary fat to starch content on fecal microbiota composition and activity in dogs1.

Dietary fat is known to modulate the hindgut microbiota in rodents; however, there is no clear evidence on the impact of high-fat diets on canine gut microbiota. The purpose of this study was to investigate the effect of feeding of diets differing in the amount of ME provided by fat and starch on the composition and activity of canine fecal microbiota. Twelve adult (3 to 7 yr of age) spayed Beagle dogs received a low-fat-high-starch diet (LF-HS; approximately 23%, 42%, and 25% ME provided by fat, starch, and CP, respectively) and a high-fat-low-starch diet (HF-LS; approximately 43%, 22%, and 25% ME provided by fat, starch, and CP, respectively) following a 2-period crossover arrangement. The higher amount of fat in the HF-LS diet was provided by lard, whereas the higher amount of starch in the LF-HS diet was provided primarily by maize and broken rice. Each period lasted 7 wk and included 4 wk for diet adaptation. Dogs were fed to meet their daily energy requirements (set at 480 kJ ME/kg BW0.75). Fecal samples were collected on weeks 5 and 6 of each period for the analysis of bacterial richness, diversity, and composition [by Ion-Torrent next-generation sequencing], bile acids, ammonia, and VFA. Additional fecal samples were collected from four dogs per diet and period to use as inocula for in vitro fermentation using xylan and pectin as substrates. Gas production was measured at 2, 4, 6, 9, 12, and 24 h of incubation. On week 7, blood samples were collected at 0- and 180-min postfeeding for the analysis of bacterial lipopolysaccharide (LPS). Feeding the HF-LS diet led to a greater (P < 0.05) fecal bile acid concentration compared with the LF-HS diet. Bacterial richness and diversity did not differ between diets (P > 0.10). However, dogs showed a lower relative abundance of Prevotella (P < 0.01), Solobacterium (P < 0.05), and Coprobacillus (P ˂ 0.05) when fed of the HF-LS diet. Fecal ammonia and VFA contents were not affected by diet (P > 0.10). Relative to the LF-HS diet, in vitro fermentation of xylan using feces of dogs fed the HF-LS diet produced less gas at 6 h (P < 0.01) and 9 h (P < 0.05). Blood LPS did not increase at 180-min postfeeding with either diet (P < 0.10). These findings indicate that feeding a HF-LS diet to dogs does not affect bacterial diversity or fermentative end products in feces, but may have a negative impact on Prevotella and xylan fermentation.

Fecal microbiota composition changes after a BW loss diet in Beagle dogs.

In developed countries, dogs and cats frequently suffer from obesity. Recently, gut microbiota composition in humans has been related to obesity and metabolic diseases. This study aimed to evaluate changes in body composition, and gut microbiota composition in obese Beagle dogs after a 17-wk BW loss program. A total of six neutered adult Beagle dogs with an average initial BW of 16.34 ± 1.52 kg and BCS of 7.8 ± 0.1 points (9-point scale) were restrictedly fed with a hypocaloric, low-fat and high-fiber dry-type diet. Body composition was assessed with dual-energy X-ray absorptiometry scan, before (T0) and after (T1) BW loss program. Individual stool samples were collected at T0 and T1 for the 16S rRNA analyses of gut microbiota. Taxonomic analysis was done with amplicon-based metagenomic results, and functional analysis of the metabolic potential of the microbial community was done with shotgun metagenomic results. All dogs reached their ideal BW at T1, with an average weekly proportion of BW loss of -1.07 ± 0.03% of starting BW. Body fat (T0, 7.02 ± 0.76 kg) was reduced by half (P < 0.001), while bone (T0, 0.56 ± 0.06 kg) and muscle mass (T0, 8.89 ± 0.80 kg) remained stable (P > 0.05). The most abundant identified phylum was Firmicutes (T0, 74.27 ± 0.08%; T1, 69.38 ± 0.07%), followed by Bacteroidetes (T0, 12.68 ± 0.08%; T1, 16.68 ± 0.05%), Fusobacteria (T0, 7.45 ± 0.02%; T1, 10.18 ± 0.03%), Actinobacteria (T0, 4.53 ± 0.02%; T1, 3.34 ± 0.01%), and Proteobacteria (T0, 1.06 ± 0.01%; T1, 1.40 ± 0.00%). At genus level, the presence of Clostridium, Lactobacillus, and Dorea, at T1 decreased (P = 0.028), while Allobaculum increased (P = 0.046). Although the microbiota communities at T0 and T1 showed a low separation level when compared (Anosim's R value = 0.39), they were significantly biodiverse (P = 0.01). Those differences on microbiota composition could be explained by 13 genus (α = 0.05, linear discriminant analysis (LDA) score > 2.0). Additionally, differences between both communities could also be explained by the expression of 18 enzymes and 27 pathways (α = 0.05, LDA score > 2.0). In conclusion, restricted feeding of a low-fat and high-fiber dry-type diet successfully modifies gut microbiota in obese dogs, increasing biodiversity with a different representation of microbial genus and metabolic pathways.

24-hour evaluation of dental plaque bacteria and halitosis after consumption of a single placebo or dental treat by dogs.

OBJECTIVE To determine whether consumption of a single dental treat with specific mechanical properties and active ingredients would provide a 24-hour effect on dental plaque bacteria and halitosis in dogs. ANIMALS 10 dogs of various breeds from a privately owned colony that had received routine dental scaling and polishing 4 weeks before the study began. PROCEDURES Dogs were randomly assigned to receive 1 placebo or dental treat first. A 4-week washout period was provided, and then dogs received the opposite treatment. Oral plaque and breath samples were collected before and 0.5, 3, 12, and 24 hours after treat consumption. Volatile sulfur compounds (VSCs) concentration was measured in breath samples. Total aerobic, total anaerobic, Porphyromonas gulae, Prevotella intermedia-like, Tannerella forsythia, and Fusobacterium nucleatum bacterial counts (measured via bacterial culture) and total live bacterial counts, total live and dead bacterial counts, and bacterial vitality (measured via quantitative real-time PCR assay) were assessed in plaque samples. RESULTS Compared with placebo treat consumption, dental treat consumption resulted in a significant decrease in breath VSCs concentration and all plaque bacterial counts, without an effect on bacterial vitality. Effects of the dental treat versus the placebo treat persisted for 12 hours for several bacterial counts and for 24 hours for breath VSCs concentration. CONCLUSIONS AND CLINICAL RELEVANCE Although clinical benefits should be investigated in larger scale, longer-term studies, results of this study suggested that feeding the evaluated dental treat may help to decrease oral bacterial growth in dogs for 12 hours and oral malodor for 24 hours. A feeding interval of 12 hours is therefore recommended.

Fat digestibility is reduced in old cats with subnormal cobalamin concentrations.

Fat digestibility is decreased in old cats for unknown reasons. Subclinical gastrointestinal diseases and pancreatic dysfunction, both related to ageing, can affect food digestibility. The aim of the present study was to elucidate the prevalence of subnormal cobalamin concentration and pancreatic disease in old cats and study the relationship between both markers and fat digestibility. A total of sixty-four cats without evident signs of gastrointestinal disease were included and grouped according to age: (1) fifteen middle-aged (MA), aged 3-7 years; and (2) forty-nine old, aged 10-17 years. All cats were tested for serum cobalamin, specific feline pancreatic lipase (fPL) and feline trypsin-like immunoreactivity. Then, sixteen of the old cats were selected and grouped according to cobalamin and fPL concentrations: control (normal cobalamin and fPL); low vitamin B12 (cobalamin <290 ng/l; normal fPL); and high fPL (normal cobalamin; fPL >4 µg/ml). A food digestibility trial with a high-fat diet (21·6 %) was performed. In the old group, cobalamin was lower and fPL higher than in MA cats. Of the old cats (n 49), 14 % had subnormal cobalamin, 8 % had a severe increase in fPL, 2 % had both alterations and 14 % had a slight increase in fPL. By contrast, MA cats did not have cobalamin deficiency or an increase in fPL concentrations. Fat digestibility was lower in low vitamin B12 cats than control cats. Decreased fat digestibility is not present in all old cats but could be a characteristic of subclinical chronic gastrointestinal disease. Cobalamin concentration, as a marker of gastrointestinal disease, could be useful for the routine evaluation of old cats.

Effect of citrus polyphenol- and curcumin-supplemented diet on inflammatory state in obese cats.

Among obesity-associated disorders, low-grade inflammation has been described. The putative therapeutic properties of citrus and curcumin polyphenols could be associated with their anti-inflammatory properties. Two diets supplemented either with hesperidin (0.05 %) and naringin (0.1 %) from citrus extract or with highly bioavailable curcumin from Curcuma longa extract (0.09 %) were fed to eight obese cats for two 8-week periods (cross-over study design) while maintaining animals in an obese state. Plasma acute-phase protein (APP; α1-acid glycoprotein (AGP), serum amyloid A and haptoglobin) levels were assessed before and at the end of each test period. TNF-α, IL-1ß, IL-2, IL-4, IL-5, IL-10, IL-12, IL-18, transforming growth factor-ß, interferon (IFN)-γ mRNA levels were determined in peripheral blood mononuclear cells (PBMC) by real-time PCR. Compared with pre-study values, supplementation with citrus polyphenols resulted in lower plasma AGP and haptoglobin concentrations, while that with curcumin resulted in lower plasma AGP concentration. There were no differences between the supplementations. TNF-α, IL-1ß, IL-4, IL-5, IL-10, IL-12, IL-18, transforming growth factor-ß, mRNA levels remained unaffected by either dietary supplementation. In contrast, IFN-γ and IL-2 mRNA levels were lower at the end of the citrus and the curcumin supplementation, respectively. There were no differences between the supplementations. The present study results show a slight effect of citrus and curcumin supplementation on inflammatory markers expressed by PBMC, and a decreased concentration of APP, which are mainly expressed by the liver. This would confirm that hesperidin and naringin or highly bioavailable curcumin extract have beneficial effects, targeted in the liver and could improve the obesity-related inflammatory state.

Effects of consuming diets containing various fats or citrus flavanones on plasma lipid and urinary F2-isoprostane concentrations in overweight cats.

OBJECTIVE: To compare in overweight cats the effects of feeding moderate-energy diets with moderate fat content but with saturated fat (beef tallow), saturated fat plus citrus flavanones, or monounsaturated fat (olive oil) on plasma lipids and urinary F2-isoprostane concentrations. ANIMALS: 20 overweight cats with mean+/-SD body weight of 5.2+/-0.2 kg and mean body condition score of 7.8+/-0.2 (9-point scale). PROCEDURES: Body weight, plasma total cholesterol and triacylglycerol concentrations, and urinary F2-isoprostane concentration (as marker of oxidative stress) were measured at the beginning of the study, when the cats were fed a maintenance diet, and after 1, 3, and 5 months of consuming test diets. RESULTS: In overweight cats, citrus flavanones supplementation of the saturated fat diet was associated with lower energy intake and with lower plasma lipids and urinary F2-isoprostane concentrations than in cats fed the saturated fat alone. Monounsaturated fat feeding resulted in lower food intake than in cats fed saturated fat. However, plasma lipids concentrations remained within reference limits throughout the study. CONCLUSIONS AND CLINICAL RELEVANCE: Although the clinical relevance of these findings is unknown, the significant differences detected indicated that lower energy intake with citrus flavanones supplementation or with substitution of saturated fat for monounsaturated fat could be good strategies for decreasing plasma lipids concentration and oxidative stress in overweight cats, even before considerable loss of body weight is observed.

Identification and evaluation of the probiotic potential of lactobacilli isolated from canine milk.

Canine milk protects puppies against infectious diseases through a variety of mechanisms. In this study, the presence of potentially probiotic lactic acid bacteria grown on MRS-Cys agar plates from milk of nine bitches was investigated. The Gram positive catalase negative bacilli identified in this study belonged to four Lactobacillus species (Lactobacillus reuteri, Lactobacillus fermentum, Lactobacillus murinus, Lactobacillus animalis), as well as one isolate that was identified as Weissella viridescens. Random amplified polymorphic DNA profiling revealed 28 different genetic profiles among the lactobacilli isolates. Their probiotic potential was evaluated through different assays, including survival in conditions that resemble those existing in the gastrointestinal tract, production of antimicrobial compounds, adherence to intestinal mucin, degradation of mucin and pattern of antibiotic sensitivity. Some strains showed potential for future applications as canine probiotics.

Plant polyphenol intake alters gene expression in canine leukocytes.

BACKGROUND/AIMS: Polyphenol compounds may explain most of the health-related beneficial effects of plants and vegetables, mainly through their antioxidant properties. The aim of the study was to assess the main changes on leukocyte gene expression of dogs caused by intake of three natural polyphenol-rich extracts and to compare them with caloric restriction. METHODS: 20 female dogs were divided into 5 groups: control fed ad libitum (C), caloric-restricted to 30% less than control (CR), and 3 groups fed ad libitum supplemented with citrus extract (CE), green tea extract (GTE) or grape seed extract (GSE). Leukocytes gene expression was analyzed in a specially designed microarray. RESULTS: CE treatment mainly downregulated genes related to inflammative (IL-8, VLA-4) and cytotoxic response (GRP 58) as well as proliferation of leukocytes. GTE induced gene expression related to leukocyte proliferation and signaling (GNAQ, PKC-B). GSE upregulated some of the genes increased by CE treatment. CR downregulated genes related with energy metabolism (ATP5A1, COX7C) and inflammatory markers (VLA-4). CONCLUSION: A chronic ingestion of citric, grape seed and green tea polyphenols is able to modulate canine leukocyte functions through changes in gene expression. CE ingestion reduces expression of some genes also diminished by a 30% caloric restriction.

Absorption and pharmacokinetics of green tea catechins in beagles.

The present study evaluates for the first time in dogs, the kinetics of green tea catechins and their metabolic forms in plasma and urine. Ten beagles were administered 173 mg (12.35 mg/kg body weight) of catechins as a green tea extract, in capsules. Blood samples were collected during 24 h after intake and urine samples were collected during the following periods of time: 0-2, 2-6, 6-8 and 8-24 h. Two catechins with a galloyl moiety and three conjugated metabolites were detected in plasma. Most of the detected forms in plasma reached their maximum plasma concentration (Cmax) at around 1 h. Median Cmax for ( - )-epigallocatechin-3-gallate (EGCG), ( - )-epicatechin-3-gallate (ECG), ( - )-epigallocatechin glucuronide (EGC-glucuronide), ( - )-epicatechin glucuronide (EC-glucuronide), ( - )-epicatechin sulphate (EC-sulphate) were 0.3 (range 0.1-1.9), 0.1 (range 0-0.4), 0.8 (range 0.2-3.9), 0.2 (range 0.1-1.7) and 1 (range 0.3-3.4) micromol/l, respectively. The areas under the plasma concentration v. time curves (AUC0 --> 24) were 427 (range 102-1185) micromol/l x min for EGC-glucuronide, 112 (range 53-919) micromol/l x min for EC-sulphate, 71 (range 26-306) micromol/l x min for EGCG, 40 (range 12-258) micromol/l x min for EC-glucuronide and 14 (range 0.1-124) micromol/l x min for ECG. The values of mean residence time (MRT0 --> 24) were 5 (range 2-16), 2 (range 1-11), 10 (range 2-13), 3 (range 2-16) and 2.4 (range 1-18) h for EGCG, ECG, EGC-glucuronide, EC-glucuronide and EC-sulphate, respectively. In urine, catechins were present as conjugated forms, suggesting bile excretion of EGCG and ECG. Green tea catechins are absorbed following an oral administration and EGC-glucuronide is the metabolic form that remains in the organism for a longer period of time, suggesting that this compound could suffer an enterohepatic cycle.

A new LC/MS/MS rapid and sensitive method for the determination of green tea catechins and their metabolites in biological samples.

A new rapid and sensitive method has been developed, using liquid chromatography in tandem mass spectrometry (LC-ESI-MS/MS) to identify green tea catechin metabolites in plasma and urine after oral intake of a green tea extract. (-)-Epigallocatechin-3-gallate (EGCG), (-)-epicatechin-3-gallate (ECG), (-)-epigallocatechin (EGC)-glucuronide, (-)-epicatechin (EC)-glucuronide, and EC-sulfate were identified in plasma, whereas in urine only the conjugated catechins were detected (EGC-glucuronide, EGC-sulfate, EC-glucuronide, and EC-sulfate). Standard calibration curves prepared in plasma were found to be linear in the range of 10.9-1379.3 nmol/L for EGCG, EGC, ECG, and EC. The accuracy and precision of this assay showed a coefficient of variation of <15%. The method allowed the detection and quantification limits (for 20 microL injection) from 1.1 to 2.6 nmol/L and 3.8-8.7 nmol/L, respectively, in plasma and 0.8-1.8 nmol/L and 2.6-6.0 nmol/L, respectively, in urine. This method can be applied for future clinical and epidemiological studies, allowing the identification of the active metabolites that will reach the target tissues.

Absorption and pharmacokinetics of grapefruit flavanones in beagles.

The present study evaluated the pharmacokinetics of three different grapefruit flavanone forms in dog plasma and demonstrated their absorption after an oral intake of a grapefruit extract; pharmacokinetic parameters of these forms were also determined. Ten healthy beagles were administered 70 mg citrus flavonoids as a grapefruit extract contained in capsules, while two additional dogs were used as controls and given an excipient. The grapefruit flavanone naringin, along with its metabolites naringenin and naringenin glucuronide, was detected in dog plasma. Blood samples were collected between 0 and 24 h after administration of the extract. Naringin reached its maximun plasma concentration at around 80 min, whereas naringenin and naringenin glucuronide reached their maximun plasma concentrations at around 20 and 30 min, respectively. Maximum plasma concentrations of naringin, naringenin and naringenin glucuronide (medians and ranges) were 0.24 (0.05-2.08), 0.021 (0.001-0.3) and 0.09 (0.034-0.12) micromol/l, respectively. The areas under the curves were 23.16 l (14.04-70.62) min x micromol/for nariningin, 1.78 (0.09-4.95) min x micromol/l for naringenin and 22.5 (2.74-99.23) min x micromol/l for naringenin glucuronide. The median and range values for mean residence time were 3.3 (1.5-9.3), 2.8 (0.8-11.2) and 8.0 (2.3-13.1) h for naringin, naringenin and naringenin glucuronide, respectively. The results of the present study demonstrate the absorption of grapefruit flavanones via the presence of their metabolites in plasma, thus making an important contribution to the field since the biological activities ascribed to these compounds rely on their specific forms of absorption.

Development and characterization of a canine skin equivalent.

The development of a complex cellular model, which incorporates the basic cell components of the dog skin, would be a useful tool to investigate the biology and pathology of canine skin and also to replace animal testing partially. The aim of the present study was to develop and characterize a canine skin equivalent. Epidermal keratinocytes and dermal fibroblasts were freshly isolated from skin biopsies from healthy dogs. Fibroblasts were embedded into a bio-matrix from collagen type I matrix protein; this built the scaffold where the keratinocytes were seeded, at air exposed conditions. At 3, 7, 15 and 21 days of culture in special growth media, skin equivalents were analysed by histological, immunohistochemical and electron microscopical techniques. At 15 days, keratinocytes underwent differentiation to a multilayer epidermis with stratum basal, stratum spinosum, stratum granulosum and stratum corneum. Expression of epidermal cytokeratins in keratinocytes was detected by immunhistochemistry, and followed the same pattern than in the normal canine epidermis. Fibroblasts from the skin equivalent expressed vimentin as dermal fibroblasts do. A basement membrane (BM) was observed underneath the epidermis; ultrastructurally, it was similar to the normal canine BM and collagen IV and laminin 5 were detected immunohistochemically as major components of this structure. Skin equivalents developed from canine cutaneous cells presented a similar morphological structure than healthy canine skin. Moreover, the immunohistochemical analysis revealed the expression of the major markers of the epidermis (keratins), dermis (vimentin) and BM (collagen type IV, laminin 5).