Iron Bioaccessibility and Speciation in Microalgae Used as a Dog Nutrition Supplement.

Chlorella vulgaris, Arthrospira platensis, Haematoccocus pluvialis, and Phaeodactylum tricornutum are species of interest for commercial purposes due to their valuable nutritional profile. The aim of this study was to investigate the iron content in these four microalgae, with emphasis on their iron bioaccessibility assessed using an in vitro digestion system to simulate the process which takes place in the stomach and small intestine of dogs, followed by iron quantification using atomic absorption spectrometry. Furthermore, the extraction of soluble proteins was carried out and size exclusion chromatography was applied to investigate iron speciation. Significant differences (p < 0.004) in iron content were found between C. vulgaris, which had the highest (1347 ± 93 µg g-1), and H. pluvialis, which had the lowest (216 ± 59 µg g-1) iron content. C. vulgaris, A. platensis, and H. pluvialis showed an iron bioaccessibility of 30, 31, and 30%, respectively, while P. tricornutum showed the lowest bioaccessibility (11%). The four species analysed presented soluble iron mainly bound to proteins with high molecular mass ranging from >75 to 40 kDa. C. vulgaris showed the highest iron content associated with good bioaccessibility; therefore, it could be considered to be an interesting natural source of organic iron in dog nutrition.

Fecal Microbiota, Bile Acids, Sterols, and Fatty Acids in Dogs with Chronic Enteropathy Fed a Home-Cooked Diet Supplemented with Coconut Oil.

Medium-chain fatty acids (MCFAs) are considered to be interesting energy sources for dogs affected by chronic enteropathies (CE). This study analyzed the clinical scores, fecal microbiota, and metabolomes of 18 CE dogs fed a home-cooked diet (HCD) supplemented with virgin coconut oil (VCO), a source of MCFA, at 10% of metabolizable energy (HCD + VCO). The dogs were clinically evaluated with the Canine Chronic Enteropathy Activity Index (CCECAI) before and at the end of study. Fecal samples were collected at baseline, after 7 days of HCD, and after 30 days of HCD + VCO, for fecal score (FS) assessment, microbial analysis, and determination of bile acids (BA), sterols, and fatty acids (FA). The dogs responded positively to diet change, as shown by the CCECAI improvement (p = 0.001); HCD reduced fecal fat excretion and HCD + VCO improved FS (p < 0.001), even though an increase in fecal moisture occurred due to HCD (p = 0.001). HCD modified fecal FA (C6:0: +79%, C14:0: +74%, C20:0: +43%, C22:0: +58%, C24:0: +47%, C18:3n-3: +106%, C20:4n-6: +56%, and monounsaturated FA (MUFA): -23%, p < 0.05) and sterol profile (coprostanol: -27%, sitostanol: -86%, p < 0.01). VCO increased (p < 0.05) fecal total saturated FA (SFA: +28%, C14:0: +142%, C16:0 +21%, C22:0 +33%) and selected MCFAs (+162%; C10:0 +183%, C12:0 +600%), while reducing (p < 0.05) total MUFA (-29%), polyunsaturated FA (-26%), campesterol (-56%) and phyto-/zoosterols ratio (0.93:1 vs. 0.36:1). The median dysbiosis index was <0 and, together with fecal BA, was not significantly affected by HCD nor by VCO. The HCD diet increased total fecal bacteria (p = 0.005) and the abundance of Fusobacterium spp. (p = 0.028). This study confirmed that clinical signs, and to a lesser extent fecal microbiota and metabolome, are positively influenced by HCD in CE dogs. Moreover, it has been shown that fecal proportions of MCFA increased when MCFAs were supplemented in those dogs. The present results emphasize the need for future studies to better understand the intestinal absorptive mechanism of MCFA in dogs.

A Novel UHPLC-MS/MS Method for the Measurement of 25-Hydroxyvitamin D3 in Canine Serum and Its Application to Healthy Dogs.

Several studies have shown the importance of vitamin D3 supplementation in small animals. In dogs, a low vitamin D3 status is associated not only with bone metabolism but also with different kinds of disorders, such as congestive heart failure, gastrointestinal diseases, chronic kidney diseases, and some types of cancer. However, it is crucial to maintain balance and monitor the introduction of this essential nutrient through the diet because over-supplementation can result in toxicity. Due to the clinical importance of assessing the vitamin D3 status in small animal patients, a quick, simple, and highly performing analytical method for its measurement is needed. In this study, we describe the development of a novel liquid chromatography-tandem mass spectrometry method for 25-hydroxyvitamin D3 quantification in canine serum. The approach was successfully validated following current European guidelines, proving excellent linearity (R2 always ≥0.996), accuracy (always within ±13%) and precision (always <10%). The application of the validated approach to samples collected from 40 healthy dogs made possible the definition of a reliable reference interval for 25-hydroxyvitamin D3, the main biomarker of vitamin D3. In addition, variations below 5% in the results obtained quantifying the same samples using a water-based calibration curve demonstrated that a surrogate matrix may be used without affecting data accuracy. Thanks to its simplicity, the proposed technique represents a useful tool for supporting clinical routine and investigating correlations between serum concentrations of this metabolite and multiple diseases. Additionally, it could enable the monitoring of supplementation in small animal patients in veterinary clinical practice.

From Nutritional Adequacy to Hygiene Quality: A Detailed Assessment of Commercial Raw Pet-Food for Dogs and Cats.

Raw meat-based diets (RMBDs) are widely used as unconventional diets for dogs and cats at different life stages, despite concerns regarding nutritional adequacy and microbial contamination. The aim of this study was to evaluate both the nutritional and hygiene quality profile of RMBDs purchased in Germany. For this purpose, crude nutrients were assessed in 44 RMBDs and compared to declared values. In addition, selected minerals were determined in 31 RMBDs labelled as complete and compared to the minimum requirement (MR) for intended species and life stages. Aerobic colony count (ACC) and Enterobacteriaceae were used to assess the hygiene quality of 37 commercial RMBDs, while the presence of Salmonella spp. was examined in 10 products. Fat and protein content exceeded tolerated deviation from declared values in 33% and 45% of RMBDs, respectively. Each RMBD showed at least one concern regarding nutrient content. The RMBDs had high fat contents (mean 69, range 33−95 g/Mcal) that were negatively correlated with protein (r = −0.74, p < 0.0001). Considerable contaminations by ACC and Enterobacteriaceae were found (2.61 × 108 ± 3.63 × 108 and 3.61 × 106 ± 8.39 x106 CFU/g, respectively). A higher count of Enterobacteriaceae was detected in a frozen RMBDs made of poultry or carcasses from different animals, compared to the thawed counterpart (p = 0.003), as well as compared to other sources, such as beef, horse, and lamb, regardless of the storage condition. Salmonella spp. were found in 2/10 RMBDs. This study confirmed that feeding commercial RMBDs might pose a risk to pet health.

Evaluation of Weight Gain, Clinicopathological and Radiographic Changes after Early Diagnosis and Treatment of Congenital Hypothyroidism in Cats.

Congenital hypothyroidism is uncommon in cats. This case report describes weight gain, clinicopathological and radiographic changes after early diagnosis and treatment of congenital hypothyroidism in three British shorthair cats' siblings. Data were assessed at 53 (diagnosis), 83, 185 and 365 days of age. Correlations between serum insulin-like growth factor-1 (IGF-1) and body weight, levothyroxine dose, total thyroxine, and thyroid-stimulating hormone concentrations were evaluated. The body weights of the congenital hypothyroid kittens were compared with those of their two healthy siblings and British shorthair kittens of the same age. At diagnosis, the congenital hypothyroid kittens showed a significantly lower body weight compared to the healthy siblings (p = 0.03). After diagnosis, oral levothyroxine supplementation was started. The difference in body weight was no longer observed after one month of treatment. The clinical signs, clinicopathological and radiographic abnormalities ameliorated after one month of treatment. IGF-1 concentration was significantly positively correlated with body weight (rs = 0.80, p < 0.002). In conclusion, resolution of the clinical signs, achieving a consistent within-breed weight, and improvement of the clinicopathological and radiographic parameters demonstrated the importance of the early diagnosis and treatment of feline congenital hypothyroidism.

Fecal microbiota and inflammatory and antioxidant status of obese and lean dogs, and the effect of caloric restriction.

Introduction: Obesity is the most common nutritional disease in dogs, and is generally managed by caloric restriction. Gut microbiota alteration could represent a predisposing factor for obesity development, which has been associated with a low-grade inflammatory condition and an impaired antioxidant status. Besides, weight loss has been shown to influence the gut microbiota composition and reduce the inflammatory response and oxidative stress. Method: However, these insights in canine obesity have not been fully elucidated. The aim of this study was to assess the differences in serum and inflammatory parameters, antioxidant status, fecal microbiota and bacterial metabolites in 16 obese and 15 lean client-owned dogs and how these parameters in obese may be influenced by caloric restriction. First, for 30 days, all dogs received a high-protein, high-fiber diet in amounts to maintain their body weight; later, obese dogs were fed for 180 days the same diet in restricted amounts to promote weight loss. Results: Before the introduction of the experimental diet (T0), small differences in fecal microbial populations were detected between obese and lean dogs, but bacterial diversity and main bacterial metabolites did not differ. The fecal Dysbiosis Index (DI) was within the reference range (< 0) in most of dogs of both groups. Compared to lean dogs, obese dogs showed higher serum concentrations of acute-phase proteins, total thyroxine (TT4), and antioxidant capacity. Compared to T0, dietary treatment affected the fecal microbiota of obese dogs, decreasing the abundance of Firmicutes and increasing Bacteroides spp. However, these changes did not significantly affect the DI. The caloric restriction failed to exert significative changes on a large scale on bacterial populations. Consequently, the DI, bacterial diversity indices and metabolites were unaffected in obese dogs. Caloric restriction was not associated with a reduction of inflammatory markers or an improvement of the antioxidant status, while an increase of TT4 has been observed. Discussion: In summary, the present results underline that canine obesity is associated with chronic inflammation. This study highlights that changes on fecal microbiota of obese dogs induced by the characteristics of the diet should be differentiated from those that are the consequence of the reduced energy intake.

Seaweed Supplementation Failed to Affect Fecal Microbiota and Metabolome as Well as Fecal IgA and Apparent Nutrient Digestibility in Adult Dogs.

The present study investigated in dogs the dietary effects of intact seaweeds on some fecal bacterial populations and metabolites, fecal IgA and apparent total tract digestibility (ATTD). Ten healthy adult dogs were enrolled in a 5 × 5 replicated Latin square design to evaluate five dietary treatments: control diet (CD); CD + Ascophyllum nodosum; CD + Undaria pinnatifida; CD + Saccharina japonica; CD + Palmaria palmata (n replicates per treatment = 10). Seaweeds were added to food at a daily dose of 15 g/kg. The CD contained silica as a digestion marker. Each feeding period lasted 28 d, with a 7 d wash-out in between. Feces were collected at days 21 and 28 of each period for chemical and microbiological analyses. Fecal samples were collected during the last five days of each period for ATTD assessment. Dogs showed good health conditions throughout the study. The fecal chemical parameters, fecal IgA and nutrient ATTD were not influenced by algal supplementation. Similarly, microbiological analyses did not reveal any effect by seaweed ingestion. In conclusion, algal supplementation at a dose of 15 g/kg of diet failed to exert noticeable effects on the canine fecal parameters evaluated in the present study.

On the Variability of Microbial Populations and Bacterial Metabolites within the Canine Stool. An in-Depth Analysis.

Canine faecal microbial populations and metabolome are being increasingly studied to understand the interplay between host and gut microbiome. However, the distribution of bacterial taxa and microbial metabolites throughout the canine stool is understudied and currently no guidelines for the collection, storage and preparation of canine faecal samples have been proposed. Here, we assessed the effects that different sampling points have on the abundance of selected microbial populations and bacterial metabolites within the canine stool. Whole fresh faecal samples were obtained from five healthy adult dogs. Stool subsamples were collected from the surface to the inner part and from three equally sized areas (cranial, central, caudal) along the length axis of the stool log. All samples were finally homogenised and compared before and after homogenisation. Firmicutes, Bacteroidetes, Clostridium cluster I, Lactobacillus spp., Bifidobacterium spp. and Enterococcus spp. populations were analysed, as well as pH, ammonia and short-chain fatty acids (SCFA) concentrations. Compared to the surface of the stool, inner subsamples resulted in greater concentrations of SCFA and ammonia, and lower pH values. qPCR assay of microbial taxa did not show any differences between subsamples. Homogenisation of faeces does not affect the variability of microbial and metabolome data. Although the distribution patterns of bacterial populations and metabolites are still unclear, we found that stool subsampling yielded contradictory result and biases that can affect the final outcome when investigating the canine microbiome. Complete homogenisation of the whole stool is therefore recommended.

In Vitro Evaluation of the Effects of Tylosin on the Composition and Metabolism of Canine Fecal Microbiota.

The present study investigated the in vitro effects of tylosin (TYL), alone or associated with prebiotics (PRE), on selected canine fecal parameters. Eight treatments were set up: control diet with no addition of substrates; TYL; Fructooligosaccharides (FOS); Galactooligosaccharides (GOS); Xylooligosaccharides (XOS); TYL + FOS; TYL + GOS; TYL + XOS. The flasks (five for treatment), containing a canine fecal suspension (prepared with the feces of healthy adult dogs) and the residue of an in vitro digested dry dog food, were incubated in an anaerobic chamber at 39 °C. TYL and PRE were added at a concentration of 0.2 and 1 g/L, respectively. Samples were collected after 6 and 24 h for analyses. PRE decreased pH values, iso-butyrate, and iso-valerate throughout the incubation; increased lactobacilli, cadaverine, and, tendentiously, total volatile fatty acids after 6 h; increased n-butyrate, putrescine, spermidine, and reduced spermine and E. coli after 24 h. TYL resulted in lower total volatile fatty acids and lactobacilli and higher Clostridium cluster I after 6 h and higher pH values, spermidine, and E. coli throughout the study. When associated with TYL, PRE counteracted some undesirable effects of the antibiotic such as the decrease of lactobacilli and Clostridium cluster XIVa at both 6 and 24 h. In the present study, TYL exhibited inhibitory effects on canine fecal microbiota partially counteracted by PRE supplementation.

Influence of dietary protein and fructooligosaccharides on fecal fermentative end-products, fecal bacterial populations and apparent total tract digestibility in dogs.

BACKGROUND: Feeding dogs with diets rich in protein may favor putrefactive fermentations in the hindgut, negatively affecting the animal's intestinal environment. Conversely, prebiotics may improve the activity of health-promoting bacteria and prevent bacterial proteolysis in the colon. The aim of this study was to evaluate the effects of dietary supplementation with fructooligosaccharides (FOS) on fecal microbiota and apparent total tract digestibility (ATTD) in dogs fed kibbles differing in protein content. Twelve healthy adult dogs were used in a 4 × 4 replicated Latin Square design to determine the effects of four diets: 1) Low protein diet (LP, crude protein (CP) 229 g/kg dry matter (DM)); 2) High protein diet (HP, CP 304 g/kg DM); 3) Diet 1 + 1.5 g of FOS/kg; 4) Diet 2 + 1.5 g of FOS/kg. The diets contained silica at 5 g/kg as a digestion marker. Differences in protein content were obtained using different amounts of a highly digestible swine greaves meal. Each feeding period lasted 28 d, with a 12 d wash-out in between periods. Fecal samples were collected from dogs at 0, 21 and 28 d of each feeding period. Feces excreted during the last five days of each feeding period were collected and pooled in order to evaluate ATTD. RESULTS: Higher fecal ammonia concentrations were observed both when dogs received the HP diets (p < 0.001) and the supplementation with FOS (p < 0.05). The diets containing FOS resulted in greater ATTD of DM, Ca, Mg, Na, Zn, and Fe (p < 0.05) while HP diets were characterized by lower crude ash ATTD (p < 0.05). Significant interactions were observed between FOS and protein concentration in regards to fecal pH (p < 0.05), propionic acid (p < 0.05), acetic to propionic acid and acetic + n-butyric to propionic acid ratios (p < 0.01), bifidobacteria (p < 0.05) and ATTD of CP (p < 0.05) and Mn (p < 0.001). CONCLUSIONS: A relatively moderate increase of dietary protein resulted in higher concentrations of ammonia in canine feces. Fructooligosaccharides displayed beneficial counteracting effects (such as increased bifidobacteria) when supplemented in HP diets, compared to those observed in LP diets and, in general, improved the ATTD of several minerals.

An in vitro evaluation of the effects of a Yucca schidigera extract and chestnut tannins on composition and metabolic profiles of canine and feline faecal microbiota.

The in vitro effect of a Yucca schidigera extract (YSE) and tannins from chestnut wood on composition and metabolic activity of canine and feline faecal microbiota was evaluated. Four treatments were carried out: control diet, chestnut tannins (CT), YSE and CT + YSE. The YSE was added to canine and feline faecal cultures at 0.1 g/l, while CT were added at 0.3 g/l for a 24-h incubation. A total of 130 volatile compounds were detected by means of headspace-solid phase microextraction gas-chromatography/mass spectrometry analyses. Several changes in the metabolite profiles of fermentation fluids were found, including a decrease of alcohols (-19%) and esters (-42%) in feline and canine inoculum, respectively, which was due to the antibacterial properties of tannins. In canine inoculum, after 6 h, YSE + CT caused lower cadaverine concentrations (-37%), while ammonia (-4%) and quinolone (-27%) were reduced by addition of CT. After 24 h, the presence of CT resulted in a decrease of sulphur compounds, such as dimethyl sulphide (-69%) and dimethyl disulphide (-20%). In feline faecal cultures, after 6 h, CT lowered the amount of indole (-48%), whereas YSE tended to decrease trimethylamine levels (-16%). Both in canine and feline inoculum, addition of CT and, to a minor extent, YSE affected volatile fatty acids patterns. In canine faecal cultures, CT exerted a marginal inhibitory effect on Escherichia coli population (-0.45 log 10 numbers of DNA copies/ml), while enterococci were increased (+2.06 log 10 numbers of DNA copies/ml) by YSE. The results from the present study show that YSE and tannins from chestnut wood exert different effects on the composition and metabolism of canine and feline faecal microbiota. In particular, the supplementation of YSE and tannins to diets for dogs and cats may be beneficial due to the reduction of the presence of some potentially toxic volatile metabolites in the animals' intestine.

In vitro influence of dietary protein and fructooligosaccharides on metabolism of canine fecal microbiota.

BACKGROUND: The present in vitro study investigated whether the utilization of fructooligosaccharides (FOS) may influence canine fecal microbial population in presence of diets differing in their protein content and digestibility. Fresh fecal samples were collected from five adult dogs, pooled, and incubated for 24 h with the undigested residue of three diets: 1, Low protein high digestibility diet (LP HD, crude protein (CP) 229 g/kg); 2, High protein high digestibility diet (HP HD, CP 304 g/kg); 3, High protein low digestibility diet (HP LD, CP 303 g/kg) that had been previously subjected to enzymatic digestion. In the in vitro fermentation study, there were six treatments: 1) LP HD; 2) HP HD 3) HP LD; 4) LP HD + FOS; 5) HP HD + FOS; 6) HP LD + FOS. Fructooligosaccharides were added at the final concentration of 1.5 g/L. Samples of fermentation fluid were collected at 6 and 24 h of incubation. RESULTS: Values of pH were reduced by FOS at 6 and 24 h (P < 0.001); conversely, low protein digestibility and high dietary protein level resulted in higher pH at both sampling times (P < 0.001). At 24 h, FOS lowered ammonia (-10%; P < 0.001) and resulted (P < 0.05) in higher concentrations of total volatile fatty acids (VFA) (+43%), acetic acid (+14%), propionic acid (+75%) and n-butyric acid (+372%). Conversely, at 24 h, low protein digestibility resulted (P < 0.01) in lower concentrations of acetic acid (-26%), propionic acid (-37%) and total VFA (-21%). Putrescine concentrations were increased at 6 and 24 h of fermentation by low protein digestibility (+21 and 22%, respectively; P < 0.05) and FOS (+18 and 24%, respectively; P < 0.01). After 24 h of fermentation, high dietary protein level resulted in lower counts of lactobacilli and enterococci (-0.5 and -0.7 log cells/mL, respectively; P < 0.05) whereas low protein digestibility tended to increase counts of C. perfringens (+0.2 log cells/mL; P = 0.07). CONCLUSIONS: Results from the present study showed that diets rich in protein may exert negative influences on the canine intestinal ecosystem, slightly increasing the presence of ammonia and reducing counts of lactobacilli and enterococci. Moreover, the presence of poorly digestible protein resulted in lower concentrations of VFA. Conversely, administration of FOS may improve metabolism of canine intestinal microbiota, reducing ammonia concentrations and enhancing VFA production.