Effects of L. reuteri NBF 2 DSM 32264 Consumption on the Body Weight, Body Condition Score, Fecal Parameters, and Intestinal Microbiota of Healthy Persian Cats.

In the literature, there are several studies showing the effects of different probiotic administrations in dogs, while there is limited information about their effects in cats. Furthermore, there are no studies that examined the effects of the probiotic strain Lactobacillus reuteri on cats' welfare, especially considering a specific breed. In this study, the effects of L. reuteri NBF 2 DSM 32264 on body weight, body condition score (BCS), and fecal parameters (fecal score and fecal moisture) of healthy Persian cats were assessed; additionally, a microbiological analysis was carried out to quantify bacterial species like Escherichia coli (for the total coliform count) and Lactobacilli. The administration of L. reuteri NBF 2 DSM 32264 showed no alteration in the body weight and body condition score of Persian cats. The fecal moisture decreased at the end of the study and the values of fecal score were improved. Moreover, at the end of the study period, an increase in Lactobacilli (p > 0.001) was observed. The data collected report the ability of L. reuteri NBF 2 DSM 32264 to improve fecal quality parameters in healthy adult Persian cats, leading to an increase in Lactobacilli and a reduction in total coliforms.

In Vitro Assessment of Postbiotic and Probiotic Commercial Dietary Supplements Recommended for Counteracting Intestinal Dysbiosis in Dogs.

Many environmental aspects influence the preservation of a beneficial microbiome in dogs, and gut dysbiosis occurs when imbalances in the intestinal ecosystem cause functional changes in the microbial populations. The authors evaluated the effects of two specific commercial dietary supplements: a combination of a postbiotic and prebiotics (Microbiotal cane®) and a probiotic product (NBF 1®) recommended for counteracting intestinal dysbiosis in dogs, on the gut canine microbiota composition and its metabolic activities (production of short-chain fatty acids). The investigation was performed using an in vitro fermentation system inoculated with dog fecal samples. Microbiotal cane® promoted a more immediate increase in Lactobacillus spp. after the first 6 h of fermentation, whereas NBF 1® promoted the increase at the end of the process only. The two supplements supported an increase in the Bifidobacterium spp. counts only after 24 h. The in vitro abilities of Microbiotal cane® and NBF 1® to increase selectively beneficial bacterial groups producing acetic, propionic, and butyric acids suggest a possible positive effect on the canine gut microbiota, even if further in vivo studies are needed to confirm the beneficial effects on the intestinal health.

Endocannabinoid System Receptors at the Hip and Stifle Joints of Middle-Aged Dogs: A Novel Target for the Therapeutic Use of Cannabis sativa Extract in Canine Arthropathies.

The endocannabinoid system (ECS) has emerged as a potential therapeutic target in veterinary medicine due to its involvement in a wide range of physiological processes including pain, inflammation, immune function, and neurological function. Modulation of the ECS receptors has been shown to have anti-inflammatory, analgesic, and immunomodulatory effects in various animal models of disease, including dogs with osteoarthritis. The goal of this study was to identify and compare the cellular expression and distribution of cannabinoid receptor type 1 (CB1R) and type 2 (CB2R) and the cannabinoid-related G protein-coupled receptor 55 (GPR55) on the synovial cells of hip and stifle joints of seven dogs of different breeds without overt signs of osteoarthritis (OA). The synovial membranes of seven hips and seven stifle joints were harvested post mortem. The expression of the CB1R, CB2R, and GPR55 present in the synovial tissues was investigated using qualitative and quantitative immunofluorescence and Western blot (Wb) analysis. Synoviocytes of the stifle and hip joints expressed CB1R, CB2R, and GPR55 immunoreactivity (IR); no significant differences were observed for each different joint. Cannabinoid receptor 2- and GPR55-IR were also expressed by macrophages, neutrophils, and vascular cells. The ECS receptors were widely expressed by the synovial elements of dogs without overt signs of OA. It suggests that the ECS could be a target for the therapeutic use of Cannabis sativa extract in canine arthropathies.

Expression of cannabinoid (CB1 and CB2) and cannabinoid-related receptors (TRPV1, GPR55, and PPARα) in the synovial membrane of the horse metacarpophalangeal joint.

Background: The metacarpophalangeal joint undergoes enormous loading during locomotion and can therefore often become inflamed, potentially resulting in osteoarthritis (OA). There are studies indicating that the endocannabinoid system (ECS) modulates synovium homeostasis, and could be a promising target for OA therapy. Some cannabinoid receptors, which modulate proliferative and secretory responses in joint inflammation, have been functionally identified in human and animal synovial cells. Objective: To characterize the cellular distribution of the cannabinoid receptors 1 (CB1R) and 2 (CB2R), and the cannabinoid-related receptors transient receptor potential vanilloid type 1 (TRPV1), G protein-related receptor 55 (GPR55) and peroxisome proliferator-activated receptor alpha (PPARα) in the synovial membrane of the metacarpophalangeal joint of the horse. Animals: The dorsal synovial membranes of 14 equine metacarpophalangeal joints were collected post-mortem from an abattoir. Materials and methods: The dorsal synovial membranes of 14 equine metacarpophalangeal joints were collected post-mortem from an abattoir. The expression of the CB1R, CB2R, TRPV1, GPR55, and PPARα in synovial tissues was studied using qualitative and quantitative immunofluorescence, and quantitative real-time reverse transcriptase PCR (qRT-PCR). Macrophage-like (MLS) and fibroblast-like (FLS) synoviocytes were identified by means of antibodies directed against IBA1 and vimentin, respectively. Results: Both the mRNA and protein expression of the CB2R, TRPV1, GPR55, and PPARα were found in the synoviocytes and blood vessels of the metacarpophalangeal joints. The synoviocytes expressed the mRNA and protein of the CB1R in some of the horses investigated, but not in all. Conclusions and clinical importance: Given the expression of the CB1R, CB2R, TRPV1, GPR55, and PPARα in the synovial elements of the metacarpophalangeal joint, these findings encouraged the development of new studies supporting the use of molecules acting on these receptors to reduce the inflammation during joint inflammation in the horse.

The Lipidomics of Spermatozoa and Red Blood Cells Membrane Profile of Martina Franca Donkey: Preliminary Evaluation.

Fatty acid-based lipidomic analysis has been widely used to evaluate health status in human medicine as well as in the veterinary field. In equine species, there has been a developing interest in fertility and sperm quality. Fatty acids, being the principal components of the membranes, play an active role in the regulation of the metabolic activities, and their role on spermiogenesis seems to be of great importance for the resulting quality of the sperm and, thus, fertility. With the application of widely used lipidomic techniques, the aim of this study was to evaluate: (a) the fatty acid content of the spermatozoa's membranes of 26 healthy male Martina Franca donkeys and its possible correlation with sperm parameters, and (b) the evaluation of the composition of the red blood cells' membrane. PUFA omega-6 are the principal components (40.38%) of the total PUFA content (47.79%) in both types of cells; however, DPA is the predominant one on the spermatozoa's membrane (27.57%) but is not present in the erythrocyte's membrane. Spermatozoa's motility (%) is positively correlated with stearic acid and EPA, and progressive motility (%), with oleic acid. These findings offer information on the composition of both types of cells' membranes in healthy male MF donkeys and reflect the metabolic transformations of the spermatozoa's membrane during the maturation period, providing a better perception of the role of fatty acids in sperm parameters and fertility.

Effects of Feeding a Hypoallergenic Diet with a Nutraceutical on Fecal Dysbiosis Index and Clinical Manifestations of Canine Atopic Dermatitis.

BACKGROUND: an imbalance of the intestinal microbiota can cause health problems in the gastrointestinal tract and in other organs. Canine Atopic Dermatitis (CAD) is a genetically predisposed, inflammatory and pruritic allergic skin disease with multifactorial etiology and multimodal treatment. The aim of this study was to assess the effect of a nutraceutical product on Dysbiotic Index (DI) and the skin lesions of atopic dogs. METHODS: a nutraceutical product was administered to 32 dogs with CAD. The product was associated with a standardized hypoallergenic diet for 60 days; the dietary regimen continued for 120 days, while ongoing therapies remained unchanged. Values of Visual Analogic Scale (VAS), Canine Atopic Dermatitis Lesional Index (CADLI) and DI were evaluated on day 0, 60, 120. RESULTS: all the 32 dogs showed a statistically significant decrease (p < 0.001) to V60 of VAS and CADLI, which persisted and increased to V120 when diet alone was continued. The decrease in the DI value was also statistically significant (p < 0.001). CONCLUSION: the intake of nutraceutical associated with diet resulted in a decrease in the index of intestinal dysbiosis, with an improvement in the subjective severity of cutaneous lesions.

The Fatty Acid-Based Erythrocyte Membrane Lipidome in Dogs with Chronic Enteropathy.

Canine chronic enteropathies (CEs) are inflammatory processes resulting from complex interplay between the mucosal immune system, intestinal microbiome, and dietary components in susceptible dogs. Fatty acids (FAs) play important roles in the regulation of physiologic and metabolic pathways and their role in inflammation seems to be dual, as they exhibit pro-inflammatory and anti-inflammatory functions. Analysis of red blood cell (RBC) membrane fatty acid profile represents a tool for assessing the quantity and quality of structural and functional molecular components. This study was aimed at comparing the FA membrane profile, determined by Gas Chromatography and relevant lipid parameter of 48 CE dogs compared with 68 healthy dogs. In CE patients, the levels of stearic (p < 0.0001), dihomo-gamma-linolenic, eicosapentaenoic (p = 0.02), and docosahexaenoic (p = 0.02) acids were significantly higher, and those of palmitic (p < 0.0001) and linoleic (p = 0.0006) acids were significantly lower. Non-responder dogs presented higher percentages of vaccenic acid (p = 0.007), compared to those of dogs that responded to diagnostic trials. These results suggest that lipidomic status may reflect the "gut health", and the non-invasive analysis of RBC membrane might have the potential to become a candidate biomarker in the evaluation of dogs affected by CE.

Inclusion of small intestinal absorption and simulated mucosal surfaces further improve the Mucosal Simulator of the Canine Intestinal Microbial Ecosystem (M-SCIME™).

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.

Untargeted fecal metabolome analysis in obese dogs after weight loss achieved by feeding a high-fiber-high-protein diet.

INTRODUCTION: In humans and companion animals, obesity is accompanied by metabolic derangements. Studies have revealed differences in the composition of the fecal microbiome between obese dogs and those with an ideal body weight. OBJECTIVES: We have previously reported that the fecal microbiome in obese dogs changes after controlled weight reduction, induced by feeding a diet high in fiber and protein. Despite these findings, it is unclear if taxonomic differences infer differences at the functional level between obese dogs and those with an ideal body weight. METHODOLOGY: Untargeted fecal metabolome analysis was performed on dogs with obesity before and after weight loss achieved by feeding a high-fiber-high-protein diet. RESULTS: Fecal metabolome analysis revealed a total of 13 compounds that changed in concentration in obese dogs after weight loss. Of these compounds, metabolites associated with bacterial metabolism decreased after weight loss including purine, L-(-)-methionine, coumestrol, and the alkaloids 1-methylxanthine and trigonelline. Conversely, the polyphenols (-)-epicatechin and matairesinol and the quinoline derivatives 1,5-isoquinolinediol and 2-hydroxiquinoline increased after weight loss. CONCLUSION: These results suggest differences in intestinal microbiome at the functional level after weight loss, but further studies are needed to determine the role of these compounds in the etiology of obesity and weight loss.

Comparison of the Therapeutic Effect of Treatment with Antibiotics or Nutraceuticals on Clinical Activity and the Fecal Microbiome of Dogs with Acute Diarrhea.

Dogs with acute diarrhea are often presented to clinical practice and, although this generally represents a self-limiting condition, antibiotics are still frequently used as treatment. The aim of this study was to evaluate the effects in dogs with acute non-hemorrhagic diarrhea of the administration of an antibiotic combination in comparison to a nutraceutical product. Thirty dogs were enrolled and randomly assigned to two groups: 15 dogs (group A) received a nutraceutical commercial product while 15 dogs (group B) received an antimicrobial combination of metronidazole and spiramycin. For each dog, the Canine Acute Diarrhea Severity Index, the fecal microbiota and the Dysbiosis Index were assessed. Both stool consistency and frequency decreased on day 2 in the dogs of group A compared to baseline, while in group B, these parameters significantly decreased at days 3 and 4. The global concern for rising antibiotic resistance associated with indiscriminate use of antimicrobials, in both humans and animals, suggests the necessity of avoiding empirical and injudicious use of these molecules in diarrheic dogs. These results suggest that the nutraceutical treatment had a similar clinical effect compared to the antibiotic formulation, representing a valid antibiotic-sparing therapeutic approach in canine acute diarrhea.

Circulating Endocannabinoids as Diagnostic Markers of Canine Chronic Enteropathies: A Pilot Study.

Chronic enteropathies (CEs) in dogs, according to the treatment response to consecutive trials, are classified as food-responsive (FRE), antibiotic-responsive (ARE), and immunosuppressive-responsive (IRE) enteropathy. In addition to this classification, dogs with loss of protein across the gut are grouped as protein-losing enteropathy (PLE). At present, the diagnosis of CEs is time-consuming, costly and sometimes invasive, also because non-invasive biomarkers with high sensitivity and specificity are not yet available. Therefore, this study aimed at assessing the levels of circulating endocannabinoids in plasma as potential diagnostic markers of canine CEs. Thirty-three dogs with primary chronic gastrointestinal signs presented to Veterinary Teaching Hospitals of Teramo and Bologna (Italy) were prospectively enrolled in the study, and 30 healthy dogs were included as a control group. Plasma levels of N-arachidonoylethanolamine (AEA), 2-arachidonoylglycerol (2-AG), N-palmitoylethanolamine (PEA), and N-oleoylethanolamine (OEA) were measured at the time of the first visit in dogs with different CEs, as well as in healthy subjects. Plasma levels of 2-AG (p = 0.001) and PEA (p = 0.008) were increased in canine CEs compared to healthy dogs. In particular, PEA levels were increased in the FRE group compared to healthy dogs (p = 0.04), while 2-AG was higher in IRE than in healthy dogs (p = 0.0001). Dogs affected by FRE also showed decreased 2-AG (p = 0.0001) and increased OEA levels (p = 0.0018) compared to IRE dogs. Moreover, dogs with PLE showed increased 2-AG (p = 0.033) and decreased AEA (p = 0.035), OEA (p = 0.016) and PEA (p = 0.023) levels, when compared to dogs affected by CEs without loss of proteins. The areas under ROC curves for circulating 2-AG (0.91; 95% confidence interval [CI], 0.79-1.03) and OEA (0.81; 95% CI, 0.65-0.97) showed a good accuracy in distinguishing the different forms of CEs under study (FRE, ARE and IRE), at the time of the first visit. The present study demonstrated that endocannabinoid signaling is altered in canine CEs, and that CE subtypes showed distinct profiles of 2-AG, PEA and OEA plasma levels, suggesting that these circulating bioactive lipids might have the potential to become candidate biomarkers for canine CEs.

Fecal microbiota in client-owned obese dogs changes after weight loss with a high-fiber-high-protein diet.

BACKGROUND: The fecal microbiota from obese individuals can induce obesity in animal models. In addition, studies in humans, animal models and dogs have revealed that the fecal microbiota of subjects with obesity is different from that of lean subjects and changes after weight loss. However, the impact of weight loss on the fecal microbiota in dogs with obesity has not been fully characterized. METHODS: In this study, we used 16S rRNA gene sequencing to investigate the differences in the fecal microbiota of 20 pet dogs with obesity that underwent a weight loss program. The endpoint of the weight loss program was individually tailored to the ideal body weight of each dog. In addition, we evaluated the qPCR based Dysbiosis Index before and after weight loss. RESULTS: After weight loss, the fecal microbiota structure of dogs with obesity changed significantly (weightedANOSIM; p = 0.016, R = 0.073), showing an increase in bacterial richness (p = 0.007), evenness (p = 0.007) and the number of bacterial species (p = 0.007). The fecal microbiota composition of obese dogs after weight loss was characterized by a decrease in Firmicutes (92.3% to 78.2%, q = 0.001), and increase in Bacteroidetes (1.4% to 10.1%, q = 0.002) and Fusobacteria (1.6% to 6.2%, q = 0.040). The qPCR results revealed an overall decrease in the Dysbiosis Index, driven mostly due to a significant decrease in E. coli (p = 0.030), and increase in Fusobacterium spp. (p = 0.017). CONCLUSION: The changes observed in the fecal microbiota of dogs with obesity after weight loss with a weight loss diet rich in fiber and protein were in agreement with previous studies in humans, that reported an increase of bacterial biodiversity and a decrease of the ratio Firmicutes/Bacteroidetes.

The Erythrocyte Membrane Lipidome of Healthy Dogs: Creating a Benchmark of Fatty Acid Distribution and Interval Values.

Molecular-based approaches are rapidly developing in medicine for the evaluation of physiological and pathological conditions and discovery of new biomarkers in prevention and therapy. Fatty acid diversity and roles in health and disease in humans are topical subjects of lipidomics. In particular, membrane fatty acid-based lipidomics provides molecular data of relevance in the study of human chronic diseases, connecting metabolic, and nutritional aspects to health conditions. In veterinary medicine, membrane lipidomics, and fatty acid profiles have not been developed yet in nutritional approaches to health and in disease conditions. Using a protocol widely tested in human profiling, in the present study erythrocyte membrane lipidome was examined in 68 clinically healthy dogs, with different ages, sex, and sizes. In particular, a cluster composed of 10 fatty acids, present in membrane glycerophospholipids and representative of structural and functional properties of cell membrane, was chosen, and quantitatively analyzed. The interval values and distribution for each fatty acid of the cluster were determined, providing the first panel describing the healthy dog lipidomic membrane profile, with interesting correlation to bodyweight increases. This molecular information can be advantageously developed as benchmark in veterinary medicine for the evaluation of metabolic and nutritional status in healthy and diseased dogs.

Yeast-Derived Formulations Are Differentially Fermented by the Canine and Feline Microbiome As Assessed in a Novel In Vitro Colonic Fermentation Model.

The current study evaluated the effect of five yeast-derived formulations (T1-T5) on microbial metabolism and composition of the canine and feline gut microbiota using a novel in vitro colonic incubation approach. This novel in vitro model allowed for growth of the entire spectrum of dog- and cat-derived bacteria from the inoculum, thus offering an excellent platform to evaluate effects of nutritional interventions on the gut microbiota. Further, yeast-derived ingredients differentially increased production of acetate, propionate, butyrate, ammonium, and branched short-chain fatty acids, with T5 and T1 consistently stimulating propionate and butyrate, respectively. 16S-targeted Illumina sequencing coupled with flow cytometry provided unprecedented high-resolution quantitative insights in canine and feline microbiota modulation by yeast-derived ingredients, revealing that effects on propionate production were related to Prevotellaceae, Tannerellaceae, Bacteroidaceae, and Veillonellaceae members, while effects on butyrate production were related to Erysipelotrichaceae, Lachnospiraceae, Ruminococcaceae, and Fusobacteriaceae. Overall, these findings strengthen the health-promoting potential of yeast-derived ingredients.

Effect of dietary antioxidant supplementation on fresh semen quality in stallion.

In this study, the effect of dietary supplementation of organic selenium, vitamin E, and zinc on raw semen characteristics was evaluated. Ten stallions with normal fertility were divided into two groups: a control group (CG), in which standard diet was provided, and a treated group (TG), in which the standard diet was supplemented with 1500 mg of α-tocopherol acetate, 360 mg of zinc, and 2.5 mg of organic selenium on a daily basis. Semen parameters on fresh semen were evaluated three times in all stallions before antioxidant supplementation (T0) and 30 (T1), 60 (T2), and 90 (T3) d after supplementation. Dietary supplementation with experimental antioxidants resulted in a significant increase in average path velocity (121.9 ± 3.1 µm/sec in TG vs 118.9 ± 4.3 µm/sec in CG), straightness (86.2 ± 2.4 % vs 82.6 ± 3.9 % in TG and CG respectively), viability (75.6 ± 10.2 % in TG vs 72.3 ± 6.9 % in CG) and total seminal plasma antioxidants levels (2.7 ± 0.5 mmol/l vs 1.9 ± 0.4 mmol/l in TG and CG respectively) while progressive motility 69.7 ± 11 % vs 62.2 ± 9.3 % in TG and CG stallions respectively) and abnormal sperm morphology (8.2±1.5 % in TG vs 14.4±4 % in CG) significantly improved in treated stallions after 60 d of supplementation. In contrast with previously reported in other species, a negative effect of antioxidant supplementation on semen concentration was recorded in the TG. A positive correlation between progressive motility and total antioxidants in seminal plasma in both treated and control stallions suggested that motility is affected by oxidative-antioxidative status, and that dietary antioxidant supplementation could increase the ability of spermatozoa to contrast reactive oxygen species or the ability of seminal plasma to reduce the oxidative stress. The improvement of semen parameters after antioxidant supplementation was not linear, and after 30 d (or 60 d for some parameters), a further increase was not noted. This evidence suggested that in our standard conditions, dietary intake of these antioxidants could be slightly under the dietary requirement and further evaluation of the actual nutrition requirements of organic selenium, zinc, and vitamin E in the stallion are needed.

High-performance liquid chromatography with post-column derivatisation and fluorescence detection for sensitive determination of aflatoxin M1 in milk and cheese.

A new HPLC method with fluorescence detection using pyridinium hydrobromide perbromide as a post-column derivatising agent has been developed to determine aflatoxin M1 in milk and cheese. The detection limits were 1 ng/kg for milk and 5 ng/kg for cheese. The calibration curve was linear from 0.001 to 0.1 ng injected. The method includes a preliminary C18-SPE clean-up and the average recoveries of Aflatoxin M1 from milk and cheese, spiked at levels of 25-75 ng/kg and 100-300 ng/kg, respectively, were 90 and 76%; the precision (RSDr) ranged from 1.7 to 2.6% for milk and from 3.5 to 6.5% for cheese. The method is rapid, easily automatable and therefore useful for accurate and precise screening of aflatoxin M1 in milk and cheese.