Comparison of the fecal microbiota of adult healthy dogs fed a plant-based (vegan) or an animal-based diet.

Purpose: Pet guardians are increasingly seeking vegan dog foods. However, research on the impact of these diets on gastrointestinal (GI) physiology and health is limited. In humans, vegan diets modify the GI microbiota, increasing beneficial digestive microorganisms. This study aimed to examine the canine fecal microbiota in response to a vegan diet compared to an animal-based diet. Methods: Sixty-one client-owned healthy adult dogs completed a randomized, double-blinded longitudinal study. Dogs were randomly assigned into two groups that were fed either a commercial extruded animal-based diet (MEAT, n = 30) or an experimental extruded vegan diet (PLANT, n = 31) for 12 weeks. Fecal collections occurred at the start of the experimental period and after 3 months of exclusively feeding either diet. Bacterial DNA was extracted from the feces, and the V4 region of the 16S rRNA gene was amplified using PCR and sequenced on Illumina MiSeq. Beta-diversity was measured using Jaccard and Bray-Curtis distances, and the PERMANOVA was used to assess for differences in fecal microbiota within and between groups. Alpha-diversity indices for richness, evenness, and diversity, as well as relative abundance, were calculated and compared between groups. Results: Beta-diversity differences occurred between diet groups at exit time-point with differences on Bray-Curtis distances at the family and genus levels (p = 0.007 and p = 0.001, respectively), and for the Jaccard distance at the family and genus level (p = 0.006 and p = 0.011, respectively). Significant differences in alpha-diversity occurred when comparing the PLANT to the MEAT group at the exit time-point with the PLANT group having a lower evenness (p = 0.012), but no significant differences in richness (p = 0.188), or diversity (p = 0.06). At exit-timepoint, compared to the MEAT group, the relative abundance of Fusobacterium, Bacteroides, and Campylobacter was lower in the PLANT group. The relative abundance of Fusobacterium decreased over time in the PLANT group, while no change was observed in the MEAT group. Conclusion: These results indicate that vegan diets may change the canine gut microbiota. Future studies are warranted to confirm our results and determine long-term effects of vegan diets on the canine gut microbiome.

Impact of fecal sample preservation and handling techniques on the canine fecal microbiota profile.

Canine fecal microbiota profiling provides insight into host health and disease. Standardization of methods for fecal sample storage for microbiomics is currently inconclusive, however. This study investigated the effects of homogenization, the preservative RNAlater, room temperature exposure duration, and short-term storage in the fridge prior to freezing on the canine fecal microbiota profile. Within 15 minutes after voiding, samples were left non-homogenized or homogenized and aliquoted, then kept at room temperature (20-22°C) for 0.5, 4, 8, or 24 hours. Homogenized aliquots then had RNAlater added or not. Following room temperature exposure, all aliquots were stored in the fridge (4°C) for 24 hours prior to storing in the freezer (-20°C), or stored directly in the freezer. DNA extraction, PCR amplification, then sequencing were completed on all samples. Alpha diversity (diversity, evenness, and richness), and beta diversity (community membership and structure), and relative abundances of bacterial genera were compared between treatments. Homogenization and RNAlater minimized changes in the microbial communities over time, although minor changes in relative abundances occurred. Non-homogenized samples had more inter-sample variability and greater changes in beta diversity than homogenized samples. Storage of canine fecal samples in the fridge for 24 h prior to storage in the freezer had little effect on the fecal microbiota profile. Our findings suggest that if immediate analysis of fecal samples is not possible, samples should at least be homogenized to preserve the existing microbiota profile.

The Effect of Dietary Synbiotics in Actively Racing Standardbred Horses Receiving Trimethoprim/Sulfadiazine.

Synbiotics are often provided to horses receiving antibiotics to protect against microbiome disturbances, despite a lack of evidence for efficacy. The purpose of this study was to evaluate the effect of a synbiotic product in horses receiving antibiotics. Sixteen actively racing Standardbred horses were randomly allocated (four-way crossover) to one of four groups: antibiotics (10 days; AB), synbiotics (28 days; PROBIOPlusTM; PBP), PBP + AB, or Control. The fecal microbiome was investigated using 16S rRNA sequencing, and fecal dry matter (DM; %), pH, and scores (FS; 0-9) were measured. Data were analyzed with two-way ANOVA. Results found microbiota differences in community membership between PBP + AB and all other treatments during and after antibiotic treatment. During antibiotic treatment, AB and PBP + AB were significantly different from Control. After antibiotic treatment, PBP + AB was significantly different from all other treatments. The few differences found in relative abundance of phyla or predominant genera were mostly in fiber degrading bacteria. The Fibrobacter population was significantly higher in AB and PBP + AB horses than Control. Unclassified Ruminococcaceae was significantly higher in Control than AB and PBP. After antibiotic treatment, PBP + AB horses were significantly higher than PBP horses. In conclusion, these data provide support for the ability of PROBIOPlus™ to maintain healthy gastrointestinal microbiome during antibiotic treatment.

The influence of a probiotic/prebiotic supplement on microbial and metabolic parameters of equine cecal fluid or fecal slurry in vitro.

The microbes that reside within the equine hindgut create a complex and dynamic ecosystem. The equine hindgut microbiota is intimately associated with health and, as such, represents an area which can be beneficially modified. Synbiotics, supplements that combine probiotic micro-organisms with prebiotic ingredients, are a potential means of influencing the hindgut microbiota to promote health and prevent disease. The objective of the current study was to evaluate the influence of an equine probiotic/prebiotic supplement on characteristics of the microbiota and metabolite production in vitro. Equine cecal fluid and fecal material were collected from an abattoir in QC, CAN. Five hundred milliliters of cecal fluid was used to inoculate chemostat vessels maintained as batch fermenters (chemostat cecal, N = 11) with either 0 g (control) or 0.44 g of supplement added at 12 h intervals. One hundred milliliters of cecal fluid (anaerobic cecal, N = 15) or 5% fecal slurry (anaerobic fecal, N = 6) were maintained in an anaerobic chamber with either 0 g (control) or 0.356 g of supplement added at the time of vessel establishment. Samples were taken from vessels at vessel establishment (0), 24, or 48 h of incubation. Illumina sequencing of the V4 region of the 16S rRNA gene and bioinformatics were performed for microbiome analysis. Metabolite data was obtained via NMR spectroscopy. All statistical analyses were run in SAS 9.4. There was no effect of treatment at 24 or 48h on alpha or beta diversity indices and limited taxonomic differences were noted. Acetate, propionate, and butyrate were higher in treated compared to untreated vessels in all methods. A consistent effect of supplementation on the metabolic profile with no discernable impact on the microbiota of these in vitro systems indicates inoculum microbe viability and a utilization of the provided fermentable substrate within the systems. Although no changes within the microbiome were apparent, the consistent changes in metabolites indicates a potential prebiotic effect of the added supplement and merits further exploration.

This research investigated the impact of an equine prebiotic/probiotic supplement on the equine cecal microbiota by utilizing an in vitro fermentation system. By using two types of fermentation systems and inocula obtained using a fecal slurry and cecal contents, we evaluated how the addition of the supplement changed the microbial function over the 48 h experimental period. Although the supplement did drastically influence the production of volatile fatty acids produced by the microbes in all systems, the microbial composition did not change. Thus, indicating the supplement did not, in this in vitro context, provide probiotic or prebiotic potential. However, the systems remained viable and the microbes actively metabolized substrate for the duration of the experiment.

Fecal prevalence of Clostridium innocuum DNA in healthy horses and horses with colitis.

This study compared the prevalence of C. innocuum DNA in the feces of healthy horses and horses with acute colitis. C. innocuum was identified in 22% (15/68) of colitis cases and 18% (12/68) of healthy horses (p = 0.416).

Fecal microbiota of horses with colitis and its association with laminitis and survival during hospitalization.

BACKGROUND: The association of microbiota with clinical outcomes and the taxa associated with colitis in horses remains generally unknown. OBJECTIVES: Describe the fecal microbiota of horses with colitis and investigate the association of the fecal microbiota with the development of laminitis and survival. ANIMALS: Thirty-six healthy and 55 colitis horses subdivided into laminitis (n = 15) and non-laminitis (n = 39, 1 horse with chronic laminitis was removed from this comparison) and survivors (n = 27) and nonsurvivors (n = 28). METHODS: Unmatched case-control study. The Illumina MiSeq platform targeting the V4 region of the 16S ribosomal RNA gene was used to assess the microbiota. RESULTS: The community membership (Jaccard index) and structure (Yue and Clayton index) were different (analysis of molecular variance [AMOVA]; P < .001) between healthy and colitis horses. The linear discriminant analysis effect size (LEfSe; linear discriminant analysis [LDA] >3; P < .05) and random forest analyses found Enterobacteriaceae, Lactobacillus, Streptococcus, and Enterococcus enriched in colitis horses, whereas Treponema, Faecalibacterium, Ruminococcaceae, and Lachnospiraceae were enriched in healthy horses. The community membership and structure of colitis horses with or without laminitis was (AMOVA; P > .05). Enterobacteriaceae, Streptococcus, and Lactobacillus were enriched in horses with laminitis (LDA > 3; P < .05). The community membership (AMOVA; P = .008) of surviving and nonsurviving horses was different. Nonsurviving horses had an enrichment of Enterobacteriaceae, Pseudomonas, Streptococcus, and Enterococcus (LDA >3; P < .05). CONCLUSION AND CLINICAL IMPORTANCE: Differences in the microbiota of horses with colitis that survive or do not survive are minor and, similarly, the microbiota differences in horses with colitis that do or do not develop laminitis are minor.

A Comparison of Methods to Maintain the Equine Cecal Microbial Environment In Vitro Utilizing Cecal and Fecal Material.

The equine gastrointestinal (GI) microbiota is intimately related to the horse. The objective of the current study was to evaluate the microbiome and metabolome of cecal inoculum maintained in an anaerobic chamber or chemostat batch fermenter, as well as the fecal slurry maintained in an anaerobic chamber over 48 h. Cecal and fecal content were collected from healthy adult horses immediately upon death. Cecal fluid was used to inoculate chemostat vessels (chemostat cecal, n = 11) and vessels containing cecal fluid (anaerobic cecal, n = 15) or 5% fecal slurry (anaerobic fecal, n = 6) were maintained in an anaerobic chamber. Sampling for microbiome and metabolome analysis was performed at vessel establishment (0 h), and after 24 h and 48 h of fermentation. Illumina sequencing was performed, and metabolites were identified via nuclear magnetic resonance (NMR). Alpha and beta diversity indices, as well as individual metabolite concentrations and metabolite regression equations, were analyzed and compared between groups and over time. No differences were evident between alpha or beta diversity in cecal fluid maintained in either an anaerobic chamber or chemostat. The microbiome of the fecal inoculum maintained anaerobically shifted over 48 h and was not comparable to that of the cecal inoculum. Metabolite concentrations were consistently highest in chemostat vessels and lowest in anaerobic fecal vessels. Interestingly, the rate of metabolite change in anaerobic cecal and chemostat cecal vessels was comparable. In conclusion, maintaining an equine cecal inoculum in either an anaerobic chamber or chemostat vessel for 48 h is comparable in terms of the microbiome. However, the microbiome and metabolome of fecal material is not comparable with a cecal inoculum. Future research is required to better understand the factors that influence the level of microbial activity in vitro, particularly when microbiome data identify analogous communities.

Gastrin and Nitric Oxide Production in Cultured Gastric Antral Mucosa Are Altered in Response to a Gastric Digest of a Dietary Supplement.

In vitro organ culture can provide insight into isolated mucosal responses to particular environmental stimuli. The objective of the present study was to investigate the impact of a prolonged culturing time as well as the addition of acidic gastric fluid into the in vitro environment of cultured gastric antral tissue to evaluate how altering the commonly used neutral environment impacted tissue. Furthermore, we aimed to investigate the impact of G's Formula, a dietary supplement for horses, on the secretion of gastrin, interleukin1-beta (IL-1ß), and nitric oxide (NO). These biomarkers are of interest due to their effects on gastric motility and mucosal activity. Gastric mucosal tissue explants from porcine stomachs were cultured in the presence of a simulated gastric fluid (BL, n = 14), simulated gastric fluid containing the dietary supplement G's Formula (DF, n = 12), or an equal volume of phosphate buffered saline (CO, n = 14). At 48 and 60 h, 10-5 M carbachol was used to stimulate gastrin secretion. Cell viability was assessed at 72 h using calcein and ethidium-homodimer 1 staining. Media was analyzed for gastrin, IL-1ß, and NO at 48, 60, and 72 h. There were no effects of treatment or carbachol stimulation on explant cell viability. Carbachol resulted in a significant increase in gastrin concentration in CO and DF treatments, but not in BL. NO was higher in CO than in BL, and NO increased in the CO and DF treatments but not in BL. In conclusion, the addition of carbachol and gastric digests to culture media did not impact cell viability. The use of an acidic gastric digest (BL) reduced the effect of cholinergic stimulation with carbachol at a concentration of 10-5 M and reduced NO secretion. The addition of the dietary supplement to the gastric digest (DF) appeared to mediate these effects within this model. Further research is required to evaluate the specific effects of this dietary supplement on direct markers of mucosal activity and the functional relevance of these results in vivo.

The influence of a simulated digest of an equine dietary feed additive G's formula on contractile activity of gastric smooth muscle in vitro.

G's Formula is a novel equine feed additive formulated to promote optimal GI function. The objective of this study was to determine whether the addition of a simulated digest of the composite feed additive G's Formula (FA) would alter the contractile response of gastric smooth muscle to acetylcholine (Ach). Smooth muscle strips from porcine stomachs were excised and attached to an isometric force transducer. An experiment was run to compare tissue contraction between tissue exposed to FA (FA; n = 8, simulated digest of FA was added to the bath) and control tissue (CO; n = 8, no additions made). Increasing concentrations of Ach were added into the bath such that the concentration increased from 10-8 -10-3  M. Based on the analysis of these data, a difference between FA and CO was observed. Therefore, another trial was run which included a blank group (BL n = 6) in which the tissue was exposed to the simulated digest without FA. More CO (n = 5) and FA (n = 4) tissue was also run. Force was compared to baseline and between groups. In FA group, mean force for 1-min following all Ach additions was higher than baseline (p < .05) and by 2-min the integral-under-force/time curve (AUC) was higher than baseline from 10-7 -10-3  M compared to lower concentrations of Ach in both CO (10-6  M for both) and BL (10-5  M and 10-6  M, respectively). By 8-min AUC of all Ach concentrations were higher than baseline in FA compared to an Ach of 10-6  M in both CO and BL. A simulated digest of FA appears to sensitize gastric smooth muscle to Ach in vitro. FA may increase GI contractility, and the functional effect of this should be studied further in vivo.

A Botanical-Based Equine Nutraceutical Reduces Gastric Smooth Muscle Contractile Force In Vitro.

The objective of this study was to evaluate the effect of a botanical-based equine nutraceutical on contractility of gastric smooth muscle in vitro. Gastric ulcers are prevalent in performance horses and negatively impact horse welfare. Gastric hypermotility has been positively associated with the development of gastric ulceration in nonequine species, and reduction of hypermotility may be protective against their development. Stomachs from 12 pigs processed for food at a provincially inspected abattoir were collected within 1 hour of slaughter. Explants of nonglandular gastric tissue were prepared and suspended in a tissue bath, attached to a force transducer, in the presence or absence of a simulated digest extract of the nutraceutical. Tissue was stimulated to contract using increasing doses of acetylcholine. Peak and mean contractile force over 1 and 2 minutes after exposure to acetylcholine were measured. Exposure of gastric smooth muscle to the nutraceutical significantly reduced contractility of the tissue. These data provide support for the use of this nutraceutical to reduce contractility of nonglandular gastric smooth muscle and may indicate a protective effect of this nutraceutical in horses with mechanically induced gastric ulcers. Future studies are needed to clarify the role of gastric hypermotility on development of equine gastric ulcers and to determine the effect of this nutraceutical on equine gastric contractility and ulcerogenesis in vivo.

A time-course evaluation of inflammatory and oxidative markers following high-intensity exercise in horses: a pilot study.

Exercise is a physiological stress resulting in reactive oxygen species and inflammatory mediators, the accumulation of which are thought to contribute to degenerative articular diseases. The horse is of particular interest in this regard as equine athletes are frequently exposed to repetitive bouts of high-intensity exercise. The purpose of this study was to provide a detailed description of the response of articular and systemic oxidative and inflammatory biomarkers following high-intensity, exhaustive exercise in horses. A group of horses (Ex) underwent repeated bouts of high-intensity exercise, at a target heart rate of 180 beats/min, until voluntary exhaustion. Baseline plasma and synovial fluid (SF) samples were taken 24 h before exercise and then at 0.5, 1, 2, 4, 8, and 24 h following exercise cessation. This time course was repeated in a group of nonexercised control horses (Co). Plasma and SF samples were analyzed for prostaglandin E2 (PGE2), nitric oxide (NO), total antioxidant status (TAS), and glycosaminoglycans (GAG). The Ex group had significantly higher plasma NO at 0.5, 1, and 2 h; and higher plasma PGE2 at 0.5 and 1 h compared with Co. SF PGE2 and GAG were also higher in Ex horses at 8 h compared with Co. It is concluded that high-intensity exercise in horses results in a rapid increase in systemic oxidative and inflammatory markers from 0.5 to 2 h after exercise, which is followed by local articular inflammation and cartilage turnover at 8 h postexercise. NEW & NOTEWORTHY In horses, the influence of exercise systemically and within the articular space remains unclear and requires further detailed characterization. In this study, we identify that an acute bout of high-intensity exercise in horses induces systemic inflammation and oxidative stress within 30 min of exercise cessation, which lasts for ~2 h. Articular inflammation and cartilage turnover were also be observed within the equine carpal joint 8 h following exercise completion.