Equine Hindgut Research Articles

Development of Electrochemical DNA Biosensor for Equine Hindgut Acidosis Detection.

The pH drop in the hindgut of the horse is caused by lactic acid-producing bacteria which are abundant when a horse’s feeding regime is excessively carbohydrate rich. This drop in pH below six causes hindgut acidosis and may lead to laminitis. Lactic acid-producing bacteria Streptococcus equinus and Mitsuokella jalaludinii have been found to produce high amounts of L-lactate and D-lactate, respectively. Early detection of increased levels of these bacteria could allow the horse owner to tailor the horse’s diet to avoid hindgut acidosis and subsequent laminitis. Therefore, 16s ribosomal ribonucleic acid (rRNA) sequences were identified and modified to obtain target single stranded deoxyribonucleic acid (DNA) from these bacteria. Complementary single stranded DNAs were designed from the modified target sequences to form capture probes. Binding between capture probe and target single stranded deoxyribonucleic acid (ssDNA) in solution has been studied by gel electrophoresis. Among pairs of different capture probes and target single stranded DNA, hybridization of Streptococcus equinus capture probe 1 (SECP1) and Streptococcus equinus target 1 (SET1) was portrayed as gel electrophoresis. Adsorptive stripping voltammetry was utilized to study the binding of thiol modified SECP1 over gold on glass substrates and these studies showed a consistent binding signal of thiol modified SECP1 and their hybridization with SET1 over the gold working electrode. Cyclic voltammetry and electrochemical impedance spectroscopy were employed to examine the binding of thiol modified SECP1 on the gold working electrode and hybridization of thiol modified SECP1 with the target single stranded DNA. Both demonstrated the gold working electrode surface was modified with a capture probe layer and hybridization of the thiol bound ssDNA probe with target DNA was indicated. Therefore, the proposed electrochemical biosensor has the potential to be used for the detection of the non-synthetic bacterial DNA target responsible for equine hindgut acidosis.

Multidimensional Approach for Investigating the Effects of an Antibiotic-Probiotic Combination on the Equine Hindgut Ecosystem and Microbial Fibrolysis.

The equine hindgut ecosystem is specialized in dietary fibers’ fermentation to provide horses’ energy and contribute to its health. Nevertheless, antibiotics are known to disrupt the hindgut microbiota, affecting the fibrolytic activity of bacteria and the intestinal immune balance, leading to diseases. This in vivo study used a general and comprehensive approach for characterizing the hindgut ecosystem of 9 healthy horses over 28 days in response to a 5-day challenge with oral trimethoprim-sulfadiazine (TMS), with a special emphasis on microbial fibrolytic activity and the host immune response. Horses were supplemented with two doses of Lactobacillus acidophilus, Ligilactobacillus salivarius (formerly L. salivarius), and Bifidobacterium lactis blend or a placebo in a 3 × 3 Latin square design. Changes in fecal microbiota were investigated using 16S rRNA sequencing. Clostridioides difficile was quantified in feces using quantitative polymerase chain reaction. Anaerobic microbiological culture was used to enumerate functional bacterial groups (cellulolytic, amylolytic, and lactic acid-utilizing). The environmental dimensions were assessed by measuring the concentrations of volatile fatty acids (VFAs) and lactic acid using biochemical methods, and changes in pH and dry matter weight. Systemic and local inflammation was evaluated by determination of cytokine and immunoglobulin (Ig)A concentrations in the serum and secretory IgA (SIgA) concentrations in the feces using immuno-enzymatic methods. Oral TMS treatment strongly altered the whole hindgut ecosystem by 2 days after the first administration. Bacterial diversity decreased in proportion to the relative abundance of fibrolytic genera, which coincided with the decrease in the concentration of cellulolytic bacteria. At the same time, the composition of microbiota members was reorganized in terms of relative abundances, probably to support the alteration in fibrolysis. C. difficile DNA was not found in these horses, but the relative abundances of several potential pathobiont genera increased. 2 days after the first TMS administration, fecal concentrations of VFAs and SIgA increased in parallel with fecal water content, suggesting an alteration of the integrity of the hindgut mucosa. Recovery in bacterial composition, functions, and immune biomarkers took 2–9 days after the end of TMS administration. Supplementation with this bacterial blend did not limit bacterial alteration but might have interesting mucosal immunomodulatory effects.

Changes of microbial and metabolome of the equine hindgut during oligofructose-induced laminitis

BackgroundLaminitis is a common and serve disease which caused by inflammation and pathological changes of the laminar junction. However, the pathologic mechanism remains unclear. In this study we aimed to investigate changes of the gut microbiota and metabolomics in oligofructose-induced laminitis of horses.ResultsAnimals submitted to treatment with oligofructose had lower fecal pH but higher lactic acid, histamine, and Lipopolysaccharide (LPS) in serum. Meanwhile, oligofructose altered composition of the hindgut bacterial community, demonstrated by increasing relative abundance of Lactobacillus and Megasphaera. In addition, the metabolome analysis revealed that treatment with oligofructose decreased 84 metabolites while 53 metabolites increased, such as dihydrothymine, N3,N4-Dimethyl-L-arginine, 10E,12Z-Octadecadienoic acid, and asparagine. Pathway analysis revealed that aldosterone synthesis and secretion, regulation of lipolysis in adipocytes, steroid hormone biosynthesis, pyrimidine metabolism, biosynthesis of unsaturated fatty acids, and galactose metabolism were significantly different between healthy and laminitis horses. Furthermore, correlation analysis between gut microbiota and metabolites indicated that Lactobacillus and/or Megasphaera were positively associated with the dihydrothymine, N3,N4-Dimethyl-L-arginine, 10E,12Z-Octadecadienoic acid, and asparagine.ConclusionsThese results revealed that disturbance of gut microbiota and changes of metabolites were occurred during the development of equine laminitis, and these results may provide novel insights to detect biomarkers for a better understanding of the potential mechanism and prevention strategies for laminitis in horses.

PSIV-14 Fecal Microbiome of Horses Grazing Integrated Warm- and Cool-Season Grass Rotational Pasture Systems

Abstract The objective of this study was to characterize shifts in the fecal microbiota of horses grazing different forage types within integrated cool- and warm-season grass (CSG; WSG) pasture systems and to explore relationships between forage nutrients and microbial composition. Eight mares grazed integrated rotational systems containing mixed CSG and one of two WSG: bermudagrass or crabgrass. Fecal samples were collected after 2–3 weeks grazing WSG, CSG, and following an orchardgrass hay diet (HAY). Forage nutrients were determined by near-infrared spectroscopy and analyzed by two-way ANOVA in SAS (v.9.4). Following DNA extraction, 16S rRNA gene sequence analysis was conducted in QIIME 2 (v.2020.2) with Kruskall-Wallis tests for alpha diversity, Spearman correlation with forage nutrients, and taxonomic assignment with Greengenes. A random forest classifier and regressor determined ability to predict forage type and nutrients based upon bacterial composition. Statistical significance was set at p ≤ 0.05. Forage water-soluble carbohydrates (WSC) were greatest in CSG and lowest in WSG; neutral detergent fiber (NDF) was greater in HAY than CSG or WSG. Species richness and evenness (Shannon Index) was greater in horses adapted to WSG vs. CSG or HAY and was correlated with WSC (rs = -0.49) and ethanol-soluble carbohydrates (ESCrs = -0.62). The random forest classifier resulted in model accuracy of 1.0 and identified amplicon sequence variants (ASV) most important in prediction of forage type. Sixteen ASV were from the order Clostridiales including Lachnospiraceae, Ruminococcacceae, and Veillonellaceae families and the genus Coprococcus. Other important taxa included Prevotella spp., Streptococcus luteciae, and Fibrobacter succinogenes. The regressor accurately predicted WSC (r2 = 0.95) and ESC (r2 = 0.84), but not NDF (r2 = 0.09). These results suggest that the equine hindgut microbiome is impacted by forage type and soluble carbohydrate content; however, further research is required to determine functional and physiological significance in grazing horses.

Development of the equine hindgut microbiome in semi-feral and domestic conventionally-managed foals

BackgroundEarly development of the gut microbiome is an essential part of neonate health in animals. It is unclear whether the acquisition of gut microbes is different between domesticated animals and their wild counterparts. In this study, fecal samples from ten domestic conventionally managed (DCM) Standardbred and ten semi-feral managed (SFM) Shetland-type pony foals and dams were compared using 16S rRNA sequencing to identify differences in the development of the foal hindgut microbiome related to time and management.ResultsGut microbiome diversity of dams was lower than foals overall and within groups, and foals from both groups at Week 1 had less diverse gut microbiomes than subsequent weeks. The core microbiomes of SFM dams and foals had more taxa overall, and greater numbers of taxa within species groups when compared to DCM dams and foals. The gut microbiomes of SFM foals demonstrated enhanced diversity of key groups: Verrucomicrobia (RFP12), Ruminococcaceae, Fusobacterium spp., and Bacteroides spp., based on age and management. Lactic acid bacteria Lactobacillus spp. and other Lactobacillaceae genera were enriched only in DCM foals, specifically during their second and third week of life. Predicted microbiome functions estimated computationally suggested that SFM foals had higher mean sequence counts for taxa contributing to the digestion of lipids, simple and complex carbohydrates, and protein. DCM foal microbiomes were more similar to their dams in week five and six than were SFM foals at the same age.ConclusionsThis study demonstrates the impact of management on the development of the foal gut microbiome in the first 6 weeks of life. The higher numbers of taxa within and between bacterial groups found in SFM dams and foals suggests more diversity and functional redundancy in their gut microbiomes, which could lend greater stability and resiliency to these communities. The colonization of lactic acid bacteria in the early life of DCM foals suggests enrichment in response to the availability of dams’ feed. Thus, management type is an important driver of gut microbiome establishment on horses, and we may look to semi-feral horses for guidance in defining a healthy gut microbiome for domestic horses.

Transporting and Exercising Unconditioned Horses: Effects on Microflora Populations

The objective of this study was to determine if transportation and exercise stress in horses affect the microflora populations in the equine hindgut. Four horses were subjected to three transport periods (0, 3, and 6 hours) with a 7-d rest period between each transport. Horses were fed 0.91 kg/day of Purina Impact All Stages 12% and had ad libitum access to Cynodon dactylon (Coastal Bermudagrass) hay. Fecal samples were collected before (0 hours) and after (48 hours) transport. In addition, three horses underwent a different standardized exercise test with a 7-d rest period between each exercise. Standardized exercise test intensity was determined by heart rate to validate if the horse was in aerobic or anaerobic work. The protocol for fecal sample collection after exercise was the same as for transport. Prokaryotic community profiling was conducted by 16S metagenomic analysis. After DNA evaluation, differences were found in the microbiome at transport 0 hours and grouped transport 3 hours time 48 and transport 6 hours time 48 (PERMANOVA P = .037) where Bacteroidetes increased 48 hours after transport and Firmicutes decreased 48 hours after transport. Exercise microbial communities showed no difference in either alpha or beta diversity when compared with controls (0 hours). In the present study, difference in microflora may have resulted from stress duration of transport rather than stress duration of exercise.

Evaluation of equine rectal inoculum as representative of the microbial activities within the horse hindgut using a fully automated in vitro gas production technique system.

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

Multi-kingdom characterization of the core equine fecal microbiota based on multiple equine (sub)species

BackgroundEquine gut microbiology studies to date have primarily focused on horses and ponies, which represent only one of the eight extant equine species. This is despite asses and mules comprising almost half of the world’s domesticated equines, and donkeys being superior to horses/ponies in their ability to degrade dietary fiber. Limited attention has also been given to commensal anaerobic fungi and archaea even though anaerobic fungi are potent fiber degrading organisms, the activity of which is enhanced by methanogenic archaea. Therefore, the objective of this study was to broaden the current knowledge of bacterial, anaerobic fungal and archaeal diversity of the equine fecal microbiota to multiple species of equines. Core taxa shared by all the equine fecal samples (n = 70) were determined and an overview given of the microbiota across different equine types (horse, donkey, horse × donkey and zebra).ResultsEquine type was associated with differences in both fecal microbial concentrations and community composition. Donkey was generally most distinct from the other equine types, with horse and zebra not differing. Despite this, a common bacterial core of eight OTUs (out of 2070) and 16 genus level groupings (out of 231) was found in all the fecal samples. This bacterial core represented a much larger proportion of the equine fecal microbiota than previously reported, primarily due to the detection of predominant core taxa belonging to the phyla Kiritimatiellaeota (formerly Verrucomicrobia subdivision 5) and Spirochaetes. The majority of the core bacterial taxa lack cultured representation. Archaea and anaerobic fungi were present in all animals, however, no core taxon was detected for either despite several taxa being prevalent and predominant.ConclusionsWhilst differences were observed between equine types, a core fecal microbiota existed across all the equines. This core was composed primarily of a few predominant bacterial taxa, the majority of which are novel and lack cultured representation. The lack of microbial cultures representing the predominant taxa needs to be addressed, as their availability is essential to gain fundamental knowledge of the microbial functions that underpin the equine hindgut ecosystem.

PSVII-35 Fecal innocula from horses fed DigestaWell® Fiber improves in-vitro dry matter digestibility of commercial hays in horses

Abstract Enhanced microbial activity in the equine hindgut increases nutrient availability, reduces fecal waste, and adds value to low quality forages. The encapsulated dietary supplement DigestaWell® Fiber (DF; fibrolytic cofactors, methionine, B vitamins) has been shown to improve in-vitro digestibility measures of common hays fed to horses. However, its in-vivo effect on dry matter (DMd) and ADF digestibility (ADFd) is unknown. The objectives of this study were to test whether fecal innocula from horses fed DF would result in improved DMd or ADFd relative to innocula from unsupplemented horses consuming alfalfa or coastal bermudagrass hays. Utilizing a crossover design, four Quarter Horse mares were fed 2% BW daily alfalfa hay (AF; 32.8% NDF, 27.1% ADF) or coastal bermudagrass hay (CB; 63.2% NDF, 35.4% ADF), with or without oral DF supplementation (32 g/d), for 14d. Fresh feces were collected to serve as inoculum in an ANKOM Daisy II incubator at 37.5°C to determine in-vitro DMd and ADFd at 48h or 72h. Forage samples were run in triplicate. Data were transformed to approximate normality, then analyzed using a repeated measures mixed model in SAS with main effects of forage, treatment, incubation time, feeding period, and significant interactions. Across forages and incubation times, in-vivo DF supplementation improved DMd (P &amp;lt; 0.01). Across incubation times, DF improved ADFd of both AF (P &amp;lt; 0.0001) and CB (P = 0.04), but not DMd. Across forages, DF improved both DMd and ADFd at 48h (P &amp;lt; 0.001 and P = 0.003, respectively) but not at 72h (P = 0.29 and P = 0.27, respectively). Taken together, and combined with previous findings, these results provide further evidence that DF improves forage digestibility, presumably due to improved microbial fermentation in the equine hindgut.

92 Digestawell® fiber improves in-vitro digestibility of alfalfa and coastal bermudagrass hay fed to horses

Abstract Enhanced microbial activity in the equine hindgut increases nutrient availability, reduces fecal waste, and adds value to low quality forages. The dietary supplement DigestaWell® Fiber (DF; fibrolytic cofactors: methionine and B vitamins), designed to increase microbial efficiency, has been shown to improve in-vitro digestibility of alfalfa and tall fescue forages after 48h incubation. The objective of this study was to determine if DF would improve in-vitro dry matter digestibility (DMD) and ADF digestibility (ADFD) of alfalfa and coastal bermudagrass hays incubated for 48h or 72h. Utilizing a crossover design, four mature Quarter Horse mares were fed 2% BW daily of alfalfa (AF; 32.8% NDF, 27.1% ADF) or coastal bermudagrass (CB; 63.2% NDF, 35.4% ADF) hay for 19d periods. DMD and ADFD were determined after 48h or 72h incubation with or without treatment (320mg/L DF) at 37.5°C in an ANKOM Daisy II incubator inoculated with 200g fresh feces. Forage samples were run in triplicate. Data were transformed to approximate normality, then analyzed using a repeated measures mixed model in SAS with main effects of forage, treatment, incubation time, and feeding period, and statistically significant interactions. Across incubation times, DMD was higher for AF than CB (P &lt; 0.01; 49.4% vs 42.1%). Across forages, DMD was higher at 72h than at 48h incubation (P &lt; 0.01; 47.5% vs 43.9%). DF improved DMD across forages at 48h (P = 0.03), but not at 72h (P = 0.47). Across incubation times, DF improved DMD of CB (P &lt; 0.01), but not AF (P = 0.79). Additionally, across both forages and both incubation times, DF improved ADFD (P &lt; 0.01). In conclusion, DF may improve microbial fermentation in the equine hindgut. Future research should investigate in-vivo effects of oral supplementation of DF on forage digestibility measures.

16 Digestawell® fiber improves in-vitro digestibility of alfalfa and coastal bermudagrass hay fed to horses

Abstract Enhanced microbial activity in the equine hindgut increases nutrient availability, reduces fecal waste, and adds value to low quality forages. The dietary supplement DigestaWell® Fiber (DF; fibrolytic cofactors: methionine and B vitamins), designed to increase microbial efficiency, has been shown to improve in-vitro digestibility of alfalfa and tall fescue forages after 48-h incubation. The objective of this study was to determine if DF would improve in-vitro dry matter digestibility (DMD) and ADF digestibility (ADFD) of alfalfa and coastal bermudagrass hays incubated for 48h or 72h. Utilizing a crossover design, four mature Quarter Horse mares were fed 2% BW daily of alfalfa (AF; 32.8% NDF, 27.1% ADF) or coastal bermudagrass (CB; 63.2% NDF, 35.4% ADF) hay for 19-d periods. DMD and ADFD were determined after 48-h or 72-h incubation with or without treatment (320mg/L DF) at 37.5°C in an ANKOM Daisy II incubator inoculated with 200g fresh feces. Forage samples were run in triplicate. Data were transformed to approximate normality, then analyzed using a repeated measures mixed model in SAS with main effects of forage, treatment, incubation time, and feeding period, and statistically significant interactions. Across incubation times, DMD was higher for AF than CB (P &lt; 0.01; 49.4% vs 42.1%). Across forages, DMD was higher at 72h than at 48h incubation (P &lt; 0.01; 47.5% vs 43.9%). DF improved DMD across forages at 48h (P = 0.03), but not at 72h (P = 0.47). Across incubation times, DF improved DMD of CB (P &lt; 0.01), but not AF (P = 0.79). Additionally, across both forages and both incubation times, DF improved ADFD (P &lt; 0.01). In conclusion, DF may improve microbial fermentation in the equine hindgut. Future research should investigate in-vivo effects of oral supplementation of DF on forage digestibility measures.

Anaerobic fungal communities differ along the horse digestive tract

Anaerobic fungi are potent fibre degrading microbes in the equine hindgut, yet our understanding of their diversity and community structure is limited to date. In this preliminary work, using a clone library approach we studied the diversity of anaerobic fungi along six segments of the horse hindgut: caecum, right ventral colon (RVC), left ventral colon (LVC), left dorsal colon (LDC), right dorsal colon (RDC) and rectum. Of the 647 ITS1 clones, 61.7 % were assigned to genus level groups that are so far without any cultured representatives, and 38.0 % were assigned to the cultivated genera Neocallimastix (35.1 %), Orpinomyces (2.3 %), and Anaeromyces (0.6 %). AL1 dominated the group of uncultured anaerobic fungi, particularly in the RVC (88 %) and LDC (97 %). Sequences from the LSU clone library analysis of the LDC, however, split into two distinct phylogenetic clusters with low sequence identity to Caecomyces sp. (94–96 %) and Liebetanzomyces sp. (92 %) respectively. Sequences belonging to cultured Neocallimastix spp. dominated in LVC (81 %) and rectum (75.5 %). Quantification of anaerobic fungi showed significantly higher concentrations in RVC and RDC compared to other segments, which influenced the interpretation of the changes in anaerobic fungal diversity along the horse hindgut. These preliminary findings require further investigation.

Effects of two equine digestive aid supplements on hindgut health.

Gastrointestinal disease is the number one killer of horses. Little is known about the maintenance of microbes in the equine hindgut and how to distinguish a healthy gut in a live horse. Utilization of internal and external digestibility markers and starch fermentation has been extensively studied in ruminants and is the basis for research conducted on horses. The aims of this study were to investigate the effects of two equine feed digestive aid supplements on hindgut health (HGH) as reflected in fecal pH and digestibility and to compare and validate DM digestibility measurements through the use of internal and external markers such as chromium oxide (CR), lignin (Lig), indigestible ADF (iADF), indigestible NDF (iNDF), and indigestible lignin (iLig). Nine mature Quarter horses (six geldings, three mares) were used in a crossover design, three feeding periods of 17 d (51 d total), using three treatments: control, no feed additive (CON), Smartpak (SP; Plymouth, MA), or Platinum Performance (PP; Buellton, CA). Both SP and PP contained a strain of Lactobacillus, whereas SP further supplied mannanoligosaccharides (MOS) and fructooligosaccharides (FOS) and PP supplied Saccharomyces boulardii. Within the 17-d period, horses were offered orchard grass hay and sweet cob grain and the assigned treatment daily and four CR cookies to deliver 8 g/d of CR for the last 7 d of each period. Total feces were collected from 15 to 17 d. Feed and fecal samples were dried, ground, and sent to ANALAB (Fulton, IL) for nutrient analysis. Duplicate samples of feed and feces were placed in ruminally cannulated cows for in situ determination of iADF, iNDF, and iLig to estimate digestibility. Estimated CR fecal output, CR DMI, and DM digestibilities were evaluated using the root mean square prediction error percentage of the observed mean (RMSPE), concordance correlation coefficient (CCC), and Nash–Sutcliffe efficiency methods. Marker predictive ability tests showed iADF to have the least amount of bias with the smallest RMSPE (4%), largest CCC (0.43), and the largest amount of random bias (error of dispersion = 0.45). Supplementation of PP decreased CR DM digestibility (P < 0.02). Smartpak increased fecal pH (P < 0.09), but PP had no effect on fecal pH. Therefore, SP had a beneficial effect on HGH that is believed to be due to MOS and FOS.

A comparison of methanogens of different regions of the equine hindgut

The diversity of the methanogenic archaea associated with the six segments of the horse and donkey hindgut (caecum, right ventral colon, left ventral colon, left dorsal colon, right dorsal colon, and rectum) was analyzed using 16S rDNA gene clone library. A total of 641 positive clones, 321 from the horse and 320 from the donkey hindgut, were examined by the RFLP, revealing 9 different ribotypes, 8 in the horse and 5 in the donkey hindgut. In both the animals Methanobacteriales (Methanobrevibacter-like sequences) and Methanomicrobiales (Methanocorpusculum-like sequences) were detected as the dominant orders followed by the uncultured Methanomassiliicoccales. The composition of the equine archaeal community was found to be dependent on the gut region. In both the two animals no Methanobrevibacter-like clones were detected in the caeca, which were instead inhabited by the Methanocorpusculum-like archeons. The Methanosarcinales were found only in distal regions of the horse hindgut.

60 Effect of Sodium Caseinate on Equine Hindgut Fermentation and Fiber Digestion.

60 Effect of Sodium Caseinate on Equine Hindgut Fermentation and Fiber Digestion.

The Effect of Moxidectin Treatment on the Equine Hind Gut Microbiome, Metabonome and Feed Fermentation Kinetics in Horses with Very Low Parasite Burdens

Seventeen horses, mean 12 ± 3.5 years, kept at pasture, with haylage provided and no concentrates. Faecal Egg Counts were conducted September 2015‐March 2016, no eggs seen, no anthelmintic given. Sampling commenced March 2016, points were 0 (prior), 16, 48 and 168 hours post anthelmintic. Treatments were randomized, nine animals dosed orally with Moxidectin 18.92 mg/g at 0.4 mg/kg bw and eight controls. Three horses from each group were randomly assigned for fermentation kinetics. Sequencing of the 16S rRNA gene was conducted on extracted faecal bacterial DNA, bioinformatics using QIIME assigning operational taxonomic units (OTUs). LEfSe (Segata et al., 2011) was used to identify differentially abundant OTUs. Bacterial metabolic profiles were characterized by 1H NMR spectroscopy (Escalona et al., 2015), from urine, analysed by Principal Components Analysis. Fermentation of hay and oats, separately, were measured by in vitro gas production (Murray et al., 2006), data were analysed by repeated measures ANOVA.

Effects of liposomal-curcumin on five opportunistic bacterial strains found in the equine hindgut - preliminary study

BackgroundThe horse intestinal tract is sensitive and contains a highly complex microbial population. A shift in the microbial population can lead to various issues such as inflammation and colic. The use of nutraceuticals in the equine industry is on the rise and curcumin is thought to possess antimicrobial properties that may help to minimize the proliferation of opportunistic bacteria.MethodsFour cecally-cannulated horses were utilized to determine the optimal dose of liposomal-curcumin (LIPC) on reducing Streptococcus bovis/equinus complex (SBEC), Escherichia coli K-12, Escherichia coli general, Clostridium difficile, and Clostridium perfringens in the equine hindgut without adversely affecting cecal characteristics. In the first study cecal fluid was collected from each horse and composited for an in vitro, 24 h batch culture to examine LIPC at four different dosages (15, 20, 25, and 30 g) in a completely randomized design. A subsequent in vivo 4 × 4 Latin square design study was conducted to evaluate no LIPC (control, CON) or LIPC dosed at 15, 25, and 35 g per day (dosages determined from in vitro results) for 9 days on the efficacy of LIPC on selected bacterial strains, pH, and volatile fatty acids. Each period was 14 days with 9 d for acclimation and 5 d withdrawal period.ResultsIn the in vitro study dosage had no effect (P ≥ 0.42) on Clostridium strains, but as the dose increased SBEC concentrations increased (P = 0.001). Concentrations of the E. coli strain varied with dose. In vivo, LIPC’s antimicrobial properties, at 15 g, significantly decreased (P = 0.02) SBEC when compared to 25 and 35 g dosages. C. perfringens decreased linearly (P = 0.03) as LIPC dose increased. Butyrate decreased linearly (P = 0.01) as LIPC dose increased.ConclusionFurther studies should be conducted with a longer dosing period to examine the antimicrobial properties of curcumin without adversely affecting cecal characteristics.

Can fecal samples be used to inform about microbial communities of the equine hindgut?

Can fecal samples be used to inform about microbial communities of the equine hindgut?

Revealing the influence of glucocorticoid treatment on the excretion of anabolic-androgenic steroids in horses through in vitro digestive simulations and an in vivo case study

Anabolic-androgenic steroids (AAS) are strictly forbidden in equine sports because of their stimulating effect on muscle growth and performance. Nevertheless, low levels of AAS have been found in some horses, untreated with AAS. Glucocorticoids (GC), used as an anti-inflammatory therapy and structurally related to AAS, might play a role in this phenomenon. In order to unravel this possible correlation the influence of glucocorticoid treatment on the excretion of AAS was studied both in vivo and in vitro. In vivo effects were investigated by analysing urine samples collected from a gelding treated with betamethasone. Additionally, multiple in vitro digestion simulations were set up, according to a previously validated protocol, to study the possibility of a direct biotransformation of glucocorticoids to AAS, by the microbiota of the equine hindgut. Urine and in vitro digestion samples were extracted and analysed with UHPLC-MS/MS and UHPLC-Orbitrap-HRMS analytical methods.A significant influence on the urinary excretion of α-testosterone (αT), β-testosterone (βT) and androsta-1,4-diene-3,17-dione (ADD) was seen. αT-concentrations up to 20ng/mL were detected. ADD was not found before treatment but could be detected post-treatment. Cortisone and cortisol also peaked (>30ng/mL) between day 37 and 48 post-treatment. The in vitro digestion results however revealed no direct biotransformation of glucocorticoids to AAS by the microbiota of the equine hindgut. This study shows that a glucocorticoid treatment can disrupt the synthesis and excretion of AAS, not by direct biotransformation upon gastrointestinal digestion, but more likely by influencing the hypothalamic-pituitary-adrenal axis.

Faecal parameters as biomarkers of the equine hindgut microbial ecosystem under dietary change

Faeces could be used for evaluating the balance of the equine hindgut microbial ecosystem, which would offer a practical method for assessing gut health and how this relates to disease. However, previous studies concluded that faeces microbial ecosystem was not representative of the proximal hindgut (caecum and ventral colon). This study aimed to evaluate if variations of the faecal microbial ecosystem were similar to those observed in the proximal hindgut. Six horses, fistulated in the caecum and right ventral (RV) colon, were subjected to a gradual change of diet, from a 100% hay (high fibre) diet (2.2 DM kg/day per 100 kg BW) to a 57% hay+43% barley (high starch) diet (0.8 DM kg/day per 100 kg BW hay and 0.6 DM kg/day per 100 kg BW barley). The two diets were iso-energetic and fed over a 3-week trial period. Samples of digesta from the caecum, RV colon and faeces were collected two times on the 10th and 20th day of the trial, for each diet to assess the microbial ecosystem parameters by both classical culture technics and biochemical methods. The variations observed in the caecal and colonic bacterial composition (increase in total anaerobic, amylolytic and lactate-utilizing and decrease in cellulolytic bacteria concentrations) and microbial activity (changes in volatile fatty acids concentrations and increase in lactate concentrations) demonstrated that the hay+barley diet caused changes in the hindgut microbial ecosystem. Similar variations were observed in the faecal microbial ecosystem. Feeding the hay+barley diet resulted in higher concentrations of faecal lipopolysaccharides. The functional bacterial group concentrations (cellulolytics, amylolytics and lactate utilizers) were significantly correlated between caecum and faeces and between colon and faeces. From analyses of the metabolites produced from microbial activity, only valerate concentration in the caecum and the proportion of propionate were significantly correlated with the same parameters in the faeces. Results of the principal component analysis performed between all the caecal/faecal and colonic/faecal parameters revealed that the total anaerobic and cellulolytic bacteria concentrations, as well as valerate, l-lactate and lipopolysaccharide concentrations were strongly correlated with several microbial parameters in the caecum (P<0.027; r>|0.45|) and in the colon (P<0.013; r>|0.50|). This demonstrated that faecal samples and their bacterial analyses could be used to represent caecum and RV colon hindgut microbial ecosystem in terms of variations during a change from a high-fibre to a high-starch diet, and thus could be markers of particular interest to diagnostic proximal hindgut microbial disturbances.