Equine Hindgut Research Articles

147 The ‘horse-side’ of host-microbe interactions and gastrointestinal homeostasis in the equine hindgut

Abstract Homeostasis is a complex physiological process that enables biological systems to maintain balanced internal environments in response to internal and external stimuli. In the equine gastrointestinal (GI) tract, homeostasis involves a network of factors that regulate gene expression, maintain the integrity of the mucosal barrier, and modulate immune responses to pathogenic microorganisms while supporting commensal bacteria that provide benefits to the host (Figure 1). These factors work together and respond to changes in diet, environment, external stress, and challenges by infectious agents or toxins. Proper gastrointestinal function is dependent on maintaining homeostasis in the system, and the dynamic interactions between the microbial population (the microbiota) and the equine host are central to this equilibrium. The regulation and interplay between the equine host and GI microbiota are evident in the distribution of distinct microbial communities within the various compartments of the equine hindgut. The contributions of each compartment to digestive physiology and the overlap with various stages of microbial fermentation in the equine hindgut are well-researched. What remains to be documented is gene expression in tissues of the equine GI and the underlying role this might play in the processes of digestion, physiology, and homeostasis in the GI. To better understand the roles that host gene expression plays in the interactions with the microbiota, it is critical that we investigate gene expression in the different tissues along the intestinal tract. Protein-coding genes are crucial in maintaining the health of the gastrointestinal tract. They direct the synthesis of various structural elements, molecular transporters, and digestive enzymes, which support digestion, nutrient absorption, and gut integrity. Non-coding genes also play an important role in regulating protein-coding gene expression, modulating immune responses, and maintaining gut barrier function. Moreover, there is evidence that non-coding transcripts regulate the composition of microbial communities in the GI tract. The presentation will focus on the current research and future directions for investigating protein-coding and non-coding gene expression to understand the mechanisms involved in the host-microbe interaction in the equine hindgut and their potential role in maintaining GI homeostasis.

145 Utilization of 16s sequencing data and physiological measurements to produce a comparative analysis of the equine microbiome

Abstract Rapid advancements in microbiome research, particularly in the technologies associated with next-generation sequencing have enabled unprecedented investigations into the composition of the equine microbiome. The large datasets produced from these studies often present challenges to researchers seeking to understand the applicability of these data to equine physiology. To that end, a multi-year project was conducted with the goal of determining the core bacterial population of the equine hindgut through analysis of rectal swabs collected from thousands of horses and applying logic to the association between these ecologies and equine health. Samples were obtained from horses that varied across a diverse range of considerations including location, breed, age, and diet, amongst other metadata points including disease state, health history, vaccination and deworming status, body condition, and pregnancy status. These data were used in the development of a comparative score, that authors defined as the Microbiome Quotient (MQ score). Rectal swabs were collected, and DNA was extracted using the Quick-DNA Fecal/Soil Microbe Miniprep kit (Zymo Research, Irvine, CA) and the V3-V4 hypervariable region of the 16S rRNA gene was sequenced by following the Illumina 16S Protocol (San Diego, CA). Sequences were denoised using DADA2 within the QIIME2 pipeline to obtain amplicon sequence variant composition. These data were filtered again in R (4.2.2) to remove samples with missing data and low sequencing depth. Sample metadata was used to classify samples as either typical or atypical. The MQ score was calculated by computing the Euclidean distance difference between a sample and the centroids representing typical and atypical microbial ecologies, and then subtracting these distances to obtain the individual score. This score quantifies how similar the microbiome in a sample is to those of typical and atypical horses. These scores were then categorized into tertiles, representing the status of the individual horse’s microbiome. These statuses indicate differences in bacterial ecology among samples and their subsequent relevance to the health of the horse. While a large portion of the equine fecal microbiome is conserved through homeostatic and other mechanisms, there remains a high degree of variability in the lesser abundant genera that may be responsible for the different physiological interactions between the microbes and their hosts. Utilizing this MQ score, feeding and management recommendations can be made, providing veterinarians and horse owners with a tool to support the health and performance of horses through modulation of the bacterial population and its functionality. Further research is necessary to fully elucidate the impact of the various metadata components on the bacterial ecology. Additionally, the function of the metagenome is a critical component of microbiome sciences. Prediction of metagenomic function is likely to provide insight into the regulation of physiological function by the microbial population residing within the host.

The effect of the fat to starch ratio in young horses' diet on plasma metabolites, muscle endurance and fear responses.

High-starch diets may affect equine hindgut microbiota and increase blood glucose levels, which may cause unwanted physiological changes, but may also elicit behavioural changes such as increased fear reactions. The purpose of the current study was to feed a high starch (300) and low fat (43; HS_LF) or a low starch (60) and high fat (85; LS_HF, g/kg of DM) concentrate within the available commercial range and investigate how muscle endurance and fear reactions of horses respond to different diets. Twenty Danish Warmblood stallions (4 years) were randomly allocated to two treatments: LS_HF (n = 10) and HS_LF (n = 10) for 9 weeks. During the two last weeks, a single step exercise test was performed, and plasma metabolites and blood gases were measured before and after exercise in a 2 × 2 factorial design. The effect of two diets on fearfulness was tested by exposing the horses to novel objects test (T1 and T2). Plasma metabolites was not affected by diets. However, plasma level of glucose post-exercise (4.9) was lower than pre-exercise (5.6 mmol/L; p < 0.001). Similarly, plasma level of insulin post-exercise (4.2) was lower than pre-exercise (13.1 pmol/L; p < 0.001). Plasma level of lactate dehydrogenase (p < 0.001), non-esterified fatty acids (p = 0.002), β-hydroxybutyrate (p = 0.001), and fructosamine (p = 0.01) post-exercise was higher than pre-exercise. Regardless of type of diets, RRR-α-tocopherol was the dominance α-tocopherol stereoisomers in plasma. In conclusion, during aerobic exercise, fat to starch ratio in horse diets within the normal range had no significant effect on plasma metabolites. However, horses fed LS_HF tended to show more investigative behaviour than horses fed HS_LF.

Gene Expression Analysis before and after the Pelvic Flexure in the Epithelium of the Equine Hindgut.

Previous research demonstrated the distribution of distinct microbial communities in the equine hindgut surrounding the pelvic flexure. The current study evaluated gene expression in epithelial tissues surrounding the pelvic flexure to characterize patterns that might correlate with microbial distribution. Gene expression was determined by analyzing RNA sequence data from the pelvic flexure, the left and right ventral colon, and the left and right dorsal colon. An average of 18,330 genes were expressed across the five tissues sampled. Most of the genes showed some level of expression in all five tissues. Tissue-restricted patterns of expression were also observed. Genes with restricted expression in the left ventral and left dorsal colons have communication, signaling, and regulatory functions that correlate with their known physiology. In contrast, genes expressed exclusively in the pelvic flexure have diverse functions. The ontology of genes differentially expressed between the pelvic flexure and the surrounding tissues was associated with immune functions and signaling processes. Despite being non-significant, these enrichment trends were reinforced by the functions of statistically significant expression differences between tissues of the hindgut. These results provide insight into the physiology of the equine hindgut epithelium that might influence the microbiota and its distribution.

23 Characterization of the bacterial and protozoal communities within the equine hindgut using molecular methods

Protozoans make up 50% of the biomass of the equine hindgut, but their role is relatively unknown in comparison to the more prominently studied bacterial community. Previous studies have found differences in the bacterial population between lean and obese horses, but similar trends have not been investigated in the protozoal population. The objective of this study was to characterize the equine hindgut bacterial and protozoal populations based on the body condition score (BCS) category of the host, to identify associations between bacterial and protozoal species with BCS category, and to determine the predictive nature of the populations with respect to host BCS. Equine fecal samples were collected from horses (n = 45) that were body condition scored using the Henneke scale resulting in n = 16 lean horses, n = 10 normal horses, and n = 19 obese horses for 16S and 18S rRNA profiling. QIIME2 (Quantitative Insights Into Microbial Ecology, v. 2020.2) and R statistical software were used for data analysis. 18S α and β diversity measures, observed species, Jaccard, and unweighted UniFrac distances did not show significance between the BCS categories in the protozoal population (P > 0.05). 16S α diversity with observed species, Chao 1, and Shannon diversity showed significant differences between the groups via a Shapiro test (P = 0.0322) and a further Tukey test found the significance between obeseand normal (P = 0.025), while the other categories were not significant. 16S β diversity measures, Weighted UniFrac and PCoA Bray Curtis, found significance between the categories with β disper (P = 0.011) for UniFrac and adonis and β disper (both P = 0.001) for Bray Curtis. Three taxonomic classifications from the protozoa family Blepharocorythidae significantly correlated (Spearman rho > 0.3) positively with the lean and obese categories and negatively with normal. The 3 bacterial taxonomic classifications that correlated with more than one BCS were the class Gastranaerophilales correlated positively with normal and negatively with obese, order Marinifilaceae correlated positively with lean and negatively with normal, and the order Lachnospiraceae correlated positively with normal and negatively with lean. The bacterial population had higher accuracy when modeling (Random forest) for the ability to predict BCS category using the population (lean 61%, normal 86%, obese 83%) than the protozoal population (lean 64%, normal 64%, obese 65%). The bacterial population had more significant differences between BCS categories and better predictive abilities. The protozoal population has limitations due to a lack of research on their role and genomes.

51 Estimation of in vitro digestibility measures within equine fecal inoculum using feedstuff fiber composition

Previous studies support the use of equine fecal microbes to simulate hindgut fermentation to calculate in vitro true digestibility (IVTD) of feedstuffs. Neutral detergent fiber (NDF) and acid detergent fiber (ADF) composition have been shown to predict IVTD, in vitro NDF digestibility (IVNDFD), and in vitro ADF digestibility (IVADFD). However, little work has been done to predict these digestibility measures within horses. Thus, the study objective was to determine correlation and estimation of in vitro digestibility measures using equine fecal inoculum from feedstuff NDF and ADF composition. Four mature stock-type geldings (8.5 ± 3.9yr; 558.4 ± 31.0kg; 6.0 ± 0.4 BCS) were used in a duplicated 4 × 6 Factorial Design. Horses were acclimated to a basal diet of 1.8% BW as-fed of Coastal Bermudagrass hay (Cynodon dactylon)and 0.2% BW as-fed of Bluebonnet® Equilene® Pellets for 21d. Feces were collected on d22 via rectal-grab for use as incubation inoculum. Dried, ground feed samples (0.5g: alfalfa hay, Coastal Bermudagrass hay, soybean meal, rice bran, hempseed meal, Bluebonnet® Equilene® Pellets), sealed in ANKOM filter bags, were placed in duplicate within 2 DaisyII incubators for 48hr. After incubation, fiber bags were removed and dried, then analyzed for NDF composition to determine IVTD. Split samples were analyzed to determine pre-incubation NDF composition of feedstuffs. Correlations and linearregressions were performed within R Statistical Program©. Both NDF (r = −0.936) and ADF (r = −0.897) were highly correlated to IVTD. However, ADF had a greater correlation to both IVNDFD (r = −0.807) and IVADFD (r = −0.848) than NDF (r = −0.684; r = −0.720). A linear regression between NDF and IVTD produced the model: IVTD (g/kg DM) = 1052.30 – 0.932 * NDF (g/kg DM); R2 = 0.930; P < 0.001. A lower goodness-of-fit was found between ADF and IVTD: IVTD (g/kg DM) = 1033.25 – 1.528 * ADF (g/kg DM); R2 = 0.866; P < 0.001. An additive model of IVTD versus NDF+ADF mildly increased goodness-of-fit (R2 = 0.953). Sample ADF strongly predicted IVADFD (IVADFD (g/kg ADF) = 881.91 – 1.595 * ADF (g/kg DM); R2 = 0.835; P < 0.001) but only moderately predicted IVNDFD (IVNDFD (g/kg NDF) = 855.15 – 1.518 * ADF (g/kg DM); R2 = 0.749; P < 0.001). These results indicate the effectiveness of using feedstuff NDF to predict IVTD when using equine fecal inoculum. Feedstuff IVNDFD and IVADFD are predicted with less precision, likely due to the lack of foregut digestion of feedstuffs that would occur in vivo. The use of in vitro total-tract digestion modeling in horses may estimate equine hindgut fiber digestion with greater precision.

Longitudinal study of the short- and long-term effects of hospitalisation and oral trimethoprim-sulfadiazine administration on the equine faecal microbiome and resistome

BackgroundHospitalisation and antimicrobial treatment are common in horses and significantly impact the intestinal microbiota. Antimicrobial treatment might also increase levels of resistant bacteria in faeces, which could spread to other ecological compartments, such as the environment, other animals and humans. In this study, we aimed to characterise the short- and long-term effects of transportation, hospitalisation and trimethoprim-sulfadiazine (TMS) administration on the faecal microbiota and resistome of healthy equids.MethodsIn a longitudinal experimental study design, in which the ponies served as their own control, faecal samples were collected from six healthy Welsh ponies at the farm (D0–D13-1), immediately following transportation to the hospital (D13-2), during 7 days of hospitalisation without treatment (D14–D21), during 5 days of oral TMS treatment (D22–D26) and after discharge from the hospital up to 6 months later (D27–D211). After DNA extraction, 16S rRNA gene sequencing was performed on all samples. For resistome analysis, shotgun metagenomic sequencing was performed on selected samples.ResultsHospitalisation without antimicrobial treatment did not significantly affect microbiota composition. Oral TMS treatment reduced alpha-diversity significantly. Kiritimatiellaeota, Fibrobacteres and Verrucomicrobia significantly decreased in relative abundance, whereas Firmicutes increased. The faecal microbiota composition gradually recovered after discontinuation of TMS treatment and discharge from the hospital and, after 2 weeks, was more similar to pre-treatment composition than to composition during TMS treatment. Six months later, however, microbiota composition still differed significantly from that at the start of the study and Spirochaetes and Verrucomicrobia were less abundant. TMS administration led to a significant (up to 32-fold) and rapid increase in the relative abundance of resistance genes sul2, tetQ, ant6-1a, and aph(3”)-lb. lnuC significantly decreased directly after treatment. Resistance genes sul2 (15-fold) and tetQ (six-fold) remained significantly increased 6 months later.ConclusionsOral treatment with TMS has a rapid and long-lasting effect on faecal microbiota composition and resistome, making the equine hindgut a reservoir and potential source of resistant bacteria posing a risk to animal and human health through transmission. These findings support the judicious use of antimicrobials to minimise long-term faecal presence, excretion and the spread of antimicrobial resistance in the environment.-NEQ1286BccpFJdhcRCuNyVideo

Fecal Microbiota, Forage Nutrients, and Metabolic Responses of Horses Grazing Warm- and Cool-Season Grass Pastures

Integrating warm-season grasses into cool-season equine grazing systems can increase pasture availability during summer months. The objective of this study was to evaluate effects of this management strategy on the fecal microbiome and relationships between fecal microbiota, forage nutrients, and metabolic responses of grazing horses. Fecal samples were collected from 8 mares after grazing cool-season pasture in spring, warm-season pasture in summer, and cool-season pasture in fall as well as after adaptation to standardized hay diets prior to spring grazing and at the end of the grazing season. Random forest classification was able to predict forage type based on microbial composition (accuracy: 0.90 ± 0.09); regression predicted forage crude protein (CP) and non-structural carbohydrate (NSC) concentrations (p < 0.0001). Akkermansia and Clostridium butyricum were enriched in horses grazing warm-season pasture and were positively correlated with CP and negatively with NSC; Clostridum butyricum was negatively correlated with peak plasma glucose concentrations following oral sugar tests (p ≤ 0.05). These results indicate that distinct shifts in the equine fecal microbiota occur in response different forages. Based on relationships identified between the microbiota, forage nutrients, and metabolic responses, further research should focus on the roles of Akkermansia spp. and Clostridium butyricum within the equine hindgut.

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.

Effects of Differences in Fibre Composition and Maturity of Forage-Based Diets on the Fluid Balance, Water-Holding Capacity and Viscosity in Equine Caecum and Colon Digesta.

Horses are herbivores, and their hindgut functions as a fluid reservoir as forage fibre properties have great impact on the water content of digesta and the milieu in the ecosystem. Our objective was to compare the effect of grass fibre maturity and legume forage on the water-holding capacity (WHC) and viscosity of the equine hindgut and the body weight (BW) and fluid balance of horses. Three diets: concentrate and late harvested grass haylage (35:65 energy ratio) (C); early and late harvested grass haylage (80:20) (G); lucerne and late harvested grass haylage (80:20) (L) were fed to six caecum and colon fistulated horses for 28 days in a Latin-square design. Total water intake and BW were higher when the horses were fed Diet L, but the digesta WHC was higher when fed Diet G. Total water excretion (via faeces + urine) and the difference in total water intake-output was higher when fed Diet L. Viscosity, measured on centrifuged digesta fluid, did not differ between diets, but the individual colon data of one horse were higher. In conclusion, early harvested forage might be beneficial for the fluid balance of athletic horses providing a higher WHC of hindgut digesta without increasing BW. The importance of digesta viscosity in relation to equine diets needs further investigations.

Integrated multi-omics reveals novel microbe-host lipid metabolism and immune interactions in the donkey hindgut.

Evidence has shown that gut microbiota play a key role in host metabolism and health; however, little is known about the microbial community in the donkey hindgut as well as the interactions that occur between gut microbes and the host. This study aimed to explore the gut microbiome differences by analyzing the microbial community and differentially expressed genes (DEGs) related to lipid metabolism and the immune system along the donkey hindgut. The hindgut tissues (cecum, ventral colon, and dorsal colon) were separated, and the contents of each section were collected from six male donkeys for multi-omics analysis. There were significant differences in terms of dominant bacteria among the three sections, especially between the cecum and dorsal colon sites. For instance, species belonging to Prevotella and Treponema were most abundant in the cecum, while the Clostridiales_bacterium, Streptococcus_equinus, Ruminococcaceae_bacterium, etc., were more abundant in the dorsal colon. Apart from propionate, the concentrations of acetate, isobutyrate, valerate and isovalerate were all lower in the cecum than in the dorsal colon (p < 0.05). Furthermore, we identified some interesting DEGs related to lipid metabolism (e.g., ME1, MBOAT1, ACOX1, ACOX2 and LIPH) and the immune system (e.g., MUC3B, mucin-2-like, IL17RC, IL1R2, IL33, C1QA, and MMP9) between the cecum and dorsal colon and found that the PPAR pathway was mainly enriched in the cecum. Finally, we found a complex relationship between the gut microbiome and gene expression, especially with respect to the immune system, and combined with protein-protein interaction (PPI) data, suggesting that the PPAR pathway might be responsible, at least in part, for the role of the hindgut microbiota in the donkeys' gut homeostasis. Our data provide an in-depth understanding of the interaction between the microbiota and function in the healthy equine hindgut and may also provide guidance for improving animal performance metrics (such as product quality) and equine welfare.

Fecal microbiome of horses transitioning between warm-season and cool-season grass pasture within integrated rotational grazing systems

BackgroundDiet is a key driver of equine hindgut microbial community structure and composition. The aim of this study was to characterize shifts in the fecal microbiota of grazing horses during transitions between forage types within integrated warm- (WSG) and cool-season grass (CSG) rotational grazing systems (IRS). Eight mares were randomly assigned to two IRS containing mixed cool-season grass and one of two warm-season grasses: bermudagrass [Cynodon dactylon (L.) Pers.] or crabgrass [Digitaria sanguinalis (L.) Scop.]. Fecal samples were collected during transitions from CSG to WSG pasture sections (C–W) and WSG to CSG (W–C) on days 0, 2, 4, and 6 following pasture rotation and compared using 16S rRNA gene sequencing.ResultsRegardless of IRS or transition (C–W vs. W–C), species richness was greater on day 4 and 6 in comparison to day 0 (P < 0.05). Evenness, however, did not differ by day. Weighted UniFrac also did not differ by day, and the most influential factor impacting β-diversity was the individual horse (R2 ≥ 0.24; P = 0.0001). Random forest modeling was unable to accurately predict days within C–W and W–C, but could predict the individual horse based on microbial composition (accuracy: 0.92 ± 0.05). Only three differentially abundant bacterial co-abundance groups (BCG) were identified across days within all C–W and W–C for both IRS (W ≥ 126). The BCG differing by day for all transitions included amplicon sequence variants (ASV) assigned to bacterial groups with known fibrolytic and butyrate-producing functions including members of Lachnospiraceae, Clostridium sensu stricto 1, Anaerovorax the NK4A214 group of Oscillospiraceae, and Sarcina maxima. In comparison, 38 BCG were identified as differentially abundant by horse (W ≥ 704). The ASV in these groups were most commonly assigned to genera associated with degradation of structural carbohydrates included Rikenellaceae RC9 gut group, Treponema, Christensenellaceae R-7 group, and the NK4A214 group of Oscillospiraceae. Fecal pH also did not differ by day.ConclusionsOverall, these results demonstrated a strong influence of individual horse on the fecal microbial community, particularly on the specific composition of fiber-degraders. The equine fecal microbiota were largely stable across transitions between forages within IRS suggesting that the equine gut microbiota adjusted at the individual level to the subtle dietary changes imposed by these transitions. This adaptive capacity indicates that horses can be managed in IRS without inducing gastrointestinal dysfunction.

PSIV-17 Impacts of high forage crude protein concentrations on the equine fecal microbiome

Abstract The objective of this study was to evaluate effects of forage crude protein (CP) concentrations on the equine fecal microbiome. Six mares were assigned to either a high-protein (HP; 24% CP) or normal-protein (NP; 15% CP) hay cube diet in a randomized cross-over design. Fecal samples were collected on the last day of each 35-d period. Branched-chain and short-chain fatty acids (SCFA) were measured by GC-MS; data were analyzed by mixed model ANOVA in R. 16S rRNA gene sequence analysis was conducted in Qiime2. Alpha and beta diversity were assessed by Kruskall-Wallis Tests and PERMANOVA. Taxonomy was assigned using SILVA. Metacyc pathways were predicted with PICRUSt2. Differential genus-level and pathway abundances were analyzed by LefSe (LDA &amp;gt;2.0). Significance was set at p≤0.05. Species richness did not differ by diet, but evenness was greater in NP vs. HP. Beta diversity metrics including Weighted UniFrac also differed by diet. Genera including Methanocorpusculum, Rikenellaceae RC9 gut group, and Eubacterium coprostanoligenes group were more abundant in HP. Treponema and Fibrobacters were more abundant in NP. Twenty pathways related to amino acid synthesis were identified as markers of NP, while 10 pathways related to amino acid degradation and SCFA synthesis were markers of HP. Pathway predictions were supported by metabolite analyses, with acetate (HP: 751; LP: 632 ± 49 ug/g), propionate (HP: 565; NP: 421 ± 43 ug/g), butyrate (HP: 341; LP: 210 ± 34 ug/g), and valerate (HP: 80; NP: 42 ± 11 ug/g) greater in HP than NP. Isobutyrate (HP: 108; NP: 94 ± 7 ug/g; P = 0.09), and isovalerate (HP: 91; NP: 48 ± 10 ug/g) were also greater in HP. These results demonstrate that consuming higher-CP forages impacts equine hindgut microbiota structure and function, leading to increased rates of protein fermentation. Further research is needed to determine physiological significance in forage-fed horses.

Effect of Donor Diet on In Vitro Digestibility of Forages by Fecal Inoculate

Previous research indicates equine fecal inoculates produce comparable results to cecal fluid when used for in vitro procedures to analyze dry matter digestibility (DMD). Equine hindgut microbial communities represented in fecal samples have been shown to be affected by diet. The study's objective was to determine the effect of the donor diet on in vitro DMD when fecal donors were fed high starch, or high fiber diets. Six Quarter Horses were used in a crossover design to compare the effects of a grain versus forage diet on in vitro digestion of forages ranging from: CP 7.7 to 16.4 %DM, NDF 53 to 72 %DM. Feces from each horse were obtained on day 22 of each period and used to inoculate in vitro fermentation vessels in order to evaluate the effect of donor diet on the DMD, neutral detergent fiber digestibility (NDFD), and acid detergent fiber digestibility (ADFD) of four forages. Data were analyzed using the MIXED procedure of SAS to evaluate digestibility differences in the diet by forage composition interaction. Fecal samples from horses on the grain diet promoted higher NDFD of forages with high NDF and low CP when CP and NDF were used as covariates (P=.04 and .03). There was a horse effect on DMD and NDFD (P ≤ .05). Findings suggest diet may influence the hindgut microbiome's ability to digest neutral detergent fiber and should be considered when selecting equine fecal samples for in vitro digestion procedures.

Comparison of the Fecal Microbiota of Horses with Intestinal Disease and Their Healthy Counterparts.

(1) Background: The intestinal microbiota plays an essential role in maintaining the host’s health. Dysbiosis of the equine hindgut microbiota can alter the fermentation patterns and cause metabolic disorders. (2) Methods: This study compared the fecal microbiota composition of horses with intestinal disease and their healthy counterparts living in Korea using 16S rRNA sequencing from fecal samples. A total of 52 fecal samples were collected and divided into three groups: horses with large intestinal disease (n = 20), horses with small intestinal disease (n = 8), and healthy horses (n = 24). (3) Results: Horses with intestinal diseases had fewer species and a less diverse bacterial population than healthy horses. Lactic acid bacteria, Lachnospiraceae, and Lactobacillaceae were overgrown in horses with large intestinal colic. The Firmicutes to Bacteroidetes ratio (F/B), which is a relevant marker of gut dysbiosis, was 1.94, 2.37, and 1.74 for horses with large intestinal colic, small intestinal colic, and healthy horses, respectively. (4) Conclusions: The overgrowth of two lactic acid bacteria families, Lachnospiraceae and Lactobacillaceae, led to a decrease in hindgut pH that interfered with normal fermentation, which might cause large intestinal colic. The overgrowth of Streptococcus also led to a decrease in pH in the hindgut, which suppressed the proliferation of the methanogen and reduced methanogenesis in horses with small intestinal colic.

33 Profiling miRNA transcripts in the equine hindgut by quantitative PCR

Gastrointestinal (GI) homeostasis in a horse results from dynamic interactions between a horse's gut physiology and the microbes in the various compartments. MicroRNAs (miRNA), single-stranded 20–22 basepair RNA molecules involved in post-transcriptional control of gene expression, have been implicated as an essential mechanism for control of gastrointestinal physiology and communication between host and microbe. Researchers have described how host-derived miRNAs influence microbial communities' composition in the GI. We have recently described how the pelvic flexure separates distinct microbial populations in the equine hindgut and subsequently have wondered if equine miRNA transcripts expressed by GI tissues could have a role in maintaining this separation. Investigations to characterize miRNAs' expression profile and other non-coding RNAs in the equine GI tract are limited. To address this, it is critical that we first know something about the expression of non-coding transcripts in the equine GI. This study investigated miRNA expression in tissues of the hindgut surrounding the pelvic flexure. RNA was isolated from the intestinal epithelium of 3 4-year-old American Quarter Horses collected from the right and left ventral colon (VC), pelvic flexure (PF), and right and left dorsal colon (DC). The miRNA transcripts were reverse transcribed using the miScript II RT kit, and relative abundance was quantified using the miScript SYBR Green PCR kit and primer sets for 286 annotated mature equine miRNAs. Biological replicates were pooled, and differential expression was determined following normalization by the delta Ct = Ct(target) – Ct(sample) method. A total of 230 miRNA transcripts were expressed in at least one GI location, with 60 expressed in all 5 locations. Twenty-eight transcripts had expression restricted to a single site, 5 of which were only expressed in the pelvic flexure. Additionally, 42 transcripts have patterns of expression across a subset of anatomical locations. Notably, 10 transcripts were expressed in the VC and PF, 5 transcripts were expressed in the DC and PF, and 15 transcripts were expressed in the VC and DC but not PF. Pathway and target analysis for the miRNAs with compartment restricted expression was performed using DIANA TOOLS. Of note, miRNA transcripts expressed only in the PF were predicted to target genes in the Hippo signaling pathway, which is associated with the regulation of intestinal tissue proliferation and homeostasis. Further investigation of miRNA expression in the equine hindgut will improve our understanding of gastrointestinal homeostasis's regulatory processes.

70 A comparative in vitro analysis of coated and non-coated yeast survivability at simulated equine gastrointestinal pH conditions

Fermentation in the equine hindgut is key to ensure the assimilation of dietary components such as vitamins, amino acids, and minerals. Furthermore, microbial fermentation products (of volatile fatty acids) provide horses with energy. Imbalances in the gut microbiota could impact the rate of fermentation leading to physiological responses such as hindgut inflammation, over production of lactic acid and ulceration. Feed supplementation with direct fed microbials (DFMs) is a common approach to restore microbial balance and ensure animal health. DFMs transit the gastrointestinal tract through highly acidic conditions in the stomach could result in a reduction of the initial DFMs concentrations. The use of coating technologies to encapsulate pH sensitive microorganisms is a way to ensure the delivery of high concentrations of DFMs in the hindgut. The aim of this study was to evaluate the survivability of a coated and non-coated concentrated yeast at pH 2, 6.3, and 6.8, to simulate pH conditions and digesta residence times of an equine stomach (1 h), small intestine (5 h) and cecum (17 h), respectively. A total of 2 replicates of Saccharomyces cerevisiae concentrate and a coated protected lyophilized Saccharomyces cerevisiae were added to MRS (selective media) broth and RPMI broth (non-selective media) at pH2; 2 additional replicates of each product were added to each selective and non-selective media at pH 7 (control). Samples were incubated at 38°C for one hour; after incubation samples were washed and media at pH 6.3 was added. Tubes were incubated for 5 h, after incubation media at pH 6.6 was added for a final 17-h incubation. pH was measured and S. cerevisiae counts were plated for enumeration after each incubation period. One-way ANOVA was used to identify statistically significant differences among groups. Counts of the coated S. cerevisiae were between 9.8 and 10.1 log10 cfu/g in selective and 9.6–9.8 log10 cfu/g in non-selective media. These counts were not significantly (P > 0.05) reduced compared with controls in selective (10.3–10.4 log10 cfu/g) and non-selective (9.7–10.3 log10 cfu/g) media. No differences were observed between coated – and non-coated concentrated S. cerevisiae. These results showed that S. cerevisiae used in this study survived the simulated transit times and pH conditions of the equine gastrointestinal tract. Concentrations of S. cerevisiae above 9 log10 cfu/g reached cecum simulated pH conditions, the high concentrations found suggest its potential use as a DFM that could enhance dietary components assimilation in the equine hindgut.

138 Expression profiling of the ventral colon, pelvic flexure, and dorsal colon epithelium by RNA-seq

The equine large intestine is essential for nutrient absorption and as a location for microbial digestion. The pelvic flexure is an important anatomical landmark that divides distinct microbial communities in the equine hindgut. Previous work from our group showed that several taxonomic groups were restricted to the cecum and ventral colon. Since no physical barrier separates these compartments from the more distal areas, we became curious about alternative mechanisms that support the segregation of different microbial groups. Physiological differences in each compartment likely result in different requirements from the microbiota at each location. This study evaluated gene expression in the mucosal and submucosal layers of the right and left ventral colon (VC), pelvic flexure (PF), and right and left dorsal colon (DC). Samples were collected from 3 4-year-old American Quarter Horses, and total RNA was isolated. Single-end libraries were prepared using the NEBNextⓇ Ultra II Directional RNA library kit and sequenced on an IlluminaⓇ NextSeq 550 platform. The sequencing generated an average of 22.5 million reads per sample (range 14.5 to 30.9). Gene expression analysis was performed using the Galaxy platform by aligning quality trimmed sequence reads to EquCab3 using the HISAT2, assembling transcripts with StringTie, and quantifying transcript abundance with Salmon. DESeq2 was used to assess differential expression between the intestinal compartments. Expression (present in 2 of 3 biological replicates) was detected in at least one intestinal compartment for 21,987 transcripts and ranging from 14,766 to 15,134 transcripts per sample. A total of 13,944 transcripts were detected in all 5 areas, and 739 transcripts demonstrated a tissue-restricted expression pattern (detected above threshold in only on anatomical location). Gene ontology was used to identify the functional roles of transcripts expressed in all or only one hindgut compartment. Transcripts expressed in all tissues were associated with internal cell metabolic processes, which are common despite cell type. In contrast, transcripts with tissue-restricted expression patterns were associated with cellular differentiation and secretion processes. Matrix metalloproteinases, transcription factors, and integrin proteins were identified as differentially expressed (P < 0.05) in the PF compared with the surrounding VC and DC compartments. Continued investigation of these transcripts in the equine hindgut will enhance our understanding of digestive physiology and support future studies of interactions between host and microbiota.

68 Effect of a marine-sourced calcium on fecal pH in horses

Lithothamnium calcareum (LC) is a red, marine algae containing calcite in its cell walls. LC has been touted for its gastroprotective effects in horses attributed to the increased surface area of its honeycomb structure allowing greater acid-buffering capacity versus calcium carbonate. Effects on the equine hindgut have not been evaluated. This pilot study aimed to determine if LC (CalseaPowder, Nuwen, France) fed to horses at a rate of 4.6g/kg finished feed would alter fecal pH. Twelve mature horses (12.5 yrs ± 1.4, mean ± SEM, 6 mares, 6 geldings) in moderate exercise in the Ohio State ATI equine program participated in a randomized crossover study. Horses were maintained on 1.2-ha fields and brought into the barn for feeding and riding sessions. Horses transitioned onto the control feed over 2 wks (CON: SAFE ‘N EASY Performance, BUCKEYE Nutrition, Dalton, OH, 1.2% total Ca, 100% added was CaCO 3 ), formulated to meet or exceed NRC 2007 requirements for mature horses in light-moderate work. Horses were randomly assigned to receive either CON or CAL (CON + LC , 1.2% total Ca, added 74% CaCO 3 , 26% calcite) during the first test period then crossed over for the second. Horses acclimated to CON or CAL for 2 wks and were offered 0.5 kg feed per 100 kg bodyweight, divided into 2 feedings (0700 and 1600). Horses had access to mixed grass forage to supplement pasture, fresh water and salt ad libitum . At the end of the first 2-week trial period, horses were brought into stalls bedded with sawdust and observed. All fecal samples were collected immediately upon defecation (within 1–3 h post feeding) from the middle of the top of the manure pile to avoid bedding contamination and analyzed. Within 5 min, approximately 10 mL of fecal water was squeezed through cheesecloth from freshly defecated samples and stirred for 60s. Fecal water pH was then measured in duplicate with a calibrated pH meter. Diets were switched and the protocol repeated with 2 wk acclimation. Statistical analyses utilized R version 3.6.3, with significant differences declared when P ≤ 0.05. A linear mixed effect model was used with pH vs. diet and horse as a random effect. A Dunnett post-hoc comparison between the 2 diet groups was applied. There was no effect of period ( P = 0.66). There was an effect of diet ( P < 0.001). Horses consuming CAL had higher fecal water pH (6.87 ± 0.2; mean ± SD) compared with CON (6.69 ± 0.2). LC may be useful in supporting hindgut health in horses, especially when such changes in pH become clinically relevant (e.g., acidosis). Future work should examine the effects of different doses of LC on hindgut pH and its effects on the microbial population.

58 Evaluation of substrate:buffer ratios for in vitro cultures inoculated with equine cecal contents

In vitro fermentation systems allow researchers to mimic the equine hindgut while providing quick, affordable, and publishable data. However, the substrate:buffer ratio within the system has a profound impact on subsequent fermentation parameters. The objective of this study was to evaluate effects of varying substrate levels on culture pH, gas production, dry matter disappearance (DMD), neutral detergent fiber disappearance (NDFD), and volatile fatty acid (VFA) concentrations utilizing the ANKOM RF Gas Production System (ANKOM Technology; Macedon, NY). Cecal fluid was collected from 4 cecally cannulated Quarter horses and used to inoculate fermentation bottles containing buffer and substrate. Substrate consisted of brome or corn included at 0.5, 1, 1.5, 2, 3, or 4 g/flask (dry matter basis) while buffer and inoculant remained constant at 125 and 25 mL/flask, respectively. Flasks were equipped with continuous gas pressure monitors and placed into a shaking incubator for 48 h at 39°C. Data were analyzed using mixed models with fixed effects of treatment, time, and treatment by time interaction, repeated effect of time, and random effects of run by horse within run. Changes in gas production, VFA production, and terminal pH were proportion to substrate amount for cultures containing 0.5 to 2 g of substrate, but were less for cultures with 3 or 4 g of substrate (linear and quadratic effects, P < 0.0001). These data suggest that cultures containing 2 g or less of substrate were substrate limited, whereas activity of cultures with more than 2 g of substrate may have been inhibited by buffer exhaustion or end product accumulation. Results of this experiment indicate that the composition of fermentative end products is impacted by substrate concentrations, and that cultures containing 125 mL of buffer and 25 mL of cecal inoculum should be limited to no more than 2 g of substrate.