ARS/SCA: The Role of Forage Carbohydrate and Secondary Metabolite Concentrations in Equine Hindgut Fermentation, Amine Production, and Pasture-Associated Laminitis

Sub-objectives: Determine whether amine production in the equine large intestine during carbohydrate overload is a consequence of carbohydrate availability or of decreased large intestinal pH Evaluate the interaction between plant carbohydrate concentrations and plant phenolic concentrations on the activity of gastrointestinal microbes. Hypotheses: Amine production by hindgut microbes occurs as a result of acidic pH, not the presence of fermentable carbohydrate. Plant phenols will reduce amine production in an in vitro carbohydrate overload model. Justification and Background: The rapid fermentation of carbohydrate in the hindgut is one of the primary events associated with acute laminitis in the horse (Garner et al., 1977; Garner et al., 1978; Bailey et al, 2004). Although early researchers induced experimental laminitis with a starch overload, a bolus dose of inulin, a fructooligosaccharide has also been used to induce laminitis under experimental conditions. Furthermore, the accumulation of fructan, a fructo-oligosaccharide synthesized by cool season grasses has been implicated as a cause of pasture associated laminitis (Longland and Byrd, 2006). The presence of high concentrations of fermentable carbohydrate in the large intestine leads to a proliferation of Grampositive bacteria. Lactic acid bacteria such as streptococci and lactobacilli predominate, causing a lactic acidosis (Garner et al., 1977; Garner et al., 1978). It is thought that the endotoxins, the exotoxins or the amines produced by these bacteria contribute to laminitis (Geor, 2010). Amines are a product of amino acid fermentation, specifically, decarboxylation (Park et al, 1996). Obligate amino acid fermenting bacteria, such as the hyperammonia-producing bacteria (HAB) found in ruminants, utilize decarboxylase and deaminase pathways for ATP production. However, many bacteria employ decarboxylases (e.g. glutamate decarboxylase) for pH homeostasis under acidic conditions (Roe et al, 1998, Flythe and Russell 2006). When the hindgut of a horse is overloaded with carbohydrate, lactic acid bacteria proliferate, lactic acid is produced, and the pH declines. The bacteria respond to this acidification by decarboxylating their intracellular pools of glutamate and other amino acids. Decarboxylation consumes protons, which directly leads to an increase in the cytoplasmic pH (Roe et al., 1998). Some bacteria also alleviate the osmotic stress associated with fermentation acid anion accumulation by expelling the intracellular pools of amino acids (Roe et al, 1998; Flythe and Russell 2006). In the case, of pasture associated laminitis the extracellular amino acids would then be available for decarboxylation by other bacteria. Because one step in laminitis is proliferation of Gram-positive bacteria, antibiotics that target Gram-positives have been used to ameliorate the carbohydrate-induced disturbances in the microbial community of the hind gut (Rowe et al 1994; Bailey et al, 2003). Recent studies have demonstrated that phenolic compounds from red clover have antimicrobial effects on Gram-positive HAB bacteria (Flythe and Kagan, 2010). The isoflavone, biochanin A, reduces deamination by mixed rumen bacteria. The effects on decaboxylation in general, and on the microbes of the equine hindgut bacteria in specific, are not known. However, it is possible that biochanin A or other clover compounds could help to mitigate the effects of laminitis. Approach: In the proposal entitled, “Factors affecting carbohydrate and secondary metabolite concentrations in Central Kentucky pasture plants” (submitted for NP101 and NP215 by Lawrence, Kagan and Smith), we propose to take samples of common cool season grasses , and two legumes (white and red clover) to evaluate seasonal and species differences in carbohydrate and phenolic concentrations. The varieties selected for the study will be representative of those commonly found in KY pastures. The data collected will provide estimates of typical water-soluble carbohydrate and fructan concentrations. We will obtain information on the concentrations of phenolic compounds in these plants and how those concentrations relate to carbohydrate accumulation. The carbohydrates discovered in the other proposal will be employed in microbiological experiments in this project. Contingency: if results from the latter project are unclear or delayed, model carbohydrates that are typical in other forages will be used (e.g. inulin, amylose). Experiment set 1: The purpose of these in vitro experiments is to determine if amine production is directly linked to carbohydrate metabolism or if it is the result of increased acidity. More simply put, why do the hindgut bacteria make the amines? Feces collected from horses will be incubated with forage carbohydrate extracts or isocaloric concentrations of monosaccharides. Treatments will include different buffering regimes and direct acidification/alkalinization to control the pH. Volatile fatty acids, pH, ammonia and amine concentrations will be monitored. This experiment will reveal if the bacteria produce amines as a result of general metabolism or acid stress response. The results will be used to generate secondary hypotheses that could include fermentation acid anion accumulation versus simple intracellular pH, and amino acid fermentation versus de novo amine production. Each treatment will be performed in triplicate with inocula from three non-laminitis-prone horses. Pertinent treatments will be repeated with inocula from laminitis-prone horses when they are available. The need for subsequent replication will be determined heuristically. Experiment set 2: The purpose of these experiments is to determine the effects of forage phenolic compounds on in vitro carbohydrate fermentation and amine production. More simply put, can we use “natural products” from the plants that horses consume to control amine production? These experiments will be guided by the results of experiment set 1. Feces collected from horses will be incubated with forage carbohydrate extracts. Clover phenolic compounds will be added as amendments. Other pasture plant phenolics that are identified in the pasture project might also be used. The production of amines and other products that are identified in experiment set 1 will be quantified to generate dose-response curves, with the goal of defining a minimum inhibitory concentration of one or more phenolic compounds. The statistical aesthetic of experiment set 1 will be applied in experiment set 2. Expected outcomes: We will learn the effect of the carbohydrates actually found in Central Kentucky pasture plants on the hindgut bacteria of the horses that feed on those plants. The project will lead to a better understanding of why hindgut bacteria produce biologically active amines when a horse takes in an excess of these carbohydrates. By understanding how pasture plant phenolic compounds affect this process, we will be able to propose interventions based on feeds or on products derived from feeds. Each experiment set will generate at least one peer-reviewed publication. Experiment set 2 could generate one or more invention disclosures or technology transfers.