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In recent years, the microbiome has become a topic of great interest in humans and across animal species, including horses. With the increasing emphasis on the role of the microbiome in equine health and nutrition, it is important to understand the basics about this rapidly evolving field of science.
What Is the Microbiome?
Microbiome is a term that refers to all the microorganisms present in a given habitat. While the term microbiome is most often used in reference to the gastrointestinal microbiota, this term can refer to the microbiota present in any habitat. For example, in nature, there are microbiomes associated with soils, surrounding the roots of plants or in bodies of water. In humans and animals, different parts of the body have distinct microbiomes. A few examples include the microbiomes associated with the skin, the eyes, the vagina of females, the mouth, and, of course, the gastrointestinal tract and the manure produced as waste from digestive processes. Furthermore, within the gastrointestinal tract, the microbiome differs between organs, with different microbial communities inhabiting the stomach, small intestine, and even between the various segments of the large intestine. In the field of equine nutrition, there is a heavy focus on the hindgut microbiome.
Figure 1. A microbiome can be found anywhere. In the horse, the eyes, skin, mouth, reproductive organs, digestive organs, and the manure output are just a few examples. Photo credit C. Williams.
The Equine Gastrointestinal Tract
Horses are classified as hindgut fermenters. After feed is ingested, it passes through the esophagus to the stomach. A horse has a one-chambered stomach, the function of which is to secrete acids and enzymes that begin the process of breaking down (or digesting) carbohydrates, fats, and proteins in feed into simpler components. From the stomach, the digesta moves into the small intestine. There, additional enzymes are secreted, and the small intestine is considered the major site of digestion and absorption of soluble carbohydrates, fats, protein, and some vitamins and minerals. While enzymatic digestion in the foregut (the digestive tract up through the small intestine) can break down these nutrients, horses, like other mammals, do not produce enzymes capable of breaking down fiber. Thus, fiber passes undigested into the hindgut (or large intestine). The first two segments of the large intestine, the cecum and the large colon, contain a large population of bacteria and other microorganisms. These hindgut bacteria are capable of fermenting fiber. Any sugars, starches, or proteins that escaped digestion in the foregut are also subject to microbial fermentation. For more information on equine nutrition. see the fact sheet FS038, The Basics of Equine Nutrition.
The Hindgut Microbiome
Microbial fermentation of fiber and other residual nutrients in the hindgut produces by-products, including short-chain fatty acids or SCFAs (sometimes called volatile fatty acids or VFAs), B-vitamins, vitamin K, and gasses. Production of SCFAs is particularly important in equine nutrition, as 50–70% of a horse's energy needs are met by SCFAs absorbed from the hindgut.
Microorganisms present in the hindgut include bacteria, archaea, protozoa, fungi, and bacteriophages. Bacteria are the most abundant, and most studies of the hindgut microbiome focus primarily on bacterial communities. Archaea are a separate domain of methane producers. Fungi play a role in fiber degradation, and while the function of protozoa in the equine hindgut is, as of yet, unresolved, studies in other animal species have found protozoa to be important for breakdown of soluble carbohydrates. A bacteriophage is a virus that infects bacteria and archaea. There have only been limited studies of bacteriophages in horses, but these microorganisms may serve to regulate bacterial populations in the hindgut (Julliand and Grimm, 2016).
Hindgut bacteria are often described and grouped based on their functional role within the hindgut community. For example, fibrolytic bacteria are those bacteria capable of digesting fiber (like in hay), whereas amylolytic bacteria are starch degraders (like in concentrated feeds). Other common groups include lactate utilizers (degrades lactate created when the body breaks down carbohydrates) and proteolytic bacteria (protein degraders).
Figure 2. Some bacteria that make up the microbiome prefer to digest feeds that are higher in starch like the sweet feed (bottom). Other bacteria break down more fibrous or structural carbohydrates like in the hay (top). Photo credit C. Williams.
Studies of the Hindgut Microbiome
With growing interest in the hindgut microbiome in recent years, it may seem that this is a new field of study for equine researchers. However, scientists have been studying the gut microbiome of horses for well over a century (Julliand and Grimm, 2016). The first microorganisms identified from the equine gastrointestinal tract were protozoa from cecal contents in 1843! The first bacteria from the equine gastrointestinal tract were later identified in 1894. Microscopy and culture-based studies of the equine hindgut microbiota continued throughout the early part of the 20th century. Laboratory methods at the time were limited to culturing of aerobic bacteria (bacteria that tolerate and/or require oxygen for growth). As the gastrointestinal system creates an anaerobic environment, little was known about communities of anaerobic bacteria (bacteria that cannot grow and survive in the presence of oxygen) in the equine hindgut until the advent of anaerobic culture techniques in 1950.
Even as these new methods expanded the scope of microbiological studies of the equine hindgut, a significant proportion of bacterial species remained unculturable and unidentified, with little or nothing known regarding their function in the hindgut ecosystem or their importance in horse health and nutrition. In the 1980s, new molecular-based, culture-independent techniques began to be developed in which bacteria in environmental samples could be identified using a DNA marker, the 16S rRNA gene. Joshua Lederberg coined the term microbiome in 2001 and, by the early 2010s, surveys of 16S rRNA gene sequences in environmental samples became a standard practice. The National Institutes of Health began the first Human Microbiome Project in 2008. However, the quantity of and, therefore, advances in, equine microbiome research are considerably less than in humans and even other species such as cattle. Studies of the equine hindgut microbiome continued to rely primarily on traditional culture techniques through the 1990s and most of the 2000s. The first use of 16S rRNA gene sequencing of bacterial DNA from the horse hindgut appeared in the literature in 2001, and limited further studies were published prior to 2010.
Figure 3. Researchers have begun to explore links between equine behaviors, such as anxiety (for example, due to extensive stall rest or confinement) and the hindgut microbiome. Photo credit C. Williams.
While this field of study has continued to rapidly grow in the years since, there is still much to learn about the structure and function of the equine hindgut microbiome. Recent studies mainly focus on understanding basic variances in the hindgut microbial community due to diet and other factors such as stress, age, exercise, and the environment. Researchers have also investigated the connection between the hindgut microbiome and various gastrointestinal disorders and disease in the horse including obesity, equine metabolic syndrome, laminitis, and colic. Interestingly, in humans and laboratory animal species, a gut-brain axis has been identified wherein microbial populations influence behavior and conditions such as anxiety. This concept has prompted emerging research into possible connections between the hindgut microbiota and equine behavior.
Influence of Diet
Diet is considered the dominant factor affecting community structure of the gut microbiota in the horse. As mentioned above, the hindgut microbiome differs between horses fed concentrates (grains) and horses maintained on forage-based diets (hay or pasture). The microbiome also can be affected by type and nutritional composition of forages or concentrates. For example, Sorensen et al. (2021) found differences in the microbial communities and SCFA production in horses fed a cool-season grass hay (smooth bromegrass) versus alfalfa hay, while Gomez et al. (2021) identified differences in microbial populations when horses were fed either a low-lignin alfalfa hay or alfalfa with normal lignin concentrations. (Note: Lignin is the indigestible portion of plant stems that reduces the fiber digestibility of forages).
In general, forage diets have been associated with greater diversity of the equine hindgut microbiota as well as stability of the microbial populations over time. Microbiomes of horses adapted to concentrate diets higher in starch have a greater prevalence amylolytic bacteria such as Lactobacillus spp. and Streptococcus spp., in comparison to horses maintained on forage-only diets higher in fiber. These bacteria also have been shown to increase following an abrupt, rapid inclusion of concentrates into all-forage diets. In the horse, these bacteria are capable of creating acidic conditions in the hindgut, and if hindgut pH drops significantly, this could lead to a disruption of the microbial community and negative health consequences in the horse (i.e. laminitis). Conversely, there is a greater prevalence of fibrolytic bacteria such as Fibrobacter spp. and Ruminococcus spp. in horses maintained on high-fiber forage vs. diets that include concentrates. In comparison, transitioning horses between different forage types, for example between hay and pasture (Garber et al., 2020) or cool- and warm-season pasture grasses (Weinert-Nelson et al., 2022; 2023), causes more subtle changes in hindgut microbial communities.
The Hindgut and Horse Health
The equine hindgut microbiome may play important roles in several nutritional and gastrointestinal disorders. Studies in other species have shown that the microbiota plays a causative role in weight gain/loss and metabolic disorders. While associations have been identified between the hindgut microbiota and both obesity and equine metabolic disease, much more remains to be learned about the relationship between shifts in the microbiota and onset (or exacerbation) of these conditions in horses.
In contrast, the role of the hindgut microbiome in laminitis caused by carbohydrate overload (i.e. a horse that over-consumes concentrate) is well-documented. As discussed above, soluble carbohydrates are primarily digested and absorbed in the small intestine. However, if a horse consumes an excessive amount, the capacity for enzymes produced in the small intestine to break down these nutrients can be exceeded. Soluble carbohydrates that are not broken down in the small intestine pass to the cecum. Soluble carbohydrates are rapidly fermented by bacteria such as Streptococcus spp., which in turn grow in number with access to more soluble carbohydrates. These bacteria produce large quantities of lactic acid as by-products of their fermentative processes, resulting in a drop in pH. Cellulolytic bacteria cannot tolerate acidic conditions and rapidly die off. As cellulolytic bacteria die, endotoxin is released and then absorbed by the horse, where it leads to the development of laminitis.
Differences in the hindgut microbial community have also been found between healthy horses and those with conditions such as colic, diarrhea, colitis, and equine grass sickness (Garber et al., 2020). Research is needed to determine whether the microbiome plays a causative role in these conditions. If such relationships are established, it is possible that the presence of specific bacteria (or changes in abundance) could be used to determine risk for developing one of these disorders.
It should be noted that administration of various medications can lead to disruptions in the equine hindgut. Antibiotics, including penicillin and trimethoprim sulfadiazine, have been found to alter the equine hindgut microbiome. Disruptions of the normal gut microbial community by antibiotic administration can allow for the proliferation of pathogenic bacteria such as Salmonella and Clostridium difficile, which are associated with development of diarrhea. Nonsteroidal anti-inflammatory drugs (i.e. phenylbutazone, firocoxib, etc.) used for pain relief can also impact the hindgut microbial community. Changes in the gut microbiota can also occur after administration of anthelmintics (dewormers).
Digestive Supplements
Digestive supplements with targeted effects on the equine hindgut microbiome can largely be classified into two categories: probiotics and prebiotics. The goals of administering probiotics and prebiotics range from increasing digestibility of feedstuffs to stabilizing the gut microbial community, as well as prevention and treatment of gastrointestinal disorders.
The World Health Organization (2001) has defined probiotics as,
- "live microorganisms which when administered in adequate amounts confer a health benefit on the host."
Conversely, a prebiotic is,
- "a substrate that is selectively utilized by host microorganisms conferring a health benefit." (Gibson et al., 2017)
Therefore, the basic difference between these two types of supplements is that probiotics are living, while prebiotics are not.
Bacteria that have been evaluated for use as equine probiotics include species belonging to groups like Lactobacillus, Streptococcus, Bacillus, Enterococcus, and Bifidobacteria (Cooke et al., 2021). Research has evaluated the impacts of probiotic supplementation on nutrient digestibility, hindgut microbial community composition, acidosis, diarrhea, and athletic performance; however, results have been inconsistent. Variable outcomes of probiotic administration are likely due to several factors that can impact efficacy, including the specific strains utilized, the formulations utilized (single or multiple strains), and the number of colony forming units administered. In addition, processing, storage, and transit through the equine foregut can also affect viability of probiotics. Conversely, supplementation with the yeast Saccharomyces cerevisiae has produced consistent results. The addition of yeast has resulted in increasing nutrient digestibility across a variety of diets and lowering lactic acid concentrations (with a concurrent increase in pH) in horses fed large concentrate meals (Coverdale, 2016). However, it is generally accepted that probiotics do not result in any long-term alterations in the equine hindgut microbiota. While bacteria in probiotics may survive transit through the foregut and are detectable in the hindgut, these microbial changes in the hindgut and any potential health benefits occur only when the probiotic is being fed and do not persist after probiotic supplementation has ceased.
Prebiotics are non-living feed ingredients that resist degradation by digestive enzymes in the foregut and, upon passing to the hindgut, selectively promote growth of beneficial microbes. Prebiotics that are commonly supplemented include polysaccharides such as oligofructose, inulin, fructooligosaccharides, and mannanoligosaccharides (Garber et al., 2020). Even some typical commercial feed additives are considered prebiotics due to the fermentation capacity they provide (i.e. beet pulp, oat hulls, soy hulls, etc.). Yeast cell walls and fermentation by-products are also used in prebiotic formulations, and even formulations including non-viable bacterial cells can function as prebiotics. Prebiotics can increase nutrient digestibility and SCFA production, increase hindgut pH, and mitigate hindgut disturbances that occur following abrupt inclusion of concentrates in the equine diet (Coverdale, 2016). Unlike probiotics, the beneficial effects of prebiotics often continue even after supplementation has stopped.
Overall Conclusions
A microbiome is the community of microorganisms living in a specific habitat. The equine hindgut harbors a large microbial community that ferments dietary fiber and produces by-products that provide a large portion of a horse's daily energy needs. This equine hindgut microbiome contains microorganisms, including bacteria, archaea, protozoa, fungi, and bacteriophages. In addition to fermenting fiber, the hindgut microbiota can also break down other nutrients that escape enzymatic digestion in the foregut. While the composition and function of the hindgut microbiome can be influenced by many factors, diet is the key driver of hindgut microbial community structure. Relationships have also been established between the equine hindgut microbiome and numerous health and gastrointestinal disorders. Better understanding of the responses of the hindgut microbiota to dietary changes and the role of the microbiota in disease could lead to the development of improved and more-targeted feed management strategies. As such, interventions, such as probiotics and prebiotics, that promote health of the equine hindgut microbial community are emerging strategies.
References and Additional Reading
August 2023
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