Through the Looking-Glass: Chapter 1, page 1

Through the Looking-Glass

a bug's-eye view of the equine gut
and what it can tell us about feeding horses

last revised 14Jan2020

Chapter 1

"What's one and one and one and one and one
and one and one and one and one and one?"
"I don't know," said Alice. "I lost count."

Horses are large herbivores who, in their natural setting, graze a wide variety of grasses and other plants, including sedges, rushes, forbs (small ‘herby’ plants), shrubs, trees, horsetail, lichen, and moss.

Wild or feral horses (Equus caballus) living in the footills of the Rocky Mountains in western Alberta, Canada, have been observed to eat over 40 different genera (genus, plural) of plants throughout the year. Their monthly diet included about 20 different genera. Even more remarkable, analysis of 10 separate diet studies of wild burros or donkeys (Equus asinus) living in the Mojave Desert in the southwestern United States found that 175 different plant species were consumed by burros in this desert landscape. The faeces (manure) or stomach contents contained an average of 33 different plant species per burro; it ranged from 11 to 54 species, depending on location and time of year.

As a supplement or replacement for this natural diet, domesticated horses typically are fed hay (dried grass and/or legume), with or without some type of grain-based 'concentrate'.

To make effective use of this high-fibre diet, horses rely on the vast population of bacteria and other microbes (protozoa, fungi, etc.) in their large intestine, particularly in the caecum and large colon. In animal nutrition circles, this part of the gut is referred to as the 'hindgut', to distinguish it from the 'foregut' of ruminants such as cattle, sheep, and goats.

Unlike horses, ruminants have their engine in the front: most of their gut microbes are located in the rumen, which is the first and most voluminous of the three pre-gastric chambers (rumen, reticulum, and omasum) which make up the forestomach (literally, ‘before the stomach’). As the rumen is the principal site of microbial breakdown, or fermentation, of dietary fibre in ruminants, they are called 'foregut fermenters'. In contrast, the majority of the gut microbes in horses are located in the large intestine, so they are called 'hindgut fermenters'.

Bacteria make up between 70% and 90% of all microbes in the faeces of healthy horses, and up to 99% of the faecal microbes in horses with acute colitis (inflammation of the large colon, resulting in diarrhoea). So because of their predominance, we'll focus on the bacteria of the equine gut, with just a brief note here and there about the other microbes.

The estimated number of bacteria in the equine gut almost defies the imagination. Let's begin with the hindgut.

Bacterial Numbers

Caecum

For some perspective, the generic or average-size, adult horse weighs about 450 kilograms (1,000 pounds) and stands about 150 centimetres (60 inches, or 15 hands) in height at the wither (the highest fixed point on the body). Although common horse breeds range from Miniature Horses and small ponies weighing less than 90 kilograms (200 pounds) to draft horses weighing more than 900 kilograms (2,000 pounds), for simplicity’s sake we’ll focus on the average-size, adult horse.

The first part of the hindgut is the caecum. It’s about 1 metre (3 feet) in length and has a capacity of about 33 litres (9 US gallons). In terms of the entire equine gastrointestinal tract, the caecum accounts for around 4% of the total length and 16% of the total volume.

Its relatively large capacity is explained by the fact that the caecum is the first portion of the gut in which microbial fermentation makes a substantial contribution to the horse's nutritional needs. An adult horse on a forage-based diet (pasture and/or hay) may meet more than 80% of its daily energy needs from the volatile fatty acids that are produced by microbial fermentation of dietary fibre in the hindgut. The caecum alone is responsible for generating about 30% of the horse's daily energy needs.

These figures refer to the maintenance, or basal, energy requirements of an inactive adult horse. Daily energy needs increase with regular exercise and with pregnancy, lactation, growth, and recovery from serious debility. The relative contribution made by hindgut fermentation decreases as the horse’s daily energy needs increase above maintenance and the forage portion of the diet is supplemented with more energy-dense foods such as grain-based concentrates. But even then, hindgut fermentation makes a substantial contribution to the horse’s daily energy needs.

So, it's quite surprising that there's not a lot of data available on the total number of bacteria in the equine caecum and large colon. Instead, research has focused on the diversity of bacteria, and particularly on how the relative abundance of key bacterial groups may change with diet and disease. Bacterial diversity is discussed later in this chapter, and the impact of diet on the gut microbiota is the main focus of Chapter 2. For now, let's try to pin down roughly how many bacteria there might be in the equine caecum.

A handful of studies have provided culture-based estimates of bacterial numbers in the equine caecum. However, bacterial culture methods are notoriously inadequate for identifying the more fastidious and downright persnickety bacteria in the hindgut, even when enrichments are added to the culture medium.

That alone tells us a good deal about how little we really know of conditions within the gut, and what the gut microbiota, as a diverse community, needs to thrive.

An often-quoted figure ("30%") is from one of those culture-based studies. Before culturing each sample of intestinal contents, the researchers performed a direct microscopic count of the bacteria in the sample. They found that bacterial colony counts after culture were only about 30% of the direct microscopic counts in samples from the caecum and large colon, and only about 10% of the direct microscopic counts in samples from the small intestine.

In other words, at least 70%, and as many as 90%, of the bacteria seen microscopically did not grow in culture, despite the use of nutrients and other enrichments aimed at encouraging their growth.

Enter the various culture-independent methods that are now the standard means of evaluating the gut microbiota. Most of these methods are based on the analysis of target gene sequences or gene products that are specific to particular types of microbes—and most importantly, they are not reliant on the microbe's specific growth requirements or ability to grow in the laboratory.

In one study using these methods, estimates of the total number of bacteria in the caecum of healthy horses ranged from 5 billion to 11 billion (average, 8 billion) per millilitre of fresh caecal contents. For some everyday context, one teaspoon equals about 5 grams, or 5 millilitres of liquids or soupy material such as caecal contents. So, there is an average of 40 billion bacteria in just one teaspoon of caecal contents.

That study included only 5 horses, but they were kept under similar conditions and fed similar diets. Note that the lower and upper limits of the range differed by more than two-fold (5–11 billion/ml). This horse-to-horse variability comes up disconcertingly often in studies of the equine gut microbiota—so much so that it's worth making this central point this early in the programme:

Each horse’s gut microbiota is unique.

...and as we'll see later, there's a disconcerting amount of variability even within the same horse.

It's also worth noting that the caecal contents in those healthy horses were about 90% water. That's normal, and as we'll see in Chapter 2, it's evidently necessary for normal microbial activity and gut motility (which, in the caecum, involves extensive mixing of its contents before they're periodically sent downstream into the large colon).

Anyway, with an average volume of 33 litres, the equine caecum probably contains between 165 trillion and 360 trillion—that is, at least 165,000,000,000,000—bacteria.

Large Colon

The large, or ascending, colon in the horse is a rather elaborate, two-storey affair. It has a double-horseshoe configuration with a hairpin bend, the pelvic flexure, that allows the two 'horseshoes' to nestle one atop the other. The colon narrows dramatically through the pelvic flexure, so this arrangement effectively creates two distinct colonic compartments: ventral (lower) and dorsal (upper).

For most of its length, the large colon is a roomy structure. In the average horse it is over 3 metres (10 feet, and up to 13 feet) in length, with a capacity of over 80 litres (21 gallons, and up to 34 gallons). These figures represent an estimated 11% of the total length and 38% of the total volume of the gastrointestinal tract. Combined, the caecum and large colon account for 54% of the total gut capacity in the horse.

As with the caecum, microbial fermentation of fibre in the large colon contributes substantially to the horse's daily energy needs. With as much as 80% of the horse's daily energy needs being met from microbial fermentation in the hindgut, and the caecum responsible for about 30%, the large colon, with its larger capacity, is responsible for generating about 50% of the horse's maintenance energy needs.

But just as with the caecum, there is an inexplicable dearth of culture-independent data on the total number of bacteria in the equine large colon. Several culture-based studies have provided estimates for various sites in the large colon, notably the right ventral colon, but also the pelvic flexure and the left dorsal colon. Bacterial counts varied somewhat (and inconsistently) with collection site, but mostly they varied with culture method—and with the horse's diet.

Some culture-based studies showed an increase in bacterial numbers of three- to sixteen-fold between the caecum and the large colon, but one study showed a decrease of more than four-fold. Rather than speculating on the reasons for these differences, let's look instead at the products of fermentation in the hindgut.

In two different studies, the concentrations of volatile fatty acids (acetate, propionate, butyrate, etc.) and lactate (lactic acid), as well as the pH, were similar in the caecum and large colon. As these products of microbial fermentation were present in similar amounts, it is likely that both the spectrum and number of bacteria are also quite similar in these sequential hindgut segments.*

* A culture-independent study of the various equine gut segments confirmed the similarity in bacterial spectrum between the caecum and large colon in healthy horses. In addition, the bacterial spectrum in the faeces was not significantly different from that in the large colon, so it was concluded that faecal samples are an adequate representation of the large colon contents in healthy horses.

Based on this indirect evidence, it is probably safe to assume that total bacterial counts in the large colon are similar to those in the caecum (5–11 billion/ml). The contents of the large colon have a similar soupy consistency to the caecum, and a water content of 90% to 95%, so we're probably within range to estimate that, with a capacity of at least 80 litres, the equine large colon probably contains somewhere between 400 trillion and 880 trillion bacteria.

Adding the total estimates for the caecum and large colon, there are well over 560 trillion and as many as 1.2 quadrillion bacteria in the equine hindgut. For some perspective, one quadrillion is 1 followed by 15 zeros: 1,000,000,000,000,000.

But wait; estimates of the total number of bacteria in the faeces suggest that this may be too conservative a figure. Again, culture-independent estimates of total bacterial counts in the equine faeces are hard to find, but one study reported a range of 200 billion to 500 billion (average, 300 billion) bacteria per gram of dry matter (i.e., results converted to 100% dry-matter for comparison). The faeces of healthy horses are 70% to 80% water, depending on the diet, so that works out to be a range of 40 billion to 150 billion (average, 60–90 billion) bacteria per gram of fresh faeces.

If we rework those numbers to reflect a water content of 90% (to match the water content of the large colon), it suggests that the large colon might contain between 20 billion and 50 billion bacteria per millilitre—or about 4 times as many as the caecum, which is in line with at least one culture-based study.

So, now we're talking about 1.6 quadrillion to 4 quadrillion bacteria just in the large colon. Even if we split the difference between caecal and faecal estimates, we're still at well over 1 quadrillion bacteria in the large colon alone.

As for the faeces, there are between 200 billion and 750 billion bacteria per teaspoon of fresh horse manure. As a point of reference, one teaspoon of fertile soil contains between 100 million and 1 billion bacteria.

Fertile soil is an interesting analogy here, as the interactions between a plant's roots and the microbe-rich soil (humus—not to be confused with hummus!) that surrounds them have some features in common with the lining of the horse's gut and the microbe-rich gut contents. In some respects, the microbe-laden—and microbe-manipulated— gut contents are the horse's 'fertile soil'.

We'll leave the rest of the lower intestinal tract (small colon and rectum) be, as relatively little digestion occurs in these sites. Instead, let's head upstream to the small intestine—a section of the equine gut that has been greatly under-appreciated with regard to its microbiota and the contributions these microbes make to digestion.

Small Intestine

In the average adult horse, the small intestine is about 22 metres (72 feet, and up to 98 feet) in length, with an average capacity of 64 litres (17 gallons). That represents about 75% of the total length and 30% of the total volume of the equine gastrointestinal tract.

In the few studies that have examined the microbiota of the equine small intestine, bacterial numbers are not consistent along its length; rather, they tend to increase the further downstream one samples, although the horse's diet may have a good deal to do with it.

Still, as the jejunum (the middle section) comprises at least 90% of the total length and volume of the small intestine, estimates of total bacterial numbers in the jejunum may be a reasonable approximation for the entire small intestine. With a total bacterial count of 400 million to 500 million per millilitre and an average volume of 64 litres, we could figure a broad estimate of total bacteria in the small intestine to be in the range of 26 trillion to 32 trillion.

There don't appear to be any culture-independent estimates of the total number of bacteria in the equine small intestine, but an estimate of 30 trillion or so is supported by the study which found that culture yielded only about 10% of the number seen microscopically in samples from the small intestine. In that study, culture yielded an average of 30 million, and up to 62 million, bacteria per gram in the jejunum, which suggests an actual figure of 300 million, and up to 620 million, bacteria per gram—for a total in the entire 64 litres of about 19 trillion and up to 40 trillion bacteria.

So, 30 trillion is close enough for our purpose here, which is simply to highlight the sheer number of bacteria in the equine gut, a rough estimate of which comes to a median of 2,500,000,000,000,000 (2.5 quadrillion) bacteria in the small intestine, caecum, and large colon.

intro