Microbiome, fermentation and metabolites: horse and human compared

What fermentation in the gut actually produces

Fermentation here means this: in the absence of oxygen, bacteria break down carbohydrates that the body's own enzymes cannot split. The main end products are three short-chain fatty acids, namely acetate, propionate and butyrate. They are metabolic waste for the microbes and an energy source for the host at the same time. A 1984 review estimated the contribution of the human large intestine to total energy supply at 5 to 10 per cent and set this against herbivores, in which the same route covers a substantial part of requirements. The figure is old, but to this day it remains the reference value that more recent work is calibrated against.

Why does almost everything revolve around butyrate?

Because butyrate is regarded as the preferred fuel of the gut lining, and clinical research has organised itself around that. A 2019 methodological review counters that this focus distorts the picture. Short-chain fatty acids act differently depending on dose, time elapsed since exposure and time of day, sometimes even in opposite directions. Anyone who measures only butyrate, and only at a single time point, captures a fragment and takes it for the whole.

The horse's caecum, the human colon: same chemistry, different design

In the horse, the fermentation chamber sits downstream of the small intestine, in the caecum and the large colon. A passage study in five caecally fistulated horses of around 562 kilograms, fed 6.81 kilograms of grass hay dry matter per day, measured retention time in the hindgut: 22.2 hours for the liquid phase, up to 26.3 hours for coarse particles of 8 millimetres. There was no meaningful difference between particle sizes. The horse therefore barely sorts its digesta; it simply holds them long enough for the microbes to do their work.

The sampling site decides the result. In a crossover study with six caecally fistulated horses, sampled every three hours over 24 hours, total concentrations of volatile fatty acids were higher in the caecum than in the faeces, while diversity was higher in the faeces than in the caecum. A second study compared digesta from the caecum, left ventral colon, right dorsal colon and rectum of eight Warmbloods: total fatty acids, butyrate, pH and ammonia differed markedly between the segments. Anyone using rectal contents as a proxy sample may well overestimate forage digestibility, the authors conclude.

That fermentation does not run the same way everywhere is shown by a data compilation covering 37 herbivore species. Relative to body size, feed intake, retention time and gut volume, equids, kangaroos and rabbits produce strikingly little methane. The familiar split into foregut and hindgut fermenters does not explain these differences. Even among hindgut fermenters, then, the horse is no standard case, and it makes a poor stand-in for other species.

When fermentation tips over: starch, pH and hindgut acidosis

Hindgut acidosis describes the acidification of the fermentation chamber when starch passes the small intestine undigested and is rapidly fermented to lactic acid in the caecum. What matters is not the ingredient but the amount per meal and how it is spread out over time. In a trial with four caecally fistulated horses, each given the same protocol-defined amount of oats and hay on every test day, feeding order alone shifted the timing of the lowest caecal pH from 170 to 280 minutes. Blood glucose and insulin showed nothing of this difference: the caecal environment responded while the blood values stayed silent.

At what amount does it become critical?

No generally valid threshold is established, and figures from single trials are not a feeding recommendation. What is established is the direction. When six individual feed ingredients, maize and oats among them, were supplemented in a Latin square with six horses at a daily amount that was the same for all and deliberately kept low, caecal pH stayed within the normal range throughout, and the treatments did not differ on average.

With sustained high starch intake the picture changes. In a study of 19 Bardigiano horses of around 14 months over 129 days, diversity in the caecum, pelvic flexure and right dorsal colon was lower on the high-starch ration than on the high-fibre one, and starch-degrading genera linked to acidosis were proportionally more common. Recovery takes weeks: after an abrupt switch to a high-starch ration over five days, fibre-degrading activity fell in twelve horses while starch-degrading activity rose; only three to four weeks after the return to the fibre ration did the faecal parameters reach baseline again.

What the full picture looks like is shown by the experimental oligofructose model, which deliberately induces laminitis. Faecal pH fell, while lactic acid, histamine and lipopolysaccharide rose in the blood; Lactobacillus and Megasphaera increased proportionally, and 137 metabolites changed measurably in all. The model describes a deliberately induced overload, not everyday conditions.

What is established in humans: from fibre to metabolite to endpoint

At the level of dietary habits the evidence is strong. A series of systematic reviews analysed 185 prospective studies and 58 controlled trials, together nearly 135 million person-years. Comparing the highest with the lowest fibre intakes showed 15 to 30 per cent lower all-cause and cardiovascular mortality, along with a lower incidence of coronary heart disease, stroke, type 2 diabetes and colorectal cancer. The most favourable range was 25 to 29 grams of dietary fibre per day. The authors rated the certainty of this evidence as moderate.

At the level of metabolites it thins out. A meta-analysis of 14 intervention groups with 205 adults found that cereal fibres raise acetate, propionate, butyrate and the sum of short-chain fatty acids, with a larger effect at a body mass index above 29. A second meta-analysis of nine randomised trials in type 2 diabetes found an increase in the total but no robust effect on the individual acids; the long-term blood sugar marker HbA1c fell by 0.18 percentage points.

What can be measured also depends heavily on the preparation. In 80 healthy adults, replacing conventional white flour with high-amylose wheat flour raised faecal butyrate excretion by around 38 per cent relative to the comparison group, while total fatty acids and stool weight were unchanged. An analysis of 61 studies on overweight, in turn, found that prebiotics lowered two inflammatory markers in the blood in humans, while a third fell only in animal experiments: an example of results failing to mirror across species even within a single research question.

At the level of the administered fatty acid itself, the picture flips. A systematic review of 46 studies with 913 participants in total found no effect of acetate, propionate, butyrate or mixtures on blood glucose and insulin. Only vinegar lowered the acute rise in blood glucose, and the authors rated the overall certainty of the evidence as very low. In eleven randomised trials with 729 people with irritable bowel syndrome, prebiotics improved neither symptoms nor quality of life; they did raise bifidobacteria, but inulin-type fructans made bloating worse.

Individual well-controlled trials, by contrast, do show effects. An ester that releases propionate specifically in the colon reduced weight gain and the increase in abdominal fat over 24 weeks in 60 overweight adults. In a 21-day crossover study with 20 healthy adults, plasma butyrate was higher on a high-fibre diet than on a low-fibre one (2.85 versus 2.02 micromoles per litre), and the numbers of certain B and T cells in blood were lower. In 54 children with obesity, 96 per cent on sodium butyrate over six months reached the predefined target of a reduction in body mass index, against 56 per cent on placebo, and in 36 adults with active ulcerative colitis, sodium butyrate lowered faecal calprotectin and the inflammatory marker in the blood.

And then there are the findings that counsel caution. A meta-analysis of eleven studies found lower values in ulcerative colitis overall for total fatty acids, acetate, propionate and valerate than in healthy people; in active disease butyrate was additionally reduced, whereas in remission it was higher than in healthy people. In critically ill patients, dietary fibre in 21 studies with 2,084 people improved gut barrier permeability and the inflammatory marker, without any measurable difference in fatty acid levels. So the metabolite does not necessarily explain the effect, and a low value is not automatically a deficiency.

Where horse and human meet, and where they do not

The mechanism is shared: anaerobic bacterial fermentation of indigestible carbohydrates into the same three fatty acids, the same fundamental dependence on which carbohydrates reach the fermentation chamber at all, and on the time they spend there. What differs is the share, the site and the consequence. How sensitively the environment reacts to rapidly available starch has been described experimentally in the horse; a counterpart in humans is not established.

What follows from this is above all what does not follow. A finding in the horse justifies no recommendation for humans, and the same holds in reverse. Lactic acid formation in the equine caecum after a grain meal has no direct counterpart in the human colon. The 25 to 29 grams of dietary fibre per day from human research is a population figure, not a number that could be converted to body weight. And a substance that works in one species remains untested in the other for as long as it has not been tested there.

Even within a single species, effects often fail to appear. In a randomised study with 20 four-year-old Warmblood stallions over nine weeks, plasma metabolites did not differ between a high-starch and a high-fat ration; only exploratory behaviour tended to diverge. An altered microbiota therefore does not automatically translate into measurable metabolic values, and an absent effect in the blood does not mean that nothing happened in the gut.

Metagenomics and metabolomics: what gets measured and what does not

Most statements about the microbiome come from sequencing a single gene, 16S rRNA. The method yields relative shares: it says what proportion of the sequences found belongs to which genus, not how many bacteria are present in total. A study that combined sequencing with cell counting by flow cytometry found microbial loads differing by up to tenfold between healthy people. The much-quoted contrast between Bacteroides and Prevotella turned out to be an arithmetic artefact of the relative-abundance representation and disappeared as soon as cell counts were used instead of percentages.

Does the sequence tell us what the bacteria are doing?

No. Functions can only be estimated from 16S data. A comparison across more than 5,000 human samples from four body sites showed that the functional profiles predicted this way deviate systematically from the reference obtained by whole-genome sequencing; the deviation could only be offset by calibration with sample pairs measured in parallel. And even fully sequenced genes say only what would be possible, not what is currently being read out.

On the metabolite side the fallacy is even more widespread. A methodological review states that the concentration of short-chain fatty acids in digesta or in faeces is no measure of their production rate. The acids are absorbed rapidly, the mucus layer at the gut wall forms a zone of its own with its own conditions, and the values fluctuate over the day. A low faecal butyrate value can therefore stand equally well for low production or for very good absorption. Without a time course and a dose-response curve, such single values are, in the review's words, misleading at worst.

• A sequencing result without a total cell count describes proportions, not amounts.
• A metabolite value without a time course describes a moment, not a process.
• A faecal sample describes the end of the road, not the site of fermentation.
• A difference between groups describes an association, not its direction.

What reasonably follows from the current state of knowledge

The most robust part of the field is also the least spectacular: a fibre-rich diet in humans and a forage-based ration in horses are well studied and not seriously in question. Everything beyond that, meaning individual fatty acids, individual bacterial strains, individual metrics from a faecal sample, is considerably less well established than the coverage suggests.

• With every study report, check first: which species was investigated, and where in the gut was the sample taken?
• Distinguish between the statement ‘the composition has changed’ and the statement ‘the state of health has changed’. The first is common, the second rare.
• In feeding and nutrition questions, what counts is the amount per meal, the spread across the day and the length of the transition, not the ingredient alone.
• Commercial microbiome analyses based on a single faecal sample answer less than their presentation suggests.
• Digestive problems in horses belong in veterinary practice, persistent digestive complaints in people belong in medical assessment.

ForschungPferd rates this topic area at evidence grade B and transferability T1: the mechanism is established in both species, the transfer of a clinical effect from one species to the other is not.

Measurement levels in fermentation research and their documented blind spots

Original analysis

Limitations and uncertainty

• The equine literature contains hardly any systematic reviews or meta-analyses on this topic. Robust statements rest on controlled feeding trials with frequently fewer than twenty animals, in some cases only four to six, often from single yards and a small number of breeds.
• Fistulated horses do allow direct sampling from the caecum and colon, but they are few, mostly adult, healthy and on standardised feeding. How representative they are of leisure, sport or pasture-kept horses is an open question.
• In humans, hardly any study measures in the colon itself. Almost all statements rest on faecal samples and blood values, that is, on indirect quantities whose relationship to actual production is unknown.
• The meta-analyses on dietary fibre and short-chain fatty acids report high heterogeneity throughout. Preparations, doses, study durations and measurement methods differ so widely that pooled effect estimates carry only limited weight.
• The oligofructose model of laminitis is a deliberately induced extreme state. It describes a limiting case and not the gradual courses that are likely to be more common in practice.
• Two central references in this article date from 1984 and 2017. They have been kept because they still provide the authoritative reference values for the energy share of fermentation and for quantitative measurement in the microbiome.

Open questions

• How high is the actual production rate of short-chain fatty acids in the colon of living humans, and how does it relate to the values measured in faecal samples?
• Is there a measurable threshold in the horse for starch per meal above which the caecal environment reproducibly tips over, or is the threshold individual and dependent on the existing microbiota?
• Do differences in microbial load, that is, in absolute amount rather than in proportions, explain part of the contradictory findings on short-chain fatty acids in humans?
• Why do equids produce strikingly little methane in cross-species comparison, and does this difference have consequences for the yield of short-chain fatty acids?

Frequently asked questions

Can I read off from a faecal sample how my horse's caecum is doing?

Only to a limited extent. In a study with caecally fistulated horses, fatty acid concentrations were higher in the caecum than in the faeces, while diversity was higher in the faeces. A second study compared digesta from four segments and found clear differences in fatty acids, butyrate, pH and ammonia; with rectal contents as a proxy sample, forage digestibility is likely to be overestimated. Faeces therefore reflect the direction of a change rather than the state of the fermentation chamber. For assessing an individual horse, the veterinary examination is what counts.

Do short-chain fatty acids do anything as a supplement?

The evidence is contradictory and weak overall. A systematic review of 46 studies with 913 participants found no effect of acetate, propionate, butyrate or mixtures on blood glucose and insulin, and rated the certainty of the evidence as very low. Individual well-controlled trials, by contrast, do show effects, for instance in children with obesity or in active ulcerative colitis. This page gives no recommendation and deliberately names no amounts; questions of that kind belong in medical consultation.

What exactly is hindgut acidosis?

An acidification of the fermentation chamber in the horse, triggered when starch passes the small intestine undigested and is rapidly fermented to lactic acid in the caecum. Studies show that feeding order alone affects the timing of the lowest pH, and that a permanently high-starch ration lowers diversity in the caecum and favours starch-degrading bacteria. In the experimental extreme case, lactic acid, histamine and lipopolysaccharide rise in the blood. Suspicion of such a process is a matter for veterinary practice, not for self-experimentation.

If dietary fibre is so good, why do prebiotics often fail to work in studies?

Because a way of eating and an isolated preparation are not the same thing. The evidence for dietary fibre comes from 185 prospective studies with nearly 135 million person-years and relates to the whole pattern of eating. Prebiotics, by contrast, deliver a single substance: in eleven randomised trials with 729 people with irritable bowel syndrome they improved neither symptoms nor quality of life, and inulin-type fructans even made bloating worse. They did raise bifidobacteria, but this microbial effect did not translate into a clinical one.

Why can a result from the horse not be transferred to humans?

Because the same mechanism runs at a different scale and in a different place. In humans, the contribution of colonic fermentation to energy supply has been estimated at 5 to 10 per cent; in the horse, the hindgut is the central digestive organ, with a retention time of around 22 to 26 hours. Acidification of the equine caecum after a grain meal has no counterpart in the human colon. ForschungPferd therefore classes this topic area as T1: shared mechanism, no established transfer of a clinical effect.

What is the difference between 16S sequencing and shotgun metagenomics?

16S sequencing reads a single marker gene and answers which genera are present proportionally. Shotgun metagenomics reads all the DNA present and answers which genes are there. Estimating functions from 16S data is possible but imprecise: across more than 5,000 human samples, the predicted functional profiles deviated systematically from the genome reference. Both methods also yield proportions, not amounts. Without a parallel cell count it remains unclear whether a genus has actually increased or whether others have merely decreased.

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