Good evening everyone. My name is Charlotte, and thank you for joining us for tonight's webinar, the Horse's Gut Microbiome, presented by Simon Daniels. The webinar Vets Equi programme has been kindly sponsored by Bailey's Horse Feeds, so a big thank you to Bailey's Horse Feeds for sponsoring our equine programme and tonight's webinar.

So a bit about our speaker today. Doctor Simon Daniels is a senior lecturer in equine science at the Royal Agricultural University in Cirencester. Simon worked in the horse feed industry before moving to academia.

Initially working on a knowledge transfer project between the University of Liverpool vet school and a commercial parasitology lab, Simon undertook a PhD looking at parasitology and the effects of anthelamentics on the gut microbiome. His area of specialism remains in equine gut health, which encompasses both intestinal parasites and equine nutrition. I wish to let you all know that tonight's webinar will be recorded and available on playback, and you'll all receive a certificate for tonight's attempts also.

Please use the Q&A box for any questions you may have for Simon throughout the presentation. And at the end of today's session, we will see if we can answer any questions that you may have. If we run out of time with the questions submitted, we will email out any responses to you in the next few days.

So with no further ado, I'd like to now hand over to Simon to start this evening's session. Thank you, Simon. Perfect, thank you, Charlotte.

Yes, so tonight's session is thinking about the horse's gut microbiome. What do, what do we actually know about it, and actually what does it all mean? And So.

What do we actually mean by the gut microbiome? I think over the last decade, there have been an explosion of studies that are looking at the gut microbiome. It's become a huge buzz, you know, we're, we're lagging behind working other livestock and and.

Humans before that. And we start thinking about the gut microbiome, and a lot of the studies are thinking about the bacterial community profile within the gut, and some of the function of those microorganisms in the gut. And.

Why do we want to know about that? And we are understanding more and more all the time from human work and from other animal models that in actual fact, the gut microbiome plays a really important role. We, we tend to think of it, it's sort of quite simplistically as well it's the digestive tract, but actually there's a lot more to understand, we have.

A much better understanding or, or developing an understanding in other models of the gut brain axis. And there is some work, and we're currently doing some work thinking about that with the horse. There's, sort of new strands thinking about the gut brain immunology axis.

So there's so much going on out there. But this webinar will think about. Why, you know, where does our basis, where does our base knowledge for the gut microbiome come from?

What did we already know before the term microbiome started appearing and we had all of these new papers coming out, . What do we need to understand about this science, because it's providing fantastic information, but we have to understand a little bit about how these studies are run and what the limitations are, and what information that we can draw. So we can, as we move forward, we understand that yes, we, we're heading in the right direction and we have a much better understanding the microbiome.

But it's not a magic bullet, you know, it's just part of the, of the process. And so that the idea is to sort of look a little bit back to start with and then look at some of the more recent work and how it's been applied and and how that might then become more useful, especially to vets in practise and to horse owners, horse managers, how might this stuff become useful in the way that they look after and manage their animals. So I suppose the first bit of it is thinking about the fact that it's important throughout the horse's life.

So whether we be talking about a young animal on the very start of its journey, that, that's growing, and before it then, you know, before, before there's any hint of, of actual work in its life. Albeit a sports horse, a leisure horse, whatever that horse is doing. So, you know, the gut microbiome is playing an important role throughout life.

I sort of like these pictures as well, cos I'm normally telling people, you know, I think horses are great, but actually I'm really excited about their guts, that's the bit that I'm really, really interested in. And so it's about sort of having a, a, a, a better understanding that it's more than just food in, energy and faeces out. Actually, it's trying to understand at all stages of life, growth, development, work, how, what sort of important role the gut microbiome might be playing.

And I suppose it comes back. Starting with the gut itself, the anastoian physiology of the gut, and the, the image you here on the screen is is a great way to depict the fact that, you know, the horse is a hindgut fermenter. When you look at the hindgut portion, it's huge.

And I suppose crudely, we've always thought, well, that's a huge fermentation tank, you know. From the for gut perspective, very similar to our own system. OK, we know that the, the stomach is a little bit unique, and we know from the fact that horses get stric suffer with gastric ulcers, that it's maybe not the best design in the world.

But it, it, it's a, a, a simple stomach, monogastric. But we maybe don't know as much about the for gut in the horse as we thought. We were often, a lot of that has been applied from pigs, for example.

But the hindgut is our import we always think of ours important area. Equally, until 20 odd years ago, we sort of made the great assumption that actually the fore gut didn't really have a, any. Bacteria worth considering, and all the microbial activity was in the hind gut.

And we now know that actually there is a for gut microbiome. It's smaller, it's a little bit more niche. But it does play some role.

We don't necessarily always remember what that is. And so thinking about it in its sort of crude fermentation tank status, the nice little diagram there from Shira Razavici, sort of gives quite a nice, depiction. In that we've got the horses designed to ferment fibre.

So cellulose and hemicellulose from our plant cells and pectin, which is effectively the glue that jams those cells together, is all nicely fermented by this cellululotic population of Fibrobactys, Clostridia, ruinococcus, and our fungi and our protozoa, and they're really the powerhouse of, of the hindgut from a fermentation. Perspective, and they give us acetate and butyrate, butyrate feeding gut cells, acetate as a, as an energy source. Now, when we're breaking down these, these fibrous products, which don't get broken down in the fore gut because the horse doesn't have the right enzymes to be able to, to break down cellulose, then, as they are, as, as fibre is fermented, we do get some sugars released.

We'll probably also get a bit of protein released as well, depending on the, the maturity of, of the plant that's being digested. The protein just acts as a bit of energy for the, the microbiota, it's no use to the horse once it gets to the hind gut. But the sugar that is, that is liberated, the oligosaccharides, is not fermented by these, cellulotic population.

It's, it will be fermented by these sacrolytic fermenters, the non-cellulotic population. And any starch that reaches the hindgut, so anything that is not digested in the small intestine that gets to the hindgut, and the same with any sugar, will also be fermented by these, these microbiota. And in the horse, they're normally the, the smaller sort of population.

And they will give us, a bit of propionate, which is fine because we can feed that directly into the glucose pathway, and that's useful. But we also get some lactate. And I suppose this is the one that we think about when we use the classic model of starch overload, getting to the hindgut, and what happens is we have an up regulation of the sacculittic fermenters, we start to shut down and lose some of the, the cellulotic population, and we get more lactate.

Now, the reason nature does have a way to mop this up, but it's not particularly functional in the horse. So there are bacteria, groups of bacteria. So within the, the veinella.

They will convert lactate back into propionate. And so in humans, in cattle, that is the mechanism. So if we think about cows as a, as an example, ruminants as an example, ruminal acidosis can probably be buffered off a little bit longer because they have this ability to be able to convert some of that lactate into propionate, short chain fatty acid.

And so it's sort of Delays the onset of acidosis. Now we know with horses, there was some work from nearly 20 years ago now that shows actually, in horses on a completely grass-based diet, the, the numbers of Balinella are are absolutely minute within their microbiome. And even those that are fed a diet with a little bit of starch, they still make up a very, very small proportion.

So it's almost like nature's failure in the horse of being able to mop up lactate. . And so that's a, that's a bit of a a a sort of a horse specific issue.

But the system is, you know, quite advanced in a way, in that we can actually produce, well, berate predominantly is is powering those colon cells, but propionate, we can feed it straight into glucose pathway, it's a great source of energy, and acetate. And the horse actually is great in that it doesn't produce much methane, and it sinks some of the carbon, dioxide and hydrogen that's, that's the byproduct of fermentation. Back into acetate and makes good use of that.

So that's a sort of in a way, well, it's a bit of an overview of how the whole system works. But as I said, we tend to have this sort of thought that food goes in to this hindgut tank or into the gut in general, and we get energy, and we tend to sort of leave it there. I mean, we've known for 30 odd years that huge amounts of energy for the horse, 60 to 70% of its energy, or more for a, you know, .

In an animal doing nothing can come from those short chain fatty acids. I suppose, from my perspective as a scientist, the hindgut is a bit of a gift that keeps on giving. We still don't really know huge amounts about it.

The more layers we unpeel, the more we realise we're still at the tip of the iceberg. But there's a lot more, you know, to understand and to sort of unpick within the horse's hindgut. And in actual fact, when you start looking at our understanding, and I sort of hinted at this already, the hindgut end we knew that there were bacteria, fungi, protozoa, and they fermented fibre, but we had no idea really what else they were doing.

And there were, when you look at the work on the horse's foregut, we already know various groups have looked at the fact that actually horses have quite poor, amylase, both concentration and activity, which does partly explain why they're not great at digesting starch. But a lot of the figures that are out there for concentrations and activity of various enzymes, actually, when you try and. Trace where they originate, the data doesn't come from horses.

It's often extrapolated from pigs. Now they're mammals, and realistically, the values should be pretty close. But in actual fact, there's a lot of sort of guesswork going on, and there are groups, our, our group's been doing a little bit of work trying to get a, a better understanding under certain circumstances as to, you know, what's normal in the horse.

Because we have known for a long time. That horses are quite delicate little creatures when it comes to their digestive system. And if we take these two quotes, thinking about how, well, too much hay, but more to the point, excessive quantities of corn, induce violent indigestion and gripes.

And new hay and oats are combined, preferably known to be injurious. Yeah, definitely. Similarly, grass is the first nor.

Of all cults after they're weaned, whereas when they're fed corn and hay, but especially the first, it exposes them to unspeakable injuries. It's, it's no surprise to me as somebody who's really excited about horse guts, that, we've known for a long time that feeding horses starch is a problem. And when we make changes to the horse's diet, we increase the risk of colic.

And I suppose colic is, in my world, colic is the sort of umbrella that that covers a lot of those boxes, and. When we look back at the work, and some of the work, sort of the, the epidemiological studies that look at risk factors associated with colic and events such as diet changes, dosing ltalin tic, those studies now from Noah Conan and from Mark Hillier's group, which is sort of late 90s, early 2000s, . In actual fact, the more work we do, a lot of the time you think, well, that, that makes logical sense, because it feeds back into, well, there's the risk factor for colic increases over, within 7 days of changing the diet or within a week of giving analmitic or or various things.

And we, there's still a lot of question marks as to why those things happen and is it actually that individual event or is it a, Selection of events that all happen to happen at the same time, or in more susceptible animals, but we know that . The horse's gut microbiome or horse's gut is, it is susceptible to change, which therefore leads to colic. And when we look back, this is really our, I suppose this is to me, this is our basis for trying to understand more about the gut microbiome, because if the more we understand.

The better we manage, we can manage horses. And if we can put really good management practises in place, then theoretically we can try and reduce the risk of colic. You know, it, it really, it's all about sort of best practise and being able to understand or.

Even to some extent, trying to unpick why for 20 odd years we we've understood that colic can happen within X window of time after making that change. But then my next question is, but why? What is it about that change that increases that risk?

And we've known about the the gut ecosystem for over 125 years in the horse, . But it still remained this sort of black box. And really the work started to advance in the 90s.

And credit here goes to, to Veronique Julian's group in Dijon. They had a group of fistulates and they've done a lot of work trying to understand the, the Heinegu ecosystem. And a lot of this was culture-based work.

And, and this gave us a great starting point, and they did various nutrition studies. They did some studies, with horses receiving analmitic, looking at the effects on the hindo microbiome, and a lot of work trying to understand the effect of starch reading, reaching the hindgut, what happens when we feed pre and probiotics, . The issue with the with the culture-based work is it has its limitations.

We can't culture everything and there are some microbiots that you just will not be able to culture. And so therefore the culture-based work is a great foundation. And it gave us quite a good starting point, but it has those limitations in that it, you can't call through everything.

Equally, having the, the fistulates gave us great access, but these days, that's not really seen as particularly ethically acceptable. I I there's one, group of, of can animals that I'm aware of in Europe, and that's it. And most people are now working with faecal samples.

Now, in the early 2000s and then beyond, we started with our, our early, . 16S studies, the 16S work really was the sort of the opening of the door of of a microbiome work, and. This starting with tanga sequencing and then moving onwards.

To, to, the work on the alumina platforms, for example, this was really the, the point where things that it was a game changer because all of a sudden, all we needed was to be able to take bacterial DNA, this, this 16S gene, and we'll have a little look at what that actually means in a second. And we didn't need to be able to culture anything. We could get an idea of the, the bacteria that were present.

But that actually left us with lots of unknowns, because we had a technology that all of a sudden meant that we could actually get a better idea of the microbiota, of that bacterial community profile in the horse's hindgut or in the faeces as a proxy for the hindgut. And I remember, probably around 2010, I, I remember having a conversation with my PhD supervisor, he was working on a project, he said, come and have a look at this, this is really interesting data. And it was really interesting data, but there were huge numbers of unknowns within the, within the data set.

And the reason for that is these types of studies, these, these microbiome studies are reliant on a database to be able to align those sequences to, to be able to tell us what those microbiota are or what they're most likely to be. And at that point, there were huge levels of unknowns because this was very new to the horse. And we're trying to align to a broad database, which would be used for predominantly human samples, but, but all sorts of, environmental samples, animal samples, and trying to align those sequences as best as possible.

And so that left us with, with quite a few gaps and quite a few unknowns. And then really, in the last 1015 years. There, there's been an explosion of the era of omics, and I, and I'm probably as guilty as anyone else and getting sort of quite carried away about, metabolomics and metataxonomics, and if I can find enough money, metagenomics.

But there are all of a sudden there are all of these various omics words appearing. And it's sort of, it's great. But it's having a bit of understanding about what it actually all means, because I, I'm quite an applied scientist really, in that I I like I really like doing all this work, but it's always about well how do we apply this back to managing horses to make their life better, how do we improve their gut health?

How do we improve horse welfare by understanding more with these various techniques. And so I think This is quite a useful sort of consideration in that the graph here we've got this sort of the axis showing genetics and environment, and our microbiome studies out on the top here are are sort of highly environmentally led and they give us a phenotype. So they, they're giving us a phenotypic profile.

There is, of course, there is a genetics element. But in actual fact, it it it's very much a sort of taking one gene, or if we're looking at 16S study, and then also considering the wider environment. The next layer in the other area that I tend to work in is the metabolo.

And the metabolo is are the metabolites present, and those metabolites might be microbial metabolites, so very close to the microbiome. They may well just be dietary metabolites or, or other metabolites in the body. And then as we keep going in through the layers, we sort of keep getting into greater layers of complexity, and they start becoming sort of quite intertwined.

So the proteome, the transcriptome, right all the way down to the genome. So a lot of the studies that have appeared and a lot of the studies we're talking about are are quite sort of out on the outer frame and they're quite phenotype driven, and I suppose that's quite an important consideration that we're we're looking at a phenotypic profile with a lot of these studies rather than the actual, genetics. The other thing that's been hugely driven by these types of studies is the approach.

We are taking a systems biology approach quite often, and there's a nice little quote here from the Institute for Systems Biology whereby we take the, the biology element, and I suppose historically that was the element that we ran with. And then we're taking the, the technology, and that could be the, the level of sequencing or whatever. But in actual fact, it's the computation element.

And as a biologist these days, I feel like I should really be a computer scientist, because there's quite a lot of time spent actually trying to mathematically model or use bioinformatics, the tools that we, that we need to be able to take the, the biological question. Use the technology, and then actually use, computation to be able to provide the data and then do the, the levels of statistics to be able to try and understand. And, and it's a cyclic process, this really, in that it often generates new questions, get new data.

We then generate a new hypothesis and we start the, the cycle again. And I think with this, we're also quite aware that, you know, for a long time, you know, our sort of gold standards have been animal work. We're doing in vivo studies.

We're doing more and more in in vitro studies, but actually, and there is some work, I suppose that's starting to go that way within animal science, is to actually model things in, in, in the land of the computer in silico. Yeah, I, I think it'd be a little while before we have huge amounts of equine work going. In that direction.

But we're doing more and more sort of, being able to understand and model and predict forward. And there's definitely been sort of computational models for parasitology trying to predict things like, the, the speed of anlytic resistance, occurring. Not specifically in horses.

That was a, a ruminant example. But, you know, I think we're, we are heading down that route as science progresses. And so it's sort of keeping up and trying to understand what, what does all this mean?

And actually, what does it mean on the ground? So when we talk about microbiome studies, a lot of the papers that you'll see, they will often be referring to 16S sequencing, and 16S sequencing is, it, it's correctly, the correct, the correct term for it is amplicon sequencing. And so what we do is we take a gene, the 16S gene.

So if this is our little bacteria here. It has a 16S gene and all bacteria have the 16S gene. And so we take this, this, this gene and we extract it from the bacteria.

We take the extracts and we use PCR amplification so we amplify up that 16S gene. We then take the PCR product, those copies, and we send that off for sequencing, and then we can then identify those bacteria in, in, in the longer term. How do we do that?

Well, there's a, a, a whole sort of selection of a computer science effectively that that plays a a role. One of the tricky things, and this is one of the first considerations when you start comparing studies, you say, well, actually I'm interested in looking at the effect of the microbiome in animals that are that are obese or the animals that suffer with laminitis. You know, I do laminitics have a different microbiome?

And you find, I, I'm, I'm sort of fictionally making some of this up now, and some of that work has been done, . You try and compare the studies, but actually one of the first things you need to know is which bit of the 16X gene did we look at? Cause studies typically don't look at the whole gene, they look at a hyper variable region of the gene.

So most studies will look at the V3 or the V4 hypervariable region. But there are studies where they decide to look at. V1, V2.

And so that's fine, and as long as it's clear in the paper, but it's just, it makes it more difficult when you're trying to draw conclusions and compare studies. So typically, in the 16S studies, we look at the, we, we, we basically amplify this, this specific region. It gets sequenced, and then we use this lovely bash language, which feels sort of very 1980s computers to me.

But, we work through a pipeline, and so, a really common pipeline is chime, or chime 2, which we take our raw sequences and we basically follow through a set of steps to stick our paired ends together. And to be able to classify the taxonomy, and to be able to analyse for diversity within and between samples, and to be able to, to do some statistics. And even statistical packages start getting a bit tricky.

They're, they're much better now than they were 5, even 10, definitely 10 years ago. Because this data is normally very skewed, doesn't follow any sort of normal distribution process. And so tools such as LeFy, for example, can be a really good way to be able to sort of robustly analyse the data and to plot out what can, what can we actually see here?

So, there are huge benefits to this. Amplicon sequencing means that we can identify bacteria that we can't culture because we don't need them to be alive. They can, you know, you know, dead bacteria, it's absolutely fine.

All we need is that little bit of DNA, and it's pretty quick. And these days it's quite cost effective. I mean, in the time that I've been doing these studies, the cost has, has fallen and fallen.

And it is really useful. It gives us, a bacterial community profile, which can be really useful, and it's definitely hugely advanced our knowledge of this, this black box of the hind gut. And if we look at some of the studies, especially some of the early work, I, I've just picked a few examples here, .

Work from the US, from Canada, and from the UK all looked to actually just trying to characterise the gut microbiome to start with. So in the horse, what is it, what is a typical gut microbiome? Is there a core?

What we know from that is the core is quite small. And there are lots of, of, of smaller groups around the edge, and it's quite horse driven, and we'll have a, a, a, a look at that shortly. Equally, at the same sort of time as just trying to get a handle on what's normal and what do we expect there, starting to become, well, well, what happens in a disease state?

What happens when horses colic? What happens if we try and model laminitis. What happens in horses suffering with equine graft sickness?

How does this affect the microbiome? And so, you know, these studies again, well, some of them earlier, and then as we were starting to get into a better understanding of this technology, started to, to give us a, a, a better insight. Some work and more since I'm sure, looking at the effect of, well, what happens when we give antimicrobials, what happens when we give proton pump inhibitors.

And, a few of us all got quite excited about, antimintics and what happens when we give different antimintics to the gut microbiome. And as I say, these studies have all provided. A wealth of information that we didn't have before, before the, the, the days of being able to very sort of easily sequence the, the, the faecal profile.

But we have to be aware of some of the, the limitations, especially to this amplicon sequencing, so to our 16S approach. Because there are various methods. So I already described that different studies might look at different areas of the, the hypervariable region of the gene.

And so that means that then studies are not directly comparable, . Now, to get the bacteria out of the faecal sample, for example, you need a kit. The early studies that I did, we used, and people within the group were using, a faecal stool kit that was really designed for humans, and that worked and still works.

But then others were using soil kits, and the faecal kits disappeared off the market, or you can only get it in huge numbers. The soil kit works absolutely fine. And somebody might have even done this by now, but we always talked about actually running a study by taking a faecal sample and using different extraction protocols, and then sequencing those samples and running the, the analysis pipeline, because you would like it would be likely that you would get differences.

In your end data set. The other thing are the pipelines. So I describe Chime 2, and I would say that Chime 2 is probably one of the most common pipelines for bioinformatics that's used for microbiome studies.

But, there are others. So, the group in Dijon use a platform called Frogs. There are other various platforms, they all do effectively the same thing, but the likelihood is you might get slight differences in the data that you get out of the other end.

So it, it does make it a little bit questionable about how reproducible it is. Now that shouldn't be impossible. Because one of the beauties of these types of studies is that people deposit their sequence their raw sequences in places like the European Nucleotide archive.

They, they're usually easy to be able to get the raw sequences, so you could quite simply take them. And run them through the different pipelines and actually see the, you know, the differences. Probably be a great student project if I could find the right student to, to, to do that, because it does start to throw out questions.

The other thing to To think about with Amplicon sequencing is most studies are 16S because that's 16S covers bacteria and some archaea. So we get some methanogens in there as well. But we don't get any fungi because they are the 18S gene.

We're not getting protozoas, so we're only getting a proportion of the gut microbiome. Another consideration is the fact that the 16S profile gives us structure. So it gives us the the structure of the bacterial community.

But what it doesn't tell us about is the function. And so my little analogy here is a bike with skir wheels anatomically is absolutely fine until you try and use it and it's the function bit that's often really important. And I think we have been a little bit guilty with some of the early studies of getting very excited when we see bacterial community profile changes, in horses, and we instantly think there's a change occurred, we've increased the risk for colic, for example, or we've reduced the risk for colic by doing X.

But if we don't measure function, and this, this approach doesn't directly measure function. Then in actual fact, there is a chance that what happens is the new bacteria that have up regulated and and taken over the space effectively, still do the same job. And I, I love this paper, it's getting a few years old now, and it's, it's focused on ruminants, and it's a very early, what we call shotgun sequencing that I'm gonna talk about in a second, that, that does give you functional pathways of what actually happens in the microbiome.

Showed that in, in two animals that had different bacterial community profiles, the pathways that the, the, of the, the, of what was happening in the microbiome was exactly the same. So they called the paper the players change, but the game remains the same. And I think that's a really important consideration.

We we have to . Understand more about function. So the structural limitations are, yeah, great, we know it's there, but we don't necessarily know what they do.

And some of those changes might lead to disease states and some of those changes might not lead to disease states, and we just need to understand more about that. We also have to have some understanding of course variation. And this is quite a problem in that this, graph here is from Tina Blackmore's work from about 2014.

And there were two, things occurring here, so there's a test and train, so they did an. Intervention in these 6 animals. And, and in an ideal world for these types of studies, your PCA plot would be, well, one corner would be the test element, one corner would be the, the train element.

But what we've actually got here are, is a variation between within each animal, but all the animals are different from each other. And scientifically that makes life very difficult. But it's a great example.

Of how you get individual animal variation, and this is really quite common in microbiome studies, and therefore makes life a little bit difficult. Now, there are ways to identify function or there are ways that we can initially predict and then measure function. So with the, the, the work using the 16S gene.

What we can do is we can use predictive algorithms. So there's, something called pie crust, which basically predicts from the 16S gene which path, which metabolic pathways, are likely to be linked. And there was a group who actually said, well, we want to make this more ruminant specific, so they created cowy, and that was hosted and believed still is hosted on a server at Aberystwyth.

Cow, pie crust had a, a, a, an update in, in 2020 and is now much better. It's still a prediction, but it gives you an idea as to what might be happening. So if you're seeing a bacterial community profile change, then, there may be a change in the microbiome in the way it functions, but it's not a, a, a definite yes it will.

Another option is to to actually look at the metabolone. And that gives us an idea of . What bacterial metabolites are being produced, and so if you have a change in the microbiome and a change in the metabollo at the same time, Then you might be able to put one on one together and potentially get two, but it's not concrete.

So metabolomics again helps us to understand a metabolic phenotype. Now a friend of mine, Ebony Escalona, when she was doing her PhD, their group then characterised what is the normal metabollo in thoroughbreds, in plasma, in faeces, in urine. And this is again being studied in normal and diseased states, and again it, it gives us a marker of function, but it doesn't give us true function.

Now finally, we have shotgun sequencing which I sort of briefly mentioned before and or or or true metagenomics. And, and this approach is much more fancy. It gives us the the structure that we really want, that we get the 16S, but it also, it instead of being one gene that the 16S gene is, it gives us every gene in the genome, which is quite complicated, but allows us to look at function.

The problem is, it's quite expensive. You're looking at about 1000 pounds a sample. So there aren't many studies that have taken this approach and where they are, they've worked on a few samples.

So I've got a couple of examples of that. So, Rachel Gilroy's work from last year, based on 5 horses, took a, a metagenomic sequencing approach, and constructed 123 high to medium quality metagenomes of bacteria in archaea. But it, what it actually left was a, a huge level of unknown, a little bit like the early, 16S sequencing in that, the databases aren't necessarily up to speed for horses.

And so what we're left with is a huge amount of unknown, so it's sort of one step forward, but almost a step back. And I had a look at at other studies. There have been a couple this year that have taken this approach, that the top one here that was looking at the effect of sacroyservicii on the microbiome during stress, but actually use this approach only for structure rather than actually looking at the the pathways within the microbiome.

One area that is becoming quite interesting, and, and I picked up three studies actually that are all sort of looking at a similar thing is trying to understand more about, the microbiome, the, the metagenome, and how that might link to athletic performance. And there's a couple of studies out there that've started to look at this and to also look at at the metabolo. And I know, some work from Chris Praman's group.

I don't think it's published yet, but it was some work, presented at a conference earlier this year, that's showing that you can actually use microbiome data to predict race placing, and also early life events from fos can actually look at er at disease risk going forward. And, a proportion of that, is, to be able to be traced through from, . From some of those early from a longitudinal study that they conducted.

But there are some issues here, working with faeces, has its limitations because it's only a proxy for the hindgut, you know, it's much more ethically acceptable, but they are it's not the same as gut content, and so we just have to consider that when we interpret the data. What does it actually all mean? So when we're trying to apply this to horses, I think we have to remember that you are what you eat.

And there are some nice studies that actually, you know, we know this across all species, but actually, Sheryl Salem's work from 2018 shows that, microbiota are, are influenced by the diet, so by seasonality, plant maturity, when we add conserved forage, and this is logical because the microbiome has to work differently. It gets more or less sugar, more or less protein, depending on these different states. And, and we've done some work within our group.

We've looked at various conserved forages, not how they act in the horse, but actually just the microbes on there, because the food gets in, but we know. That I, I, I actually microbes can get through that protective barrier in the stomach and therefore, what effect does this have? We know that that soaking hay has it increases the, the number of proteobacteria.

Therefore, potentially the, the level of lipopolysaccharides that are actually getting into, the horse's gastrointestinal tract. We don't yet know the effect of that. And clearly, it's so lots of people soak hay and it doesn't have massive effect, but it would be good to understand more.

Similarly, our work looking atantlemantics, which doesn't seem to have a huge effect when the horses are, are very low, Parasite burden, but, or groups of horses with low parasite burdens, but what we can see are some effects and what we can actually do is take a a a combined effect of understanding the, the microbiome, the metabolo in in a culture-based setting. So we did some in vitro work and some vivo work together. So, the microbiome and horse management, I think one of the things that's really important here is this is a research tool, OK?

What we don't really know at the moment is what is normal. And I think there's always a, a concern that we could be trying to correct to a norm when we don't really know what normal is yet. We know that the microbiome is really complex and.

We have to consider what good husbandry is, and if we're making, if horse owners are showing or performing good husbandry, then everything should be OK. And so it becomes difficult with client horses, and I think there are probably companies out there that offer to you know, provide an owner an idea of what the horse's gut microbiome looks like. But as I said, one of the big difficulties at the moment is we don't really know.

What normal looks like. We, there, there is lots of animal variation, and, we often looking at the structure, but we don't necessarily know that much about the function. So we need to think about what's the purpose of looking at an individual animal.

It's a great tool looking at broader, From a study perspective, trying to get a broader understanding of various states, diets, disease states, but I'm not sure yet for individual animals it's particularly useful. But we've come a huge way on our journey, and we understand so much more about this mystery black box than we did in the 90s. But I think that the work that's come out in the last sort of year or so actually shows that we're just at the tip of the iceberg, you know, and the more we uncover, the more we realise we don't actually understand at all.

So, sort of some conclusions really. Omics tools have definitely greatly advanced our understanding of the gut microbiome, and it's an excellent research tool. But currently, there should be, some, we should focus a little bit on on using it as a research tool.

There are lots. Of different platforms, animal variation, and lots of things that make interpreting this quite tricky. And I'm sure that studies will provide real novel insights into intestinal disease in horses, and in time, it could actually become a diagnostic tool.

But right now, I think really, most of our microbiome understanding is very much focused on the broader, bigger picture. Hopefully, I went through that a little quicker than I expected, that wasn't too much of a gallop, but I would be open to taking any questions. Thank you, Simon.

We haven't got any questions, come through. I'll just quickly double check, make sure we haven't got any more at the moment. And no, we haven't got any questions at the moment, but obviously, if we do have any come through later or anyone wants to email us, then we're obviously more than happy to pass that on.

Thank you again, Simon, for an informative session, and thank you again, Bailey's Has Feeds for sponsoring tonight's webinar. We hope you all enjoyed it, and we hope to speak to you soon. Thank you.

Thank you, everybody.