Good evening everyone and welcome to tonight's webinar, which has been kindly sponsored by Bailey's and my name is Sofia, and I'm from the University of Edinburgh. Before we get started with the webinar, just a few housekeeping things for those that aren't familiar with these webinars. If you've got any questions or any technical difficulties, then feel free to put them in the chat box, and somebody will be able to either address your technical difficulties, or if you've got any questions for the speaker, then I'll be able to ask them at the end of the talk.

OK, so, I'm really pleased to introduce tonight Doctor Simon Daniels. So Simon, started out as an animal scientist, and then spent time working in the feed industry before he moved back to academia. He worked for the University of Liverpool Vet School, on a non.

Exchange project, particularly looking at equine parasitology, and then when he completed his PhD on equine and film and tics, the efficacy and the effects on intestinal health. And this was with Professor Chris Praman, who's currently now at the University of Surrey veterinary school. So Simon has been in academia for the past 9 years, and as well as teaching, he is actively involved and very passionate about gut health research.

He's now currently a senior lecturer at the Royal Agricultural University, where he specialises in equine gastrointestinal health. So, without any further delay, over to you, Simon, talking about the black box of the horse. Thanks, Sophia.

Good evening, everyone. Yeah, so, you've gathered from my bio. Kind of there from Sophia, but I get quite excited about horse guts.

And I think there's, be it from the parasitology angle or the general function and the health of them, there's a huge amount there, which is kind of led to this idea of, of the black box. I'm kind of go into that a little bit as we, as we go through tonight's session. So we're gonna think a little bit tonight about the, gastro gastrointestinal tracts, anatomy and physiology, more on the physiology side of things and what we know.

Or we don't know, and even on just sort of relatively basic function, and thinking a little bit about the gut microbiome, and I suppose in, in, in the world we live in today, and especially in the, in the world of science, so the gut microbiome is huge, it's a very sort of, well, it's not new anymore. We we're looking at this for 10 years or so, but it's still a pretty sexy term out there, and this is. The area that everybody's kind of looking at, and we're now starting to think about the gut microbiome actually as a virtual organ itself and starting to just peel off the surface of, of some of maybe the, the systems this may link to and and other functions and, and this might, you know, in the future actually help us try and understand and answer some of these problems where we're still scratching our heads thinking, well, why does this happen?

And I think it's thinking a little bit about the, the hindgut as an ecosystem, about the components of it, and thinking about how it works a little bit more, and then how can we actually maximise gut health for horses? What can we do practically, because all the science. Is great, he says it's a scientist, but actually, quite often we then want to take this down to practical application.

What can our understanding and our continuing understanding of gut structure and function, how can we maximise gut health for horses by improving this, this knowledge? So, the, the horse's guts, a nicely, stripped down version here, but actually thinking about the black box or maybe maybe the black boxes. So even fairly recently when I thought about guts, I got relatively excited about the, the high.

Go in there it's nice, large anatomical or anatomical structures, huge volume, this massive great fermentation tank. And it really is a black box. Food goes in, energy and faeces come out, but we don't really fully understand how it functions and how it does all the things that it does.

So that was really my sort of, inspiration for this, this black box. We don't really know the workings of it. We just know what goes in and what goes out.

But then when you spend a bit of time looking at the four guts, so anything from the oesophagus all the way down to that ileocecal junction, you start to take things a little bit for granted. OK, well, we know the, we know the secretions when it comes to digestive physiology, we know the functions. But actually, how much of this do we know from the whole And when you start to delve into some of the work that's been done, quite a lot of the work is extrapolated.

So we may know exact enzyme concentrations and activity in pigs, and then quite often we borrow that idea across the horse, but it hasn't actually been looked at in detail, or some have, some haven't. Some bits have been done in in slightly isolated. So it's really, the more we look, I actually start to think, well maybe the fore gut also falls into a bit of a black box and then we put food in and we get energy out and it goes on down to the hindgut, but how much do we actually know about the workings and mechanisms of these structures?

So with regards to the for guide itself, and this is an area that we've been doing a little bit more work, . Recently, as I say, people have started to look at some secretions in the horse, but, it tends to be a little bit isolated. And until fairly recently, we never really accounted for a for microbiome.

Make this lovely simplified assumption that in our horse's stomach, where we have get down to a nice PH2 down in the pyloric region, that we wouldn't expect to find bacteria there, and OK, the pH increases a little bit higher, especially up in the in the cardiac region, but Are there bacteria there? What do they do? What is their role?

And this is something that we're starting to scratch the surface with, a little bit, but it needs maybe needs a little bit more consideration. And when it comes to the fore gut, we, we know with regards to certain disorders, we probably think about small intestines and colic and maybe, maybe parasites, and actually right up at the the forefront really is gastric ulceration. And, you know, it's an area that we are scientifically making some traction on to some extent, but we still don't really fully understand everything that's occurring and why.

And, and I think this is quite interesting in the, in the scientific literature because we can't always explain, but by looking back over the studies in the past that we have horses that may be kept in what we would. Believe would be ideal conditions. And yet they, for avoiding ulceration, and yet they still have ulcers.

So there are lots, there's a lot of work there to, to do to try and work out exactly what's going on. But we do know that, the horse is a trickle feeder, and it's designed to have food trickling through all the time, and maybe that plays, partly into the role of, of the function. On the gastric ulcer side, sort of thinking about our own little group at the REU.

There's I've got colleagues who work very much in in behaviour and in brain physiology. And with the, in, when we look at the, the works we general stereotypy, there's a a lot of, links in the literature to horses that that. Formal stereotypic biting, appearing to be, or believed that they have a stronger link to being prone to gastro pulses, but there seems to be little to no empirical evidence of this.

So we actually ran, a couple of studies or a study to see, just very simply, looking at the horse's stomach. Was it, was it possible that these horses, that display or stereotypies, did they have, altered, gastric anatomy? Was it possible that they could produce more, hydrochloric acid than non-crib biting horses?

And, the work was done with the cadavers, so it was a little bit by proxy. But actually, if we looked at the gastric content and measured the pH. Was it more acidic in the horses that had been crib fighters, compared to the non-crib fighting horses, and it might have been a huge surprise, but actually what we found is, yeah, there, there was no difference in the anatomy or physiology of these horses that display or stereotypic behaviours to non-cribbiing horses.

But it also gave us a bit of a handle to be able to compare, well, OK, if we look at numbers of gastrop producing cells through via, histology, how does this compare to other mammals? And it's probably not hugely surprising that actually, down in the yoric region, you know, it's actually pretty similar. So, With regards to the, the forega and anatomy and physiology, it's kind of an interesting inner area, and I think that probably shows that those working on, gastric ulcerations, there's definitely quite a lot more work to do, but especially those working in the behaviour world, maybe you need to consider that maybe there's not a, a, a direct link or not anatomically or physiologically with with these animals.

With regards to the for guts and the for gut secretions, this is an area that we have been working on a little bit recently. Because we've been trying to do a little bit more work in vitro and looking at how we could look at potential new feedstuffs and how we could, start to, to test this in a lab situation before we had to feed new horses. And then we realised that actually we don't have good data on for gut secretions because, as I mentioned before, er a proportion of that is actually extrapolated from pigs.

So it then becomes quite difficult to actually empirically say, well this this happens in the horse, and maybe some things are happening in the horse and we don't understand them because we don't actually understand the enzymatic activity. We know it's there, but we don't understand the activity or the concentration. So, some pilot work, that I was doing with a student over the last year, we were actually having a look at the Pepsin concentration, and activity, in the horse, which is quite interesting and we were looking at, Heps in concentration, using predominantly cadavers and some ex-clinical, samples from a, a local practise.

And actually, the, whether the stomachs were full or partful in the cadavers or from the, the fastest, stomachs from the horses that have been gastroscoped. There wasn't a huge gift. It didn't actually make a huge difference.

There was less, Pepsin concentration in the fasting horses, as we'd expect, but not significantly less. And actually, the values fell into, normal, normal mammalian range of. About 0.5 milligrammes per mal, which is, maybe as we'd expect, but it wasn't something that we actually knew.

So when we're sitting down trying to work out how do we create a model to try and do a little bit of this work in vitro, well, we didn't actually have a value to start with. And then that sort of starts to, lead to what is there anything in the horse that maybe is a little bit odd or interesting that we don't necessarily understand? And there's some, there's some interesting work.

We've basically set about characterising every, four guts, enzymatic secretion, which is, have been quite a challenge, but, quite good fun all at the same time, especially if you, if you like playing with guts. But looking back at the other work that's already been done, there's a study back from 2007 that identified that lipase was the most abundant. Pancreatic enzyme in the horse.

This is quite interesting, especially because this supports from the where we have been, or where we're at with our own work trying to look at, enzyme activity and, concentrations. We also seem to find a lot of lipase, . To some extent, this makes a bit of sense to previous work because we know back from the 1990s that horses can digest fat very efficiently and over an adaptation period, they can, it has a a glycogen sparing effect, you know, from, excess physiology type studies, using muscle biopsies that We can convert horses to predominantly fat metabolism and then save that that glycogen store for when they really, really need it.

And we also know from work in the early 2000s that we could feed horses a large, a really good proportion of their, Diet as fat, you know, up to 44%, and it seemed to have no negative effects if we if we feed them a high starch diet, which won't surprise many of us, it, you know, it does have a negative effect on diet and gut health. However, a high fat, Diet didn't seem to do so, but it left a bit of a question. And actually now trying to put these pieces together, it does appear that we can feed horses high fat because they appear to produce a lot of lipase.

It doesn't actually help with the head scratching point of horses are designed and have evolved to eat low quality forage, which doesn't have huge amounts of fat in it, so I'm, I've absolutely no idea why horses seem to produce so much lipase, but it does help us to understand that from . A dietary perspective, because I get very keen into the feeding side of gut health as well as the parasite side, then actually, we can use some of these energy sources, and, they, they appear to be, digested very effectively and don't appear to have a negative effect on, on gut health. So, it's, it just shows that as we start to try and unpeel these layers and the things that we think we understand pretty well.

Actually, the reality is probably we don't understand anywhere near as much as we thought we did. And we're, again, those with an interest in nutrition probably have some awareness already that we know that in the horse, ayela activity, and concentration tends to be relatively low, that they're not fantastic at, digesting starches intestine, hence why we often end up with starch finding its way down to, the hind gut. But it's quite interesting that actually it appears that we can, improve this through, supplementation, and this is some work that was, done by Chris Prawell back in 2015, and they were looking at, they actually looked at the hindgut and I sort of end up paying back to this when I talk a little bit about the hindgut, but they're actually looking at if they could try and feed a a supplement.

Designed to improve starch digestion, how would that influence the the effect on the hindgut, which as I say, I'll come to when I talk about the hindgut, but there are ways and means to try and improve this so we can maybe improve the, the, the activity. So it just shows that maybe we can't directly always be extrapolating from other mammals because actually maybe the horse has some areas that seems to have a lot of lipase. It doesn't seem to have much in the way of .

Of amylase. Similarly, quite interestingly, we know that the horse has gastric lipase from work back in the, the late 80s, that the gastric lipase is identified as present in the stomach. But we don't really know how much this is again something we've been working on characterising concentrations.

We also don't really know how important it is, how, how do we need to start fat digestion in the stomach? What is happening with regards to, to fat digestion in the stomach? And currently we don't have a particularly good answer to that, we just know that it is there.

And then we I mentioned before we got microbiome. And actually, there are a little succession of studies that have looked at this, or this area. If we go back to 2005, 1 of, my colleagues, Mary Wo Collier, who's a nutritionist, was looking at, using, from cadavers, stomach, An oculum to ferment feed in vitro, and successfully managed to model fermentation in an in vitro situation, showing that the bacteria that were in the stomach content of these cava stomachs was perfectly capable of fermenting feet.

And then we move forward to 2012 and and Perkins and others, identified that actually there is a diverse for gut microbiome. Now, there are a lot of sugar fermenting bacteria. So, laxilli, streptococcus, things that will rapidly ferment starch, but they are present in the horse's stomach.

But then that begs the question, this must lead to the production of volatile fatty acids. But are they useful? How much do they produce?

Are they absorbed, do they leave with the rest of the time and go into the small intestine? Are they absorbed somewhere? We actually have no idea.

We just, so we assume they're being produced, but we don't know if there are any value to the horse, do they play any role in anything else that goes in the stomach? We really just do not know. When we look at the the microbiota and the small intestine, by the time we got to sort of 2012, 2013, the, the use of these sequencing type studies, and, and it's another favourite area of mine, that the OIS area of being able to identify bacteria from profiling the the 16S gene, it gives us a way Being able to identify things that we can't actually culture.

And if we look at Kirsty Dougal's work, we can see, the, the profile of microbiosa in the ileum. And it's definitely much stronger in the ileum just before we then get to that, that hindgut area. And it's a slightly different profile again, to the different regions of the hindgut, but they're there and they're present.

So how important are they? What other roles do they link into? And so then when I'm thinking about behind the fore guts, the area that I would have originally put is less of a black box because it's relatively straightforward.

The food goes in, we know the enzymes that are there, we know how . Foetus digested. There have been a lot of studies looking at things like transit time and in the sort of the early 2000s, but actually, the reality is the full gut is still a bit of a black box because we don't know no microbial activity particularly well, or what it's doing.

Some of the enzymes that are present, we don't know or don't have good evidence of how much is there, what they're actually doing, how active they are, if they're different in the horse to to other mammals. So actually the fall guy itself is really a bit of a still remains a bit of a a a black box. So then we get to the hindgut, and I think the hindgut really is as it it's unreal sort of black box, but it is particularly interesting.

And I think it's about thinking about it really as an ecosystem. In the, in the hindgut, you know, we've got billions of. Fungi, bacteria, protozoa, all there, symbiotically helping the horse to by, digesting cellulose, because as we all know, mammals don't produce cellulose.

So they, that we tend to think of the, the hindgut as just a fermentation tank. And actually, there's, there's a lot more to it. But it's great probably to think a little bit or refresh our minds as to how it really works.

So we have all of these bacteria in there, and the predominantly they are the cellulosic population. So they are there degrading cellulose, pectin, and hemicellulose. And And then we have our, from bacter, our ruinococcus, there'll be some fungi and protozoan there as well, stria.

And as they're slowly degrading these plant cell walls and getting into the pectin, and degrading these structural components, that does release a little bit of sugar, the osaccharides. So they actually are fermented by Other groups of bacteria are non-cellulotic, and any other starch and sugar that is making it to the hindgut is fermented by these sacroletic fermenters. And both groups are cellulotic, and our, sacrolytic fermenters are both producing fermentation products.

And, and the three key ones that we that we all want are acetate, butyrate and propionate. Propionate especially because it feeds nicely into glucose pathway. Actually, a huge amount of the horse's glucose comes from high gut fermentation.

So. So, these are all really important outputs, and we get, we get some, obviously we get some gas as well. Spirokeets are particularly good at being able to, turn gas back into acetate.

We get, and then the methanogens do a little bit of work there, but we don'ts don't produce huge amounts of methane. The really interesting bit is where we've got the, the little red circle here. So if we take ourselves or cattle and we compare to the horse, there appear to be some differences.

So where we get some starch sugar, these os, lots of osaurs, but predominantly starch and sugar, lots more starch and sugar appearing in the hind guts. They, and the fermentation products, we can end up with lactate. We all know lactate is extremely acidic.

But if we look at one, group, the, the via species. Paineella are really useful in that they can convert lactates into propionate. Now in the horse.

The vanilla make up about 1% of the microbiome and from different studies and looking back at Christian Daley's work from back to 2012, you can identify that, in completely grass kept horses, virtually undetectable, it would, in, in the microbiomeerella. Now with ourselves and in cattle. And and our ruminants, in ruins in general, as you increase the starch content of the diet, the numbers of failings seem to increase, so they have there's a bit more capacity to mop up lactate.

And we haven't found this in horses. So it's almost like they don't have this innate capability to be able to mop up lactate, and this may be a reason why the horse's hindgut is so sensitive, because if we get more starch and sugar there, and therefore we produce. Producing more lactate, they have, less ability to be able to mop this up as effectively as we do, and ruminants do, and therefore, are more likely to, to lead to hyacidosis, because it can't, we can't mop up the lac lactate and do anything useful with it.

So, I just think that's really, that's something that's really quite interesting and maybe helps us to get a little bit of a, a handle on why the horse's hindgut is so sensitive. We can also see on the other side of the screen, some more of Kirsty Dougal's work, looking at this characterization of different regions of the horse's gut. So we've got ilium secum, right and left ventral colons, left and right dorsal colons, small colon, and faeces, which is really quite interesting.

So we can see the profile in small intestine definitely differs to the other areas, but there is, there is a difference. So, in the secum, definitely slightly different to, to the large colon. And actually, that differs slightly again, especially as we get down into small colon.

And faeces, and, and those of us that are often doing sort of gut health and nutrition studies and looking at, sequencing as a proxy for the high microbiome in a non-invasive measure, we're using faeces and we know that it's not a perfect representation, but it gives us an an approximate idea of of what is going on there. But it's, it is definitely variable, and we can see that we've got, you know, bacteria that are more focused on, fermenting fibre, and they tend to find the seco, and then actually we tend to find a few more that are, a little bit more tuned in to a little bit more sugar, and they tend to be more further through into, into the large colon. But this is still again a huge area of our discovery and understanding, and I remember, looking at some data with Chris Pram back in about 2013, and actually there were huge, just amounts of unknown sequences because we, we didn't know what these bacteria were.

We're getting more of a handle of what is actually present in the horse's gut, but it's still, a lot of it is just a, a big unknown. And if we continue on with Kirsty's work, Kirsty was actually looking to, identify, is there a core community in the horse's hind gut? And what their, their conclusions, their group's conclusion was actually.

The core community is made up of many low abundance operational taxonomics units. And just so operational taxonomic units are basically not clusters of sequences that are 97% similar or greater, so we can therefore classify them or identify them as certain groups of bacteria. So, in the, the figure at the top, the light blue bars are not the core.

The core is identified, and this is from their their study in the ilium, the secum, and then moving through the hindgut right down to the faeces of these tiny little bars at the top. So it, it would appear that the horse has a very small core community. And maybe that's also why it can, it's not always particularly stable.

It also seems to be very diet driven. And if we look at Tina Blackmore's work, this particular study looked at, feeding horses and looking at their, their microbiome and looking at the same horse. Over time, so sampling over different time points.

And this is a perfect example in that the the graph is maybe not particularly clear, but the, you can see that the ponies represented 6 ponies are represented by colours and then there are different time points. What they tended to find was pony 1 would be similar, the different sampling time points would be all similar to each other, but pony 1 might not be anything like pony 5. The pony 5 time points are all similar.

So there with these types of studies, there's actually a lot of horse variability. Which can actually make these really interesting studies quite challenging. And they tend to, they need to be intervention type, experiments for us to get useful information, because we need to look at what was there originally and then how that animal changes over time.

Because actually, There's often quite a lot of animal noise. So that just shows us that each animal's core might then be slightly different because it's made up of these different, small or small numbers of these low abundance OTUs. So it's a really complicated system.

And we've got a sort of a rough handle of what's going on in there. But again, it's, it's hugely variable. And I couldn't talk about, my favourite area without actually bringing in a little bit of parasitology.

And I think this is where, when the last slide looked complicated, because we're trying to get a good handle on what's there and still trying to then link into that, what the function is, we then start to think a little bit about, what else is part of the microbiome? And I think it's been very easy through, past, antrolytic, programmes from the 1960s onwards that were, you know, very heavily focused on treatment, to, to think that, you know, worms equal colic, or the horse owner definitely. Worms, colic.

And that's been a, you know, obviously a huge me there's a lot of work and I'm trying to, to identify that. And we probably forget a little bit that actually, worms or macrobiota actually evolved to live in the gastrointestinal tract. So we have microbiota and macrobiota living together.

Now, over the last few years, we have not just identified the gut microbiome, but the nema biome, and definitely some work, over in Calgary, Ryan Amenko's work looking in cattle at the nema biome and using the nemabiome as a way to be able to identify a tic resistance. So, That's been a a a a a a a a big eye opener, but actually we now realise mammals as well, that the nema biome can interact with the microbiome. So there's this huge area of complexity in within inside our black box.

And that actually there are the bacteria inside the worms are communicating with the bacteria inside the gut. And maybe that starts to help us understand a little bit maybe the survival mechanisms behind some of these parasites, maybe some of the unknown areas of their, their life cycle. And, and this is sort of really quite interesting because when I was doing my PhD, we were looking at, trying to identify that while alimatics are really important for parasite control, there's equally a risk factor for colic following.

Implemented treatment, especially microcytic lactates. So we were having a look at the effects on, microbiota functional fermentation using in vitro experimentation in horses that had been given alemente. Looking at the bacterial community profile of faecal samples as a proxy for hindgut, structure, and also looking at the metabolites being produced.

And at the same time, as always in science, there are a couple of other groups doing some similar work, and we've managed to all compliment each other, which, is always nice. So Laura Peachy's work was really interesting in that Laura identified. That horses with 100 eggs per gramme or greater of Saxonians had a different gut microbiota composition compared to horses that had less than 10 eggs per gramme.

So the macrobiota appeared to influence the microbiome. Some more recent work, Nicky Walsh's work, is looking at, changes in gut microbiota following an antimantic administration. They were really also looking at, inflammatory markers to see what Was there a link between, especially treating horses with either, 5 differentbendazole or moxidectin, for insisted tachistones, was there a link there to, to gastrointestinal disease?

And again, had some slightly interesting findings that they, what they couldn't identify was, was the effect that they were, they were picking up post-treatment linked to the antromantic. Or was it linked to the effect on the parasite because they had a group of horses that were all identified with insisted South Estonians using Jackie Matthew's Eliza. Now, our study, we had horses with very low, parasite, load, so they were mature animals that had, no egg faecal counts over a period of time.

And so what we found was that there was, while there, these horses did have a bit of a parasite, they would have had some burden, and one of them did excrete some earth stones following treatment. Our, the only changes we saw were a few days following treatment, which appeared to be potentially linked to some insisted burden actually working its way into the gut lumen. So it looks from this collection of studies that actually our focus needs to be on the effects of parasite death on the microbiome and maybe that link, that's our, our link to colic.

So it it's an area again that's continually growing our understanding, but it still to some extent leaves us scratching our heads a little bit about, well, what does this all mean and, and how does this all come together? And I think It it we then start to look at what else do we know about higher microbated change. And actually a really nice study that came out of Liverpool in 2018 from Shevl Salem and others, which it followed a group of mares that were out at pasture over a season using again, bacterial community profiling.

Predominantly as a way to, look at the, the structure of the higher microbiome from, from proxy. And this is really interesting work in that probably, again, doesn't maybe don't seem hugely surprising, but the structure of, or the composition of those bacterial communities were influenced by the season. They were influenced by weather.

They were influenced by the grass growth. And in the winter, when the horses were really low on grass and they were supplemented with some forest, this this particular horses were supplemented with haulage, then they sort of changed in that composition again. Which really shows the strong link to diet, and again, we appear to have quite a small core.

So it fluctuates and it changes. And actually the very simple things of managing horses of changing diets very gradually. Is linked to this idea of, well, actually, that, you know, you, you, you are what you eat and your gut microbiome is hugely influenced by, the food that is going in, and, and, especially in the horse, where it doesn't cope well with change, you know, there's a definitely a really strong link there.

And again, as I mentioned before, Chris's work. Using a supplement to increase the amyolate activity in the small intestine influenced the hindgut microbiota with the probability then that actually that these horses that had had, and these were therapist in training, so they had had would more likely had Starch finding its way to the hindgut, which would alter the microwave's composition because we don't have the ability to mop up particularly to lactate, and we alter that, that hindgut population. Well, if we can actually prevent that and actually get the, the for the fore gut working more effectively, then the hindgut can become a little bit more optimised.

So there's a really strong link, and but it would seem unsurprising, but definitely to me because my whole life revolves around horse guts. But, you know, actually, if we can optimise some of these areas, then the gut can work as it's designed. Similarly, if we're thinking about guts, we definitely can't leave, disease out there.

And we're starting to look at more and more studies, and I've just picked a couple of little examples here, you know, and there are a lot more studies out there looking at this, but, a friend of mine, Joy Lang, who's down at the University of Surrey, was looking, doing some work looking at echo grass sickness, looking at the, bacterial composition from the faecal samples of, of horses with, echogras sickness compared to, case controls, and they were also looking at the metabolone. So they were looking, when I talk about metabollo, I've mentioned this a couple of times, we're thinking about the Bacterial metabolites. So if we alter the way these bacteria are behaving, does it alter their, their metabolite output, their, their metabolic phenotype?

Does that change under certain conditions? And it's, it's really interesting, and I'm probably gonna go off on a tangent now, but actually, we've spent a lot of time over the last nearly 10 years, getting very excited about gut microbiota. And actually, definitely in other species, again ruminants, we get hugely excited about changes.

We do something and gut microbiota change. But just because the structure changes, that doesn't necessarily mean that the function changes. And there was a really nice study using a a next, a full, shotgun sequencing approach which allowed them to infer the the metabolomic side, against the bacterial structure in some cattle.

And actually what's happening. Was the bacteria profile appeared to change, but the their metabolic output didn't, didn't change. So while some of the bacteria were changing, actually the metabolites being put out remained the same.

And if anybody's particularly interested in this study, it's by Taxus and others from 2015. The paper's got a great title, it's called the Players Change, but the Game remains the same, so it's not one that you'll forget in a hurry. But it it it's trying to understand by using multiomics approaches, what is the structure and what is the function.

Some more recent work over the last couple of years has actually been looking at obesity in horses and probably not hugely surprising that actually obese animals have a a a different function in their in their gut microbiome. And again, the, the laminitis, again, probably not hugely surprising and we think of laminitis, especially with starting with maybe behind acidosis. And this is quite interesting because not only can we see the trigger factors of if we wanted to, Amy Biddle's work in this particular study site, they're actually models, inducing laminitis in an in vitro situation.

So using faecal samples and putting starch, in and altering that, that bacterial composition. And these laminitic ponies also seem to have altered bacterial activity and some work by Katherine Hale identified that actually if you fermenter forage with er using faecal samples. From, ponies that have had laminitis compared to non-laminitics, then actually, there are differences in the, in the function.

You get differences in the, the fermentation of vitro, which makes you think that something must be occurring in the, in the high duct microbiome, even when they're not actually display laminitis, they have laminitis, but they, they've had lainitis. Something about the, the metabolism in the hindgut is a little bit different. So again, I, I'm just all I seem to be doing this evening is giving you more and more questions and maybe not really giving you any answers.

So this is the, the true academic in me here, but what it makes us think is we tend to think. Of the horse's gut, the hindgut is just a hyper fermentation tank, but actually that hindgut microbiome is so much more than just fibre fermentation. And so probably one of the, the newer elements actually is this idea of the microbiome as or the gut microbiome as a virtual organ.

Now we're actually thinking about this now as a as a really as just that, a virtual organ. And it's actually having a much better understanding of the gut brain access. So, I've borrowed this from personal communication, this slide here from, Andy Burnett, who, works at the University of Oxford, and Andy works with, humans.

And also in his area of specialism is schizophrenia, and he's also working a lot in rodent models. And they've been really focusing on this link to the gut brain access, and how the gut brain, how the effect from the gut can influence behaviour, can influence mood. And actually it's, it's, it's quite exciting in that our gut bacteria and the outputs from our gut bacteria, they can have an influence and they travel up to the brain.

Similarly, if we, if we swap this round, in a stressful situation, . In producing stress, we produce glucocorticoids, we end up with a, a, a leaky gut. These bacteria from that are when we're stressed, leak out in from the gut, and they, activate and work we end up with a pro-inflammatory cytokine response, which then affects back on the brain and we have a a a negative effect and this seems to influence behaviour.

Now, with with a good healthy gut microbiome, actually a lot of their work in rodents have started to try and translate this into humans, and that it starts getting a bit more difficult is actually with using prebiotics, and they've done some work with probiotics. Predominantly prebiotics, this leads to production of neurotransmitters, and actually, we end up inhibiting these pro-inflammatory cytokines and where they can cross the blood brain barrier and we end up altering the mood. And you might think, OK, well, how does this thing translate to horses, and there have now been a couple of studies.

again, very recently in this last year, and, specifically, Louise Bourne's work, actually looking at, the effects of starch feeding and, ponies fed starch. It alters their dopamine, and one of my colleagues has been working on a, non-invasive marker for, dopamine, which is inferred by, blink rate. And found what Louise's work showed was actually that starch-fed ponies has increased dopamine markers and were more hyperreactive than ponies fed on a fiber-based diet.

Which again is inferring this link to the gut brain axis. And, and we could continue because we that we now have an understanding that actually our gut microbial and this virtual organ has therefore a a a bigger influence on our immune system. So keeping the gut healthy, let's think about maybe some some practical application, because all I seem to, all I feel like sitting here, I've given you this evening are lots and lots of questions, but actually there are some practical things that we can do and we can keep doing.

So horses are designed to run on forage and they like consistency. So actually the message to give out to clients really is. Still, that we want to try and keep horses on a forage-based diet.

And we, especially in the nutrition world, are getting much better at providing in creating diets that are more forage-based, that are lower starchs, that are higher fat, so that we can get the energy levels we need with lower starch. Because we also know that starch alters the composition, alters the behaviour. And that link to the gut brain axis.

And we need to do it consistently. There's the simple pain rule, changing the diet over, 7 to 10 days, you know, is important because it gives our microbiota, a, a, a, a chance to change to this new feed stuff. And we know from work done back in 2002, that the, the longer you, the slower you make the change, the lower the risk factor for colic.

We can see that fat is really digestible. Other good things about fats, things like omega 3 fatty acids, they work on, anti-inflammatory pathways, which is great. They somehow play into our gut brain axis.

I have no idea. But that's, but definitely fat is useful, and, and, especially our nice omega 3 fats, and prebiotics. In other mammals seem to support, we, we have an idea that we can support gut microbiota.

We don't know in horses well yet if this can have any effect on behaviour. There's huge amounts of work to be done here, but they definitely seem to play a role, and they, and these prebiotics are important for feeding these, the gut microbiome to make sure that it stays nice and healthy. So our black box, the reality of the situation is I don't think I'm going to in my career lifetime run out of using the term, the black box in that the horse's gut remains a black box, but we're definitely getting closer to understanding more about normal for gut function and The hindgut really is a gift that keeps on giving.

It's still a black box, but we have, we've moved in the last 10 years, a huge, huge step in being able to have a greater understanding of this bacterial ecosystem and all these microbiota and macrobiota and how all these things are are are linking in together. Just pop that one in there that have no financial, or non-financial relationships with any of the products or services in the presentation. And at this point, if anybody has any questions, I'm more than happily go answering those for you.

That's great. Thank you very much, Simon. Really, really fascinating, talk.

Has anybody looked at donkey microbiota looking at those sitting there? Now that is a good question, and I, I actually, I've been very naughty here because again this is one of my colleagues, Professor Mary Moore Collier, this is one of her, her pictures, but it's a great . It is a great question answer.

And actually, yes, there is somebody, who was based over at Barkan, who was actually working with a donkey sanctuary, and they were looking specifically at donkey diets, and actually their angle was actually looking more at fungi and and looking at how fungi, the role that fungi play, which is something that we've tend to stay away with We've all been looking at bacteria, almost forgetting about everything else, but there has been some work looking at because again, we usually say, oh well, it's an award, and we treat it the same as a horse when actually we know it's really not the case. No, they can be quite different, can't they? Oh, definitely.

Yeah. So, so just a couple of questions. So, one kind of the altered microbiome in disease states.

I mean, how much, how convinced, how convincing is it that the alter microbiome might be, is it a cause or is it actually effect? So are these animals predisposed because they have an altered microbiome or is the microbiome altered because they have a disease? From the studies that have been done, it, it all points to that it's the disease that alters the microbiome.

It's something linked to disease that actually, and actually, that joys work with the car sickness stuff actually if we think about the fact that, you know, this is, we're looking at a neurotoxin here that's shutting down the gut, actually it's not almost not hugely surprising that then it's altering that bacterial composition. The gut's not functioning normally, so therefore, it, it's altered. So it, it would appear with more in the disease states that it, it, it's, a, a cause rather than an effect, but it would be interesting to, yeah, really interesting to delve a bit further with the sort of the obese and the laminitic animals to we we again.

There are groups Karon Argo's group in, Scotland are definitely sort of been working on this area with obesity and and laminitis, but to have more of a handle on why maybe these laminitics appear to have a slightly altered, activity. And again, I think it comes down to this more, it's more about actually what the the microbiome is doing rather than its structure. Yeah, OK.

And So I guess moving on, I suppose the probiotics and things, I guess if we are trying to alter it, then well, I'm assuming we we now have a reasonable understanding of what it should be. Do, would you say that? So?

Well, sort of, but not. This is where it's quite tricky because. Again, because there's so much horse variability, and we tend to get excited and think, oh, well, let's look at this disease state and see what it does to the microbiome, but actually we still are trying to get a good handle on, because everything is so diet influenced, what is normal, what is the core, we've got a rough idea, but definitely it would appear that Prebiotics seem to, especially in, in other mammals, seem to, have an and it's more this sort of gut brain axis idea of actually the sort of the, effect on the mood and the effect on, on everything else that that where this is really sort of kicking in.

But obviously, we've been trying for a long time to look at using things like pre and probiotics, and could that, influence, effects of fe starch and that type of thing. Yeah. So, so actually this into horse variation.

So there's studies where they, they showed that, there were so many, there was quite a large variation between individuals. I, I'm assuming they, were they all on the same diet for that study or? Yes.

Yes, yeah. OK. Yeah.

And this is where it gets a bit, a bit tricky in that you can sometimes, I mean, that, that was a real sort of good good example. And, and often you find maybe they're not that far apart, but they, they definitely sort of Cluster to each other. So, with my, my, intervention study where we gave the horses and Mick and and moved them over, actually, and, and looked at the gut microbiome, actually all their even their metabolo, you could see that they clustered.

So the, the metabolic profile of the horses stayed over time, stayed similar to, their previous, results, but not necessarily similar to the horse that they're also sharing the same grazing with. Yeah. So great, well, yeah, really fascinating area.

that that's all the questions I think I'll stop there before we. Go down many avenues. But thanks again, for a really, really interesting talk.

We'd like to thank Bailey's, again for sponsoring this webinar, and we'll hopefully, have you back on a webinar soon and hopefully, we'll, have some work on webinars in the next few months as well. OK, bye, everyone.