[Music] okay I hope I'm at the right conference because I will actually speak about chickens so I'm the first one so what I will try to do is just give you some kind of an overview of a lot of studies that have been going on on butyrate and dodging us and exodus butyrate and intestinal health in chickens so normally I don't start with pictures of chicken meat and so on but this time I do just to tell you that actually well what people are willing these days of course is well safe products consumers they really want safe products Salmonella free Campylobacter free they want to eat happy chickens well animal welfare is becoming much more important they do not want residues they want less antibiotic usage in animals and so on so there's a lot to do these days about sustainable food and production of animals what you see here for example this is just a picture of chicken breast of the US market and you can read on the label there no antibiotics you mainly raised cage-free so on of course cage free because it's broiler but still there's a lot of things going on on sustainability and you know well probably I've been listening to this kind of talks quite a lot these are the three pillars of sustainability social sustainability which is clearly about how people look into meat consumption it's also about welfare antimicrobial resistance and so on then you have of course the environmental sustainability and luckily chickens have a low carbon footprint at the end but still and of course also yeah then you have the economical sustainability and of course this is getting more and more important and actually when you try to raise chickens broiler chickens for example we of course go for optimal animal performance and this is somehow sometimes conflicting with some of the other sustainability pillars because by producing animals with a very fast grow tree you actually make them much more prone to diseases and this leads to welfare issues and to antimicrobial usage and so on so this a little bit of conflict sometimes there so one of the key questions or challenges is of course how we can improve animal health animal welfare without affecting animal performance I mean it's fast growing animals that are healthy as well that's basically the main challenge for the future of course well the first thing is safe products I'm just keeping going to give you a few examples of how butyrate is actually being involved in someone our control these are actually some kind of older studies we have been publishing already it's already close to 14 years ago but still just showing you something that well at least for the working on butyrate and chickens this is all how it started so at those days in the early 2000s about a lot of issues of course it's someone a lie it's still the case but at that moment it was a huge pandemic and actually someone eliza bacterium that is residing in the seeker it is always in the hindgut and this is of course the place where fermentation of sugars is also occurring and at a certain moment we well-coated we used good products with different assets in vivo trials formic acid acetate propionate and butyrate and we could see that if you deliver butyrate to the hindgut of the animals to the seeker of the animals that actually that has quite a effect on someone a lot colonization the more beauty rate the higher the puter rate levels in the seeker the less Salmonella you had at that organ and this is the place where some analysis I mean so it's actually quite important what you see here this is just data from an infection trial okay you just inoculate animals individually with high doses of Salmonella and this is the titers in law colony forming units so that means a control population with about ten thousand two hundred thousand bacteria program and if you add butyrate you see that it goes down with about 100 fold - 1000 fold in colonization level so this is quite something this is Salmonella shedding butyrate supplemented group and a control group so actually again showing you the same effect the reason why this is the case is because it's butadiene oh it's produced by a lot of bacteria but it's for pathogens it's very often some kind of a signal to reduce virulence we don't know actually how that really specifically is working but some pathogens sends Beauty rate even at low concentrations and then they the pathogens reduce villains and it's a very specific action if you just take a test tube and you have butyrate added to the test tube and Salmonella you will see that only the virulence genes are actually down regulated are not of global metabolism so it's not really anti bacterial but it's switches down bacterial virulence what you see here someone Allah is an invasive pathogen meaning it goes into epithelial cells and then just goes to the bloodstream okay and this process of invasion is very critical if salmonella is not invading epithelial cells it will not colonize so it's a very important aspect of the pathogenesis and that's what you can see here if you have a normal Salmonella strain and you will knock elate a bird you see that this is the colonization pattern even after 30 days you still have high concentrations of Salmonella if you use a non-invasive strain knocked out in a certain villainous gene you see that this is not colonizing anymore I'm just telling you that this process of intestinal epithelial cell invasion is crucial for the pathogenesis of a colonization and this is actually what butyrate is doing it specifically down regulates villain's gene expression and it specifically down regulates invasion gene expression so that is how this system is working you have similar effects and Campylobacter you have also have other pathogens that react by reducing virulence and toxin production for not going to focus too much on this but this is actually a cell culture as say showing you that if you pretreat some another bacteria which propionate or butyrate that you have much less bacteria inside the epithelial cells and for acetate you have more or less the opposite effect so it's all about ratios between fermentation assets if you have lot of acetate and almost no Beauty rate propionate you will have some nelec colonization going up if you have a lot of propionate and butyrate you will see that colonization is going down and also invasiveness is going down so this is just a small story on how we started working on now butyrate because of course at that moment we just used well a lot of companies start using co2 butyrate products but of course a lot of bacteria as well produce beauty rates and we started gaining interest into the potential of butyrate and the potential of adding products to steer towards beauty rate fermentation so as I just mentioned also animal welfare less antibiotics is of course a key issue for future poultry production and in fact this is not always easy because of course that means that well you have a lot of intestinal pathologies and intestinal syndromes and diseases in these fast growing animals and if you want to work with less antibiotics we will need to preserve of prevent diseases and this is what I will be focusing on in the next part of the the slides just how can we promote health in the animals and of course I will mainly go into detail on Beauty rate production you see a lot of pictures on what I call production diseases ok diseases that are really only occurring in animals that are produced in a way that brokers are being produced ok you will not have a lot of these issues in your backyard chicken simply because they don't grow so fast and what you see here well there quite a lot you see frogs don't provide striping which is a myopathy due to hypoxia to fast growth and even the capillaries of blood vessels can't even reach all tissue anymore so you get some some cell damage also pathologies such as femoral head necrosis or other Kandra necrosis osteomyelitis and this is also something skeletal disease that have to do with fast growth definitely and if you isolate bacteria from this kind of lesions these are gut bacteria that you isolate you isolate ecoli enterococci and so on so this is also something that has to do with or intestinal integrity and then of course the real intestinal pathologies coccidiosis I will not focus to it too much today Clostridium perfringens a necrotic enteritis and a lot of other enteritis syndromes but important is that most of the antibiotics used in poultry practice in broiler practice is for to treat intestinal problems 60 to 70 percent is for intestinal pathologies just focusing bit on aquatic and to write this you know well this is the typical picture you see this focal ulcerations sometimes a little bit more and this is caused by castilian perfringens okay this is a toxin producing bacterium that is suddenly massively multiplicative in the small intestinal tract producing toxins and causing gut necrosis and again we have done a lot of studies on trying to figure out how we can use alternatives to antibiotics for this specific pathology and actually also if you use an experimental model in which you try to induce this type of lesions you also see that coated butyrate products are actually very effective what you see here this is a control population in which about 50% of the animals developed necrosis when you start adding butyrate at different concentrations you actually see that numbers of animals that have necrosis in the gut or actually much lower okay so as I just mentioned the crud can - right this is affect well it's an old massive multiplication of castilian perfringens in the small intestine and this is effect the most severe well case of what we call these biosis and this videos is something that's often used and misused in the field I think as well if animals are performing not well a lot of people say okay they have these back to uses whatever it may mean so but co-sleeper fringes is definitely a dysbiosis because if you sample the god of these animals you only have Clostridium there but more important I think is all these types of intestinal syndromes that are like affecting performance which you if you take samples of the intestine you see some kind of inflammation okay and in problems it's very it's very often the case can be due to whatever mycotoxins may be sometimes a little bit of coccidiosis a lot of viruses I don't know viruses real viruses may be diet composition or whatever but then what you see is quite often this congestion of blood vessels so that points to inflammation and what you see here is in fact just astrology on an intestinal sample and all the brown dots here these are lymphocytes these are lymphocytes that immune cells that have been infiltrating the gut wall so this is inflammation you see the normal slender long villi here you see the thick blunted have used villi and all the inflammatory cells and this is if you take samples from different flocks you see this kind of excess of information quite often a few months ago we took a lot of samples and different water flux in the field and we just gave these samples to the pathologists and they thought these were diseased animals because this massive inflammation of yours very frequently in broiler flocks now focusing a bit on this and to write this this what I just said this inflammation you will get and I will the main issue there is epithelial permeability because inflammation always means that there's some kind of defect if you have some kind of defect there that means that some antigens will cross that barrier make contact with some receptors and start an inflammatory cascade so epithelial integrity is the key issue to avoid inflammation and inflammation is linked to performance defin they cost a lot of energy for these animals that they have to spend and cannot use to wane gain weight so just well if you have damage to any whatever the trigger is it's a virus it's a micro toxin it's a coastal toxin it's even subclinical coccidiosis normally a host can cope with that okay if you have a little bit of gut damage or epithelial cells damaged normally it is repaired very fast normally it's not a problem at all yes well you have many different strategies to maintain this epithelial integrity cells can control if rate very fast even small gaps can be sealed by just cells that connect again no problem at all if the damage is not too too severe tight junctions are very important also in this regard and also of course a lot of anti inflammatory responses can be important and if you look at this slide you will see that what you see here and this is something that was in the first talk of FA I think already in the beginning they are indeed if you look at increases of cell proliferation of cell differentiation as well and inflammatory responses you know that butyrate is actually doing this so beautyrx it seems to be a very important molecule also in maintaining intestinal health because it is very well it's very strong in maintaining well in helping to maintain and restore in epithelial integrity I just give you just one some data of a vitro experiment just to show you actually what Beauty rate can do this is just cell culture system this is a transverse system which you actually grow epithelial cells just on in the growth medium and you can measure electrical fluxes through these cells if you have cell damage the electric flux is much higher that's basically what we do and this well it's just a very tightly sealed epithelial cell layer this is just an in vitro system in a cell culture with all the cells that really connect with each other using these tight junctions as you can see here okay you can damage this cell layer by different means even by adding toxins or whatever but you can also just add some cytokines so you damage this layer and you will see that you have reduced resistance electrical resistance so the permeability is actually increasing that's what basically it is is saying if we add these cytokines but if you at the same moment also add butyrate you see that actually this integrity is completely maintained so this is one of the most powerful chemical molecules in this kind of in vitro model systems so it's it's it's really very affecting in preventing this integrity losses and that's something you can very easily show in vitro this is actually published this this was a gut paper of a few years ago that we have been publishing this kind of work the same with metabolites produced by a lot of these beauty raid producers and also quite a lot of other metabolites in general that have this kind of effect you can also measure well cytokine expression so this is il8 which is really one of the first cytokines produced by epithelial cells to attract granulocytes so immune cells and actually you can do exactly the same story here this is the basal level of il8 secretion in normal epithelial cells you can again induce it look at the scale it's going up to 1,000 times whatever but if you add butyrate in the same medium you also see that it's not going up that high so butylated strongly anti inflammatory that's what I want to point out here it helps to maintain epithelial integrity it is very strong anti inflammatory component you can show that in vitro but also in vivo it's something that is very well documented so beautyrx 8 is doing these three things it also helps to work for cell differentiation as well so this is a quite important to note this is I already have mentioned this I'm just want to explain a bit about and it's good that beta I gave a lot of introduction already on microbiota because it has been very well documented in in mouse models and in humans what beneficial microbes are to some extent of course for chickens and some people have raised the question already or the microbes the same in chickens and in humans basically this you find more or less the same gene around species of course it will be other strains but you find very similar patterns there and what we have been doing is just looking into inflammation models in chickens and then figuring out how the microbiota composition looks like okay and this isn't and well this is a model this just a model ok we have been using in which we just give some kind of poorly digested diet we give a bacterial cocktail after we have been depleting the market or biota some coccidia and damages to sampling and microbiota composition analysis it's just a model you can use any kind of model you can use a coccidiosis model a diet induced inflammation not less wound it's just an example okay but what you see and this is what we want to achieve in these kind of models is of course bodyweight depletion that is the animals grow less and also they have some lesions coccidiosis lesions but also some microscopical but a gut wall appearance that doesn't look completely normal if you look at histology in this kind of models you see of course Phylis length decreases and it's quite well it's 25 percent decrease in venous length and you see a lot of immune cells that infiltrate the gut wall you see that here this is a gut wall that is of these cherish conditions that is really full of immune cells so this is just an example of an inflammation model in broilers and what I want to come to is if you look at microbial composition here kind of models you see that actually also butyrate producers are often depleted and it can be because of the inflammation because the information also yields a lot of well oxygen radicals and so on that maybe also ensures that this strictly anaerobic bacteria are depleted somehow close to the epithelial cells so in the mucous layer okay so this is just we have been mentioning for carrier bacterium already well we have a lot of data showing microbial composition shifts I just pick out a few of them what you see here is the callee bacterium pros nits i abundance and actually if you plot the correlation it's statistically not the best way to go here but still if you do that you see that the abundance of this germ is going up around village length is going up and especially this one is important that if you have a lot of inflammatory solid flux if you have any flame cut that the abundance of this germ is is very very low and I just picked out the calla bacterium because it's an it's an important one you can also do that for other ones this is battery Secaucus this is one that we have been isolating many years ago you also see the same trend okay and you can actually do that with a lot of bacteria out of this room in a cooker say I family also some of these lack of spirits they are family and these are all butyrate producers and I fully agree with Pedro when she says that you can't just look at LACMA spirits say I remain a cooker say you have to pick out different genera and than just because sometimes you don't see this kind of bacteria going up or down but others take over and others are depleted so it's really it's probably because they the function is quite redundant between some of these bacteria but anyway so it seems to be the case that a lot of these beauty raid producers are actually when the abundance is low you get an inflammatory cell influx in the gut wall and this is what is already mentioned a few times a lot of these bacteria are coming from these two families and these two families contain a lot of butyrate produces but also others not all of them are actually producing butyrate but basically well if you look at chickens if you look at swearing at humans and so on you find a lot of these germs that's basically it so the principle mechanisms of beneficial microbes versus opportunistic pathogens that whole concept is just everywhere the same also over all the different animal species this is something you cannot reach but it doesn't matter yeah these are a lot of strains that we have been isolating chicken from chicken Sica before and actually also Peter Lewis was involved in some of these papers but what I just want to point out here is that all the strains that you isolate from chickens actually they fall into the same genera families than the ones you're isolate from humans some of them you see here we have been also isolating from chickens salsa ficarra bacterium patristic caucus and so on and if you use these strains this is chicken eyes a lot of bitter Secaucus if you use these strains in rodent trials as a model for human inflammatory bowel disease they are very very active as well so they are not only active in chickens they all is also very active in models for human disease and also fatality bacterium pros need selfish or actually one of these strains we just has been out licensed to a company for use in humans so this is an easy chicken strain so okay just just give you some data again this is the necrotic enteritis model in which we use cost vedyam to induce intestinal pathology if you use this kind of strains as a feed additive you see that numbers of animals that develop lesions is actually decreased these are for independent trials the blue is the control population area for example 55% of the animals that develop regions and you actually decrease it if four independent trials and also it works with some other strains and it's it's a bit weird maybe that you just administer one strain and this will not be because this particular strain is colonizing to a very high level is producing a lot of butyrate that's not a case but it also changes the whole microbial composition when you just add enough so if you start sequencing microbiota of an animal in which one of these trains have been administered you see that there is a global microbial shift because you have to imagine that in this kind of models you really have a severe dysbiosis you have a lack of butyrate but user but just administering them again you change the global microbial composition again and this is really well this is a little bit shown here this is again a trial in which we have been administering this battery Secaucus bacteria at very high concentrations and this is just a snapshot of some data coming from small intestinal microbial composition analysis using 16s sequencing and this is a trial in which we have been using just body digestible diets and what you can do if you administer this kind of bacteria in these models is lowering feed conversion so digestion is much more efficient and in this kind of models diet induced inflammation models you of course see again also this issues with fillers length immune cell infiltration and so on and by just administering this bacterium you see that well actually lactobacilli populations go up but also anti bacterial populations go down and this is adding quite important because this enterobacter CI these contain each coli not some one Allah probably in this case but equal I like organisms that are actually opportunistic pathogenic and we often see if we take samples from chickens in these inflammation models that if we induce inflammation enterobacter see I go up butyrate producers go down and actually you can play with feed additives to try to restore this particular balance that's basically the thoughts of all these studies you can see that here eco lies going down enterococci going down some hydrogen sulfide producers or going down and so on and butyrate producers go up this is just a small snapshot of all these data now coming to something very practical again how to increase butyrate and maybe cecal but intestinal you know that there are a lot of different compounds available to do that go to beauty rate esterified butyrate and so on and so on you have quite a lot of options there prebiotics in principle but there was one question already on enzymes and also this is something that actually has an effect on microbial composition I will just show some data in a few slides this is a slide while effect this has already been shown by pedra just slight on how these acids are produced it's it's a bit simplistic it's a simplified shame just to show that of course mostly the fermentation assets are a consequence of polysaccharide breakdown using many different bacterial species or ginger add that all generates some steps in this breakdown process but if you look at this graph here if you think about raw products or food additives or alternatives to antibiotics there's quite a lot in here you have prebiotics you have maybe the pure acids itself maybe you can add some bacteria that produce some of these metabolites and so on and also of course you can start playing around with enzymes because if you help in breaking this down maybe you produce some kind of oligosaccharides that I have this prebiotic potential so basically first thing or enzymes just that we just well have a lot of papers in progress using Salinas's and other enzymes and looking at the effect on on butyrate production and microbial populations and simply said if you just take a test you play you take a fecal or sickle slurry just add bacteria and give it some substrate and you add the enzymes in the test tube we will see that also in a lot of cases butyrate production in this test tube is going up so it means that at least it can be of value to add enzymes to diet of animals but it depends on the enzyme of course and on the what it is really cleaving off from what is and products of this enzymatic reaction and just show you some data often of paper we published two or three years ago this is just well this is just showing you of course the Arab Enoch silent structure and this is the breakdown of this Arab Enoch silence or Arabic silence from plant cell walls if we degrade it using different enzymes you end up with Arabic silent oligosaccharides and one of the questions we have been asking ourselves before is what would happen if you really administer this kind of prebiotic substances to deployers and actually if you start doing this you will see that well this prebiotic approach you will see that actually it stimulates gut health to some extent and in this particular study here we have been again using some kind of diet induced inflammation model this is the feed composition you will see that this is not normal there's a lot of riders also rapeseed sunflower meal just to induce some state of well digestibility issues bacterial overgrowth and and inflammatory cascades going on in the small intestine and then we just used Scientologist acolytes at a 0.5% concentration in the diet of these animals what you get is this here so actually well this is published by Celine the mascot three years ago and actually what you see is that also here feed conversion is going down if you administer this sauce so this means that digest abilities is improved also body weight is going up but also village length is going up and this is all significant this is that day 26 so about three to four weeks of age and this is the age to which mostly in this kind of in vivo models the animals are most vulnerable or susceptible to this well drops in performance so this is what you see here is an sila oligosaccharide induced increase in absorptive surface longer feel I saw more epithelium sells and it's very nice that beta already has commented on this particular enzyme this is one of the key enzymes in microbial butyrate production and you can somehow quantify a bit using quantitative PCR using degenerated primers and if you do that with the samples from the animals that received Xylo oligosaccharides as a supplement you see that well it's much more homogeneous okay so this is a quantification in love units of the gene that encodes this enzyme involved in butyrate production it's like a marker of well the capacity or the potential of a microbiota to produce butyrate it's simplified again it's not it's not fully theirs but it's it's it's it's a good marker for that what you see is that if in the control population there's quite some variability some animals seem to have much less of this beauty rate producing community others seem to have a bit more but if you add Scientologists acolytes you seem to enhance this kind of population okay that's basically what this slide is saying and even within the same diet huh you see that there's quite some some variability and this has already been mentioned in one of the previous talks one man is not another man but also one chicken is not the other cheek and although they get the same diet okay and they are in the same room so even then you have this kind of variability here and that's also something F when you look at microbiota composition analysis even within the same population on the same diet in the same room you have these differences but adding this kind of substrate seem to induce the capacity of the microbiota to produce butyrate and actually these are just some data you see that specific groups of lactobacilli are going up this axis is not good it's not completely valid but lactobacillus q status for example in colon in Sikkim you see that some specific genera or species even of butyrate producers are going up and actually what we tend to think that is happening is that you get some kind of a mechanism in which these silo oligosaccharides that actually are used by some lactobacilli to produce lactate and then this lactate is being consumed by butyrate producers to produce butyrate so it's this whole concept of cross feeding and that's why you need this high diversity okay that's basically what this slide is is telling you and actually you can you can model that in vitro so if you take this strain and you add sila autogas decorates it will produce lactate if you take this one it's beautiful user and you add sila oregon sucker right in a test tube nothing happens okay it needs the lactate that is used by these bacteria that is produced by these bacteria to further convert it so it's really a cascade of events that lead to two butyrate that's why maybe if you start with chickens that only live for five weeks and not have a fully developed microbiota and are under a lot of antimicrobial use that maybe this full cascade of this microbial variability and and all these different groups or maybe not present and maybe the microbial composition of the animal it's not adult enough to have a complete breakdown of all these components and that's why maybe we need to help them with with enzymes that could be a theory again a slide on salmonella and this is just showing you that these silo oligosaccharides or even other stuff that is fermentable to butyrate if you use someone election models actually that is also something that seems to help in reducing colonization by this pathogen maybe it's because of the butyrate that has been produced as well because what you see here this is just what you see here is actually if you take a sickle dropping or fecal sample you add it in a test tube very simple very simplistic as well and you just add one of these components at different concentrations in a test tube you see that actually this the bacteria are producing butyrate out of it we don't see that with Solano's in the resistance store for example in the test tube of course but if you also have an in feet edition of this cuz I don't sucker right so you do infection challenges with Salmonella I also see that the colonization level in the Sica so the place of fermentation towards Beauty rate is going down again so it seems to be like there's a general concept of having more Beauty rate production less enterobacter SEI less Salmonella less inflammation and so on there seems to be a very good correlation there between all different pathologies or intestinal issues that are relevant for project production just a final slide already just to mention to you that anyway well there are quite a lot of studies showing that Beauty rate is important there are quite a lot of studies showing that microbiota that is producing butyrate are very important but to weigh how to enhance butyrate production in the gut can be very variable there are a lot of different methods that can be used to stimulate endogenous Beauty rate production I think the problem we have a bit is and in contrast to what is going on in in human medicine it's a completely different thing if we speak about health in chickens very often or if veterinary and speak about health in chickens it sometimes means that they perform less and actually I don't think that's the same as health if they perform less why wouldn't they be less healthy okay I think that's a major difference between what we are doing in chickens and what people are doing in humans to actually have very different definitions about what health is actually what else is really so maybe one final remark here you also have quite a lot of other molecules butyrate is very well known but I think in the near future there will be a lot of other metabolites being published produced by a lot of different bacteria that actually targets maybe the same receptors as well butyrate is doing or maybe other receptors that are also very relevant for maintaining intestinal health so this is last thing I wanted to say so thank you [Music]