Philip asked me to talk about microbial metabolites and how they can interact with the broiler got Walt to improve performance well first of all I should say what we know about microbial metabolites that's a that's what I'm going to talk about but there's a huge amount that we still don't know so keep that in mind please there was a paper out actually in in in 2000 fixed 15 that predicted that antimicrobial use in food animals would increase by 2030 fortunately I think this prediction will hopefully and probably be wrong because we see that very recently that there's a there's a move all over the world to stop using growth promoting antibiotics in animal feed it used to be only Europe some crazy politicians in Europe decided in 2006 to stop this use of growth promoters but now this seems to be worldwide and this is probably one of the major reasons why we are sitting together here today in indeed we have been in the poultry industry we have been probably spoiled for many years using growth promoters to sort of alleviate any possible problems due to shifts in the microbiota composition in the intestinal tracts following the you know following the kind of nutrition or the the changes in nutrition which are being implemented so so when microbes were not of interest to nutritionists at all in the past to the point even that they they used ileal fish duration to study the effect of nutrients completely ignoring what was happening downstream below the ileum so cecum and colon were of no interest at all and now it seems to be the other way round c-command : seems to be the focus of everything but anyway when we come to think of it dr. Moore already said earlier on that chickens are coprophagic so it seems quite logical that they pick up on average around about 1 million bugs per gram of anything they pick from the environment but also this is on average taking the average of drinking water feed and pecking the environment so it's probably a little bit lower in humans the number of perks we swallow per gram of whatever we swallow but still we should realize that any animal including humans are ingesting quite a number of microbes all the time and and the thing is that in principle of course bacteria ourselves built of building blocks that are essentially similar to the building blocks that are used to build a eukaryotic cells which is amino acids nucleic acids and and you name it and and the consequence of that is of course that to some extent we may compete for the same nutrients and so in in the intestinal tract this is a problem because you don't want your competitors to run away what you with your nutrients so when so in in the animal in the intestinal tract of the animal it is clear that the crop and the proven triggers play an important role because we know that there the feed is acidified and the pH drops to about say between five and six in the crop and and a lot lower again in the province rigorous and it was always thought that this was meant to to kill the microbes in order to have Morris more or less sterile environment I personally don't believe that anymore because if if you want to kill microbes you don't use acids no to the country you preferably use alkaline because that's a much better disinfectant but what acids do actually they do kill a number of microbes but other microbes actually by getting into contact with acid they enter into what is called a viable non cultural state and a viable non cultural state is actually a situation in which the bacteria still are alive but they cannot multiply they can survive but they don't multiply and that's the trick nature plays to get the small intestine especially at the level of the duodenum and jejunum in a you know not sterile but at least in a situation where there's no competitive competition for the nutrients the number of microbes that are generally present in duodenum and lemon and jejunum are are around about log 3 which is the concentration of microbes you get on average in tap water so it's it's quite clean but of course the the microbes actually can be controlled like this kept in check at the level where absorption takes place but then by the time the microbes have moved through the the small intestine into the lower intestinal tract they recover from this viable non culturable stage and they start multiplying again and and that's that's a that's a good thing because in there in the Sica of course the number of microbes is extremely high as as dr. Moore already pointed out to you so Sica is actually the fermentation vessel of the chicken where fermentation takes place and you know it I have presented this already in the in the previous meeting in in Sao Paulo but I think it's it's a good starting point to to refer to what we are talking about the microbes which are present Sika and in the mammals and in humans and present in sequent colon as it's usually is often the case of most of the cases the vast majority of of the microbes that are there are FEMA coots second most important group are back through Adidas and it's it's more handy I think to look at at functions of these microbes and essentially we can say that the most of the fema kills belong to the short chain as fatty acid producers they are called secondary fermenters because they many times they cannot start fermenting the nutrients that are there by themselves so they in many cases they need what is called the cell wall decorators the bacteria detest to help them enter bacteria cir there as well and we heard something about it already this morning and and you also have this mutant if the decorators and I think musingly graders are very important because they they they live they thrive of the mucus produced by the host so we should realize that mucus production by the goblet cells in the intestinal tract is not only meant to create a protective mucus layer on top of the epithelium but it is also meant to feed the microbes and that is important in episodes when the intestine is empty in episodes of starvation now talking about the metabolites which will be important for the host I think we should think about what comes down the intestinal tract that is not absorbed at the level of the small in this that is what is what becomes available for the microbes to multiply and to produce their metabolites and first of all of course what comes down is those things that cannot be digested by the host because it hasn't got the enzymes available to digest it and these are the so called non-starch polysaccharides which essentially are plant cell wall components plant cell wall components which cannot be you know absorbed because the enzymes are not there are readily available for the microbes to thrive on and this is considered to be the the physiological situation so nothing else should come down at the level of the seeker which is that can be digested by the host but unfortunately in many situations we see that there is also some of the protein that arrives down the lower end of the intestinal tract and and this is not my finding definitely not this is from literature we see that metabolites produced by the microbes that are present in the cecum and colon of animal of mammals and humans and also that are present the seeker of of chickens they are based on the metabolic conversion of the non-starch polysaccharides and of the residual protein by the microbes and so my talk will be focusing on metabolites based on the use of the non-starch polysaccharides and metabolized based on the use of residual protein now this slide I've presented many times before so for those of you who have heard this talk before they will be a little bit bored now maybe but when we look at the non-starch polysaccharides they have to be broken down to oligosaccharides and monosaccharides and then they are further converted into different metabolites and what I want to do now is just give us an illustration based on a work of one our P one of our PhD students who looked at what happens when you provide the only size oligosaccharides directly to the animal helping it's helping the microbiota to break down the non-starch polysaccharides into oligosaccharides because this is the hard bit of the work you know the hard bit of the work is actually these large polymers which are difficult to access because they are in these plant cell walls which are very dense and very hard to you know attack by the enzymes produced by the microbes so if you provide them the only go Sakurai's directly then it may help the microbes to use these and this is actually the idea behind most of the work done on prebiotics because most prebiotics are actually illegal saccharides that are non digestible for the animal and so when the work that said ended was actually we're just adding 0.5% of china' oligosaccharides to the feet of broilers and she could clearly show that this had a beneficial effect on daily weight gain and on feed conversion so this is not you because this has been published before but what we wanted to find out is what exactly does it do to the microbes and as I like the discussion earlier on about about microbiota in the small intestine and microbiota and the large intestine actually in the chicken the the ileum and colon go together and they are you know in the movement and the dynamics of the nutrients passing through the intestinal tract ileum and colon are before the Sica it's its contents from the ileum and colon that shift up and down that are taken up by the seeker and fermented then and Seigle contents is at the end of the fermentation process cycle contents is directly passed into the environment so the colon is actually before the seeker and that's a bit odd when you think of mammals where the cecum is obviously before the column so colon or ileum you may consider it as one one functional unit in the intestinal tract and in this colon with these kind of oligosaccharides we saw an increase in lactobacillus iya which seems rather logical of course but we didn't tease lactobacillus a I it was only one species only one species that was increased and that was lactobacillus crisp artists and then when we looked at microbiota in the in the seeker there was an increase in the cluster 14a strains this is a family of butyrate producers and these this family was increased and within this family there was only one species that was increased that was the Amero steepest beat ear Atticus now we were we happened to have these both of these strains in culture in the lab and that's always very handy so what's it in then did was she took the an iris deepest bit erotica so the butyrate producing microorganism and culture dudes in the presence or not of desire of the Exile Orioles oligosaccharides and she looked at production of lactate and production of butyrate and when she did that she saw that adding the sauce or not did not make any difference the and iris deepest bit ago Atticus had a basal level of production of beauty rates so then looking at the lactobacillus crisp artists adding the sauce gave a spectacular increase in lactate production which seems quite logical but then Biko culturing these together she could show whoops sorry she could show that no more increase in lactate because all the lactate was consumed by the under Estevez beautyrx Atticus and converted into beauty rate and I think this is a very important aspect of the cross feeding that is going on in the intestinal tract when you look at the breakdown of of non-starch for evil non-starch polysaccharides so with sauce you actually stimulate you favor this particular pathway now the effects of butyrate I don't have to come back to because I think everybody has been reading a literature about this there is stimulation of butyric production is considered to be a beneficial effect so when one of the important messages I think is that one of the effects of butyrate is that is it is stimulating tie junctions and by different ways reducing inflammation not suppressing inflammation but reducing the need for an inflammatory response more than anything else now the the lactate producer and we were hearing already a number of things about lactate produces this morning the lactobacillus in the in this in this case you know the lactate production increased you to rate production but it can also increase propionate production by by cross feeding however it is clear that when the lactate is not consumed by the butyrate and protein protein it producers there's a problem because quite a you know not at all but the the vast amount of of lactate produced by the by the lactobacilli and the bifidobacteria is actually d lactate and d lactate cannot be used by the cells of the host which means that when it's accumulating it simply results in a drop of the age and excess lactate will actually make the ph dropped from say around about 6 to around about 5 and the big challenge is that that at pH 5 the Firmicutes which are essentially the butyric producers can no longer survive so you get a ethic destruction killing of firmicutes due to the pH drop and that causes a crash of the whole system and of course this the consequence of that will be inflammation right going back to our to our scheme I didn't say much yet about about propio bits propionate is is an interesting metabolites it's a favorable man with a blight no doubt because it can be used as an energy source also by the epithelial cells and it has a number of a number of favorable effects as indicated here and one of these favorable effects is that it in it stimulates satiety and maybe you're not convinced that societies should be stimulated in our animals but I think it should because when we look at the modern broiler this broiler has been selected so much two words feed intake that over feeding is becoming a problem so at the moment it looks like producers are struggling more to reduce feed intake by prowlers rather than to having to stimulate it so society is actually you know in improving satiety feeling and responses is is actually an important objective coming back to the propionate production there has been an elegant study showing the different pathways of protein propionate production but what is important to remember is that two of these pathways are only minor in terms of quantity in terms of importance quantitatively but this one is the quantitatively the most important one and this one is actually mediated by the battery details and battery details if you ask yourself the question are these important that is favorable for the intestinal tract or not yes of course they are very important because they are the the first the first group to attack the fibers but on the other hand you should not forget that battery details are gram neg bacteria and this brings me to a point which I want to stress here that is that all brand negative bacteria as they multiply and die off and we should realize that dying off of microbes is something that is continuously going on at it at a huge rate in the intestinal tract as they die off lipopolysaccharide is released and of course we cannot consider this truly as a as a metabolite of the of the microbes which is the topic of this thought that I should still warn that lipopolysaccharide is actually a very powerful trigger of inflammation and it's a powerful trigger also of leakage of damage to the tie junctions and damage to the epithelial barrier so excessive amounts of lipopolysaccharide released in the intestinal lumen may be harmful under certain conditions so we've been looking at some aspects that's some important metabolites derived from the non-starch polysaccharides but I want to share with you also some thoughts about about the metabolites derived from residual protein and I think residual protein can be important especially because we tend to overfeed protein to our animals and well I at least have a tendency to eat too much protein as well because it's palatable you know we have special taste receptors for proteins so we like protein and animals also like protein so where excess protein is is something very common and so when you look at at the end metabolites derived from proteins without entering too much details this is just a short list of what you can find in the literature and I want to focus and stress some on some of these metabolites together with you and the first one I want to have a short look at is ammonia and when you look at the effects of ammonia ammonia is actually a gas but in in the liquid it will be ammonium hydroxide and ammonium hydroxide especially when it's present in in relatively high concentrations it will reduce the receptors for bitter eight which means that production of butyrate will no longer have sufficient effects on the host cells and release conditions and more importantly even ammonia will reduce the energy production by the mitochondria which means that the epithelial cells run out of energy and the important consequence of this is that the most energy requiring activity of epithelial cells is maintaining the tight junctions between the cells and these tight junctions constitute the gut barrier so if the tight junctions are not maintained the gut barrier is disrupted and leakage of plasma plasma fluid but also plasma proteins from the host into the intestinal lumen is taking place and the constant of practical consequence of that is wet litter so when so energy production by epithelial cells is very critical and anything that that inhibits the energy production by the mitochondria in the epithelial cells will cause serious damage so what else is there well there's a lot to do in in the human medical world about the biogenic amines and some of them you definitely know peterson cadaverine and and a number of them i don't want to go into too much detail about them because they're considered to be very very important for human health because they may be involved directly but probably mostly indirectly in in development of pathology of the intestinal tract which is typically for whom for humans and that is colon cancer so so biogenic amines are not of a major interest at this moment but it may be worthwhile to investigate this is further I think also for or poultry nutrition and then we come to the in doubles and the indoles are something very controversial because when you read the human literature indoles are are considered to be potentially harmful and we know about the number of these molecules you've all heard about indolence cattle and you know they names so the sound already it it when you think of it it's it's considered to be not very favorable because they are also involved in going off of foods and feed and feed stuffs so when they are considered not to be very favorable in at least in in the conditions where we have been looking at them for mostly but when you look at the the role of in dole in the intestinal tract it actually seems to have a number of very beneficial effects so so in dole seems to protect against violence mechanisms of of pathogenic microorganisms it helps stabilizing those tight junctions which I was referring to earlier on and it reduces intestinal inflammation so so in dole although it has a very bad name because it's a it makes food go off its and it the flavor is is actually revolting but in the intestinal tract the effects of endo seems to seem to be rather positive and this will again have an effect on inflammation actually reducing inflammation again in humans as I said there are effects which are considered negative and even devastating but these are effects in long term and we see that especially in humans the toxic effects in the long term on kidney function and heart function have been focused on a lot in the human medical world so this is what you get when there's excess in don't produced in the intestinal tract and then dr. more already referred to the neurotransmitters it's a it's a fascinating area a fascinating area it's there are these are metabolites of the microbes in the intestinal tract and they are produced by conversion a very specific amino acids into neurotransmitters the neurotransmitters the important neurotransmitters that are produced are serotonin dopamine and gamma amino butyric acid which are actually neurotransmitters in the nervous system of humans and that means that these could have an effect on the brain but they definitely can also have an effect on the neural system of the intestinal tract which could influence first autism which could have enormous consequences for efficacy of nutrient digestion so neurotransmitters produced from amino acids may have an important role but they need to be further investigated what I wanted to point out to you also is that the lactobacilli are the most important producers of gamma amino butyric acid and so this is one of the beneficial effects of lactobacilli okay this gamma amino butyric acid has a number of documented effects in the intestinal tracts reducing inflammation stimulating the production of propionate and butyrate and also under heat stress condition it has been shown that it has a clearly documented beneficial effects right and just to take this one step further I want to go to another another study which civilian did a number of years ago and that was a study which in which we we had a relatively acceptable feed formula nothing special about this feed formula but what we did is we replaced some of the soy protein by other protein sources which in the industry are known to be less easily digestible so by only you know changing part of the protein by a source of protein which is less attacks or less easily attacked by the enzymes of the host create a situation where we expect that there's residual protein passing into into the Sica and and we compare it to where to more readily digestible digestible protein sources and so what we saw is that in the grower face the slowly digestible protein as I shall call it had a negative effect on daily weight gain and a negative effect on feed conversion so it means something so when looking at the microbiota we unfortunately didn't see much however when we took this one step further and we looked at at the function of the microbiota we used it to to qPCR s1 is actually quantifying the genes present coding for an enzyme which is a key enzyme in the butyrate production that is the beautiful Kawai acetate Kawai transferase and the other one is a qPCR you know quantifying the presence of genes coding for a key enzyme in hydrogen sulphide production which is the B sulphate reductase and as you can see here the ratio of capacity to build on beauty rate production versus capacity of hydrogen sulfide production or shifted to words more capacity of hydrogen sulfide production so there was a functional change which actually which actually led to increased capacity of hydrogen sulfide production hydrogen sulfide production is a characteristic of the proteobacteria and of the sulfate reducers so this is what X excess protein also does it stimulates hydrogen sulfide production and where does this hydrogen sulfide product come from well it does not only come come from the the sulfur containing amino acids methionine and cysteine but it also comes from the sulfated mucins which are produced by the host and it also can come from sulfides that are present in additives so we should be careful using sulphide additives in animal feet because this may potentially increase the production of hydrogen sulfide but the question is of course is the production of hydrogen sulfide is that a problem so when if we look at in our scheme we should definitely add hydrogen sulfide as one of the effects of protein digestion but the question is there about the the value or the the negative effects of this and what we can find in the literature is that hydrogen sulfide actually has a large number of beneficial effects which are all listed here and don't want to go into detail because that would take too long but there are there's definitely there's definitely a role a beneficial role for hydrogen sulfide in the intestinal tract the problem rise arises however when there is excess hydrogen sulfide produced with excess hydrogen sulfide you get inhibition of a a key mechanism in energy production which is the mitochondrial cytochrome C so here again there is there is blockage of the energy production in the intestinal epithelial cells with excess hydrogen sulfide and the consequence of that is of course again problems with intestinal barrier so excess hydrogen self they sulfide inhibits mitochondrial energy production but it has a also a number of other of other harmful effects so we should be very very careful about hydrogen sulfide because in high concentrations it will again trigger inflammation there's a little little detail which I want to share with you with which i think is although it's a detail it may have considerable importance that is in in this in these conditions when when sulphide groups and sulfate groups are converted into sulfide hydrogen sulfide is produced but under these conditions when there's inflammation in the intestinal tract this will also be converted in tetra thionite and for the microbiologists amongst us this rings a bell because theta thionite is actually used in the microbiology lab as is selectively a selective chemical to select it to have a selective medium for salmonella so indeed it retire Nate allows Salmonella to multiply while inhibiting most other bacteria and that is a problem because if there's too much protein in the intestinal tract then you may end up with an animal that will become more susceptible to salmonella and there's one other organism that is also allowed to thrive when there's theta at I innate round and that whoops so it's missing here but I can tell you that is Campylobacter so an inflammatory condition in the gut with too much protein will stimulate the multiplication of Salmonella and Campylobacter so one point which I want to address here but very shortly is that we should not forget that amino acids can also be converted in propionate and butyrate and then there's this one other study which I like to to go through together with you very briefly and that is a study in which we added a small amount of of amorphous cellulose or cellulose powder to the feed of day-old chicks 0.5 percent in the feet of day-old chicks and then after that we we switch to one percent so when you know with my Maya even when we were starting a study was this will dilute the nutrients in the in the feed of the day-old chicks so you will have a negative effect on performance but the opposite was true actually what we what we could see is that the birds that got these cellulose in their starter they had a significantly better weight gain and that continued during the grower phase and when we looked at the feed conversion ratio in the starter these birds also had a better feed conversion ratio so what I'll be looking here what I'll be looking at in this case what happens with the Solano's it is obviously non-digestible for the host it enters the seeker and and what happens in the seeker well if we look at if we take sickle content and we take it in the lab bring it in contact with a number of of non-starch polysaccharides of with resistant starch and we measure the capacity of the cycle microbiota you know as as a whole as a community to to produce Beauty rate and you can see that some of these non-starch polysaccharides allow the microbiota to produce more beauty rates because this is measuring Beauty rate production but cellulose doesn't do anything so it's not through increased Beauty reproduction that these animals were performing better so what happens with the cellulose it doesn't stimulate this pathway clearly and then we looked at the microbiota in these birds and it turned out that there was just one genus that was influenced so there was not a major shift shifted the microbiota it was just one genus and and it turned out that this genius Alice deepest was clearly significantly stimulated by the presence of the cellulose in the seeker this is a genius that belongs to a family with didn't sound very familiar to me at a time these recognized OCI they belong to the bacteria Dita's and what is known about is steepest genius is that it is a strict anaerobe of course it's a gram-negative because it belongs to the bacteria detest non spore-forming but it has a powerful saturdays so on its own it can enzymatically break down cellulose even if we know that in nature in the environment regular cellulose requires a network of microbes in the intestinal tract obviously one single genius can do the job and then what we also know about other steepens is that it's present in the core microbiome of the chicken but also in the core microbiome of humans and under pathological conditions it can get lost and and also I like the story the publication of the group of toric and I should be grateful to where to all the studies which you did drop in there in Australia with respect to comparing well-performing versus poor performing birds some of these things really popped up in these studies and and now we are beginning to see the light whether you know what what is actually the meaning of this so a list appears is associated with better performance and it's its main metabolic end product is actually succinate and of course section 8 brings a bell to us because that can be used in the the krebs cycle and we could show in the lab that indeed this deepest when you bring it in contact with cellulose it will grow better and and of course when it grows bathroom it will produce more more more 16-8 so when so the cellulose breakdown in the intestinal lumen is is the work of a single genius and and what it actually does is is it stimulates the production of section 8 but then then it becomes a little bit puzzling because it doesn't seem to move further towards towards butyrate although although 16 it can clearly be converted at least in in propionate and that has been shown in the literature that succeed it can be converted into propionate by a number of different microbes so so this pathway can be stimulated but what is also remarkable I think is that there's recent publications coming out that in the intestinal tracts the epithelial cells have receptors for sex in it so they can take up the section eight from the intestinal lumen and take it up into the epithelial cells so it can provide the section eight directly to the host and these receptors are expressed at least it has been shown in the mouse to my knowledge it hasn't been shown in the chicken yet they are expressed in the mouse they are expressed both in the small and the large intestine on the epithelial cells and that is just the immunohistochemical image from from those studies and and so we know that inside the epithelial cells the citric acid cycle can can use this is section eight but to our surprise there has been a study coming out very recently with very elegant study using knockout mice and this study could document that the section eight which is taken up from the intestinal lumen into the epithelial cells is not immediately used as energy source for the epithelial cells but it is converted into glucose and this glucose is transported into the blood so this was a mouse study but I can imagine that this could all equally well have been the case in the chicken explaining probably why we see these better performances so to wrap up because I think I've I've eaten up my time mr. chairman I think there's a number of metabolites which we know and which we we're starting to get a picture of and I think we should also take into account the quantities because quantitatively there's an enormous difference also the the power of the effects should be taken into account and and we know of there are literature data with respect to quantities of a number of these these metabolites under physiological and under pathological conditions not all of them have been investigated in in much detail but what we should conclude and that is my last last slide is that some of these metabolites have beneficial effects clear beneficial effects some have clear harmful effects some we are not too sure I mean lactate is definitely okay but if you have excessive amounts you may and end up into trouble hydrogen sulfide small amounts seems to be okay but large amounts seem to be harmful so we're I think we should be careful with hydrogen sulfide and maybe we should put it on the in the right column sixteen eight we still don't know for sure but it looks like it has may have beneficial effects and what about methane I think there's still a lot of work to be done but what I mean here is at this moment we and many other people are investigating the methyl metabolites produced by the microbes and we know there are powerful tools available which are called metabolomics and metabolomics are at our fingertips now I mean they can do produce very fast results as long as you know what you're looking for so targeted metabolomics are available and they can give fast results so what scientists need to do at the moment that what we are doing and many other people is trying to find out what metabolites should be present in what concentrations to have a favorable effect on the intestinal tract and my my personal view is that I think that within five or six years veterinarians will be going to the to the chicken house and instead of taking blood vessels and looking at antibody titers or looking at blood biochemistry they will take seagull samples send it to the lab get the next data metabolomic profile and by comparing these to the data that have been generated on the normal concentrations of these metabolites I think they will be able to find out whether the microbiome may have beneficial effects or whether you're in a condition where the microbiome is in trouble thank you very much [Music]