[Music] well thank you very much and thank you for the invitation to come here and talk about fiber fermentation and the possible role of butyrate and pickles I'm afraid that Bruna the silver promised you a bit too much about what I'll talk about in relation to sound nutrition so I guess I will disappoint you here instead I will remind you on the last presentation you heard before lunch by a llama yet because what she percent is actually very much could be a good set of four what I'm also going to present for you today there's been a lot of focus in the last decades on the possible possible health benefits of short chain fatty-acids and particularly on butyrate being the main fermentation products of fiber fermentation in the in the god of humans and animals but the question is what is the potential role in pig nutrition I'll try to wrap out on that on how we can try to translate some of the data or the information we know from studies in in vitro cell models rodent models and in human studies on how that can could impact peak health because a lot of the knowledge we have so far about the mechanisms on and on the short chain fatty-acids and in particular on butyrate is actually related to human nutrition and particularly there's been a lot of focus on the effect on on cancer obesity and diabetes in human trician within the last year's and as i mentioned a lot of this come from in vitro cell models rodent models but also pic and human studies where short chain fatty-acids have actually been either given in the feet or the diet or infused directly in the god or even in the veins and to study the mechanisms by which the short chain fatty-acids could have an effect and this review here from last year is just a mention of how you can sometimes you have to be sure you don't jump too fast into conclusions on the effects because what this review here says that it will summarize and critically evaluate previous studies on the effect of butyrate and its derivatives on animal performance with the effects on human health as a reference and you have to consider yet that maybe you sometimes we jump too fast into conclusions on what the actions and mechanisms are because can we be sure that the buter rate or shortened fatty acids that we provide in the feed will actually be the same as if they are produced in the gut of the animal so there's just a reminder to you so here are some of the different effects that you to rate is now currently believed to have on the cellular level and you can see here there are so many different effects that butyrate is believed to have some of those mostly relate to what is happening in the gut and helping the intestinal homeostasis and maintaining gut health whereas others are actually working even in the in the systemic level and cellular level where it can affect for instance insulin sensitivity satiety and so on and I'm going to take you through some of these mechanisms so beyond butyrate as being an energy source for the animal I want to discuss a little bit on how fiber and microbiota play a role in this but that has already been dealt with before lunch so I'll do that fast and then I'll talk a bit about the mechanisms by which butyrate can affect internal barrier function got inflammation and how it can affect the peripheral inflammation and metabolism and finally try to pose some questions to you on how you think that could be translated into Pig nutrition well it's well known that if you take isolated purified fibers that if you inoculate them with a fecal flora or God flora for instance in this case from humans you'll get a different fermentation profile here as a proportion of escheat propionate and butyrate that that these different sources will give different profiles and what is coming out as giving the most beaut the oligosaccharides like fructooligosaccharides collector alga right collector oligosaccharides an inland type of fibers and that was also what founded the prebiotic concept in the mid 90s because these carbohydrate sources also the ones that are providing more bifidobacteria and more butyrate however please note that even if you use such a simple system in different studies using the same type of fiber you can actually have different proportions coming out of your study the other thing I want to mention but this is that that fiber fermentation and the end products that you get from it is actually more complex when you come to a real diet because there you are not feeding isolated carbohydrate sources you are feeding combinations of different carbohydrates so the short chain fatty acid profile in the gut of a human or a pig or a rat or whatever a pulled chicken will depend both on dietary composition the site of the gut and also some time of adaptation and you don't see that in these simplify in vitro studies so here we have an example from a study with it some years ago with ileum cannulated pigs and we fed those pigs different sources of carbohydrates we fed them raw P poly raw P and raw potato starch in combination with the wheat brain and the idea was that we knew that these carbohydrate sources have a different start structure and we also believed and we showed that that will give a difference in the proportion of future rate and acetate and propionate in the sequel content so you can see here that because the start structure is different pea and potato starch will give rise to higher butyrate levels when we heat treated or extruded these sources we could see that the butyrate production went down and that was because the starch became more available for digestion in the small intestine but what you can also see is that when we come to the colon here that the the butyrate proportion is different from what you see in the cecum so it goes down actually here with the pea and the potato starts with it whereas it increases with the raw barley and that's probably reflects that some of the the bacteria nicola is now starting to ferment some of the fiber sources rather than the starch so it's just to illustrate that is a dynamic system and that is illustrated here and was also touched upon before lunch if you have a diet you have a complex mixture of different carbohydrate sources some are insoluble some of those will become soluble through microbial digression by some groups of bacteria these soluble polysaccharides can then be either metabolized by the the greatest or by other groups of polysaccharide the greatest and these can also again be transformed into oligosaccharides or free sugars and be used by another group of bacteria the intermediate metabolites metabolites that are produced by these degraders or utilizes can then be used by other bacteria to produce short chain fatty acids and other compounds and that was also shown nicely this morning and an example showing this is here from healthy volunteers here you can actually see that the different Feli of bacteria are differentially associated with the different fractions of the the faeces so roseberry and rum okay they are more associated to the particulate face a particular face of the of the feces whereas other groups like Bacteroides and the protea better are more associated to the liquid face now showing this in a more complicated way but very nice and simple this morning it is very important to see that you have different groups of bacteria that are involved in microbial metabolism of carbohydrates the primary carbohydrate the graders are typically producing intermediate products like pyramid and acetyl coenzyme a which are then resulting into lactate and acetate and these metabolites can then be further transformed into propionate and butyrate so the the outcome of of that is that you can have different resulting products or fermentation products depending on the substrate you have meaning the type of feed or feed ingredients that you have in your diet but you can also have that even the same substrate will give rise to different metabolic products depending on the fermentation pattern or the fermentation route and that will depend on the rate of supply than physiology and the environment of the bacterial cell so in other words the environmental conditions for the bacteria finally it's also important to note that acetate can be transformed into butyrate so even if we see a high acetate production then that might eventually end up in a high future rate production so finally these are the end products as you all know acetate propionate and butyrate are the main products if you're a protein fermentation you additionally will have branched chain fatty acids and that is why if you have a high content of branched chain fatty acids in your gut content or in your feces that indicates a high protein fermentation once the show ten fatty acids have been introduced they need to be absorbed by the cells either the the butyrate or the short chain fatty acids are diffusing through the cell in the undissociated form or they are actively taken up by transported coupled to hydrogen or to sodium and in in the cell think they can then be metabolites to provide energy to the cell or they can be transported to the peripheral system through another transporter and it's important to note this because I'll come back to that later that these transporters are very important for the active transport of short chain fatty-acids now to illustrate an example on how you can mod manipulate the production and absorption of short chain fatty-acids by a diet we could perform the study some years ago and this is not a typical Pig diet it's a diet that is simulating a more human diet we call it a western-style diet it contains very high amounts of fat very high amounts of digestible carbohydrates and they're a little fiber so it's what we also in daily life calls and McDonald diet we compared that to two diets where we increase the fiber content either by including a resistant starch or our penis island in the diet but as you can see here the fiber level in the two diets was about the same but the fibrous sources were different so in there are being a silent rich diets we had whole grain rye and we had enzyme-treated wheat bran and the other diet we increase the level of fiber by including high amylose corn starch and raw potato starch and we wanted to look at the fermentation profile the absorption of short chain fatty-acids and also the metabolic response to these diets so we fed them to ten intact pigs per treatment for three weeks to weekly blood and fecal samples and let's slaughter we collected content and tissue content for measuring different parameters of inflammation and metabolic response at the same time we also made a study with Cathedral texts where we were able to study the absorption of glucose and short chain fatty acids to the liver from the gut using this this model here and we then set them in a crossover design so that we could check three diets with a limited number of pigs so what we saw in this study was that we were able to manipulate the composition of microorganisms in the gut particularly we saw that we had an increase in pewter rate producing bacteria with the Urbanus island diet compared to the control diet and with an intermediate increase with the resistance tarts diet so there are being a silent diet was more efficient in increasing the number of of butyrate perusing bacteria we also saw an increase in bifida bacteria and lactobacillus it also led to an increased production of butyrate in the gut in the different segments of the gut here you have the end of the small intestine the cecum and the three parts of the colon and you can see here that we have an increase in puter a production with air penis Island diet particularly intermediate with the resistant starch diet what you also can see is there that they production decreases throughout the colon as we come from the cecum down to the more distal parts and finally what we showed in the study was that we had an increase in butyrate acetate propionate and butyrate that was absorbed to the liver from the gut and particularly we had a three point six fold increase in butyrate absorption from this diet so if we try to summarize this you can see here that the concentration in the large design is different between the diets you had to know the unit here if I had to put it on the same scale this would be one 1,000 units about 1,000 units higher then you will see in the bloodstream so that's why I have put it in millimole per kilo these are micro molar per liter you can see here the concentrations in the polar Lane that is the block that is going to the liver from the small intestine you can see here that you have a market marginal or big increase in butyrate going to the liver then on the other side of the liver in the hepatic grain you can see that the concentrations of butyrate are far far lower but they are still different between the diets and finally in the arterial blood which is in the periphery you'll see an even lower concentration it's very important to bear this in mind now when you think of potential actions of butyrate and the systemic level is that the concentrations there are actually quite low that is illustrated here in this graph where you can see that when you have the gut lumen here you have a mix of acetate propionate and butyrate but as soon as you're passing the liver the concentrations of butyrate and propriate up becoming very very low or even very limited it means that the remote tissues like the brain muscle pancreas and fat tissues are not exposed to very high concentrations of butyrate or propionate so anyway one of the roles of short chain fatty-acids as shown in one of the first slides is that it can act as a signaling molecule for a range of the different metabolic processes and in order to do so the they need to be recognized by the different cells and until now three receptors are known for short chain fatty-acids one of them is DP r41 or called if ffs are free it recognizes acetate propionate butyrate and Valerie and as you can see here it is located on a range of different cells throughout the body both in the intestine on the intro intro intro crying L cells but also in other cells in the body the other one is gpr-43 or FFR - it recognizes formic acid acetic acid propionate butyrate and Bala rate and again you can see here this receptors located on different cell types particularly some of the immune cells and the intestinal epithelial cells and finally and probably the most important one who comes to the colon and fermentation is the GPR 109a receptor which is also called an ITIN Nissen receptor because it recognized no hacen but it also recognizes butyrate and that is located on the colonic cells epithelial cells on the macrophages and dendritic cells that are located underneath the colonic cells another important issue or another important aspect of short chain fatty-acids is that they are known as histone deacetylase inhibitors histone acetylation and the acetylation are very important aspects of gene regulation it means that when the histones are deacetylated the the DNA becomes inactive and cannot be transcripted into mRNA and two functional proteins however a short chain fatty-acids can act on inhibiting deacetylases and in this this results in in in an effect where the chromatin is open and transcriptionally active so in a way you can say that short chain fatty-acids then can affect the mRNA and micro RNA expression and it can prevent cell silencing and that is one of the roles that is now very much studied - in terms on how short chain fatty-acids can act in cancer prevention and cancer treatment this scheme here is quite similar to the one that was shown this morning just before lunch where you can see the role or how they got is protected against invading pathogens and antigens both by the mucous layer and also by the immune cells underneath the epithelial cells so when diet is passing through the gut the first antigens are dietary entities but as we move along you'll see that the exposure to antigens is coming more and more from the microbiota it also means that the panel of immune cells that are involved in maintaining gut homeostasis are changing I won't go more into details than that because I think you've already been noting to that but one important thing to notice here is that here's a schematic view of what can happen if you have an imbalanced diet with lack of fiber if you have a fiber rich diet that fiber will be degraded by a fiber grading degrading microbiota and they will keep the mucus degrading microbiota in balance and keeping them them down so that you can maintain an intact because a layer however if you have a fiber poor or fiber free diet these mucosa tech degrading bacteria will take over and it will erode the makohe mucus layer leaving the epithelial cells open for mucosal pathogens and that will cause a barrier dysfunction so it's just an illustration on on the balance that we'll need to have as mentioned this morning as well tight junctions are also extremely important for the maintaining of a god barrier function and it prevents that invading pathogens toxins and other antigens enters the systemic circulation so maintaining this part here healthy by by appropriate tide is very very important and there's one of the measures that are being used now to see how fiber or short chain fatty-acids can affect intestinal epithelial barrier function so from this general introduction I'll now show you some examples on how this has been shown in in science on the effects of different short chain fatty acids and particularly on butyrate how they affect the function of the cells this is a study from in vitro cells colonic human colon cells that has been stimulated with the elf ears and compared to a control diet in this study they added sodium butyrate at 0.1 and 0.5 millimolar and what they showed was that these short chain fatty acids or butyrate in this concentration which used the pro-inflammatory cytokines it stimulated anti inflammatory cytokines it activated p-part gamma and it suppressed the NF kappa-b C pathway which is a crucial step in the cells response to inflammation and it inhibited these histone deacetylation as I mentioned before so that is is a general effect that you see that you have this inhibition of this effect however this is an in vitro study it's a cell model and if we look at this these concentrations here are far beyond what you would see in an inner cell in vivo so the question is how relevant are they when it comes to Aviva's situation but that is why we also have other models or other choices this is an example again related to human health and disease a rodent Kalidas model you can induce acute colitis by dextran sulfate sodium and that is done in this upper case here you have a control group and then you have had this vehicle vehicle of DSS and in the lower panel you have a model of chronic colitis and that is a Aisle 10 deficient mice this is a wild-type mice and you can see here that the histological score that means that damaged of the tissue is really really much higher than in the control mice here but if you add in the 20 or 100 or particularly 100 milligram per kilogram of butyrate you'll see a reduction in the histological score or an improvement of the cell tissue at the same time they showed that this phosphorylated I Chi beta alpha which is again the cell responds to inflammation and histone is acetylation was improved in in in the same way in the acute and the chronic model another example on how it has been studied how short chain fatty-acids can improve health is by a recent study where they added antibiotics to helium cannulated pics for two weeks but they wanted to do was that they actually wanted to abolish the effect of fermentation by the feet itself they didn't manage to do that but they managed to reduce the production of short chain fatty-acids to about one tenth of what they had in a controlled diet after two weeks they had daily infusions of acetate and propionate and butyrate in the helium of the pigs and looked at the response of what happened interesting what they found was that in the ilium and the colon they had a reduction in cell proliferation index when they added the antibiotic but when they added had this infusion of short chain fatty acids they increased the cell proliferation again even more than you had in the control diet at the same time they showed also by gene expression that that the short chain fatty-acids could either reverse or even improve a pop ptosis and cell cycle arrest it increase the amount of short chain fatty acid receptors in a gene expression level increased barrier function through improvement of tight junctions and muse in production and it increased the number of nutrient transporters in the gut a final interesting paper that I want to present in this in this respect was a recent study also where they actually infused butyrate directly into the jugular vein of pigs they were fed a conventional corn soybean meal based diets but then they infused these this butyrate directly in the vein they had no effect in this study on the concentration of short chain fatty-acids in the colon however what they did find was an increased enrichment of the bacterial community and at the filler level they saw a tendency for reduction in Bacteroides and an increase in the firmicutes and at the and at the class level they also particularly saw an increase in trust radialis Romney cocky precedium and in a row I can't see truncus and the reduction in adult pervert Sara and subdue Lingam and blouse yeah so that's quite interesting that by changing systemic levels of short chain fatty-acids you can actually affect the flora of the God what they also showed was that they had an increase in short chain fatty acid transporter they had a reduction in histone deacetylation and they had an increase in the immune Fox function of the mucous membranes at the same time they also showed that they had a reduction in pro-inflammatory cytokines and an increase in anti inflammatory cytokines so I think this particular study is quite interesting because he could show it's not only the microbes that affects the host but it could also be the host that affects the microbes and so coming back to what I started out saying that most of what has been done in pics and pictures at least is by the addition of butyrate to the diet as a feed additives and what I've seen so far and I've only taken a few of them is that the results you see in literature is actually quite wearing sometimes you do have an effects on everyday daily weight gain and feed efficiency sometimes you don't sometimes you have even negative effects on on the on the Isaac's for instance expression and so on the reason for this could be that it might depend on dietary composition it might depend on God maturation and it might depend on on the settings where the church interests have been studied I pick out one particular study here rather recent one where they fed 450 milligrams of coated sodium butyrate to four we go piglets house in groups of six the treatment they had an increase in a high in daily game they also saw an increase in the lose height and they saw an improved barrier function they also showed a lower mast degranulation and lower master coronation is then associated with the lower mucosal content of mast cell mediator sees an anti-inflammatory these are pro-inflammatory cytokines and they also showed that the gene expression of these muscle mediators were reduced and they linked that to an inhibition of the day in Kasich million which is related to apoptosis of the god cells another example is where treble terrine was added to piglets and these piglets were challenged either challenge or not with acetic acid in in the rectum and in this study they showed that 1.1 percent tributing alleviated the the acetic acid injury induced by or the injury induced by acetic acid by increasing the oxidative status reducing apoptosis improving tight Junction formation and improving EGFR signaling of the pigs so this our diets of effects of adding butyrate to the diet but the recent study or recent review here actually showed that butyrate might be important even if we don't try to manipulate it or not this graph here shows the changes that you might have when the piglet is going from lactation into weaning what they demonstrate here is that you have an increase in in every through in likeness pair Asia I'm not good I'm not a microbiologist and a Rousseau pillow Trisha and you have an increase in bacterial community but they lead to a reduced butyrate production and by the reduction in butyrate then you'll have a reduction in the short chain fatty acid receptors for instance the gpr-43 that will lead to increased cell apoptosis increased levels of pro-inflammatory cytokines and reduce cell proliferation that will cause damage to the intestinal cells and it will reduce the level of digestive enzymes reduce food intake and body weight however when the pick then go from weaning into the post winning period and is adapted to a new feed then you will see an increase in particularly lactobacillus and Romelu Lukaku you'll have a reduced bacterial diversity but you will increase butyrate concentrations and then you'll have the exact opposite effect as we saw before here when you go from from milk into the weaning phase you'll have an decreased apoptosis reduce pro-inflammatory cytokines and increased cell proliferation improving the health of the god so the strategy here is to make this weaning transition so smooth as possible so if you can find ways to promote butyrate production to maintain Apatow she's a proliferation balance they're good and minimize intestinal and from an inflammation that will be the way to prevent post waning diarrhea here's an example of a recent study where they tried to do that by adding different fiber sources to weanling pigs they added 5% of different fiber sources corn soybean hulls wheat bran corn brine and then they showed that they could manipulate the butyrate production they could increase the feed efficiency and by changing modulating the microbiota composition of the diet now going back to our human study what did we find there we also looked at some of these transporters and the effect on immune system what we did find was that the enhanced short chain fatty acid production we saw with their penis island and resistance diet before increase the expression of of the of the hydrogen coupled receptor in the large intestine so we were able to to to change the transporter system however for our short chain fatty acid sensing receptors our results were quite conflict they were actually inversely related to the butyrate pool in the cecum but not in the colon and the effects of our Venus Island and resistance tarts were were diverse so we don't have this very nice link to what is heppa theis hypothetic of a fiber in this in the study also we only saw minor effects on the intestinal barrier function in spite of this very big increase in in in fiber and future production and we did not down regulate pro-inflammatory cytokines in this study unfortunately now the interesting thing about this study is that this is a collaboration we had but with with with the Institute of clinical medicine at always University and they actually wanted to use this study for a model for what is happening in humans so they were working with slight Lupe's pre-diabetic persons and we wanted to see if we could get similar results in the pixon and the humans but for the human study we had to modifying our diet so we had a western-style diet and we also had what we call the healthy carbohydrate diet and that was a combination of resistance starts and our Venus Island in that diet so we wanted to see if we could get similar responses in the pigs and in the humans interesting in that study what we did find was that we modulated the microbiota composition of the fecal flora of these patients however if you look at this side this is full change from when they actually started the study so for the most of the bacteria actually saw a reduction in in some of the friends the firmicutes with which we are interested in in increasing whereas we had only an increase in the per feed of bacteria the group of us see no bacteria I City no bacteria so that was quite disturbing you would say but the reason is that the inside actually quite healthy they eating a very nice fiber-rich diet so many of these persons here actually had a very healthy carbohydrate died from to begin with so that's why we were not able to induce positive changes in the microbiota composition we did change the short chain fatty acid content of the thicker content of the patients you can see here both in short chain fatty acids and acetate and butyrate and we did see that the healthy carbohydrate diet actually improved intestinal barrier function and reduced markers of inflammation so apart from this effect on intestinal barrier function which appears to be very very important to maintain both got health but probably also systemic health and this is an example here of these ceases in human medicine where damage to the epithelial layer could have an effect and you can see here that it's diseases like ulcerative colitis Crohn's disease iliac disease obesity biddies and so on which are not relevant for human productive or animal form pig production I would say but you could imagine that there were many of these responses in inflammatory diseases and metabolic disorders that would also be relevant in pig production so apart from the effect on the barrier function a short chain fatty-acids themself might have effects even on the remote tissues as I mentioned here in the beginning please note on this slide here which is from 200 2015 that some of these dashed errors here means that it is not confirmed in in vivo studies and it might only be hypothetical mechanisms by which the short chain fatty-acids thought to have an effect so again you can see here the receptors are very crucial and important for the proposed effects that you can have both on the liver on lipid tissue on muscle tissue and even on the brain and on society so I've come a little bit back into that shorten fest yes it's might affect adipose tissue function by increasing the stories of triggers rise in the adipose tissue it might reduce a release of free fatty acids into the body and it might reduce a secretion of pro-inflammatory cytokines and chemokines in the fat tissue and reduce a local mic macrophage infiltration so that is definitely something that has been a huge interest in related to obesity in the skeletal muscle metabolism it is now currently believed that it can reduce lipid storage due to a reduced supply it can increase fatty acid oxidation it can increase the glucose uptake and glucagon could trade in storage and one of the mechanisms by which it could do that is that they can increase pyy and glp-1 and that will lead to an increase in insulin sensitivity and glucose metabolism so I just found a study that was published recently on how butyrate might affect meat quality and that might be linked to some of these effects on on these receptors and remote effect that you will have by adding butyrate this is a study where they supplemented 0.3 percent sodium butyrate two barrels that were housed two by two in 8 replicates what they showed in this study was that they had effects on the shear force they had soft the meat they had a higher Marling score they had a slightly higher intramuscular fat content and they had darker meat when they added the 0.3 percent sodium butyrate what they also showed in this study was that they had a change in gene expression of the different types of muscle fibers from from a mites hg one was increasing was am i m-i h c 2b was decreasing and that was also reflected in the protein expression of these two and finally it actually ended up in a higher percentage of low fiber which could probably explain that you have the darker meat another possibility in which in a way we're both butyrate could affect the portent is another example of an experimental study I would say where they have changed the starch in two different diets one of them was fitted gelatinize starts the other one was with raw potato starch and the argument of using this during this study was that apoptosis that meaning the slowing of the cells of dead cells from the god might be one of the factors that can affect a boar taint so if you could stimulate the flora and reduce apoptosis then you might be able to reduce for taint so that was the argument for doing the study and what they did show was that they had reduced apoptosis when they fit the group with the potato size because it contains a lot of resistance starts which they will then feed the microbes in the God and they also showed that they had a enormous reduction in schedule in the thesis of the pics that were fed potato starch and also in the plasma of the pigs however you have to be aware that this is experimental diets and it might not have much of a effect in in more real life in situation so I'm going to wrap up so I was just going to comment on that it could possibly also have any effect on the satiety and that is shown in this study here where they have increased the amount of fine growing pigs they changed in microbial community but they also changed the relative expression of some of the genes that are involved in the society like it you why why yes okay so the final slide I'll give to you is then all these data that I presented now what are the relevance to to pick nutrition well as I mentioned many of the studies are related to eight or lifestyle-related diseases in humans and they are mechanistic mostly intended for clinical science and this is not the case we have in in pig production however both the local and systemic effects that we have uncut homeostasis and there are monological health are definitely relevant for pig production particularly when we think about infectious diseases post weaning diarrhea long disease and so on the question is are the effects similar for short chain fatty-acids produced by gut bacteria or when we give it as a feed additive are the doses that I used physiological relevant then there is the second issue and the systemic effect on peripheral tissue and appetite and nutrient regulation and that brings me back to a presentation of bruno de silva here on the satiety on the south it might be that definitely by adding the right fiber and in stimulating butyrate production you can have a better appetite regulation you can have less aggression maybe in both slaughter pigs and in the south and the last question could be the effect on meat quality so with that I want to thank my colleagues who have contributed to the work that I've been presenting and I'm also very much open for questions [Music]