[Music] we've all worked hard today already so this is the last talk and I will try and keep it short and simple so well our eminent hosts published in 1996 an important paper which has been cited many times already today and they were stated that as soon as the temperature passes over 30 degrees that this means heat stress and when you look in the literature and we've heard it several times already today it's obvious that there is a difference between acute or even repeated acute heat stress versus chronic heat stress and it makes a lot of difference and I think it also relevant to different conditions in different countries I think in Europe we have spells of heat stress but they are short only during short period of the day and whereas in other conditions of course this is more like chronic and of long duration and so when I actually when going through the literature of course I came up with the same sort of data which have been shared with you already several times today and these data they are they come back they come from different papers and this sort of sums up the most important observations that were made in the literature and a lot of them are directly related to to intestinal health so where I would like to focus on on a few of these with respect to what they mean when we're looking at the health of the intestinal tract in the animal and one of them is definitely the reduced antioxidant capacity and of course that brings us to the the previous presentation we all know that antioxidant capacity and and you know oxidative stress is very important for muscle but we sometimes forget that actually the most metabolically active cells in the body are the epithelial cells of the intestinal tract so if there's a cell in the body that is very susceptible to oxidative stress it's the intestinal epithelial cell and of course we know that the protection against this oxidative stress is mediated by a number of mechanisms that are incorporated in the cellular metabolism and one of these new metabolic pathways if is of course related to the superoxide dismutase had this superoxide dismutase and it really is key in the protection of the cells against against oxidative stress and of course we know that in the intestine in the intestine when there's oxidative stress and there's damage to membranes this will inevitably lead to inflammation and so inflammation is one aspect which is directly related to heat stress inflammation in the intestinal tract luckily for us the intestinal tract has been you know coping with inflammation for other reasons as well so this is just one one trigger of inflammation on top of all the other triggers that are already there but that is also probably the reason why under hot tropical conditions when you look at issues like dysbiosis and intestinal stress they seem to be much more severe and and and probably this is the reason why we are in Europe we are have maybe an easier life in trying to cope with these bios's and trying to cope with with other stresses to the intestinal tract because in tropical heat stress conditions you have this additional pro-inflammatory factor that comes on top of all the rest so in the intestinal tract there are a number of feedback mechanisms which which sort of dampen down inflammation and I think we should keep this in mind heat stress is just one of the different stress factors that affects the intestinal epithelial cells and so the way we handle them is to some extent similar as the way we handle other stressors of the intestinal epithelial cells and that is also for instance through supporting the dampening down mechanisms so the negative feedback mechanisms on the intestinal inflammation and one of these dampening down mechanism pathways is the P power gamma pathway which which reduces reduces intestinal inflammation through suppression of the NF kappa-b pro-inflammatory pathway and this is just a simple practical list of known known feed additives feed ingredients that can act on the P power gamma pathway that have documented effects supporting the P power gamma pathway and this can you know like dampen down inflammation the intestinal tract and one of them I want to stress because it'll come back in some more slides and I all will also want to show you draw your attention to this one to the glutamine which was mentioned earlier on by dr. Michael kit as an important factor so when is indeed something that seems to be quite universal then there's the aspect of heat stress increasing susceptibility to disease and we actually worked together with a group in Greece some time ago in using our model of necrotic and righties and using repeated acute heat stress we could show that indeed the animals become more susceptible to necrotic enteritis this is just numbers of birds that have almost no lesion or extremely high lesion and you can see the red bars show you that there's more birds with high lesion score in the necrotic and rightist model in birds that have been repeatedly exposed to heat stress which means that under heat stress conditions necrotic enteritis might be more severe so indeed each stress is increasing susceptibility to disease but one one of the most fascinating aspects I think is that heat stress is known to increase intestinal permeability so it induces gut leakage as as we were told this morning already and this is not something which is unique to the chicken because there's a lot of interest in in human medicine looking at athletes you know athletes doing extreme efforts body temperature rises due to the efforts and then you also see increased intestinal permeability now gut leakage due to heat stress and then you come to think of gut leakage can also be induced by say starvation if you starve the birds for even only twenty two hours you already increase the permeability of them of the intestinal mucosa so here again heat stress comes on top of different other stressors at the level of the the epithelial cell and so that brings me to my favorite topic and I apologize for those who have heard me before because I tend to repeat myself but it's very fascinating to see that what is happening actually in case of heat stress is the tie junctions between the epithelial cells are damaged for the simple reason that due to oxidative stress the epithelial cells have a hard time of producing enough ATP to maintain that maintain the tight junctions high junctions maintenance is actually what costs the epithelial cells most of their energy and so when there is a leakage of the tight junctions that means leaking inside out towards the intestinal lumen but also leaking outside in which means that there's compounds that are present in the intestinal lumen that pass in between the epithelial cells which they should not do in case of ty junctions being closed that should not happen but when this happens there's fragments of the microbes that are present the intestinal lumen that come into contact with the basolateral side of the epithelial cells where you have two toll-like receptors that sense the presence of bacteria and for our body the presence of bacteria in between the epithelial cells is a sign of danger and this sign of danger will induce the cascade of NF kappa-b activation inflammatory response consequence of that is a vicious circle of ty Junction disruption so ty Junction disruption leads to activation of the NF kappa-b cascade pro-inflammatory signals activated inflammatory cells release of pro-inflammatory signals again and these pro-inflammatory signals in turn again will damage the tie junctions you can measure this there is a tool available which I think is really useful but technically very tough and that is using icing chambers you take the mucosa of the intestinal wall of the chicken you you create an electrical electrical charge and you measure the resistance of the mucosa and that is a direct measure of tie junction tightness and by using this you can measure effects on tight junctions and this is just a simple list of of nutritional factors that have been shown in the literature to stabilize tie junctions and to me this is important to realize that tight junctions tape stability is essential for the defense of the animal against a lot of pro-inflammatory signals but also against the negative effects of heat stress on the intestinal barrier integrity so here again we come up with luta mean and with beauty rates and then this one I think is also very important was mentioned already this morning leaking from the intestinal lumen in between the epithelial cells will result in lipopolysaccharide from gram-negative bacteria which is a very stable molecular complex leaking in between the epithelial cells entering the bloodstream and then you get endotoxemia fortunately the chicken is very resistant to endotoxemia it will not die from endotoxemia but the endotoxemia causes an inflammatory status and so that adds to the inflammatory signals which were there already and so what happens then is in case of leakage not only LPS like a polysaccharide will pass through but also fragments of bacteria and intact bacteria can pass in between the epithelial cells and enter the bloodstream we call this the Kiril translocation bacteria when they enter the bloodstream blood from the understan goes directly to the liver through the portal vein when it arrives in the liver these bacteria ending up in the liver they normally should be eliminated by the cooker cells the macrophages of the liver but because of the increased bacteremia increased bacterial translocation there are areas in this liver where the bacteria are maintained and where they cause necrosis and the white spots which you can see in the slaughterhouse when you go in the slaughterhouse you can see these white spots on the liver of the chicken that's actually necrotic fahsai due to bacterial translocation so to me it's very important that nutritionists should go to the slaughterhouse they should not stay behind their computer go in the slaughterhouse look at the livers of the chickens you will immediately know when there's a problem with leakage of the intestinal wall and then the other one I think which is important is that not all of the bacteria are eliminated from the bloodstream in the liver so they start circulating and it's fascinating to see you know at one point we did an experiment in which we injected a Salmonella bacteria intravenously to broilers injected in the bloodstream and then we took blood samples at different time intervals and we continued up to 14 days and after 14 days we could still isolate Salmonella from the blood stream which means that the broiler actually tolerates bacterial translocation tolerates bacteremia and that means that bacteria that are in the bloodstream they can end up they can end up in the bone marrow especially in the bone marrow of the femoral head and then you get this femoral head necrosis and you can see this when you open up the bird and you dislocate the hip joint the femoral head will break that's due to bacterial translocation histologically you can find colonies of bacteria in this femoral head so what is an the effect on the intestinal microbiota because pierre andre asked me explicitly to comment on this and there is surprisingly little information in the literature I found one paper which i think is very fascinating don't look at this because it's it's a it's not very clear but the take-home message is is very simple the pink here is Firmicutes so under terminal neutral conditions in the in the seeker of broilers you have a dominant population of firmicutes under heat stress conditions there is shift less firmicutes more lactobacilli and then people say oh lactobacilli is good for you here are the fema coots here are the here are the Proteobacteria the lactobacilli are the ones that produce lactate this lactate needs to be further converted into propionate and butyrate if that is not the case you get accumulation of lactate and that is very harmful so if you have too many lactate producers it can be a problem and indeed I came across this study where people were looking at at at heat stress conditions and they were measuring serum D lactic acid that's lactic acid produced by the microbes because the host produces a lactic acid if you find the lactic acid in serum it means that there is a excess of Lehi production of lactates in The Seeker that spills over into the bloodstream so it means that there's overgrowth of lactic acid bacteria and not enough Firmicutes to convert the lactic acid into Beauty rate and propionate so that's what happens under heat stress conditions too much d lactic acid which may even cause lactic acidosis and so d lactate is no problem as long as it is converted into Beauty rate and propionate and it will fuel the beauty weight production through the Clostridium cluster 14a strains which can convert d lactate into Beauty rates but when there's excess lactates then the pH drops then the firmicutes die-off because at a low pH and the Sica Firmicutes cannot survive they die off and there's less Beauty rate production and that results in inflammation so if I had of time mr. chairman for a couple more minutes I would just like to you know there's different ways in which we can stimulate bit rate production in the Sica which is important for the stability of the tight junctions reiterate reinforces the tight junctions you can add it to the feed or you can enhance the endogenous production and I would like to to comment on a on a study which we did and which we which was published actually last month in poultry science what we did is we we we took weeds soaked it with water either incubated it or not with a exile anaise complex and then took the water-soluble flat fraction so the the fraction of what you know the Cylons that have become soluble and then we could show that actually the treatment with the enzyme shifted the degree of polymerization the degree of polymerization to a band which is like between 15 and 30 and this this was then used we use this as a as an additive you know the the extract from the weed was then added to a feed formula which already contained a lot of wheat so it was a wheat soy based formula and we added only 0.1% of this fraction which was like obtained from weed being treated with enzyme in vitro you know in the lab then added at one point at 0.1 percent to the feed and we could see that these actually stimulated feed intake during the start appearance and as we have heard today feed intake is an issue under heat stress conditions and this resulted in a significantly better body weight gain so this wheat extract at just 0.1 percent significantly increased body weight gain and an Fein take in this starter period and we could also show that by simply looking at the t lymphocyte infiltration as a as a parameter for inflammation we could show that there was a significant reduction in the inflammatory response at the level of the intestinal mucosa and on top of that to me the most fascinating aspect is the you know the L cells these are endocrine cells producing glucagon-like peptide one and glucagon-like peptide two which we heard already today are controlling feed intake and are controlling village length in the small intestine and it turned out that by adding is just just simply 0.1% to the feed we could significantly increase the number of these endocrine cells these are cells that are sitting in between the epithelial cells and they produce this glucagon-like peptide one and glucagon-like peptide two and you increase their density so this explains the mode of action actually of this in their study then looking at at the microbiota we could show that it was a significant increase in lucknow spirit CI and reno coca CA which means that we restore we restore the firmicutes the butyric producers and we could also show that was an effect on on short chain fatty acids especially butyric acid and acetic acid production so to conclude i think the the most important aspect to take into account when you talk about heat stress is that it this is an oxidative stress it's an oxidative stress so it's a pro-inflammatory stress and so what you what you see is that you lose beauty rate producing firmicutes and that's the key problem under heat stress conditions and we know that beauty rate is so important with respect to its anti-inflammatory and antioxidant we you can protect against heat stress using different tools oops sorry using different tools but I just illustrated one of them and I think we won't make it with just one - I think the future is - to go for the right combination keeping in mind that you know this shift in the microbiota is very important under heat stress conditions thank you very much thank you Richard really saved a lot of time thank you very much so the questions do you want to read it here so it's propionate production of parameter as important as butyrate production probably propionate and butyrate both are energy sources for the epithelial cells in the lower intestinal tract the epithelial cells of the upper intestinal tracts don't use propionate or Beauty rate they use glutamine so we've seen in several presentations today how important glutamine is under heat stress conditions and this may be part of the explanation because glutamine is the energy source for the epithelial cells of the duodenum and the jejunum if you compare propionate to butyrate both are energy sources for the epithelial cells of the lower intestinal tracts but on top of being an energy source butyrate also is a histone deacetylase inhibitor which means that it shifts the expression of the genes in the epithelial cells towards in an anti inflammatory phenotype should I take the next one also yeah thank you yeah next one this means giving lactobacillus as probiotic could increase lactate and then decrease Beauty rates I didn't say that I said I said that excessive lactate producing by bifidobacteria or lactobacillus can be a problem when you use when you lose lactobacilli as a probiotic what you use is actually should we say ten to the ninth ten to the eighth not more than that you're talking about numbers that are like way below the numbers that are naturally present there and then taking into account the lactobacilli they don't produce spores so 95 to 99 percent of them will die passing through the pro ventricular you know passing through the upper part of the intestinal tract so what you add in terms of numbers will not cause excessive lactate production I'm not saying that it will not have an effect I will I'm only saying that it will not cause excessive lactate production thank you the last one is a really good concluding question sir feed intake is decreased with heat stress is there evidence of the role of gut microbe microbiota and feed intake the answer is definitely yes because the L cells which I was talking about they have two receptors for Beauty rate which means that they are influenced by the firmicutes that are present there so if your numbers of Firmicutes reduce there's less Beauty rate signal going to the L cells and that is probably one of the mechanisms by which because you know L cells produce glucagon-like peptide 1 and glucagon-like peptide 2 and glucagon-like peptide 1 is very decisive for feed intake but I'm not I'm not telling that this is the whole explanation this is just you know tip of the iceberg probably thank you very much please so anything [Music]