[Music] good morning good afternoon good evening ladies and gentlemen wherever you are to our live welcome to our live webinar today and as you'd say to your TV program fictional dot online it is our first live webinar on digital today and like usually each webinar starts with a basic introduction on the science and then we have a roundtable to allow you to question our speakers around the table do not forget to visit our fictional your fictional platform to revisit recent events you will see related to the topic of today that we organized last year and the year before some advances academies on strengthening the intestinal frontier on the development of the immune system and so on and there are still a lot of question today on my computer and we decided to focus this webinar on resilient microbiota and what could be the link with performance we have also recently launched a new type of program on feed channel dot online it's what we call the short educational videos we will do that also on microbiota to clarify some point but actually we start with a net energy to peak the objective is to give the scientific and clear basis of any point through episode of three minutes three to four minutes with expert of the topic and we started on net energy theory with dr. Jean Dubreuil who initiated and largely contributed to the development of a net energy in swine and now also more in poultry it is our tense fictional live webinar today and we are pleased to welcome lots of participants I just remind you that you can at any time ask your questions through feed channel dot live at gmail.com it will appear regularly on the screen to get the to remind you to ask your question [Music] president is somewhat a northern recent word used for a lot of things what do we mean by resilience in animal production it's a capacity of an animal to be minimally affected by a disturbance and to rapidly return to the normal states statutes of the animal and the with resilience were often linked with what we call stability what we calls to resistance robustness turnarounds and so on but what about micro biota and today we are focused on resilient micro biota how to define how to set up a resident microbiota what could be the factors that influence a micro biota and we are invited to well-known scientist Professor Richard neuchâtel from the Department of Pathology biology and avian disease at Ghent University in Belgium and he will talk later on and also professor even rich leak from the veterinary Research Institute from Brno in Czech Republic I let them introduce later on when they will start to talk as you will see our invitees largely involved today in the relationship between microbiota and the performance and so on to illustrate a little bit more I wanted to show this this figure to show that in fact in the normal state we are the anomalies in homeostatic state and there is some variation according to the situation and there is when there is a challenge either the status is changing so it goes from homeostasis to gallows disease and come back to the homeostatic States and when the challenge is too strong in fact it goes outside of the instances and go to the disease and to simplify that it's a little ball in the valley the ball can move up and down in the interval a to show that it's maintaining the status but sometimes when the channel is too strong in fact the ball goes in the next Valley and then has difficulties to come back to the initial stage so next slide explain what do we mean by resilience in fact and we already organized in 2018 and 2019 some seminar on strengthening seborrhea functions a gut integrity how to develop the reactive immune system but we wanted to focus today on what we call the resilient my computer what does that mean when we talk about the equilibrium between microbiota and host so now I would say what do we mean by resilient microbiota and I will last even to start to set up the frame of the microbiota in that I don't do everybody who is listening right now thanks for joining us it is good to realize what happens in chicken intestinal tract when the chicken is hatched and in this figure I am going to describe what is happening in the chickens he come soon after hatching and during the whole life in this experiment we follow the development of seeker microbiota in egg-laying hens in hay lane chicken line and we as indicated on the x-axis on regular intervals we collected three chickens from one particular job initially it weekly intervals slightly later on in monthly intervals and immediately after with the remind composition of seeker microbiota by 16s RNA sequencing and we observed that week the first week of life is characteristic for e.coli in the chicken cecum ecoli and Enterobacteriaceae dominate but the dominance equal oil asked only for the first week of life soon after during the week two three or four equal I is nearly completely replaced by gram-positive bacteria belonging to pyramids and different shades of green indicates different families within this within this view the most important numerically the most dominant feel our families are those which are named co-con say but fathers like rocks video say lucky bath talked about CMOS they are present as well usually with certain delay in this particular experiment when the chicks were one month old a new group of bacteria are highlighted with the magenta color with Port Alfred horn appeared and this bacteria belong to film of gram-negative bacteria feed on bacteria death as different remedies appear and disappear but you can also see that in this particular flock the development are still all for cat microbiota equal cat microbiota lasted for 20 weeks of life and when the chickens reached sexual maturity maturity numbers of gram-negative bacteria deters and gram-positive M equals nearly equalized and then remain in the same ratio nearly till the end of the till the end of the egg production of this flock when death of this rock so Brazilian microbiota looking at this slide I would say Brazilian microbiota is characterized by more or less equal representation of filming feuds and bacterias however exact timing when I said one month or sexual maturity week 20 this may change from farm to farm from plot of pocket it really depends on the external conditions as we will be discussing later on during this presentation when I introduced the development of a thicker microbiota and particle in one particular flopper my first fight it is true it happens like this is in most of the commercial flocks all over the world different people different research we will be grease on it except except for the exact timing but it is it is highly specific to the commercial conditions and when you realize that commercial current commercial conditions hatching raising of chicks are completely different from what from the situation to which chickens have evolved during the evolution then you may come to an experiment which we perform next and this is highlighted in shalida slide we realized something very trivial and simple the chickens have got to be hatched in a nest in an intimate contact with adult an adult hen as a source of gut microbiota so we just did what happens with the development of chicken gut microbiota when the chicks are raised in the presence in the inner contact with an adult hand as a source of normal healthy adult gut microbiota and the results were quite surprising or the contrast how different it was how the different microbial had development in the contact chicks on the right part in the right part of the slide and chicks which were raised in the absence of an how contrast differences we observed normal chicks normal enmity innocence of commercial production without without any contact with an adult hen without any source of half an hour type of microbiota during the first 6 or 12 days of their age their secret microbiota was dominated by gram-positive Hippocrates in a total agreement with a previous fight but when we placed chicks in a contact with an adult hand within a few days within 24 hours within single day gut microbiota under adult hand was transferred to the chicks and chicks on day 6 all day 12 of their life hard work already the microbiota of ideal type so when we are running a commercial production it's fine it works but we are losing something we are waiting for resilient microbiota to be established for longer period of time then it would happen in the nature if the chicks were hatched in a nest and I think even when you talk about the attachment of the microbiota with hen with the proximity with en it's quite interesting to see how in fact it helps also to get a more quicker resilient michel butor so I'll let you maybe talk about these points but I introduced what happens or compared would happen to chicks which are raised in the absence or presence of an adult hand this is in a direct consequence with chicken resistance to Salmonella challenges r1r infection so in the current this is this in this type of in this experiment we compared what happens if chicks on a day one on a day hatch on a day one of their life what happens if they are inoculated with seeker contents from one-call chickens three weeks all chickens 1628 or 40 way forty weeks called hands for chickens and we let the microbiota to develop dinner you know the name related chicks for seven days or one week and when the chicks inoculated chicks were seven days of age we turned them with salmonella four days later we check for Salmonella comes and we observe that control chicks which were not provided any microbiota we're highly susceptible to the molecule the challenge with Salmonella Salman accounts in the cecum reached 10 to the 7 of per gram of sickle content equally sensitive to Salmonella challenge where the chicks which we only inoculated with microbiota from 1 week old Tourneur chicks so one we gold one we gold microbiota is not res it doesn't it cannot be called resilient but once we in curated chicks on day one of their life with microbiota originating from Zika of three weeks old or older chicks chickens or hands these within four days within one week within four days they become resistant highly resistant to Salmonella challenge so concluding from this type of experiment having someone our challenges some kind of indicator of resilient microbiota microbiota which is usually present in three week old chickens or order can be already called as a resilient it's I think it's very interesting point so recap we introduced first the evolution with age and normally we wanted to give you the floor to complement what even side so please Rick take the floor thank you so luckily I heard what you said and I think it's a it's very very clear that at at one point the microbiome becomes resilient and can also confer confer resistance to facultative pathogens as even said but of course we need to ask ourselves the question where do these microbiota come from and how come that they are so efficient at colonizing specifically and the this filer that are associated with the intestinal tract it's always the same for in predominant phyla that come back how is it that that this is so constant over time constant over you know chickens in any environment will have these same four phyla dominating we should realize that of course these microbes are taken up orally from the environment that and it is estimated that on average which with each no that is swallowed the birds will take roughly 1 million bugs and this passed through the esophagus into the crop and in the crop because of the acidity that is present there a number of the microbes will be killed due to the acids and so in the crop the numbers drop and they further drop then in the province rigorous but we should also realize that you know the acid which is present which is produced there in high concentrations it does definitely not kill all the microbes fortunately not a lot of the microbes enter what is called a viable non cultural state which means that they survive but they cannot multiply and so they pass through the small intestine through the small intestine in these well so-called low concentrations in the way that they are low in in activity but they a lot of these microbes can pass through the small intestine enter up in the seeker and there they become active again metabolically active again so a lot of microbes are passing through the small intestine in a viable and cultural state but a lot of microbes are also killed and are present in the small intestine as dead microbes and it is estimated in humans that about 90% of the microbes in the small intestine and in a chicken that's essentially duodenum and jejunum that the microbes are present in as you know dead killed bacteria if we go into the to the next slides and just to join in with what even said regarding early colonization and you know when in contact with the hen the chicks are obviously colonized by bacteria deters from the adult hen but when there's no adult hand around and when the chicks are just you know set in an environment which has been cleaned and disinfected it is remarkable to see that it's not just random colonization and we should realize of course that cleaning and disinfection does not mean sterile environment there is always some microbes that are still hanging around and that is of course essentially the case for spore forming from Austria Syria and as you can see on the left top of this slide the first colonizers are actually clostridial sensu stricto which means in in other words it's mostly Clostridium perfringens so that means that a perfectly healthy normal chick will first be colonized by Clostridium perfringens and then in the middle at the top of the slide you can see its ratio which is essentially issues you're coli so we should realize that the newly hatched chick which is put in a clean stable is first colonized by two acidic buffer engines and issues a coli and that sounds very frightening but it does not mean necessarily that there will be serious problems the thing is that if fish are coli if it's not a strain with specific violence factors it will will help to reduce the oxygen content in the Sica and so it will make the seeker anoxic and that makes it makes the seeker available for the colonization afterwards by the Firmicutes especially by butyrate producing and we have taken here fecal a bacterium as a as an example genus these are the ones that are strictly honorable so they need the you know the reduced and the elimination of the oxygen by the ash ratio Jesus to be able to colonize and you see that they come up when the environment is strictly anoxic so free of oxygen but as you can see that we can confirm here the point that was made by even that that it takes quite a bit of time before these anaerobes which are essential for the period production of Beauty rates actually can start expanding and become a viable population and and on the bottom on the left hand side you can also see what happens with respect to lactobacillus and it's interesting to see that the lactobacilli rotifer but they are quite early in colonization of the seeker simply from the environment so a little bit random from the environment of Amedeo cheeky in the next slide we go if we go to the next slide and we can see here that this is just taken from an experimental this disruption of the normal robust and resilient microbiota by a range of different stressors and these stressors were coccidia and were antibiotic treatments you know a real harsh stressor and and on the all the blocks the left and block is each time the control group and the right-hand block is the the group that was challenged and that had disrupted its its resilient microbiota you can see that loss of resilience is characterized by loss of richness which means that you know a the numbers of microbes and numbers of different microbes is is used and below that you can see that there is also a lot of a lot of diversity which means that you know the different species different general numbers of different species general families is is reduced and and so resilience is actually characterized by a rich and diverse microbiota now if you if you put on the next slide and this is tip this next kill so the the green line on top the green line on top is the is the control group and the red line is again the group that had a disturbed distorted microbiota by the different treatments and the the when the block when the dots are blue it means that there's low abundance when the dots are yellow or even orange it means that there's high abundance and the only point I want to make here is that in a in a healthy resilient microbiota there is a high abundance of many different organisms so you see a lot more yellow dots than blue dots so initially you see the RIC that there is a resident micro biota and so on and you can also link that to the function of the micro biota you were mentioning in fact the polysaccharide degraders and the bit rate predictions then we after yeah we had already we had already even talking about the Salmonella colonization which made sure my computer so I think I give you the floor now even to explain what you had in mind with the antibiotic therapy and the consequence to show how would mean resilience also regarding the antibiotic treatment few minutes ago I had been talking about Brazilian microbiota it can be on a resume a resident microbiota that can be defined as introducing or raising chicken resistance to solve our challenge another alternative might be to characterize Brazilian microbiota based on their responsiveness to antibiotic therapy in the experiment which is due in the experiment which is shown right now on your screen we administer recycling who adult hands and the tetracycline therapy lasted for two days only and we determined composition of microbiota in fecal it in faeces in FIFA droppings and as you can see two days after the therapy diversity of microbiota dropped considerably each line each colored line represent one article microbial genus so two days after the therapy after introduction of the therapy all different bacteria are decreased in their abundance and the only bacteria which apparently increase in it abundance was a white line Enterococcus then we stop with the therapy or natal of experiment we ceased with therapy and let the microbial community to re-establish and as you can see on day 14 indeed that 12 days after stopping the therapy the microbiota neatly returned to its original complexity tool to its original divergence but it was not exactly the same as before the whole experiment because when we then reapplied again Tetris I'll import additional day today is from day 14 to day 60 of the experiment we again decreased the diversity of microbiota but when we then stopped with the therapy on day 18 the microbiota composition and microbial communities were unable to return to the original status and after second repeated anteater traffic in therapy we recorded overgrowth of e.coli fusobacterium or streptococcus and empresses so yes the robust resilient microbiota was present in the gut micro in the gutter in the cup of the hands included in this experiment before they spent two days that long to traffic in therapy affected negatively but composition and complexity could restore apparently but secondary of the second antibiotic therapy was already too much for the chicks in their microbiota and after second therapy with second cycle of tetracycline microbiota was no longer able to return original back to the original status and Composition that's good that's very good to clarify in fact what we means by your resident microbiota now how can we influence our car how can we start with resilient my computer and I let you event to continue on that aspects fine so it is a it is relatively simple to effect an effect composition of chicken gut microbiota but the problem problem is transition previous slide please this one thing the problem is to the transition from experimental conditions to the farmer ago and it expire under experimental conditions as you can see on the left part of the slide we can very easily a nicely handle one cheek by one order administration is absurd we have a control over the volume of probiotics or bacteria which we use for interpolation and once you enter the farm you experience growth much provider issues and problems distribution via drinking water of different back probiotics especially those which are strict anaerobes is not too efficient because bacteria are dying due to the oxygen present in the water you are imprisoning water so currently we are testing spraying of edging eggs or egg hatching in cheeks approximately 24 hours before the chicks are transported to the farm so still in doing craters still in the actuaries are spraying and this way of administration seems to be quite effective and we observed why homogeneous homogeneous colonization of all the chicks present present in the in the incubator apart and if I can now go to the next slide you should be pretty careful or one should be very careful on the selection of probiotics and I am now going to present pretty contradictory data or results contradictory to all our human experiments and experience here in this slide this is a collection of our of our gut anaerobes which we have been pure cultures and for which we have determined their whole genomic sequence so you know these bacteria are quite in a detail and those and these are phylogeny and I mean arranged according to 16s rRNA sequences but more important than the dendrogram and the number of strains are dots which are external to the dendrogram wherever there is a dot red or green we use that particular bacterium for oral inoculation of newly hatched chicks and seven-day lasts seven days later we check for the presence of the particle bacterium in a serum of inoculated chicks an inter dot is red this means that although we inoculated pure culture on day one of life to the chicks individual and carefully we didn't find this bacterium in the cecum one week later if the dot is green such bacterial efficiently colonized chicken intestinal tract so we administered orally on day 105 seven days later when we sacrifice the chicks and look at the microbe microbiota composition in the cecum we found this bacterium as nicely colonizing chicken cecum and to our big surprise for example seven different lactobacilli all originating from chicken intestinal tract when we inoculated chicks on day one of life seven days later we didn't find these bacteria this lactobacillus species present in the cecum we only we did not find any isolate of Actinobacteria as efficiently colonizing chicken intestinal tract after single-dose experimental or our inoculation and we also failed with persistent colonization with any of the tested isolates pyramid of family home in oka cart a very common bug gut microbiota members and common butyrate producers we failed also with experimental colonization with any isolate belonging to family atmosphere arts fair and instead we were successful with different representatives belonging to family to feel factory address and we were also quite successful it is quite easy to colonize chicks chicken intestinal tract with different isolates from family by only heart cell and I have discussed before earlier that these bacteria are characteristic by the presence of outer membrane on this informing as a part of their cell wall okay thanks even it's very interesting to see the difficulty in fact to implement the right microbiota and so on so knows only to manipulate or to change the microcut eyes maybe it's for the feed which are and can you comment on that what we can do with the feeding approach the beneficial microbes through the feed or to throw feed formulation or whatever we should first of all realize of course what do these microbes live on when they are present in the intestinal tract and and of course if you you know if you if you just take one step back and think about about the natural environments and nothing to do with human intervention you should realize that wild animals wild animals are continuously challenged for you know you know finding finding feeds and finding enough to eat and we should realize that in the wild ninety percent of the time the intestine is empty and so when it would be a really challenging conditions if each time that the intestine is empty the microbiome would get lost so nature has found this solution for for this issue that um you know the lower intestinal tract especially at the level of the seeker is producing lots of nuisance you can when you do histology you can see that almost one in two cells is a goblet cells or mucous producing self and so this large amount of mucus is actually meant to feed the microbes when the intestine is empty however when the intestine is full the microbes need to switch to another source of nutrients and this other source of nutrients are the fibers the indigestible non-starch polysaccharides which are not absorbed in the small intestine by the host now if you make a feed formula and is deficient in fiber then you see that the microbes again switch to consuming mucus but they are more numerous under these conditions and they excessively break down the mucus layer which means that the intestinal epithelium is in direct contact with the intestinal content without a protective mucus layer where normally defense molecules are present to protect them in the epithelium so so that we should keep in mind that the feed should contain fiber and then in the next slide if we can you know I can have slightly thank you we have done a relatively naive and simple experiment to look at what happens in these newly hatched chicks for which even clearly show that when they have not been in contact with the hen the development of the microbiome is delayed and is crippled what happens when you provide these chicks these young chicks with a source of fiber which is sufficiently readily available so what we did is we we mixed a low very low concentration and I'm mentioning here 0.2 percent of of similars this was amorphous syllables in in the feed as a source of fiber and we looked at what happened to the microbiota and it turned out that this is several rows actually supported the expansion of a family which even already showed in one of his slides that is the family Eureka not a CI and it's surprising to see that there is very scarce literature on Rican Elysee I so nobody is really interested in the RIC another CI and we didn't this family it was only one genus namely the genus Elizabeth that was expanding and that was becoming significantly more abundant when cellulose are present in the feet this is remarkable because his MST page is actually a dominant component of the microbiome of rabbits for instance which are by exquisite consumers of fiber so when it turned out that these these on this defense they can break down cellulose they convert the cellulose into six in eight and this section eight is taken up by the intestinal epithelium it's not used as an energy source by the epithelial cells because the epithelial cells in the seeker they use preferentially u.s. butyrate as their energy source so that don't usually the six and eight but they convert the 68 into rico's and they pass this glucose into the bloodstream so in this way the cellulose that is in the feed is actually supporting the glide glycemia in the in the cheek which is important in the prevention of hypoglycemia which is one of the major causes of mortality in young chicks now in the next slide I want to take this effect of this you know the nutrients for the microbes in terms of fibers one step further and we know that in in for instance cereals especially for instance wheat contain an Aspie fraction of of cereals is mainly composed of silence are a banal silence and these are vinick's islands are the source of substrate for the multiplication of the microbiota in the seeker there are the dominant fiber fraction but of course in this underdeveloped microbiota in the early stages as even clearly showed you and the composition the the composition of the microbiota is insufficiently mature to be able to really efficiently make use take advantage of the presence of these urban ox islands so we try we just checked here whether you can you can simply give help these microbes a little bit by enzymatically treating the in a species of weeds so we in you know in inner I mean in vitro condition we put any you know a complex of enzymes which is sent mostly contained exiling leaves this in contact with wheat and be simply harvested if the fiber fraction that had become a soluble so and then we added this soluble fraction at the concentration of just 0.1 percent to the feed of newly hatched chicks and we could show that the enzyme just broke down the arabic silence not to the mono mears not to the Audion not to the origami but to a degree of polymerization of around about twenty between fifteen and twenty so slightly longer but soluble and and then when we added this fraction to the feed of the Perales and we compared the soluble fraction from the wheat without enzymatic treatment versus with enzymatic treatment we could see in the right column of the three multicolored columns the right column you see that there is a significant increase in abundance of two families which if i'm already explained to you namely the Rubino khoka Singh laughter Spira CI which even experienced as being very difficult to you know inoculator to colonize the chicks experimentally but when they are present you can stimulate their expansion by providing them with suitable you know and I've slightly partly degraded fiber which is more readily available for them so what we do is actually here we compensate for the lack of lack of battery detests because the battery date is which even explain you all so these are the primary degraders of the fiber as these are lacking you can compensate for this deficiency by treating enzymatic treatment so they the enzyme is actually taking the place of what the enzymes of the battery deters would do and you make the fiber available for the butyric producing room the coca CI and love the spear CF and in the next part is just confirming that this also reflects in the right column you can see that this also reflects in the end products of the bacterial metabolism which is acetate and iterate so you get more production of acetate butyrate and these are very important metabolites which are sensed by the host and I would like to add here if I may that in this study we also looked at a particular type of cell in the epithelium of the seeker which is the L cells and we could be super counted L cells and so by this treatment we could show that these chicks had a higher density of L cells in their in sickle epithelium and it happens at these L cells they are the producers of glucagon-like peptide 2 which is the chicken hormone that stimulates the development of the small intestinal villi or in other words by stimulating the Beauty reproduction in the seeker you can indirectly through a hormone a natural hormonal pathway stimulate the development of the villi in the small intestine which will definitely resulted in better absorption of nutrients so that is the reason why enzymatic treatments may improve performance of of the animals and so what we do is actually shown here in this simple cartoon what we do is we stimulate the cross feeding pathway breakdown of Horace Horace saccharides non-starch polysaccharides into oregon saccharides which are then used by the microbes to produce acetate and finally butyrate and that litter it is then signal that is perceived by the host and that is resulting in better functioning of the small intestine even if the microbes are essentially present in the seeker as I told you earlier so if we go to the next slide and it's like this is just confirming that microbiota are also influenced by probiotics and that brings us of course to - to another topic of or another way of of supporting and stimulating the the microbiome that is through the use of of probiotics there are a number of probiotics that are directly or indirectly influenced the microbiota composition and you see here some examples here by which a probiotic is actually withholding the development of of firmicutes and and butyric producers and actually supporting the resilience of the microbiome but this is phenomenal we can take it even one step further and in the next slide in the next slide we see the effect of a new what I would call a new category a new family of probiotics you know the issue of probiotics is sometimes a bit controversial because in the past and with probiotics you have believers and non-believers I think we have passed this episode now and now we know that there are probiotics that actually indirectly influence the microbiome in the seeker but you also have probiotics that well for which you can expect that they won't have any effect any direct effect on on the microbiota in the seeker simply because the number of these probiotics are aerobic for instance the SilverStripe probiotics these are aerobic microbes so they need oxygen and oxygen is absent in the lumen of the seeker so you won't expect these microbes to install themselves and to maintain themselves in the seeker but there is oxygen present in the small intestine so this category of probiotics it it is it has its effect on the small intestine and what what I want to illustrate here is that well one of the probiotics which we were testing was a bacillus which remarkably we looked at its metabolome which means that we looked at which are the essential metabolites produced by this microbe and it turned out that there were two key metabolites that were produced by this microbe the first one is haibach's unting and if you wonder what is haibach something it is a degradation product of nucleotides now there's not many bacillus strains that produce haibach something but this one is producing a lot of hype ox hunting and we were wondering what could be the role of this hype ox something produced by the bacillus we could show that it was producing this hype ox anton in the lumen of the small intestine and then it turns out that it is well known in the literature that the epithelial cells of the small intestine they produce an enzyme called something oxidoreductase nobody is telling you what this secretion of something oxidoreductase actually does but it it is clear that something oxidoreductase can convert haibach something into something and into uric acid and so the conversion of ipok something into something into uric acid at each step there is release of one molecule of hydrogen peroxide so what happens is actually the epithelial cell is secreting something oxidoreductase close to its epithelial to the surface of the epithelium that means that very locally on the surface of the epithelium of the small intestine there's production of hydrogen peroxide and hydrogen peroxide is a perfect is infected which means that all bacteria that come too close to the epithelial cells that are involved in absorption of nutrients are killed and you support this protection of the small intestine by using a probiotic like this one that is generating a high pox on tea a second metabolites produced by this microorganism is actually niacin or nicotinic acid so that is a B vitamin but the important thing about niacin is that is efficiently taken up by the epithelial cells there's a receptor specifically for niacin and inside the epithelial cell niacin is predicted protecting the cell from oxidative stress or in other words the hydrogen peroxide produced on the outer surface and outside the cell will not damage the cell because it is predicted by niacin so it is remarkable to see that one probiotic strain is actually protecting the epithelium and helping the host to kill microbes that could interfere with absorption of nutrients and so other than that on the left bottom side of the slide you see another remarkable phenomenon that is that you know that the dark grey dots on the top line of the right bottom part of the slide dark grey dots are the control and the light gray dots are the birds that were receiving this probiotic in their feed and as you can see there is there is this line the third from the top line is alternating orange yellow orange yellow it clearly shows that the probiotic is actually suppressing the expansion of proteobacteria and that is indeed a very important phenomenon in the in the stabilization form of a resilient microbiota you don't want large numbers of proteobacteria in an adult microbiome so in the next slide in the next slide and you know we we were looking at at the the production previously we were looking at the production of PT raid by the energetic microbiota and the question is always raised by nutritionists okay rather than relying on hoping that the microbes will produce sufficient butyrate why don't we just put butyrate in the field and that is indeed a possibility you can put bitter eat in the field but the thing is of course that you need to be to the Petri to be released in the region of the intestine where the beauty rate is naturally a physiological released which is the lower part of the small intestine especially in the seeker and so when what we did in this experiment is that we protected experimentally the bitter rate with a special wax that allowed the major part of the bitter a to be released in the ileum and the seeker and as you can see on the horizontal lines the longest Horizonte line is actually the effect on the microbiome when the birds receive these wax protected Beauty rates in their in their feed you can see that there is a a clear support to the expansion and the abundance of the left-most PR CI which are butyric producers or in other words providing the bitrate at the level of the seeker is like a signal for the microbes to produce even more batteries so you get this amplification effect and and you can consider that bit rates are like a well you can compare it with a quorum sensing effect you know the microbes respond to the production of the bit rate and in this case they respond by expanding bit rate producers and on the right hand side you can see that due to the high production of butyrate the birds are better protected against salmonella thank you and I think my computer all the factors and design factors that can also modify push to well the more resilient my computer we have also the external factors like C ambient temperature fine sounds that could that could that may be in very quick words maybe Rick you can synthesize on the effect of the hot conditions and we will try to move on after briefly with respect to each stress we should realize that heat stress is on its own and oxidative stress and so when as such it will influence the microbiota you know the the to block horizontal blocks here they show you the difference between in stress birds and non heat stress birds with respect to microbiota it only tells you that heat stress on its own will create a shift in the microbiota and this is a destabilizing effect so it makes the birds less resilient and this you can see very well done in the next slide if we see the next slide and UK this is just an experiment where we challenged heat stress birds with Clostridium perfringens and so the red bars are the I'll show you the severity and the severity goes from 1 to 6 so left is that the small numbers and small areas of necrosis in the challenge model for necrotic and riotous and six is like complete necrosis of the intestinal wall due to necrotic and right is so same for sitting perfringens trial a challenge you can see that in birds and their heat stress necrotic and dry this is much more severe so that's one thing we should realize that this is necrotic enteritis as a model but it simply means that the intestinal tract is less resilient and that the the birds will experience a challenge as being more severe than their under heat stress conditions by the way when we try to relay resilient Maquoketa in performance even there is a lot of information and contact the contradictory information today on the on the price on the literature so I give you the affluent that nobody able to associate correct or ideal performance with the composition with ideal composition of gut microbiota if we go for the next slide here we are this is one table from a paper indicated shown in he Jordan shown on the same slide and you can very easily a nicely see the different papers each line is output of one particular paper inch line in this table and you can very easily see the different papers end up with different conclusions so lactobacilli for example are sometimes associated with good performance high performance and there are also reports and studies which report on associated with association of octobots along with poor performance with low productivity so it is really rather difficult typically is i think also of a material nature if you realize that intestinal tract cecum is populated by approximately 1,000 different bacterial species and many of these species many of the senior strains and species they have similar met whirring function so they can replace they can overlap they can replace each other and if one particle bacterial species is upset when the other for 1,000 is present nothing serious will happen in terms of productivity instead in terms of assyrians in terms of in terms of resistance to challenge with different pathogens but if I am going I am forced to come to some kind of conclusion then I would agree that those families and bacterial species belonging to the families which are highlighted with red Arabs so families remain ochre cartel atmosphere outside take Holly Valentina's they carry bacteria these are certainly bacterial toxin which can be associated with good Kevin good and correct development of chicken gut microbiota especially in the early days of life and ourselves were our own purposes on earth never published but um for our own purposes we indeed use real-time PCR specifically bacterium as a marker of positive development or indicator of positive status of chicken gut microbiota and we use several negative markers and these are crossed video perfringens for the bacterium or TV room and e-coli so this is more or less consistent with the table which is shown in this slide right Riccardi bacterium has a positive marker equal across tritium per fission as a negative and this slide this father confirms the how complicated is the whole situation method which you can microbiota high performance is Australian colleagues in three independent trials picked up from a1 frog but on the free independent occasions chicks which were over high body broilers which were of a high body weight so of indicator over high performance and chicks from the same floor chickens from the same flock which were over loud performance of the lowest body weight and compared gut microbiota between these two extremes so they avoided average average chickens and they didn't find any unifying pattern any unifying formula which would distinguish different she ate highly performing fertilize the performing chicks and their microbiota in the cecum in comparison to the poorly performing pads so indeed all although we can indicate some of the bacterial species but the attacks are which are usually associated with good performance final conclusion is very very difficult to draw nearly impossible to draw at the moment and I'm afraid that it is not a question of our current knowledge due to the complexity this will remain a challenge even in the future if we know thank you even and if we try to summarize what we say so we took a little bit more time but I I think it's good to wrap up a little bit so so key points we discussed and maybe a Rick you can address that all you mean one major one major conclusion is I think that maybe it's not only resilient microbiota which we want to look at but also resilient microbiota function microbiota function is indeed and you know there's there's a major functional major function of the microbiota which come back all the time and one of these major functions is definitely beauty reproduction so as I said earlier on there will there may be an effect on the L cells which then has indirectly influences the absorption of the variants in the small intestine and as even already pointed out and there's not such a thing like an ideal microbiota and because of the redundancy in there in their function one functional group may be replaced by another group that that fulfills similar functions and you know this a couple of years ago we developed the qpcr for a key enzyme in in butyrate production which is actually quantitatively measuring the the presence of at the gene coding for pity really acetyl create transferase so that is another possibility to address functional you know activity of the microbiota and and of course as as was discussed earlier on also it's not only resilient microbiota but in the intestine and we need to realize that the microbiota has an influence of the immune system of the intestine and so the tolerance of the immune system to words towards proteins in the feed and towards proteins of the microbes present in the intestinal lumen is of critical importance to allow absorption of nutrients so it's not only microbiota resilience its resilience of the entire intestinal system including the immune system of the host and also of course the third component which is critically important is the intestinal barrier the tight junctions in between the epithelial cells if there is leakage of these tight junctions the entire system of you know separating the intestinal lumen from the host the host of tissues and this is destroyed and that creates a condition which we call gut leakage gut leakage which is also a critically important factor in the entire in the entire environment and so nutritionally and it's it's very important that we support a balanced microbiome and there are definitely possibilities to to help the microbiome throw through nutrients and through additives that is for me the main conclusion Thank You Rika we will take a few questions and for all the questions that we could not we are not going to answer we will put that on the web right so we you'll get a response anyway I will challenge the speakers to answer to all the questions maybe the first question is that relevant to look for my computer marker it's good after what you say the rakin even just at the end is that relevant to look for my computer my backers of performance can we be confident on the reliability so who wants to start answering that it is pretty difficult right and probably there wouldn't be any universal marker here in a universal marker we if considering markers right I was talking about bacterial species and Rick was correctly talking about what about function so I stress that we use is he are specific to fake or bacterium and we usually the higher the more positive the PCR the healthier or the more positive indicator of healthy status of we believe that it is right but the same freckle bacterium of is a potent butyrate production producers and therefore the weekly rate transferred bit rate Kuwait transferase gene for this hornets enzyme is present in the genome of a fecal bacteria so following Rick's advice you would end up in the same needle in at the same comb or the similar similar conclusions so this is both is possible and instead of thinking of positive markers positive target hide the genes or bacterial species preparing more reliable results will be to look at the negative marker so if you're negative microbiota members so if you pick up and this is also what we do right Ecola as a one of not really directly negative but indicator of something something mistaken too much of e.coli an intestinal tract and usually indicates these biases so e.coli either quantitatively by college all by specific real time or any other piece connotative type of pcr Ecoline might be a nice negative marker of early performing or usually bullish performing parts Salmonella can be described for 3d embarrassing ends man might be the same and we also have positive experience as a negative marker with huzzah bacteria this bacterium is usually enraged although we do not believe that it is a direct origin it is an indicator that something is going wrong in the intestinal tract and you can see them so negative markers might be more reliable than looking and thinking of positive markers of good performing chicks and I have one one remark that is when you look at microbiota marker we should question ourselves whether we are looking for markers of performance or we are looking at markers for intestinal health and I think this is not the same I mean you can you can if if you feed a birds a very poor feed with a lot of fiber the bird may be perfectly healthy but would not be performing then on the other hand if you want to maximum performance especially with modern breeds these have been these birds have been selected they have been selected for generations and generations based on the performance which means that if you want to grow fast you need to eat much that's what they say to the children as Welli if you don't eat you won't grow now these birds have been selected for feed intake to the point that they are over eating all the time over eating means that the absorption capacity of the small intestine is exceeded and that means that digestible nutrients come available to the microbes of the seeker and there and the seeking the seeker and the ileum of course and there did the most versatile microbes will take advantage of this and then of course if you have the choice between darn bread and cake and you know you will prefer the cake and you will eat a lot more of it that's what happens with these microbes like e.coli they can switch from one substrate to another easily and also the lactobacilli and I think that may be part of the explanation for what you said earlier on if I'm that expansion of lactobacilli it can be a good thing because the lactate may be converted by the electrosphere CI into butyrate and that is definitely advantages but when they lactobacilli are expanding too much it's it's an issue and they they can switch they can switch from fiber to too rich protein rich to whatever substrate because they are so versatile and they have so many enzymes available so I think we'll we'll have to take into account that you know markers for intestinal health is not synonymous with markers for performance good point maybe you're more practical questions maybe not easy to answer should we formulate feed taking into account my computer parameters and how to do that we wants to start well it's it's an actual question because it's in it's a practical question but then on the other hand it's a little bit of theoretical question also because so far you know formulation of feed has been based on least costs linear formulation which means that you you put in your requirements of the birds and then you put in you know the cost of the different ingredients which you want to use and the composition of these ingredients with respect to nutrients and that makes the formula but in in this linear formulation nobody has ever put in a requirement for NSP to the contrary usually people look at reducing NSP as much as possible because it's considered to be a diluent you know if you have a lot of NSP there's less room for nutrients in in the feed composition so so if we want to take an SP into consideration it's not easy it's definitely a big challenge for the nutritionist and it's to some extent it's probably impossible so the alternative solution at the moment is look what is present in your formula and and be informed at least even if you don't steer your formula with respect to the NSP composition you can easily see the NSP content and then depending on the dominant fraction of the NSP whether there will be a lot of cellulose or whether they be there will be a lot of arabic silence whether the Arab in excellence will have a lot of a lot of branched side chains of Araby nodes or less branched and the side chains of Araby knows whether there will be more monomers in defeat or not you can make a choice with respect to what you will add to support the use of these NSPS by the microbiota and in that sense I'm not I'm not really pleading for using lots and lots of enzymes but strategic use of the right enzymes to to provide a substrate that can be used by the microbes will probably be easier for the nutritionists then actually formulating based on the requirements of the microbes so in my opinion it's it's it's much more feasible to to add enzymes or even to add three biotics as a source of substrate of of the microbiota rather than then completely changing the formulation because that may make the feed too expensive it's an interesting point Rica it's managing compromising I think event want to compare my complete mountains that answer this question should we so my answer would be weekend but I shouldn't right I should I would do I wouldn't place this in among my priorities right to really address or make a feed which would select for a particular bacterial species present in the gut microbiota and the reason is that there are so many different feed producers so many different ingredients and so many different gut microbiota members that you never will met all the combinations and you will solve one particular feed its composition with an ideal microbiota but just in the neighboring district in a neighboring farm this will not be working because they will be using feed produced by another producer of slightly different composition and squad so this way of thinking probably I wouldn't it is possible but I wouldn't place this into into among my priorities however and we denies disagreement with is good enough in a nice contradiction we are running experiments like this we are considering experiment like this at the moment for our and all the experimental purposes and how to and to learn more about microbiota and assembly of such experiments is quite simple we take vehicle material thicker or sicker materials or figures from microbiota and with this we inoculate feed produced by different producers of different composition and if you subculture this anaerobic leaf for two days and if you perform several passages you start to enriched enrich the growing bacteria supernat Unwin bacteria which are positively selected by particular feed of its particular composition so you can do this you can prepare microbiota mixtures which are ideal is set to digest or to deal with party with feed of particular composition but as I say in particular cases this might be interesting this might be useful but in general in a broad scale in a broad business sense it doesn't make any any real sense except words very very specific cases so it's an interesting area but I probably do not go for this very interesting even we focus mainly on on broilers but we have also question on layers and because you say that other birds are more resilient microbiota but what is a variation of microcut in laying hens is there any critical stage what about prolonging laying cycles beyond hundred weeks you know that it's a good very frequent topic in conference today and would that affect sensitivity to sanitary challenge or patents I wouldn't be considering any any stage is a really critical right each day each week each month might be critical if something mistaken what happens if feed is contaminated with with fungi with anything may happen otherwise the development of the commercial conditions will be well constant in the middle anyway end up in the final robust resilient microbiota which is characteristic for for adult hands and their feed composition we observe certain variation at the time of around reaching the sexual maturity and around the egg production but although there is some there are some disturbances in changes in variation after all its we do not consider this as extremely important and serious so if we always consider providing probiotics or competitive exclusion products at the very first days of life and then usually you do not need to be to worry that much about particular periods of life except for the situation following the antibiotic therapy or following different kind of infections which may happen in your flocks but otherwise I probably would not pick up any any any period of life as a extremely critical and specific for for egg laying hands yeah Rick maybe maybe I don't completely agree that's good to have a debate because of our some experience which we had here in our experimental stables where we do Salmonella challenge you know we have a reference strain of Salmonella enteritidis which we use as a challenge strain and many years ago we checked how what a dose of Salmonella enter it it is of this strain was needed to to colonize the seeker of 100 percent off of birds and so when so it was when we did this in inverse of less than one week old and as you have also shown their microbiota is is immature and they are extremely susceptible to salmonella and indeed we could show that in those days we showed that less than one colony forming unit was enough to have each and every bird colonized in its seeker so so the early post hatch period is both in broilers and in layers is a critical period but then very recently we we we or more recently we we have this challenge model where we look at them at susceptibility of birds later on in life and when you were when you challenge say birds of ten to twelve weeks old with Salmonella and and here now I'm looking at intravenous challenge so you injects a Brella and thread this intravenously and when you do that with broilers of five weeks old you inject log nine which is like 1 billion Salmonella see a few of salmon are intravenously they they are perfectly normal so they are not ill they they they continue eating and nothing happens and so we thought well do they are they so fast at clearing the Salmonella from the bloodstream and the answer is no because we could culture the Salmonella from the bloodstream and we tested this for up to a period of two weeks and sit still after two weeks we could still culture Salmonella from the bloodstream without the birds showing any signs and that I think is also a consequence of of broilers and being selected for low responsiveness to gram-negative pathogens so they they seem to be tolerant to to endotoxin so so that period of the life of the birds they are not very susceptible to some another at all and now very recently we did a challenge trial and more recently with a the Challenge trial where we injected laying hens intravenously with Salmonella enteritidis of lot 9 and we collected eggs from these birds the first week day the egg-laying was really down so they didn't suffer to some extent from the Salmonella challenge more so than the broilers but the second weekday they started producing eggs at the normal level again we collected these eggs and and this was just to look at whether a vaccine was either or not protecting against against egg colonization and we could show an effect of the vaccine but still the birds were you know the susceptibility of the birds were relatively limited this was birds in in lay and now only a few month a couple of months ago we did the same challenge with birds and pointedly so at the round about the time they were laying their first egg and then we saw that all these birds become became within within hours they became extremely ill and they were starting to die so we had to put him down and when we did necropsy this was a Salmonella and rittany strain you know but they did damage to the to the intestine the internal organs not only to the intestine but much more so to deliver to the ovary and to the oviduct was extreme so I would say that point of lay is definitely another critical period in the life of the birds probably because of the stress of the first egg that is being laid and because of the the change in the endocrine system of the birds that this is a critical period and then of course there is the American Studies you know gassed and co workers they have worked a lot on the molting period so at the end of the first laying cycle the birds become much more susceptible to some another again that has been documented clearly elegantly documented these American Studies so I think extending the lane cycles is a risk factor in the way that you get close to the mole things and that the birds will become more susceptible while at the same time the protection that can be afforded by vaccines may be fading away thank you Rick I've got a long list of question but I will take the last one received look she is a guy who posted the last one because I do I took his question is a [ __ ] equation is there a possible negative effect of selection of i producing strain on the microbial microbiota composition and ecology so again we had we had several questions on genetic link between microbiota in genetics and you know in the last publication there was some difference but I would say a quite interesting question is there a possible negative effect of selection of high producing birds on the microbiota composition in ecology who wants to do answer first when we are talking about resilient microbiota we should be aware of a couple of physiological points and usually at least me I am associating resilient robust microbiota with the prolonged ability of chickens to survive to reproduce to live for a long period of time however this is not the case in the broilers in which case chickens live for something like thirty five days and in that case you may not be interested in highly robust microbiota in that case if you secure if you ensure that your farm will not be entered by by any pathogen if you're a thorough hygienic rules in the hygiene your hygiene is at a very very high level then you do not need that complex microbiota resilient perhaps you may also consider that the less complex microbiota the lower will be the competition for nutrients world microbiota members when to yourselves right then you'll also use something out of the nutrients which are consumed by the cheek from the feet of the cheek so high producing highly producing large chicken lines perhaps s lower average form of dysbiosis may even result in their high production and this might be also the effect of antibiotics as I used as a crow crow bottom they suppress full widespread distribution multiplication - of microbiota forming of the complex and nutrients present in the feed are all available available to the chicks chickens so that's one possible view and second we are now testing mixture of ten different bacteria are different from lactobacilli different from the Phaedo bacteria are mostly common mostly consisting or containing different species from Bacteroides genus or mega Moniz genus and this mixture of ten different bacteria we use equally for egg players as well as for boilers as well as for meat types meat lines and although there is some variations there are differences how efficiently each of these ten different species presenting our probiotic mixture colon is either egg layers or broilers again my feeling is that these differences are more dependent on other circumstances other environmental factors present in death or this or that particular farm rather than chicken line definitely chicken line will also contribute but in most of the cases other particles of an environmental origin feed composition will affect the microbiota composition and they're associated with different line chicken lines move to greater extent in microbiota itself after all inside the intestinal tract there is a constant temperature around 40 degrees centigrade strictly on aerobic conditions and this is the same both in broilers as well as well as in eclairs although they might be smaller physiological differences these factors will dominate over the others right oh yeah so maybe Rick you wish to complain mountains it sure I fully agree but even maybe I can add the following it is obvious that the slow growing birds have less issues with dysbiosis compared to the high-performing strains so where there is definitely an effect of of of the strain and of the this growth curve and it's difficult of course to distinguish between growth curve and actual genetic backgrounds of the animals because you always have the influence of both and but of course what we should also realize is that high-performing strains they are slaughtered earlier because they grow faster so when that means that you are actually slaughtering at an immature immature animal which for which the stabilization of the microbiota may be less advanced because they are younger but then on the other hand you could say okay this is a problem so when what can we do about it well I think on the other hand of course what you get is you get a window of opportunity as even pointed out earlier during this webinar the you know the development of the microbiota takes time and and that can be considered as a major disadvantage but we should realize of course that it is not always possible to have contact with with the laying hens definitely not so when so we should you can consider this as disadvantage but you can also consider this as a window of opportunity in which you can by using you know it's the right ingredients and the right combinations and steer the microbiota in in a direction where you wanted as for instance was the case when we were adding this cellulose to defeat or when we were adding his enzyme treated treated soluble fiber to the feed that is these are opportunities which I think we need to take advantage of and which can really help especially in moreso in the fast-growing the brie breed stand in the slow growing breeds all the question will be on swirled on the form on the webinar so really visit www.imtcva.org and we will have special recording also and also I mentioned earlier short educational video series starting with net energy and we have others in preparation so just visit you a fictional dot online website and again thanks everybody don't worry we will answer to the question and before to final close please on behalf of the whole team here we wish you good health and take care thank you very much bye-bye [Music]