[Music] good morning first off I'd like to thank the folks at a deseo for inviting me here today and thank you for challenging me at least to as we say set the stage for a lot of today's discussion right as we was mentioned already we're focus really is trying to understand this frontier of the intestine and really this dichotomy if you will and nutrients supply and what we know at least in terms of how it responds as a barrier function I think the main thing I want you to take from my message one is to set the stage for the rest of our speakers this morning in this afternoon but to to realize that the intestine is what we call a very plastic organ it responds to the environment to which it has to in terms of nutrients in terms of ingredient supply and in terms of challenges that are presented to it either from pathogens or other types of substances that cause it to respond now as Megan mentioned in general we think of the intestine is a very large maintenance type of organ system it requires essentially one out of every five calories for maintenance if we think of it from turnover of protein it turns over at a rate of 50 to 75 percent per day and one out of every four calories produced or one out of every four proteins produced by the body is synthesized and really secreted into the gut and these are very general types of maintenance numbers if you will and they readily change again as that dynamic of the intestine is put forward so we're going to hear a lot about those different types of functions and adaptations throughout today as to what those responses by the intestine are so as an example if that intestine has a pathogen affecting it it's response is going to be to try and rid the body of that pathogen more quickly so the way that it does that it changes rate to peristalsis we get reductions in the amount of dry matter that's actually absorbed by the body if a cell or an entero site is infected its response is to essentially move up the rate of turnover of that tissue and try to replenish those those effective entero sites more quickly the secretory immune response is going to be upregulated to try to produce more musin to try and move things more quickly through the digestive tract we see changes to overall functions and cellular and transport functions in the name of para cellular barrier proteins changes to secretions changes to gene expression and we'll also hear later on this morning about some of those immunological influences and microbiological influences and alterations that are affecting so I want you to realize that this is a very dynamic system and it's very hard to ascribe a singular type of response and this response will be changing and that's why I think a number of different people throughout the scientific community have begun to essentially try and look at this very complex system and complex reaction and I'm happy to see a number of collaborations across disciplines beginning to occur much more than what we have seen in the past so for my presentation I'd like to just go through a few of those ascribed functions as you see here to the intestinal tract and just give you some examples of those dynamics that are actually occurring so again all of these changes really change them as we saw in the very first slide the protein and energy demands for that tissue so first of all let's let's think about this change in peristalsis it's challenged by different pathogens it's going to change the rate of reaction so I put up the slide just as an example of one of those challenges that we tend to see and realize that the change in response by the intestine to any pathogen is really going to be dependent upon load of the pathogen virulence of the pathogen and we also see variants and responses across individuals within that population so it's very hard to ascribe and predict as a nutritionist what I need to supply because we never know those three things the load of the pathogen the virulence of that pathogen and how that individual is going to respond so as an example just to give you changes in digestibility that may occur due to presence of a pathogen one of the ways we used to do this in our lab was just by using a coccidia vaccine and what you see here is a twelve times normal dose of a coccidia vaccine given at 14 days of age and we made our measurements at 21 days of age if you see on the energy side here we see a dramatic reduction and the amount of energy use by the animal if we look at the on the the right hand side of this graph the white bar is indicating the dry matter digestibility reduction roughly from a seventy-five percent down to about sixty percent and our nitrogen apparent nitrogen retentions from about sixty-five percent down to below forty percent and I mentioned this and I'm going to come back to this particular study as we move through the presentation just to reiterate reiterate some of the examples of where we're looking at what is happening so as we think about this focus mainly now on that change in dry matter digestibility alone going back to the change in peristalsis and moving things more quickly through the digestive tract if nothing were to change in terms of absorptive epithelia we do get this change at least then and drive at a retention the other thing that became interesting as we looked at this particular challenge we were trying to ascertain changes to standardized amino acid digestibility and with that what change in endogenous amino acid production and flow was happening within the digestive tract so I put this just more as an example of what was occurring and you'll get a chance to look at this a little bit later on in the presentations and you can pause and take a look at that but I just wanted to get you a flavor of what is happening what seems strange to me as I put this slide up here I was expecting the endogenous amino acid production and flow to essentially be higher in the challenged state versus the non challenged State in fact the reverse was actually true we had a twenty to forty percent reduction in the terms of being present with the challenge across our essential amino acids but I think the thing that is important to point out is that our measure is per unit of dry matter intake right so our endogenous amino acid production per unit of dry matter intake was going down let's relate that then to what that means at least in terms of standardized amino acid digestibility essentially taking into account that basal endogenous amino acid flow again our dry matter is going through more quickly right on a per unit basis that was a little bit less in terms of endogenous amino acid flow but our digestibility was going down at least by about 15 to 20 percent so when we go to a correct for that on a standardized ileal digestible basis you can see that we have a dramatic change at least in terms of amino acid digestibility and here I only put the essential amino acids dr. Kidd we'll talk a little bit later on this morning about how some of those in particular are responding the one thing I think is critical to to understand if we're thinking about the ideal amino acid concept that ideal protein may change as we're going forward to respond to some of these challenges right we know that the acute phase proteins and I'll talk about them a little bit later on the profile of those changes during the course of an innate immune response and that response to respond to some of these challenges and invasions within the digestive tract and as you look at some of those that are are more rate limiting you can see that that total at least from a digestible standpoint change quite dramatically and this is the numbers here as a percent of the overall diet again back to that particular level and in response with that particular challenge so one of the things if you've seen me give talks before I really like to to understand and challenge dogma if you will and one of those dogmas out there really is that the turnover of an epithelial cell within the intestinal tract you ask anyone typically they will tell you two days so I wanted to go back in history of why we think that is essentially two days and the sad thing is our dogma really comes down to one chicken from the 1960s so I wanted you to understand where some of this is coming from because this is a very dynamic system and that change that happens is very dynamic so if we think about this epithelial turnover really to renew the epithelial surface we need to produce new cells through mitotic division as those cells mature they differentiate along the the crypt villus axis before they essentially go through program cell down and are sloped into the the lumen of the digestive tract and this process of birth growth and death is essential to the integrity of that epithelial surface so here's that paper that one chicken that made history right so what they did a montien bird back in 1966 essentially injected traded thymidine into the parrot name of the gut and then began harvesting birds at different points in time and as you would expect those labeled cells through audio radiography begin to be labeled and move up the length of that crypt Phylis axis so essentially they took birds at 3 8 18 24 and 48 hours after injection of that Tritiya determining and collected samples from the duodenum jejunum and ileum and i know this is probably very small for the people in the back of the room so I apologize but the one thing to realize that 48 hours 87% the the cells had traversed 80 7.5% along the the length of the jejunum and the summary and the abstract and that's what a lot of us tend to focus on said that nearly all of it had been gone in the terms of the jejunum by the end of 48 hours but again realize that going back to the statistics each of these time points represented one bird so a lot of our dogma at least did not focus on the duodenum irrilium but it focused on the jejunum because that 48-hour time point most of the the length of the crit villus axis had been traversed so that one famous chicken I tend to think of this a little bit more and I wanted to present this because it is a very dynamic system and to actually calculate the overall turnover is really what we need to begin thinking of if we look at the duodenum and this very healthy chicken was taking 5.2 days in the duodenum to turn over two point four days in the jejunum and 4.9 days in the ileum very very different than that two days at least to which we think about and ascribe that to so hopefully I'm changing your thoughts through this process and it's imperative to understand this essentially as we think of these different challenges and to what that maintenance call to the tissue actually is now back in the 1970s and 1980s we began to try and understand the influence of sub-therapeutic antibiotics and changes to how that bird becomes more efficient and a lot of what we understand in terms of that gain in efficiency is due to lowering the level of microbial load and therefore lowering the turnover of that epithelial surface and therefore improving the efficiency and lessening the maintenance cost to that tissue so if we look at Mary Coates back in 1980 and she did a number of different studies looking at conventionally reared animals versus those in a germ-free environment one of the things to realize here in the bottom line this is the percent of ileum traversed by those reared in a germ-free environment the red line is that of birds that were conventionally reared obvious difference at least the same methodology of looking at the percentage of how far that labeled antara site had migrated along the [ __ ] villus axis in the conventional animals we were looking at a replacement rate of about 26 percent per day turnover of about 3.8 days versus its germ-free counterpart taking nearly nine days to essentially turnover with them the digestive tract or essentially a ten percent turnover per day a two point four fold difference just because of presence of microbes within the digestive tract so let's fast forward a little bit to what we understand about presence of challenge again I realized that this is not new literature right so this was back from 1973 but yet I think a lot of what we need to understand is really to fully encompass a lot of that literature and basis so what fernando of McCraw saw back in 1973 they were using Anna Marius or lino challenge again Ameria sir if you line up for those of you that are unfamiliar tends to be pathogenic within the duodenum itself and indeed as we look at these challenges in turnover really we were seeing a little bit over 30 percent turnover per day in the duodenum upon challenge versus ten percent in the controls but I think what it was interesting was that that turnover even though that issue is not the one that typically is going to be infected has an influence on turnover of the rest of the epithelia throughout the length of the gastrointestinal tract even into the jejunum and into the ileum again that tissue is responding in terms of trying to turnover and shed itself of an infected cell changes again to peristalsis changes to turnover now all of those changes then lead to changes in maintenance function Bob Teeter who retired from Oklahoma State University in the US had one of the few metabolism chambers to essentially study what we know in terms of energetic use and one of the things he went in was into non challenged and challenged Birds went through a captured their maintenance energy needs upon challenge went in then a looked at severity of that challenge then after they're conducting the studies to ascertain whether they were subclinical lesions of what we term one or very clinical lesions now he did this two different pools of chickens at various different ages from 20 days of age up to 48 days of age so the one thing I just wanted to include in this line is the change in maintenance needs to that animal are quite significant again based on severity of that challenge one going from non-infected to infected you see the change in caloric needs on a daily basis but to think about this in terms of as that animal ages the changes to those additional maintenance needs are going to increase quite dramatically because we have a bigger bird a bigger digestive tract and therefore changes to that overall load that changes and impacts the global poultry industry differently right whether you're rearing a two kilo or less bird versus us in the u.s. in some cases rearing over a four plus kilo bird right so again when that animal is challenged and having to respond to those challenges that maintenance need may be very very different so as we look to other changes again as we ascribe changes to turnover part of what is relayed by that animal in terms of functionality will change as well if that cell is not alive as much it may not differentiate as much as some of its other counterparts that are around a lot longer the example I wanted to give you here was really for that of intestinal carbohydrates and I apologize because I'm using a mammalian example but theoretically I wanted to present this and then I'll give you some data from that of the bird so we know as an example the time at to which that Antero site is alive it will take on different functions in terms of intestinal carbohydrates those cells further down the creek villas axis will produce more glucoamylase and begin producing maltase and farther along in their life producing more sucrase and then lactase so we know the dynamics to which if you were challenged the speed to which that cell is going through this process of differentiation if it is not alive long enough it may not produce some of those carbohydrates which are tend to be expressed more towards the top of the [ __ ] villus axis and in the case of say a wheeling pig experiencing different types of infection whether it be Clostridium or a viral infection we know in many cases it is not going to express as much lactase therefore go through some secretory diarrhea as part of that price process when that lactose enters into the hindgut and then will perpetuate some of that with that undigested lactose being present jaha balloony back in the 1990's did went through some very eloquent work in trying to look at those different fractions along the crit Villas axis Oh to set you up with these slides she did these partial digestion from the crypt regions and collected fractions all the way to the villas tip which is represented on the left half of this axis she went in the within each of those fractions then should just look at Maltese on the top graph and sucres activity on the bottom graph I think the important thing to understand from these these two graphs really and this is in a very healthy state really is that those upper fractions of that villus really is expressing 30 to 40% more Maltese and sucrose activity so again if that cell is not alive a long enough time to express these activities we're going to also get a change in their differentiation state what they are able to express and then therefore affecting the digestibility of those nutrients as it progresses through the length of the digestive tract now we've talked about pathogens but we have other things that challenge our animals as well one of which is oxidized fat and I just wanted to present this because it is a non pathogen type of change to the dynamics of this particular system so Julia dinner did these studies back in the 1990s as well again use it radiated actually in this case it was a fly Medina analog at least that they were using but you can see in those animals fed a control fat they did not as far as those that had fed been fed and oxidized fat so the non-nutritive or non-pathogen types of challenges can also change this dynamic within the digestive tract as well wanted to make sure that we went through some of these other types of changes the other one i which i would like to talk of and changes to those responses is that of mutant production we know that mucin is one of those that is very endogenously produces a large amount of endogenous cost to the animal in the case of the pig we know that threonine and this is about five to seven percent of endogenous amino acid losses in the chicken I know at least of that endogenous amino acid loss 30% of that is as mucin if we look at the case of the pig we have not necessarily quantified that in the case of the chick but it should be easy to go back and do that so why is mucin important we know that it is the first line of defense it becomes then home for natural antimicrobials secretory IgA lysosome defensins and it maintains tissue hydration lubrication and it really facilitates digestion and the composition of that musin is imperative to become residents for that that micro flora that has both positive effects on the digestive tract but it's also a home than in those though that network or web if you will for pathogens so we know at least in terms of feeding different types of ingredients relay different functions to that amusement the literature is pervasive with our understanding and that's partly why we have a number of different exogenous carbohydrates is coming into the diet ooze that essentially change that level of viscosity presented to the digestive tract in the case of feeding barley or wheat or those other cereals with high levels of non-starch polysaccharides now accounting for that response becomes very interesting because we know that other compounds and giving other compounds can have an influence on that composition and how that responds in the environment of the intestinal tract one of those compounds that we are understanding it now has an influence on responses to production of mucin and also a composition of that museum is that of some plant-based central oils such as carvacrol thymol we know that one of the responses we can see is just an upregulation in terms of numbers of gaba cells and one of the other studies that we did in our lab a number of years ago really was to looking at functional composition in different environments fed different cereals whether it be corn or whether it be wheat so the left three bars on this graph are corn fed animals the right three are from wheat fed animals now we also supplement it in carvacrol or thymol to these animals and that's what you see with the second third and fourth or fifth and sixth bars with that those graphs the top graph is essentially log transcript of mucin to the mute to the primary secretory musin and what we see is that that predominant secretory museum is transcript transcriptionally regulated essentially by the essential oil thymol but what was most interesting if we look at the hydration state or the hydration capacity of this musin and as we would expect the wheat based diets versus the corn based diets we saw that it became more viscous or essentially not being able to hold as much water again base to that that a post translational modification that glycoprotein that is present but I think what was interesting was that in the wheat based diets we were beginning to see some some changes to that modification of that that mutant allowing a little bit more water to be held into that music this again without any enzyme being supplemented in the diet so we can and we're beginning to understand what that really means in terms of not only water holding capacity but then what that relays at least on what microbiota can essentially live within that particular environment and I mentioned musin especially because we know that as we have removed sub-therapeutic sub-therapeutic antibiotics from the diet we have gone through a change to essentially what microbiota will live some of those microbiota such as Clostridium perfringens really favored and become mucolytic and try and have that in touch and respond so in the case of Clostridium perfringens this is probably not a good thing because then they really up regulate and take advantage of that process and create the the biofilm that is occurring so we know that the the gastrointestinal tract is chronically processing these antigens and inherently it has quite a cost to the animal if it's you if the microbiota get ahold of that carbohydrate which in gross energy contents has for kcals per gram and turned it into a volatile fatty acid at 2.8 kcals per gram we've essentially lost 30 percent of that gross energy potential right it still can potentially utilize some of that VFA in the hindgut but inherently we've lost the ability of that animal to recoup that differential in and of itself so can we account for some of these challenges i wanted to go back to this particular study again that I showed you earlier on again this was a particular level of challenge that being the twelve times normal dose of a coccidia vaccine so our first question was could we account for it from a dietary perspective to recoup some of that differential and we just did this as a paper exercise because we had roughly a difference between three thousand two hundred and seventy kcals per gram in the unchallenged Birds and our metabolizable energy from our challenged Birds was 1780 five a difference of nearly 1500 kcals per kick if we were to try to energetically account for that differential we'd have to put about eighteen and a half percent fat in that diet not really practical right so the other back to the amino acid side we question could we actually account for the amino acid side alone we know we maybe could not do that from an energetic perspective the question was could we do that from an amino acid digestibility standpoint so we created a second experiment same type of challenge to which we supplemented that differential in standardized digestibility by adding an additional lysine methionine threonine isoleucine tryptophan isoleucine and valine I think the thing that point out was that we were unable to account for this in terms of body weight we did pick up a little bit of body weight differential but not completely to the unchallenged controls however if we look at the feed conversion we were able to get it in between the challenge birds with the Supplemental level it picked up a little bit of efficiency statistically not different than the unchallenged controls but we did pick up a little bit so in the essence it helped but only a partial gain in that terms of response but again it becomes very difficult from a nutritional standpoint to again try and predict how much of that challenge is the bird going to be exposed with how virulent that challenge is and what is our understanding then of that individuals response which we know is different in the terms of the digestive tract and across different animals within the population I'm going to skip through these and really go to my take-home messages the thing I wanted to leave you with today really was that the gastrointestinal tract is adaptive to the environment that it is exposed to in terms of ingredient matrix but also in terms of that bacterial environment to which it is is exposed to and those exposure to different of environments will alter the potential maintenance cost as well as that nutrient in the energy digestibility capabilities that that tissue has the other thing I wanted to relay in prediction of that really those three things again that add adaptation and type of response in terms of pathogen really is the amount of pathogen the virulence of that pathogen and other predisposing factors that relay the capabilities of response and lastly that immunological response which we'll hear a little bit later on and that recovery from response can really vary the nutrient and energy needs in terms of efficiency and overall changes to maintenance function of that tissue so with that I'd love to turn it back to our our air for today dr. Kishan yeah God says that we can also you can also want to quick questions so the question is what types of parameters were measured after injection of the tributed thymidine so I've done a number of these different studies and what they were measuring essentially if they're doing creative thymidine they're measuring the the using autoradiography through histology and essentially looking at where that labeled cell actually is so the tricky part about this is it will only incorporate into those cells that are going through mitotic division right so this is the main thing they're looking at histology looking to see where that leading edge of that labeled cell is along that crib Phylis axis there are some assumptions you have to make with that one of those assumptions is where is that cell when it originally was labeled we know that some work from from my lab when I was early on in my career as well as the Giovanni in Israel is the cells can undergo mitotic division at different places along that crypto less access typically we think they're down in the crypt region you know themselves but that's the main parameter that they're looking and then just measuring distance if you will how far they traversed along that crew Phylis axis thank you very much [Music] you