[Music] well good morning everybody hear me okay so I was asked to come up with defining what we call an ideal you know how do we do find an ideal feed additive you know what is that and and then and furthermore how do we assess that we've heard a lot of talks this week about different feed additives you know what they do from a functional standpoint that sort of thing and one of the things that I think we have to keep in mind is is that you know what kind of criteria are we going to use here obviously there's several different factors we need to consider and so we have precedent for this interestingly enough is if we go back to antibiotics I came across this bullet and I was cleaning out a former professor's office at one time and there was this book from Pfizer that pamphlet was put out in the 1950s and it actually is the blueprint for what an ideal food additive is they were obviously selling terra myosin and it was all about terramycin applications for food animals and so they went through growth performance gut health in a number of other things that we've been talking about all week and so I could probably stop right there and we could just read this pamphlet and it would tell us what we would need to know in terms of you know what an ideal feed that feed additive looks like of course we all know what happened is with antibiotics is it is is at the time ideal was easy to use cost-effective consistent and that was the point that they were making in the pamphlet over and over again we know since then obviously that antibiotic resistance has emerged in human clinical disease has continued to increase in climb and so now we worry about Mar and multiple antibiotic resistant bacterial pathogens that's led to public perception and certainly in the u.s. that's been a driver's public perceptions we don't want antibiotic fed animal we don't want anta by transitioning pathogens etc and so what we've seen now is a complete push from the market standpoint to take antibiotics out of pretty much all food animal systems the McDonald's of the world the fast foods of the world that basically told their suppliers we want to market meat products fast food products etc that come from animals that have not been fed and bikes and so at least in the u.s. anyway I mean antibiotics are pretty much gone so what's happened in conjunction with that is is we've seen a big jump in livestock production without antibiotics and so of course we have antibiotic free from a conventional standpoint the large organizations such as the Tyson's of the world or certainly marketing antibiotic free type chicken products etc pork products the the rise of natural raised products poultry pasture flock free-range poultry has become a more and more of a thing in terms of a market of course the organic production in the US has organic production standards as does most of the rest of the world for organic produce products that do not again have antibiotics and back to our Pfizer bulletin I mean the criteria are pretty much the same I think that we have to consider that it needs to be easy economical and consistent in other words it needs to be affordable to be used on a mass quantity it needs to be easy to administer worked in and a feed mill etc and you have to have a consistent response in other words every time you put it into that feed those animals need to respond the same way the same in the same fashion every time and then of course as we've been talking about this week the other factor then is gut health pathogen reduction antibiotic resistance reduction and this one is more of a new thing and the idea that some products are now able to reduce antibiotic resistance within that gut microbiome and that becomes a part of the factor as well and again we'll talk a little bit about some of the mechanisms on that but we're going to start with as far as criterion what I'm going to talk about today is mostly on the pathogen reduction side and part of that's reason because of all the different criteria it's probably the easiest one to assess on the front end in terms of making decisions in terms of where you want to go with a feed additive and how you want to screen those feed additives so let's talk a little bit about alternative alternatives to antibiotics and I've put them into two broad categories and there's others obviously we've talked about this week that would fit under one of the other of these categories the first group I would call would be inhibitory to pathogens these might be ones that you would administer to clear out a pathogen that has already been colonized and so we go from what I would call fairly broad spectrum which would be essential oils and related compounds fairly nonspecific could affect a multitude of different bacterial species within a gut microbiome or within other microbiomes organic acids are also probably somewhat nonspecific you would expect anaerobes for example that produce organic acids to be more resistant than say you know a facultative organism or whatever back to your oceans which are going to be somewhat more specific and probably target only certain groups of organisms potentially such as gram positives and then finally bacteriophage which would be highly specific good example of Salmonella and back to your fudge you mean you have 2600 seer of ours you have fog that are specific for particular serovars and in some cases maybe even just for specific strains and so you have a multitude of different specificities in terms of your compounds that are inhibitory then we have another set that I refer to as preventive vaccines would stimulate hosts immune response you work with an attenuated strain of a particular pathogen you inject that or you administer it orally and then the host response to that immune response the idea is then of course when the wild type Salmonella for example comes in then the host is able to amount of specific immune response to that probiotics which we've seen a lot of discussion on that this week and you know live good bacteria that are added externally to the gut with the hope or plan that they establish in the gut and and help to accentuate a quote-unquote healthy type microbiome prebiotics which I'm actually going to spend quite a bit of time on them because that's what we work with mostly feed additives select four already in place good gut good gut bacteria some of these could be things that you would typically find in probiotics as well but generally these bacteria are already established in the gut and the prebiotics basically select for them and then finally that I do the concept and it's kind of a natural projection to symbiotic swear you take a prebiotic you administer that simultaneously with the probiotic and ideas as a probiotic and use that prebiotic as a substrate and that helps it get established in the gut so I'm not going to spend a lot of time on probiotics you've seen quite a bit of discussion on that in the past and in this meeting basically the who definition that was laid out a few years ago it's basically live organisms which when administered confer quote-unquote a health benefit on the host and I mean this has been around a long time of over 100 some years and so you know the antibiotic era somewhat displaced that but now we're back into the air again of really looking hard at probiotics again and you know it certainly ivory emerged common probiotic bacteria Bifidobacterium lactobacillus and of course bacillus have been talked about quite a bit this week in terms of germination schemes and those sorts of things give bacillus established a multitude of health benefits and a lot of these are related to human health benefits that's a very partial list I mean resistance towards internal pathogens of course as a driver combats diarrhea relief of lactose intolerance serum cholesterol improved immune function which has been talked about quite a bit this week cancer prevention but there's a it's a very long list some of these are more anecdotal than others others are more better documented I mean you'll see certain reviews will come out and say one reveal sale combat diarrhea another one will say it will combat constipation so you kind of have to you have to sort of kind of pick through this and figure out which one's of these are anecdotal and which ones of these are more mechanistically sound or make sense from a you know a biological standpoint so let's go to prebiotics prebiotics and you probably are very familiar with this already but these are basically we call non digestible food or feed ingredients they give detectable host benefits they're not absorbed or utilized by the host this was the definition that was a little rigidly laid out by Glenn Gibson back in 1995 and we've kind of used or worked off of that as a model ever since then there's a class of oligosaccharides that falls under that fructooligosaccharides Galacticos saccharides and then manage that re cell wall compounds all of these can be utilized by expect what's quote-unquote good bacteria such as Bifidobacterium and lactobacillus again this concept of probiotic where you could feed one of these to a pro bite along with the probiotic strain get a symbiotic type effect that would help the probiotic to get established in the gut and you are basically supplying in a specific substrate that it can use the question gets to be and we started to realize is is it's not just lactobacillus and Bifidobacterium that's probably responding to a lot of these products that are probably other gut bacteria as well and that are capable utilizing these prebiotics and probably do different things with them as well not just the sum of the metabolism we understand what our lactic but actually do some other fermentation products etc and there probably is an overall gut microbe microbe population response as well and I'll get into that a little bit later in terms of what we mean by that in terms of overall gut response so obviously sequencing has dramatically changed how we approach and look at these things and we've had a lot of talks this week where people have used these types of approaches to understand and get a better idea of you know a comprehension and what's going on and from a gut microbiome I I mean I came up in the era where we still did anaerobic culturing to characterize bacteria and up into the 70s and into the 80s that was our primary way that we did it and if you go back into that literature it still has utility to this day I mean a lot of what we saw in that early literature we actually are picking up on some of the sequencing that we do now in terms of identification of species and that sort of thing and so I think there is some reason to go back to some of these methods on some of this to help document or supporter or fill in some of the blanks and what we're learning from the microbiome standpoint and of course the denatured gradient gel electrophoresis based off of 16s was used for quite a while is another way to get at shifts and populations and that sort of thing but all of this was clearly limited information of main cultural methods probably only captured about 10 to 15 percent depending on the estimates and how good your culture techniques were you know and how well you captured the strict anaerobes it was in that population with microbiome sequencing obviously we're able to get much better pictures of overall bacterial populations much more comprehensive analysis and then you know the key thing here is in-depth tax identification both known and unknown when we can start to distinguish unique genetic sequences and say yeah this organism we may not know what it is but we do know it's a it is and it can be classified as a species or a Jing or a genus so this is some work we did several years ago now where we were looking at pasture flock sequel contents and this kind of gives you an example of the kind of data that you can generate we used an Illumina hi-c con this which gave us a little bit more resolution and the things that came out of this was is that we definitely saw a temporal switching in terms of specific microbial groups lactobacillus changed out species as we went along and that's something we've seen on some other work as well Bacteroides and actinobacteria communities increased as as birds became more mat or which again is not really a surprise I mean some of that we've seen with other methods as well the key thing that we took out of this and we looked at fos and gos and then we had plum fibers kind of a non prebiotic control just a little bit more of a neutral type compound to see if there was distinct prebiotic effects there's a prebiotics tend to accelerate maturation of the bacterial community and therefore presented a barrier to sell Manila and that's a key point as I think you know we talked some in these sessions about the importance of age and the development of microbiome and I think one of the things we have to think about with a lot of these feed additives how do they impact the development and maturity of that microbiome as it goes along and you can vary that in berge to some extent and you know and that's why some of the ANOVA applications these days are starting to be looked at more closely with some of the prebiotics and probiotics as well as because the ideas is let's get an earlier head start in terms of establishment of a more mature gut and a more mature of gut microbial population the other thing that we picked up on this is as I said the barrier cell Manila because we have early development contrary to that Campylobacter like the fact that we developed an earlier microbiota as well because Campylobacter actually coincided with these increases in these bacterial specific groups normally we see Campylobacter say maybe a couple of weeks we started to see it earlier with some of these prebiotics and some of these where we saw a faster rate of maturity in terms of the microbial in there we also saw increases the capital vector in early age so it's a little bit of a so depending on the pathogen it's a little bit of a challenge sometimes to get one size that fits all and one of my take-home message is early on is that say I don't think it's possible I think we have to really take this a pathogen by case-by-case to some extent in terms of how we strategize and apply these sorts of types of feed additives so a different study along these lines we looked at some yeast prebiotics in commercial flocks and here we compare we compared that with antibiotic the antibiotic we looked at on these studies was basic tracing which was commonly used in some of these commercial flocks at the time and this gets back to my maturity pointing a little bit that I was emphasizing in the last slide is again here what we notice what the antibiotic feeding was is the Campylobacter significantly increased in the antibiotics versus both the control in the pre biked the prebiotic and in control pretty much behave the same way lactobacillus on the other hand significantly was reduced by the antibiotic of two weeks versus control and prebiotic now note that everything really kind of stabilized by four weeks I mean all nothing really much different by then or by six weeks I should say and which is something we see a lot of is is there's a window of development and changing the microbiome but once you hit 42 days or towards the end of that broiler things really start the kind of even alledon so your populations often times at the end are really not necessarily that different in regardless of the treatment and one of the things we feel pretty strongly about you really do need to look at age when you're looking at developing these micro microbiological pilot population because things happen in here that you would never pick up if you just went 242 days for your samples so all the treatments were the same by six weeks the question gets to be is as their shifts and other groups as well and what if we change out prebiotics is that going to change and different GI t GI tract groups are going to be affected the other thing I would point out here is is that we do see some of an antagonistic relationship between lack the Misses wouldn't Campylobacter we've picked this up on other studies as well as where how the lactobacillus population behaves somewhat dictates what Campylobacter is going to do significant there may be a bit of an antagonism between the two and we've continued to explore that some and kind of want to confirm that and start to understand why that might be to some extent so let's revisit our prebiotic definition again and this was a review paper that came out from Hopkins group up in Nebraska two or three years ago now and and this is an I this is a good example of how our resolution and capabilities of doing sequencing and characterizing micro problems has really changed our thinking in terms of feed additives in general and certainly specific ones such as such as prebiotics their point was is that that basically more compounds probably have probiotic type properties okay it's not just your short list of gos fos and some of them the Mannings but there probably is a much larger grouping of things that you could somewhat considers prebiotics or at least have components in them that would be behave like prebiotics and so technically the definition doesn't change it's detectible hosts benefit and i put the word in detectable because if you can't detect a host benefit then it starts to be questionable as to whether it truly is a prebiotic or not so you need to define what that host benefits going to be and it needs to be detectable and so so soluble and insoluble fibers potentially qualify as at least being sources of prebiotics resistant starch etc and there's some controversy on this some people would disagree with this and be what I would call more purist about and say no these things aren't technically prebiotics but they do impact the microbiome and they do elicit some at least in quotes prebiotic type properties so the other thing to keep in mind here is once we start getting into these complex sources of prebiotics we're looking at a what I would call components within that source versus the composite of that source and we'll go through a little bit of what I mean by that in a minute but the point is is when you get into complex sources such a cereal green or whatever how the microbiome responds to that entire entity versus how if you start fractionating it out varies and it's and it's somewhat unpredictable as well and sometimes it's a requirement of that composite you need that composite to get a complete microbiome effect I mean if you start pulling out individual carbohydrates in that sort of thing you start to lose some of that effect so there's some suggestion that there's synergism within the components of a particular source of prebiotics and that's something that we have to consider as well what it boils down to is is that prebiotics elicit what I call a complex functionality and I mean I think that's some of the take-home message from some of the work and some of the reviews that were put out in recent times so we still see a modulation of the microbial community both structure and function in other words composition changes certainly to some extent but it doesn't stop there I think one of the things that we lose sight of with microbial populations and you know anaerobes in the Sica whatever we're looking at is most of them are capable of multiple fermentation pathways I mean under some circumstances they might make lactate other circumstances they make acetate propionate and there's a whole lot of other potential in products they make and those change depending on substrate availability passage rate other organisms around that might you know potentially across speed for example and we have to take all those things in consideration when we're looking at this composite microbial population so yes we see selectivity in terms of impact on microbial composition but as that periodic structure or a source becomes more complex we also probably have a much more complex consortium response as well more organisms are involved there's more interactions going on so on and so forth and so we end up having to classify this response both as direct and indirect by direct organism C's probiotic organism hydrolyzes probiotic organism ferments prebiotic and produces Emprise but then we have a whole bunch of other organisms who are there as well who take some of those end products whether it be say something like succinate for example and then utilize that succinate to produce propionate and so this whole concept of cross feeding is something that we probably need to pay more attention to as we get to know our organisms better that we're looking at on some of these sequel contents and so and i trained as a room in microbiology so some of this comes natural to me because in the room and we see this all the time there's constant cross feeding between organisms I mean you'll have cellulite --ax which primarily hydrolyzed cellulose but then you'll have a whole bunch of organisms that can use those soluble products and basically cross feed and somewhat interact with the primary polymer degraders and I think we have to think that way in the Sica as well I think there's a lot of that going on that we're we lose sight of or we miss when we're trying to classify and characterize what's going on functionally so what that means is is that yes we need to not just do microbiome sequencing and characterization but we actually need to have what I would call a metabolic system system and systematic screening approaches we need to not just collect up and learn you know we'll find out what the metabolites are but we need to start putting that in the context of what might be going on here for example and we'll go through a little bit of an exam some examples of that so this is sort of what we've come up with in our anyway when we start the screen or when we start to try to characterize a probiotic and try to assess whether it's a viable candidate for that we're gonna use it using in a poultry setting so generally we start with pathogen reduction I mean it's the easiest thing to determined I mean this is does the presence of the prebiotic help to reduce the pathogens in whatever you're looking at in our particular case it would be sequel contents so we start out with in vitro because that allows us to do high throughput where we can screen a lot of compounds fairly rapidly and that's not a nun standard way to do things I mean it allows you to you know do some dosage determinations etc and do that fairly rapidly and get an idea and narrow down your focus to the things that you want to then go in and do what we would call the in vivo side of things where you actually challenge Birds the cell Manila bee and the birds were being fed the particular candidate prebiotic that you're interested so we generally start there and following that are simultaneous with that we also look at the rest of the microbial community because we want to know what the interaction is with the microbial community with the prebiotic itself and then can we also characterize that in parallel with what's going on with salmonella I mean in other words there's a lot of cause-and-effect going on here yes someone L is reduced but does that relate to the microbiome that's being selected and does it relate to the fermentation that the Michael Brown was doing in the president since that prebiotic and so these two things are to some extent interconnected now once we have microbiome analysis of course we can go in and determine taxon and determine who's there and are there particular signature organisms for example that are responding to that particular prebiotic that you're feeding etc and then with that you know when you want to start comparing different periodic sand compared with control then then the diverse the analysis makes a lot of sense because then you can go in and say okay does you know birds being fed prebiotic a this do you see a change in the microbial composition over time versus birds that are being fed the prebiotic B for example and how did that how does that compare with control how does that compare with other different types of feed additives say a probiotic or an organic acid or whatever you want to compare it to and so both of these kind of go hand-in-hand and then finally obviously the hosts gutters Pontus metabolomics transcriptomics and proteomics you know you want to know housing what sort of plate is the immune system having this and the immune system of course interacts with the microbiome and vice versa and so again and some prebiotics can be immuno genic as well and so you have to this allows you to start factoring some of these things in as well and it's kind of a three-step process basically you start out with what we call a pathogen screen and then we get into the gut ecology stuff here and then finally the ultimate is bird performance and something I think we need to keep in mind here is is that is ideally you'd like to have both you'd like to have pathogen reduction and all of this supports that pathogen reduction in other words you have explanations as to why you're seeing pathogen reduction and you enhance bird performance in the real world you're not necessarily going to accomplish both I mean you get pathogen reduction that's as I said that's an easy beginning point decision to make and what you hope for is at the very least you don't negatively impact performance I mean if you can show that you can maintain bird performance but now you're getting pathogen reduction that's still a win and I think we have to keep that in mind to some extent is is you're not necessarily gonna hit a home run and get all of this going at the same time ideally you'd like to and you certainly want to try but and we're talking a little bit later about how we need to think about that strategically in terms of how we might want to play this out so I'm going to go through a series of studies that we did with to kind of give you an example of how this works and you know don't get you know there's a lot of data here and I'm just gonna give you some of the take-home messages out of this and how we process this data and how we use it and we got into a project a few years ago where where you look we're looking specifically at rice bran and rice bran is a good choice for us in Arkansas because Arkansas is the number one rice producer in the United States and so there's vast quantities of rice bran available it's pretty much a byproduct not a lot done with it it's used in pet foods and some of those some of those things and Arkansas is number two in broiler so it was a natural marriage between you know rice byproducts there's excess vii and we've got a lot of birds so it makes sense to put those two things together and see you know is there some benefits here you know could this be used as feeding green and we didn't go into this naively as well there had been a lot of evidence from a collaborator we had out in Colorado State Elizabeth Ryan who had shown in mice models etc that some rice bran was inhibitory to salmonella at least in mice and there was a certain other host responses that rice bran was beneficial too and so on as part of the collaboration it was a natural thing to say well let's see if we see some of these things in a non mammalian system such as Birds and which would have some really practical implications so that's kind of what got us started on this and this is our sequel in vitro model I'm not going to go into a lot of detail it's pretty straightforward when we use a sequel in document an anaerobic and dilution solution we use a koi anaerobic hood we have you know the usual redox indicators etc so we know that we maintain it under anaerobic conditions and we've used this model for a lot of years it's kind of a what we call an initial screen the nice thing is we can run run a lot of things at the same time and do a lot of comparisons and use that to do some range finding in terms of different sources of prebiotics dosages etc and we can spike that with cell Manila and then and use that as a way to quickly screen whether certain prebiotics are going to be supportive of decreasing cell Manila in under sequel type conditions and if it approximates to some extent what you see in the cecum and close enough for us to at least get an idea of what potential is gonna work in the birds when we go to that second phase so we inoculate with a sequel microbiome on on time zero we inoculate a marker strand most of the time we look at someone no type of maryam but we've also looked at in aridity we're looking at other other serovars now as well most of them tend to behave pretty much the same way in this system so that's not been a concern and then off of that we can pull off cell Manila numbers we can characterize the microbiome and then we can also do metabolite responses as well you know fermentation patterns etc to try to put those pieces together in terms of what's going on and in this particular study we looked at three different cultivar sources of rice brand jasmine red walls and Cal rose and these are most of these are California variety's actually they weren't arkansas ver items later on we did look at some arkansas varieties and when we look at the numbers here the thing to look at here is is that so we have treatment vo feed plus equal plus the rice brand and then of course feed buff seagull seagull only feed only and the big take home here is is that only one of these cultivars cal rose was actually fairly inhibitory to send one no the others we really didn't see in fact in fact we actually saw some increases in here in terms of Salmonella and so right away jumped out at us that there is a cultivar specificity to this and there have been evidence from ryan's group and others that that was gonna that that would be the case that there be some cult of our differences and so we used that as our beginning point to start to try to characterize okay what's going on here we've got a complex source potentially of prebiotic type properties we want to characterize that in terms of you know how is that affecting the microbiome and how is that affecting then the you know this whole seagull ecology as we go forward the other thing I would point out here is is it and I'll come back to this a couple of times is is is adaptations required one of the things we do in this system is is that and we've kind of this was trial and error a little bit is lots of times you start an in-vitro system time zero where you inoculate the Salmonella and you also inoculate the seagull inoculum at the same time and generally what we've observed is we see a lot less inhibition when we do that or a lot less effect and instead what we started doing and we can get some early studies to compare the effects is we do what we call an adaptation where do we take it out 24 hours where we just incubate with the prebiotic then in that at 24 hours then we go in and spiked with cell Manila and that seems a little bit artificial but if you think about it from a practical standpoint it makes a lot of sense because from a bird standpoint generally those birds are gonna be consuming that diet for a while so they're see cos gonna be their microbiome is gonna be adapted to whatever the prebiotic isn't so this sort of simulates well more of an adaptation period and again we see much more inhibition as you notice here I mean we got down the point we're at XL Manila at that 48-hour period over here I mean we got quite a bit inhibition even to 24 hours and so that adaptation and giving the chance for the microbiome to interact directly with the prebiotic and start fermenting and producing fermentation products is a pretty critical part of the process to really get a realistic viewpoint of what's going on from an overall cecal ecology standpoint so this is what the tax that looks like and again I'm not going to walk through all of this the take-home messages or pretty much this and this was done two or three years ago with chime 1.9 I mean we were we do time to the two levels now so we've gotten away from this we looked at we used a variable for region hyper variable region every variable region has a little bit of advantages and disadvantages in terms of how you want to screen things the v4 region generally work pretty well for us for these types of populations and microbiome characterizations and we characterize from pilot in general and we use green jeans for 16s gene D database on this to identify actual organisms some take-home series is that 28-day sequel in ocula the major Jenner was Sica only we mostly see in or back to save when we add it in feed we'd start to see room to caucus and Austin spire are coming make sense room the caucus is more of a fibrous carbohydrates type degrade err whereas also fires more of a starts to greater and so it makes some sense that you probably start to see those become prominent when Kara Rose was adding on top of this we also saw a big climb in fact a fecal bacterium as well go out to four to two days again pretty much the same trend for the secant only now we're seeing some internal back to say as well as room to caucus with feed Cal rose also spire and Lachman spire and lactose Myers an organism or a group of organisms we've seen off and on quite a bit and we're trying to track that down a little bit in terms of what lacking inspire can do and why that might make some sense Carol rose we sold nearly a hundred fold Decree decrease in a neural back to say which lined up with their cell Manila dated that I'd reported earlier now a point I want to make here is is that we've published a couple papers on this is one of the things that you have to do when you standardize EC column your methods is you really have to be careful of what age of birds you take the inoculate from because as this data points out here is we saw differences between 28 day birds and 42 days birds in terms of the source of a knock on the neck and that I mean it's a kind of a common-sense thing it makes sense because this this is probably a more mature seagull microbiome here then it will be at 28 days and we've seen it with live birds and but I would also say that both are important because we get back to this whole maturity change in microbiota and that being an important component to consider when we're evaluating feed additives we do need this information here because we need to know what's going on in those intermediate periods is that microbiome develops and how that responds versus what we see at the endpoint at 42 days and frankly we've also looked at seven Dave inoculum sources as well and my and we're still working out I'd actually like to look at even earlier birds as sources of inoculum as well because I think each different age is going to give you different responses in terms of what you see and I do think that translates some to overall sequel development as things go along so this is what the diversity looks like and again principle coordinate plots Venn diagrams to show you what the shared micro microbiome is between the different days and the incubation times alpha and beta diversity 42 days no surprise were more diverse than 28 days that fits with what we've seen with live birds as well and in vivo studies beta diversity we saw some separation by treatment but no difference in the incubation time which was a good thing to know because one of the things you worry about with individual incubations you worry about okay the incubation conditions going to when you're gonna lose organisms over time if things aren't in a row because you'd like them to be or whatever and so this indicated that our incubation time was not going to be a problem in terms of being able to accurately reflect what we're seeing in terms of microbiota Venn diagrams we saw 60 percent shared Jenner for all treatments so actually quite a bit of the same organism and this gets back to the point I'm gonna raise in a bit about functionality and firm and metabolite responses 22 unique Jenner can be so of Cal Rose incubation we're still kind of sorting it out a little bit and who's there if that makes sense 50% of the Jenner we're common for all incubation times so there's a lot of overlap here and I think that's something that you have to really take in context when you're looking at this type of data is is is you're not going to get hugely diverse populations you're gonna give a lot of overlap and you know it's in fact we were a little surprised we saw that many unique Jenner in this in this in this makeup in this comparison so let's look at the metabolites data and we we did metabolomics on this with a GC mass spec we looked at feed plus Seco only and then versus versus Cal Rosa added an incubation time of 0 versus 24 hours these are only from 42 de birds and we classified with both CAG and pub can pubchem databases so results we had over 578 metabolites and detected more than half are unknown in other words we really couldn't classify them based on the chemical databases that we had and we were able to identify 211 and this kind of gives you just an idea most of them that showed what we would call significant increases in term versus control some take homes here is that nitrogen metabolism you saw a pretty good spike in glutamate Glu mates and amino acid you typically find them pretty high concentrations in the CK anyway so that wasn't a huge surprise my finding I don't really have an explanation of why we saw increases Metheny but that was interesting at Cal Rose seemed to support that or anything in ala TOEIC acid are both reflection of uric acid degradation and so apparently something's go on in here to where we're in some way affecting uric acid degradation cycles that are going on in the seek and therefore affecting Nitra metabolism beta-glucosidase inhibitors pentathol and hex eat all those actually were probably coming from the rice bran one of the things that we learned as we got into this is and I should have known this from a plant standpoint is plants tend to have any inhibitors for beta beta-glucosidase enzymes as they go along because as they develop cell wall structure cetera they need to start inhibiting those enzymes so that they can start stabilizing those cell walls and so it made sense that we were picking these up and we were picking these up in the metabolite analysis fatty acid synthesis malonic and path paths authentic acids were picked up some of these reflection of some of the sort chain fatty acid pathways need to expect such as butyrate formation etc and as I said most of these are all tenfold increase of calories versus of control I would like to go back to this point just a little bit here I think one of the things that we keep need to keep in mind when we get into these metabolite analysis is there's a lot of contributions coming from the feed and substrates and those sorts of things that come into these metabolite analysis and how things like this impact the overall secret microbiome we need to take into account I mean does the do these things inhibit beta galactosidase and enzymes in the bacteria themselves possibly the other thing that I didn't point out is we also so sawing quite a bit of phytate in here as well coming out of that which agains are not another surprise so I think as we and I'm guilty of this I mean I think I focused mostly from this from a fermentation microbial metabolism activity when I started to realize this is that there's a lot of substrates coming in from the feed materials and the other things that are coming in as well that are coming into this metabolite analysis and I think going forward what we have to do is we need to start taking a step back and actually doing what I would call some kind of kind of some kind of calculation in terms of counting for the nitrogen and carbon that's coming into the sink and and trying to account for where it's coming from and how that relates to microbial protein synthesis etc I mean we used to do that quite a bit in the room in microbiology and I think it's relevant and the chickens seek as well I think we need to start you know not just do these vascular metabolite arrays but we need to actually start tying in what's a plant source what's at my Kerrville source and how that balances out from a total carbon and nitrogen flow and I think that's a direction that we need to consider going in the future so overall conclusions on our rice studies certainly potential prebiotic source but very cultivar specifically our screening other cultivars now and we're still continuing to see this some cultivars are very effective others are not in other gut ecosystems we see other effects we've had some intriguing preliminary data and indicate that we see methane methane inhibition some of the rumen incubations we've done and that's something we certainly want to follow up on as well because that's a whole different type of metabolism and that was a surprise to us to some extent it appears that at least in vitro we certainly support unique Seigle bacteria we inhibit cell Manilla causes shifts and metabolite profiles we're of course back to the decision tree or the flowchart that I put together earlier where we're at next is actually to going and doing vivo studies now with the dosage and the cultivars that we've defined as being most effective we want to exam and the other thing I would put here is we want to examine the entire GI tract I'm guilty of mostly focusing on the Sica we I need and we're encouraging our folks in our program to do this is we need to look from crop down because for example in the crop we have a heavy dosage of lactobacillus etc there are some indications that certainly in early young birds there's a lot of overlap in terms of compartments in microbial development and I think if we're going to evaluate things and their true effects on the gut we need to start from the top all the way to the bottom and we need to look at all the compartments and see how those things are affected and how development is affected over time and then and then how that influence is eventually sequel composition in the makeup and obviously and we're getting into this now as we want to get into transcriptomics and them in logical responses start being able to add a layer of host response on top of what we're seeing from a seagull microbiome standpoint so let me wrap this up by saying back to our original question and that is you know what isn't the ideal feed additive for boilers and how can we assess this and I think we have to accept the mark and demand reality what I mean by that is antibiotics are gone they're not coming back and so we have to we have to take the lessons we've learned from antibiotics and start to apply them to what we're developing now as our new generation of feed additives and so we need to have the same concepts as we've had before it's both detectable and consistent improvements in path of control and or animal performance ideally we want both if we could get it but certainly we need to be able to whatever we get it needs to be a consistent response we get the same thing every time and the producer out there that's growing those broilers are going to demand that and if we don't have that if it's variable or it's anecdotal it's probably not going to market well expectations and in quirements it should be not only effective consistently but it should be easy to handle things that we don't talk about is is how does it handle in the feedmill will it stand up to peloton well let's stand up to other heat treatments will it stand up if you want to combine say an organic acid and a prebiotic can you do that and not lose efficacy with rice brand one of the things that we're working now with is is with an egg engineer because milling affects how you recover brand from that cereal grain and in how you recover that brand probably dictates what its efficacy is going to be and so we have to work those factors in when we start to consider these things and so it's you know what all it does is make it even more complex but the ultimate goal is to have something that's very consistent over time I I'm a big proponent of this and I always have been as we got we've got to know what's going on but have to lose it elucidate those mechanisms we've got to know okay if we add this in who's gonna respond how are they gonna firm in it and what's gonna be the outcome of that and then how does the host respond to that we have to connect the dots between those two to all those different points so we need to know how they work but I would say just as importantly we need to know when and why they don't work because when we're out there making management recommendations and things and it doesn't work isn't a management problem or is it a problem with the product and so knowing mechanisms allows a person to predict well yeah if you do this it's not going to work because of this and I think that's an important point that we have to make and finally I think you know I'm a little bit biased on this perhaps being an academic but I think peer-reviewed science is a critical part of it I think we have to you know we have to have that open trans open availability of the data all the way to the raw data I mean the nice thing with microbiome data and now is there's a lot more journals demanding that you deposit the data and it so an access etcetera and I think that's all for the good we need that we have to have that to be to make progress so I think the future in terms of expanding criteria for ideal feed additives is and I'm guilty of this as well we've got to get me on Pat just pathogen reduction and prevention we need to start focusing on some of the other things that have been talked about in the conference you know gut health immune status inflammation you know how does those things or how are those things impacted system standpoint you know performance welfare feed conversion the economics all that needs to be factored in I mean there's ways to calculate okay if this product costs this much how much economic impact that we're gonna get out of that I mean is the cost-benefit ratio makes sense and then I think one of the things I've started to realize I think is we've got to take a multiple hurdle approach for to meet what I call multiple criteria back to this pathogen versus performance thing is probably what we need to start thinking about is is that okay maybe we need to do combinations that kind of help us accomplish both maybe one particular feed additive does help you with pathogen reduction but then you add in a second feed additive that maybe helps on the performance side and perhaps there synergism between the two likewise you know back to our earlier point about feed additives being inhibitory versus preventive combinations of both may make sense now where'd you put inhibitory in such as a fudge to knock out a pathogen population but then you follow that up with a preventative agent that prevents that pathogen from reestablishing and I think we have to have start having that kind of thinking when we when we start applying those things and with that I will thank you very much for your time and it's been a complete pleasure being here thank you [Music]