[Music] and so yeah like we said yesterday we discussed a lot about how variation between ingredients is really notable and it has a direct effect on digestibility by performance and bird uniformity and so at the moment we're still struggling with handling this variation in feed formulating and today I'm going to talk about how we can maybe use enzymes to help combat this issue so before we start even thinking about putting enzymes in we need to have a good understanding of what our undigested components in our feed is so no feed ingredient is a hundred percent digestible um so even things like starch lipids proteins that the birds have endogenous enzymes for we're still looking at about twenty to thirty percent that is not digested and it's just excreted so that's wasting money so yeah we've still got that proportion that's undigested and we have three different sorts of substrates that we need to think about so we have our substrates that we have the endogenous enzymes available for so the birds can use those we have our substrates that the birds don't have the endogenous enzymes for so they can't use them for things like cellulose and then we also have our substrates there as well as not being digested by and broken up by endogenous enzymes they're also having anti-nutritional effects so things like our phytate and our n ESPYs are basically healing some stuff and so when we're considering estimating how much of our substrate can be hydrolyzed by enzymes we have a number of limitations so first of all we have coefficients of digestibility of many of our substrates are not available so they're not in our and our C values and they're also difficult to analyze so for example our NSPS hard to analyze we don't have as much data as we do for substrates that are easier to analyze differences and how nutritionists approach their formulations so often we think we're all meeting the same trying to get the same energy and protein requirements and we might even be using the same reference values but as a nutritionist how you're handling this information and what you come up at the end is very different so we're going to end up with different substrates my variable feed ingredients so globally we've got a lot of variation between our feed ingredients so generally our lower cost feed ingredients have more anti-nutritional factors in and our ingredients that are lower in digestibility tend to be have more variation and be more variable and I think the biggest one is at the moment we still don't completely understand the molecular structure of the substrates we've got so we're not sure how to handle them with enzymes so starch and protein that is not digested properly will reach the hindgut and will be fermented and that will result in lower energy yield and also these undigested components will feed the pathogenic bacterium so the issue that we have with starch digestion and why it's incomplete and when we've got starch in pelleted form it's not as easily accessible so the birds can't access it as well in pelleted diets but we're feeding pelleted diets also our faster growing birds now are not as efficiently using starches are slower growing birds we're so we've got that genetic component that makes not as good at using starch and yeah we we have changed the genetics of the bird too much now with our protein and our amino acids and we've got an issue with the species and the gene variety of the ingredients is freed and also an issue with thermal processing so what happens when we heat them up is that for example when we're heating we can get sugar moieties that bind to lysine and make it less available so then we've got more undigested lysine in the tract that's not being used properly and can feed our pathogenic bacteria so when we we talked a bit about yesterday about micro voter so these undigested components in the trapped are going to make the microbiota proliferate so then what happens is these nutrients are feeding the micro flora and they're acting as competition for the host and we've also got how we're changing the structure of that digester so the digester structure and the chemical components in the digester are going to be changed by our diet ingredients so how our diet changes so yeah the diet effects that we're having on the digester the structure the chemical composition will determine what micro votes are going to have in there so we can use enzymes to modulate and mediate the microbe odor so we and yeah we change it with the enzyme and then we yeah we change the chemical composition and that will change what microbe out or that so one thing I've been working on in my research is developing a non-starch polysaccharide database and so I've been measuring the soluble and insoluble NSP levels in different ingredients around Australia so these values here represent at least 15 different samples of each feed ingredient so this shows you how much range we've got in soluble and insoluble NSP and so as you can see it varies quite a lot and we need to consider both the soluble and the insoluble NSP because they have different anti nutritional properties so our soluble NSP mainly it's concerned with viscosity and whereas our insoluble NSP axes Dilli one so it deletes my diet down and it also has a caging effect so it traps nutrients in it so then they're not able to be utilized so corn and wheat cells have pretty much the same cell wall structure but just their arab their NSP contents very different so wheat has a lot more arabinose on and and beta glucan so that's why we have a lot more viscosity issues of wheat than we do with corn so just to put it in context a bit a typical wheat soybean meal diet has approximately 9% NS peas in it and forty forty seven percent of this nine percent is made up of cylon in a corn soybean maze night we're looking more at about 6 percent NSP and about 43 percent cylon enough so when we think about NSPS we may need often think about viscosity and when we're putting in LSP yeas is it's often a main focus and so how it works with viscosity so it's our soluble NSP s that are causing these viscosity issues and mainly arabinose island and bt glucan but we focused more on a Rambler xylem because like I just showed you there's more of it in the diet and also it's comparatively more resistant so we worry more about rammnews islands and so what happens is the soluble NSP s they thicken the rate-limiting unstirred water layer in our mucosa so then that prevents nutrients being diffused and then being absorbed and so in response to this we also have more undigested nutrients in the tract so then we've also going to have more mutant secreted because our undigested products cause fermentation and they stimulate the microbes that then stimulate the mucosa so then we get more museum present as well and then due to a change in the oxygen tension in the tract that's when our fermentation happens so when we think of it's got a tea insoluble NSP we usually mainly focus on its physical effects so how the sort of way nursing is physically changing transit rate and ability of us to stir it so but actually this overgrowth of microbiota is equally as important as the physical effect of viscosity so when we're putting in enzymes to combat viscosity were also manipulating the microflora and so that we're feeding the beneficial flora and not so much feeding the pathogenic ones so we're still not completely clear exactly the relationship between viscosity and the microbiota but we think it's may need to do with this effect on oxygen tension and changing oxygen levels within the tract so in that way we're changing the environment from being aerobic or speculative aerobic anaerobic through to just anaerobic so that'll change what microflora I can flourish because depends on environment and they call them and this was an interesting study that I read recently and what they did is they fed conventional birds and germ-free Birds NSP so they used highly methylated citrus pectin as a source of NSP so they were giving the beds the same diets the same NSP levels and the effect on viscosity was exactly the same in both birds it was only in the conventional birds that this translated to an effect on digestibility nitrogen retention and performance so this suggests that it isn't just the viscosity itself that is causing the problem it's this idea that changing the viscosity in the oxygen levels in the digester is changing what Michael Flores got which we talked about yesterday so yeah when we're talking about our soluble NSP s we can we should be thinking more about its impact on microbiota and not just physically on the expecting the tract also in some of the research we've been doing we've seen that viscosity tends to increase until the birds reach about 21 to 28 days and then not so much an issue so this suggests that as the bird ages it is able to change in soluble NSP into soluble and a species able to slowly by slowly Bligh's it so as it grows we have more soluble NSP present in the tract that's causing our viscosity issues but then at this age the microbiota isn't then also able to to convert it again into useful products so it's not until about day after day 21 to 28 though the birds microbiota is adapted to be able to use this soluble NSP Wow and so again this illustrates that with viscosity it's about the microbiome and this suggests that maybe in older birds we're able to use more viscous grains more we can use them more and not worry so much about the physical effects of viscous grains and older birds and and it also suggests that when we're putting sila knees into grower and finisher diets that the focus isn't on changing the viscosity so much it's more to do with this act of breaking up our long chains islands into our smaller and ego saccharides and how the birds can use those as a prebiotic so yes suggest maybe we should be looking at it from a different angle when we're putting violin A's into but diets and older birds so obviously at the moment due to feed costs there's a big interest in using alternative feed ingredients now the issue at the moment that's hindering this it's being able to characterize their nutritional value so particularly when we're looking at NSPS and it's very costly and difficult to analyze NSPS and using what technology is we can use on our ingredients to mitigate the risks so mister guaranty nutritional factors and our mycotoxins and that sort of thing and also the economic benefits so being able to formulate with these alternative ingredients so they're getting cost benefits so this is where our NS piezas can come in so the potential to use alternative ingredients is increased when we're able to combat these issues using enzymes so then once we've got it locked down what's actually our undigested components are what our issues are then we can go on to step two which is selecting our enzymes and it only makes sense to use an enzyme if you know that you've got the substrate in the diet and you know that's resulting what's going to result from enzyme substrates together is going to be absorbed and used by the birds so it's this indiscriminate use of enzymes that is making us have inconsistent results so yeah when we think about the value of enzymes another issue we have is that we need to divide the value between what cost benefits were going to get and also what bird performance we're going to get so just looking at the cost benefits alone when using enzymes is where we end up with inaccurate formulations using them so we need to put value on both bird outputs and also how much cost and feed we're going to save so when using our enzymes we think about their different modes of action so first of all their ability to hydrolyze chemical bonds to make our starch lipids proteins etc more available and we want to eliminate our nutrient encapsulating effects so for example with our insoluble NSP so like I said I only want to see traps nutrients in the matrix and we want to release them so we use enzymes to get rid of the insoluble and see yourself and that's a big thing when people think about NS piezas they're mainly focusing on just the soluble NSP and these viscosity effects but the influenza the P is also equally important if not more sometimes so we also need to be thinking about how we can get rid of this cage effect of our insoluble NSP s and breakdown of anti-nutritional factors for example our fighting in our phytase is together like I said where we want to be able to soluble eyes are in the soluble NSP because if our NSP is soluble it means they can be fermented in the hindgut and provide energy so we want to try and soluble eyes are insoluble NSP s and also this idea of using enzymes as a complementation in young animals so young animals that don't have enough amylase and proteins in my pays giving them so yes bird ages their release of pancreatic amylase gets less and less so when we actually need lots of amylase is in the grow and finish your phase and that's when the bird is actually making less of it so we need to think about how age impacts what enzymes are available so there's often this thought that using enzymes means that we're increasing the quality of a low-quality ingredient or we're reducing the amount of difference between a low and a high quality ingredient and they do but it's really not as simple as that so there's a lot of factors that we need to consider when we're thinking about our substrates if we want to be able to increase the precision of our feed formulations so it's not as simple as just here's the substrate is the enzyme and we need to be thinking about the location of the substrate within the ingredient matrix and its solubility any other limiting factors there are and it's accessibility so this whole concept of how susceptible your substrate is to your enzymes and as well as just thinking about that we also like Rick talked about yesterday think we need to consider the quality of the birth so a poor quality bird is going to react more to an enzyme than a high quality bird because we're changing the microbiota for the better so yeah our low quality birds are going to respond more so if you've already got really good baths then you're not going to see as much of an enzyme effect and pretty well known but enzymes must also be accompanied by a change in the feed composition so if your diets already really high in energy and protein is this idea of four cups already full you're not going to get more out of it so we need to manipulate the diet so that we can get the full benefits of the enzymes so this is where we have to apply our matrix values so we work out how much extra digestibility we're going to get from our enzymes then reduce the protein and energy of our diets and then put our end fans in because otherwise we're not going to get any cost benefits from using around the times and how all idea is that they're helping us use the protein and energy from the diet better so yeah we need to when we're putting in enzymes also think about how we need to manipulate the feed so we're actually getting cost benefits of using it and it's very rare just to stick an enzyme on top of a diet because we're not going to achieve it so we need to assign it its own nutrient value so with most enzyme substrate relationships we can measure the substrate that's in the diet we can put the enzyme in and measure what comes out but this isn't possible with NSPS and this is to do with how complex they are how much variety there is an NS p between different ingredients like I said how susceptible they're going to be to your enzymes and how exposed they are so how easy it is to access them and and yeah we can't we can't measure its conformation as well so we can't measure exactly what substrates we're gonna get as a result so then that makes people might get a bit confused at NSP enzymes like you can't measure exactly what's coming out so yeah but so how exactly are we going to measure it so what we need to do is we need to elucidate the molecular structure of our polysaccharide fractions that come out it's the only way we can really do it so we can only we can look at what polysaccharide fractions we've got coming out at the end and then just rely and all the other measurements and the bads that we usually do so I've been doing some research as well as well as the NSP database I've also been working a bit on a database of asylum levels in the feed ingredients so again these are the means from at least 15 ingredients 15 different batches of each ingredient and you can see why basically this is illustrating my xylan A's works in both corn and wheat so with wheat we have pretty high in soluble violin so like I said the insoluble xylem is the ones that's trapping our nutrients and we've also got pretty high volleyballs islands we've also got these viscosity effects so that's why xyla knees work so well on week because we're combating both issues with corn however we know Corden doesn't cause any viscosity issues because it's got barely any soluble NSP and barely any slowly pulls iodine in it but it's got really high in soluble xylem so when we're putting silent eases in corn based diets this is what we're focusing on we're focusing on getting rid of this cage effect of our in Sullivan ESPYs and this was just so I did a completely unrelated study it was an AME study with seven different diets but I just franciene measuring the cylon in them and so what I thought was interesting from measuring this island so these are just completely they were formulated to be pretty much the same but completely different ingredient levels so they had different Xylon levels and but what was interesting is that for the soluble xylan we're getting pretty high digestibility by the ileum but with our insolubles iodine with God as low as four percent of it being used so these diets didn't have any xylan eizan so again this illustrates that our focus is usually always on combating viscosity but really is this in syllables I learned that we can be getting all the benefits from putting xylan eizan so like I said to be able to we need different ways of being able to measure the outputs from putting our enzymes in so one way I've been looking at measuring how good our xyla lasers are working is to measure these smaller size fractions so we've got our Long's island and we're crunching it up with our enzymes and then when I've been looking at the smaller oligosaccharides so the ideas I'll talk about in a minute but these oligosaccharides can be used as prebiotics so we're putting a cylon aids in the diets anyway to try and come about all these nutrients Kosti things but we're also getting this added bonus of these prebiotics that are coming out of it so what I did in this study is I just took weeds from different areas different states around Australia and for those of you that haven't been to Australia is such a huge country that everywhere has pretty much different weather so we've got different growing conditions in all the different states so for example in Queensland it's pretty tropical and humid whereas in West Australia it's a bit more dry and desert E and then South Australia it's pretty gray and rainy so we've got completely different growing conditions in all these different areas so I took ten different batches of wheats grown in all these different areas and just slap some violin aids in with them and measured the oligosaccharides that came out using GCMs and i'm always interesting is just how different the amount of oligosaccharides was that was coming out and also which size they were so we think for poultry for this prebiotic effect we're mainly aiming for x2 next threes society BIOS to Xylo units together and threes are lazy knits together but we're working on that moment to work out exactly what size they like um so yeah so uh what what we examples we use will determine what an ego psyche words would come out in the presence of silent ease so this is why we're not always getting the same consistent effects with putting a signer needs in two different diets and there's all this also this focus on reducing as a way to reduce variability using the idea of phytate free nutrition so like we talked about yesterday this idea of super dosing so if we can get rid of all the phytate in the ingredients and we and more of a level playing field with them because they've all gone no fighting um so yeah putting super doses in so we can get rid of pretty much all the phytate and also get the added added benefits of our myo-inositol so they've been shown to help with translocation of glute 4 transporters and have insulin type effects and also like we talked about phytate has an impact on protein digestibility so it increases newson secretion and nitrogen and doggedness losses so if we've got rid of the fight eight then we can also improve protein digestibility so again we can reduce the variability a bit with our protein digestion and also there's interest still into alkaline phosphatases so using them to break up the lipopolysaccharides in the membrane of our ground- materia which has an impact on improving nutrient utilization so going back a bit to the prebiotic oligosaccharides again this is something I'm super interested in at the moment and I ran a little pilot study where I've said revenues Islands sigh low oligosaccharide that I'd made in the lab and said or revenues Islands and viola knees together so basically I was trying to compare if it was better for me to make the oligosaccharides in the lab and feed them to the chickens so the works already done for them or if they're better at making them themselves and 2% is really high we wouldn't ever normally put 2% in we'd put yeah tiny amounts in them real life I just wanted to make sure that we could see what was going on clearly and what we found was that we did have improvements in the short term fatty acid production as a result of our oligosaccharides but what was interesting is for all the individual short chain fatty-acids there was always a consistant difference between feeding arabinose Island and feeding the oligosaccharides that I made in vitro but there wasn't always for when the birds make it themselves so this suggests that the birds can use them better if we make them for them so highlights the potential of yeah making oligo saccharides as a food additive as a prebiotic additive and it wasn't significant but we saw increased lactobacillus levels as well which suggests that the Bears were able to use them and improve and they manipulated the micro flora so other studies have also shown that these silo oligosaccharides can improve colon function immunity villus lengths yeah so there's it's a pretty hot topic at the moment and it looks quite promising so again this highlights the added benefit of putting in our enzymes because we're getting these added benefits of these prebiotics coming and we can also use enzymes to help us with diseases so it's well known that more insoluble NSP means more viscosity and trap in the tract and more likely that we're going to have issues like necrotic enteritis because Clostridium perfringens favored soluble NSP and also like I said at the very beginning we've got more undigested nutrients in the tract because of this so especially amino acids the Australian four engines love eating amino acids so if we've got more undigested amino acids in the tract then they're more likely to flourish so I mean gonna found this when he put sila knees into wheat based diets and saw that it made clustering for engines not present and people have also seen similar with salmonella so once we've look we've sorted out what enzymes we need we can then go on to start developing and so much more so we haven't harnessed the full potential of enzymes yeah so even in really digestible corn soy based diets we've still got 10 15 percent of undigested products in them and yeah so you any one of our students has recently done a study and we found as high as 33 percent in wheat based diets that's undigested and so there's still a lot of potential and we can't ever get 100% but we expect our enzyme is to be able to break up at least twenty to thirty percent of this undigested material so when we're developing the enzymes it's important to define and exactly what we want it to do and under what conditions so we're in the gastrointestinal tract that we want to be working and the biggest issue we have is assays to measure them so this is also an issue in a lot of journals that I've found it's hard to see how much activity their enzymes had or sometimes even what family of enzymes they used and yeah so there's often not a lot of detail and journal articles about the enzymes and South but and when we want to develop a new assay we need to consider the balance between how how well it represents what's going to go on in the bird and also the costs associated with it and the throughput so how difficult it is to do because if you yeah if you want to be able to do it on farm or whatever it needs to be easy and cheap to be able to measure what's going on with your enzymes and the process that we go through is that first the enzymes will be tested in vitro then tested in animals and then we need to ensure that it's stable and the entire distribution feed chain so we need to make sure that it's fine with being stored that it's fine it's good for being that we can formulate with it and that it can be fermented so we can make large batches of it and we also need to determine optimal production process so how yeah how we're going to make it what's our best way of doing it and how yeah how we can ferment it how we can produce them and also how are we going to be able to formulate with it so as we know and there's a lot of interest in enzyme cocktails so often people in a wheat soy based diets just think about putting xylan aids in but like I said there's a lot of other NSP products in there as well that could benefit from having cocktails of enzymes and so yeah we can we should be thinking more about getting the most out of all on NSPS not just Stars Island lasers and also looking more at fight aces and NSPS together which we're doing quite a lot at the moment for example in this study Mike Harrison and Adeola and they had a marginally deficient corn soy diets a slightly low in metabolizable energy calcium and phosphorus and then they even fed Thielen aids amylase and proteins together xep or phytase or they put them all together so when they have them as two separate treatments they saw about six to seven percent improvement in body weight but when they put all four enzymes together they saw 14 percent improvement so pretty much doubled by putting them all together and so yeah this is we've doing a lot at the moment looking at cocktails but we also need to consider maybe the negative effects of putting them together as well so maybe when we put them together they're not making the substrate ideal for each other and consider what levels we want to put them in so that they weren't really well all together so this is some information that assayer gave me showing how they use their rave of byproducts so here we're looking at cocktail of a silo needs be to glucose pectin ease and malaise together and here you can in the top graph you can see that we have three different types of wheat they all had different AME levels to begin with but when you put the enzymes in they're all pretty much the same so this shows how we can use enzymes to reduce variability in this graph below they fed 60 birds with the enzyme 60 Birds without and observed the number of birds that had the same AME so as you can see you putting in the enzyme meant we had more consistency so there was less variation within the flock about how much AME they had and similar with protein digestibility so they fared either a classic diet so just a wheat soy full fat soybean meal diet or a complex diet so it had those three ingredients plus rapeseed meal sunflower meal meal we D digest barley and rye and they found that when they did the enzyme to the complex diet it made digestible protein similar to our classic wheat soybean diet so that suggests again that we can be using more alternative ingredients and yeah we can be using more variety of ingredients if we're coupling it with enzymes so it helps us open our doors to more ingredients so in conclusion the gain in improving variability we can gain from enzymes is we can neutralize our anti-nutrient effects we're releasing nutrients from our internal indigestible components which are causing us a lot of issues at the moment and then produce bioactive nutrients so for example oligosaccharides and from these components thank you very much [Applause]