[Music] indeed this is a subject that having worked in this area now for about 47 years is really the primary area of research that I'm still involved in and I am for exactly the reasons that Brian has just indicated in the United States we've seen all of these products on the market at one point or another there's a couple of them that have dropped out of sight in part because of the golden method if you will that we have recommended and and as a result of some of the research that we had done with their product if you take a look at these products you're going to recognize especially over on the right hand side with the Metheny in supplements that we're dealing not only with protected DL methionine products but also with some liquid products here as well my categorized HM bi or meta smart in there in the box with Ruben protected Matheny because that's exactly how we use it it's really the only product up there that's a dual function it behaves as a protected methionine supplement in the sense that about 50% of that product is going to get absorbed that which is not absorbed does not end up in the feces like some of the protected Matheny products do but instead what is not absorbed is going to be available in the rumen and will behave in the very same way as the other methionine analogues that are up on that slide so I'm going to review the methods real quickly I want to at least have some time here for discussion we can categorize the methods that have been you to evaluate amino acid products by in vitro methods that would be the first one the early methods basically just looked at ammonia release then we have the three-step method and then just the amino acid release method which is sort of an offshoot if you will of the Cornell approach that was developed to estimate digestibility of RUP so you can modify that procedure or to also evaluate amino acid several months debbie rouse who's one that developed that really doesn't like to use a method for this purpose and there's some good reasons for that that doesn't mean that some people have not used it it's not one that we would recommend at all it is an in vitro method however and for an analytical lab if they want a method to use and there's a few labs in the u.s. that are using that method but I'm not going to say any more about that because it's not an animal-based bioassay now the differ or the challenge or the shortcomings if you will of all of those methods there is as is that there's no influence of animal effects and I'm sure that all makes sense to you because these methods does not involve the animal in any way shape or form I'm just mentioning two references here where they've used the three-step method and both of these abstracts were funded by a genome Odle the top one was a method as used at the University of Minnesota and the one at the bottom is a modified method of that now the only reason that I'm showing you this and while we don't support the use of these in vitro methods to evaluate these are amino acid products the beauty of the second procedure here and I'm showing the results of a study where they evaluate a six different protected lysine supplements and by using the different time points that they did it at least allows one to look and try and figure out if there is a lot of loss of the amino acid from those products before the animal would ever see it where does that loss occur is most of that loss occurring in the rumen or is most of that loss occurring in the rumen or in the small intestine where is that loss or is it ending up in the feces so what if you look at this closely you will see there's a difference in those six products there in terms of the release in rumen fluid okay so one reason that some of those products now would not have a very high bioavailability is because a lot of that product is being lost in the rumen on the flipside you can have products that are very stable in the rumen but then they're also very stable in the small intestine and they end up in the feces so this method here would allow you to do a preliminary analysis of a number of different products and just to see really what's happening here before you would ever feed them you know to any animals so that is an advantage of this method here now we move on to the Institute methods I think you're all familiar with the insta to approach in terms of estimating the value of protein supplements or NDF or whatever you might be measuring the disadvantage here with these with this method is you're reliant on loss of the product from the bags any loss of product from the bag you have to assume then that would have been digested or released either in the rumen or in the small intestine so there's no effects of eating and rumination in any of these Institute's of course that would be true for the in vitro methods as well disappearance from bags beans degradation passage rates are needed if you're really going to estimate right if you're gonna have to mate a ruminal escape disappearance from mobile bags that are put into the duodenum and collected in the feces means absorption well not necessarily but that's what we have to assume also the projects then are going to be subjected to hindgut digestion and lastly you cannot use with fine or soluble products so there's a number there's a long list of disadvantages here and why we would not advocate its use however I am going to show you a couple slides here where one product has been evaluated using that method and you would be familiar with that product and that product is meat prawn m85 now if you go back into the literature and I don't know what the company suggests to you as the bioavailability of methionine in me prawn is but because I don't know that's the reason I know I wanted to show you these results so 1996 Tom Overton at Cornell University had published this data that appears on this slide and what it shows is the losses of methionine out of the bags in the rumen what ended up in the feces and by difference what would have been lost or absorbed if you well but not really absorb because again we're just measuring release from the bag so you pick your poison here if you want to estimate a bioavailability you say well on average maybe me prawns going to last in the room and average retention time is going to be 12 hours okay if you make that assumption then you're going to have to accept the fact that 22 percent was lost in the room and 26 percent ended up in the feces and 52 percent was available in the small intestine if you think that every retention time is 24 well then you're going to use those values the last line up there is just a calculation showing them the disappearance of the methane that was entering the room so 25 30 % loss in the rumen of that which made it to the small intestine about 66 67 percent would have disappeared in the small intestine or would be an estimate of digestibility okay that's the first study on me prime here's two more experiments they both came out of Canada one in year 2001 and year 2001 in the first experiment they just wanted to look at the rate of loss out of the bags in the room and notice that at one hour there was 4% loss at two hours there was 9% loss and you can go down the list if you say well the average room in retention time is 12 hours 37 percent loss now even the company would tell you that this is a slow-release product it's designed to be a slow-release product so the product is doing exactly what you would expect for the technology that is used experiment 2 now they used the mobile bag technique and cows they had a loss of 16% that was after four and a half hours they just used four and a half hours in that particular study and four and a half hours 16 percent was lost faeces 36 percent ended up in the feces 37 percent then was absorbed this study reminds me of the fact that years ago and that was at the time that I was working on NRC 2001 and I called dr. Ruka and I said I have a question for you I said tell me about me from is it a good product or not and he says one-third one-third one-third I said I don't understand he said one-third lost in the room and one-third is going to be absorbed and one-third is going to end up in the feces and he was so close to being right totally consistent with these results then there was another study that is and all these studies are reported in the Journal of Dairy Science two studies here by these authors 25% bioavailability in the one study and 34 or 22 percent depending on the dosage in the second study so for me front if you take all of the results and you put it together you'd say about a third or maybe less of that product will be absorbed based on these Institute techniques now we'll move on to the invivo methods so we're looking now for a gold standard here what are we going to use as a gold standard we're not necessarily suggesting you can't use an in vitro method to screen products or to categorize products or to maybe even to rank products but there are not going to be the gold standard so we need an animal bioassay so what are we going to use are we going to use changes in milk protein content as a method a week when I use the plasma free amino acid doha area under the curve approach or are we going to use the plasma free amino acid dose response approach well we have looked at using changes in milk protein content and so here's one study we fed five levels of methionine coming from smart M&M medicine art or just hmb which at the time was wrote him at 80 88 and we fed all three of these at five levels we fed it to cows that were being fed of Athenian deficient diet okay we wanted to see will at work or not was smarter to mean em know what does we got a nice linear increase in milk protein concentrations up to a point and then a plateau it off so there was a linear response but there's also a quadratic response and notice that that leveled off at the third level of smarter mean M so we only had three points now from which to draw dose-response line for hm bi we needed more HM bi more meta smart to get that increase in milk protein but we still got that increase in milk protein we got the same increase as we did was smarter mean M but it took all five doses to realize that with HM be I wrote them at 80 88 nothing Zippo now we've done this before and you can go through the literature you cannot I defy you to find a study in the scientific literature showing a consistent effect of feeding a llama or any form of HM be I on either plasma Metheny and levels or on milk protein concentrations so when there was nothing there so if you want to look at the plots and there are the plots so we come up with a calculated bioavailability of 0 on Rotem at 80 88 and we got a bioavailability of 42 percent on meta smart notice the scatter notice the standard errors here and I'm going to say a little bit more about this method later here's a the area under the curve approach and this is something that at us started advocating years and years ago they used it for many years to compare our efficacy of different protected Matheny products and by and large this method works pretty well however the shortcoming is that in order to do this you're giving an animal a slug dose of the product all at once they're not consuming it over a 24-hour period it's one slug and you're looking at the accumulation and of Metheny in the plasma in this case the area under the curve for me prawn was 25% of smarter me now and the two and the two MHA products were 3.2 and 1.8 okay we're not saying that this method does not work however we don't believe it to be consistent with how we feed animals or how the products would be fed to animals so if you go back in the literature you can find examples of studies and I'm not going to show you all of them but again this came out of rule Kim's lab and he demonstrated that there's a nice linear response in lysine and methionine to increasing amounts of infused lysine or Matheny so the more you infuse the higher the concentration in the blood and that response is linear it is linear when you're dealing with cows that are being fed conventional diets here's another set of studies looking at the relationship between increases in plasma and increasing amounts of infused lysine again linear and I could show you many of these studies where the responses have always been linear nobody with ruminant animals has shown anything other than a linear response to increasing amounts of either absorbed lysine or of absorbed Matheny and now we've done work with histidine we've done work with some other amino acids responses are all is Leonard okay so that tells us now we got ourselves an animal bioassay here so the first paper that was published came out at the end of 2017 from the University of New Hampshire where we use a technique to evaluate room and protected lysine products we're just finishing up a paper now doing the same thing with methionine products so what's our method we use lactating cows we use high producing lactating cows the average milk production in all of our studies is about forty to forty three kilos of milk so we're using cows just a way you'd feed these products out in the field they're fitted with ruminal cannulas for Avila mason infusion then of the unprotected amino acid we use Latin Square studies we started off using two or three week periods we found that that was not necessary that we could shorten that to seven-day periods with one day of clean out five days of infusion or treatment with collection of blood samples on the last three days so just a standardized the protocol we're using seven days the rumen protected amino acid supplements in the last four or five years now our office mix in the total mixed ration allowed to incubate in that ration for eight hours before it gets fed to the cows and there's a reason for that which none of the other methods can consider and that is what happens to these products when you mix them in a TMR the cows don't eat them right away right it's mixed in the TMR TMR is put out in front of the cows the cows are fed once or twice a day so what happens well here's what happens this is a study out of the Minor Institute where they evaluated six different protected lasing products the products were mixed in the TMR and then the TMR was sample for various periods of time after mixing and the loss of these products were determined notice oh and then they use low dry matter or high dry matter tmr's notice there's not a big effect here of differences than dry matter content of the tmr's you look at amino shirelle they're at 16 at 18 hours what there's about 4 or 5 percent loss up to about a 10% loss in the TMR before the cows they read it you go to Megami now notice more loss of the lysine from those products metabolize looks pretty dang good right pretty stable in that TMR you say wow that's a good product oh now that's a product worth some consideration it's stable in the diet same thing with a cheaper look at ads you Pro is stable as a button not much is happening to it excellent excellent what about Lacey pearl not so good not so good 50% loss after 18 hours in the TMR USA lysine even worse 50% loss after 6 hours there is no reason why any company is they're going to introduce a product can't at least do this simple test have they showed you that type of information I don't think so I don't think so now what if you take those same products and you incubate in the feed for a while and then you subjected to the Institute of just incubation in the rumen notice that USA lasing down at the right hand side after six hours of a six hour exposure to the TMR six hour exposure in the rumen it's gone it's all gone any wonder why we fed that out in the field and we get disappointed because we don't really think is working very well well no it probably isn't it probably isn't if we take another extreme look at ad G Pro same test 80% of it is still where that means 80% if you just want to use some assumptions here 80% of that edge you throw that was fed it's gonna make it to the small intestine the question is is 80% going to be absorbed or are we now dealing with a product that it's protected very well in terms of rumen but what about intestinal digestion is it a is it gonna be a hundred percent digested or is it going to be something less the answer is something less so I'm going to wrap up just where we've used this method now to evaluate some products here we evaluated the first generation of Ag pro l6 treatments our write negative control infused 30 grams of lysine in few 60 grams of lysine fed 30 grams of lysine and AG pro 45 and 60 end up with two dose response lines you take the slope of that line for the product which is point zero zero six eight you divide it by the slope of the infusion line which is point zero one eight three you have a bioavailability value of thirty seven percent pretty select here we wanted to compare generation to generation three of Ag Pro L up against infusion generation one was 37.9% bioavailability generation two is 42.4 how about the old smarty mean ml anybody in this room remember the old smarting me now now all that at a sale head back I don't know 15 years ago or so remember that yeah Robert Bennett remembers that Brian Sloan remembers that and we decided Brian in this wisdom one day when I gave him a call I said I think we need to evaluate a product that's on the market because they're claiming a really good bioavailability and I said I think we need to compare that up against old smarter mean ml I remember Brian saying or you have any of that left we said yeah we had some of that left so we ran a study you can see what the bioavailability was of that lysine and the old smart amine ml that had been setting in our lab for about 14 15 years bioavailability was 87% in the other product it was Tim the company was claiming 64 after six months the company had no choice but to pull it from the market here we compare the bioavailability of lysine and the new smart amine ml up against generation 3 of Ag Pro got a 80 point seven percent bioavailability of lysine in the new ml and a 45 46 in the third generation edgy Pro product this technique has been used by other labs now across the world this is one still in the states where they wanted to compare lyse e pearl USA lysine along with just straight lysine in some sheep here are the results no difference no difference in the bioavailability estimates of the two products as compared to straight lacing in these go and in these sheep moving on to Matheny a few years ago we four or five years ago now we want to evaluate bioavailability of methionine and smarter mean M here is the study we got a bioavailability of eighty two point eight percent here's the bioavailability of methionine and smart amine ml that's a combination products got both methionine and lysine again we got an 81% bioavailability value so the new smart I mean ml our estimate so far show an 80% bioavailability of both the lysine and methionine in the product we have looked at other products such as meat prawn Meno Shoreham the UDS is the smart amine em out of their old plant the original plant smart up is the smart you mean em that's coming out of their new plant that was a mention yesterday notice absolutely no difference two different facilities but it's still smarter mean m and it performs the same way that's pretty nice me prawn [ __ ] me prawn me prawn is 27 percent relative to smart you mean M so the me prom date is very consistent study after study and sit - or the plasma me no acid dose response method here this is a study that was done down in West Virginia looking at me brown and nova met again using the same technique and results just like we get you and h what about meta smart you know at a sale has been saying that's 50% right Brian's been saying that for years so we decided to use this technique first thing we had to do is it just infuse HMB into the small intestine and see do we get a linear response and solve for amino acids and by gum we dead so we said great this technique is going to work for that as well so we evaluated meta smart and we got a bioavailability of 50% so in spite of some of the shortcomings of the area under the curve method that they use to get their original 50% value that was a good value to use so my last slide here and this is my take-home messages I think the best thing that we can do is help in terms of helping the dairy industry and nutritionists to say do not feed do not hold your guns do not feed any of these products unless you've seen confirmed estimates of bioavailability using this method pure and simple whatever the marketing people tell you ignore it unless they can show you this data there is labs around the world now that will do those studies for you also confirm that the supplement was mixed and consumed with the rest of the diet that is important based on that minor Institute data right and last do not accept comparative milk production data as proof of claims of bioavailability do not such data always favors always in quotes is going to favor inferior products why well I tell you why up there on that slide cows respond to increased absorption of a nutrient only if that nutrient is the most limiting factor for production therefore in a comparative comparison of different sources of that nutrient all cows fed the best product must remain deficient in that nutrient and no other factor can be limiting production and I defy you to run an experiment and convince anybody that you can do that to ensure that that nutrient is still deficient after you've supplemented with the best product and that it remains deficient and all the cows within the group impossible so that isn't the case then the superior of the best product won't be seen and the inferior products relative to the superior product will look better than they are thank you [Applause] [Music]