Guests: Dr. Clay Zimmerman, Balchem; Kari Estes, Balchem Dr. Zimmerman presented a Real Science Lecture webinar on December 12th, 2023, titled “Not All Rumen-Protected Products Are Created Equal.” You can find the webinar recording at balchem.com/realscience.
Dr. Zimmerman presented a Real Science Lecture webinar on December 12th, 2023, titled “Not All Rumen-Protected Products Are Created Equal.” You can find the webinar recording at balchem.com/realscience.
Clay outlines four attributes of a good rumen-encapsulated product. They are feed and TMR stable, ruminal stable, nutrient bioavailability, and good efficacy biologically in the animal. (6:21)
Kari describes a TMR stability test that Balchem has been perfecting based on a paper published in 2016. One to two grams of a rumen-protected product (based on the nutrient composition) is mixed with a half pound of TMR in a Ziploc bag, then the mixture incubates for 0, 6, 12 or 24 hours (based on feeding 1x, 2x, or 3x per day). Once a sample is finished incubating, it’s placed in a strainer bag in one liter of distilled water for one minute. Then, the amount of nutrient that was leached into the distilled water is measured. She describes some of the observations and trends they’ve seen from using this technique on different products. (8:24)
Mark asks about the impact of abrasion during the mixing process on encap stability. Kari describes a mineral mix technique using a small ribbon and paddle mixer. In this case, 5-10 pounds of encap product are mixed with 90-95 pounds of a mineral mix for three minutes. Then a sample is analyzed for damage to the encap. Clay does not recommend pelleting any encapsulated product because that will only reduce efficacy. It may not be 100% damage, but it will be significant. (12:41)
Scott asks about the freeze-thaw stability of encapsulates. Clay mentions that all of Balchem’s encapsulated products are freeze-thaw stable. If a product is not, there will be cracks in the coating and some ruminal stability will be lost. (19:34)
When it comes to ruminal stability, matrix encapsulates tend to have lower stability in the rumen, but it varies widely. Some have no ruminal stability; some lose less than 10% in the rumen. Encapsulation is a complex process and there are tradeoffs between some of the steps. For example, between TMR stability or rumen stability and bioavailability, the goal is to find the perfect mix of these to make a high-efficacy product on the farm. Kari describes a rumen stability test that can be conducted on-farm for protected choline and lysine products. Mark describes in situ experiments for rumen stability testing using small Dacron bags in rumen-cannulated animals. He mentions that creating an encap with high rumen stability and high intestinal digestibility is key. (19:58)
Bioavailability is key, but methodologies for assessing bioavailability are a limitation. Kari and Mark discuss the pros and cons of various in situ/in vivo techniques, including mobile bag, abomasal pulse dose, and stable isotope. (29:25)
Clay mentions that in vitro techniques are a key piece to product development and testing, but may give erroneous results compared to in vivo testing. Kari describes an experiment she conducted with Mark comparing in vivo and in vitro techniques. She suggests that there may be an argument for creating specific in vitro tests built for different types of protected products. For example, for a pH-sensitive product, a step mimicking abomasal enzymes would be important. For a fat-coated product, a step mimicking intestinal enzymes for fat breakdown would be important. Clay cautions that a product with only in vitro data should be regarded with skepticism. (44:25)
Biological response in the animal is the key final step. Ultimately, you want independent, peer-reviewed data to prove the efficacy of a product. Mark reminds the audience that even if animals don’t respond to a product, there are a host of different issues that could be causing that unrelated to the product being tested. Things like water quality, water quantity, stress, cow comfort - there’s a whole laundry list of things to consider. (50:39)
In closing, Kari recommends that when picking an encap product, ask for the research that hits the four pillars: TMR stability, rumen stability, bioavailability, and animal performance. Mark suggests that you can’t make a bad encap good, but you can make a good encap bad if you aren’t careful. Clay agrees that the more data, the better. Lastly, we need more work on the feed stability pillar which has been overlooked. It is a critical piece to encap products being effective in the field. (55:13)
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Scott Sorrell (00:06):
Welcome to the Real Science Exchange, the pubcast where leading scientists and industry professionals meet over a few drinks to discuss the latest ideas and trends in animal nutrition. Hi my name is Scott Rell, and I'm gonna be your moderator tonight. Tonight's featured guest is Dr. Clay Zimmerman. Clay presented a real science lecture webinar back on December 12th, 2023, and the title of his presentation was Not All RU protected products are created equal. Now, I'd recommend if you haven't done it so far, please go back and, and watch that webinar. And you may even wanna do that before you listen to this podcast. You can find that at balchem.com/realscience, and you just simply have to scroll down until you get to the December 12th presentation. Now, clay, you're in a little bit different chair tonight. Usually you're in the co-host chair, but tonight you're in the hot seat. So I guess my first question to you is gonna be what's in your glass tonight?
Dr. Clay Zimmerman (01:09):
In my glass tonight is my favorite diet beverage, actually,
Scott Sorrell (01:13):
All right. That doesn't sound too exciting, clay. No,
Dr. Clay Zimmerman (01:17):
Not, not really exciting, but that's what I have tonight.
Scott Sorrell (01:20):
Alright, well, clay, I I see you've brought a guest with you. Maybe she has something exciting in her glass. Would you mind introducing her and telling us why you selected her?
Dr. Clay Zimmerman (01:29):
Absolutely. So our guest tonight is Kari Estes. Kari is our research manager. So she, she manages all of our, all of the external research that, that we do in our animal business in, in biochem. And she's really an expert in this area, you know, when it, when it comes to different methodologies for for assessing bioavailability. And we'll talk some about feed stability testing as well. So she's, she has quite a background on that and brings a wealth of knowledge to the conversation here tonight.
Scott Sorrell (02:09):
Excellent. Well, welcome Kari. Glad to have you here. And as the same question I asked, Clay, what's in your glass tonight? Any cherry vodkas?
Kari Estes (02:17):
No cherry vodka, definitely not. And no soda either. Mno diet beverage, but only the best juice box ever, which is Apple and Eve. Fruit Punch.
Scott Sorrell (02:32):
So good. Did you steal that from your kids?
Kari Estes (02:34):
Maybe, maybe.
Scott Sorrell (02:39):
All right. Well, I still want to hear the cherry vodka story. Maybe, maybe Mark can help us with that. So my, my co-host this evening is Dr. Mark Hannigan from Virginia Tech. Mark, welcome. Glad to have you in the, the co-host seat tonight. What, what's in your glass tonight?
Dr. Mark Hanigan (02:55):
I think there's a little Maker's Mark that might have snuck in with the diet Coke that was in there, so,
Scott Sorrell (03:01):
Oh, that's awesome. I like Maker's Mark. Tonight I'm actually drinking a Blade and Bow. I had that at Saratoga last year at the Dairy Exchange, and I found it in a state store, and I just had to get some, I liked it then, and I like it now as well. So, cheers, everyone. Looking forward to a great podcast.
Dr. Mark Hanigan (03:22):
Cheers.
Dr. Clay Zimmerman (03:22):
Cheers.
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Scott Sorrell (04:34):
Alright, Clay, now you're the director of technical services at Balchem, and you oversee our research as well. Can you tell us, kind of get us started here, what makes Balchem an expert in room protected nutrients?
Dr. Clay Zimmerman (04:49):
Yep, great question, Scott. So first of all you know, as a company, we've been around since 1967, so we're, we're, we're 57 years into this now really from the start. One of the, one of our starts back in 1967 was as an, as an encapsulation company. We moved into the animal business into the animal space in the late 1990s with, with our flagship product in the ruminant space. ReaShure was introduced in the late 1990s. That was, that was our first foray into the animal business was ReaShure. So we've, we, we've been encapsulating as a company for 57 years. We've been, we've been involved in encapsulation for ruminants for well over 25 years now, and have developed a lot of products in that space and continue to do lots of, lots of internal r and d related to new products and improving existing products
Scott Sorrell (06:03):
Very well. Thanks for that, clay. Now Clay, during your presentation, you outlined four basic attributes of a, an effective nutrient or rumine encapsulated nutrient. Would you mind kind of just giving us the treetop overview of, of those attributes?
Dr. Clay Zimmerman (06:21):
Yep. There, yeah. There, there's really four pieces to it. So if you wanna start in front of the cow, the first one is feed and TMR stability. These encapsulates they need to, they need to be able to withstand mixing in the feed mill. First of all handling and transportation to the farm. And then TMR stability, they need to be able to survive in a TMR mix for, for a length of time, depending on how long that TMR sits there. So that, that's the first piece. The second piece, if, if we're developing a, a, a rumen protected nutrient is we need good ruminal stability. I always say if you're losing a lot in the rumen, you lose the game right from the start. So we need good ruminal stability. The third piece is we want good nutrient bioavailability. So we want to get as much as we can pass the rumen and then have that cow absorb as much of those nutrients as possible. And then ultimately and this is where carry really comes in, we run, we run the animal studies to prove biological efficacy in, in, in the cow. We need to be able to show that these products can get through the first through steps, first three steps, and then, and then actually perform in the cow to to result in performance and and income for the producer.
Scott Sorrell (08:02):
Alright, clay, so why don't we start, you said you, you start in front of the cow, it's gotta be stable in, in the, the, the TMR and in the feed. So how do you go about determining whether a product is stable in the feed and the TMR? And maybe that's where we bring Kari in to talk about that. Yeah,
Dr. Clay Zimmerman (08:21):
Yeah. I'm gonna defer to Kari on this piece.
Kari Estes (08:24):
Yep. So actually working some with Dr. Hannigan here, we've been working on I would say perfecting a TMR stability test, at least for women protected choline and lysine products. We're working on methionine, we'll get there eventually. It's just a little bit harder of a molecule to work with that we're finding out. But this technique we have based off of a published paper technique out of the minor institute, I think that was published in 2016. It's really a very simple technique and easy to run. So essentially we take one to two grams of the room protected product based on what the nutrient composition is, and we mix that in with about a half a pound of TMR in just a Ziploc bag. We mix it and we let it sit for an extended period of time. So we've done previously a zero hour time point, right?
Kari Estes (09:37):
So where we just mix it and read it, and then we do a six to 12 and a 24 hour, and we pick those time points based on feeding schedules, right? So if you're feeding one x two x or three x per day once they're done being incubated, then we put them in a strainer bag. It's actually just a paint strainer bag, and we let that sit in a one liter of distilled water for one minute, that's it. And then we measure how much of the nutrient was leached into that distilled water. So it's really a simple technique, but it, it's very interesting the, the results for sure, based on the different technologies that these different products utilize.
Scott Sorrell (10:27):
So can you tell us a little bit more about that, Kari? So you, you mentioned technologies what kind of differences have you seen?
Kari Estes (10:36):
So on some of these tech or some of these products, I guess these commercially available products, some of them I feel like we understand well what the technology would be. Some of them maybe not so much so, but I think when we test them in these, in this TMR stability, you can really tell the ones that maybe have more of that matrix technology utilized, right? Where some of that nutrient, some of those nutrients might be exposed on the outside surface. Right? It's not a true encapsulate like Clay mentioned in his webinar. Those products tend to release more in the TMR over time than a true encapsulate would. That's the trends that we're seeing at least.
Scott Sorrell (11:29):
And, and how much then would you say is leached out, Kari? What percent is actually lost in the
Kari Estes (11:35):
Yeah, it, it definitely varies by product for sure. So we've had some products that by 24 hours just release maybe 10 to 15%, but others release pretty much completely a hundred percent, 80 to a hundred percent by 24 hours. So it's all over the board.
Scott Sorrell (11:57):
Okay. So just kind of, just to clarify, if it gets leached into the TMR, it's not gonna get into the cow, right? Because it's gonna be degraded by the microbes once it hits the rumen,
Dr. Clay Zimmerman (12:09):
It it, it'll make it into the cow, but it's not making it through the rumen intact
Scott Sorrell (12:14):
Yep. Yep. Okay. And then, and so is it just simply the moisture that we need to be concerned about with the TMR? Or is it the, the acids or maybe some of the microbial activity there? Do, do we have a sense for that? What's degrading these end caps?
Kari Estes (12:32):
I would say we don't know for sure, but all of those things that you mentioned are definitely possibilities. 100%, yes.
Scott Sorrell (12:40):
Okay.
Dr. Mark Hanigan (12:41):
What, what about abrasion though? Do you think that the mixing, like if, if you mixed for 15 minutes, you know, while you're eating, drinking coffee and eating a donut, is that that gonna be too much or is that not a problem?
Scott Sorrell (12:54):
Great question.
Dr. Clay Zimmerman (12:56):
So, so Kari, maybe, maybe talk a little bit about the feed mixing work that we, we've done that for a number of years, right?
Kari Estes (13:04):
Yeah, yeah. So for the TMR stability, right, we're just putting it in a Ziploc baggie and gently rotating the bag to mix it. So obviously we're not taking that important piece into consideration. But previous to this TMR test, we've done mineral mix experiments, right? So that's actually using, we use a small mixer that's a ribbon and a paddle in it, and we're using minerals, which is gonna be obviously much more abrasive than ATM R. So we mix we combine, again, depending on the nutrient content of the ribbon protected product we're testing, we'll take 90 to 95 pounds of a mineral mix, and then we will add five to 10 pounds of the room and protected product. And we mix it in that ribbon, paddle mixer for three minutes sample, and then we analyze that for, for damage.
Dr. Mark Hanigan (14:06):
Now, is that mixer sort of running at a similar speed to the large mixers that would be in a, in a commercial mill?
Kari Estes (14:14):
Hmm. I might defer that question to Clay.
Dr. Clay Zimmerman (14:18):
Yeah, it, it, it's pretty close to that. Yeah.
Dr. Mark Hanigan (14:22):
Okay.
Dr. Clay Zimmerman (14:23):
That’s a good question mark.
Dr. Mark Hanigan (14:24):
So the sheer force and so forth that it would be subjected to should be somewhat similar to a commercial mill?
Dr. Clay Zimmerman (14:29):
Yeah. And you know, we, we, we, we choose to do it in, in mineral mixes because that, that's sort of the equivalent of taking sandpaper to it, right? Yeah. It's about as harsh as you could do as far as a, from a feed mill situation. Okay. But, but yeah, of course the, the TMR is is also mo the moisture and pH is gonna vary there, right?
Dr. Mark Hanigan (14:57):
Well, so I, I gotta ask another question that I see, you know, happening with some regularity. How's it hold up to pelleting?
Dr. Clay Zimmerman (15:09):
That is a great question mark. We would, I would not recommend paling any encapsulated product because all you're going to do is reduce efficacy. It may not be 100% damage, but it's going to be significant. So we, that's, that's amazing. We do not recommend paling.
Dr. Mark Hanigan (15:33):
That's amazing how often you see that. And it's like, my goodness, that's sort of an expensive ingredient to do that too.
Scott Sorrell (15:39):
Yeah. So Clay, you don't recommend pelleting. I think that's the right answer. And yet there are companies out there that do recommend Pelleting and I don't, I don't know what you can say about that, but maybe I'll take a shot at it, right? These, these are all lipid products. They melt when, when, when you apply heat, they also crush when you apply pressure. And so to believe or to think that there's an advantage of one lipid encapsulate over another, is it, it, it's, honestly, it's, it's, it's just not correct. And so that would be my answer. I don't know what else you'd say I, but
Dr. Clay Zimmerman (16:24):
Well, no, I, I would agree with all of that. And, and, and they can vary in fragility as well, right? I mean, just think about the shape of the shape and size of some of the products, right? It's, you put 'em through a pellet mill, you're, you're gonna break most of the pieces of, of some of these end caps as well. So they're, there are a lot of different forces here that are, that are, are certainly gonna be, be a negative for an end cap. So we, we do not recommending, we do not recommend pelleting in capsule. It's, you will only, you, you'll only hurt the efficacy of a product.
Scott Sorrell (17:00):
Yeah. And then that one final word, clay. So it will damage the efficacy. We don't know how much we believe a lot, but it's gonna be wholly dependent on the parameters of your pelleting equipment, right? How much pressure, how long temperatures, all of those things. So you're going to lose some of it. So my advice would be why lose any, there are ways to get end caps into cows without pelleting them, right? Right. Let's get it in the PMR, let's get it in the TMR one of those two, then you don't have to worry about it. So in my opinion, that's the answer, don't pellet, put it in the PMR and TMR, but that'll get down off my stump and
Dr. Clay Zimmerman (17:42):
I'll tell you. Well, or or in a meal feed, right? A meal feed. Better feed, right? Yeah,
Scott Sorrell (17:47):
Absolutely.
Dr. Clay Zimmerman (17:48):
A lot of these, they do need to be mixed at a feed mill, right? Because the inclusion rates are so low, but doesn't have to be in a pallet,
Scott Sorrell (17:56):
Right? Yeah. Great discussion, mark. I'm glad you asked the question. Yeah, I've got one item I'm not sure is feed stability, but it certainly happens before the cow, and that has to do with freeze thaw stability. And what do we know about that clay? What have you seen?
Dr. Clay Zimmerman (18:15):
Yeah, so we, we've done a lot of work on that through the years. So our, our products are, they are all freeze thaw, stable. We, we test, you know, we've tested competitive products that occasionally will come across some that are not. One reason why that's definitely a must in all of our products is we do a lot of encapsulation for the human side of our business. Actually a a lot of food applications, lots of baking applications even. So it's very critical that our encapsulates survive free thaw processes for our, for the human side of our business. So we do not we, we do not produce any encapsulates that are not freeze-thaw stable. If they're not a true encapsulate, you'll, you'll crack the coating on it. If they're, if they're not freeze-thaw stable, you can actually see that we actually have some good pictures of that under the microscope where you can start to see the cracks in coatings that would occur. You are definitely losing ruminal stability if they're not freeze haw stable and you start to damage the coating at that point.
Scott Sorrell (19:34):
Yeah. Alright. Very well. So then the next attribute is room and protection. And so, clay, in thinking about this, you would think that that would be the easiest part of the puzzle to solve, right? Just, just wrap the nutrient in, in a bunch of hard fat and it's gonna go past the Roman, and yet that's not always the case. Can can you talk a little bit about that and what, what are some of the things that we've seen?
Dr. Clay Zimmerman (19:58):
Yeah, so, so when it comes to ruminal stability, some of this we definitely talked about in the real science lecture back in December. So, and Kari alluded to this already, the true encapsulation versus matrix encapsulation or, or, or the spray shield products. So the in general, the, the matrix end caps do not they tend to have lower ruminal stability in general. Now they can vary quite a bit. And c Kari can attest to this, some of these products have basically no ruminal stability. And, and others have very good ruminal stability, you know, less than 10% loss in the rumen. So there, there are some pretty major differences in al stability, but it's, it's, it's, it's not as simple as you would think. I mean, really this whole process, there's a lot of science behind it. We've been doing this again for 57 years and there it it's a very com complex process and there are trade-offs between some of these steps too. You know, there, there may be some trade-offs between ruminal stability or feed stability and bioavailability. And it's really finding the perfect, the perfect mix of these to, to make the best product from, from an efficacy standpoint for the farm.
Scott Sorrell (21:28):
Yeah. So, you know, again, of the, of the, the four steps, this is probably the one that a nutritionist or dairy farmer can test on his own farm right before he puts it in the cow and gain an understanding of whether or not it is rum or protected or not. Maybe I'll ask Kari, can you speak to that and talk a little bit about that test?
Kari Estes (21:50):
Yeah, so the water test, again, another pretty easy simple test that anybody can run, and that only uses, all you have to have is one gram of the product that you're testing. So it would have to be a ru protected choline product or a RU protected lysine product because we you, you would be measuring choline in the sol, I mean, not choline, sorry, chloride in the solution. So you can't use it on methionine, that's what I'm getting at. But so you take one gram of this product that you're testing and put it in a hundred grams of distilled water and let it sit for as long as you would like, pretty much. Usually one hour is a good time or longer. And then you can use these chloride test strips that you just put in the water and it'll give you a reading on there, PPM, so how much chloride is released from those products into the distilled water. And then you can do a simple calculation to figure out how much of that product was released into the water, how much of it was damaged.
Dr. Mark Hanigan (23:01):
Well, it's an unacceptable level.
Kari Estes (23:04):
Definitely a hundred percent, I would say 50%, right? Because then, well then you've gotta think like, this is just water, right? There's no microbes in here, so this is gonna be probably best case scenario, right? So I would say you'd wanna see something, I would like to see something less than like 10%.
Dr. Mark Hanigan (23:26):
So do you have like a, do you have a calculator or anything that you can give people to help with that calculation or not?
Kari Estes (23:32):
So we do have, oh, go ahead. You wanna go, Scott?
Scott Sorrell (23:36):
No, I was just gonna say we do, we've put together these little test kits and we've got the formula put on there of how you convert the reading on the test strip into how much of the product you've actually lost. But again, you know, we would caution that that's one of three attributes that, that we're gonna be able to measure with that it may be able to pass that room and stability test with flying colors or not. But then it may also then fail the next step, which is the intestinal release, and we'll get to that in a little bit. Clay, anything to add to that?
Dr. Clay Zimmerman (24:16):
No, I just wanna re re reiterate again, if you lose a significant amount of product in the rumen, there is no way you can have a high level of metabolizable nutrients in that product because
Scott Sorrell (24:33):
It's gone,
Dr. Clay Zimmerman (24:33):
Right? Metabolizable nutrient is rumen bypass times the bioavailability of the product. So if you don't have a good room and bypass, you're not, no matter what the bioavailability is post ruminal, you're gonna have a low number on metabolizable nutrient. So again, you kinda lose the game from the start
Dr. Mark Hanigan (24:57):
So you're saying I can't eat my cake and have it too.
Dr. Clay Zimmerman (25:00):
That's right, that's right.
Scott Sorrell (25:02):
Yeah. So Mark maybe a question for you, and this might be maybe an a bit of an unfair question, but you've done a lot of, of bioavailability testing, and we'll dig into that in a little more detail in a moment. But I'm just kind of curious if, if you've looked at it in regards to these different steps, have you looked at room in stability versus intestinal release feed stability, those kinds of things?
Dr. Mark Hanigan (25:27):
Well, first off, it's probably worthwhile clarifying that the, the eye have tested is probably not accurate. Okay. I have a group that I leave that does the testing. Okay. Your mainly insight supposed be like.
Dr. Mark Hanigan (25:43):
And you know, it's the students that do the testing. Not me, but Yeah. Okay. We'll, we'll we'll, we'll use the you know, the the royal wee and say, yes, we, we do do that. And we, we've typically used, you know, an Insitu type approach. So put put the end caps in a small Dacron bag, you know, with a, a small enough pore size that you know that there's minimal leakage out of the bag. And either do a time exposure, you know, and, and pull out bags over time so that we can get an idea of how much leaves immediately and how much is leaving, you know, over an eight hour period, for example. Or we just choose like eight hours or something that we think, I mean, we don't know how long it stays in the room, and for sure anyway, you know, we think it's summer between like six and 12 hours, but we don't know exactly.
Dr. Mark Hanigan (26:33):
And so we can, as we can assess that, and it's it's not a perfect unbiased test there, there's some bias in it that we identified when we were doing the, the nasa, you know, work. And I don't know if that, you know, estimates that we had from that apply directly to an end cap or not. But certainly with regular feeds, it underestimates, you know, some of the, the, of the loss in the rumen, but nonetheless, it is a marker, right? So if you're gonna compare a couple of end caps or multiple end caps to one another, yeah, the ones that have high loss are worse than the ones that have low loss. And so you can certainly rank them. And then when you get the bioavailability number, that's actually an aggregation of the loss in the rumen and the intestinal adjustability. So, you know, to clay's point, if you, if you have a 30% loss in the room and you're down to 70 already as a maximum bioavailability, if it's a hundred percent digested in the rumen, and they're typically not a hundred percent digest, they might be 90.
Dr. Mark Hanigan (27:40):
So then you'd have 90 times 70. So you're down to like 63% bioavailability. So if you have another one that, you know, looks like it's losing half of its amino acid in the rumen, well, it's never gonna be 63, it's already down to a half. Okay. And so, you know, it just adds up. And, and what we've seen, you know, at times, and, and Kari I think was probably the one that was doing the work at that time, was, yeah, when we get, like, typically when we get 85, 90, 90 5% ruminal protection, we get pretty much zero intestinal digestibility. So we, we can definitely, we've seen products where, you know, you guys and others have done a really superb job of al protection and also a very superb job of intestinal protection as well,
Dr. Clay Zimmerman (28:27):
Of, of total bypass, not just room bypass.
Dr. Mark Hanigan (28:29):
Exactly. Yeah. So if you're looking to dump nitrogen on the pasture or the, you know, the field or whatever, this is a good way to achieve that. I might suggest that the cost of that nitrogen is a little bit expensive.
Dr. Clay Zimmerman (28:43):
That's right.
Scott Sorrell (28:45):
Yeah. Good point. So just to kind of summarize where we're at so far, right? We've talked about feed stability, we've talked about room and stability and the need to be able to protect that nutrient during those first two phases. If you don't, you're already folded and you're out of the game, but if you're able to do that now you go on to the third step and that's intestinal release. So now you've gotta be able to release that payload that you've been protecting and the previous two steps. Clay, what do you, what can you tell us about that step and the importance of it and what are maybe some of the attributes to a a, a quality room and end cap that makes a successful room and end cap at that third stage?
Dr. Clay Zimmerman (29:25):
So Mark alluded to some of this o obviously you can, you can either overprotect it in the Ruen or underprotected, it is a balancing act again, and there there are different designs and, and manufacturing options in end caps that, that, that can lead to differences in bioavailability. I think one of the challenges, quite frankly, is methodologies to assess bioavailability. And, you know, that that's really where, you know, mark and his team have really done a, a lot a lot of work through the years to, to, to try to improve those methodologies. And, and I think I'll defer to Mark and Kari may maybe to discuss that some of these different techniques that, that have been used to, to assess bioavailability.
Scott Sorrell (30:20):
You gave a presentation recently at the Tri-State Nutrition conference talking a little bit about measuring nutrient bioavailability and some different methodology that, that you've used and, and tested in the past. I'm wondering, could you maybe kinda walk us through maybe even a little bit of history where, where did you first start, you know, when you first started trying to measure bioavailability, what worked, what didn't? And then, and then how have you transitioned and where are you today?
Dr. Mark Hanigan (30:48):
I, I think Kari can tell that story better, as good as I can because she, she was at the beginning. Okay, so this is when she was a, a young woman, you know, that a num a few years ago, may maybe four or five years ago, something like that.
Kari Estes (31:02):
Yeah, yeah. Not too, I know. Well, that's what I was thinking is, you know, when I started volunteering in your lab as an undergrad, the techniques that we were using and how that progressed until through my master's work, we went through quite a few techniques. It's pretty impressive actually. So in the beginning we were doing work with the mobile bag technique. So that was using du waal and ileal cannulated steers, and then we also had a ru cannulated cow. So we would take these Rubin protective products, incubate them in the rumen of the ru cannulate cow for eight hours, and then we would, and little bags, right? Yeah, yeah. Little dacron bags. Yep. And then we would put those, then oh no, we would do the Abba Masal incubation. Right? We'd have to do that on the lab bench. So that was a two hour incubation and a hydrochloric acid and pepin mixture.
Kari Estes (32:14):
And then we would introduce one little baggie at a time into the duodenum and attempt to catch them at the ileum. So putting them in at the small intestine or at the beginning of the small intestine and catching them at the end of the small intestine. So that technique is really cool, right? Because it's very segmented. So we can test for just al stability or release and then abba masal and then intestinal, right. But it definitely had its challenges for sure. I will say that the cannulas are hard to maintain on our animals that we had. It's doable. They just require a lot of attention. And the transit times was another big problem. So bags would pass each other in the intestine, which was interesting. Even though you're metering them in every 30 minutes, sometimes you wouldn't catch them at the ileum. And so they come out in the feces, which is fine, but that might be 24 hours till they come out there. So those, I would say were some of the biggest drawbacks from that technique. Anything else you can think of on that one, Dr. Hannigan?
Dr. Mark Hanigan (33:34):
No, I think those are the big ones. I mean, it, it was difficult to get reproducibility. Yeah. So we would use soybean meal as a control, and we would, we could get numbers for soybean meal from 30 to to to 120. Okay.
Kari Estes (34:02):
So then we did the Abba Masal pulse dose trials. So that one's also pretty interesting in that it's segmented also. So we incubate the products again in Dacron bags in the rumen for eight hours. And then we using those same rumen cannulated cows, we introduce an infusion line into the abbo masum. And after it's incubated in the rumen, then we take the contents, the remaining contents and of the products, and we dose them into that Abba Masal line. And we measure blood over time, so before, during, and after that infusion, and analyze that for the amino acid that we're interested in. So methionine, lysine, whatever it is. And we can look and see how the products release over time or how they appear in the blood. And we use an area under the curve equation to calculate bioavailability of those products. So
Dr. Mark Hanigan (35:05):
We, we compared that to a, just a, a regular infusion of unprotected amino acid, right?
Kari Estes (35:12):
Yes. Get the maximum
Kari Estes (35:15):
Yes. And so for that technique, it also has its pros and cons as well with like the mobile bag. But one of them had to do with the infusion of the raw because we weren't quite matching the release curves between the raw and the products that we were infusing, which could bias the results some. So we did, we did modify that, and I think we made the technique better from that perspective. But that technique, at least in my opinion, seems to favor some, some encapsulation technologies over, over others. Like a pH trigger product right, is going to hit the Abba Meum and release very rapidly. And so it's gonna have a much different looking curve to it than a product, like a fat coated product that is gonna have a slower release as it moves through the intestines. So you'll have two very different curves that you could be comparing to one another. You have any other thoughts on that one, Dr. Hannigan?
Dr. Mark Hanigan (36:33):
No, I, I, I mean, in the end, we found that it, it seemed to always rank the various products correctly, but the absolute values were not, you know, did not match up our, into our, you know, other ways of assessing things.
Kari Estes (36:47):
Yeah.
Dr. Clay Zimmerman (36:49):
That, that was sort of, that, that was sort of our feeling, you know, internally too. It, it it, to use it as a ranking tool basically.
Kari Estes (36:59):
So then the next technique is the, or that we worked on at least was the stable isotope technique. And that one is a completely en vivo technique, unlike the mobile bag and the ABBA Meel OSE trial. And we actually, at least in the experiment I ran, we did use Rubin cannulate cows for more of validation of the technique. But I know now I think Dr. Hannigan, you're not even using ru cannulated cows for stable isotopes. So that's one Plus
Dr. Mark Hanigan (37:36):
We, we do, we do, sometimes it depends on whether people want that number or not,
Kari Estes (37:41):
Right? Right. but essentially we, we would feed the product to the cows. The cows do have to be in steady state condition for this technique to work. So we feed the cows the products, and then on the last day of each period, the cows receive an infusion of stable isotope. And essentially that's used as a tracer. So we look at the dilution of that isotope in the plasma of these cows to calculate by availability after they've been fed these products. So I think it's probably one of the better techniques, certainly that it's totally in vivo. I know Dr. Hannigan, you've done a lot of experiments since I have been there further perfecting the technique, which has been good, especially for women protective products as well.
Dr. Mark Hanigan (38:44):
We're, it's generally still the same. We're we're just using longer infusion periods now.
Kari Estes (38:48):
Yeah. Yeah.
Scott Sorrell (38:51):
So I'm curious, would you consider then this, this last test that you've evolved to? Is this pretty much the penultimate? Is this is, is this the methodology? Is it, is it as accurate as it can get or do you continue to see further enhancements or, or perhaps different methodology down the road?
Dr. Mark Hanigan (39:13):
Well, you know, we still, it, it's quite a bit of math involved with it. And, and it's, it, it always is a lot of work to, to get the data calculations all done and, and have confidence that we have the right numbers. So we've, we've talked with, you know, clay and, and Kari about helping develop this a little bit further. And so the idea would be that if we could get away from the infusion, that would help, you know, and, and if we could take the cows and put isotope in 'em long enough for them to reach steady state, it would get rid of all the calculation complexity. So that means we need to get a cheaper source of isotope. So rather than using the purified amino acids, you know, that they, that we purchase, they actually derive those by growing algae under a carbon 13 labeled CO2 atmosphere, until they get labeled up to like 95 or 98%, and then they have to kill 'em and then isolate all the protein out of it, and then hydrolyze the protein to amino acids and clean it all up.
Dr. Mark Hanigan (40:19):
So there's a lot of cost to getting that. So what we thought was, well, maybe we could just protect, you know, it doesn't have to be perfect, but protect the algae itself and then just feed the algae that, and then it's cheaper. It's a lot cheaper to buy the algae than it is to buy the, the isolated amino acids. So we're thinking that maybe that might allow us to do more animals, which would give us more precision. It would allow the technique to be simplified because of steady state conditions being reached with the isotope as well. It's not different. It's, you know, it's still the same concept. It's just trying to simplify it and also get some more replication to try to make it even more precise.
Scott Sorrell (41:04):
Yeah. Good conversation.
Dr. Mark Hanigan (41:06):
We don't know the answer yet to that. Okay.
Scott Sorrell (41:08):
Yeah. Yeah.
Kari Estes (41:09):
But that is like a little bit more like the Len methionine technique.
Dr. Mark Hanigan (41:15):
Exactly. That would be very, very similar then. Yeah. Right. We're using a naturally occurring, sort of naturally occurring, if you consider that adding a little extra carbon, 13 to the environment is natural. But there is net carbon 13 already there. We're just enriching it. And same thing with the, you know, the yeast, you know, you're, you're selecting a line of yeast that tend to, and you're giving them a lot of selenium trying to coax them to putting more into the methionine, which they do. So the advantage of this approach would be that we can then look at any of the essential amino acids where the l'methionine is only going to be . We don't really have any other ways of doing it. Right.
Scott Sorrell (41:56):
And can you only do that with amino acids, or could you apply that technique to choline, niacin other, other nutrients?
Dr. Mark Hanigan (42:04):
We think we can apply it to other nutrients, which is one of the other reasons we wanted to use, we could have used like purified protein from the algae. It would've been slightly more expensive, but we wanted to try the algae 'cause it will have all nutrients labeled in it. And so we're thinking that there are probably additional, you know, opportunities to try to assess bioavailability of those other nutrients, such as you mentioned. Yeah. There's a lot of work that has to go into that. And I'm not going to be the person that's doing that
Scott Sorrell (42:56):
Hmm. Great. It's good to hear there's somebody to pass the baton to. Mark. You've done a great job. Clay, we've talked a lot about bioavailability. Let's talk a little bit about the importance. Why is it important to get that right? We've spent a lot of money, a lot of effort getting that right? So why is it important?
Dr. Clay Zimmerman (43:14):
It's, it's very important, right? Because as, as, as we put products into the market, you have to be able to model those in the nutritional models. So the bioavailability is a really key part of that to, to, to make sure we're feeding these products at the correct rates for, from an animal, animal performance standpoint. So we, we, we put a lot into assessing bioavailability particularly on the amino acid side of things, you know, where, where we're actually balancing for that as a nutrient in the diet.
Scott Sorrell (43:56):
All right, so we've perfected some methodologies for measuring bioavailability, but yet there's companies out there still using in vitro models. We use in vitro models, but not necessarily give us bioavailability. Can you talk a little bit about that clay? Is, is maybe where are some of the downsides to the in vitro models? How do we use 'em? And, and then Kari, I think you had some comments as well.
Dr. Clay Zimmerman (44:25):
Yeah. So they're are very key. These in vitro techniques, they're a very key piece of product development. You you cannot you really, you cannot economically get products to market. If you have to put every prototype through an animal study, it's, it's, it's impossible to do that. So it's very important to have some methodologies to rank prototypes and to get your initial assessments. So we do, we, we do our initial screenings with in vitro techniques to to try to do the best we can mimicking what would actually happen in the cow. So that, that's a very key part from a product development standpoint. But it's not the final answer. The final answer is measuring these in vivo and then really ultimately measuring performance in the animal. That's where it all comes together. But Kari, I know part of your, part of your master's work was actually assessing some of these in vitro techniques, so maybe you can expound upon that a little bit. Mm-Hmm.
Kari Estes (45:49):
Yeah. So, yes, as you mentioned, Dr. Hannigan and I worked on, we have a paper that was published in translational animal science comparing some in vivo techniques to two different in vitro techniques. One being a three step procedure, and another one being a pepin digestibility assay. So in that paper, we, we looked at blood meal feather meal, and a room protected lysine prototype. So with that prototype, it was tested in a, a lactation trial, a pulse dose trial. So those were the two in vivo experiments. And then in this three step procedure, which is performed at, I know it's at least offered at two commercial laboratories. So we were not actually running the in vitro tests. We sent the samples out to a third party for analysis. So and basically what we found was that the in vitro test did grossly underestimate the availability of that prototype when compared to the two in vivo methods.
Kari Estes (47:05):
So I think there's some reasons behind that, primarily when we look at what the enzyme cocktails are that are used in the procedure itself. So, and again, I think these procedures can favor certain technologies as well, right? So if you have a pH sensitive product, then all that really matters for you would be that there's an ABBA masal step, right? But for fat coated products, you're gonna wanna make sure that that intestinal piece at least has the enzymes that would be in the animal to break down fat, right? So we did, we explained some of that in, in the paper if people are interested in, in reading it. But I think they're, it could be improved upon, I think in vitro techniques are super important for our industry, right? They're quick, they're easy, they're cheap. But we can do better, I think, to have them match more of what's happening in vivo or we just use 'em as a ranking tool.
Dr. Clay Zimmerman (48:21):
The, the other part I would add to that as well is, you know, these, the in vitro techniques, they're not take, they're not taking into account the, the feed and TMR stability piece of this. So, you know, you, you, you can draw some pretty erroneous conclusions here.
Scott Sorrell (48:43):
Yeah. Clay, would it be a step too far to say that one of the other attributes is sometimes they're just flat wrong, right? I mean, we've had times when we've been testing products and, and they've let us astray. Yeah. we've been doing this for a very long time. So anyway, it's yeah, you gotta be very cautious with them. There's probably four different ones that we use, different ones for different nutrients and yeah. Proceed cautiously anyway. Mm-Hmm.
Dr. Clay Zimmerman (49:20):
I, so my caution would be if, if, if you see products that are only showing in vitro results, I'd be very skeptical, right? Yeah.
Dr. Mark Hanigan (49:32):
definitely.
Scott Sorrell (49:35):
Yeah, good point.
Dr. Mark Hanigan (49:36):
I I think that's a, you know, we've already talked about that, but there's a difference between ranking. I mean, if you have a hundred end caps that you wanna, you know, prototypes that you wanna screen as Clay said, you can't afford, and we, we charge like 20,000 an end cap to, to, so I, I don't know, you have to do that out, but I mean, it is a lot of money, right? And a lot of time it would take us years to do that. Okay.
Dr. Clay Zimmerman (49:59):
It'd only be $2 million, mark.
Dr. Mark Hanigan (50:01):
Yeah. So you can't, you can't do that, you know, in, in real time and make any progress. So you wanna screen that a hundred down to three or four or five, right? Using something that maybe is not accurate, but hopefully is ranking correctly. And even if it's, even if it's off a little bit, okay, well maybe when you go test 'em in the cows, you know, three of 'em rank like they're supposed to and the other one's just way off. Okay. Well, you're still way better off than trying to test all one hundreds.
Scott Sorrell (50:28):
Yeah. Great point. Mark, clay, your, your last step was biological response. What do you want us to know there?
Dr. Clay Zimmerman (50:39):
So that is the key last step, right? That's where, that's where you could take all of this into account then, right? The, the feed and TMR stability, ruminal stability, and intestinal availability. So that's ultimately where we end up is running these animal studies to prove efficacy of the product. So I would caution, I'd, I'd caution people again, don't, don't rely just on, on vitro data, but I, I was always skeptical when I was presented with products that like, just had no animal data behind them, no performance data. I wanna see that too. I always want to see that as, as a nutritionist.
Scott Sorrell (51:34):
And so talk a little bit about animal data. Clay. Is all animal data created equally as well?
Dr. Clay Zimmerman (51:41):
No. No. It, it, it all depends on, it all depends on how the trials are set up and, and, and really, you know, who's running the trials, right? So ultimately you want, you want independent peer reviewed data that's out there. So yeah, there, there are big differences and, and in studies that are so-called studies that that can be done. And there's really kind of a pyramid be behind that. From an animal study standpoint lots of studies obviously are preferred like we have with reassure, right? We have, we now have over 41 peer reviewed publications on our reassure product, for instance, that that's that's huge, right? To have that kind of data behind products. But we, we do all of our work with, with independent researchers, either universities are, are private research institutions.
Scott Sorrell (52:41):
Yeah. Yeah. Kind of what I was getting at too, clay, is, is field trials, you'll see people show up with with some field trial data and say, Hey, our stuff works wonderful. And you know, as you know, we, we just simply won't do a field trial as research. There's just too many things that can and will go wrong each and every time. Never seen one work yet. Clay, maybe, maybe you're you're better at it than,
Dr. Clay Zimmerman (53:06):
Well, no, there's, they're not controlled animal studies.
Dr. Mark Hanigan (53:09):
Exactly. You don't have a, usually don't have a control, right? Or, or if you try to do a control, then the farm sort of gets grumpy about all the extra work the feeder does, right? And, and sort of doesn't, you know, follow the protocol. And so without a control, I mean, God, it's just a crap shoot whether you can actually cease it or not.
Dr. Mark Hanigan (53:28):
Yeah. So I, I would just add one more comment on the, you know, on the, on the biological response side, the product could be fine, right? And, and, and if the cows don't respond, you know, is it because there's a water quality issue or water quantity issue, you know, a stress issue, a a, you know, are the cows comfortable? I mean, there's a whole laundry list of things that could be going on that might compromise the response in a cow, and it's not the product's fault. Okay. So I tend to, that's a good point,
Scott Sorrell (54:19):
Yeah. Yeah. Great point. Well, this has been a great discussion but I think it's time me to call last call. I usually wait until I've run out of bourbon, but that happened about 30 minutes ago, I think. I was just kind of excited about the conversation. And anyway we'll move on from that. But let's, let's call our last call and, and Kari, why don't you start us off. What's kind of a few takeaways that you think the audience should take away from this discussion?
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Kari Estes (55:13):
Not all in caps are created equal mic drop. Just kidding.
Dr. Mark Hanigan (55:19):
Nice. Nice.
Kari Estes (55:21):
No, I would say that, and I don't know, I guess I think about from a nutritionist standpoint or somebody who's, you know, trying to pick which product they're gonna be using in a, in their ration, you know, asked to see the data that these companies would have and does, does the research hit these pillars that we just went through? Right. And I would say pay attention to the techniques that are used as well.
Scott Sorrell (55:51):
Yeah. Well said. Kari. Mark, would you have anything to, to add to your, your star pupil? Well,
Dr. Mark Hanigan (55:59):
Well, I would go to I, I've forgotten who is credit or who, who stated this, but I do remember that Gordon Thompson, who I worked with for a number of years, had a really keen sense of humor, and he had this posted on his desk. Anyone who thinks something is foolproof simply hasn't met a sufficiently talented fool yet,
Scott Sorrell (56:33):
Yeah, well said. Clay, final words,
Dr. Clay Zimmerman (56:38):
Final words. I've been at biochem 10 and a half years now, but for 22 years before that, I was a nutritionist, right? For, for two feed companies. So I was part of the meat market, right? You're bombarded with products all the time from companies. So there are, you're constantly being faced with making these product decisions. What product should I use? So I think Kari said it well. Look a ask for the data needs to be credible data. The more data the better. And I, I think we're only starting to scratch the surface on the feed stability piece of this. We, we need a lot more work in that area. It's been really overlooked, I would say. So that's a, that's a key, that's a key p piece to these products being effective in the field.
Scott Sorrell (57:42):
All right. Very well, clay. Well, clay, you did a nice job being our featured guest this evening. Unfortunately next time we'll have to put you back in the co-host seat, but that's all right. Mark Carey, it's been a great conversation. I really appreciate you guys coming along. To our loyal listeners once again, as always, thank you for coming along with us. We hope you learned something. We hope you had some fun, and we hope to see you next time here at the Real Science Exchange, where it's always happy hour and you're always among friends.
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