WHAT'S GOING ON IN THE GUT? EXPLORING THE SCIENCE BEHIND RUMEN HEALTH - BALCHEM CORP

Hello everyone and welcome to the Real Science Lecture Series. I'm Scott Sorrell, Director of Global Marketing here at BALCAM. Today it's my pleasure to welcome Dr. Kirby Krogstad of The Ohio State University for a presentation titled, What's Going On in the Gut? Exploring the Science Behind Rumin Health. Rumin health is something we talk about a lot in the dairy industry. And when cows aren't performing as expected, the rumen is often one of the first places we look. But despite its importance, there's a great deal we're learning about how the rumen functions and how its environment is regulated. Today, Dr. Krogstad will take us inside the rumens to explore what we know, what recent research is teaching us about rumen physiology, and where important questions remain. Just as importantly, he'll connect that science back to the farm with practical considerations for feeding and managing cows to support rumen health. I'd now like to introduce Dr. Krogstad. Kirby is an assistant professor of dairy nutrition at the Ohio State University. Kirby's research focuses on rumen physiology and rumen health. He also investigates forage and non-forage feeding strategies, nutrient and ingredient interactions, and the role of specific nutrients in combating inflammatory signals. In addition to his research program, he conducts an outreach and extension program that focuses on enhancing animal health productivity through improving nutritional program management. He also leads an internship program that brings research and farm management opportunities to undergraduate and DVM students. Dr. Krogstad, welcome to the Real Science Lecture Series. Thank you. Thanks for having me. You're welcome. It's wonderful, wonderful to be here. I appreciate the invitation. And I think, as always, on a day like today, it's Election Day. So if you're one of our American listeners, make sure to get out there and get to the polls and vote. And we will get started on such a positive note. So I want to thank you again for the kind introduction. Scott, does the screen look good, everything as it should be? Yes, looks good. Awesome. And so we're just going to dive in, talk about some basic physiology and basic science, and also hopefully some applied lessons that I've taken from the lab and projects that we've run, but also from a little bit of field experience having grown up in the business that I hope will be helpful to all of you, give you some things to take home to your operations, to your business, to their clients. I have to start out with recognizing the students that really do the actual work. I get to sit here and do things like this and pretend I'm all important, but they're the ones that actually put the blood, sweat, and tears into the work. So in the picture here, you'll see Tita Vandervoort, Irie Maciel, and Andy Miepski, all of whom contributed to a lot of the work I'll share out of our lab. I want to give a special recognition to Tita VanVoort as she's been leading the room and health piece of this, the room in physiology piece of these projects that we're doing, and she's completing her master's degree next summer. Irie is an alum of the lab, works now for Jeffo, and Andy is now doing her PhD at Iowa State University. We tried our best keeper of Buckeye, but she sought other pastures. So we wish them well, but we really want to acknowledge them for their contribution to the work I'm going to talk about today. So we'll start out getting everybody on the same basis. We're going to talking about a little rumen structure, tissue regulation, how those things work. We'll talk about how we can manage the rumen and the gut with what we know. And finally, hopefully close with some bigger picture ideas of what's missing, what do we need to learn, and a couple of take-homes for the applied listeners who need to understand how to manage cows better at the farm gate. So I want to start with defining gut health, but even then I want a step back with a comment I received from a producer and it really surprised me, but I was in a room full of about 20 dairy farmers who milked anywhere from 600 to 20,000 cows. And I remember sitting there asking him like, well, what are the challenges you guys are still facing that, you know, the academy and our research needs to address? What do we need to be paying attention to? And are we even paying attention of the right thing? And I was really surprised. One of the younger gentlemen in the room shot his hand up. He said, honestly, the thing that drives me nuts is we still have that high producing, really good mid lactation cow who will be, she'll be cruising along. She'll be making good milk, eating a lot of feed, good rumination. And then overnight you come in the barn in the morning and rumination is zero. She's making no milk. She looks terrible. And maybe she has HBS or maybe she has some other gut issue or some problem that we really still don't know how to fix yet. We really still do not know how get after it. And that comes to this idea of gut health. Like what is it? How do we manage it? how do we fix it? And I want to start with defining it because I think like we said in the intro, we throw gut health around a lot, and I don't think we have any clue what we actually need. And I think it's sort of become a catch-all for challenges we face. Like, oh, you know, my cow's lost a couple pounds of milk. It must be a gut health problem. And I, I think that's just because we don't have a really good answer for what it may actually be. So my working definition is that gut health is the functional status of the whole gastrointestinal tract that includes interactions amongst each local epithelium, each local luminal contents, and the local microbiomes that reside within that specific site of the gut. So as you'll see on this image, we have the rumen, obviously, and the rumin microflora that we have studied for decades. But within that, we also have the tissue resident microflor that we know much less about that's directly interacting with that rumen epithelium. And we get into the hindgut and the hind gut, whether it be the large intestine or the segments of the small intestine, each of those has unique flora as well. And they interact with a very different epithelia structure than exists in the rumen. So when we talk about gut health and livestock, that's all of the interactions that we have to start cornering and start understanding and start digging at. And it's going to take some time to dig into all these details. But I think with some concerted effort, we'll start really learning some interesting things about how to manage these livestock for a really healthy, resilient gut barrier. So we'll star with the rumen. And this is one of my favorite slides I've ever made because I love Shrek. And being the millennial that I am, Shrek was a big part of my childhood. And the rumin, like an ogre or like an onion, it has layers. It's got a lot of interesting things going on. So if you look at the image on the left of the screen, we have an outer corneum layer. That's what directly faces the rumen contents. It's kind of a layer of dead cells. They're heavily keratinized. They provide this, we think they function to provide this protective barrier from all of those microbes and all of the compounds that are in the rumens just swimming around. Beneath that we have a stratum granulosum and the granulosium you'll see has still a flattening of cells, but it also has the beginning of those tight junctions and desmosomes. You'll see little railroad tracks between each of those cells. Those are those tight barriers that keep that tissue from being permeable. It stops stuff from passing across paracellularly. Then below that, we have the most metabolically active and dividing layers, the spinosum and basal. That's where our cell division is occurring. That is where a lot of mitochondria are, which you'll see illustrated in this figure from Dr. Steele's review in 2016. And that's also where a lot of our BFA metabolism occurs, where we'll be converting butyrate to BHBA, for example. And so that's what that tissue structure looks like. And because of those four layers, we presume we have quite a bit of vigorous protection there, that that barrier is highly functional and can handle a lot different things, which it can. But my contention is we probably don't actually have a great understanding of what signals are regulating that barrier, what stressors can do to that barrier and how we can manage to optimize that barrier's function. Now, the important thing to recognize is once we get past the rumen, the cow's gut looks much like any other mammal. The lower gut and the small intestine is a single columnar layer of epithelial cells. That leads some to think that, or that would lead many to think that that is putting that part of the gut at really high risk. But that set or segment of the gut has a very different system of defenses. It has mucosal layers that the rumen does not have. It Has an outer mucus layer that's populated with microbes and which helps digest feed, but also can help provide defense and exclude other problematic microbes. Then we have an inner mucus player, which really serves as this exclusion zone that's kind of adhered to that intestinal epithelium. And it has a bunch of antimicrobial peptides within there and positive microbes that are going to help keep that gut healthy. And then we get to that single layer of epithelia cells that's also immune cell rich and has a whole bunch of stuff going on that the rumen does not possess. So it's a very different type of defense, a very different structure that's going on there. Now for today's talk, though, I'm going to focus on the rumen, but I think it's important just to illustrate these are the two segments of the gut that we're dealing with and that the cow has to manage. And we have to allow nutrients to pass while providing that barrier that keeps problematic microbes, pathogens, mycotoxins, those kinds of things out of the animal. So when we think of rumen health, the first and biggest thing we've studied for years is rumen acidosis, rumen acidosis, subacute rumenacidosis. All the different things that we can call it. And there is good reason for that. But as you'll see as we go through, my contention is that this is not the only game in town, and we should be looking a little bit deeper. But I do want to start with this basic information. Now, when you look at it, there's various thresholds for SARA. We have a definition of below 5.6 for three hours a day, or below 5 point 8 for five hours a week. The definitions vary depending on the citation source and the data that they use to come to that conclusion. But I want to set us there. So we have a long understanding that low pH for longer times is problematic. But why? What is the data to say that that is a problem? Now, the first thing is, do cows actually experience this? Does Sarah still exist? When we look across the literature between a recent review and some older papers, somewhere between 1 in 10 and 1 in 4 animals will have some prevalence of Sarah. The O'Grady study highlighted there in green, I wanted to make sure to include that today, especially because that's a grazing situation, a pasture situation. So those of you that are in countries where we're going to be relying almost exclusively on pasture-fed cattle, whether that be Ireland or New Zealand or some of the other northern European countries, you still do have some risk of sera, especially early in that season when the grass is hyper-degradable and very lush. So there is some prevalence of it there. But why does that sera matter? Well, first of all, sera depresses fiber digestibility. Excuse me. It depresses cyber digestibility, and this is a study from 2002 that shows it quite nicely across forage family. We have a cow with sera, a cow without it. We put these feeds in them, incubate them and look at degradability. When we have a serra incubation, we lose 10 to 12 units of fiber digestibility. So we know that reducing pH alone can depress NDFD. And it does seem to be at least partially attributed to pH and pH alone. So this is some nice data from calcium amygdala. and as you move from 7.2 down to 4.8 you can see those NDF digestion curves decline as pH declines but you'll also see two sets of dots. Those dots are about the substrate that's included in the inoculum so it is not just pH that can reduce fiber digestibility it is also some substrate effects like starch versus fiber and that actually is borne out in the most recent NASEM version that modeled this, which demonstrated that for every one unit increase in dietary starch that we feed to these cows, we can expect six-tenths reduction in fiber digestibility. And so you'll see that here in the NASEM data, the 26, dietary starch of 26% is at zero. And that's because that's our base diet in the NASA model. And as we progress up to 32, we lose about three and a half points of NDFD. So when we consider a ration formulation scenario, increasing starch, we expect possibly to increase milk production or increase total energy to the cow, but we have to remember that there is a trade-off there that we lose that fiber digestibility. And part of that may be because of ruminal acidosis and sera, but part of it is likely just that substrate competition between fiber and dietary starch. So we need to keep that in mind that it's not just pH that is driving some of these responses or maybe a problem in the gut. The other changes are probably related to the microbes. And this is an area where a colleague of mine has been really leading us for the last 20 or 30 years, Dr. Jeff Perkins. And when we reduce pH, we also see increases in provatella, increases in proteobacteria, increases in E. coli in the rumen, all of which can probably change the ability of those microbes to degrade different carbohydrates. And there's a lot of this is a quote from Dr. Perkins that I think sums up nicely where we need to be heading with some research, which is that more research is needed to explain differences among microbial populations, and they're expressed caseins as affected by pH independent from changes in substrate and growth factors. And that could be things like branched chain volatile fatty acids or the starch versus NDF versus sugar discussion. We need to understand how those microbes express carbohydrate degrading enzymes. That's that casein complex that he's referring to. So we get the reduced pH, we get reduced fiber digestibility. pH alone causes some of that. substrate competition causes some of that. Microbial shifts cause some of that. Now, to get more to the rumen health and tissue health part of this, we need to pause and realize all the things that come along with that reduced ruminal pH and subacute ruminal acidosis. We've talked about the reduced fiber degradability here, which you'll see on the slide. We talked about altered ruminal microbiome that we just discussed. We also get an altered volatile fatty acid profile that can accompany reductions in pH and higher grain feeding. And finally, we get to this idea of increased lipopolysaccharide and increased biogenic amines that can also potentially be problematic, that accompany cells lysing in the rumen when pH is reduced, which leads to this broader question of, does this LPS increase cause inflammation, or is it causing tissue level or systemic inflammation? And that's where my interest started in my PhD program at Michigan State with Dr. Bradford, and we started digging into some of this. And the first paper I read that got me kind of piqued in thinking about this gut health question was a review paper from Zabelli et al. 2012. And they summarized a bunch of studies feeding various levels of dietary starch, mostly, I will say, from ruminal acidosis challenge studies. And their trying to identify if there was a relationship between markers of inflammation and ruminal LPS and dietary concentrate level. So in this graph, you'll see three panels. There's ruminal LPS in the top, plasma haptoglobin in the middle, and plasma serum amyloid A on the bottom. And for each of those, they did a breakpoint analysis. And at about 45% of the diet is concentrates. And so that's concentrates, that's not starch, that'S not sugar, it'S not any specific nutrient, it's just concentrates so that could include non-forage fiber, it could include corn, and it could include soybean meal, things like that. As we pass across that level and go above 45% of the diet is concentrate, they suggest that we have risk of systemic inflammation and increased LPS. But again, that's mostly challenge experiments. And for those of you that are applied nutritionists and myself that I try to get out and work with dairy farmers and veterinarians, I look at this and become concerned because most of our diets to high producing cows, at least here in the States, are probably going to be over that 45% number. They're probably going to be 50, in some cases 55, or even 60%, depending on geography. So it's like, well, shoot, do we have a bunch of cows at risk for a gut-derived inflammatory challenge? And that's what our team wanted to dig into. But the question is, well why does that even matter? Why is this balance between concentrate and forage important? Well, because we know if we push concentrates like corn and increase starch into a diet, we can often drive milk and milk protein. And milk protein, obviously right now is driving our milk chickens, driving our economics. I haven't, I didn't check the milk protein price this morning, but I thought it was $2.80 a pound or so. But we're looking at 80 to 100 gram a day benefit when we substitute starch in place of non-forage fiber in these two studies as an example. So the reason this question is so important is because we have to balance that economic return of feeding more starch and driving milk protein with the rumen health and gastrointestinal health of that animal. So it is a balancing act that we need to consider. And in this case, what we're up against here is revenue of 50 to 60 cents per head per day from that additional dietary starch. So I set that up, or I want to provide that data to frame a little bit of this discussion to understand that it's complex, it's nuanced. We've got trade-offs, we've got things we've gotta consider. So as we started digging in more to this question, we wanted to understand not just blood biomarkers of inflammation, but also tissue level responses to changes in starch. So during my PhD with Dr. Bradford, we ran our own acidosis challenge experiment. We had a control diet that you'll see on the left of this table that was relatively high forage, moderate starch, pretty typical of an upper Midwestern cow, getting a corn silage, haylage diet. Then on the right, we did a Sarah challenge diet where we reduced the forage and we increased the starch. And the starch came from wheat and barley. So we switched from a corn-based diet to a wheat-barley-based grain mix. And so we're doing two things. We're increasing total dietary starch concentration, but we're also increasing the degradability of that starch source. We wanted to determine if that was going to cause some issues. And we really thought we did this. We were thinking this was an extreme diet. Forage NDF was 16, starch was 32. We thought we were really going to cause some issues with these cows, and the cows taught us otherwise. We increased starch with wheat and barley in this case. The cows ate 2.1 more kilos a day. They made a kilo more milk. The astute viewer is going to look at these slides and be like, wow, that's pretty terrible feed efficiency, and you're right, it is, but I would caveat that by saying these cows were 290 days in milk. We didn't want to do this to mid-lactation cows because this was the first time we were running a challenge study like this, looking at rumen tissue. So we started with a lower risk group, a group of really late milk cows. So instead of getting the severe challenge we expected, we actually increased intake in milk from that increase in starch. But it actually, in my mind, turns out to be a perfect illustration of the challenge and balancing act we're discussing, which is additional energy into that rumen, additional degradability against the ability to maintain a healthy gastrointestinal barrier. So the question then became, did we even achieve our goal of causing sera. So on that control diet, our room pH was about 5.9, which is already low. Our fecal pH was 7.2, and they spent a little less than two hours below 5.6. When we looked at the sera diet, we did reduce room pH, we reduced fecal PH, and we did increase the time spent below 5,6. So by this measure, we didn't increase and probably cause sera by some of the definitions that we observed earlier. So does this sera condition result in tissue level inflammatory responses? That was really our question. So we started with the blood measures that everybody gets on these studies. They get a blood tube, they take a sample from the tail or maybe a jugular sample or whatever, and they measure some acute phase proteins. And we did the same. We looked at haptoglobin, serum amyloid A, lipopolysaccharide binding protein, and another marker, uh, D lactate. And what you'll see is we had a baseline period. And then the challenge period is in gray when we switched them to the high grain diet. And there was a slight increase on haptoglobin from day two to day three, that was significant statistically. But if you look across those responses, there's really just not much there. There's nothing too dramatic, nothing too crazy. So this got us thinking, well, you know, this high starch diet may not be so much problem or Sarah might not be so much of a problem. But one thing I want to caveat here is this was only nine cows and you'll see those nine cows listed on the slide. And the response was dramatically different from individual to individual. So you'll See one cow, um, on this slide, 52 77 had an enormous increase in her plasma haptoglobin following the switch to the high grain diet. So there is likely some level of individual variation that we don't appreciate or We don't understand whether that be that animal's microbial population, the tissue adhered microbes, a basic immunophenotype in the rumen that we don't understand or something else in the gut. She had a huge response to this increase in dietary starch. Now, we tried. And so remember, that was plasma. That was blood systemic. It could have come from anywhere, not necessarily from the gut, and we really wanted to look at rumen tissue specifically. So one thing we did on this study is we evacuated the rumen tissue contents and took tissue papillae samples. And in this case, we looked at mRNA transcript abundance of some inflammatory markers to try to understand if there was a shift. And you'll see, as we go from control to sera, there were some changes. Only one of them was statistically even a trend, and that was a reduction in IL-1 beta, which was actually counterintuitive. We didn't expect that. We expected to see an increase in IL-1 beta, which is a pro-inflammatory marker. And you'll see IL-10 seemed to increase, but that wasn't significant, but tremendously variable. IL-17 seemed to decrease, again, tremendously variable, but this was largely driven by two individual cows who had big changes in their tissue transcript abundance. It was not a consistent shift across cows. So again, we come back to that idea of individual variation. we also did just some basic histology which is always tremendously useful and you will see we saw some big changes in that 26% starch diet with corn we had a very organized ruminal epithelium a very tightly bound stratum corneum layer and a very organised tissue structure but then if you look at the high starch diet you'll see that that internal area that innermost portion of the tissue expanded as a proportion of the total. The stratum corneum thinned, which I don't know if that means greater risk or if that mean nothing at all. It's hard to really say. But there were changes in that tissue that happened just within a few days of the dietary switch. So that rumen is dynamic. It is responding. And whether that thinning in tissue changes risk factor is hard to say. But there are substantial changes in that tissue. What we wanted to really investigate, which was the more novel part this study was trying to understand if there's an immune cell presence within that papillae and whether it's a certain type of cell that may be regulatory or pro-inflammatory or help us understand how this tissue actually maintains itself. And what we observed is that there is an immune cells presence in the tissue. In this immunohistochemistry plot, you'll see this blue in the upper left is staining of nuclei. Then you have the green and the red. The green is staining CD3 positive cells, which is a T cell. The red is staining a global immune cell stain, it's CD45, it's expressed on every leukocyte. And then the bottom right-hand corner of that plot is co-staining. So those are the ones that express both. And you'll see they're heavily overlapped, which tells us nearly all of the immune cells we're finding are T cells. In this case, we also did flow cytometry. We took the tissue out, we digested it in the lab and tried to harvest immune cells. And from the cells we could measure, 85% of them were T lymphocytes. And we did, this has been seen by some other investigators. I wish I could take all the credit that I'm the only one that's ever known this, but in 2020, there was a paper, I think it's Johansson et al. And they looked at CD3 staining in rumen tissue of cows that had acidosis and not, and they also observed a presence of T cells in their, in their livestock, but didn't dig into it quite to the same degree that we're doing now. So I'm really excited for what this could mean. But this is one of those regulatory levers in the rumen where these cells are probably helping maintain this barrier, but we really don't know much about how. So now we've done this a few different times. You saw that first study of Krogstad et al in 25, but now we have Vandervoort 26 and Maseau et al 26. We've gone across a few studies and we're starting to see consistently that there is a prominent T cell population within this rumen that likely provides some regulatory help to that tissue. We did see in one study that it's mostly gamma delta T cells, which is really interesting because in plasma, and in a few different studies in calves and cows, that cell type promotes resolution and regulation. It usually secretes cytokines like IL-10 that help to heal things and maintain an anti-inflammatory posture, if you will. Do they do the same thing in the rheumatic tissue? It's hard to say. And I will say that was in one studies where we had evidence of prominent gamma delta population as we dig into it in a current study we're doing right now we'll see if that holds up and so stay tuned for that data coming out probably at adsa next summer um there are some myeloid cells present which would be things like macrophages and dendritic cells but in our initial study they were about seven percent of that population so a lot more work to be done there and t that my student would tell you is we need to understand the function of these cells the function must be elucidated to better understand what role these t cells are play. So one thing we wanted to do is find a better stress model. So we did that diet, we did the head acidosis challenge that didn't really work because the cows ate more, they made more milk. So what we did is we dropped the forage NDF to 12, really dropped it. This is really, really low. And then we increased starch from 23 to 33, really trying to push these cows, really trying To stress out that gut. And you can see the diet specs there in the table. The first thing I want to start with is we saw no change in room and pH in this study. So any shift that we're seeing in the tissue or in digestibility or production is not related to changes from room and pH. So all the room and PH is usually what we think of with gut health. It is not the only game in town. And this time we added a wrinkle to this study. We harvested that tissue. And instead of just doing the flow cytometry and immunohistochemistry staining and looking at T cells and lymphocytes, we decided to look at that tissue, digest it, and measure inflammatory proteins and cytokines in that tissue. So in a panel of 15 cytokine, these four were significantly increased as starch increased in the diet. And if you're on gamma, IL-1 alpha, IL-4 and IP10 all increased as we increased dietary starch. And you'll see that the most dramatic shift seemed to be when we crossed over 30% to that 32 or 33% dietary starch ration. So in this case, we did measure blood inflammatory biomarkers. We had no change. We did measure tissue biomarkers. We have these changes you see here. So changes in the tissue are going to preclude any changes in the broader systemic circulation. So if we want to understand gut health, what I'm saying is we need to get to the tissue. We have to go beyond just circulating biomarker. But this is the first evidence we have that increasing starch in the diet may cause some baseline stress, but it seems to occur most when we cross over 30% diet dry matter to 32 or 33. for those interested in performance in this study this again was a small study only six cows crossover design first thing you'll notice is they ate a ton of feed they ate about 80 pounds of dry matter a day 35 kilos a day and as we increased dietary starch we tanked milk fat from about 3.9 down to 3.3 and our energy corrected milk was greatest on that low starch diet at about 53 kilos. So one of the take-homes from an applied setting is if you're worried about gut health and tissue stress from increased dietary starch, you will probably get a milk fat depression response before you have significant tissue stress from dietary starch when you're feeding dry ground corn. So I apologize for all the caveats that go along with that, but that's how science works. It moves slow, it moves stepwise. So in this case, if you're feeding dry ground corn, increasing starch, you will probably have a milk fat depression response before you get to the tissue inflammation that we observed here. So the next step that we wanted to look at was green source. So this first step was dry ground core in a low forage diet. Well, what about barley? Barley is a little bit more rumen digestible or rumen degradable than corn. Does that cause issues? So we ran a study. It's accepted in Journal of Dairy Science Communications. It will be coming out probably in January as we change journal publishers. But what you'll see here, first of all, is there was a pH response. As we substituted in barley for corn, pH was reduced, which we would expect. Barley is more rumen degradable than corn. It should cause a pH depression. We measure this with the smack stack ruminal boluses, which gives us that instantaneous measurement over time. So it shows a fairly granular level that we got the response we expected. But as we increased starch from 22% to 27% of the diet dry matter, we didn't get much change within grain type. So grain source really did drive a pH response. Starch concentration, not so much. But then how is that related to actual tissue inflammation? So we did the same thing. We looked at tissue level inflammatory markers, tried to understand if there were any changes. The corn is in red. The barley is in gray. Low starch is the crosshatch pattern. the high starch is the solid pattern and what you'll see is we didn't have a ton of consistent or dramatic responses um actually when we went to the higher starch diet in some cases it seemed that there was less inflammatory stress occurring in that diet but more probably needs to be done here the caveat being this study was a truncated design so it wasn't quite as powerful as we would have hoped but the take home here was for grain type there was no consistent or dramatic shift in that rumen tissue. When we provided adequate forage NDF, these diets had 20% forage NDF and we were at moderate starch levels of that 22 or 27%. Now rumen pH did respond, but tissue inflammation in this case did not. The other interesting thing that we've been learning that we're really excited about is that we are starting to dig into the regulatory cytokine network within the rumen compared to what we see in blood. And what stuck out there is the IL-1 family of cytokines seems to be very prominent. So on the left, we have IL-1-alpha, and on the right, we Have IL-36-RA, which is IL-360 receptor antagonist across two projects now. The first paper or the first marker, IL-100, in plasma is less than 1% of the total measured cytokine in this panel. But in the rumen, it's between 4% and 8% of total. So you see a pretty dramatic increase in the proportion of the total cytokines we detect. Now, if we look at IL-36-RA, we can see it's 10 to 15% in plasma, but then in rumen, it's nearly 90, 95%. There is a little bit of variance cow to cow, but even cow to cows, these changes are very clear. So the rumen seems controlled, seems to be controlled and regulated by IL-1 family of cytokine. So when you dig into the basic literature across species, that's not tremendously surprising. Across species, we've seen that these two cytokines, IL-1-alpha and IL-36, are very prominent at epithelial barriers. IL-2-alph is especially interesting because it is always expressed in the tissue. It is not expressed only when an event happens. It's always there. But when something goes wrong, that IL-3-alp can be released or can be located in the membrane that causes a stress cascade, as you see in the illustration on the left. So what may be interesting in future research is understanding how IL-1-alpha's physical location within the tissue may change when diets shift. Now, that's a pro-inflammatory signal. IL-2-1 alpha, if it is released by a cell or if it is located in the cell membrane where other neighboring cells combine to it, it will drive an inflammatory and immune cell response. IL36-RA, on the other hand, is the opposite. It's a receptor antagonist. It blocks IL-36 signaling, which is an inflammatory molecule that drives immune cell responses. So my thought is, is that IL-36 is likely playing a big role maintaining that tissue that is constantly bombarded with microbial signals and with LPS and biogenic amines like we talked about, but still needs to maintain function and maintain healing and resolution. So I think IL- 36 is likely putting a big roll in that by blocking some of those signals and allowing that tissue to continue functioning as we would expect. So, I think some really cool basic biology that our team has been working on and that my students are digging into. Like I said, we should have some more data coming out on that in the next year or so. But I go through all of that and I want to just pose the question of what are we missing? We've looked at how dietary changes then transfer to the cow. How do those dietary changes affect something we can measure in the cow? But as I said at the beginning, we have this rumen microbial and tissue adhered microbial population that's likely going to mediate most of, if not all of that response. So the next steps we really need to dig into are understanding how is that tissue, how is that response mediated by the microbes that are present? So we're hoping to start answering some of that question with our ongoing project that we have right now. So keep in mind that those microbes and those tissue adherent microbes, which is a distinct population likely need to be investigated further with those dietary changes to understand that individual variation that I mentioned earlier in the presentation. So that was the basic biology half. Now I'm going to close in the next 10 or 12 minutes to talk about how do we manage this from an applied perspective. So I think about this from an implied perspective. We can manage the concentration of starch in the diet, the source of starch, the particle size of the grain that we feed. We can manage forage concentration and we can manage the bulk. How do those things affect the health of the gut? So this is a paper Dr. Bradford and I wrote while I was in my PhD and we looked at, we did a literature review where we were looking at whether feeding starch contributes to systemic inflammatory risk in cattle. So we wanted to understand is the starch concentration associate with biomarkers? Does starch in the hindgut increase stress biomarker and is there a clear dietary cutoff to consider? So when we dig into the literature, we found that increasing starch does not consistently alter systemic inflammation. So on the left, we have haptoglobin. You'll see that black line that just popped into the screen. That's kind of our current cutoff for a healthy and unhealthy animal or a low-risk and high-risk animal in the postpartum period. And the reason I put that in there is because increasing dietary starch did not change any risk status of any group of cows. You'll see two studies where there was a significant increase in haptoglobin when we increased dietary starch, but those changes need to be considered with two things. Number one, the McCarthy et al. study, they increased haptoglobin going from 20 to 20 or 22 to 28, I think is the number, dietary starch. But they also increased production, reduced ketones, and had a bunch of positive responses to accompany that. So I'm not sure we can say that was a bad change. The increase in the Albornoz study was only when we fed high moisture corn, and it also had a time response in that first 21 days. So I would encourage you to dive into that Albornos 2020 paper to understand that response in a more nuanced fashion. Serum amyloid A, there was one study where we increased serum amylloid A as we increased grain, and that increased grain was when we were increasing barley up to like 40% of the diet. So that was the only significant response that we observed. So our takeaway from this is dietary starch concentration alone is likely not driving inflammation to a degree. So it's about finding that optimum concentration. If we're too low, you're likely missing out on protein and milk protein production and milk production. If you're too high, you'RE going to suffer milk fat depression, ruminal acidosis, and potentially inflammation. But like I said, you'll probably get milk fat suppression before you would get some gut-derived inflammatory response. This is some of that Alborno's data, where I would say my takeaway from it is less degradable starch, especially in that immediate postpartum period, likely means less risk for that cow. So here we have LDP on the left, haptoglobin in the middle, TNF-alpha on the right. And you'll see we have low starch, high starch, and then dry ground corn or high moisture corn. And in that low starch diet, when we go from dry ground to high moisture, we're going to have a lot of risk for the cow. And in the high moisture diet, we seem to reduce biomarkers of inflammation. When we go to the higher starch diet, the high moisture corn seemed to be more of a problem. So some of these responses were significant, some were not. But in general, I would say less degradable starch in that postpartum period would be less risk. What about particle size? This is a big one that I want to think about more thoroughly and get more data on for industry partners to use. We know that we can reduce grain particle size and increase digestibility. digestibility. Sorry, my title's cut off there, but you can see that in the plot. As we move from right to left, we reduce particle size, we increase digestibility, but only to a point. Once we get below about a thousand microns, starch digestibility does not seem to measurably shift, and this is from a meta-analysis by Dr. Luis Ferreto. But what we need to remember, and what other species have done a better job of, is that particle size can be a problem. And this is from poultry and swine literature is that there is a limit to the fineness of particle size as very fine particles negatively affect gut health. And in another paper, there's a very similar quote straight out of the text that fine particle size of the diet negatively affects health of the gastrointestinal tract. From a ruminant perspective, I don't think we've done a great job of considering that. In general, we say smaller particle size, the better for grain. Now at the Hialeah soybean discussion, particle size is a question there as well. So we really need to take the time to understand, okay, what is a good particle size for performance, but also not so small and so problematic that we cause stress in the gastrointestinal tract. That needs to be considered. We talk about forage concentration. Is that a risk? Is that an issue? And we need to think about that in a holistic fashion. If we reduce forage, we increase intake, generally speaking. And one of the reasons I think that's important to remember is because if we have, say, a feed hygiene issue in the diet and we have mycotoxin and we reduce forage, that cow might eat more. She may take in more toxins. She may take more of that feed hygiene problem. That could be a risk. Reducing forage also reduces ruminal pH. But as we've seen, ruminal PH is not the only game in town, but it is accompanied by changes in the bacterial population. It is accompanied by change in fiber digestibility. Then finally, as I said, we reduce forage. We generally reduce NDF digestibility because of that increased intake, reduced passage and things of that nature. The effects also in the literature so far are generally confounded by starch intake and concentration, which usually change with the forage concentration. But I just want to throw that out there that reduced forage concentrate is at risk, perhaps. But there is one study that in the postpartum period demonstrated quite nicely the potential here. And I think they did some follow-up work, but it hasn't come out yet. But this is Engel, King, and Oba 2024. And what they did is they provided one group of fresh cows with a TMR. and then they provided another group of fresh cows with that same TMR but with a couple pounds of Timothy hay top dressed onto the diet and what they observed is those cows for the first few days of lactation that got the Timothy hay had a 38% reduction in plasma haptoglobin concentrations following calving and I think it's reasonable to expect that that may have been a result of some gastrointestinal effect of having grass hay in the diet. Now this is a systemic marker it's non-specific markers, so we can't say that with any certainty, but I think that's a reasonable assertion at this point and deserves a little closer investigation. But the grass hay did reduce haptoglobin in this instance, and it did not change milk yield during that transition period. Another question that often comes up is variation. Diet variation, ingredient variation, feeder variation. How does that affect gut health? How does dat affect the ration? I harp on my producers, about feeding at a consistent time every day, pushing up at a consistent time, every day partially because of room and health, but mostly because of consistency from the human aspect, less about the animal aspect. But that does have variation affect animal health. And there's a couple of studies I want to talk about. First of all, this has got at all 2015, they did a really neat approach where they took a low starch diet. They had a high starch diet, and then they looked at a third treatment, which was an acidosis induction treatment where the cows were fed control low starch ration, but then suddenly increased starch at two instances about a week apart to see if there was a measurable stress response. And I think this is perfect because it reminds me of growing up when my uncle would feed the cows Monday to Saturday, and my dad would pinch hit and feed cows on Sunday. Since my uncle was doing it every day, he was better at feeding. He was more consistent. He got the ingredients added at the proper amounts. My dad did not do it every day. So he was not as talented at getting the right amount of silage in, the right amount of corn in. So what if he dumped in too much grain one day? That's what I think this study is really illustrating. And so they observed that serum amyloid A did increase on that sera diet, those abrupt dietary changes did seem to cause a measurable shift in a plasma biomarker of inflammation. Now, whether this was like biologically relevant and production relevant is another question, but it's meant to illustrate that we do have measurable shifts in that cow when we do Have some variation. But there are also differences in how cows respond in the acidosis literature that I think relates to variation as well. Now, this is two different studies from the same group from the group up in Canada. And they have two different grain challenge approaches. They have one where they'll feed a control diet, which is in the top half of the screen for five weeks, then they suddenly switch the cows to a serra induction diet with a lot of grain. So you can see five weeks of the same diet, overnight switch to a new diet. They also have an alfalfa pellet induction approach where they stepwise remove alfaufa hay and increase alfafa pellets. And they do that over six weeks. And what you'll see that when they do this, they get similar responses in ruminal LPS. They had similar responses and time below 5.6, whether using the alfalfa or the grain approach. So the traditional markers of acidosis are the same according to these two methods. But what is very different is the stress response from an inflammatory perspective. The alfaufa treatment, where they are adapted over time, has no response in haptoglobin. While the grain approached, we have a dramatic response in haftoglobin in this one set of data. So that gets me thinking, is it the grain or is it alfafla? Maybe it is the grain. or is it the model they're using and the sudden switch in diet that caused a change? Really need data to consider. And I think that diet change is part of the question. But when you look across all the literature, including non-challenged scenarios, this is a review in Applied Animal Science from Dr. Weiss and Dr. St. Pierre. And this is aligned from that paper. Although, or sorry, not a review paper, but they looked at diet variation. And although treatment did not affect any production measures during the 21-day experiment, diet variation did increase day-to-day variation in performance and intake in milk. But the cows responded to a change in diet, but the diet was never bad enough long enough to matter. So does variation impact the cow? Yes, we do see more variation in cow performance if we vary the nutrients delivered day-by-day. But as Dr. Weiss and St. Pierre mentioned here, it was never bat enough long enough to matters. So, so long as you're managing it to a reasonable point, you're probably going to be okay. So let's bring it home. Gut health, what matters, what doesn't? This is my current working risk gradient. Greater starch, probably more risk, but you got to get pretty high, like 32, 33, 34. More degradable starch, possibly more risk. Smaller particle size, probably more risks. Sudden increases in degradable starch, as we observed in the GOT study, probably More risk. The opposite of those, obviously probably reducing risk, along with a more stable feed composition, probably reducing risk. What about a competitive feed bunk? I would assume increasing risk, hard to say. Heat stress, again, I would assume increasing risks. Mycotoxin, direct-fed microbials, sugar, fresh pasture, as we talked about earlier in the slide. How do those fit into the same gradient? So that's where I stand right now on how we can manage gut health in these cows. So to bring it in, to land this plane, the rumen is a dynamic structure with a complex regulatory network that currently appears driven by IL-1 cytokines. We can manage carbohydrates, and that can be important for room and health, especially looking at starch, degradable starch, and forage concentration. And consistency is important, but the data are inconsistent. Pardon the wordplay, but I do think that is a valid take home today. So I want to close with thanking my team. In the picture, you have Andy, who I said has graduated. Michaela Robinson, who's now a master's student at UC Davis. Irie, who I mentioned earlier, Tita, who I mentioned before, and Bradley Gatto, who is a senior at Ohio State looking to go to that school. So I want to thank them for their hard work, thank my sponsors and financial supporters of this research, and with that, close it out and have some time for discussion. First question comes in from Daniel. He's asking in the field, how can we separate the effect of high starch causing inflammation or reduced cow health versus a reduced fiber slash forage level? Yeah, that's a good question. So I would say the main thing is based on the data we have now, I don't, I should say, I do not know how you can separate them effectively, but I would says based on data we now, if you have north of 17, 18% forage NDF and your milk fat is strong, you probably do not have a major rumen health inflammatory risk, especially if feeding a dry ground source of uh of starch if you're feeding a high moisture source or silage with a lot of starch the risk increases but so long as forage fiber is say 18 20 22 you're you're likely okay all right uh schuyler would like to know how fine is fine when it comes to corn particle size that's a good question and i um i have this i think everybody in the audience has their own answer, I would imagine. You know, in my mind, fine is when we start getting under 500, 300, 250. And I know a lot of nutritionists who are really driving to get that corn grain really, really beat up. So two parts of that. Number one, from a digestibility perspective, so long as you're under about 900 micron, you're not going to gain much more on starch digestibility based on the published literature. There's a whole bunch of anecdotal data. We've got a whole bunch of fecal starch data that the labs give you. But based on the published literature, get under 900, your starch digestibility is okay. But in a ruminant, we don't know what happens to the gut if we get under nine hundred. We don't Know how the tissue handles that. So a lot of work to be done. All right. Dr. Jim is asking, how does small particle size affect the gut health aside from its higher fermentability affecting room pH? Yeah, that's a good question. I think um in a ruminant the fermentability changes in ph changes in microbial population would be my primary concerns in the other species there is some different effects there with the physical size in like a chicken per se with a gizzard and different things going on there um causing ulcers but yes in a cow i do think it it still comes down to the fermentation the degradability the increased rate that piles up that acid more quickly that could cause risks um and then whether that gets to the small intestine then and could cause issues within those billi and within those um uh tissue folds uh the grain can settle in there and then get fermented in a very small pocket and potentially cause some issues so it's a good question but it's one that i don't think we have the answer yep next question comes in from manly uh any specific strains for feeding dfms so um i i laugh because i have i have written some proposals to some dfm suppliers to do some gut health work and and i'm still chasing that rabbit so i would tell you at this point i don't have an answer on gut health but i can tell you that um there's a meta-analysis out there for yeast published in like 2009 that shows if you feed yeast you get a little bit of a ph bump and some fiber digestibility. So yeast continues to be beneficial. The recent bacillus data is encouraging across a few different examples, but that's one where I have a student actually working to summarize a little bit of literature right now, and we'll have that at Tri-State. So please stay tuned and join us at the Tri-STATE Dairy Conference for a summary of the bacillis data. And then there are some really novel products out there, you know, the ruminative microbe products that have some data out there as well. But from a gut health perspective, we don't have it yet. All right. Next question's from Lester. If we have to go to the tissue to understand gut health better instead of solely trusting blood biomarkers, what are the analysis that you would suggest? Transcriptomatics or focusing on specific genes? Is there a cheaper option as well? Yeah, great question. So there's two halves to that. First of all, I would say from the applied side, we hope to develop a cow side blood marker test or something that can be like, yes, she's high or low risk for a gut issue. That's going to take a lot of time, a lot of cows, and probably some kind of omics approach. In my research program, we're doing a couple of things. We take tissue samples and we do histology, which is cheap and easy, and it still physically shows you how that tissue changes. We look at immune cell staining to try to understand where those immune cells are located within that tissue complex. And we're also doing the protein cytokine analysis, which is cheaper than some of the omics, but, you know, relatively still expensive. Like for me to buy a plate for that cytokines analysis, like 3,800 bucks or something. And then we're going to add some transcriptomic sequencing and RNA sequencing to try to really get at some individual cell functionality. But for a budget, like if you're going run a study on a pretty tight budget, I'd say the cytokine, the tissue cytokines, the mRNA transcripts and histology would be very useful. All right. Next question comes in from Trinidad. What are your thoughts on systematic inflammation coming from the gut during the transition period? Yeah, that's an excellent question, Trinidad, and I appreciate the thought on that. My current view of the transition period inflammatory inflammation is that it is mostly going to come from the most obvious source, which is the uterus. And the cow just pushed out a calf. She just did a whole bunch of stuff. I think that's where most of that inflammatory response is going to come from. You know, they used to do it. And Scott, I think you'd remember going through this where the steam up diet was all the rage, right? We're going to get that rumen prepped. We're going to give that cow ready and help that gut be healthier. But the data for that is not, it's just not there it's really really not supportive so i i don't think the gut is a huge source unless you have like a toxin issue or a mold and yeast in your silage issue or some other problem then i think it becomes a could become a risk factor all right next question let's pick one here high air uh asking sarah shifts fermentation toward propionate and often lowers methane per kilogram of dry matter, but at the cost of fiber digestibility, as your data show. How should we interpret low methane results in trials where rumen pH is not monitored? Should pH or LPS be standard safety endpoints when evaluating methane reducing additives? That is a good question, Hire, and phrased well, I would say you're totally right. We shift towards propionate. That's a hydrogen sink. We reduce methane. I don't know if LPS or pH is a safety endpoint per se. I do think so long as like we have the clinical health outcomes are adequate and, you know, don't have measurable shifts in plasma, then you don't have a whole lot of reason to worry. But some of those compounds, we know specific compounds could be problematic, like the bromaform-containing compounds can be problematic. In that case, I'd still want to know what the tissue looks like, because not only do you have shifts in propionate, you might have shifts and hydrogen, shifts in oxidation reduction potential and chemistry of the rumen. That still could cause tissue stress. Do I think it needs to be standard? No, but I do think case by case, depending on the chemistry of compound, we still want to look at some of those tissue responses. I hope that answers Hire's question. All right. Very well. Next question is from Alexis. Did you measure starch KD of your starch sources, either seven-hour starch or calculated starch Kd for multiple time points from in vitro feed lab analyses? Yes. Thank you for the question, Alexis. And I think we've had that discussion off the air. I'm certain of who that is. We did not measure that in these studies. We did measure particle size across all of our studies. So we're feeding grain sources that were in the six to 800 range across all these studies that were from my lab. And we are going to start investigating more on those rates of degradation. All right. Dr. Kumari asks, when high starch or rapidly fermentable diets thin the stratum corium, does this reflect an increased rate of cell disquamation due to chemical irritation, or is it a sign of impaired cellular proliferation in the stratium bacilli? that is someone who has done their homework um the quick answer is i don't know what i would like to do so we have a store of tissue samples across three or four studies now what i would like to is do some staining to try to look at cell turnover and proliferation but we don't have that yet and so i have to cop out and say i don t know all right next question comes in from eh my jovi i hope i got that right thanks for your presentation in one slide you showed cows ate 37 kilograms of dry matter per day with low starch diet is that correct 37 kilograms it was so that was in the main thing to remember there was a very low forage diet and those cows were earlier in lactation they were about 70 80 days in milk when we started the trial so the the forage level was 12 and a half percent it was a corn silage and alfalfa heavy diet. And yes, that is correct. They ate roughly 85 pounds or 37 kilos of dry matter on that treatment. And we're doing another study right now where we take forage MDF down to that same level of 11 or 12. And those cows are eating 36, 38 kilos of dried matter. So the takeaway there I think is when you don't have forage and fill in the diet, especially in that mid-early lactation, most cows will eat and eat and eat. That's crazy. All right. Paul's asking from a modeling standpoint, do we have adequate studies and data to predict acidosis based on the diet, i.e. starch and NDF content? You know, there are a couple, excellent question. There are a few papers that look at some dietary inputs and it's been a while since I've read those. In general, the big ones are going to be forage NDF starch and starch degradability. And I think from those three, if we don't have the modeling estimate now, we probably should be pretty darn close. There was a paper in 2024 that was a big review on acidosis induction, and I think they based some of their estimates on rumen BFAs, which is not a practically feasible approach, but I think you should be able to get pretty close with forage fiber starch degradability. Okay, very well. You had mentioned HBS earlier in the presentation. Kind of curious, how common is that in the United States right now? From like an incidence perspective, I don't have a good number for you. I know there was some recent epidemiology data globally that suggests its incidence is increasing. But the only evidence I have today is that anecdotal conversation with that group of farmers where the one raised his hand and said it still happens way too often and every one of those farmers in the room was nodding in agreement and like it was that moment of group empathy where everybody's yes it's a problem and it still sucks um but it's not a huge incidence it would probably still be in that you know one two percent or less all right very well lynn would like to know uh Between grain sources and starch concentration, which one significantly reduces rumen pH as seen with barley versus corn? And does high dietary starch consistently trigger symptomatic inflammation? So, yes, grain sources is a challenge or not a challenge, but it does moderate rumen PH. So barley, wheat, oats are highly degradable sources. when fed at the same dietary starch concentration all things being equal will cause reductions in ph compared to corn as to the dietary starch question so long as you're my current thinking so long is you're under 30 of total dietary starch and you're feeding a dry ground source prominent mostly a dry brown source like dry ground corn you're not going to have a gut derived event from the starch alone. If you have some kind of issue or challenge, I would assume it's either heat or feed hygiene or some other issue that then caused a clinical symptom. So long as you're under that 30%, I don't see starch being an issue for clinical outcomes. All right. I got another stumper from Dr. Kamari. When evaluating grain source effects, how do you account for shifts in liquid versus solid associated microbiome fractions adhere directly to the rumen epithelium when measuring localized inflammatory triggers? The question in the past, or in the past, I haven't. In the study we're doing right now, we are actually going to look, we're going to do an approach called dual RNA sequencing, where we will sequence the tissue adhered microbes and the tissue and try to understand both the microbial response and the host response so the answer to your question is we're doing it now we haven't done it in the past yeah all right kirby lots of questions uh we have crossed the top of the hour so i'm going to make this our last one coming in from key are there any key points to consider when evaluating the use of anti-inflammatory agents what are some examples of agents related to cytokines mentioned in the presentation that that's a good question um i'm going to skew towards the feed additives so like the non-pharmaceutical agents we can use um there are growing data on some plant polyphenols and some phytogenic compounds that can reduce either albumin to globulin ratio or haptoglobin or things of that nature in a cow some of those compounds in a petri dish can reduce specific excited guys so i do have my eye on that space i don't have any specific compounds for you today but there's a lot of active investigation going on there all right very well well thank you uh kirby this has been a great presentation and q a session i also want to thank everyone for attending today's webinar if you have additional questions please submit them to a and h dot marketing at balchem.com the real science lecture series continues with educational topics each and every month, including our next few. Next week, on November 10th, we will be joined by Austin Thurl. He's the executive director of AFCO, and he's going to be discussing recent AFCO regulatory updates. Then on November 24th, we're proud to host our fourth installment of the Real Science Lecture Series for our EU customers. Joining us will be Dr. Aline Burgers of Wageningen University, who will discuss extending lifespans and lactation length. Finally, on December 1st, we welcome Dr. Francisco Penegaricano of the University of Wisconsin covering reducing methane emissions from dairy cattle, progress, challenges, and opportunities. Visit bowelchem.com slash real science for details on future webinars and to register for upcoming events. Bal Kim's podcast series continues to offer a deeper dive into our webinar topics. Log on to your favorite podcast platform and search for Real Science Exchange or simply visit balkim.com slash podcast. And if you want a really cool Real Science Exchanges t-shirt, just subscribe to the Real Science Echange. Send us a screenshot along with your address to nh.marketing at balkim dot com. We'll get that off to you right away. And on behalf of Val Kim and Dr. Kirby Krogstad, thank you for joining us today.