Joining together for the third episode of the New Revelations in Transition Cow Nutrition from the 2022 Cornell Nutrition Conference four-part mini-series to discuss animal nutrition requirements are remarks from Dr. Barry Bradford, Michigan State University and topical insights from Dr. Clay Zimmerman from Balchem.
Guests: Dr. Barry Bradford, Michigan State University
On this third episode of the New Revelations in Transition Cow Nutrition from the 2022 Cornell Nutrition Conference four-part mini-series to discuss animal nutrition requirements are remarks from Dr. Barry Bradford of Michigan State University and topical insights from Dr. Clay Zimmerman of Balchem.
Bringing forward recent research on transition cow studies, Dr. Bradford began the conversation by highlighting the 2022 Michigan State University study that aimed to understand metabolic physiology better. 3:30
While the focus is usually on refining nutritional strategies, Dr. Bradford suggested the bigger question is how dairy farmers will leverage efficiency and productivity in the future. 4:31
Focusing much of his professional career on better understanding nutrient requirements and capabilities in dairy cattle, Dr. Bradford remains dedicated to helping the industry understand ways to achieve the next five pounds of milk sustainably.
When limiting cow health problems, Dr. Bradford shifted his focus to analyze epidemiological studies on lactation sustainability and the impact of conditions during milk production. 6:15
Influencing the microbiome continues to be a metric for achieving higher production levels, but what are other strategies?
Dr. Bradford shared that choline continues to impact phospholipid synthesis and transportation to the mammary gland positively. He added that the focus of choline in transition cows directly affected liver health and suggested analyzing choline concentration in early lactation stages. 12:27
Within the study, Dr. Bradford mentioned research showed active choline cells became less responsive to inflammatory stimuli. He mentioned that choline concentration not only increased mRNA, but overall he saw an 80% increase in colostrum yield from the supplemented study. 25:27
Just a week into lactation, Dr. Bradford’s research showed cows produced nearly 100 pounds of milk daily. But what were some additional effects of choline on the cows?
While there weren’t any negative impacts on productivity, Dr. Bradford mentioned the study showed baseline research on the anti-inflammatory significance and body circulation through plasma TNF. 33:31
Dr. Bradford added they also looked at carryover effects of lipid protein (LP) in days 22 to 84, sharing they saw milk yield peak with added LP and choline supplementation. He shared that the LP carryover yielded another five pounds per day and remained steady for another two months post-challenge. 43:33
Wrapping up the conversation, Dr. Zimmerman summarized key strategies Dr. Bradford shared and mentioned the value in protecting choline during transitional periods to not only impact herd health but increase milk yield overall. 54:55
If you would like to review Dr. Bradford’s webinar from the 2022 Cornell Mini Symposium, you can view all four webinar series at balchem.com/realscience.
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Scott Sorrell (00:08):
Good evening everyone, and welcome to the Real Science Exchange, the podcast we're leading scientists and industry professionals meet over a few drinks to discuss the latest ideas and trends in animal nutrition. Hi, I'm Scott Sorell, one of our hosts here tonight at The Real Science Exchange. Tonight we're sharing the third and four podcasts recorded at Baal Kim's mini symposia prior to the Cornell Nu Nutri Nutrition Conference. This event focused on the new revelations in transition, cow nutrition and practical implications of this new research. Let's first welcome my always entertaining co-host, Dr. Clay Zimmerman. Clay, thanks for joining me tonight and in the spirit of our pub theme here. What's in your glass tonight?
Dr. Clay Zimmerman (00:51):
Well, in the, on, in honor of the state of Michigan, I, I have some hard side or tonight back, back, back to an old favorite here tonight.
Scott Sorrell (00:59):
Back to an old favorite. Yeah. That's awesome. Yeah. Good for you.
Dr. Clay Zimmerman (01:02):
Yeah. What's, what's in your glass tonight, Scott?
Scott Sorrell (01:05):
So, tonight, and I don't know if you guys can see this, but this is a frosty mug pulled directly from the freezer and delivered just moments ago by my son. This has become my favorite beer. It's called Blood Rain, and it's, it's brewed by the Eerie Brewing Company in Erie, Pennsylvania, where Noah goes to school. So, in honor of my son, I didn't, you know, I was gonna drink whatever berry drinks, but I didn't know what he drinks, so I decided to have a blood rain instead. So clay let's toast. Toast to another great podcast on transition Cow Nutrition. Cheers, my friend.
Dr. Clay Zimmerman (01:43):
Cheers.
Speaker 3 (01:44):
Tonight's podcast stories are brought to you by Reassured Precision Release Choline reassure is the most researched, encapsulated choline on the market today, consistently delivering results to your transition cows of higher peak milk, reduced metabolic disorders, and even in utero benefits to her calf leading to growth and health improvements. Visit alchem.com to learn more.
Scott Sorrell (02:06):
So as we kick off this third in the series of four podcasts from the many Symposium BS sponsored at the Cornell Nutrition Conference in 2022, we welcome Dr. Barry Bradford from Michigan State University. Dr. Bradford's talk on transition, CALT nutrition research from the university covers a lot of ground, so we'll be breaking in at a few points during the presentation to discuss some of the primary findings. Clay, can you highlight some of the primary findings Barry will hit on during the presentation?
Dr. Clay Zimmerman (02:37):
Yeah, so he will he will talk about some collage from data that they, that, that was captured during the study. There's actually been an article published on that already. He will hit on some of the some of the milk production data, you know, during lactation. He will talk some about semantic cell count because the, the study was really designed to look at, at immune function of room protected choline in these transition cows and their, and their calves. And and he talks a little bit about fi findings in the calves that were born to these cows that were supplemented with rumor protected colon.
Scott Sorrell (03:27):
All right. Thank you, clay. I think we're gonna enjoy this one.
Dr. Barry Bradford (03:30):
Happy to share today a little bit of, lots of data that we have from a study that we completed here about a year ago at Michigan State University. And I'm presenting today mostly on behalf of Dr. Turner Schwartz. Turner's a postdoc in our group and was the point person behind this study. So, I, I wanna start off with kind of a bigger-picture question. And that's, you know, something that I think nutrition consultants are getting all the time from their clients is, you know, it's a tough industry to make a living in. We've gotta always be pushing the boundaries in terms of either efficiency or productivity or both. And, you know, one question, you know, for strategic thinkers out there running dairy farms is to be pushing their nutritionist, how are we gonna get the next five pounds of milk? How are we gonna compete with the guys that are always getting better?
Dr. Barry Bradford (04:20):
And, you know, people are used to thinking about that in nutrition. And you know, what we're usually used to thinking about is well, how can we refine our nutrition strategies or do a better job of putting up forage so that we have feeds that are more digestible, better-delivering nutrients to the animal so that we can meet her nutritional requirements to make milk, right? And that's kind of the basics about how we think about this. And there's nothing wrong with that. And I think some of what I'll talk about today is around that. So there's plenty of room to keep working on that. But I think one of my big thrusts in my career is to help nutritionists to think about other ways that they can help farms get the next five pounds of milk. Okay? So one of those ways might be instead of just trying to get more delivery of certain nutrients to the mam gland to meet her needs for that, what if we can help the farm achieve goals to limit transition cow problems or health problems that impact the productivity of maybe on an average farm, about a third of the cow, sometimes even more.
Dr. Barry Bradford (05:24):
And one of the reasons I bring that up is we have some good evidence from epidemiological or observational studies that when cows go through, even if it's a short window of time that they're dealing with adversity at the start of lactation, it affects that cow for a long period of time after that. So this is data collected from a number of published studies, just looking at, again, cows that had no research intervention, just cows that had either mastitis, metritis or ketosis at the start of lactation, and then were followed compared to herd mates that did not deal with those problems. And you can see that these conditions were associated with anywhere from 700 to 1100 pounds of less milk production compared to peers in the same herd. Okay? You can't necessarily say from this for sure, that it's a cause and effect relationship, but it's a little hard to explain otherwise.
Dr. Barry Bradford (06:20):
Okay? So we have these short-term windows cows go through, and yes, we can get most of them through those challenges, but what's harder to measure is these incremental losses and milk for the whole rest of the lactation that actually costs us a lot, if it's 20% of our cows going through this, all right? So it may not be top of mind all the time because it's just a handful of cows at any one time. But of course, if all the cows are going through that over the course of the year, it's a big hit.
Dr. Barry Bradford (06:49):
The other thing we can think about, and it starts to get further and further away from what we think about our jobs being as nutrition consultants, but I think we can have an influence on these things. And the third one that can have a huge impact on productivity of a herd on average is to have older herds. Well, how do we do that? Well, typical herd that I see today that I'm looking at their records still most herds are upwards of 35% first lactation cows. We all know those first lactation cows are gonna produce substantially less milk than the older herm herd mates. And so having these, these young herds actually has a big impact on how much milk we're actually shipping. And if we look back at that transition cow sort of observational studies that are out there maybe the bigger impact of those early lactation risk factors is not so much the lost milk for the whole lactation, but the massive increase in the risk of those cows leaving the herd.
Dr. Barry Bradford (07:49):
So in these studies, these cows made it through these conditions, but then if you actually keep track of those cows over the next 3, 4, 5, 6 months, either from poor fertility or from getting hit with another round of disease or whatever, they have a much greater risk of leaving the herd. And of course, again, compound this over a third of the cows over years, and pretty soon we don't have a huge fraction of our cows reaching lactations three and four. Okay? And it's surprisingly hard to find a graph on this, but this is data from Korea where they looked at 1,290 lactations just to show the simple data on how much milk do cows make with advancing parodies. And these cows aren't as productive as a typical American herd, but it makes the point that we really aren't hitting that cow's lifetime potential for peak lactation yield until about lactation four.
Dr. Barry Bradford (08:42):
And of course, we have a very small fraction of our cows in the US reaching lactation four. So if we can move the average cow in our herds from 2.2 lactations to 3.2, for example we would expect, you know, by dropping the fraction of first lactation animals for one thing we could expect a substantial bump in herd level milk production. And Albert Dres has done some really excellent work, in my opinion, on this question, of what should be our optimal average lifetime in a herd, given the trade-offs between genetic progress with, you know, using very young animals and turning those genetics very quickly versus the cost of giving up the milk by u having a younger herd and all the other costs risk of death and that sort of thing rising with older animals. And you can see his analysis suggests that we hit peak profitability at about lactation five.
Dr. Barry Bradford (09:40):
And we are a long ways from that. Okay? So again, what can we do in nutrition to help push the needle on this? And maybe that's where our next five pounds of milk comes from on some herds. So how do we go about doing that? Well, again, this seems like the domain of veterinarians and certainly they play a role as does just good herd management. But I think we shouldn't pass the buck as a nutritionist in terms of playing a, a role in this effort as well. This is from a review I and a couple grad students wrote six years ago. We were kind of reviewing some major developments in nutrition. And a lot of the point that we make here is that these opportunities to improve health and longevity of our cows really do bring in nutrition because of all these new ways that we now know nutrition can influence physiology.
Dr. Barry Bradford (10:30):
So it can do it through influencing the microbiome, which of course we've known for a long, long time. But the ripple effects of that on health are really coming to the forefront in the last 10 years or so. Nutrients acting as signals. I'll talk about that more today. Nutrients interacting with genetics, I'll have a little bit of information on that as well. And then just interaction with other physiological systems, with the endocrine systems in the body, with the immune system and all that. Ops opens up lots of questions, and they're difficult to answer, but it does at least give the potential that by different, using different feeding strategies we can allow animals to be more resilient to challenges and not succumb to these disease pressures that, again, strip away productivity and also decrease longevity. Okay? At least that's the pipe dream, right?
Dr. Barry Bradford (11:24):
That's what we wanna work towards in the long run. So we're gonna talk a little bit about coaling today, and of course there's a lot of good work that's been done on coaling. But it doesn't hurt to step back and say, you know, why is there so much focus on this in transition cows? There's a number of reasons for that. Part of it is just because we know that's a window of time where liver health is a challenge, and choline is an important metabolite for supporting healthy metabolism in the liver, particularly when it relates to fatty acids. However, there's also just pretty good evidence that we have different availability of choline if we have the same, like a stable feeding scenario in terms of dietary choline. So this is data showing pretty clearly that at least in this scenario choline concentrations are much greater in late lactation and even mid lactation compared to cows in the first three weeks of lactation.
Dr. Barry Bradford (12:27):
That doesn't necessarily on its own mean that that's a problem, but at least suggests that there is opportunity to try to normalize circulating concentrations of choline in early lactation and potentially change function through that. And I should point out here, this is plasma total choline. One of the challenging things about studying choline into metabolism is it gets converted to many different compounds, okay? And it happens quite quickly. So this is just actually a somewhat simplified version of choline metabolism showing in color different pathways that this can go down. So Cho is a methyl donor, okay? So it can be converted to beane and through that be used in methionine es sile, homocystine senile methionine cycle. So we have these one carbon metabolism interactions and it can also be converted to phosphocoline and through either of those routes generate phosphate tito choline which becomes an important precursor for signaling molecules like sphingomyelin.
Dr. Barry Bradford (13:41):
And these kind of compounds that are brought up a lot when we talk about liver metabolism and fatty liver and, and handling with lipoproteins. But also this third pathway here where choline is used for acetylcholine. And this talked about more in human nutrition when it comes to neural benefits of choline and potential, you know, brain effects. But it hasn't really been considered a whole lot in the bovine side. But anyway, what happens if we feed this is we see sort of slight increases often in a number of these different subcategories. And so looking just at naked choline, I guess doesn't necessarily show us big shifts by feeding it, okay? But it doesn't mean we're not seeing changes in metabolism through these metabolites. So specifically thinking about acetylcholine and its potential contribution to how cows respond to choline there's some pretty interesting information out there from the mouse world where it's been shown that different immune cells will actually express different types of acetylcholine receptors.
Dr. Barry Bradford (14:55):
So there are subtypes called muscarinic acetylcholine receptors, and then there are nicotinic acetylcholine receptors. And based on what's been done in mice so far, at least there's reasonable evidence that niacin can have either pro or anti-inflammatory effects, depending on which receptor subtype is expressed on different cell types. Furthermore, through those types of receptors as well as acting on even intracellular receptors in mitochondria there is additional evidence that we can actually change expression of cytokines in immune cells through this acetylcholine signaling through these different pathways. Okay? So all that to say this is one metabolite of choline that we haven't thought about a whole lot in the bovine world that may actually open up opportunities to consider choline to be potentially immunomodulatory.
Dr. Barry Bradford (15:55):
So a few years ago we did some work in this space really led by Dr. Miriam Garcia when she was working in our group. And we, we just simply asked, what are the impacts of choline on bovine immune cells if you take them out of the complexity of an in vivo situation. So rather than feeding choline and trying to understand how immunity is affected in that situation here, to sort of set a baseline, we asked what if we pull those immune cells out of the body and ad choline at that point, and then sort of test the functionality of those cells in response to that.
Dr. Barry Bradford (16:33):
So what we did in this study is we took blood samples from either early or mid lactation cows, nine in each time point, and we used the traditional typical way of isolating different types of immune cells. We centrifuge the blood with a, a compound called FICO that has a very specific specific gravity that lets us separate cells based on their density. And by doing so, we get red blood cells and polymorph nuclear cells at the bottom which then by lysing or exploding the red blood cells and washing them, we can isolate the pmn, which is about 90% neutrophils. There's a few other cell types in there, but most of what we see is gonna be from neutrophils. Okay? So neutrophils are a really key innate immune cell type. They're the primary cell that we rely on to move out of blood and into tissues to a site of infection and either engulf bacteria and destroy them or carry out what's called NETosis, where they shoot out these nets of DNA and protein that latch onto bacteria and kill them that way.
Dr. Barry Bradford (17:45):
So that's called extracellular killing. Okay? So the main point is this is the main innate immune way that the body will kill bacteria. So they're a very important cell type in the immune system. In addition, then we have a second fraction that comes out above the fico which contains the mononuclear cells, so just cells with a single nucleus, and that's primarily made of lymphocytes which are adaptive immune cells, okay? Or monocytes, which is another innate immune cell type. And so we actually studied each of these three cell types in different manners, okay? So what we did is we isolated these cells, sort of stabilized them, incubated them with choline, and then we added another stimulus that we know causes a response in these cell types. So in the top graph here, you can see responses of monocytes. These are precursors of macrophages and other cells and they are responsive in, in blood, but they're typically on their way to developing into a different cell type.
Dr. Barry Bradford (18:52):
So they're not typically as mature or responsive. So you can see then we looked here at TNF alpha secreted into the cell culture media. So this is an inflammatory cytokine that demonstrates at least one aspect of how this cell is responding to the inflammatory stimulus of LPs or lipi lippo polysaccharide. So we're mimicking a gram-negative challenge to these cells, and as expected, they at least do increase the TNF alpha in response to the LPs. Although it was a mild response in this cell type. Okay? And what we found here is a tendency for a reduction of TNF alpha response as we increase choline from a concentration of three micromolar to eight and then to 13 micromolar. Okay? We saw a more obvious reduction in mRNA abundance of tolike receptor four, which is the receptor for LPs and the transcription factor N fkb one.
Dr. Barry Bradford (19:55):
Okay? So what does this all mean? In the presence of choline, this cell type seemed to become less responsive to an inflammatory stimulus. So choline seemed to have anti-inflammatory effects in monocytes, in neutrophils. Again, these are more mature, they're more ready to respond violently to an inflammatory stimulus. When we hit them with lipo polysaccharide, we get a very dramatic TNF alpha response as expected. But we also saw a much clearer reduction of that TNF alpha response with increasing choline concentration. Okay? On the mRNA side, we didn't see anything as obvious here. Although I will say that neutrophils, once you take them out of the body and start messing with them, they're on the path to dying. So a functional assay, like looking at secreted TNF alpha, I think is probably more meaningful for this cell type. So for both of these innate immune cells, it appeared that increasing choline concentration had anti-inflammatory effects on these innate immune cells.
Dr. Barry Bradford (21:01):
Okay? Is that good or bad? It's context dependent, most likely. But interesting finding when we then look at the adaptive immune cell that we had to play with, which is the lymphocytes. And to be a little more specific, we didn't characterize these lymphocytes, but mostly what you find in circulation that we would've isolated is T lymphocytes. So we think these are mostly t and there's lots of subsets of T cells, but some of them are involved in what's called cytotoxic killing. Okay? So the adaptive immune system can kill pathogens through this cytotoxic toxicity mechanism as opposed to what we usually think about, which is antibody production. And that's mostly coming from B cells, okay? Anyway, one of the ways that we assess lymphocyte functionality is to, again, stimulate them and then look at how well they proliferate. So lymphocytes are meant to replicate if we stimulate them with something that mimics a sign of a bacterial or, sorry immune system activation.
Dr. Barry Bradford (22:10):
So we use con A as most people do, and you can see as expected, we saw proliferation response compared to the NO con A controls. And although it is a little bit hard to see with the naked eye here, statistically, there was a highly significant linear effect of choline. So as we went from three to 13 micromolar concentration of choline those lymphocytes proliferated at a more rapid rate in response to con A. Okay? So this is sort of in contrast to what we saw with the innate immune cells. They were sort of dampened in their inflammatory response to L P s, but then when we test a proliferative signal for adaptive immune cells, they're responding at a greater rate. When choline is on board at higher concentrations, we lo I mentioned a few gene expression responses that we looked at. In addition to that, we looked at expression or mRNA, abundance of enzymes and transporters involved in turning on these different pathways that I talked about a little bit ago.
Dr. Barry Bradford (23:19):
And by and large, choline cons, increasing choline concentration increased mRNA, abundance of many downstream enzymes and activators and receptors for these different metabolites. Okay? So it seemed to be a feed forward mechanism. Choline is activating its own metabolism, its own signaling downstream, which again, is sort of interesting because this is all in immune cells, which hasn't been studied very much. So, at least at the mRNA level, there's evidence that immune cells can metabolize choline and take advantage of these downstream pathways. And those are, again, upregulated when choline is on board. And to summarize the immune cell responses here at the bottom, we seem to see at least evidence of some anti-inflammatory responses in neutrophils and monocytes in a greater proliferative response in lymphocytes. Okay? Now, I'll be the first one to say that there is value, in my opinion in cell culture experiments, but it's not the same thing as affecting a whole animal.
Dr. Barry Bradford (24:29):
Okay? So this really got us interested in saying maybe there's more to dig into even than all the work that's been done in transition cows. Maybe we should be looking at this in a little bit different way because it can maybe have these direct effects on immune cells. So we put together a study, again, Turner led the effort here, and we carried out a study where cows were started at about 24 days pre-cal with either zero 30 or 45 grams per day of ruen protected choline. Now at least at the time we were doing this, this was a somewhat novel, slightly different formulation of bams products. So it's not exactly the same thing that's on the market right now. And that's, to my understanding, still the case. But it's very similar. Okay? And basically these doses were intended to deliver 20 or 14 grams per day of choline.
Dr. Barry Bradford (25:27):
So we supplemented those again, the last 24 days before calving, and then through 21 days post calving. And, you know, one of the novel pieces here is, again, we're wanting to see is some of the benefit of choline that's been reported. Is it really because we're shifting sort of immune response or inflammatory conditions in fresh cows? So we didn't want to go too early and cause a complete train wreck. We decided to challenge cows with inter memory LPs at 17 days in milk. Okay? So we'll walk you through some of the responses we saw here. First of all, and we didn't necessarily anticipate this, we did carefully measure colostrum yield, and we saw a pretty substantial effect independent of dose of choline. So choline increased colostrum yield, as well as colostrum protein yield. And the intermediate dose choline 30 also significantly increased colostrum fat yield. And this magnitude of this change is about an 80% increase compared to control. So I was actually quite surprised by at least the magnitude of that. In IgG content we assessed by bricks, and we didn't see any evidence of a difference there. So, which again, means that the yield of IgG G would you know, be increased by roughly the same proportion as the total volume. So that's an interesting thing. It wasn't really our focus, but surprising side finding.
Scott Sorrell (26:55):
So, clay, we're gonna break in here and talk a little bit about the colos from response that Barry experienced in that trial. That's, that's quite a number. What can you tell us about that?
Dr. Clay Zimmerman (27:08):
Yeah, it was, it, it was a really good response with increased colostrum yield and, and these reassured supplemented cows. So they, they actually observed an 80% increase in, in colostrum yield in the, in these reassured supplemented dams. So really really great response as far as increased colostrum yield. But the other key point to that is it was done without any reduction in colossal quality. Be, you know, normally you might expect with an 80% increase in yield that you might cut down on the percentages of, you know, IgG G or some, some of the milk components. That's not what they observed. There was no reduction in colossian quality at all. So, so these were really really great findings. The other piece of that and there was, and there was a paper published on this, it, it was May of last year, may of 2022 in in, in JDS communications. And they also measured choline metabolites in the, in the colostrum as well. So there was a, a tremendous increase in all the cho in the yields of all the cho metabolites in the colostrum as well, so that, you know, that's gonna be transferred, you know, from this colostrum to those calves. And, you know, potentially, you know, I improve health, health and growth of the calves.
Scott Sorrell (28:49):
Yeah. Great summary, clay. Let's get back to the presentation. Now
Dr. Barry Bradford (28:52):
As cows transition post calving, we did look at blood calcium for the first seven days. We didn't take samples like hourly or anything like that, but at least from the time points we looked at, we saw no evidence whatsoever of a treatment effect here on postpartum hypocalcemia, we saw kind of the standard dip in blood calcium on day one. Most of the cows recovering by day three, and nearly all of them by day seven, although some of them were probably still marginally lower than you'd like to see. But anyway, no evidence of treatment effects there. So this is milk yield. So again, we're showing the first here, just the first 16 days in milk. We see the characteristic rise over that time, and these cows were doing pretty well approaching nearly a hundred pounds of milk per day, even just a week into lactation.
Dr. Barry Bradford (29:43):
But what we saw very clearly here, as has been reported in some other studies, is a clear and significant increase by choline regardless of dose. I was maybe a little bit surprised that we saw no evidence at all of a treatment by time interaction. Those cows were already different almost immediately. And the separation between the black line and the two treatment lines here stayed pretty uniform, at least through that first 16 days in milk. So that's, I'm just gonna use this number cuz it's, it's pretty much, right? That's about a five pound per day response in milk. Slightly more than that perhaps. I'm not gonna show a graph of energy corrected milk. We didn't have energy corrected milk for every single day, so we had to do it by week. But basically the story was exactly the same. It's, it's basically, we didn't see any clear significant effects on milk components. Okay? We also I maybe a little bit surprised by this, saw no clear evidence of a change in dry matter intake. So despite giving five pounds per day more milk with similar components, these cows were eating basically identically across treatments.
Dr. Barry Bradford (30:55):
Okay? I wanna spend most of the time on the more novel part of the study, which is the l p s challenge at day 17. So this gets thrown around a lot. I just wanna make sure that people understand what we're actually trying to do here. So if you look at the gram negative bacteria, and I'm just gonna use e coli as an example here, because that's actually what we use in this study. The part of the, the outer wall of that bacteria contains these lipids with sugars attached to them called lipo polysaccharide or LPs. It's also called endotoxin. And basically we purchased endotoxin extracted from e coli from a particular strain, and then we administered that endotoxin to challenge cows intra memory. So we challenged two quarters in this study, again on day 17 in milk. And just to give sort of an overview of what typical responses we expect in after that I'm gonna show some memory specific and some whole body responses.
Dr. Barry Bradford (32:02):
So first of all, somatic cells score. This is a score, so it's on a log scale. So we saw a very dramatic increase, again, as expected in somatic cells, and it kind of peaks at about eight hours. Although we didn't have samples between eight and 24 hours, but that's kind of what we'd expect from past literature. We did see a significant effect of treatment here but with contrasts that was really just a 30 versus coline 45 difference. And if you look at the time series they sort of overlap each other at different times. I don't think there's much of a story there. Basically, we didn't see any impact on somatic cells. And again, if you don't know this those somatic cells after a challenge like this primarily represent neutrophils that have migrated out of the bloodstream into the meric gland.
Dr. Barry Bradford (32:56):
So again, comparing to our in vitro study if choline has a very significant sub repeatable, anti-inflammatory effect on neutrophils, we would maybe expect some diminished recruitment into the MAM gland, but we did not see that here. The second thing we anticipate seeing is certainly in the local, in the mam gland itself, we'd expect production of cytokines by all the immune cells that have moved in. And then to a lesser extent, we expect some of those cytokines to reach the, per the whole body circulation and to be influencing other tissues through that mechanism. Now, this is plasma TNF alpha. These concentrations are actually quite low. We do see a shift over time. We do see a response at four to eight hours. But it's not a huge magnitude. And basically these cows in terms of at least T n f alpha had a fairly subtle response.
Dr. Barry Bradford (33:57):
Okay? Now, what was surprising when you look at that is to look at the fever response. We had cedars put in these cows, so we had eye buttons that were collecting body temperature intravaginal temperatures continuously throughout this challenge, which gave us some really nice data. And these cows really responded in terms of fever. So the mean response at this basically six hour time point was over 106 degrees Fahrenheit if you're used to that scale. So these cows had a raging fever, but as we'd expect, they got that under control really within about 16 hours, and were mostly back to baseline then from there forward. Now I don't really know what to make of this, but we did see a, an effective choline at the higher dose after that returned to baseline. So they remained slightly elevated in terms of body temperature in that recovery phase. Okay? And I'll show you moving forward, we didn't really seem to tie that to anything of super relevance in terms of negative effects on productivity. And so again, we're not quite sure what to make of that.
Dr. Barry Bradford (35:06):
I've talked about this a lot. Probably many of you have seen this. But downstream then of these initial inflammatory signals like cytokines or lipids that we didn't measure we would expect some systemic response. And one of the easier to measure aspects of that beyond fever is to see the liver shift its expression of proteins or its production of proteins towards these positive acute phase reactants. So those include haptoglobin, C reactive protein, serum amyloid, a, et cetera. We only measured haptoglobin here, but again, we did see that systemic effect where there was a significant increase in haptoglobin by 24 and 48 hours. And again, you see the characteristic delayed response before that signal can move to other tissues and have that effect. There's no significant effect of treatment here. It was log transformed for analysis. And so these differences that look to be big on a normal scale are not really meaningful just because of the great dispersion among individual animals. Okay,
Dr. Barry Bradford (36:16):
So then perhaps further downstream of the immune system response we start to see some metabolic disruptions or changes. So we have done some metabolomics in this study. I don't have anywhere near enough time to go through all of it with you, but there's some highlights I wanted to pull out. So one of the thing that's been discussed a lot in the last few years Lance Baumgart and others, and actually Matt Waldron did some great work on this 15 years ago or more is when we cause an inflammatory response in these cows. We expect the immune system to shift its metabolism to where many of these cells when activated preferentially are gonna use, utilize glucose and at a much higher rate than normal. And one of the reasons they do that is they shift to this anaerobic glycolysis where they're just taking glucose, metabolizing it to lactate and kicking the lactate out instead of burning glucose all the way to carbon dioxide.
Dr. Barry Bradford (37:15):
All right? So that allows them to shuttle through the the glucose much faster than normal and basically produce ATP at a high rate, even though it has some inefficiencies with it. And that allows it to carry out, for example, an oxidative burst response to make reactive oxygen species to kill things. Okay? So not too many of these studies, some, but not too many have reported lactic acid in circulation, which is what you would expect to increase if what I just described is actually going on. And here, in fact, we did see in the first eight hours post challenge more than a doubling of blood lactate. Okay? So that, again, fits very well with that story of we're shifting glucose metabolism to this process that's gonna utilize what's called the chori cycle. So push glucose to immune cells, maybe to muscle to partially metabolize it to lactate and then kick the lactate back to the liver to be regenerated into glucose. There was actually a significant effect of treatment here, but it's worth pointing out that treatment difference actually existed before the challenge. So we're not quite sure what to make of that. It's statistically, again, very significant decrease in lactate with choline. And we still need to dig in this more. This is not very old data, so we've gotta think about it further.
Dr. Barry Bradford (38:36):
Another met metabolic disruption that has been reported multiple times with l p s challenges is a drop in many essential and non-essential amino acid concentrations in plasma. And again, I've seen this before, particularly I've shown some data in beef cattle, but the magnitude here is kind of astounding to me. So blood methionine plasma methionine drops by two thirds in the eight hours following this LPs challenge. That's a massive shift in availability of a really key essential amino acid to other tissues, including amam gland of course. And so but almost equally as impressive as that recovers by 24 hours. So still very curious. I don't personally know exactly what's going on to drive that dramatic shift, but pretty interesting. No treatment effects here. So it's just an overall response to l p s. One metabolite that's kind of interesting that did show a treatment effect here was glycine.
Dr. Barry Bradford (39:37):
So glycine in, involved in antioxidant defenses and choline had at least the higher dose actually resulted in, in greater glycine concentrations following LPs. But again, that difference was there prior to challenge as well. Okay. So it's just maintaining that gap between the treatments and all of the cows had a decreased glycine concentration following the challenge. Okay? So lots of interesting metabolic shifts as well. What about dry matter intake? Perhaps the most important metabolic measure host challenge, again, as we expect to see with a disease challenge like this dry matter intake dropped substantially by roughly a third host LPs challenge and took roughly three days for most cows to get kind of back to normal. However, we, again, we saw no interaction of LP s with treatment, which is way down here well treatment by LP s here, treatment by LPs by time here, not significant, and no evidence whatsoever of an overall treatment effect.
Dr. Barry Bradford (40:48):
Okay? So it's, it's not necessarily helping cows through this by getting them eating quicker. Like likewise, if we look at milk yield following challenge a pretty substantial decrease, maybe about a 40% drop in response to LPs, but a pretty quick recovery, again, much like previous studies. And if we dig into that again, the treatment effect is still there. Although we give up some power here cuz now we split into six groups instead of three. But the overall p value is a 0.08, so at least the higher dose is still better than control. But again, no interaction with LPs. So there's, it's not like the drop with LPs was diminished by choline.
Dr. Barry Bradford (41:34):
One interaction that we found that was just kind of interesting is this treatment room and protective choline interacted with genetics for mastitis. So on the X axis here is the predicted transmitting ability that P t a for somatic cell score. So cals with a lower PTA would be expected to have lower somatic cell score on average. And this is just, you know, from herd records. So it means not just during disease challenge, but all the time cows up here would be expected to have a higher s scs. And in fact, what we saw in response to the LPs challenge, I don't know if this has been done before, a pretty market difference. So the cows with I guess better mastitis genetics hit a peak of about 4.7 million somatic cell count in response to LPs the cows with worse genetics for mastitis hit about double that concentration.
Dr. Barry Bradford (42:33):
Okay? So on a log scale that's like, not necessarily doesn't look huge, it's about half a log, but if you put it on an actual scale, that's a lot of difference in cell number. And what's kind of interesting here then is that interaction with treatment. So at the lower end with better genetics, not necessarily any obvious difference, although maybe the choline 45 is different. But up here in those cows with the genetics that would give them a propensity to have higher somatic cells. The two choline treatments reduce somatic cell count by somewhere in the neighborhood of 2 million cells, okay, per mil. So about a third reduction in somatic cell count. So in one way of thinking about it is cows with poor mastitis genetics were helped by this choline treatment.
Dr. Barry Bradford (43:33):
And this may or may not be related, but we also looked at carryover effects of treatments and L p s in days 22 to 84. Okay, so up through about peak milk yield. And what we saw here was a, a remaining significant effective treatment and a remaining significant effect of l p s, where even after we stopped feeding the choline, we kept that roughly five pound per day benefit of choline, at least through another eight weeks, eight or nine weeks post feeding. And likewise LPs caused a hangover of about five pounds per day for about two months post challenge. We, again, didn't see any interaction here, so those effects were additive. So the best cows didn't get challenged with LPs and they were fed coaling and the cows that had the lowest yield had no coal and did, and were challenged with LPs. So between those was a pretty big gap. So kind of interesting,
Scott Sorrell (44:38):
Clay, we're gonna break in here again. Dr. Bradford just told us about a nine pound increase in milk production that he, he, the response from, from feeding reassure that's much higher than than we normally expect, right? That's a big number. What do you think attributed to that?
Dr. Clay Zimmerman (44:59):
Yep. It's a great question. It is, it is higher than the mean response. We've had lots of studies where we've seen, you know, four and a half to five pound milk responses with, with reassure supplementation. They saw nine in this case, and, and it held, it, it it held throughout they went out through 84 days of lactation, meas measuring these responses. And those, those differences held again, the reassure it was only supplemented 21 days pre to 21 days post. And, and Dr. Brad Barry refers to it as the hangover effect of being, being supplemented with, with the colon during the, during the transition period. So the, the magnitude of response, it's I don't, I don't know that I have all the explanation for, for that. Obviously we know we were improving, we're improving liver health on these cows that certainly will explain some of this.
Dr. Clay Zimmerman (46:12):
You know, we're, we're, we're certainly we we'd be increasing glucose production on these cows and, you know, you need glucose to make lactose to make milk. I'd be speculating on the rest of it, but, you know, you have to wonder, are we, are we making more more secretory cells in the mammary gland to see that kind of response? I will say, and this is pretty rare to get this feedback we always blind the treatments in the study so that the the researchers don't know which treatments are which. But the response was so noticeable that they wondered what was in that treatment
Scott Sorrell (47:00):
They,
Dr. Clay Zimmerman (47:00):
If it was very noticeable that these cows were, were producing higher on, on the treatment and they were very curious cuz again, the treatments are blinded, but it, it, it was a really noticeable response. The other, you know, the other piece of it, and again, the initial design of the experiment was really to look at you know, potential immune responses in the cows to protected colon. We didn't clearly see those responses here, but I, I, I do think, and, and, and Barry speculates on this as well, that that we probably are having some beneficial effects on the immune system of these cows and the calves as well.
Scott Sorrell (47:47):
All right, thanks for that, clay. Let's let's finish the presentation. Now,
Dr. Barry Bradford (47:52):
One other genetic interaction I wanted to share with you is that overall, if we looked at the entire study period, and again, looked at genetics on the X axis, so the PTA for a total milk yield if we look at the cows with poorer milk production genetics, we saw a really market increase in energy corrected milk yield. So this is on a daily basis now, it's about a 10 kilo difference, over 20 pounds of milk in the genetically inferior cows. Whereas in the, the most genetically superior cows for milk yield, we didn't see an obvious treatment effect. Okay? So again, there is an overall significant treatment effect, but also an interaction with genetics. So pretty interesting novel insight there.
Dr. Barry Bradford (48:36):
So before I run out time, I just want to touch briefly on, we also studied the calves. And one thing I want to make really clear, we're only studying the effects of gestational exposure or prenatal exposure to co choline here because the calves were all fed colostrum replacer so that we didn't have to deal with differences in treatments of colostrum. At least it was uniform and they all got milk replacer after colos, sperm replacer. So treated the same postnatally. And we measured a whole bunch of things, including some measures of immunity that I don't have time for today. But I'll share some sort of metabolic outcomes. And if you're interested in this, this art, this piece of the study is now an article in press in jds, so you can access that open access. So first of all, growth arguably the most important outcome no real obvious effects.
Dr. Barry Bradford (49:33):
You can see there was a significant sign, significant effect of dose in the first week postnatal for average daily gain. But if you put it on a body weight scale over time they sort of reconverge. And I don't know that that's anything too meaningful there. One of the things that we were particularly interested in here because of evidence that choline can act as an antioxidant or support antioxidant systems is whether we could see differences in reactive oxygen and nitrogen species here on the left and on antioxidant potential, the capacity in blood to control oxidative stress. And there was no significant treatment effects in each of those two independent measures. But often in this space, people look at the ratio of those two and call it the oxidative stress index. And when we express it that way at least the intermediate dose, the choline 30 dose did have a significant reduction in this proxy for oxidative stress compared to control.
Dr. Barry Bradford (50:33):
So maybe some evidence of a carryover benefit for oxidative balance in the calves if their dams have been fed choline. Likewise, if you take the next step and look at inflammatory markers, we saw no evidence of an effect in bulls. But in the heifer calf specifically with an interaction of treatment by sex there was a reduction in postnatal haptoglobin concentration in the ca in the heifers from cows fed the highest dose of choline. Okay, so maybe there's something there. Lastly, on calves there's a number of really interesting correlations between dam measures pre-treatment, okay? So roughly 24 days pre-cal and the corresponding calf measures. So here, this is reactive oxygen and nitrogen species in the dam was linearly related to those measures in calves postnatally and kind of the inverse of that, the antioxidant potential. So a positive measure as that increased in dams pre-treatment, that also increased in their corresponding calves postnatally.
Dr. Barry Bradford (51:43):
So some we would call that maternal imprinting of postnatal metabolism. And there was one interaction here that was interesting. So we also saw a relationship between dam nifa and late gestation and calf nifa, but that relationship was influenced by treatment. So we had an interaction here where the control animals showed a pretty tight linear relationship, higher Neen dams, higher neen calves, and the choline treatments seemed to disrupt that relationship where calf nifa was pretty much uniform regardless of dam metabolic physiology here. Okay, so kind of interesting. So as I'm running outta time, let me hit the highlights again from this study where we had a ton of data, pretty interesting stuff. Number one, that 80% increase in clot yield, I was surprised by, and I think it's worth exploring. I wouldn't necessarily take that as gospel true for always choline is going to do this, but I think it's worth looking at in some commercial herds.
Dr. Barry Bradford (52:43):
Choline increased milk and energy corrected milk substantially, again, about five pounds per day post calving. But it didn't do that by diminishing the negative impact of L P s. So I'd say if that was our core idea going into the study that didn't really pan out, the genetic interactions, I pointed to a couple of these I think are super interesting to me and maybe point to some possible research on precision feeding. Maybe some animals would benefit from ke feeding choline further in lactation, depending on their genetics. I think this is the first study to demonstrate in a randomized controlled study that if you give cows early lactation LPs challenges, that the, the hangover if you will, goes for at least two months. And that we probably really are affecting whole lactation milk yield very substantially with those early lactation mastitis cases.
Dr. Barry Bradford (53:36):
And lastly, just some sort of exploratory data linking gestational phenotypes of cows and then neonatal phenotypes that point to lots of areas for investigation about imprinting of calf physiology. Okay, so again, coming back to these big picture ideas I don't know if this study really drives home the point all that well, but I do think I encourage nutritionist out there thinking not only about meeting the needs of the memory gland, but how do we help cows be healthier? Because there's other ways to get more milk on these herds. And with that I'll call it quits and wanna share contact information both for myself and Turner who would be an extra resource on this data. Thank you.
Scott Sorrell (54:24):
So Clay that's the end of the presentation, and this is last call as we wrap up this podcast. I'm gonna ask you, what should nutritionist take away from Dr. Bradford's presentation?
Speaker 3 (54:37):
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Dr. Clay Zimmerman (54:55):
So a few things. I really like how you started off the presentation where he talked about where, you know, where will you get your next five pounds of milk? Very intriguing, start to the presentation and you know, it's not just nutrition. He hit on the fact that you know, there are benefits to keeping cows in the herd longer. And I, I think we lose sight of that at times. But you know, it's, it's been known for a long time. Cows don't really reach, you know, their, their tr their full maturity until about the fifth lactation. So he hit on that, you know, improved longevity in the herd. He, he, he hit on improved health of these cows. That's all part of keeping cows in the herd, and it certainly is a piece of increasing peak milk. So certainly the study that he talked about, you know, feeding, feeding reassured during transition we saw tremendous responses there.
Dr. Clay Zimmerman (55:59):
And again, if you, you know, if you look at the rumor, at the rumor protected colon meta-analysis, it's, it's clear that that, that all cows are responding to to rumor protected colon during the transition period. And he cl he clearly saw that here and the the, the tremendous response that he observed in, in colostrum yield. We, there's a lot of interest in that. I, I, I've got, I get calls every year, multiple calls about you know, why, why is this her dropping in colostrum yield? And you know, per, per, perhaps perhaps this is is a strategy we that nutritionist should look at to help with that.
Scott Sorrell (56:49):
Hmm, clay, thank you for that summary and thank you for joining the podcast once again. We now have three Cornell Podcasts complete. And one more yet to go. For our listeners, watch for our next episode with Dr. Heather White from the University of Wisconsin. She'll, she'll share new insights from the, the university Transition Cal research program. And as we said in the beginning, there's so much new research around coal from many universities. So stay tuned for that and catch all the podcasts on your favorite podcast app, Toya loyal listeners, thanks again for coming along for 60 episodes and sticking with us as we explore more topics. 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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