Guests: Kari Estes, Balchem; Dr. Mark Hanigan, Virginia Tech, Mingyang (Charlie) You and Dr. Joe McFadden, Cornell University, Dr. Tanya France, University of Wisconsin, Maria Torres de Barri and Dr. Geoff Dahl, University of Florida Balchem sponsored several abstracts presented at the 2024 ADSA Annual Meeting. This episode consists of five segments, each focused on an abstract.
Balchem sponsored several abstracts presented at the 2024 ADSA Annual Meeting. This episode consists of five segments, each focused on an abstract.
Segment 1: Evaluating the total mixed ration stability of rumen-protected lysine products.
Guests: Kari Estes, Balchem; Dr. Mark Hanigan, Virginia Tech
This research compared the TMR stability of a Balchem prototype, several commercially available rumen-protected lysine products and a positive control of unprotected lysine. (3:39)
A sample of TMR and the equivalent of one gram of lysine from each product were mixed and placed in a plastic zip bag for 0, 6, 12, or 24 hours. After each time point, the sample was placed in a strainer bag, dipped in distilled water, and drip-dried. The solution was collected and analyzed for free lysine content. (5:28)
About 85% of the unprotected lysine was recovered at 0 hours. After 24 hours, around 50% was recovered. The rumen-protected lysine products varied widely; one product released nearly 87% of its lysine in 24 hours, while another only released 9%. TMR stability should be taken into account when determining feeding rates and handling of rumen-protected lysine products. (7:19)
Segment 2: Evaluating the total mixed ration stability of rumen-protected choline products.
Guests: Kari Estes, Balchem; Dr. Mark Hanigan, Virginia Tech
In this experiment, Kari evaluated TMR stability of five commercially available rumen-protected choline products, along with a positive control treatment of unprotected choline chloride. (14:04)
At 0 hours, about 80% of the unprotected choline was recovered and 50% was recovered at 24 hours. Results for the rumen-protected choline products were highly variable, ranging from 5% release to 100% release at 24 hours. Rumen-protected choline products should be evaluated for TMR stability in addition to rumen stability and intestinal release. (17:25)
Segment 3: Effect of dry period heat stress and rumen-protected choline on productivity of Holstein cows.
Guests: Maria Torres de Barri and Dr. Geoff Dahl, University of Florida
The experiment had four treatments: heat stress with and without rumen-protected choline, and cooling with and without rumen-protected choline. Cows in the cooling treatment were provided shade, soakers, and fans, while cows in the heat stress treatment were only provided shade. (24:45)
Heat-stress cows had higher rectal temperatures and respiration rates than cooled cows. Heat-stress cows also had lower dry matter intakes, shorter gestation length, lighter calves, and produced less milk. (29:36)
For cows in the cooling group, choline supplementation increased milk production. However, cows in the heat stress group supplemented with choline produced less milk than cows who did not receive choline. (31:04)
Dr. Dahl suggests that not cooling cows in heat-stress environments when they’re receiving choline will not result in optimal results. (33:49)
Segment 4: Effects of dietary rumen-protected, ruminal-infused, or abomasal-infused choline chloride on milk, urine, and fecal choline and choline metabolite yields in lactating cows.
Guests: Mingyang (Charlie) You and Dr. Joe McFadden, Cornell University
This experiment evaluated early and late lactation cows supplemented with choline via three different methods. Each treatment had 12.5 grams of choline ion provided daily: fed in rumen-protected form, continuously infused into the rumen, or continuously infused into the abomasum. (36:29)
Choline bioavailability was influenced by the delivery method of choline. Fecal and milk choline concentration was only observed in early lactating cows with abomasal infusion. Abomasal infusion increases the choline metabolite betaine in feces and urine. These results suggest there is potential saturation of choline metabolism in the lactating cow. (40:53)
Segment 5: The metabolic fate of deuterium-labeled choline in gestating and lactating Holstein dairy cows.
Guests: Dr. Tanya France, University of Wisconsin; Dr. Joe McFadden, Cornell University
Dr. France explains that choline can be metabolized via two different pathways. Using deuterium-labeled choline (D-9 choline) allows researchers to know which pathway is used. If D-3 or D-6 choline is measured, the methionine cycle is used, and if D-9 choline is measured, the CDP choline pathway is used. The hypothesis was that the physiological stage (late gestation vs early lactation) would influence choline metabolism. (51:06)
Dr. France found that both choline metabolism pathways were used in both physiological stages. This experiment also confirmed that choline is a methyl donor and that choline recycling can occur. The research also evaluated the relative amounts of choline and choline metabolites in each pool. (53:40)
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Scott Sorrell (00:00:07):
Good evening everyone, and welcome to the Real Science Exchange, the pubcast where leading scientists and industry professionals meet over a few drinks to discuss the latest ideas and trends in animal nutrition. Hi, I'm Scott Sorrell, gonna be your host here for this episode of The Real Science Exchange. Tonight's episode is gonna be just a little bit different at the 2024 ADSA conference. Balchem presented eight abstracts during the scientific sessions, and tonight we're going to record short segments with each of the presenters or the researchers, student researchers for each one of those abstracts along with their advisors. And then we're gonna put 'em together in one podcast. Now, this allows us to do two, two things, right? It allows us to kind of showcase some of the rising stars in the industry and hear about can we just love to do that? And also gives us an opportunity to showcase our research. So enjoy!
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Scott Sorrell (00:02:23):
In this segment, Clay and I will be reviewing a poster that was given at this year's ADSA and the title of that poster was evaluating the TMR Stability of Rheumatoid Protected Lysine Products. That poster was presented by Kari Estes and her advisor was Dr. Mark Hannigan. Now, Dr. Hannigan, I, I don't believe this is the first time you've been in an advisory role for Ms. Kari, is that right?
Dr. Mark Hanigan (00:02:51):
Well, I'm not even sure what year it was, but she started working in my laboratory as an undergraduate student, I believe a sophomore. And she worked on some of the techniques we were trying to develop for assessing bioavailability of amino acids at that time. And, then she decided that the project she was helping the graduate student on could be done better. And so as an undergrad, she then worked on it some more, and then she worked as a technician for a year, I think, and then we got Balchem to fund her, you know, as a graduate student for, I don't know, a couple more years. And, and then we, we sort of hung onto her a little bit, you know, because she'd come and help us for a few years.
Scott Sorrell (00:03:32):
And Kari, why don't you just give us an overview of the poster that you presented there at the ADSA?
Kari Estes (00:03:39):
Yeah. So with any room-protected nutrient, there seems to be a lot of focus on room protection and bioavailability or intestinal release, right? But I think a piece that is or can be crucially missed is the feed or the TMR stability of these products. Right. That's gonna be the first environmental challenge that the products can incur. The moisture and the acidity, perhaps, of the TMR could be quite damaging to some of these products. So we wanted to evaluate five commercial rumen-protected lysine products and a chem prototype for TMR stability. And we didn't develop the technique by any means. It has been published already in the Journal of Dairy Science from the Minor Institute, but we did make a modification to it which was adding a positive control treatment, which was an unprotected lysine treatment.
Clay Zimmerman (00:04:46):
I wanna point out as well, so we don't lose sight of this, this, this poster was actually picked for Spotlight. The committee picked, you know, a few of these favorite topics and it was also presented as a spotlight where, where, where Kari did a three minute summary of the poster in front of an audience.
Kari Estes (00:05:11):
Yeah. So I tried not to do an oral presentation, but I still got one out of it.
Clay Zimmerman (00:05:17):
Exactly. Exactly.
Kari Estes (00:05:19):
Yeah. Yeah. So if you want, I can briefly tell you how we ran the experiment.
Scott Sorrell (00:05:27):
That'd be awesome.
Kari Estes (00:05:28):
So we took a sample of TMR and the, we took the equivalent of one gram of lysing from each of these products, right? The products ranged in protection technology and lysing content. So we took that into account by equalizing that piece of it with the one gram of lysine. So we gently mixed 'em together for 30 seconds, put 'em in a Ziploc bag, and they sat there for 0, 6, 12 or 24 hours. And then at the end of each time point, we took the contents, put 'em in a strainer bag, just dip them in water for distilled water for one minute, allowed them to drip drive for two minutes, and then we took a sample of the solution and had that analyzed for free lysing content. We had that done at a third party lab. Yeah, so it was, it's very, it's a very quick procedure, which is nice, but yields some very interesting results for sure.
Clay Zimmerman (00:06:30):
Very quick, but very labor intensive for that period of time. Correct?
Kari Estes (00:06:34):
That is true. That is true, yes. Yes. When you're talking about one or two samples, it's very quick, but you know, that number jumps up pretty quickly when you run everything in triplicate.
Clay Zimmerman (00:06:44):
So Kari, why did you pick the time points that you selected?
Kari Estes (00:06:49):
We picked those based on the typical, maybe, well maybe not typical, but potential feeding times at on farm. The 24 hour time point would be a one x feeding. The 12 hour time point would be two x feeding, and then the six hour time point would be the four times feeding.
Clay Zimmerman (00:07:14):
Thank you. So Kari, you want to want to discuss the results?
Kari Estes (00:07:19):
Yes. So interestingly, with the unprotected lysine, right? That was our first objective, right? Are we able to recover all of that free lysing that we put into the TMR? And we found much to my surprise that we were unable to recover all of it. And as the incubation time increased in the TMR, the recovery actually decreased. So even at time zero, right? So that was just sitting in the TMR for 30 seconds, we were covered about 85% of the free lysing, and then by 24 hours it was down close to 50% of recovery.
Clay Zimmerman (00:08:03):
So, Mark, do you have any thoughts around what might be occurring where we're not recovering? All of that? All of the unprotected lysine HCL?
Dr. Mark Hanigan (00:08:14):
Yeah, I mean, there's feed has a lot of different compounds in it or, or components and, you know, a lot of those molecules that are in there have charges on them. And so they're able to bind to something like lysine, which is, which is positively charged, right? And so if there's a negative charge anywhere on that feed, it's gonna bind lysine and not wanna release it again very readily when you go to wash it. And we know there's lots of binding sites all over feed. That's not a new finding. So it's just soaking up a bunch of the lysine that gets put on it and it's not releasing it very readily.
Clay Zimmerman (00:08:55):
Thanks. So, Kari, what did you, what were your findings with the rumen protected lysine products?
Kari Estes (00:09:03):
Yeah, so after we found those findings with the unprotected lysine, we did create a correction factor that we applied to the data for the Ruben protected products, right? Because any of the lysine that they could be releasing could undergo that same fate as the unprotected lysine. So once we applied that correction factor, we saw that there were very different stability values among these commercial products. So ranging from, there was one product in particular that released close to 87% by the 24 hour time point versus the other extreme, we had another product that had much lower release, which was only 9% at the 24 hour, and all the other products kind of fell in between that. So quite, quite large differences between the products.
Scott Sorrell (00:10:04):
Kari, I'm curious if you have a theory as to the ones that were not well protected, what might have caused that? Was it something in the structural design of the endcap, or any thoughts related to that?
Kari Estes (00:10:16):
Yeah, that would be my first inclination would be that the encapsulation or the protection technology maybe is not as good as other ones. So that moisture or acidity coming from the TMR is just able to break down that coating and reach the lysine more easily than others.
Scott Sorrell (00:10:41):
Alright. Very well.
Dr. Mark Hanigan (00:10:41):
So does that relate, do you think the solubility too of them or not? I mean, 'cause those products have different, a fractions, so to speak, right? The soluble fraction that's readily available in the rumen
Kari Estes (00:10:55):
Could be, yeah. Especially at that, yeah. Zero hour time point for sure.
Clay Zimmerman (00:11:03):
So Kari, how, how do these findings compare to, you know, you mentioned at the beginning there were some previous work done and published. How does this, how do these findings relate to that previous work?
Kari Estes (00:11:21):
Yeah, so I think there were maybe two products in our experiment that were also tested in that published paper. Yeah. And they matched very, very similarly. Yep.
Clay Zimmerman (00:11:35):
So what about is it possible that some of these products could have passed through the bag when you were doing the protocol?
Kari Estes (00:11:45):
That's a very good question. So the strainer bags that we use are a 250 micron. So I will say visually, I didn't see anything come through that bag, right? But the very last step of taking the sample of the solution, it is filtered in a 0.45 micron filter. And I can guarantee you that none of the products will go through that filter.
Clay Zimmerman (00:12:20):
What are the implications of this potentially for nutritionists and dairy producers?
Kari Estes (00:12:27):
Yeah, so I think based on this data that we've, what, what we found is that we definitely need to be taking into account TMR stability when we are trying to determine feeding rates or even how we should handle some of these products, right? Maybe some of these products we need to handle differently one versus the other. But they'll all be in ATM R, I guess, eventually. So yeah, we just need to take it into account for sure.
Clay Zimmerman (00:13:00):
And, and, and maybe something people need to think about, right? As, as they're presented with research, you know, did these products actually go through it, you know? Were they fed in the TMR?
Scott Sorrell (00:13:14):
Mark and Kari, thank you very much for joining us today.
Scott Sorrell (00:13:23):
This episode clan and I will be reviewing a companion poster to the one that you just previously listened to, and the title of this one is Evaluating the “TMR Stability of Room and Protected Choline Products”. Again, that was done by Kari Estes and Mark Hannigan in an advisory role. Kari wanted to start us off with kind of giving us an overview again of why it is important to measure the stability in a TMR, right? When we think about rumen protected products, we're thinking about protecting them in the rumen. Why is it important to protect them in the TMR as well?
Kari Estes (00:14:04):
Yeah, I think it's a piece that is, that can be missed. For sure. These products can sit into, sit in A TMR for up to an extended period of time, right? Perhaps 24 hours. And so we need to be looking at the stability of those products before they even make it to the rumen, right? They could be damaged and damaged completely, even for all we know, so,
Scott Sorrell (00:14:31):
Right.
Clay Zimmerman (00:14:32):
Yeah, we, we always say right there, the the three legged stool of a good ruminate end cap feed, stability, ruminal stability, and intestinal digestibility, you, you truly do lose the game from the start before the cow ever eats it if you're losing it either in feed mixing or in the TMR.
Scott Sorrell (00:14:54):
Yep. Good point. Kari, why don't you kind of give us an overview of the objective of this study and what were some, what, what was the protocol of the study?
Kari Estes (00:15:04):
Yeah, so we wanted to look at the TMR stability of five commercially available rumen-protected choline products. And this one was a little bit different, right? We did lysing before or in our previous poster, but as I had mentioned before, that technique had been completed before and published in the Journal of Dairy Science by the Minor Institute, but choline had never been done before. So we really just wanted to see if this technique would work for choline. I figured, well, we figured it probably would because it seems to behave pretty similar to lysine in terms of the hydroscopic of the nutrient, but we figured we would give it a whirl. So we still did make that modification though, where we added the unprotected choline chloride to make sure that we were able to, well, not even to make sure, we just wanted to see how much we'd be able to recover, right?
Kari Estes (00:16:09):
See if we were able to recover a hundred percent of it at the different time points. So the protocol was still the same, where we took the TMR and the equivalent of one gram of choline from these products, mixed it together for 30 seconds, and let it sit in a Ziploc FA bag for 0, 6, 12 or 24 hours. We put it in a strainer bag, dunked it in one liter of distilled water for a minute, let it drip drive for two minutes, and then we took a sample of the solution, filtered it, and analyzed it for choline content. So, very similar protocol.
Clay Zimmerman (00:16:43):
So Kari, maybe explain again, why those particular time points?
Kari Estes (00:16:49):
Yes. So we picked the 24 hour time point because that would mimic a one x feeding per day. We pick the 12 hour for the two x feeding per day, and then the six hour for four x feeding per day.
Clay Zimmerman (00:17:06):
And certainly, you know, with fresh diocese closeup diets, it's very common to only feed them once a day, so that 24 hour time point's pretty critical, especially with protected choline. So Kari, what were your findings here?
Kari Estes (00:17:25):
Yeah, so again, for our first objective, that was looking at the recovery of the unprotected choline, and we saw very similar results to the lysine, and that we weren't able to recover a hundred percent of that choline at any time point. And it mimicked very similar to the lysine in that we recovered close to 80% at the zero hour time point. And then as incubation time increased, the recovery decreased, and then we recovered close to 50% by the 24 hour time point.
Clay Zimmerman (00:18:01):
So, Mark, would you care to speculate on why, why we did not get complete recovery of the unprotected choline chloride in the TMR?
Dr. Mark Hanigan (00:18:12):
Yeah, as we, you know, talked about before, it's binding sites that are all over that feed, and particularly when you cut it up, you know, and open up the inside of the cells and stuff, I mean, there's all sorts of things that are gonna bind to choline. And so it would've been really surprising if you got a hundred percent recovery. And, and I think looking at some of the stuff that's in the literature, I mean, they failed to, you know, assessment, right? It's not that this is bad that it happens, it's just something that we have to correct for in the assay. And if you don't correct for it, you're going to way overestimate the loss during TMR your TMR stability test.
Scott Sorrell (00:18:52):
You know, I was wondering as well, in maybe the 24 hour one, would it be possible that we might be seeing some microbial activity or fungal activity that might be degrading the nutrients at that time point? No thoughts?
Dr. Mark Hanigan (00:19:10):
I’m not sure you're asking, but yeah, I think, I think that's a possibility as well. Particularly if you're leaving it for, you know, 12 and 24 hours, that's a long time for a microbe. So yeah, there's, there's lots of microbes in the feed and they could be chewing it up as well if it's free. If it's protected, then presumably not, but obviously our, our, our reference point is the free concentration. So,
Clay Zimmerman (00:19:34):
So Kari, what were the rest of the results that you found on the rumen-protected products?
Kari Estes (00:19:40):
Yeah, so again, like the lysine, we saw a very vast difference in the stability of these products. So at the 24 hour time point, we had quite the spread. There was a product that had a hundred percent release by that time point, and then we had a product that was about 5% release at 24 hour. And then all of the products, you know, the other products were in the middle of those two. So quite, quite the large spread there.
Clay Zimmerman (00:20:16):
So what, what would you attribute those differences to?
Kari Estes (00:20:21):
I would say the protection technology or the encapsulation technology that we're, that is what is utilized on these products, for sure. Some of them definitely are providing more protection than others for that moisture and potential acidity or microbes to get through that coating and actually get to the choline.
Clay Zimmerman (00:20:47):
And cold and chloride itself is very, it's, it's very soluble, correct?
Kari Estes (00:20:51):
Yes, yes.
Clay Zimmerman (00:20:53):
So would is some of that difference just due to solubility in the encapsulated products then?
Kari Estes (00:21:02):
Yeah, I would think so.
Clay Zimmerman (00:21:05):
So Kari, what are, what are the implications of this?
Kari Estes (00:21:11):
Definitely as I had mentioned before, I feel like it's a, a component of room and protective products, not even just of choline, right? That can be overlooked. We focus a lot on room instability and intestinal release, but we need to be evaluating these products for TMR stability. 'cause This is gonna be the first environmental challenge that these products are going to endure. And that's even before the cow consumes it. So we, we need to be looking at this piece very critically.
Clay Zimmerman (00:21:47):
So, Kari, what's next?
Kari Estes (00:21:50):
Well, I definitely wanna continue testing lysine and choline products as they come onto the market, right? New, new products. And, but I definitely want to modify this technique to be able to test room and protective methionine products. That's my next goal, maybe next year at A DSA.
Dr. Mark Hanigan (00:22:14):
What about stability in the mixer?
Kari Estes (00:22:18):
That's something that we've looked at before. We have a small batch mixer and we've done some mineral mixed ability testing, and that also has some very interesting results based on product and encapsulation technology.
Clay Zimmerman (00:22:40):
Yeah, and I do, I do, I do want to add that, you know, the, these both the feed mixing protocol and now the TMR stability protocol, that's a lot of te you know, Kari does this testing as we're developing new products as well, so that, you know, that that is all part of our product development process is, is looking at looking at feed and, and mixing stability.
Scott Sorrell (00:23:10):
Yeah. Yeah, good point. Well, Mark and Kari, I want to thank you very much for coming and discussing this very important topic. And we're back. And in this segment, I will be reviewing an oral presentation that was given at the ADSA. The title of that talk was called “The Effects of Dry Period on Heat Stress and Rumen Protected Choline on Productivity in Holstein Cows”. And that was presented by Maria Torres de Barri; her advisor is Dr. Geoff Dahl from University of Florida. And Geoff, this is not your first time at the Real Science Exchange, so welcome back, appreciate having you back here.
Dr. Geoff Dahl (00:23:55):
Thanks, Scott.
Scott Sorrell (00:23:57):
Would you mind kind of introducing your student and telling us a little bit about her?
Dr. Geoff Dahl (00:24:02):
Sure. Maria is a master's student with me. She did an internship year plus ago with me, which is usually sort of the, the first battle testing of students. And she survived that and decided that she would agree to come start her master's. She is a native of Brazil, is a veterinarian from Brazil and has been working on this project oh, just about a year now.
Scott Sorrell (00:24:30):
Okay, excellent. Well, Maria, welcome. It's glad it's nice to have you here at the Real Science Exchange. Why don't you start off by telling us the objective of the study and then what were some of the methodologies that you used in the study?
Maria Torres de Barri (00:24:45):
So, as our objective in our study, we know, as we saw in other research, the heat stress has a negative effect on the memory growth in dry cows comparing to the cooling cows. And when you see the some other research about the cooling effect on, for example, memory growth in in vitro studies, we see the colon could reverse the growth of those memory epithelial cells. So as our hypothesis, we'd like to test if the supplement with choline in, during the entire dry periods in all cows could reverse the negative effects of the memory growth and function in the cows.
Scott Sorrell (00:25:34):
All right. Very well. And in, in, what was some of the methodologies used? How did you do this?
Maria Torres de Barri (00:25:39):
So we had 50 ones, 51 multiple cows that we enrolled it then around 47 days before cv. So in the day that they dry off, they dry it off, we enrolled in the trial and we split then in far treat in far treatments, cooling, heat stress, both receive RPC, that is when protected choline or control. So for the cooling group, we provide to them shade, soakers and fans. And for the heat stress group, we just provide shades During this, during the entire diet period, you have dry matter, dry matter intake collection daily and also respiration rate and rec temperature. Once they cal, we stop with the supplementation with choline, and we keep them in the trial until six days in milk. And we continues tracking the dry matter intake and the co yield and milk yield during those 60 days.
Clay Zimmerman (00:26:44):
So, Maria, at, at calving, were those, were the cows still cold or heat stress? How were they how, how were those treatments applied then?
Maria Torres de Barri (00:26:56):
We haven't had we haven't had treatment a temperature treatment. We, we keep getting a cooler pen. We fan soakers and shades.
Clay Zimmerman (00:27:09):
Okay.
Dr. Geoff Dahl (00:27:10):
So during lactation, the cows were all cooled.
Scott Sorrell (00:27:14):
Okay. And did I understand correctly that they did not have choline after calving? It was all pre fresh?
Maria Torres de Barri (00:27:22):
Yes, correct.
Scott Sorrell (00:27:23):
Yeah. Yep. Okay.
Dr. Geoff Dahl (00:27:25):
But for the entire dry period.
Scott Sorrell (00:27:27):
Okay. Yep.
Clay Zimmerman (00:27:30):
And what is, what is the period of heat stress in Florida?
Dr. Geoff Dahl (00:27:33):
Forever.
Clay Zimmerman (00:27:39):
That's right.
Scott Sorrell (00:27:40):
I I was curious as well, were you documenting the, the heat stress index with this as part of the study?
Maria Torres de Barri (00:27:51):
Yes. We, we had, we have the data from the THI and, but they were not different among the control and the a among, among the heat stress and the cooling and the cooling side was 72, and the heat stress side side was 73. The THI,
Dr. Geoff Dahl (00:28:13):
Essentially between June and October, we don't have an hour that we would have a THI less than 72.
Scott Sorrell (00:28:24):
We've only got partial results so far from the trial, is that correct? Has the trial ended?
Maria Torres de Barri (00:28:29):
Yes, we finished the trial, but we haven't taken a look all the data that we have yet.
Scott Sorrell (00:28:35):
Okay. And so you just presented some partial data at, at the ADSA this year, then?
Maria Torres de Barri (00:28:43):
Yes, we have like next analysis that we want to perform as a corzo analysis. Also, we performed ma epithelial biopsies. We also want to take a look on that to see how is the rate of proliferation in apoptosis in those cells for our cows compared to comparing the treatments. And also we want to track the calf development data. So we also have biological material from calfs that we want to take we want to analyze.
Clay Zimmerman (00:29:28):
So, so Maria, what, what are the results so far that we're presented at the ADSA meeting,
Maria Torres de Barri (00:29:36):
As we were expecting, we saw a negative effect on respiration rate and red temperature in the heat stress group. So they have a higher rates for those variables. They, the heat stress compared to the cooling, they, they eat less. I mean, they decrease, they dry metal intake also, they had a shorter gestation length. The calves were born lighter compared to the cooling animals, and the dry period was shorter compared with the cooling animals as well, which stress with the, with the cooling animals and the milk ke we have decreasing milk killed the energy corrected milk. Compared we have a negative effect of heat stress decreasing energy corrected milk compared to the cooling cows compared the cooling group
Clay Zimmerman (00:30:40):
About, am I correct about a, about a six kilo difference in, in energy, correct? In milk yield,
Maria Torres de Bari (00:30:48):
Yes. About six seven kilos.
Clay Zimmerman (00:30:51):
Seven kilos, yes.
Dr. Geoff Dahl (00:30:55):
And that was for the heat stress versus cooling as main effects. Now the choline did what?
Maria Torres de Bari (00:31:04):
The choline, when you compare the cooling cows that were treated with the choline, they produce two kilos of Milky Mart, the cooling cows without supplementation. And what about the heat stress cows? So we had a we had the heat trusted cows, the heat trusted cows supplemented with cooling. You had a decrease in milk production compared to the heat stress control group. So what do we see for the energy corrected milk? When you compare the heat stress with the, the heat stress that were supplemented with choline is the choline didn't combat the, the colon didn't mitigate the negative effects of the heat stress in terms of milk killed. And when you take a look on the control group for cooling and the, the animals that were supplemented with choline just a minute. So when you take a look on the, the heat stress side, the heat stress cows that they were supplemented with choline, we didn't see a reverse compared to the, the control heat stress control group. We didn't see a reverse in the effect in the negative effects of the heat stress in terms of milk production. But when you take a look on the cooling cows compared to the cooling cows that receive the cooling, the, the cow supplemented this colon had a increase in two kilos of milk production milk daily
Maria Torres de Bari (00:32:48):
Of in terms of energy corrected milk.
Clay Zimmerman (00:32:57):
And that, that's a, that's a really good finding because this was only prepartum feeding of the choline.
Maria Torres de Bari (00:33:03):
Yes. This is prepartum feeding. Yes.
Dr. Geoff Dahl (00:33:09):
And we are increasing our numbers because that is trending the right way. It's trending towards a positive effect of choline, but we need to increase our numbers to get a better estimate.
Scott Sorrell (00:33:26):
and I think Geoff, part of the I mean the, the, the main objective of the study is to take a look at the mammary development and, and you've done the, the, the mammary biopsies. And so that's kind of exciting to see what that data's gonna look like when, when can we expect to have that data available to the public. Is that something we'll present next year at the ADSA?
Maria Torres de Bari (00:33:49):
Yes, we are, we want to start to work on this as soon as possible. I just start a new trial to have more data the same as I mentioned here, we are doing a new trial with less amount of animals. But as soon as I finish this one, the plan is start with the memory particular cells analysis, and then to present in the SA next year.
Scott Sorrell (00:34:20):
Perfect. Any other questions from you, clay?
Clay Zimmerman (00:34:24):
So what's any take home messages at this point based off of what you're seeing so far?
Dr. Geoff Dahl (00:34:33):
Well, I would say the practical implication is if you're not cooling the cows when they're getting choline, you may be not maximizing or optimizing your benefit. So it's not a silver bullet to overcome the effects of heat stress by any means. But if you don't cool 'em, you may not be getting as much of the advantage or any advantage to that. The other important part, I think, is the calf, right? What are we seeing with the calf? And it appears, at least from our initial look at the data, that there is a reversal of the negative impacts on the calf with the choline supplementation to heat stressed animals. So if that holds up, then that in itself could be a huge practical implication.
Clay Zimmerman (00:35:34):
Yeah, that's, yeah, that's great.
Scott Sorrell (00:35:45):
And in this segment, we're gonna be reviewing an oral presentation that was given at the 2024 ADSA meetings down in Florida. The title of the presentation was “Effects of Dietary Rumen Protected Ally Infused or Abnormally Infused Choline Chloride on Milk, Urine, and Fecal Choline and Choline Metabolite Yields in Lactating Cows”. That presentation was given by Charlie Yu from Cornell University. Charlie's here with his advisor, Dr. Joe McFadden from Cornell. And give us a treetops, what exactly were you trying to understand with this study, and then maybe just kinda a little bit of how you went about that.
Dr. Joe McFadden (00:36:29):
Now we saw, we had a really big question I think that stands and, and something that's very difficult to study is choline by availability and to understand how much choline is used by the animal for different functions. I mean, it's such a complex metabolite in the sense that, you know, the animal can convert that choline into a variety of other metabolites and current evidence suggests that stage of lactation and could certainly influence choline utilization. So we, we really wanted to try to better understand that, to try to understand choline use in the cow, but, and, and then try to unders sort of provide some estimates or at least work towards a way to better understand bioavailability for choline. Now this particular study is one of two studies that we performed in recent time here to try to assess this. And this was more of the economical way of doing things.
Dr. Joe McFadden (00:37:26):
I'm sure that there are, there are a variety of approaches to look at choline bioavailability. And, but in this particular case, we had eight rumen cannulated cows, and then we sort of had two, two groups. We had, they were either blocked as either being late lactation or being early lactation. They differed by roughly 150 plus days in milk. In terms of the difference they were in a four by four Latin square. So fairly minimal study design in terms of having just five day experimental periods. You know, a lot of questions about is five days are treatment long enough? And there's been consistent studies now to show that by day four, day five, you can get a plateau in some of the choline responses. And so, yeah, we, we think it's more than adequate. And there's been other studies that have shown that a, a couple day washout after is, is, is more than enough to get things back to baseline.
Dr. Joe McFadden (00:38:23):
And so we have these five day experimental periods and we had sort of four treatments. We had a zero grams per day of choline chloride, so that's our control. And then we provided unprotected choline chloride at 12.5 grams of choline ion per day, and that was infused directly into the rumen. It was a continuous infusion. And then we had a rumen protected choline also providing 12.5 grams per day of choline ion per day. And that was delivered in as, as frequent meals throughout the day. And then we had an amaal infusion also providing 12.5 grams per day of choline ion delivered as a continuous infuse eight. And, you know, they're, you were fed a, a typical typical sort of northeast diet. That diet though, in terms of their intake was matched through their baseline. So we didn't want to see the dairy, the daily sort of variations that you can see in intake with such a small population.
Dr. Joe McFadden (00:39:25):
So we took their baseline intake, and then that's what we basically fed them for the next five days and throughout that experimental period. And that's what that does is, is it really helps stabilize sort of baseline choline and choline metabolite levels in the different pools. It, it does help to sometimes see differences and, you know, we did measure milk production composition, but this was a such a small study population. I, I don't think we can infer too much from that, that data. But yeah, they we did collect the urine, we collected the feces and we, we, we sampled milk. So we knew their daily milk yields. And the goal there was to try to again, look at the choline and choline metabolite concentrations in those different, you know, pools in the fecal, the urine, and in the in milk and also plasma. What, what I, I think these, you know, the two bioavailability studies that we've done, this was being, being one of the, the we're putting out, we're looking at urine more so than we have in the past. I'd like, I think we like to think that something is bioavailable once it gets absorbed, it's used by the animal and that's it. And I think we can't ignore the urine after these two studies anymore. And yeah, so maybe, maybe we can get into the results.
Scott Sorrell (00:40:46):
Results, you know, that'd be great. And, Charlie, would you like to kind of walk us through some of the main results you got from the study?
Charlie Yu (00:40:53):
So first of all, it's evident that colon bio availability bioavailability. It, it was influenced by the delivery method, like how the colon was fed, either unprotected, either RU protected or either apple masal infused and also lactating stage as well. And long story short fecal and the milk choline concentration was only observed in early lactating cows only with apple masal infusion and also amaal infusion increased some of the choline metabolites like phosphocholine and the fe fecal and the urine beane and but not the end product of choline metabolism like phosphocholine, the end product. It was not changed. And it simply suggests there are potential saturation of choline metabolism in the lactating cow. And also, like in lay lactating groups, we saw that supplement with amaal infuse the increase of fecal TMA and urinary TMA levels.
Dr. Joe McFadden (00:42:02):
Yeah, I guess we'll add to that and say that a couple of findings that I thought were interesting were that, yeah, again, the choline response that you see in milk was evident in the early lactation group and not, not in the late lactation group. I think that's, that's really important to, to emphasize. It's something that was sort of supported by the increase in phosphocholine as well. And so phosphocholine and choline concentrations were enhanced in early lactation and, and were not so in late lactation in response to the ABBA Masal choline infusion. I think that sort of speaks to how the, the high choline demand that that the mammary gland has during early lactation. That's really something we've seen repeatedly. We definitely keep seeing the beane response. Somebody always, I always get this question like every six months, like, oh, what's the metabolite that should be the most responsive to choline?
Dr. Joe McFadden (00:42:59):
It's beane. Okay. no surprises there. And, and we had substantial increases in beane, and, and when you looked at the urine, you could really see that, yeah, that, that, that goes into urine eventually. It does go into milk too, but yeah, the urinary beane concentrations went up really, really high. You know, we were hoping to try to see in this particular study we didn't see any response in, in milk TMAO. So TMAO is a biomarker of, of gastrointestinal choline degradation. But when you, we looked at fecal choline metabolites I don't know if this has been shown before, but maybe, maybe we're the first, but the, the treatment of the choline to the ABBA aum, so this is post ruly. Keep in mind, we did see an increase in fecal trimethylamine concentrations. And so, you know what, what I wanna emphasize here in, in, and our by availability studies are showing this, is that there is post ruminal degradation of choline to trimethylamine.
Dr. Joe McFadden (00:44:04):
And there are, there are bacteria in there that have an enzyme called TMA liase, and it will convert choline to trimethylamine. And that, that would certainly negatively impact the bioavailability of choline. Our other study does a, I think a, a, a better job at trying to provide some estimates of that degradation, but here we're looking at sort of single digit kind of percentages in terms of how much choline is degraded postly. But we need to be able to understand that because there's certainly a use of in vitro tools to estimate intestinal digestibility. And those, those assays don't necessarily account for, or they don't account for the role of TMA lice.
Clay Zimmerman (00:44:44):
So if, if you've, if you're finding TMAO in the cow, do you, do you know, is that coming from ruminal degradation or postal degradation?
Dr. Joe McFadden (00:44:54):
Yeah, on this particular study, the TMA we have, again, we had a fecal TMA response. And so I, I'm sort of perusing the data here and I'm looking for the TMA, and you definitely see the increase response to the ABBA infusion in, in urine. And so that is sort of a good, you know, it's, it's about roughly 5% difference, maybe 10% difference relative to the other treatments, but it's certainly significant. And in that particular case, yeah, that's ABBA Masal, right? That's post ruminal, that's all coming from the the role of teammate ice past the rumen. So yeah, there is a role there. You will see it in urine. And we, we showed that before that 99.9% goes in the urine, and we didn't see that response in the in the milk. We did not see a change in plasma TMAO response in this particular study. But we have in others.
Clay Zimmerman (00:45:54):
So how, how early in lactation were the early lactation cows?
Dr. Joe McFadden (00:45:59):
They were, so I had of a mean sort of time of 97 days in milk.
Clay Zimmerman (00:46:07):
So can you speculate on what, what you may have seen if these were actually transition cows?
Dr. Joe McFadden (00:46:15):
I would suspect that well, I wanna be clear that most of the studies that we've done in the past that I can think of that, and these were, I'm looking at the age of the late lactation cows, 270 days in milk. But an amaal infusion typically I would've said, increases the choline levels, but earlier in lactation, the greater the response if it's, if it is bioavailable. And, but like I wanna emphasize that typically when you do these studies on the equivalency of choline ion across treatmentsandn a missile infusion of choline, we've done this in mid lactation before and you get a choline response. It's, it's, it's, it's very typical. The I would've, I would've expected an increase in at least milk phosphocholine concentrations earlier in lactation, perhaps greater than what was observed in in this particular study. I do wanna emphasize here that the plasma choline response was enhanced, not the milk in late lactation animals, but in the plasma choline response, we had more plasma choline in the amaal infusion treatment relative to the others. But that was the same, whether it was earlier, late lactation, it was only specific for the milk or the early lactation animals had greater milk choline levels.
Scott Sorrell (00:47:42):
Joe, you started out by saying that you know measuring choline bioavailability is very difficult. Have you found that method yet, or is there still more work to be done?
Dr. Joe McFadden (00:47:53):
Oh, we found it. It is the other abstract we talking about with Tonya France. It is a, it is, it has its own limitations, which we'll talk about. That said I think we're getting closer. I think one of the things I learned in this exercise is that, you know, we have the ability to look at choline levels in, in fecal, you know, when you look at rumen-protected choline products, that's a, that's, that's a difficult thing to do when they produce a lot of feces. And so then you're trying to develop a proper sampling protocol to ensure that what you're measuring in that feces and as the actual measurement, right 'cause you're taking something, you're feeding something at these micro inclusion levels and then you're trying to, you know, see if it's actually ending up in the feces. That that is a real challenge. I worry that potential limitation of, of our study is that we could be we're providing an estimate. I'm not sure if you know, I, I think we, we, we put the potential to be underestimating, just be depending on our sample protocol.
Scott Sorrell (00:48:57):
Alright, well, we'll be looking forward to the next segment with Tanya. Charlie, I wanna thank you for joining us today. I understand that you're a PhD student at Cornell. What's gonna be your next steps here? Do you, do you have some more research to do before graduating?
Charlie Yu (00:49:13):
Yes, I have a big cell work coming up. It's basically investigate like how c induced insulin resistance in myo tubes, like from sheep over myo tubes.
Scott Sorrell (00:49:27):
All right. Very well. Well, good luck with that. And thank you again for joining us, Joe. Thank you as well.
Dr. Joe McFadden (00:49:32):
Thank you.
Charlie Yu (00:49:33):
Thank you all.
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Scott Sorrell (00:50:07):
Right in this segment we're going to review an oral presentation given that the ADSA this year. And the title is “The Metabolic Fate of Deuterium Labeled Choline and Gestating and Lactating Holstein Dairy Cows”. That presentation was given by Tanya, France. And Tanya, welcome back. This is this might be your third time back at the Real Science Exchange. I believe. So welcome back. We appreciate you coming to visit once again. And I will say that we must say that that Joe in, in the previous segment kind of gave us a prelude to, to this segment. So I'm looking forward to it. 'cause He said that you guys have come up with a, a viable way to measure choline bioavailability. So he kind of, kind of teased out a bit for us. So looking forward to this Tanya, why don't you just kind of give us an overview of the study and the objective?
Tanya France (00:51:06):
Yeah, so we were really interested in looking at the metabolic flux of choline in the dairy cow because as we know choline can be metabolized via two different pathways. And so in order to do this, we used stable isotope methodology, which has been well established in human and mice models. And we did this using this deuterium labeled or D nine choline. And so we were able to measure the D nine choline and the different metabolites. So if it was D three or D six choline, it would be utilizing the methionine cycle or one carbon metabolism. Whereas if it has a D nine attached to any of the metabolites, it would be using the CDP choline pathway such that it would not be donating any methyl groups. So our hypothesis was that physiological state, so either late gestation versus early lactation will influence choline metabolism, including the methyl group utilization in the dairy cow.
Clay Zimmerman (00:52:22):
Anya, since we can't see the pathways and so forth. Yeah, what, what do you mean when you say D nine labeled called?
Tanya France (00:52:33):
So that means that there's nine deter atoms, which are replacing the hydrogens on each of the three methyl groups of the choline molecule. And so if it's a choline is, if a methyl group is being donated, it would lose one of those three deuterium atoms, and that's how we can measure it.
Dr. Joe McFadden (00:52:55):
Perfect. You know what, you know, what's makes this study, you know, so unique is that, you know, again, you have these methyl groups that are on choline and there's three of them. And so when you use this kind of tracer technique, you can sort of understand where each methyl group is going if it's labeled, and that's a powerful tool to understand how the colon is converted into those other metabolites. And then when you can quantify them in different pools, then you can provide some strong estimates for how dietary choline is utilized.
Scott Sorrell (00:53:28):
Were there any surprises once you saw where the choline is being utilized?
Dr. Joe McFadden (00:53:36):
Tanya
Tanya France (00:53:40):
No, I mean, I guess I was glad that both pathways were being used during both late gestation and early lactation, but I, I wouldn't say I was surprised by that.
Dr. Joe McFadden (00:53:51):
Well, I was sort of surprised. I mean, I was certainly pleased to see the responses that we saw. Again, there was so many big questions surrounding choline biology that I think this study has answered. And, and in reality the study itself is a rather simple study and relies a little bit on the use of mass spectrometry, obviously, but, you know, to provide definitive evidence that choline is a methyl donor is something that we, you know, we talk about it, we take, we say, Hey, it's methyl donor, but you really need some concrete evidence once in a while. And this study does that, it actually shows that it is indeed behaving as a methyl donor and providing methyl groups within the methionine cycle. So the, that that's something you we wanted to see and we saw it. And then yeah, it's nice.
Dr. Joe McFadden (00:54:39):
You know, Tanya mentions that being able to look at the pathway utilization we certainly gained a lot of value there to try to understand that. And I think there's so many other things. I mean, I don't wanna steal the thunder away from Tanya Tanya's the one who's partly discovered this, but, you know, one of the cool findings was, is that we discovered so we, we wanted to better understand this, the, the postural degradation of choline and trimethylamine and TMAO. And one of the metabolites that we discovered was D three trimethylamine and not getting too sciencey for everybody, but basically this is a, demonstrates provides evidence for this, this choline recycling that can occur, that choline enters the body of the animal. It can act as a methyl donor, it can be converted back into choline containing phospholipids. And then those phospholipids are very, could be entered into bile and go back into the intestines and be acted upon again by the bacteria postly. And you know, it's just, we provide evidence for this recycling like evidence for it. And it's certainly something that we've read in textbooks about before, but it's nice to actually be able to measure it and see it.
Scott Sorrell (00:56:01):
Are there any follow-up studies then? Or, or any plans now that you've got this tool? How do you plan on using it?
Dr. Joe McFadden (00:56:10):
Well, I think we're, you're bypassing some big stuff here. Okay. Alright. Sorry. We, there's this study has some major conclusions that we can't necessarily ignore and we really wanted to try to look at, you know, how much of the choline is being used in these different, different pools. And I don't have the numbers exactly in front of me and Tanya, you can correct me if I'm wrong, but depending on stage of lactation, we're seeing anywhere from five to 9% of the choline that gets post ruly is getting converted to trimethylamine and tmao. And so something as high as nine or 10%, that is certainly, I think significant when we're trying to provide some estimates or thinking about metabolizable choline supply. And we need to, we need to remember that and we need to think about that moving forward and, and try to develop approaches that are less costly than this study to allow us to include that as a potential correction when we want to calculate bioavailability. The other one was on how much of the choline was actually used by the mammary gland. And so it depended on the metabolite involved. And I think we, we estimate around, I think it was 12% of the choline that was infused was actually used by the mammary gland for milk choline. But when you added up all the metabolites, so I think Tanya, what was it, 24,
Tanya France (00:57:30):
It was 2025
Dr. Joe McFadden (00:57:32):
Was being used by the mammary gland. So that's significant and it just emphasizes that it's not just choline that it's all these other metabolites that we need to think about.
Clay Zimmerman (00:57:46):
Were there, were there differences in the late gestation cows versus early lactation?
Dr. Joe McFadden (00:57:52):
So we did, I mean, Tanya can provide some comment here, but we did see changes in the plasma concentrations of these metabolites, which is what we would expect. We did see in numerically lower milk choline, plasma choline concentrations. But one thing that I think was really important here is again, we saw evidence for methyl group utilization by methionine cycle, and we see that the metabolites that were involved in that PEMT pathway, those metabolites were are increasing sort of early and early lactation. You know, some of these p values are significant, others are tendencies, but overall it looks as though that there is an increase in the utilization of choline as a methyl donor in early lactation. That said, it didn't say that the D nine, that the other pathway wasn't important. It was also important as well. And, and I think when you look at the non-ruminant data, there's plenty of evidence to suggest that actually plays a much stronger role in providing phospholipids. But yeah, it's a combination of the both that appear to be very relevant and an early lactation animal is important for that animal.
Scott Sorrell (00:59:07):
All right. Anything else? Clay
Clay Zimmerman (00:59:11):
So now I think it's time to ask what's next.
Scott Sorrell (00:59:14):
Now I can ask that question.
Dr. Joe McFadden (00:59:16):
What do you wanna do next? Tanya
Tanya France (00:59:24):
I mean, I think a big part of it that we were unable to measure and account for was how much of that choline we infused, how much of it was going to the fetus. So there's a lot of evidence of that happening in humans. And they were able to do that with D nine choline and there was a part of a large part of the PC that was made that had the D nine D three attached to it going towards the fetus. And so that is something that we weren't able to account for in this study. However, I think it would be really cool if we were able to somehow do that in the cow. It would require infusing this leading up to and while they cabin, but so,
Clay Zimmerman (01:00:19):
So how, how many days were the infusions?
Tanya France (01:00:24):
They were five days.
Clay Zimmerman (01:00:26):
And and how long did it take for, for the concentrations to pee? During that five period?
Tanya France (01:00:36):
At day three, we saw it kind of start to plateau. So we measured the last two days, so day four and five. But over the course of time it started at about the third day.
Dr. Joe McFadden (01:00:52):
Yeah, in terms of, I mean, I have so many ideas but I'm gonna, I'm gonna need a massive budget to start to answer some of these. If anybody wants to look at the cost of a labeled calling, you'll understand why it hasn't been done before. But that said, yeah, it's such a powerful tool and I think there is opportunity to, you know, answer some other questions about choline utilization and their interactions with other nutrients and other forms of you know, life stages of the animal environmental stresses, things like that. There, there's a lot of opportunity there to answer some big questions, you know, relatively quickly if you, if we have a, some, some investment to support it.
Scott Sorrell (01:01:35):
Okay. All right. Very well, great. I wanna thank everybody for joining us today. It's been quite interesting and looking forward to the next time. Tanya, we, you be joining us once again.
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