Hello, my name is Daniel Luchini, head of Ruminant Research and Innovation of Adisseo. Welcome to the Amino Acid Balancing for Lifetime Performance webinar series. The topic of today's webinar is strategic use of methionine to impact reproduction. And with that, we have two great minds that are experts on this topic. Doctor Phil Cardoso is an associate professor at the University of Illinois, Urbana Champagne. He received his DVM and Master of Science degrees from the Universidade Federal do Rio Grande do Sul (UFRGS) in Brazil and his Ph.D. from the University of Illinois. Phil's Dairy Science program aims at placing students in applied positions and academia. Phil and his students have published over 90 peer reviews, manuscripts and three invited book chart chapters. His academic program builds from questions asked by dairy producers and focuses on having the dairy cows diet as a medical prescription for performance, health and reproduction, and strives on linking the impact of nutrition on metabolism and reproduction and health in dairy cows. Pn the other side, Mateus Toledo has an extensive background in dairy management, nutrition and reproduction with emphasis on amino acid nutrition. In his current position as dairy nutrition consultant for Purina/Land O'Lakes animal nutrition. He provides dairy farm nutrition management and technical support throughout data analysis. Mateus is a published author of several scientific articles out of his work to obtain his master of sciences and Ph.D degrees at the University of Wisconsin in Madison. Mateus offers a unique blend of reproduction physiology and nutrition knowledge to dairy farmers. In order to warm up with this webinar, I just have a question in terms of what happened with reproduction and just to set up the system here. I will try to explain what are the embryo and fetal losses from insemination to calving when calving rate is about 30%. Which is a unexpected number for any given dairy on today's market. These are calculations estimated by Dr. Jose Santos, and it's a clear explanation of the risks and the high early and late embryo mortalities that we have. When we inseminate cows, there are unexpected, 10% to 15%, failures in the fertilization. So with those 80% to 85%, to 90% now fertilize early embryos, there is about a 57% embryo mortality in the first 28 days after fertilization. From day 28 to day 45, which is essentially what a normal commercial dairy would do, which would be between the first and second pregnancy check. You have about 15% embryo losses, late embryo mortality losses. And after day 45 there are about 10% fetal losses. That exemplifies, highlights the big problems that we have in terms of these high losses. But the real point that I need to point out, I want to make up here, is that even in normal conditions, we pay our attention into the embryo losses between day 28, day 45 or the first and second pregnancy checks. Most of the embryos are lost before placentation, that is before day 28. And the reason why this is important, is because of the high cost, what that means to the dairy producer. According to Dr. DeVries in 2006, and these numbers have not changed that much, there is a loss of about $150 to $360 of pregnancy losses, due to pregnancy losses in the first month of pregnancy. So with this set up, I will just let Dr. Phil Cardoso explain the basics of why methionine, which is known as a required amino acids, may help to alleviate these issues. And after Dr. Phil Cardoso basic explanations, we will have Dr. Mateus Toledo, give us an explanation of the work that he did at the University of Wisconsin, his Ph.D degrees and also will bring us down to practices of how he uses this knowledge to help dairy farmers in today's market. Welcome Phil. Welcome Mateus. Phil, the floor is yours. Thank you Daniel for the introduction. Thank you Adisseo for inviting me to be part of this webinar series. Share a bit of the information we have generated but also connect that information with everything else that has been done in this area of amino acid, more specific methionine on reproduction. So throughout the years, we have been able to deliver pieces of information. And one of the things with the challenge of this webinar and trying to be as concise and give them as much information in one package as possible. I've asked my former student Anne R. Guadagnin, that is doing a post doc now at the University of Wisconsin, to put everything in one slide. And this is the challenge and I think it came out pretty interesting. So this is a summary of three of the papers that we have published. And one of the information or one of the pieces of information we wanted to achieve was how can we connect information from the uterine tissue but also connect with the ovary and also with the oocyte and then the embryo. So on that early lactation cow, I think we've done a lot of research during the transition period. With that fresh cow, early lactation cow, what are the impact of those nutrients that contribute to the communication among the uterus, the ovary and the embryo. The concept as it comes after fertilization. So we were very interested in that. I'm still interested in that, in combining all that information. So one of the things that we were able to establish is that the uterine environment is changing according to methionine in the diet of those cows. And when I'm saying methionine is that and we can go later on how do we decide or define what should be the amount of methionine or concentration of methionine in that diet. But we believe when we give the amount of methionine that is necessary for the cow to perform, those are the results that we were able to capture so far. One thing that I'm gonna be up upfront is that only by manipulating feed ingredients, we were not able to get to that number. We have to add that rumen protected source. So when we do that, we achieve and again, that's gonna be very different according to someone's ingredients, someone's diet, that's gonna change. But when we achieve what we want, we think that we change the uterine environment into the better. So one of the things that we were able to see was that there's this influx of PMNs or those Polymorphonuclear neutrophils, early on into the production cycle of that come into the uterus, but not later on when fertilization needs to happen, what we believe it's something very positive. So here it's a graph for that section and that's what we are seeing when we are doing is the Cytobrush. You can see that in the beginning of that lactation at 15 days in milk, cows that had methionine to the correct concentration in the diet, they have this higher PMN. However, when we see it at 73 days in milk, that was different. If cows don't have the fever, they have no impact in intakes negatively. I believe that that higher influx of PMN is actually needed. Another thing that we are able to see it's needed for that uterine involution to be able to happen with success. So one of the things that Daniel was mentioning is that all this embryonic loss that we have and if we go to the very practical number, that is conception rate at first breeding and we expect farms to be at 40-45%. Most often, the reason for them not to be at 40-45% is maybe because uterine involution or the uterine environment is just not ready for that pregnancy. And that's where we see farms, maybe 10 years ago, the voluntary waiting period of all farms was at 45 days in milk. Nowadays, we are talking about 70-80 days in milk. Not only recognizing that the uterine environment needs to be ready, but also some other factors that we can talk later about, ways of synchronization and timing where we have so good progress rates out of synchronization protocols that we can allow the cow to recoup better. So that's one of the things that I believe we are being able to do that when we are sitting with methionine. Can I ask you one question Phil, I guess that you're going to get into this throughout your presentation. But how do we help or assure that the cow, that uterine environment is ready, such a dynamic process, so many things happening at the first 15-20 days. So I guess I maybe I'm getting ahead of myself, but how do you help the cows for assuming that ? That's a very good point. And then here when I'm showing the graph, when I'm talking about the uterine environment, and I go back and I talk about the Cytobrush. So that's something that usually farms, they are not doing this, right? So farms, they are not actively outside, most of them are not going actively performing something similar to an AI to get that information. So oftentimes the information that we have, it's gonna be out of vaginal discharge. We can kind of classify that a little bit different. So here we are talking about endometritis, that's not necessarily very correlated with that vaginal discharge. So if you think about the uterine tissue or the uterine horns, then we have the cervix where I can go back to that one. So I have the uterine tissue here. I have the cervix and then I have the vagina. So when I'm at a commercial farm and I'm walking behind the cows and that's the only way I use to identify metritis is that I'm assuming that whatever is coming out of the vagina of the cow that could be a purulent or not secretion that's gonna be coming from the uterus. There's some research that may say, well, that's not a lot of association there. Yes, sometimes but the type of the secretion may tell you if maybe it's just coming from the vagina or actually coming from the cervix or actually, it's coming from the uterus and we have all those negative impacts. So I think the way we are now more established for that I would, and maybe Mateus can step in and tell his story here, but it would be to go actively, you know, there are tools that we can use. Now, we use a lot in the research setting the metric check. So pretty much a stainless rod where we actively go there and scoop all the material that is coming from the vagina. Because if you think about it just walking behind the cow, maybe that material contaminated is there, but it's not coming out of the cow. If we get that as access to that material, especially in the first two weeks of the post calving. Once a week if we check that and actually, if we just smell that secretion and if that secretion has a smell and the cow has fever or has a temperature above the normal, that would be something that is related to this cow is not well. She needs to be treated and then there's a lot of conversation about the treatments, but that means that that's not happening. So the evolution is not happening well, then it can be happening for many reasons. So that would be one thing right there, a picture of that herd at that moment. Now, like I just said, if your conception rate at first breeding is not happening at the 40-45% rate. That could be an indication that that uterine environment is not happening as well. And that's usually something that we will affect pregnancy rate. So your pregnancy rates are low and you start digesting why they are low. So why some farms are at 30-35% and some farms are at 20%. Sometimes it goes back to all this uterine environment and we believe that cows being fed to what they require, it's important because now cows at 15 days in milk, they can have that influx of PMN without causing fever, without causing metritis or that infection to be able to help with that involution. Mateus do you have any suggestions here? No, I think that's interesting data. I agree with you. I was just thinking more, methionine is one of the most functional amino acids and there's so many ways that it could be affecting eating environment. Any thoughts on how methionine could be regulating those PMN cells or you think that's more an interactive effect on the health of those cows. That's a very good point. I think everything comes back to having healthier cows and that goes through the liver and goes through other impacts. But actually, when we see that and one thing that we have is that when we go there and we collect those cells, we have the PMNs but we also collect a piece of tissue. So we do a biopsy of that uterus. We can see there are genes there that are being regulated differently. And actually, we are able to put in its light and see the histology and actually see how those glands and how the uterine is performing, that's changed. So for example, in the beginning, you have cells that are much bigger, taller, so more volume of cell and we are not used to do that a lot in dairy, but in equine, they do a lot of data association of, if I have a more metabolically active tissue, my cells are gonna be different. And that happens at 15 days where you have these larger cells, glandular cells. But that doesn't happen at 73 days for example. So it's something that that tissue is more receptive and we also know that that tissue has different composition as well. So I think it's just a reflection of what the cow is needed. And maybe some of this transports or even some of the nutrients in the uterine tissue, is able to generate that response. Maybe a huge speculation here, but if we think that inflammation it's driven by glucose. So let's say it has a preference for that type of substrate. Then if I can have more receptors of that glucose in the uterine tissue that tells me that maybe here PMNs can work in a better way or they can come to this tissue because now I have the source where they can thrive on and we've seen that you have higher expression of gluten receptors in the uterine tissue of cows that were consuming methionine versus cows that were not. So, I think it's interesting. That's kind of where we think it's coming from. It probably could be a direct effect, like you're saying, or a combination of things that those cows could be eating more and being healthier and then get more nutrients to the eaters as well. And let's see, I have here the ovary. So I think that there this information from the uterus where we are able to identify this gene expression from that tissue and alluding to some of those differences. Then we can go back to the ovary. What's the role of the ovary to that cow getting ready? And one of the things that we explored was we follow cows after calving, and we divide those cows in two groups. One, we just wait for them to ovulate. So we follow the ovalution from the first follicular wave, starting at seven days. We start ultrasounding those cows as soon as they ovulate, then we stop that daily or every other day ultrasound. Because that's another thing that can impact conception at that first breeding. That is resumption of cyclicity. So some cows, they delay that resumption most of the times that's related to nutrition like the negative energy balance. But also maybe this negative protein balance if we think about changing or just changing the concentration of that amino acid in the diet. So one of the things that we were able to do, one of those cows, we follow them and we see if they ovulate or not. And again, our data sets with 40 cows, that's not ideal for that. We get some information from it, but it's not the best data set for that. That would be better in a commercial farm, a lot of numbers. The other side is that some of the 40 cows we say, OK, a hosting cows, she will ovulate. Usually multiparouse cows that we are talking about, at 16 millimeters of that follicle size. So when cows achieved that 16 millimeters, we went there with a needle and we aspirated that follicle. So we're not looking at the ovocyte. What we were looking at was the fluid and all the cells that compromise the follicle. And that's what I have here. So this is what we see, of course we would see one follicle, in this case you're seeing more than one, but those would be the follicles, they have this granulosa and those theca cells, that's it. And within that fluid, that's what starts to expand. And that's when cows ovulate and then you have fertilization. So we are a little bit before that, but we know there's this whole pathway here that happens before we can have estradiol in the follicular fluid. So cows, if they're gonna show asters, they're gonna have that concentration of that hormone pretty much coming from that follicle. The moment she ovulates that full fluid estradiol stops circulating. There's no signs of asters anymore in that cow. But one thing I want to highlight is that lipids or cholesterol, they are involved in this cascade that is gonna go through estradiol but also progesterone. So when I highlight this progesterone, it needs to be metabolized in a certain way in that follicular fluid. So we can get it to estradiol. And we have these enzymes here, they are responsible for different steps on that cascade. And one thing we did was so we were able to do some gene expression of those cells from the follicle. We can't say if it is granulosa or theca cells, we didn't separate those, but we were able to see that the cows that were receiving methionine, whenever they had methionine and here is a bigger scope of a project that had choline as well. But we can see that this enzyme that is responsible for that is one of those steps was in a higher concentration or was highly expressed. So we believe that there is a change in that genesis pathway that is being impacted by methionine. And when we check for concentration of those amino acids or some amino acids, this tree here, inside the follicular fluid, then we can see that methionine is in a higher concentration in that follicular fluid. So for some reason, we are feeding that, that's going through the cow. And now it's showing up at that follicular fluid and that's what's gonna be, perhaps be important for the development of that embryo. So here we are still in the ovocyte stage, in that follicular fluid. We didn't find any difference for lysine and we found a tendency for histidine to be higher. We didn't check for arginine, we didn't check for other amino acids. There was a very limited amount of, you know, it costs quite a bit to do this analysis. So we didn't have a vast panel of those amino acids. But then we see that maybe some of the other ones and when we went and we checked for those cows throughout lactation in the serum of those cows. So we have information from minus 10 until 73 days. You can see that consistently if we don't feed for for rumen protecting methionine, we are gonna have a lower concentration throughout the time. It was just not a few points, we were able to do that where we can see that consistently. Those cows were above 25 micro molar in the serum and cows were below. That would be kind of the our ovary story, where I think there is still a lot to grow on that area. So we didn't check the tissue of the ovaries and what may be impacted. What we checked was the cells from the follicle. So we are talking about the dominant follicle. We are not talking about pre-entro follicles or where we would believe that this follicle that we are talking about, we start growing a few days or a few months before and in that environment. So I think that there is still a lot of things to do that. Estradiol is gonna prepare the uterus for conception, right? So you saw an increased expression of genes there for production of estradiol. How do you think the uterus is gonna look like if you're having more methionine in there, you have a higher expression of genes that control production of estradiol there. Did you have any thoughts on uterine environment at that time with that dominant follicle there? That's a good point. So usually, and I don't have here the number, but usually our cows are gonna be ovulating. So the cows that we just leave them to ovulate by themselves. Our first ovalation has happened between 15 and 17 days. So we didn't see a difference. So days to first ovulation between cows with methionine or not, they were not different. So that information of 15 days that we have from our uterine biopsies, that's what we are seeing, the change in that uterine tissue that gland eithelial cell in the gland of the uterus. So I think that's a good point that that estradiol can be causing that change for that cell to be more metabolically active differently, right from control and methionine. Even though I'm having both ovulating at the same time, the quality of that follicle or ovulation it's different. Yeah, I think that's a good point. Then one thing I was gonna ask you Mateus, I know that you did some different analysis or more perspective on serum concentration of amino acids. Especially methionine, because here, to be honest, I think we collect that sample at the same time all the time, and we provide a top dress to the cows in our trials with that methionine and we see this kind of standard pretty much. But of course, here there's a lot of cows that's an average standard deviation, but we never looked into within the day or relative to the top dress or when the cows are eating more the protein source or the methionine if that's changing. I remember you did some of that, didn't you? Yeah, there was a while back on our first study, when we top dress in a commercial dairy and we did not see difference in the first three hours and it starts to go up for the cows top dress with rumen protected methionine. And it started to go up with six and a peak at 12, and at 18 it starts to come down again and control was really flat. So that was an interesting profile when you talk top dress that we see. And in our second study, there was a GMR and then we don't see that pattern anymore so it is flat throughout the day. It's interesting, I don't think at this point we know what would be the effect of that profile and production or metabolism, but that's something that needs to be explored further. I don't know if you have any thoughts on that. I think it's interesting and we assume that, you know, the rumen gives some consistence of delivery of that profile of that protein being absorbed. But you're right, I think the way we are providing maybe in a different, in a bolus or different concentrations throughout the data that I'm not sure we don't know exactly. I mean, if the plasma concentration goes up because tissues are not uptaking, right? So they're not using, then it's accumulating in the blood. Is that going to another tissues instead of mammary gland, for example? And then the embryos is being able to use more methionine. I'm not sure if that will be the case. We took a look at the plasma amino acid concentration at different time of the day when the methionine is offered in a TMR. We have that information and essentially what happens is that as you know, the first feeding of the day, the cows consume more than the rest of the day, right? Independed how you try, as much as you try, to have equal amount of dry matter intake throughout the day, the cows would consume more at some times than others. But nevertheless, when we did check for that plasma amino acid concentrations, there was perhaps a time point that the plasma concentration was higher during the day. But the differences were not as high as you feed it as once. So the points that you are making both of you are is that yes, there is a difference. But if you feed the methionine that once top dress or if you feed in a TMR, but when you feed in a TMR, that variation you have throughout the day is not as high. That's the point that I wanted to make, it looks like more like this specific figure that you are seeing here looks more like that. And we have that information. It's interesting because I think one of the studies we published, we just graph individual cows and we saw a huge variation with cows at time zero at the time of feeding being at 50 micro molar. And then six hours later, there were 30 micro molar. But you know, you can't control intake over time as well. So we don't know what's happening there. We just know that there's a huge variation during the day. And from cow to cow too, of course. So that's why when you look into this, this is as Phil was kind of pointing out too. There is so much information that we still need to gather in terms of how these dynamics of plasma amino acid concentrations throughout the day. And this information, despite the fact that there is plenty of it, there is more that needed to be made because of initially and it's sad to say, but the cost of having plasma amino acid concentration is extremely costly. And if you're looking into 40-50 cows and several times a day that a sum of money that is unmanageable. I think we are trying to get newer technologies that would allow us to unearth that type of information and hopefully will come up soon. Just gonna have one question for Phil in that graph there. Do you think your control cows, it looks like they're going down after calving, do you think that shows that your control is really deficient in methionine? Because I think we, I'm just jumping ahead here, but I'll show that one of my studies that it looks like after calving controls cows really goes down in terms of methionine concentration in plasma. Where the methionine cows stay flat. I think so. I think the fact of the milk output for methionine is a big one and I think that's what's showing there. I think also that, we've tried or we've fed methionine prepartum in a certain way. Today, we are feeding in a different way more associated to what's happening, lactation. We have association of energy. But we also don't know if it's correct or not. And so I do think that after calving, there's a huge impact of that deficiency of methionine mainly because of milk protein output. And I think that's a big one. But I also think before calving that difference may be kind of important as well for some of the things that are happening, especially cholester genesis, that I don't think we take it into account. And we, I think the whole dry period on that evolution of the mammary gland, we were just talking about the uterine tissue with epithelium cells, but the mammary gland with another type of epithelium cells, I think it's something we're gonna have to relate a lot as well. And I think, coming back to the swing of the methionine, that needs to be a little bit broader on the sense of, you know, if we think about technologies like metabolomics where yes, if I have a spike in insulin, maybe I can use better that higher methionine. If I don't have the insulin, that will be a different issue. So all hormones that are gonna be fluctuating throughout the day of within the cow that it may be associated with how this amino acids may be absorbed and used by the tissue. So I think it's a very complex question. One thing Daniel, I think I even discuss in this paper here, but you've done some work with embryos and you came up with a number. So it's very hard to say, right? So we have the requirement that we try to understand for methionine. But then you say, hey, if I go out there and I collect a blood sample from a cow and I want that cow to be, I know she is getting enough methionine and we talk about this 20-25 micro molar and say if I don't feed methionine that's what I get here, in the midwest. I would say that's what you have a 3 to 7 ratio with lysine. And you're gonna get to that maybe below 20. And when you see the cows that were fed methionine, you are above 25 all the time, would you dare to say that we should be always beyond 20? And can you relate to that embryo work you did with a long time ago? Well, that's a good point Phil and thank you for bringing this up. In fact yes, in this study just for the audience to be aware of what we did. Essentially we just collected eggs and then we did in vitro fertilization with different concentration of methionine in the media And the very concrete conclusion that we had is yes, methionine is required for those embryos to grow. And then we did increase concentration of methionine in the media. And the conclusion of that paper was that the methionine had to be at about 14 micro molar or 15. After that, it really didn't matter much. Now, a couple of questions out of that, is the methionine that we were using was L-methionine. And here we're looking into DL-methionine. So keep that in mind because having 20 or 15 micro molar here is DL, not just L. So judging by this, I would say, heck you are in danger. And also, again, going to the blue line in here that you are borderline and this is 50% of your cow, right? Roughly speaking. So a lot of your cows are at risk of not having enough methionine to at least assure that an embryo will have enough, as to warrant that at least you have that nutrient as you guys are alluding to. Well, it's a combination of other nutrients, right? You have to have glucose, you have to have fatty acid, you have to have a healthy cow and so on and so forth. To answer your question, Phil, we do know out of that trial that methionine is required and there is a minimum requirement. I really honestly cannot tell you what that minimum requirement is. But I think in this case, at least if we have 25 that would be my out of sleep answer to that question. And I think to finalize here. So we talk about the uterine tissue, we talk about the ovary. I think another thing that I wanna touch base is this work we did with embryo and it connects with Florida again with Peter Hansen. So we collect the embryos, we super ovulated those cows. So from pre calving, prepartum, until 72 days in milk, they were being fed methionine or not, the group control. We check the quality of those embryos, we were trying to address some of the methylation. Well, there's no difference there. What we saw was this, we can do embryo by embryo with immuno to chemistry. We can highlight the fat or the lipid inside the embryo. So that's why I have it there. It's arbitrary unit we have no concentration, we're just comparing two things. So I'm comparing the embryos coming from cows with methionine and the embryos coming from cows that did not have that methionine. That blue line that we saw before. And you can see that there was a statistical difference. So here we don't say that the embryo is fat. We are saying there is different lipids than the control. That's what the trial said. Now, why was that? At that moment? We didn't know, is it because the embryo is reserving or making more reserved for energy? Does that, because I don't know, is this inside the embryo? Is it on the outside of the embryo? So it's more communication of that membrane we didn't know at that time. So one of the things that we did as well is some of those embryos from those cows. We also had it analyzed by one talked technique that it's called MALDI-MSI. So we collaborated here with Purdue where they go embryo by embryo, they can shoot a laser and they see the profile of those lipids. So we have metabolics, well there is lipidomics so we are talking about lipids here. So actually the type, not just the amount of the fat, but the type of fat of the fatty acids. It's a single embryo, so we're not pulling embryos, we're doing one by one. So we don't have the chances of stretching and learning a lot. But we can see that there is more of this polyunsaturated type of change. You have a little bit less of monounsaturated to have more of this polyunsaturated types of fatty acids. And if you think about it, that type of fatty acid here in the embryo could be also related to what's happening in the uterus. So remember we had also a sample from the uterus where that embryo is coming from and that uterine tissue now we have more ability to profile. So at 15 days in milk, no difference. But at 30 and 73 days in milk, you see that change for that higher lipid and perhaps this more unsaturated type of lipid. And we know that that type of fatty acids remember needed for that progesterone secretion in the tissues or in that environment. But also it's kind of can counteract that prostaglandine F2alpha. So all that prostaglandine that could be, you know, causing abortion or that early embryonic death. Perhaps that is something that is also changing. And that's why the uterine tissue could be, the embryo could be this reflection of that change in the uterine tissue. So I think that's very interesting, where I would think that this is a more a positive change. That may be because now we have an amino acid that can build a protein that can transport lipids from the liver, from adipose tissue to the uterine tissue. We have the chance now of the cow changing the profile like we would have from feeding different types of fatty acids to the cow. Like we see with, we don't do that anymore but let's say omega-3, omega-6, we don't do the fish meal but you see all those components kind of happening here perhaps because the cow has the ability to mobilize and transport. Because now we have that protein specifically, I'm not showing here, but we've seen differences in apolypoprotein being higher expressing the cows that have methionine in the diet. So it's all about that transport. I think this is very interesting and we see that in milk, even though we're not talking about I think the previous seminar or webinar, we were talking about this milk fat effect of methionine where it would be something. Well, we don't think about that, but it's not happening only in the milk, it's happening in the body, in the tissues of the cow. And here we are talking about the uterine tissue. Phil thank you. I mean, this is a good really basic explanation and linking the importance to the gene expression level. I think it's a good link in between the two. Thank you. This was great, Phil. In terms of the importance of linking, providing the right nutrients to the cow, the cow will actually grab them and actually will act accordingly. So this is great. Thank you. Mateus, I will give the floor to you. Thank you again for being part of this and I think that you're going to be discussing a little bit bringing us back to earth and to reality of what's happening at the real cows, not at the gene level. Thank you Adisseo and thank you Daniel for the invitation. I'm happy to be here and talk about some of the studies I did when I was a grad student. So some of the studies I'll show here there was a companion study with Cornell University and the University of Wisconsin, in collaboration with Adisseo as well. So a lot of people were involved on these studies. I just wanna start talk a little bit about methionine. Methionine is one of the most functional amino acid. It's not only about protein synthesis, it's not only about milk protein. And you can see here in the diagram just showing how many functions amino acids can have. And then I just wanna highlight some of the methionine here, for example, for protein initiation mTOR, antioxidative defense, methionine is involved in glutathione and taurine which could be involved in health. Phagocytosis and oxidative bursts, which also involved with the immunity and health DNA methylation which we discuss a little bit later, it could be involving reproduction as well. And then also synthesis of other compounds such as choline, carnitine and polyamines. And reproduction, which we're gonna be talking about today. It could be involved with embryo development. I'll show some data on pregnancy loss and then time to pregnancy. This was the first study we did top dressing rumen protect methionine in a commercial dairy from 30 days postpartum to 126 days. So not transition period after the transition period. We had 309 cows. So not a huge group of cows to analyze binomial data. But it give us an idea what's going on in here. You can see a picture here of the top dressing on your left, on your right few, I think this is what we were discussing before, the platinum methionine. No effect 0 and 3. And then in 6, 9 and 12, we peak at 12 and then starts to go down with 18 and 24. And then you can see the control is pretty flat. So kind of an interesting profile here when we talked methionine. One of the things here for the sake of time, I'm not gonna show production data but just know that in this study, feeding rumen protected methionine improved percent protein percent fat. And there was only multiparous cows, no effect in primiparous cows. Some of the first data I want to show about reproduction is amniotic vesicle and embryonic size by parity. Look here on your left primiparous cows and on your right, on which multiparous cows. You can see for primiparous amniotic vesicle there was not a difference in primiparous cows, but in multiparous cows, there was difference. So for those multiparous cows that were fed rumen protected methionine, they had greater amniotic vesicle. And then along with these, if you look at the embryo below here, except for crown-rump length, but the abdominal diameter and the volume was also greater for multiparous cows that were fed rumen protected methionine. One of the things I want to highlight here, if you look at control the size, let's keep amniotic vesicle, for the control here, you have 479 and then for primiparous cows you have 617. You know, one of the thoughts you had maybe those multiparous cows they were deficient just looking at the size of the amniotic vesicle in those other cows, multiparous cows. Now, the interesting data, you know, pregnant loss by parity as well. We're looking at primiparous here on your left. It is the first pregnancy check and then the second pregnancy check at 28 days was by blood and then 61 and 32 was by ultrasound. And then for primiparous cows, we did not see an effect, not a huge number of cows in here, but no effect in pregnant losses in primiparous cows. For all their cows, those multiparous cows, when you look at 28 to 61 there was an effect of maintaining those multiparous cows that were fed methionine. They had less pregnancy losses, ok? And then there was 68% 70% and then for 32 to 61 that was about the same and not a huge number of cows in these, but that was about the same. So think about the embryo size, you know, just an effect on the older cows, multiparous cows. And that was very intriguing for us. This was consistent multiparous cows again for pregnancy loss, there was decrease. Remember the production data that I mentioned in my first slide for this study. So those multiparous cows, they have increased percent protein, percent fat, embryo size was improved and then they had less pregnant losses and then primiparous cows, no effect at all. So that was very intriguing for us. Just an explanation here, thinking a little bit about multiparous cows and primiparous cows, those mature cows they produce more protein, right? So they need more methionine, I did not show this data before. But when you look at circulating methionine, those multiparous cows they have less concentration in blood compared to primiparous cows, which maybe would suggest those cows are more deficient. So you have less methionine going to the uterus, less methionine for embryo growth, right? And then you impair embryo development and you have increased risk for pregnancy loss. Now, when you feed more methionine you improve protein and fat percentage, right, you have more methionine going to the uterus and then that improve embryo development and reduce pregnancy loss. So that was our speculation why you have that difference in primiparous and multiparous cows. So with that idea that only mature cows responded to methionine in terms of reproduction, we decided to do a large study, and this was a companion study with Cornell University now feeding from prepartum until the second pregnancy check. So we fed for the first two months of pregnancy. So the difference from the first study, there wasn't a TMR and then after cows are out of the trial on the second pregnancy check, we follow those cows until 350 days in milk. So we had 470 cows, that was a 10 days study. And again, a companion study there between UW Madison and Cornell University. I think if you show these for this study, I just wanna highlight some of the things that we saw here for our study as well. So those are samples just from UW Madison. So we analyzed 225 cows individually. So those were more than 1000 samples analyzed. And then you see day relative to parturition and concentration of methionine. And it was interesting that we just saw a tendance at day 7 for cows fed methionine to have increased methionine, no difference at the time of parturition. And then it really goes up in blood, 46% increase on day 7 and then it stays flat until day 21. Interesting the control on day 7, after day 7, it starts to go down and it's lower also on day 21. Which maybe it suggests those control cows were deficient in methionine Do you think that could be association with, NEFA. From 0 to 21 days, we would think that cows would have higher NEFAs than maybe later on. And then maybe that's again that protein, not only milk, but it's also that methionine may be needed for that. Dealing with that whole lipolysis from adipose tissue to the liver. And that could be something that it's also going down not only in milk, but also there is the need for protein to be used as a fat transport because of that moment, you know, NEFA is so prevalent on that moment. No, I agree with, you just did not measure that. I think, at that time, after calving cows are using a lot of amino acids for not only production, but there's a challenge with health as well. Like you're saying, there's protein and amino acid used for acute phase proteins, right? So for health. So there's a lot of demand of methionine and other amino acid too. And that would be one of the reasons why those control cows are more deficient. We measured body condition core, we did not see any effect in body core in this case. One of the interesting things that I would say here, we measure other amino acids. Well, all the essential and non essential and interesting the cows fed methionine they have lower concentration of some other amino acids which maybe could suggest some body protein mobilization as well. You know, we have those cows fed methionine producing more protein. They need a greater pull of amino acids and that's why they have lower concentration of some amino acids which are being used not only for protein, but maybe other health functions as well. But that will be interesting, if you had more data in terms of health to try to see what was going on in here. In terms of reproduction different from our first study, we did not see just for the sake of time, I'm not showing here, but we do not see an effect in pregnancy loss or conception rates. But we thought that maybe there was a correlation with methionine and health. So we look at interaction of reproduction and health and how methionine would interact with reproduction. Methionine with lessen some of the negative effects of health disorders on reproduction. The first data you're seeing here is cows without health disorders. And then you're looking at time after parturition and in proportion of cows pregnant. So when you look at just the healthy cows, there was no difference in control in rumen protected methionine. So both of those group of cows were getting pregnant at the same time. Interesting when you look at cows with health disorders now and all those health disorders, you know, DAs, ketosis, clinical mastitis, RP, respiratory problems, lameness, and then cytological endometritis most of those they happened at the first 30 days that we recorded. Those are records from dairy coop, when you look at reproduction here, time to pregnancy, there was a difference between treatments. So those cows that were fed rumen protected methionine, they got pregnant earlier, right? And then you can see the difference here about 24 days. So those control cows were delaying to get pregnant. They got pregnant by 149 days and those cows got methionine that was earlier by 125 days. So it seems those cows with health disorders, they have a demand for methionine and that impacts reproduction. So if you feed more methionine for those cows with health problems, they will respond and they will get pregnant earlier. Mateus, I just want to point out a thing, this particular point in here, which is I think very important is to make the connection because if I understood you correctly, the majority of this health event happened within the 30 days in milk, right? And now you're looking into this days to get pregnant, which is 125 and 149 days, right? So you are linking the impact of what happened the very first days after calving to 121 to 150 days in milk later. And that's an important connection that is very easy to lose when you are in a any dairy, in any commercial dairy. And you're having 400, 500 or 600 cows, how do you remember what happened with that cow that was 60, 80, 90 days earlier. So I think this is an important point to really bring up because the connection of what happened, what happened in the first 30 days after calving is not staying there. I mean, you're going to see it later on and I think this clearly shows that, sorry to cut you off Mateus. No, that's all right. That's an interesting thought. I think every day I go, I mean it's clear if there's something going on during the transition period, they're gonna have problems with reproduction. So everything that happens with cows during the transition period, it will affect later lactation, not only reproduction but new production too. And I'll show that we maybe affect culling too. You know, those cows will leave the herd sooner. They're not staying for too long in the herd. So the last slide that I wanna show it's about herd exit and then looking at control and methionine in the proportion of cows that were sold and die and left the herd. This is by 350 days so after those cows left the trial. So they were moved to a common herd diet. And then we followed those cows until 250 days. What we saw was a tendency for those cows fed methionine to have a lower proportion of cows that were sold. No difference in die and no difference in proportion of cows that left the herd. And below here, you can see just over time the two lines, what happened the time after parturition and in proportion of cows that were sold. So I think these might be an effective reproduction or when you go to herds, cows are culled because of low milk production or they're not getting pregnant. So they're becoming DMB or they had a healthy issue before. So they're now making a lot of milk and then producers just gotta cull that cow. It might be, you know, these might be a consequence of reproduction. You know, those cows in the control group that they delayed to get pregnant and then they ended up being culled later in lactation. So they're not staying in the herd for too long. Thank you, Mateus. Phil I don't know if you have any comments or anything to add to this excellent summing up explaining, I mean, my objective for this webinar was to show to the audience. the basics why, explaining why methionine as a required nutrient, the functions of it go beyond milk production, milk composition, milk fat, milk protein. But it can help to ease these issues that are affecting the cows. As Mateus has you pointed out healthy cows, they are fine. Perhaps, you know, in a herd that you are not feeding methionine and all the cows are well, then you don't need methionine. But the tricky part is that what is that what you need? And to assure that by supplying this nutrient, you are going to at least put the cows at ease. The fact that normally a big proportion of the cows do have these health issues after calving. And what we know from what we learned today, methionine is not going to solve your issues, but it's going to at least give the cows the tools to fight against those issues. I hope that with this, the audience gets more knowledge about in terms of why many times they should consider feeding or not methionine to these cows. Phil I don't know if you have any comments or Mateus, any final comments? I would say that, you know, in my work as for herds that have a lot of problems, you know, during the transition period. It might be that the problem, it might be a major problem that they're having management or other nutrition problems. But you know, having a high level of methionine in there will be kind of insurance as well that may help those cows in terms of production and reproduction. So at least that's what we saw in this study here. Perfect. Thank you. You know, I think it's fascinating the data you know, recently there was a paper where we were showing control energy diets and high energy diets and we only considered the healthy cows and then there was not a big difference. So I think some of these strategies like, I think methionine or control energy, they are actually reducing the amount of cows that are getting sick or that is kind of having a greater chance of leaving the herd. And I think maybe that's more palpable to the farmer than maybe intake. Is there a cow, you know, maybe access to dry matter intake and other things, it's a little bit harder. But I think the number of cows leaving the herd by 60 days or at some point that they may consider, that's something that we have that impact of that nutrient, that there is no way where the cow is gonna take it from. The only source that she will have is from her own body. That's it. There's no way it's essential amino acid. That's what we are talking about. It's not gonna come from any place else besides the feed or she needs to pretty much eat herself. That's it. So I think that's the huge impact on all those things that we are talking about. One thing that I also mentioned, you know, for producers it would be more ideal if you can keep your older cows in the herd, just think about it takes about two lactation and a half, three lactation to pay off that cow. So the 4th, 5th and 6th lactation that cow is pay off. So every milk you're getting, I mean, just paying for nutrition. But the rass is your, it's the producer income, right? So you wanna try to have a lower culling rate for your 3rd and 4th lactation, maybe fifth lactation. And then keep keeping all your older cows in the herd. So you're gonna make more milk, you're gonna be more efficient, right? Absolutely. Mateus thank you very much, for sharing this with us. It is very obvious that there are needs for the cows that we need to do whatever we can to give the cows the tools to fight. These stresses that they are experiencing and today's cow that we are milking, the genetic potential is so high and they are geared to produce as much milk as they can. So we need to make sure that from the nutrition point of view are not only methionine but all the other nutrients we need to assure or at least try to make our efforts as much as we can to provide those nutrients, so they can express the genetic potential and keep those cows, Mateus as you were mentioning, try to keep those cows in the herds as much as we can. Thank you both of you for this webinar and hopefully, this is part of a series of webinars. There are more to come, hopefully, we will keep you guys coming up. Thank you very much. Good day. Thank you. Thank you.