Hello I am Daniel Luchini, head of Ruminant Research & Innovation of Adisseo. It is my pleasure to present the second webinar of a series of six to be presented throughout the year under the umbrella of a series of webinars called Amino Acid Balancing for Lifetime Performance. The title of this seminar is Ensure a good start of the lactation cycle and it is my pleasure to have with me, Dr. Erminio Trevisi and Dr. Johan Osorio. Dr. Trevisi obtained his Ph.D. at the Università Cattolica del Sacro Cuore in molecular biology in 1992. And since then, he dedicated his career to work in the area of nutrition and physiology of dairy cattle. Dr. Trevisi is a full professor and director of the Department of Animal Sciences, Food and Nutrition at the Faculty of Agriculture, Food and Environmental Sciences at the Università Cattolica del Sacro Cuore in Piacenza, Italy. Dr. Johan Osorio received his Ph.D. in Animal Sciences from the University of Illinois in 2014. His work is focused on the link between applying nutrition and molecular biology in transition dairy cows and their offering. His research interests are on improving animal health by advancing the understanding of nutrient gene interactions and the link between metabolism and the immune system during the transition period. Dr. Johan Osorio joined the Department of the Sciences Faculty at Virginia Tech in the spring of 2022. During this webinar we want to address the issues during the transition phase of the lactation cycle. And as an introduction to the topic, I just want to call your attention on the remarkable milk production increases in the top 10 high milk producing countries over the last 60 years. It's not only the amazing increase in milk production per cow per year in each country, but also to point out the linearity of milk production each in each of these countries. All of them grow at different pace, but all of them grew. And for example, for the last 60 years, each year in the US, cows produce 130kg more milk than the year before, which means that we don't have to go even as far as 60 years ago, cows that we are milking today are very much different than cows that we were milking even 10 or 15 years ago. So the underline question that we have today and we would hope to address is that this increase in milk production, health issues and culling by lactation stage are heading into the same direction as the milk production. Are they going in opposite directions? For example, again, going back to the US, according to the National Animal Health and Monitoring System, those trends are not going in the same direction. For example, cows culled by mastitis issues indeed, they actually look like they are an increasing pattern, the same lameness. Some other aspects of issues related to health culling systems are going into somewhat the right direction. For example, reproduction where less cows are culled by reproduction at least, cows are 150 days in milk. And perhaps this is because the wide use of time inseminations. But again, even in those trends that are going into the right directions, the pace of increase in milk production are at least at a similar level or even going into the opposite directions. So the underlying issues here is how many cows are removed from the herd. And again, from the same data set from the National Animal and Health Monitoring System. In the last several years, cows that are fewer than 50 days in milk are being culled and this is in 2014, which is the last data set that we have at hand, 20% of the cows have been culled. The cows that are being culled between 50 and 199 days in milk, 24% of the cows are being culled. So you can see here that number and I would call these two lines forced culling and this is my own interpretation of this data set is that 40% to 44% of the cows in any herd, at least in the US are being culled before 199 days. And the reason why I interpret this way is because I don't think anybody would want to kill a cow during the time that she produces the most milk in the lactation cycle. So here, there's the question how we can help resolve these issues or this undesired culling. Dr. Trevisi and his associate are being leaders not only in Europe, but in the worldwide scientific community, in trying to understand and looking into indexes to assess not only the gravity of these issues experienced by cows during transition, but also try to implement some management and feeding practices to resolve these issues. So here's a question for you Dr. Trevisi, how did you start in this area of research? Many thanks to invite me to speak about this topic that is really important. Not only in the last year but for a long time because, the transition period is the most important that should be considered when we have a herd. I started this, as the first topic in my research several years ago, try to understand what's happening in the animal that are present in a farm that has a low frequency of diseases. In comparison with another farm that have a higher frequency of diseases. The diet was more or less similar, we think that the most important things should be linked to the energy mobilization. But we observed different conditions. So the worst condition in the animal with a higher frequency of diseases, when we remove it from the group, the animal with clinical symptom was linked to a possible presence of liver lipidosis. And the second important point that the causes was not mainly related to and acute liver damage, but a general stressful condition around calving that interfere with the liver metabolism. So this was the problem that complained me for several years, me and my group and we try to put this information in the general overview of all the critical point observed and seened during the transition period. In this slide, I try to summarize the variation in the immune-competence, the inflammatory like condition that we can observe just immediately after calving, the marked negative energy balance (NEB) that we know very well, the oxidative stress, and the hypocalcemia. We do not have time to discuss the link between all these different critical point. But in our opinion, at the beginning it was very important to understand why the animal showed the inflammatory like condition just after calving. Dr. Trevisi just out of curiosity, when you went into these herds, you have a good looking herd health wise, one herd that experiences some issues. You went there, pull blood samples, look into the blood and you say, we have some indications here, some of the cows, are telling us something by these markers, for lack of a better word at this point, right? I mean, that's what you were looking for. And then you selected some of these markers that you are showing up in here, right Dr. Trevisi? But at the beginning, obviously, we considered a lower number of biomarker than now. But we consider the most important was NEFA, beta-hydroxybutyrate, glucose, urea, and transaminase from the liver. But our curiosity was that some of these parameter was not so different as expected when animals have a clinical problem or they show an over performance. So we say why we have to consider only NEFA for example, or beta-hydroxybutyrate, that are not able to signal early the problem in the animal. And the problem is that when we try to consider all the markers, our curiosity was that some of the positive acute phase protein are or seem to have an important role in the different response of the animal. At the beginning, we have not the possibility to measure haptoglobin, that is the most important positive acute phase protein, we measure only ceruloplasmin. But as you can see in this graph haptoglobin and ceruloplasmin show more or less the same behavior. The most important changes between the two biomarker is the reduction of ceruloplasmin after the peak is slower. And so we have a prolonged effect, it seems from this marker. But the problem is when we analyzed a lot of animal that all the animals show at this peak after calving and the peak in our opinion, showed different behaviors, different pattern of change. And in particular, our curiosity was that some animal prolonged this high level for more time, but the peak was not so different between the animals in general. So we think that the severity and the duration of the response could be key to the response of the animal after calving. So, we try to consider the factors that could affect the inflammatory response during the transition period. And in this slide, we can see that a lot of factors can induce an inflammation, a short, a long inflammation. This is different, it is difficult to say, but in these factors, we have a pathogen or a damage associated molecular pattern. So it means that we can have a virus bacteria but also a lot of other factors that are not considered so dangerous for the animal. For example, the activity of the animal should be enough to induce an inflammatory response like a dystocia, for example, or overcrowding of the animal. But these factors have been recognized several years after. Another important point is the nutrition, all the mistake in dry period and at the beginning of lactation are enough to induce an inflammatory response. But what links all these parameter together is that the response is probably induced by pro-inflammatory cytokine. So, all these factors are able to induce in different parts of the body, the release of pro-inflammatory cytokine, for example, overcrowding produce or induce the production of an increase of this mediator in the brain and then the animal start to show an inflammatory response. And so we can have some changes in the liver because in the liver, we have the acute phase reaction. So the liver is the organ where the body tries to modify the homeostasis, to recover homeostasis and produce the positive acute protein. And these are called positive because they increase, but at the same time, and this is probably the most important thing, we have the reduction of some common proteins that are produced by the liver, and this protein are called from some colleague that worked in this area before, negative acute phase protein because they reduce the level, sometime it's an absolute reduction. In other cases, it's a lower production than required for the animal in a specific physiological phase. So, when we have an induction of inflammation, we can have a look like that, we can have some protein or related parameter to the positive acute phase protein that increase. And some other that showed a more or less reduction. So, the liver is not damaged in the short period but is impaired. So our idea is why we don't use these different parameter to measure the severity of the inflammation. when we consider the activity of the liver it's important, not to see that the liver is inflammated in a specific moment, in our opinion it is important to have a good parameter to verify if the response is uncontrolled, is reiterated or the resolution, that is the last component of the inflammation, is delayed. To do that, we think that a negative acute phase protein, at least in the peripartum, is more sensitive than a positive acute phase protein. We try to put it all together, this information and we suggested some parameter, some summary of the parameter that could explain the phenomenon and could also give some information for the normal herd, not only for the research. And so we suggest that, as the first time, one indexer that we call a the Liver Activity Index (LAI). And this index is a combination of three different parameter, albumin, cholesterol and vitamin A. It's interesting to note that cholesterol and vitamin A are not protein, they are not directly produced by the liver, but are index of some protein that are produced by the liver and that are more difficult to measure in the plasma. And so cholesterol is an index of lipoprotein and vitamin A is an index of the carrier of vitamin A in the blood that is retinol binding protein. So at the beginning, but this was only for research proposal, we take three or four measurement in the first month of lactation, we transform the unit of standard division of this data, and then the final score gives us the possibility to rank the cow, from the cow in a good condition to a cow in a bad condition. But in this case, we have a some limitation, we have more time to do this analysis, particular retinol binding protein, Vitamin A is a more expensive analysis. And we have the possibility to work only in one herd. The result was very interesting, we related the rank, so the score of this LAI with the fertility. So the animal with higher level of a LAI have better fertility, it was a very interesting and so we decided to move in a more practical index that we call "liver functionality index". We reduced the number of bleeding, only two in the first months of lactation, about three to 30 day milk. We considered another parameter, more easy to determine that is total bilirubin, instead of vitamin A. And we use the good animal with a good LAI level as a standard of the good variation of this parameter in the first months of lactation. And so we propose this formula and the final score in this case is more easy to understand because it allow to classify the animal inside the herd, but also between herds. So we can have a good benchmark of the animal during the transition period. It's interesting that we have a very good correlation when we calculated LAI and LFI. So we think that this is a good system to classify animals around calving. This is just the changes in the transition period of the three parameter in a cow with high or low LFI. I have one quick question on this. So, when you suggest to measure these at three and 28 days in milk is so that you can look at how high those cows are getting into all the level of functionality of the liver. And how much is that change? Or is there is some lingering cow that don't have a good resolution of the inflammation by 28 days in milk? That's kind of what the rationale for those two time points it is ? Yes, thanks Johan. So I come back a little bit, when we calculated this index, we consider it the starting point. So the three day after milk for us is the best level to judge what's happening around calving. We tried to use also one day today, but we realized that several animals showed a higher variability of this data. And so it was not possible to judge the animal. And the 30 days in milk, we use a 20 days at the beginning. So four weeks because we can consider how the liver is able to produce more protein, more common protein that are needed to produce milk at the end because this serve to the mammary gland to have the nutrients to synthesize milk. So the formula include the shape of this cure. So we consider it a good shape the animal that we consider that in the first experiment with a good value, obviously, this level could be also evaluated. But I don't know if it's so important to change again the value. Now, the LFI can change from -5 or 6 to +5 or 6. This is the changes that occur in different herd in Italy and also in other countries, it is more or less the same. So Dr.Trevisi, following up on this, perhaps if I understand this correctly, then it's not so important, the intensity or the degree that those different biomarkers change. But the length of the time that they change, if you assess in your assessment, that those biomarkers come back to similar concentrations that they were at the beginning, that cow may have passed through an inflammation process, but they came back and that leaves back to whatever it needs to be. Inflammation is obviously a normal process that it will happen to every cow. It will happen no matter what we do. The important thing is not to assess how high that is, but to assure that those cows are back to normal within this period of time that you said 30 days. Is that a correct interpretation? Yes, but I had also another information that is very important, in a healthy cow the real inflammation lasted no more than one week. So this is important to allow the liver to produce then more Apolipoprotein and more carrier for vitamin. It is interesting that also the carrier of cortisol is produced by the liver. So, animals are not able to counteract all the problem that can occur during this longer period. Because in our opinion, this is also the factor of some challenges that occur before calving. So, we can see after calving probably an addition of a different strange situation that can occur before It's interesting also to consider the other parameter that usually we try to measure after calving when we have a high or low LFI index. So in this slide, I try to summarize all the data that we have measured, that always in all the experiment, but is confirmed in different experiments. So we can have a milk yield that increased in this animal. But sometimes the increase is not so evident in the first 30 days of milk. In our opinion, we can explain this because animals are able to support the lack of dry matter intake by the lipid mobilization. Indeed, these animal eat more and produce more or less 10% more. So, it is important to reduce the negative energy balance. This animal have a higher frequency of rumination, have a better immune response, several parameters. So when we calculate the negative energy balance, we observed a reduction of about 20% and also the 30% of nitrogen balance. So we can conclude that this animal reduce the energy of maintenance. But we all know that usually we consider this as a constant, but this is not true. Probably the animal with a low LFI spends more energy, more amino acid to support the immune system. So when we calculate the efficiency of this animal, we have an increase of efficiency in the first month of lactation. But I'm sure that this could be confirmed when we extend the controller to more weeks and the animals stay better, have an improved fertility. Also with LFI we have a good relationship with a high LFI and the fertility. So animal have lower problems, lower metabolic and other kind of diseases. And in this case, we have also a reduction of greenhouse gasses if we compare this to the amount of milk yield of milk produced production. So one point to make here, Dr.Trevisi this is a really interesting information that you can see, this is a prolonged or a really positive consequence of looking into the LFI that it goes beyond that period in terms of just simply in linking, which is nowadays I think the industry accept that fact that cows are healthy in transition, have better reproduction. But this is confirming that issue, which is very encouraging. Sorry for interrupting you Dr. Trevisi. No this is important. At the beginning when we published the paper with liver activity index. And we found that these association with the better reproduction. The idea that we try to transfer was that if the animal started in a good way in the first week of lactation, the resumption of the ovary is better. But at that time, we have not other evidence because we have not controlled the ovary before we try to do that with other colleague. And the beginning, it was a very difficult to support this idea. Now, fortunately, we have a more data for group that work mainly in reproduction and confirm that the quality of the ovocyte produced that when we have a better condition from energy point of view. So, negative energy balance are useful to have a better functionality of the ovary recovery of the new ovocyte and also these are more, the dominant one are more fertile. So this is a confirmation. So this confirm what we suppose that several years ago that when we are able to identify subject with subclinical problem, at 30 days, we can have more time to pay attention and care to solve possible problem in view of the new lactation, the new pregnancy. Also because if you observed that animal with a lower LFI have a greater risk of culling, also independently of the infertility because there are animal that can have other problem during the lactation. So, in another point we can use this index as an index of adaptation of the transition period. And so can have a meaning for the welfare of the animal. If the animal have a good transition period, stay better, it can suggest a better general condition of all the farm. In this case, it is possible to identify, I call it, the breeding problem because when for example, you observe a low level of lipoprotein in the animal, you have to think that several conditions are not good. And then we can compare animal within the farm and between farms. So we can decide which are the best condition of the animal. I say before that in our opinion, LFI is also the consequence that started, for the condition that started weeks before. And this graphic demonstrated that when we have a subsequent inflammatory challenge, the response of the animal can be modified. And in particularly for the cellular immune response, we can have a progressive reduction. So the hypothesis that we confirmed recently with some trial studying the animal from the dry off to the new lactation is that if we have a problem that start two weeks before, for example, with a not very good procedure for a dry off, not very good resolution of the mammary after the dry off, we can have a more frequently problem and then this animal can show in the new lactation, more problem and perhaps a lower LFI, sometimes we demonstrated that in other cases that we have not the statistical evidence, we can add the new information or new protocol to to detect the animal with problem in a earlier phase than 30 days milk. This was a frequent question that we had in the past. Indeed LFI work but 30 days milk, in some cases, we have already a lot of animal with problem in the first four weeks of lactation. So we try to to change the indicator and to produce an indicator in the first week of a lactation or before calving. So, before calving, I think we can have some suggestion but it is very difficult to foresee with a good precision what's happening at the beginning of a lactation. But we can have a good suggestion to have some alarm in the farm. I propose now for the first time, I call it the instant LFI of the herd, because in this case, we can with just one blood sample, take a sample of the animal in the farm in the two critical point. So a 3-7 days milk and about 30 days milk, we suggested to take at least 5 or 6 animals per group and then we can calculate an LFI. In this case, we have no variability, we have just one data but could be useful to have an idea in comparison with the time or the other farm. I just show what's happened when we collected in the same day, also in other time point that could be considered important in the farm. And it's interesting to see that animal with high instant LFI show at an intermediate level of albumin, higher level of the cholesterol and a lower level of bilirubin after calving. So this means that it is a very simple approach, but we can have an idea of the farm and probably if we need a more time to do other analysis to understand another important parameter. Very simple is the ratio albumin and globulin, in this case we observed that this ratio remained more stable than other parameter for a longer time. So we selected animal with a high intermediate or low ratio of these two indicator. And we observed that this calculation, the indicator calculated before dry off are able to classify the animal for milk yield, but also for response inflammation in the new lactation. So this could be another simple parameter that could be used in the farm, in the practical condition. Now, we are working with this new approach, we call it the post calving inflammatory response. We add some new parameter, we have for oxidative stress introduced the relative metabolite for inflammation lactoglobin. For the response for negative acute phase protein, we introduced paraxonase and albumin, we rank the animal according to the good level in our condition just in this short period, first week after lactation. And again, we observed a good forecast of the animal for the interior lactation. But we have a lower correlation with the LFI and this suggests that probably we lost some animals that could have a problem, probably at the end of the first week and in the second week of lactation. So, work in progress about this aspect. So when you have a high, let's say you have a high LFI in a farm, would you recommend to do more particular test on the blood or how would you go about it? You know, for a farmer? No, at the moment, the use of LFI in the farmer in the normal herd is not so popular. We have some colleague that work in the farm that use that. But they normally try to understand if the farm have a low LFI not a high LFI. I suggest also to use this simple tool to verify if the condition are good. So in this case, you can say, OK, I work in a good manner, I can try to improve something but probably the transition period is not so bad. This is to be an approach that I try to suggest, but it's difficult that farmer take a blood to do this test when the condition is good. But it is important LFI because you can compare this also in different situation. So, if the farmer can measure this, I don't know, 2 or 3 times in the year, you can see if the situation is stable or is not good. Or you have a seasonality for your LFI that might change during the year. You know? Yes, there could be in particular if, so LFI for example, can be modified by heat stress. And so you can have not very good tools to reduce the impact of the high temperature. And so you can use this to verify if the tool is OK, if the distribution of the ventilation is ok. For this aspect, there are also some other indicators. So it is not the most recommended in my opinion. But usually in the summer, we have a reduction of LFI. In Italy we have not the possibility to use this in the normal commercial farm. But we use it as a strategy to control when people suggest that something is wrong. So we added this information but in this case, in my opinion, it's not enough to analyze only LFI because if there are problems, probably you have to understand also if for example, you have a two intensive lionization or you have other problem in the farm. But it's important to verify if you have an exaggerated inflammation in the farm. It can be used also Dr. Trevisi, because of the consequences of not only the immediate consequences but the medium and long term consequences in the herd. If you go to a herd and look into this, you may know already that there are problems in there, right? But you now assess it correctly and then you can say you are going to have six months from now, you're not going to have this many cows pregnant or you're going to have these issues. Plan ahead, come back six months later, do it again and see, see you resolve the problem, you are ok. Now to go, I think it can be used as an assessment tool on how the farm is working out. Yes, I agree in my opinion at this moment, it is not so popular this tool but some biomarker in the blood could be, should be used to see if the situation is ok or if you have to improve something before, you know, the problem for fertility, for example or for some problem that could start in the first weeks of lactation, but then can be more evident weeks after, for example, lameness in this case we can detect if you have some subclinical condition. Obviously, we are not able to identify lameness with this indicator. But if you have a low albumin or low lipoprotein, I'm sure there are problems. And so, in this case, it is necessary to verify the good resolution of the uterus after calving, to verify the mammary gland performance, not only somatic cell count but also for example, the presence of some microbe and for the feed to, to see if the situation is correct or if there are some other problems. So now with the precision livestock farming, we can have also some other tool that we can combine. But in this case, we have an additional information, and sometimes we have noted this a PLFI tool. So we have to combine all the information to manage better before that big problem appear in the farm. So the prevention I think is important that we have to introduce this approach to prevent more specific problem. So, another important point in my opinion is that diet is one of the most important, one of the most potent factor that can induce or can resolve the inflammation, not obviously if we have some some pathogen, but because it controls the gastrointestinal tractor and can add the good nutrients to reach the homostasis in case we have this metabolic condition. So LFI could be also a tool used to verify if the changes in the diet that we have introduced are effective or not. So I show just this data that I think would be interesting. When we compare the animal with high LFI or low LFI I said before, we observed that animals eat more and produce more. But a very interesting thing is that it has higher level of total amino acid in the blood. So we can say, OK, this is related to the dry matter intake, but the difference started very early after calving. And another important point, I showed just a few amino acids but it's interesting the relationship between cholesterol, albumin and glycine, threonine, isoleucine and valine. So better liver functionality means also high gravability of essential amino acid and non-essential amino acid. One interpretation could be also that animal that have a good liver function, spare amino acid for the other activity of the cow in particular, obviously for milk production. And I think that there are several data that then confirm that when we can, when we are able to change, it is a factor, we have a better condition of the animal. All right, thank you Daniel for inviting us to this webinar. So today, I'm gonna go through some of the slides showing like as you said, some of the data that we have collected over the years related to this topic and transitionary cows. And let me just start by kind of echoing what Dr. Trevisi was saying regarding the problems that occur on cows in those days postpartum and this data from Denmark that clearly, it is a clear example of how much this is a problem and the incidence of disease in cows in the first few days. Postpartum, it is more pronounced for third lactation like older cows. So that's part of where all the importance on animal welfare, you know, morbidity in dairy cows or across dairy farms. It is coming from, because it's a big issue for the dairy industry and one of the reasons or one of the main reasons for this is that as soon as the cow calves, they're gonna start bruising cholesterol even before bruising cholesterol. And in those first few days, the cow has to adapt to produce this much amount of milk. So that's gonna be a draw, a lot of nutrients to go through the cow. And because of this reason coupled to this is the depression or the decreasing intake around those first few days around calving. So that can just exacerbate the problem. And because of this reason, the cow doesn't need as much as it needs to sustain that lactation when you always have a negative energy balance period commonly known during the first four weeks postpartum. So there is a tissue mobilization due to this negative energy balance. And as a consequence, there is always a decrease in body condition score in those cows during the first few weeks postpartum. What we have been doing here is trying to put together some of the transition cow studies that we have been working on in the past years. We just wanted to simplify here is that dry matter intake, as I mentioned before, there is a dip or a decrease in intake in those days around calving. And if this is even more meaningful when you start comparing that increase in dry matter intake, postpartum against the milk yield. So you can see there is a much more pronounced production or kilograms coming out of the cow than kilograms coming into the cow. So that's kind of 11 of the one of the roots of this whole situation that the cow is involved in the first few days postpartum. Now, if you put this in terms of energy, energy intake and energy requirements for sustained that milk production is even more pronounced. And I always reminisce on the conversation that I had with Dr. Ingvartsen in Denmark that he always talk about milk acceleration. So it's the amount of, or the change in milk production from one day to another in those cows in the first week postpartum, so the more pronounced that acceleration is the more there's a greater predisposition of those cows to, you know, become ill or have some sort of a condition that can lead them into a path that that will not allow them to have a smooth transition into lactation. Because of that, there is a lipid mobilization, that is fairly common in transition cows, we know that there's gonna be an increase in NEFAs and subsequently BHB because of the partial fatty acid oxidation in those transition cows. So these all of these can lead to some other issues like fatty liver and decrease in liver function that we always talk about. But also which we have been in the past year, we have been moving from just talking about negative energy balance, but there is also a problem with protein. So it's a negative protein balance or we always say negative metabolizable protein balance. And subsequently, we can go down the road into talking about negative amino acid balance. And in this case, this data from Maryland that when they were looking at cows before parturition and after parturition minus two weeks, five and 12 weeks relative to parturition. And in yellow is the body fat content of those cows and in red is the protein body content of those cows. And you can see that just as much as there is a decrease in, or a reduction in body fat, there's also a decrease in body protein in those cows regardless of the amount of crude protein in the diet. So just as much there is a negative energy balance, there is a negative protein, metabolizable protein and amino acids. And in the case of methionine there is a methyl donor, there is also some conversation into what it is a negative material donor balance. And just as an example here, this calculations made from Bell back in 2000, when he look at or he report as much as half a kilogram of negative metabolizable protein balance in the first week postpartum for those cows and same pattern as negative energy balance once the cows start eating more and get more adjusted to the lactation. That balance is gonna come to zero and then positive. The need for these metabolizable protein or amino acid is obviously protein synthesis, but also glucoses and other compounds. So, like S-adenosylmethionine (SAM) one of the primary body donor, methyl donors and glutathione and taurine there, antioxidants that I'm gonna talk more about glutathione later in this slide. So here is just to show you some of the diet profile for a close up diets that were used on those three experiments that were conducted at Illinois. We have a fairly 15% group protein in those close up diets. And we also had metabolizable supply, metabolizable protein balance that is a positive, it's fairly common. But the main point on this is that you can see that by supplying rumen protected methionine in those diets, we're able to reduce the lysine to methionine ratio to around 2.7-2.8 in the close up diet, but also in the lactation diet. So this is the lactation diet, obviously the crude protein was increased on those diets. However, obviously, postpartum as we just mentioned, there is a negative metabolizable protein balance across the board on those trials or in treatments regardless of that. Like I said before, we were able to decrease that lysine methionine ratio to around 2.7-2.8 postpartum and just a quick summary from those trials, we observed a very consistent increase in dry matter intake, postpartum around 22kg increase in dry matter intake. Same as milk yield, there was a fairly increase around 3kg of increase in milk production for those cows that were supplemented with rumen protected methionine. And subsequently energy corrected milk also was impacted, positively impacted by increasing the energy corrected milk in those cows that were supplemented with metionine, in the case of milk fat there was not much of an effect there. However, in the milk protein percentage there was an increase in milk protein percentage. Now because there was a greater increase in milk yield, we also had an increase in milk fat yield across the board in those experiments. And also it's pretty obvious that milk protein yield is gonna be increased when we supplemented those cows with methionine. Now, some of the benefits for methionine, Dr. Trevisi was talking about the importance of liver function. We see here that methionine can be a very important factor for several steps that can help the liver to be become more functional for the animal in those particular weeks when there is a lot of alterations and adaptations that the cow has to go through. So in this case, methioine can be utilized for phospho- lipid synthesis. That is if you will the cover of those VLDL very low density level proteins, there are molecules that the liver uses to pack fatty acids coming from the APO's (apolipoproteins) tissue and pack it into triglyceride. And then within the VLDL, you can export that out of the liver and can go to the mammary gland or other peripheral tissues. So, that's one mechanism that deliver, would prefer to use or will be beneficial for the animal to remove those uh fatty acids from within the liver. However, we know that based on a lot of data from Illinois, we know that the liver will prefer to esterify and just, you know, store that fat has triglyceride. We by providing some of this methionine we might be able to shift that preference in those first few weeks postpartum and then can have a big impact in the final outcome, health outcome for those cows. Another function for methionine is the glutathione can be served as a precursor for these antioxidants that I'm gonna talk a little more about those later on. And methionine can also be useful for apolipoprotein synthesis. That is some of these proteins that are required for the assembling of this VLDL. And also as an amino acid, it can be utilized in the protein synthesis of important proteins like albumin and there is a negative phase protein that Dr. Trevisi mentioned earlier on. Another important effect that we observed in those three experiments that were conducted at Illinois is that prepartum once we start feeding rumen protected methionine in the beginning of the close up by 10, 12 days, post- feeding, prepartum, we are seeing an increase in the concentration of glutathione in the liver across the board in all three experiments that caught my attention and I have been kind of almost being like a preacher of this but broken record talking about this. But it's because it's kind of a very interesting effect that we observed from this experiment. And we also run another trial looking at subclinical mastitis that I'm not gonna talk about for this presentation. But we also observe an increase in glutathione in the liver. So it's a pretty consistent factor for methionine to improve the synthesis of glutathione in the liver of dairy cows. Now, what I did taking back on that looking at that effect, what I did was to run a meta-analyzise if you can call it analysis of two studies. But I use data from my trial back in Illinois and also from Dr. Zhou experiment. We combined those and we break down cows, they have higher liver group down minus 10 days and low liver glutathione at the same, minus 10 days. And you can see there is some little overlap here because what I did was to divide cows within each study because there might be some differences on the way that people runs, the way that we run the kits or any variation between studies. So I prefer to do that within the study, anyway at the end, we observed that there was an increase in dry matter intake for those cows. They have a greater glutathione at minus 10 days, by the time that they start the lactation, they start to expressing a more behavioral eating, as much as 1.4kg per day. And what was also pretty particular was an increase in milk production, 4.5kg increase milk production on those cows. They have a greater preparable liver glutathione. So, in a way that we think about this effect is that by providing, in this case, methionine and full disclosure on this, some of these cows, they have a low glutathione prepartum. There were cows that were on rumen protected methionine treatment, so that means that there is some other factors that are on play in this whole glutathione synthesis and also storage because the glutathione can be synthesized but also being utilized by the liver or being exported out of the liver. So it's more about how do we understand how we can preserve that glutathione prepartum. So we can have all these very wonderful beneficial effects postpartum. Johan, can you say more about this aspect? So do you think that to restore a good level of gluathione requires more time? For example, of addition of precursor like methionine or some other product or do you think that it could be more related to a specific condition of a liver? And so perhaps it's not enough, the nutrients, to restore a good level. So I think, that's a good point, the idea is that because not all the cows, the majority of the cows that were on the rumen protected methionine treatment were on the high, but not all of them had a high. There were some of those from a protected methionine in cows were on the low glutathione concentration. This tells me there are other factors, could be that the level of the diet, like the energy and concentration in the diet might be a factor. It could be some other explanation like overcrowding, you know how much the cows have access to the feed. But there is something else going on and some of these might be related to this relationship between in the liver and body condition score that I'm gonna show you some of those data. Also the fact that for glutathione synthesis, if you look at the whole pathway that is required and both steps in the synthesis require ATP. So there might be other underlying factors that are needed to have like a optimal storage of glutathione prepartum. I think Dr. Trevisi brings a really good point, Johan, because even in these cows, as you pointed out, some of this cows were in the protected methionine diet, you took these samples 10 days before calving, right? If I'm correcting those cows have been fed only for 11 days. How about if you fed, methionine longer period, would those cows had a chance to actually increase the glutathione? It's clear the glutathione is very important here. So the goal is to increase gluathione, I guess that what I'm trying to assess is how we can increase that level of gluathione in these trials perhaps it's too early? I don't know how long it takes the liver to actually increase the glutathione content. So that's a good point. I don't think we have that kind of data to look at. OK, we feed in 10, 20 and 30 days rumen protected methionine and we're gonna see what's the level of glutathione in the liver? I haven't seen that kind of data. We typically do, start a close up minus 21 days and then at 10 days. So we're looking at 11 days supplementation, in the other experiment that we run for subclinical mastitis and the effects on rumen protected methionine, we also fed cows for 11 days to replicate this effect and we had an increasing liver glutathione. So at the very least, I can say that 10, 12 days is a good time to have an effect when you're supplementing rumen protected methionine However, the closer you get to calving, the more alterations are gonna occur. There are other hormonal things that are gonna start popping up on those days. I think there's this story, it might come up to be more complex than what we think. Well, Dr. Trevisi back to you. You are the expert on this, I mean, you've been working with this for perhaps longer than Johan was alive. Do you know how long it would take believer to accumulate glutathione? No, unfortunately, I have not the response because we usually don't take a biopsy of the liver because in Italy, it's more complicated to do that. So we work mainly with the blood and the other tools that we can help to understand the physiology of the liver. Now, we try with other colleague to have some information for echography from the liver. But we are too young to have a good information from practical point of view. But there are some data that demonstrate a good relationship between echography and this aspect. So I have no idea about the level of gluathione but I suspect, so it is just an hypothesis that this animal with a very low glutathione and also when they receive, for example, for some day methionine, could have a condition like a chronic stress that would be interesting to see if the animal have a contemporary higher level of a negative or positive acute phase protein or low level, very low level of negative acute phase protein. Just to understand if the liver are not in a good condition in this case, weeks before the calving. So obviously, at the calving time, these animal are an animal that respond very bad for all the challenge that they will receive. In addition to the challenge that probably they already have because probably we have also to define an animal in a not good condition where the level is too low. So if in this case, receiving a methionine has solve the problem from this data, I think that most of the animals that receive methionine solve the problem. Also when they started with a very low level of a glutathione, this is another way to read this data, but not all, so means that in this case probably they should start the methionine, to receive methionine two weeks before or more to have some other more challenging treatment. This is my opinion, but unfortunately, I have no data on glutathione in the liver. If you think about it, a lot of abroad data, they call it the sustain storage in the body or like amino acid storage in the body, that's how they describe the glutathione. Perhaps if there are inflammatory insults like heat stress, waves during the dry off that require the liver to kick in, start an inflammation, it's gonna draw some of those amino acids to utilize and to produce over some of these acute phase proteins. And then it's gonna draw out the amount of liver glutathione in the liver before calving. We still need to get more data on this. But at the very least, there's some interesting effects so far. In the same way, from these 2 studies, we observe an increase in albumin that we have been discussing this. So it's pretty consistent with a good liver function during the transition period and also a reduction in reactive oxygen metabolites, lower oxidative stress, greater liver function at the very least, this data is a good indicative at trying to solve this. This way or having an optimal way that we can store the liver glutathione prepartum might be a good way to go or could be an additional tool for dairy farmers to promote a barrel transition on those cows. Now, this is the effect that I was commenting before, if you look at the body condition score, there is no effects prepartum statistically speaking, that means you can see there is more greater body condition score, but it is not statistical. But postpartum you can see that there is a greater body condition score in those cows, they have higher glutathione prepartum, which is very interesting because it pairs really well with what we're seeing in the current experiment that we're running here at Virginia Tech. How to explain this, for me it's too soon, we're at this for the experiment that we just finished. We're collecting more data on how to explain this and I can show you the data from here from Virginia Tech. So, in this case, this is the study for Ana Flavia for her Ph.D. program and what she's doing or what she did was to take a liver biopsy at minus 10 days of the expecting calving date and follow those cows up to 30 days postpartum from minus 21 days prepartum. So, typically transitional cow studies, we follow all those cows, took blood samples, liver biopsies also at seven and 21 days. But what we did is once we finish, we look at the liver glutathione minus 10 days, and then based on that concentration, we separate cows in high and low liver glutathione groups. And what we're seeing here, saying a greater separation in this case for these cows, we're seeing a trend for a greater milk yield 2.9kg, so it's to that extent it's consistent with what we were seeing with the prior two studies and also uh an increase or a trend for an increase in milk protein concentration that if we start to pair out this, you're very likely we're gonna have a greater milk yield as well. So she just finished this experiment last week, this is kind of fresh off the oven, still warm data. And this is the data on body condition score, you can see that again, there was no effects, but you can see it start seeing a separation on those cows by the end of the close up period before the calving. And very consistent with the other two experiments, there is a greater body condition score postpartum on those cows that have higher glutathione 10 days before calving. To what extent does this means, I don't know if this means that we have more questions at this point than answers. But could be that whether the cows they know they're gonna get fatter, and a greater body condition score. So perhaps the body is trying to prepare for a greater lipid mobilization postpartum, to have enough antioxidants already, so they can counterbalance the amount of oxidative stress or the potential oxidative stress that they might have once they start to shedding all these body condition postpartum. At this point, we have a lot of metabolites, I'm sure we're gonna come back and give you a better picture of what's going on with this data set. But so far this, this is what we have from this trial. And what I mentioned before, glutathione synthesis requires ATP, so there is likely an additional factors, besides having a good amount of methionine because methionine is a methyl donor for SAM but also down the methionine cycle, it serves up as a precursor for cystine and also for glutathione synthesis. And an interesting factor for this, glutathione is a tripeptide. So, some of prior research from Illinois suggests that some of this system might be utilized by the mammary gland in when there is some interference between amino acids as they are transported into the mammary gland because there's some overlapping in the transporters in the mammary gland. Maybe this glutathione that is absorbed by the mammary gland is degraded and is releasing these free amino acids that can supply or support some of that milk protein synthesis. So that's kind of where we're going with this line of research at this point. But for sure we're gonna keep you updated on this. I think that anything that we found in terms of what we're talking about, that could be some differences in energy level in the diet or some management that can help us to not just synthesize but also maintain that accumulation of glutathione that might be very useful for transition cows. Thank you. Thank you very much Johan for your presentation and Dr. Trevisi for your introduction and highlighting the importance of biomarkers. Looks like here we are unearthing perhaps one that we can look at and Johan I think your task could be how to make the correlation between some not so invasive process to get the piece of the liver to understand perhaps how the cows are going to react after postpartum. But I think it's clear that the importance of nutrition throughout this transition period, as Dr. Trevisi pointed out, it's also clear that we have some biomarkers that we can use to assess that, which is great. It's really nice to see that there are a lot of things that are being done by different research groups to understand, to resolve this issue, which is a real issue to dairy producers in today's market. Of course methionine is one of those solutions that we can apply for now. Hopefully, we are going to come up with either even better solutions to the producers. Thank you both of you for taking the time to participate in this webinar. Thank you very much.