Hello everyone, and welcome to the Real Science Lecture Series. My name is Scott Sorrell, Director of Marketing at Balchem Animal Nutrition and Health. Today, we welcome Dr. Kelly Nichols from U C, Davis to discuss how protein nutrition can aid in the sustainable dairy industry. Dr. Nichols earned her BS and MS from the University of Guelph and her PhD from Wageningen University in the Netherlands. With a passion for advancing dairy nutrition, she's since worked with leading nutrition companies and universities in research and teaching roles, joining the University of California, Davis in March of 2024. The Nichols lab studies how dairy cattle digest and metabolize nutrients to better understand dietary protein and energy interactions, mammary gland function, and nutrient conversion in the milk. The research includes tissue level metabolite flux, energy and nitrogen balance, digestibility, and milk production in response to nutritional interventions. With strong collaborations with global animal nutrition companies and top-tier university research groups, the Nichols lab offers students exciting opportunities for international experience through research exchanges and industry partnerships. Dr. Nichols, welcome back to the Real Science Lecture Series. The floor is now yours. Great, thanks Scott for the introduction and the invitation. Welcome everyone who's tuning in. We're going to talk this morning about what I've called exploring flexibilities in protein nutrition for a sustainable dairy industry. Here we have, of course, a very flexible cow able to ride this bicycle. I'll explain what I mean by flexibilities in protein nutrition throughout this presentation. Before I get started, I just want to acknowledge the groups, many of those that Scott already mentioned, that I've worked with, had the privilege of working with over the last 10 years or so. The University of Guelph, Trouw Nutrition, where I was working before joining UC Davis, and Wageningen University in the Netherlands. Of course, my team here at UC Davis, this is sort of a preemptive acknowledgement. A lot of the topics I'm going to mention today and talk about, those will be threads that my new team here will be pulling over the coming years. A shout out to my new group here as well at UC, Davis. To get started, kind of set the scene, why focus on protein nutrition? The ruminants are incredible. They have a lot of ability to be flexible in terms of the feedstuffs they consume, how they're able to break those down, work with them post-absorptively to make milk or put on body protein. Why do we care about how protein nutrition fits into this picture? We feed protein as much as possible. We want to come out as milk protein, but there will always be a certain amount of loss through feces and urine contributing to manure and nitrogen. This is not news, I'm sure, to any of the listeners. There are substantial environmental concerns associated with nitrogen excreted from manure of our ruminants. We have ammonia emission, nitrous oxide being a very potent greenhouse gas. The implications for water bodies in terms of nitrogen runoff and of particular importance and growing concern in some regions would be nitrate pollution in drinking water. I focus on feed protein to sort of look at all of these balances here because imported feed on farm is usually the largest contributor to whole farm nitrogen balance. This is a good starting point if you're considering how can we mitigate some of this loss in terms of this whole farm system of nitrogen balance. Often it is actually more than any imported fertilizer nitrogen. That feed is contributing the most to the starting point of nitrogen balance. Depending on where you're listening from, I suspect many of you are somewhat familiar in one way or the other with what has been kind of dubbed the nitrogen crisis in Europe. I spent the last 10 years living in the Netherlands. I'm not going to go through the details of this. That has been growing for decades and decades, but you've probably seen headlines such as this or photos in the media, farmers' protests happening in the Netherlands and Ireland and some of the other European countries. The point of sort of framing this up is what this represents is basically the impact of very intense pressure to fix what became an urgent environmental issue. This nitrogen deposition onto the land in these very intensely populated areas with animal production systems. You end up with farmers feeling betrayed by the government and basically alienated by a general public that has, as many of you I'm sure acknowledge, lost sort of that connection with where their food comes from. You end up with a lot of intensive policy and a lot of just general social unrest. I've titled this not just a European problem because you're starting to see sort of growing intensity around this nitrogen issue in terms of pollution here in the U.S. These are two figures that I've pulled. The one on the right talks about the probability that groundwater systems in the U.S. are violating nitrate regulations, where the blue are areas with low probability of this, the orange are areas that have a high probability of violation, and the red dots are areas that have already been observed to violate nitrate groundwater regulations in those areas. The figure to the left, and just to note, this is relatively recent data. The figure on the left here highlights areas that I like how this is framed as opportunities to fix nitrogen pollution. These are areas across the U.S. that have the greatest cropland nitrogen surplus. One thing for all of our dairy-inclined listeners, if you're looking at these figures, you can probably see that a lot of those red dots or these red regions associate closely with areas where we also have a lot of dairy production systems. Along the West Coast and along this region, which I'm not from the U.S., but you guys call this the Midwest, I'm pretty sure. There's a lot of cows up here and, of course, here in California. You start to see some of these headlines, for example, this one from the Natural Resources Defense Council, starting to mimic a little bit of what we've seen in Europe over the years. All of this is to say we recognize that while ruminants do deliver a lot of efficiencies with respect to transferring human inedible feedstuffs into human edible food, human edible protein, and other nutrients, there's also a lot of inefficiencies associated with that system. This is something that we have to manage. As I said, while I find that particularly the figure on the left in the previous slide to be quite optimistic in terms of framing it as opportunities to improve these potential problem areas, I do think that there's going to be a growing emphasis on things like nitrogen discharge regulations and nitrogen source air pollution. We talk a lot about methane, but I think the nitrogen discussion is only going to grow and become more intense, particularly here in the U.S. We're left with this question of how to deal then with manure nitrogen surplus if we start to limit what can be deposited onto the land. One of the easiest ways to do that is to think about how do we reduce that manure nitrogen content. The other side of this, of course, lies with issues around resource availability, which is something that is a real concern, a growing concern these days in terms of, you can think about that in terms of cost of our protein-rich ingredients being really variable over time. Also the public perception of what we're feeding our production animals, that sort of longstanding feed versus food debate, GMOs, these types of discussions that can influence ultimately all the way back down to the farm what we're thinking about feeding our animals. How I place sort of this idea of understanding metabolic flexibility in our ruminants, that lies at the heart of basically being able to determine strategies to improve protein efficiency that align with the particular goals and requirements of individual producers. Farm A might have completely different pressures in terms of sustainability targets, motivations from their supply chain of targets that they need to hit, limitations in terms of the ingredients that they have available. That could be completely different than farm B in a different state or a different country or even just a couple of miles down the road. Understanding kind of the scope of the flexibility that we have to work in can allow for solutions for a variety of groups of dairy cows or beef animals that are being managed. What do I mean more specifically by flexibilities? Like I said, we have our feed protein going in, milk protein going out in our dairy cows, and we have manure nitrogen. Within the animal itself, I put this into three buckets of areas. We have the rumen, of course, we have the post-rumen digestive tract, and then we have the post-absorptive system. Within all three of these areas, there is flexibility within. Flexibility, for an example, would be the ability to derive the same metabolites out of different resources. We have flexibility within, but we also have the option for these compartments to interact. An example being urea recycling, where we have the post-absorptive system feeding back into the rumen and the potential for that cycle to continue, which is something we'll talk a little bit more about later in the presentation. Today I'm going to touch on three areas that I think illustrate this idea of metabolic flexibility really well. That's the impact of energy source on protein metabolism, the capacity to think about rumen nitrogen balance in a little bit of a different way, and also mammary gland amino acid metabolism, where we've shown there's a lot of flexibility involved. First, we're going to talk about the impact of energy source. To describe this, this is a figure from a colleague of mine, Jean-Baptiste Daniel, at Trouw Nutrition during his PhD. This is a meta-analysis looking at the impact of incremental increases in protein supply. A unit increase in metabolizable protein supply here on the x- axis, and then incremental increase in protein yield on the y- axis. The lines that you see coming across here represent, again, incremental increases in energy supply in these diets. As you expect, across all of those lines, we get this classic diminishing returns curve as we increase metabolizable protein supply relative to what goes out in milk protein yield. What you see across, as we start to increase energy supply to these animals, we see an increase in milk protein yield at any unit of metabolizable protein supply here. Why is that? Our protein synthesis is an energy-expensive process. By providing more energy, we can drive that production a little bit more. Amino acids themselves are glucogenic, so they have the potential to be used for glucose to be used as energy. There can be a bit of a sparing effect if you provide more non-amino acid source energy. Of course, we have the fact that increasing energy supply regulates these endocrine signaling responses that ultimately regulate protein synthesis itself. This is clear. We know this well. It's been characterized well over many years. A big question that hasn't really been sort of pulled apart is what about the source of that energy? We talk about energy, but what does it matter where that energy is coming from? What I mean by that, energy source, two sort of buckets that we can think about, that would be glucogenic energy. In practice, we're looking there at things like starch-rich feedstuffs. We have some of that corn silage, corn grain in concentrate feeds, wheat in concentrate feeds. Under an experimental setting, we can test this by things like post-ruminally infusing glucose or ruminal infusions of propionate. You see those types of models in the literature. We have lipogenic energy. Practically, this looks like our rumen in our fat supplements, feeding more fiber-rich diets that are going to generate more acetate, for example, in the rumen. In our experimental models, we see examples of post-ruminal infusion of fat or fatty acids. To describe, sort of set the scene in terms of this energy source question, this is some data from 10 years ago or so now that I always like to use to start this off. This is some data out of INRA, France, where they really did a great job looking at this question of the difference in energy source. The figure here, nitrogen intake on the x- axis and milk nitrogen yield on the y- axis, the symbols here represent milk nitrogen efficiency. That transfer of nitrogen intake into milk nitrogen, where the square symbols are a low crude protein diet, 12% crude protein, and the triangles are a higher 16.5% crude protein diet. The dark filled-in squares or triangles were a starch-based diet, so a corn silage-based diet, whereas the open symbols were a more fiber-based diet. You can see very clearly here, I've calculated the milk nitrogen efficiency for these clusters. The lipogenic diet had a lower milk nitrogen efficiency than the glucogenic diet across those levels of nitrogen intake. That kind of sets the scene here. We dove into this in more detail during my time at Bath and Engel, where we, this is data from a study where we used an abomasal infusion model here, where we looked at a low metabolizable protein situation and a high metabolizable protein situation that was done through post-ruminal infusion of essential amino acids. We infused either saline, glucose, or palm oil as a lipogenic energy source. What we saw was very similar to the previous slide. Milk nitrogen efficiency increased with that glucose infusion, independent of protein level. When we look at the impact of fat on milk nitrogen efficiency, just to describe the second study here that we did, this was a feeding trial. We used a hydropalm, so a rumen inert fat source, where again, we had a low protein situation and a high protein situation, and then a factorial with fat inclusion in there as well. What you see is that milk nitrogen efficiency, if we look across both of these studies, it tended to increase particularly at low protein levels when we included fat. With this infusion, you see the numerical increase here in this infusion study with the infusion of palm oil. You see this trend or an interaction where supplementing hydropalm increased milk nitrogen efficiency at the low protein level. There has also been quite a bit of work trying to understand what goes on post-absorptively to sort of generate this shift in amino acid partitioning and ultimately efficiency of nitrogen utilization. What we've seen and some of my colleagues is when we feed more glucogenic energy or infuse more glucogenic energy, what you see in the blood, so this is the arterial concentrations, you see that increase in glucose. We see a very distinct decrease in our branched-chain amino acids. Usually, we see also a distinct decrease in lipogenic precursors like acetate and BHB. What is kind of happening in response to that? Glucose is stimulating this insulin cascade, which while it does stimulate some turnover in muscle, we're getting a pretty big response in extra-mammary lipogenesis. That is turning on that system. What we're seeing is actually, those lipogenic precursors, acetate and BHB, are being partitioned towards that extra-mammary lipogenesis, so adipose tissue. Those branched-chain amino acids, specifically compared to the other groups of essential amino acids, they can serve as fatty acid precursors. We see them also being directed towards adipose. On the mammary gland side, what we see then in response to this sort of shift in partitioning of amino acids of these fatty acid precursors is we see an increase in mammary blood flow quite distinctly in these studies to maintain that output of milk protein and milk fat. Mammary blood flow increases to pull more of those milk precursors into the udder in response to this shift in partitioning that's happening post-absorptively. We'll talk a little bit more later about how mammary blood flow plays into this kind of concept of flexibility. We've done the same analysis for studies where we compare with lipogenic energy. What we see is that it doesn't stimulate this cascade of post-absorptive amino acid repartitioning. We don't get this big shift. We don't get this big mammary gland blood flow response when we feed more lipogenic energy versus glucogenic. Just to summarize on this part of metabolic flexibility, our glucogenic energy pretty consistently stimulates milk protein yield and increases nitrogen efficiency. It also stimulates this insulin cascade that can have consequences for partitioning of certain amino acids and energy metabolites. We do see this shift in mammary gland metabolism in response. On the other hand, lipogenic energy looks like it can have some positive responses in milk nitrogen efficiency, particularly at low protein levels. I have to underscore the fact that we really haven't, there is not a very robust body of literature that really looks at feeding fat and the impact on nitrogen metabolism. I would still consider this a fairly big gap in our knowledge base. There's no insulin response, at least in terms of how it impacts amino acid repartitioning. A take-home there would be, what is the consideration of this for dietary ingredient inclusion, especially when we think about more novel ingredients or our byproducts, where we don't always have a great idea of the nutrient composition or the variability that might exist there. A question could be, does this offer more glucogenic or lipogenic energy? What could be the downstream implications for protein metabolism? Like I said, I think on the lipogenic side, this is a pretty big gap still in our knowledge. Let's talk about the second sort of bucket of flexibility that I wanted to share with you. This is around rumen protein balance. By rumen protein balance, depending on what sort of formulation system you're working within, this might be defined slightly different. In general, across the systems, when we talk about rumen protein balance, what we're describing is the difference between the maximum possible microbial protein synthesis that could be derived from the nitrogen available in the rumen relative to the energy supply available in the rumen. You get where a zero is sort of the perfect synergy between that rumen available nitrogen and energy. A negative would actually mean there's a deficiency in nitrogen, whereas a positive value would mean that there's excess of nitrogen. This is some data, again, out of INRA, looking at an incremental increase in rumen protein balance and how that impacts urinary nitrogen excretion. What we see here is the slope of that curve suggesting that around 80% of any additional rumen available nitrogen is basically being excreted in urine. Everything above that zero line you can think about as excess. Something that this makes me think about is how many of our diet formulation parameters focus a lot on this idea of ruminal synchrony between protein and energy supply. A limitation that I see to that is that this assumes that nitrogen metabolism in the animal, nitrogen recycling, and the utilization kind of between those dynamics are constant. In many cases, this ignores the potentially substantial contribution from endogenous nitrogen sources. This figure here on the right represents the proportion of gastrointestinal urea nitrogen entry. That's urea coming from the liver relative to the digestible nitrogen intake of the animal. What you can see, something that jumps out at me from this data is, one, the variability in that proportion, particularly in sheep and non-lactating cattle, and how much less variability there seems to be in our lactating cattle. Part of that has to do with the diversity of diets that are fed to these more growing animals versus our lactating cattle and just the number of experiments in this data set. The second thing that jumps out at me is there's far fewer studies looking at this sort of dynamics of urea recycling in lactating cattle. In practice, what this sort of suggests to me is that there might be some potential here to rethink how we consider rumen protein balance, and it might be leading to these excessive safety margins, you could say, for rumen nitrogen supply if we don't have a good handle on some of these dynamics around nitrogen recycling and the potential utilization from this source. This is some data out of Cornell, a relatively recent study published. They looked at a few different diets, but I just focus here on two of their treatments where they have a base diet and then a base diet with the addition of urea. This rumen nitrogen requirement is based on some values from CNCPS version 7 that I've flipped around to represent rumen nitrogen requirement. They've added in urea here to basically make this 100% of the rumen nitrogen requirement being met, whereas the diet without urea is lacking a little bit in predicted rumen nitrogen requirement. You see the impact on the crude protein content of these diets where the urea inclusion increases the crude protein content slightly. If we look at these two situations where you have a base diet and a base diet where you've just added some urea, the impact on dry matter intake, there was a tendency for slightly higher dry matter intake with that urea. We'll talk about why that possibly is a little bit later. No difference in energy-corrected milk yield or milk protein content or protein yield. With this increase in rumen nitrogen balance, as one would expect, we did see it. They saw a significant increase in milk urea content. Based on calculated milk nitrogen efficiency, this is my own calculation based on their data, a lower milk nitrogen efficiency, as you would expect, alongside milk urea nitrogen. We have an increase in plasma urea nitrogen. Based on CNCPS, there was a shared predicted urine nitrogen output being, of course, higher with that urea. A non-significant but numerical increase in NDF digestibility when we have that extra urea likely explains that slight increase in dry matter intake. If you look across sort of this response to just simply adding urea to meet some kind of target in terms of rumen nitrogen requirement, a takeaway that I have from this is that balancing for estimated rumen nitrogen requirements, in this case by adding urea, a rapidly degradable rumen nitrogen source, likely increased nitrogen excretion. I say likely because this is predicted urine nitrogen, but we see that back in milk urea nitrogen and plasma urea nitrogen. It's likely that's happening with no positive impact on energy-corrected milk yield and milk protein output. If we're in a situation where we're trying to reduce nitrogen excretion, do we really need to be supplementing this little bit of extra rumen degradable protein to meet an estimated rumen nitrogen target? How much then, on the other hand, can we actually rely on endogenous urea to compensate for a slightly lower or providing a slightly lower rumen nitrogen balance that we are kind of in control of? How much can we let the animal regulate rumen nitrogen balance versus what we try to manipulate with the diet? What we know from mostly studies with non-lactating animals and a few studies with lactating animals is that the proportion of hepatic urea output that recycles back to the gastrointestinal tract increases as dietary crude protein content decreases. Something that is a little bit less clear, particularly in more intensive dairy cow diets, is the impact of the source of rumen undegradable protein versus rumen degradable protein and the balance there and how that impacts this relative recycling of urea as we start to decrease dietary crude protein content. In reality, we're not really so much worried about the optimal crude protein content anymore in practice. We're worried more about what is the composition of that protein and how is it serving the rumen, the post-rumen, the mammary gland. These are the things that we care actually more about. What are the underlying mechanisms that actually might be inhibiting greater return of urea to the gastrointestinal tract? Particularly, like I said, in sort of more intensive dairy production diets, I think there's a lot of open questions around why they don't necessarily capture as much urea as they potentially could based on what we see in our non-lactating animals and those types of diets. There's a lot of knowledge gaps there. This is really an area that my team here at the UC Davis is going to be looking at over the coming years. Some comments to summarize on this idea of reconsidering rumen protein balance: cattle can use endogenous nitrogen to support short-term periods of ruminal nitrogen insufficiency. It's possible that depending on where we're operating at, we might be able to reduce some of that, so to say, safety margin that we add in in terms of rumen available nitrogen. This can be a route to reducing dietary crude protein content and nitrogen excretion. If you're in a situation where you do have pressure on reducing that manure nitrogen content, this can be an area to consider. As I mentioned, I really think this is an area where we need more understanding of this mechanism in lactating dairy cattle to really understand if this is something we can rely on going forward to improve nitrogen efficiency. For our last topic in this metabolic flexibility discussion, we're going to move to the mammary gland and talk about amino acid metabolism. I'm sure most of you are all aware that when we think about the digestible amino acid profile, of course, the most substantial portion of that is our microbial crude protein. With this, we generally assume that that is a fixed amino acid profile. We can argue whether that's valid or not, but that's what we're currently assuming. We also have, of course, rumen undegradable protein. This is the fraction that we can work with in terms of what can be changed based on the ingredients that we're feeding. The goal to optimize nitrogen efficiency through this means would be to aim for rumen undegradable protein amino acid profile that's going to complement that microbial amino acid flow in some way. The question has become, what is then that complementary profile? There's been a lot of work looking at this. This table, I'm going to build it out. These are studies where different profiles of amino acids have either been infused post-ruminally or fed in a rumen protected form. This column has the dose of the amino acids in grams per day. This column represents the marginal efficiency. By marginal efficiency, this is the efficiency of use of every extra unit of supplemented protein, so the efficiency of every extra unit of protein into milk protein yield. That's what this number represents. In this first study from 2010, you see the comparison between infusing a complete essential amino acid profile where they're used with a marginal efficiency of 0.31 versus the non-essentials, which are basically not used efficiently in any way. When we add them together, you see that sort of moderate marginal efficiency, where basically what we conclude from here is that supplementing extra non-essential amino acids in terms of milk protein output are not being used efficiently. This is some work that I did in the Netherlands where we infused different profiles of essential amino acids. We had what was sort of isometabolizable protein doses, so the same amount of amino acids in all of these mixtures just composed of different groups of essential amino acids. The complete profile, what I've called group one amino acids, so that's histidine, methionine, phenylalanine, and tryptophan, plus isoleucine, leucine, and valine, so what you'd know as the branch chain amino acids, then those group ones with arginine, lysine, and threonine included, and then just the branch chain amino acids on their own. You can see here, again, a very distinct difference in marginal efficiency. This complete profile compares very well with the complete profile here from Hélène Lapierre and colleagues. You see that start to decrease essentially as the profile becomes further away from casein, which in both of these experiments, these complete essential amino acid profiles were formulated relative to their profile in casein. This is some more recent data where in this case, this was a feeding study where we were feeding rumen protected histidine, methionine, and lysine and comparing that with the same amount of digestible amino acids coming from rumen protected soybean meal and rapeseed meal. In this case, you see a significant difference in marginal efficiency. One, because the dose, particularly here with just histidine, methionine, and lysine, is relatively low. As we start to bring in these plant protein sources, we're bringing one non-essential amino acids into the mix. You also have to consider the fact that the amino acid profile in these sources doesn't match that of casein, whereas the formulation of this histidine, methionine, and lysine combination was also done in the profile of those amino acids in casein. You see this pretty stark difference in efficiency of use. The takeaway here is that marginal efficiency increases as the digestible amino acid profile more closely resembles casein. Every unit of extra supplement protein that you give, the efficiency of that use will be higher as that profile more closely resembles casein. There is the potential for even greater efficiency if we prioritize the essential amino acids within that profile. We are going to look a little more closely at the infusion study I mentioned in the previous slide where we infused these different groups of essential amino acids. This is the milk protein yield response from that study where we saw that these incomplete infusions where we did have the group ones present, so histidine, methionine, phenylalanine, and tryptophan, resulted in the same level of milk protein yield as our complete infusion profile. If those group ones are present, there is some flexibility apparently around how well the mammary gland can use these other essential amino acids to produce milk protein. What are the sources then of this flexibility? How can this happen on the mammary gland level? In the mammary gland, we have blood flow moving across, and we talk about amino acid uptake from blood. That represents the irreversible loss of amino acids into the mammary gland. The vast majority, around 90% of those amino acids, end up being used for milk protein. This is a number that's relatively consistent across a variety of dietary conditions. I always say we talk about ruminants, dairy cows being relatively nitrogen inefficient, depending on how you look at it. The mammary gland itself in isolation is actually extremely nitrogen efficient. Some of those are going to be used to build back up constituent protein, and some of those amino acids that come in are going to be transformed. They can be used for oxidation to contribute to lactose and fatty acid synthesis. Then they get transformed, and in that way, actually contribute back to milk protein. This is sort of the system that we are working with when we talk about mammary gland amino acid metabolism. That blood flow is kind of the first step as it influences then our estimation of uptake. What we see are fairly strong responses in blood flow to changes in essential amino acid profiles. Things like amino acid deficiency can increase mammary blood flow. This is some data from a study where we removed histidine out of a complete essential amino acid infused profile. What we see is this very distinct increase in mammary plasma flow to try to account or try to adjust for this removal of what is a very important group one essential amino acid for milk protein synthesis. Similarly, we can talk about this in terms of great imbalances of amino acids that are supplied. This is, again, that data or that experiment that I did with different amino acid profiles being infused where when we infuse just the branched-chain amino acids in that very high load, we see an increase in mammary plasma flow. My guess is to try to accommodate or try to adjust and pull in more of those other essential amino acids that would be required to maintain milk protein synthesis, not just the branched chains that were abundantly available in this case. When we look within the mammary cell, there are several ways that we can consider intramammary flexibility. I'm going to use an example again from this study with these different amino acid profiles where when we looked at what we call the uptake to output ratio of these different groups. Uptake to output ratio represents the amount of an amino acid that got taken up into the mammary gland relative to the proportion of that amino acid that ended up out in milk protein. These group one amino acids, methionine, histidine, phenylalanine, and tryptophan, they typically are taken up in a one-to-one ratio. For every one unit of methionine that comes into the mammary gland, we find one unit of methionine back in milk protein. That is what we saw consistently in this experiment. These other groups of essential amino acids fall into a category where we typically see them being taken up in excess relative to what comes back out in milk protein yield. That's because they contribute to, among other things, the synthesis of our non-essential amino acids. Some of that uptake goes directly to milk protein as itself, and some of those amino acids get transformed into non-essential amino acids and then contribute into casein synthesis. In this experiment, when we looked at the uptake to output of the essential amino acids that were being infused, what we saw was this great excess of that ratio for the groups that were being provided. To put it the opposite, the groups that were not included in the infusion. In this case, when we infused the group ones with isoleucine, leucine, and valine, what we saw is that those other essentials that were not included, arginine, lysine, and threonine, went more directly into milk protein as themselves. Their uptake to output ratio was relatively closer to one. The uptake to output ratio of isoleucine, leucine, and valine was much greater. Relatively more of those were kind of taking that load of contributing to non-essential amino acid synthesis for maintaining that same level of milk protein output. We saw the opposite when we infused the group ones with arginine, lysine, and threonine, where the branched-chain amino acids were contributing, you can think of it, more directly to milk protein. Arginine, lysine, and threonine were then contributing more to non-essential amino acids to maintain that same level of milk protein output. To summarize around mammary gland amino acid metabolism and that flexibility, the profile of digestible amino acids impacts the efficiency of use of any supplemental protein. Practically, we should be looking for ingredient profiles that could potentially enrich digestible protein with essential amino acids that match the profile of casein. This intramammary compensation for nitrogen and carbon between our non-group one essential amino acids, this is still a really interesting question. We've started to look at it in this way, I would say across the amino acid metabolism research that's being done. A big question I still have is what is the scope of this flexibility? How does that potentially change or persist across lactation, for example? We can start to think about this as considering the profile of these amino acid groups, not necessarily individuals. If we understand that there's flexibility within the group, can we think about them instead as digestible amino acid targets for the group versus the individual? Might that give us more space, more flexibility when supplementing rumen undegradable protein? Some take-home messages to summarize across all three of these areas of flexibility that I've discussed this morning: energy source matters with respect to improving protein efficiency. In this case, we talked about the difference between glucogenic and lipogenic energy. They elicit different effects on post-absorptive amino acid metabolism. Both have the potential to improve nitrogen efficiency, but specifically around lipogenic energy, so fat supplementation, there are definitely some open questions around the interaction with improving nitrogen efficiency. We can think about minimizing rumen degradable protein balance as a route to achieve moderate increases in milk protein efficiency and possibly substantial decreases in urinary nitrogen excretion if this is something that is a target in your situation. To a degree, based on what we understand now, recycling of endogenous urea may compensate for periods of low rumen nitrogen supply, but its actual utility at this point, the dynamics are not understood well enough, particularly in lactating dairy cattle, to really rely on it at this point. The mammary gland is very flexible in how it uses amino acids. Considering these digestible amino acid groups as a focus alongside balancing for individual amino acids might open up some space for improving nitrogen efficiency based on the ingredients that you have available. With that, I'll thank you for your attention, and I'll turn it back over to you, Scott. I'm happy to take some questions. All right. Thank you, Dr. Nichols. Before we get started with the Q&A portion, we'd like to share a brief video, and then we'll be right back to answer the questions you've submitted during today's presentation. Balchem has invested decades perfecting the art of nutrient encapsulation. Now, new AminoShure- XM is setting a new standard, delivering 35% more metabolizable lysine than leading competitors. With AminoShure -XM, you can provide your cows with a more consistent and higher quality source of metabolizable lysine, leading to improved and more dependable results. A recent meta-analysis found that feeding rumen protected lysine led to an increase in milk fat percentage and 4.4 more pounds of energy-corrected milk. Feed new AminoShure- XM to your cows and have confidence that you're using the most consistent, reliable, and cost-effective source of lysine on the market today. Learn more at balchem.com/xl. As a reminder, you can still submit questions through the Q&A tab at the top of your screen. Dr. Nichols, your first question I'm going to give to Dr. Zimmerman. Give him the honor. He's asking, first of all, he says, excellent presentation, Dr. Nichols. Can milk urea nitrogen be used as a proxy to monitor nitrogen efficiency? If so, what are the recommendations for MUN levels? Yeah, that's a common question. Usually, what I say in response to that is it can be used as a tool to monitor over time within a herd. In that sense, it is a starting point for in the same group of animals or animals coming in and out of that lactating group, you know what the diet is that you're feeding them. If you're seeing changes over time, that can be an indicator. If you're trending down or you're trending up, that can definitely be an indicator of potentially urinary nitrogen excretion and certainly nitrogen efficiency. It becomes more difficult when you're using that to compare across different dietary situations because there are different dietary factors like sodium, potassium, that type of thing that can impact how that urea is being partitioned into milk and urine. It's difficult across different groups of animals, but I would say within one farm, as a tracker, it can be a good tool to start considering. In terms of values, again, it kind of is more about the trend that you're seeing. If you make a dietary change and it goes down and animals are still performing well in terms of other targets that are of value to you, that's kind of your starting point. All right. Very well. Next question comes in from Dr. Hutchins. Do you look at economic responses to dairy farmers along with protein efficiency? I don't, personally. Of course, the economics is super important. Where I see this, at least from what I understand in the U.S. in particular, it drives a lot of the decisions around what you're going to do in your ration in terms of potentially improving protein efficiency or not really caring about it. That's kind of the context when I started this work when I was still in Canada. The discussion was always around protein being an expensive ingredient, protein-rich ingredients being expensive. Get the best bang for your buck and make sure it ends up in the tank, not under the tail of the cow. When I lived in Europe, sustainability was always second under that economic discussion, whereas in Europe, that was the headliner. How we really need to reduce nitrogen excretion, how can we manipulate the diet, cost being a major factor in what is an option to even consider. The main thing was make sure that that transfer is efficient so that we don't have as much coming out under the tail of the cow so that you're not being penalized based on your manure nitrogen. I think it plays a central role in the decisions that a particular operation might make around protein efficiency and the ingredients that you might choose to use. All right. Thank you, Kelly. Next question comes in from Augustine. Any information on how low the fermentable carbohydrates alter urea recycling to the rumen and its practical implications? Yeah, we know that higher fermentable carbohydrate diets, generally, the proportion of urea that gets recycled is higher, especially when paired with a relatively lower rumen degradable protein diet. Those are kind of the two combinations or the two factors that you would expect a relatively higher proportion of urea recycled to the rumen. Those are realistically the two things that you're playing with: protein level, nitrogen, degradable nitrogen, degradable protein in the rumen, and degradable or fermentable carbohydrate, and that balance between the two. Of course, you can lower protein and have a bit more urea recycling, but if you also increase the fermentable carbohydrate, there's the possibility that can increase even further. Why that happens and what are kind of the thresholds there, I think are still question marks in terms of really getting into the transporters and what's driving that pull, so to say, into the rumen. All right. Thank you. Dr. Aldrich is asking, are there specific amino acids or groups that are more effective than others in stimulating milk protein synthesis machinery, i.e., MT or C1 versus as substrate? Yeah, so those group one amino acids, histidine, methionine, being the two big ones within that category, phenylalanine and tryptophan, we just have so little data on those specifically. Histidine, methionine, it's no secret that there are some pretty positive responses that have been seen when you supplement those, even individually. Methionine in particular has sort of non-protein related effects as well. For example, you see fat response with a lot of amino acid supplementation, but also some of the benefits through transition that we've seen with supplementing methionine. From a milk protein standpoint, methionine and histidine are those group ones. By the definition of that one-to-one uptake to output ratio, there is inherently less flexibility there. You can think of that as going more directly into milk protein. What we consider sort of group twos or those other ones, the branch chains, arginine, lysine, threonine, what we see not only in my own work, but the work of others who've worked in this space is that there is more flexibility once they're in the mammary gland and potentially more opportunity for either some inhibition at the transport level, how they interact with each other. Those group ones, those are for sure a priority. The group twos, so to say, it does seem like there's more flexibility across those. Interestingly, lysine is in that group. I think it's a great tool in the toolbox to have a lysine product that can be fed. My interest is understanding when we're doing that, how is then that impacting kind of the scope of that flexibility with those other essential amino acids in the mammary gland to get the responses that we see? All right. Jope would like to know, in Europe, how can I increase digestible histidine in the diet without increasing total dietary protein? Yeah, so that's about looking at the composition of the ingredients that are being fed. You have a certain amount of protein. You don't want to exceed a certain level of dietary crude protein. You're looking then at what's contributing to that. Your more rumen undegradable sources, what are those bringing? Unless you're using protected products like a rumen protected rapeseed meal or a rumen protected synthetic amino acid, it's more challenging to estimate what that digestible profile is going to look like. That's an area that I think needs to expand a bit to kind of pair this idea that we know what they need post-absorptively. We have a little bit of a limitation in terms of understanding what's actually available to potentially be absorbed. That's an area that we're actively working on. Just keeping that in mind in terms of shifting the total amount of crude protein, what's coming from rumen undegradable, what's coming from degradable, and consider how histidine plays into that from the rumen undegradable standpoint. The histidine potentially that's bypassing the rumen and ending up in the small intestine to be absorbed from those ingredients that are in that diet. All right. Thank you. Adele says, excellent presentation, Dr. Nichols. What are your thoughts on supplementing antimicrobial compounds to reduce ruminal deamination and urolytic bacteria activity while providing a standard crude protein diet of 16% to lactating cows? Oh, yeah, when I hear 16% crude protein and increasing urolytic bacteria, I would question a little bit if we might be exceeding the threshold of rumen nitrogen requirements. Again, requirement is a difficult term to use there because it depends a lot on what the carbohydrate composition of the diet is as well. In general, why the idea of reducing rumen nitrogen balance is interesting with respect to improving nitrogen efficiency is the goal there is to reduce the amount of excess ammonia that's available that ends up going through the rumen wall, ending up as urea from the liver and actually not getting recycled back to the rumen, but instead ending up in urine. You want to minimize that as much as possible. From the perspective of, I'm not sure, maybe you're getting at like, could that potentially enhance urea recycling? Yeah, high levels of ammonia in the rumen typically depress that. Like I said, the dynamics across the rumen wall of the role the bacteria are playing that are sort of more epithelium adherent to influencing urea ultimately ending up as ammonia to be reused, recaptured by microbes in the rumen. That's an area that we have still a lot of open questions about how that really looks in practice. Yeah. All right. Next question comes in from Han. To what extent do you find is acid-base balance important in considering the possibilities to reduce nitrogen excretion as both a more acid environment in the rumen and the body can influence in partitioning and urea synthesis? Yeah, another one that's interesting to think about. I honestly haven't given that a ton of thought. Perhaps that is another space for metabolic flexibility, but definitely something interesting to consider. I don't know. All right. I see that we have passed the top of the hour. Do you have a few more moments? We've got a few more questions. Yeah, yeah. All right. Next question comes in from Rolando. It says, Dr. Nichols, based on your work and the data you presented on lipogenic diets, I wanted to ask your opinion on the use of calcium soaps enriched with essential fatty acids as a fat source. Would you expect the nitrogen efficiency response to be similar to that observed with saturated long-chain fatty acids, or would you anticipate a different outcome? I would, in general, anticipate something. I have to say I would anticipate something similar to what we saw, for example, feeding the hydropalm. Like I said, the base level of crude protein in the diet seems to have an influence, again, in the very limited data that is available. Because it's limited, the implications of the source of those fatty acids, from what I've seen, there are none, if very few, studies that have really paired looking at nitrogen partitioning with the work that they're doing around fatty acid profile. There's a lot of great work going on about fatty acid profile, but usually not paired with implications on nitrogen metabolism or mammary gland amino acid metabolism, those types of questions. All right. Next question comes in from Francisco. What should our practical safety margin be for rumen available nitrogen? That's a tough one to give a number. A good starting point would be based on, for example, the new NASEM 2021. What's written in there, if you look at the studies that are used for those recommendations, it's pretty consistent with kind of what I've seen in the studies we've done. I think those are good starting points. I don't think inherently those are excessive. You have to be careful when you speak about that because it's all relative. Like I said, it's relative to the fermentability of the carbohydrates in the diet. That's a big one. Where this idea of refining rumen protein balance started to come from was talking to nutritionists who had an idea in their mind that they had to meet a certain target for that balance. In the Netherlands, for example, they do talk about it on this scale of like zero is perfect balance, and then anything higher is surplus. A lot of the school of thought is it needs to be at least, let's just say, 100 on a grams per day basis or 150. It can't drop below that, or you'll drop digestibility, dry matter intake, milk production. What we've seen is that that doesn't need to be such a high threshold. There is the capacity to reduce it, but those thresholds were set in people's minds before we understood well the ability for them to use endogenous nitrogen. It's more about questioning, do I sprinkle extra urea in there as a safety margin based on the targets of this farm, potentially limitations on manure nitrogen that they might be facing in terms of deposition? Is that a smart... Do we need to do that for these particular animals? It's more to stimulate that question in your mind of why am I putting that in there? Do we need it? All right. Thank you. We'll entertain one last question. When is a marginal efficiency too high, A.K.A. they were deficient? Yeah, that's one way to look at it, right? I would say this is an interesting question because a lot of this marginal efficiency work comes from relatively short-term studies. A lot of them are from infusion models. I've started to bring it into some of the feeding studies that I've done. Again, in a real-life sense. You're feeding across lactation, using that marginal efficiency to actually set targets for potentially dietary changes. That's something that we, I am working on in a collaboration together with Balchem, is to actually make that marginal efficiency a factor that we consider for making dietary change as the animal responds to it. In theory, if you have a very high marginal efficiency and they're actually deficient and you start to see negative impacts of that overall lactation, you might, in those cases, we may be able to see, okay, that's not a good efficiency. Potentially, it's hard in these studies. A higher value is good because that transfer of that supplement is happening very efficiently. Were they deficient? Potentially, that could be why the efficiency is so high, but the implications of that we can't really discern from the studies. All right. Thank you, Dr. Nichols, and thank you, everyone, for attending today's webinar. If you have additional questions, please submit them to anh.marketing@balchem.com. The Real Science Lecture series continues with educational topics each and every month, including our next two. The second Real Producer Exchange on August 26, where we'll explore the process of consumer-based colostrum products and how to manage dairy operations in multiple states with the producer, Rob Deepersluit. On September 9, we'll be joined by Dr. Phil Cardoso from the University of Illinois for his presentation titled Optimizing Health and Reproduction Through Amino Acid Balancing in the Transition Period. Visit balchem.com/realscience for details on future webinars and to register for upcoming events. Balchem's podcast series continues to offer a deeper dive into our webinar topics. Log on to your favorite podcast platform and search for Real Science Exchange or visit balchem.com/podcast. If you want a really cool Real Science Exchange t-shirt, just subscribe to the Real Science Exchange. Send us a screenshot along with your address and shirt size to anh.marketing@balchem.com, and we'll get that off to you right away. On behalf of Balchem and Dr. Nichols, thank you for joining us today.