Hi, everyone. Thanks for coming. My name is Woongbi Kwon. I am a postdoc researcher at the University of Illinois. Today, I am glad to share with you some data on effects of dietary valine, isoleucine, and tryptophan supplementation to excess leucine diet for growing pigs. Before we get started, let’s talk briefly about branched chain amino acid. Isoleucine, leucine and valine these are categorized as branched chain amino acid. Of course, these are indispensable amino acid for swine. When you look at the chemical structure of these three amino acid, they have similar side chains. Because of this structural similarity, these three amino acid can be metabolized exactly the same way in animals. Therefore, there must be an interaction in their metabolism. For example, there is an antagonistic effect especially from excess amount of leucine. Let’s take a look at more details about effects of excess leucine. Previous studies indicated that it pigs consume excess leucine diet, leucine reduced their gross performance, especially for their average daily feed intake. And they concluded that the troublemaker in excess leucine diet was KIC, KIC is alpha keto iso caproate, which is the metabolite of leucine. This compound can stimulate the enzymatic degradation of all three branched chain amino acid. So excess leucine can reduce availability of isoleucine and valine for protein synthesis as a result of stimulating effects of KIC. Bottom line is excess leucine has negative impact on growth performance and nitrogen utilization in pigs. Therefore, it is important to know the effects of excess leucine in swine and it is necessary to know how to overcome this problem. When we look at the results from our previous research, presented here in the Midwest meeting two years ago, we concluded that excess leucine reduced growth performance and reduced serotonin concentration in the brain and we also confirmed that excess leucine increased degradation of all three branched chain amino acid and eventually excess leucine reduced nitrogen retention in growing pigs. But based on that serotonin result, we could partly explain how average daily feed intake was affected by excess leucine because serotonin is a neurotransmitter that is regulating feed intake. Since tryptophan is a precursor of serotonin, we’ve conducted second study by adding crystalline L-tryptophan to excess leucine diet and we concluded that adding more tryptophan increased serotonin concentration in the brain, and it also increased average daily feed intake and average daily gain. But this couldn’t fully overcome the negative effects of excess leucine. And then last year, we had another presentation here in the Midwest and we concluded that valine supplementation could alleviate some negative effects of excess leucine. However, all these studies were not very practical, because we use crystalline L-leucine as a source of excess leucine. No one will use crystalline L-leucine on the practical condition because it is not a limiting amino acid in most swine diet. However, we’ve been tested the extreme level of excess leucine, which was three times more than the requirement in order to confirm clear and negative effects of excess leucine in this previous trials. So, we ended up having more practical excess leucine condition in the current study. Here’s a graphic for lysine and leucine concentration in several ingredients. We know corn and corn by product has high leucine concentration. But recently, there are a lot of novel sources of Corn Protein from ethanol industry, which easily contain almost 48 to 50% crude protein. For example, here I have NexPro and ProCap DDGS, these two sources have almost two times more leucine than the conventional corn DDGS. Many producers in the US try to use these sources to replace some portion of soybean meal from their diet. Of course, it could be a good strategy for saving feed cost, but we don’t really know how excess leucine impact pigs growth and amino acid utilization.
In this study, we use the NexPro as a source of excess leucine in the diet. Therefore, the objective of this study was to test the hypothesis that inclusion of dietary valine, isoleucine and tryptophan will alleviate negative effects of excess leucine from Corn Protein on nitrogen balance of growing pigs. So, we utilized 72 growing barrels with initial body weight of 33.9 kilogram, and they were housed in metabolite crates, and they were assigned to eight dietary treatment. Each treatment had nine replicates. When we look at the dietary treatments, we formulated a basal diet based on 70% corn and 26% HP corn product, which contained 171% SID leucine relative to the NRC requirement. In order to determine the effects of valine, isoleucine and tryptophan supplementation. We had two levels of valine, two levels of isoleucine and two levels of tryptophan by using 0.1% Crystalline L-valine, and isoleucine and 0.05% Crystalline L-tryptophan. Therefore, we ended up having two by two by two factorial treatment arrangement from this study. One thing I want to point out here is 70% SID valine, 52% SID isoleucine and 18% SID tryptophan to lysine ratio, these numbers are considered as requirement level recommended from NRC 2012. After five days of adaptation period, total amount of urine and feces were collected for five days according to the marker to marker procedure. We put second marker in the diet on day 11 and finished fecal collection on day 13. Blood samples were also collected on both day one and day 13. Let’s check the response criteria and statistical analysis before we jump into the results. We had a nitrogen balance data including biological value of protein from urine and fecal collection and we also had plasma urea nitrogen data from blood sampling. And these data were analyzed using mixed procedure of SAS, valine, isoleucine, and tryptophan levels and interaction among them were included as main effects. But there are no three way interactions from this two by two by two factorial treatment arrangement. So I’d like to show you the results from next slide based on two way interactions and main effect. And unfortunately, we couldn’t find any significant effect on plasma urea nitrogen data. So let’s see the nitrogen balance data. Let me explain the slide setup first. Both the yellow bars represent requirement level of valine which is 70% SID to lysine ratio. Blue bars represent the group of 80% SID Valine to lysine  ratio, for both of them, there are two levels of SID isoleucine to lysine ratio 53% and 63%. As you can see 53% is requirement level. So, from here, we found the interaction between valine and isoleucine and tendency of valine effect and isoleucine effect for fecal nitrogen output, adding more valine tended to reduce fecal nitrogen output, but adding more isoleucine tended to increase fecal nitrogen output even though there were no differences on feed intake or nitrogen intake among the treatments. But because of the interaction, we can only see the increase from here with valine level requirement which is 70% SID valine to lysine ratio. This result indicates that feeding extra crystalline L-valine or L-isolucine over the requirement to the high leucine diet could change nitrogen absorption from small intestine, but for the research is needed to confirm this. How about nitrogen retention? There was a significant interaction between valine and tryptophan. Unlike the previous slide, here we have two levels of SID tryptophan to lysine ratio and 18% is considered as requirement level.
Adding more tryptophan increased nitrogen retention, but because of interaction the increase was greater when valine level was at the requirement. And we also found isoleucine effect on nitrogen retention. When we added more isoleucine nitrogen retention was decreased. This result indicates that excess amount of isoleucine in the diet has negative effect on nitrogen retention on the excess leucine condition. When we look at the biological value of protein there was a significant interaction between isoleucine and tryptophan. Because of the two way interactions, every slid has different set up. Now we have two groups of SID isoleucine to lysine ratio 53% and 63%, and two levels of SID tryptophan to lysine ratio 18% at the requirement and 23%, which is over the requirement. So adding more tryptophan increased biological value, and based on that interaction, the increase was greater when isoleucine level was at the requirement. Based on the nitrogen retention and biological value of protein results, adding more tryptophan seem to be beneficial for nitrogen balance, but in this case, we should consider isoleucine and valine concentration in the diet because of the interaction. 
Let’s make conclusions. Fecal nitrogen output was reduced by adding valine, but increased by adding isoleucine if valine was at the requirement, if you add more tryptophan, nitrogen retention only increased, but increase will be greater if valine is at the requirement. But adding more isoleucine has negative impact on nitrogen retention. For biological value, adding more tryptophan was beneficial if isoleucine was at the requirement. Here’s the take home message from today’s presentation. If you want to use high protein corn by product in your diet formulation for replacing some soybean meal, first, check leucine concentration in the source and how much excess leucine will be in the diet. And second, use more crystalline L-tryptophan above the requirement if available. Third, isoleucine level in the diet should be close to the requirement level. Then you could minimize negative impact of excess leucine on nitrogen retention in your pigs. With that, I’d like to thank the sponsor Ajinomoto North America for the financial support. And I want to acknowledge all the members in Stein monogastric nutrition lab. All the comments and questions about this topic are welcome. And if you want to know more about our research, please visit our website or please check my contact information in this slide. Thanks for your attention.