Hi everyone. My name is Woongbi Kwon from the Stein Monogastric Nutrition Lab at the University of Illinois. Today, I’m glad to share with you some data on effects of dietary leucine in growing pigs. Before we get started, I want to point out why we investigated dietary leucine effects and why leucine is important in swine nutrition, especially when we use corn or corn-coproducts such as DDGS as the main ingredients in the diets. Since the price of DDGS is often lower than other major ingredients and increasing availability of different DDGS sources from the ethanol industry, many of pork producers in the US are trying to use greater amount of DDGS as the replacement for energy and protein sources in their diets. This is an example of formulating diets using conventional DDGS by replacing 20% of soybean meal for 25 to 50kg pigs. X-axis represents each indispensable amino acid and Y-axis represents percentage of amino acid concentration relative to the SID requirement. So, 100% means amino acid level at the requirement. But in this case, about 50% of excess leucine over the requirement will be generated because corn and corn DDGS has high leucine concentration. If we use the HP DDGS, which is the high protein DDGS source, this will be exacerbated. Of course, for both diet formulation, several synthetic amino acids will be required to meet the amino acid requirement. But in this case, about 70 % of excess leucine will be generated. Many of the literature indicated that the oversupply of leucine by using a greater amount of corn coproduct will have a potential problem on growth performance especially on their feed intake and amino acid utilization in pigs. As you can see here, if we use greater amount of sorghum or sorghum DDGS, excess leucine will be generated as well. Leucine is one of indispensable amino acids for swine. Along with isoleucine and valine, they are categorized as the branched-chain amino acids because of the structural similarity of their side chain. For this reason, they have unique feature that these three amino acids share the first two steps of their catabolic pathway. Let’s take a look at the metabolism of branched-chain amino acids. Like other amino acids, absorbed branched-chain amino acids from small intestine are directly going to the liver through the hepatic portal vein. In the liver, these three amino acids will be used for protein synthesis or will be transferred to other tissues for protein synthesis. Then the surplus of these three amino acids will go to the skeletal muscle and can be metabolized by transamination enzyme BCAT. This transamination in skeletal muscle is unique, because all other amino acids will be transaminased in the liver. So this enzyme, BCAT, produces branched-chain alpha-keto acids: KIV, α-ketoisovalerate, from valine, KMV, α-keto-β-methylvalerate, from isoleucine, and KIC, α-keto isocaproate, is from leucine. And then, these branched-chain alpha-keto acids will go to the liver for the second step of their metabolism by branched-chain α-keto acid dehydrogenase, BCKDH. This step produces three different acyl-CoA from each α-keto acid, then these acyl- CoA will be used for glucogenic or ketogenic functions. But in this second step of metabolism, KIC, which is the metabolite of leucine, can stimulate activation of branched-chain α-keto acid dehydrogenase. Therefore, it is possible that excess dietary leucine may affect metabolism of branched-chain amino acids by increased KIC concentrations. By doing that, excess leucine may also affect nitrogen balance or protein synthesis and growth performance in pigs. Tryptophan is another indispensable amino acids for swine. It has been considered as an important amino acid because it is involved in feed intake regulation by enhancing serotonin synthesis. However, tryptophan and all three branched-chain amino acids are categorized as large neutral amino acids, so they share a common uptake transporter (LAT1) across the blood-brain barrier. Therefore, it is possible that excess leucine may result in reduced tryptophan uptake into the brain due to competition for transporters, and this results in reduced serotonin synthesis. And this possible theory might be the answer of why excess leucine has negative impact on feed intake in animals. Therefore, the objective of this experiment was to test the hypothesis that excess dietary leucine affects nitrogen balance and growth performance, and excess leucine reduces tryptophan uptake into the brain due to competition for transporters, resulting in reduced serotonin synthesis. And last hypothesis was that excess leucine increases metabolism of all 3 branched-chain amino acids by increased activation of BCAT and BCKDH. Now we are moving on to the materials and methods. Forty growing barrows with initial body weight of 30 kg were housed in metabolic crates and were assigned to five dietary treatments. To avoid naturally generating excess leucine from the ingredients, we formulated basal diet using less amount of corn and soybean meal and greater amount of wheat and barley to contain 100% of requirement for SID leucine. In order to investigate the effects of increasing level of dietary leucine, we used crystalline L-leucine as the source of excess dietary leucine in the diets. Finally, we formulated five diets containing 100, 150, 200, 250, or 300 % of SID leucine relative to the requirement. To maintain constant crude protein level for all diets, we used crystalline glycine. After a 7-day adaptation to test diets, total amount of urine and feces were collected for 5 days according to the marker to marker procedure. By considering processing time of marker, we concluded our fecal collection on day 14. For sample collection, all pigs were euthanized on day 15 by electrocution. Blood samples were collected on both day 0 and day 15. Tissue samples of brain, liver and muscle were collected right after the euthanization. For growth performance data, initial and final body weight were recorded on day 0 and day 15, and the amount of feed consumption was recorded daily. Let’s move on to the results. This first graph indicates the average daily feed intake for 15 days. blue bar represents SID leucine at 100% requirement, the orange bar represents 150% SID leucine, the brown, gray, and navy bar represent 200%, 250%, and 300% SID leucine relative to the requirement, respectively. As I mentioned earlier, excess leucine has negative impact on feed intake in animals. The average daily feed intake linearly decreased as dietary leucine increased. Same pattern was observed on average daily gain data. Linearly decreased average daily gain was mainly due to decreased average daily feed intake. Taken together, gain to feed ratio also linearly decreased as dietary leucine increased. Same trends were observed in the nitrogen balance for 5 days. A decreasing trend for nitrogen retention was observed as dietary leucine increased. In addition, biological value of dietary protein linearly decreased as dietary leucine increased. Because of excess leucine, pigs had less nitrogen retention from their diets. Unlike growth performance and nitrogen balance data, a linear increase in plasma urea nitrogen was observed as dietary leucine increased. Plasma urea nitrogen is considered as a rapid parameter of efficiency of amino acid utilization in pigs. Increased plasma urea nitrogen means reduced efficiency of amino acid utilization. Even if all pigs were fed isonitrogenic diets, because of excess leucine, the efficiency of amino acid utilization was reduced. Our second hypothesis was: excess leucine reduces tryptophan uptake into the brain and this results in reduced serotonin synthesis. So we analyzed serotonin concentration in hypothalamus. As we expected, linear reduction in hypothalamic serotonin was observed as dietary leucine increased. Based on this brain serotonin data, we could also explain why feed intake was linearly decreased as dietary leucine increased. Like I mentioned before, there are 2 common enzymes in catabolic pathway of branched-chain amino acids. The first enzyme, BCAT, is mainly in the skeletal muscle. And the second enzyme, BCKDH, is mainly located in the liver. So we analyzed the abundance of mRNA of these two enzymes using quantitative RT-PCR. We found a linear effect on BCAT as dietary leucine increased. The linear effect on BCAT might be due to the increased dietary leucine as a substrate for this enzyme. However, even though we analyzed all 3 subunits of BCKDH in the liver, we couldn’t find any effects on BCKDH abundance as dietary leucine increased. We also analyzed concentration of branched-chain alpha-keto acids in skeletal muscle and liver. As we expected, KIC, the metabolite of leucine and the key metabolite of stimulating BCKDH enzyme, increased linearly as dietary leucine increased. Same pattern was observed in the liver. KIC concentration in the liver linearly increased as dietary leucine increased. Based on these results, increased KIC concentration by excess leucine may increase catabolism of valine and isoleucine and therefore, it creates amino acid imbalance for protein synthesis. In conclusion, excess dietary leucine reduced growth performance and protein synthesis, and excess dietary leucine reduced hypothalamic serotonin concentration. Excess dietary leucine also affected metabolism of all three branched-chain amino acids. I would like to thank the sponsor, Ajinomoto Nutrition North America for the financial support, and I want to acknowledge all the members in the Stein Monogastric Nutrition Lab. And if you want to learn more about our research, please visit our website. Thanks for your attention.