Hello, everyone. 
My name is Su A Lee. Today, I’ll be discussing effects of dietary protein concentration on digestibility of amino acids in diets fed to growing pigs. 
This work was presented in 2024 ASAS Midwest meeting.
To avoid the impact of microbial fermentation in the hindgut of pigs and because almost all amino acids are absorbed in the small intestines, digestibility of amino acids has been determined at the end of ileum. 
In addition, by correcting the apparent ileal digestibility (AID) for the basal endogenous losses of amino acids, standardized ileal digestibility (SID) values can be calculated, and these values are believed to be additive in mixed diets fed to pigs. One of the reasons values for the SID of amino acids are additive is that the values are not affected by level of amino acids in the diet. 
However, when we evaluate feed ingredients, sometimes we use diets containing a wide range of dietary protein. And it has not been known if levels of protein or amino acids actually affect the SID of amino acids, even though that’s what we have learned in the textbook.
Therefore, we hypothesized that the SID of AA would not be influenced by dietary protein even if dietary amino acids are below, at, or well above the requirement of pigs.
To test the hypothesis, 4 series of experiments were conducted.
Data from the first 2 experiments were already presented in the 2020 Midwest meeting.
However, to help you paint a bigger picture, I am going to present all data from the 4 experiments today. 
The initial body weight of pigs used in all experiment was approximately 20 to 24 kg.
In the first experiment, SBM was used as the only source of AA in diets. 
There were 4 diets containing 4 levels of SBM from 10 to 70% to create 5 to 33% CP in diets. 
A nitrogen-free diet was prepared to determine the basal endogenous losses of AA.
Linear and quadratic effects of increasing SBM were determined in this experiment. 
Before we further discuss the SID data, let me show you data for the AID, which may be interesting for you.
Data from all 4 experiments indicated that there were quadratic increases in the AID of AA as dietary protein was increased. This was in agreement with what had learned. 
Are you familiar with this figure? Yes, this figure is from the terminology paper published by Dr. Stein and his colleague in 2007 and the AID data from all experiments showed similar pattern, indicating that as dietary AA increased, the AID values, represented in orange, went up and the increases slow down.
This is because the proportion of endogenous AA in AA in ileal digesta is greater when dietary protein level is low. However, the proportion of endogenous AA is reduced as dietary protein increases, which results in an increase in the calculated value for the AID.
Jumping to the SID results, let me set up the slide first. 
The horizontal axis represents inclusion rates of SBM in diets: 10, 30, 50, and 70%. The vertical axis represents the SID of AA in SBM in percent. 
Results from the first experiment demonstrated that as SBM in diets or dietary protein levels increased, the SID of Arg, Ile, Met, Trp, and Val was not affected. This was also what we expected because we have learned that values for the SID of AA are not affected by dietary protein levels. 
However, the SID of His, Leu, Lys, Phe, and Thr presented here was reduced or tended to be reduced by increasing SBM in diets. 
This is because when we increase SBM levels in the diet, not only AA are increased, but also other nutrients are increased.  
For example, oligosaccharides such as stachyose and raffinose levels are also increased by increasing the inclusion rates of SBM. 
In this experiment, the analyzed stachyose and raffinose were approximately 5 and 1.3%, respectively. Therefore, it is possible that increasing SBM in diets increased the levels of stachyose and raffinose in the diets, which reduced the SID of AA.
To eliminate the effects of oligosaccharides from SBM, Experiment 2 was conducted with SPC because to produce SPC, the low molecular weight sugars are removed by alcohol extraction. 
Compared with oligosaccharides in SBM, the analyzed oligosaccharides in SPC were much lower, 
and the analyzed stachyose and raffinose in SPC were approximately 3 and 0.4%, respectively.
However, the analyzed trypsin inhibitor in both SBM and SPC was around 3 unit per mg, which indicates that trypsin inhibitor was not removed when SPC was produced. I will get back to this later.
Let’s move on to Experiment 2. A total of 6 diets were prepared. First 5 diets included 10 to 50% of SPC to create 7 to 33% dietary CP and a nitrogen-free diet was also used.
The linear and quadratic effects of increasing SPC in diets were determined. 
Moving on to the results, the horizontal axis represents the inclusion rate of SPC in diets and the vertical axis represents the SID of AA in percent.
The results showed that values for the SID of Arg, Ile, Leu, Met, Phe, Trp, and Val were not affected by dietary protein levels if AA were provided by SPC. And we can see that there were more amino acids that are not affected by dietary protein level in Experiment 2 compared with Experiment 1. And this can be explained by reduced oligosaccharides in SPC compared with SBM.
However, there were a few AA that were affected by increasing SPC in diets. The SID of His, Lys, and Thr quadratically or linearly decreased as protein levels in diets increased. Results from both Experiment 1 and 2 indicated that the SID of some AA is affected by increasing SBM or SPC in diets fed to growing pigs. The reason the SID values were reduced may be because the capacity of pigs for absorption of peptides and AA into the enterocytes has a limitation or because there are still antinutritional factors present in SPC such as trypsin inhibitor. Again, the analyzed trypsin inhibitor in SPC that we used in this experiment was similar with the analyzed trypsin inhibitor in SBM.
Then what if we use soy protein isolate (SPI) which contains almost zero oligosaccharides and is very low in trypsin inhibitor?
In Experiment 3, we prepared 5 diets containing 5 different levels of SPI to create 6 to 30% CP.  An N-free diet was also used.
The analyzed stachyose and raffinose in SPI used in this experiment were close to zero and the analyzed trypsin inhibitor was below the detection limit. You can see that most antinutritional factors from SBM and SPC have been eliminated in SPI. 
Results from Exp. 3 indicated that among 10 indispensable AA, the SID of His, Ile, Leu, Lys, Met, and Trp was not affected by dietary protein if protein was provided from SPI. 
However, there still were some AA that were affected by increasing dietary protein, even though the difference among the diets were not too big. 
Again, it is possible that there is a reduction in the capacity of pigs to digest or absorb AA if dietary protein is well above the requirements for pigs or a few antinutritional factors still exist in SPI. 
Therefore, we conducted an experiment where casein was used because casein does not contain most anti-nutritional factors that soy products have.
In the Experiment 4, we used 5 diets containing 5 different levels of casein to create 6 to 29% CP in diets and an N-free diet was also used. 
Results indicated that the SID of all indispensable AA was not affected by dietary protein. 
In the first experiment, we started with SBM, which contains soy-related antinutritional factors including oligosaccharides, fiber, trypsin inhibitor, and so on. Therefore, the effect of oligosaccharides was eliminated in the second experiment by using SPC. The next experiment was conducted with SPI, which did not contain any oligosaccharides and lower trypsin inhibitor. However, the results from the three experiments indicated that the reason the SID of AA is changed by increasing soy products is that these feed ingredients contain fibrous components in addition to trypsin inhibitors and unique protein structure that hinders the AA absorption, even though SPI contains very low antinutritional factors.
Moving on to the conclusions of this work, the AID of AA was increased by increasing dietary protein. This applied to all feed ingredients that we used. 
This increase in the AID of AA can be explained by different proportions of endogenous AA in the ileal digesta when protein level was increasing from low to high, which results in an increase in the AID of AA.
Results from Experiments 1, 2, and 3 indicated that the SID of some AA in SBM, SPC, and SPI was not affected by increasing dietary protein from around 5 to 30%, meaning that pigs appear to have a high capacity for digesting peptides or AA. 
However, there were other AA that had reduced SID when the soy proteins were increased in diets.
I have listed the indispensable AA here that were affected by dietary protein in each experiment. Please be aware that the SID of these AA is reduced when dietary protein is too high. 
In addition, I would like to point out that Thr was mostly affected by increasing the inclusion rate of the three soy proteins and from this result, we can tell that the reductions may be related to increased endogenous losses because Thr is the most abundant AA in mucin proteins. 
However, when casein was used in diets, the SID of most AA was not influenced by dietary protein.
The implication of this is that the direct procedure may be used to determine the SID of AA in a wide range of feed ingredients if dietary protein is not too high. 
Therefore, values for the SID of AA in feed ingredients may be determined below, at, or slightly above requirements without impacting results, even though the feed ingredients contain some anti-nutritional factors. 
Thank you for listening to my presentation and I would like to acknowledge everyone from Dr. Stein’s lab. 
If you want to learn more about our research that we are conducting in the Stein Monogastric Nutrition Laboratory, please visit our website.
See you next time~