Hi everyone. My name is Woongbi Kwon from the Stein Monogastric Nutrition Laboratory at
the University of Illinois. Today, I’m glad to share with you some data on effects of
dietary leucine and tryptophan in growing pigs. And this is also part of my Ph.D.
dissertation work. 

This is the outline of today’s presentation, starting with a brief introduction why we
investigate leucine and tryptophan effects, and I will explain about the experimental
procedure and results. And then, I will wrap up and give you the overall conclusion at the
end.

Why is leucine important? If you formulate a diet based on corn and soybean meal plus 20%
of DDGS, you can make a diet that is almost perfect and all amino acids are exactly at
their requirement if you add synthetic amino acids, except for leucine. When you look at
the leucine concentration here, it stays up at 147% of requirement without any
supplementation of synthetic leucine. This is another example of diet formulation. If you
want to fully replace soybean meal using 30% HP-DDGS which is the novel corn protein
source from the ethanol industry containing high protein, you will be over 180% easily for
leucine in your diet formulation. Of course, you should put more synthetic amino acids
than the first diet formulation, but in this case, much more excess leucine will be in
your diet. We all know that leucine is indispensable amino acids for pigs, but if we use
these kinds of diet formulation, then we should consider excess leucine in the diet,
because it could generate amino acid imbalance. 

The reason is if you look at lysine and leucine concentration in several ingredients here,
especially for corn, it has almost four times more leucine compared to the lysine
concentration, and you may know that the requirement for lysine and leucine is about the
same 1:1 ratio in all diets. Wheat and barley also have higher leucine than lysine
concentration, but it is not extreme. Of course, soybean meal contains a lot of leucine,
but it also contains a lot of lysine in the source. When we look at more recent data, not
only corn and corn DDGS but also sorghum and sorghum DDGS has high leucine concentration.
HP corn DDGS, which has high protein concentration, has much more leucine compared to the
conventional DDGS. Therefore, like I mentioned earlier, if we use these ingredients in our
diet formulation, excess leucine will easily be generated.

As you may all know, leucine, along with isoleucine and valine, they are all indispensable
amino acids for swine, and they are categorized as the branched-chain amino acids because
of the structural similarity of their side chain. For this reason, these three amino acids
can metabolize in the same way by sharing the first two steps of their catabolic pathway.
Let take a look into their metabolism.

Like most other amino acids, absorbed branched-chain amino acids from the small intestine,
they are directly going to the liver through the hepatic portal vein. In the liver, these
three amino acids are being used for protein synthesis or being transferred to other
tissues for protein synthesis. Then the surplus of these three amino acids will go to the
skeletal muscle and deaminated by transamination enzyme BCAT. This deamination of
branched-chain amino acids in skeletal muscle is unique because all other amino acids will
be deaminated in the liver. So, this enzyme BCAT produces branched-chain alpha-keto acids.
KIV, α-ketoisovalerate, is from valine, KMV, α-keto-β-methylvalerate, is from isoleucine,
and KIC, α-keto isocaproate, is from leucine. As you can see here, the arrows for both
sides—this is reversible. And then, these branched-chain alpha-keto acids will go back to
the liver for the second step of their metabolism by branched-chain α-keto acid
dehydrogenase, BCKDH. Again, this is also a common enzyme for all three branched-chain
amino acids. 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, will stimulate this enzyme BCKDH.
That means more and more of isoleucine and valine also metabolized, even if there was not
enough available isoleucine and valine in pigs. Therefore, it is possible that excess
leucine can create a deficiency of isoleucine and valine.

Research has been conducted to investigate the effects of excess dietary leucine in
growing pigs, and it turned out excess leucine reduced average daily gain, average daily
feed intake, and gain:feed ratio; reduced nitrogen retention; and increased branched-chain
amino acid metabolism by stimulatory effects of leucine. And because of this stimulatory
effect, which can create deficiency of isoleucine and valine, protein synthesis also
decreased. Interestingly, excess leucine reduced serotonin concentrations in the brain.
Based on these serotonin results, we could partly explain why average daily feed intake
was decreased. 

Leucine also goes to the brain like other amino acids. Another amino acid that goes to the
brain is tryptophan. Tryptophan goes into the brain using the transporter called LAT 1,
and a small part of transported tryptophan will be used to synthesize serotonin. Serotonin
is important because it is involved in feed intake regulation. But the problem is all
three branched-chain amino acids also use the LAT1. So, if you overloaded leucine into the
blood, that means the LAT1 transporter is busy transporting leucine into the brain and
therefore, you will get a reduced tryptophan uptake into the brain, therefore less
serotonin being synthesized. Now you have lower serotonin because of that excess leucine.

Let’s take a look at more about serotonin. Serotonin is being called as a “happy hormone”
even if it is not exactly a hormone. Serotonin is a neurotransmitter that has several
regulating functions in the body. The real name of serotonin is 5-hydroxytryptophan. And
tryptophan is a precursor of serotonin. In the body, more than 90% of serotonin is located
in the gut produced by gut enterochromaffin cells. Only 5% of serotonin is secreted from
the hypothalamus in the brain, and this 5% is involved in sleep, cognition, and appetite
regulation.  

Interaction between leucine and tryptophan is well described by Anna Wessels in 2016. When
you look at this graph, there was negative correlation between plasma leucine and
hypothalamic tryptophan. In addition, there was positive correlation between hypothalamic
tryptophan and hypothalamic serotonin from the same study. 

Therefore, it is possible that adding more tryptophan in the high leucine diets can
overcome the negative effects of excess leucine on growth performance by increased
serotonin synthesis in the brain.

So, our hypothesis of this study was tryptophan supplementation to high leucine diets is
needed to prevent drops in serotonin concentrations in plasma and hypothalamus and to
maintain growth performance of growing pigs.

Now, let’s look at the materials and methods.

A total of 144 growing pigs with initial BW of 28 kg was used for this experiment. There
were 2 pigs in each pen, and there was a total of 72 pens equipped with a nipple drinker
and a feeder. Each treatment had 8 replicate pens. 

For experimental diets, we formulated a basal diet using less amount of corn and soybean
meal and greater amount of wheat and barley to contain 100% of requirement for SID
tryptophan and leucine. The reason we used a lot of wheat and barley for this diet was to
avoid naturally generated excess leucine from corn and soybean meal. In order to
investigate the effects of increasing level of dietary tryptophan and leucine, we used
crystalline L-leucine and L-tryptophan as the source of excess dietary tryptophan and
leucine in the diet. We had 3 different levels of tryptophan up to 28% SID tryptophan to
lysine ratio and 3 different levels of leucine all the way up to 300%. Finally, we
formulated 9 diets containing 9 different ratios of dietary tryptophan and dietary
leucine. To maintain constant crude protein level for all diets, we used crystalline
glycine. 

Experimental period was 21 days. Blood samples were collected on day 0, day 11, and day
21. After the bleeding on day 21, pigs were euthanized by electrocution in order to
collect the brain samples. Using blood samples, we analyzed plasma urea nitrogen, plasma
amino acid profile, and plasma serotonin. Using brain sample, we analyzed hypothalamic
serotonin. For growth performance data, initial and final BW were recorded on day 0 and
day 21, and the amount of feed consumption was recorded throughout the experimental
period.

For the statistical analysis, a second-order surface response model was used. The model
includes the linear effects of tryptophan and leucine, the quadratic effect of tryptophan
and leucine, and interactions between linear and quadratic tryptophan and leucine. We
started with the full model and if a term or an interaction was not significant, the model
was reduced by removing the non-significant terms. Eventually, we used this model here you
can see for all data.

Now, let’s move on to the results.

All the slides of results have a similar setup, so we have in the light blue we have 100%
SID leucine to lysine, which is the requirement level of leucine; in orange, we have 200%
SID leucine to lysine ratio; and in dark brown, we have 300% SID leucine to lysine ratio.
So those are the 3 different levels of leucine. And here in the horizontal axis, we have
three different levels of tryptophan from 18 to 28% of SID tryptophan to lysine ratio. So
here you see average daily feed intake data for 21 days of growth performance. As we added
more leucine in the diets, there was a pretty dramatic reduction in feed intake. As we
added more tryptophan, we saw increase in feed intake particularly in excess leucine
condition, but it was not enough to catch up to what we have here with leucine at the
requirement. Excess leucine for sure reduces feed intake of pigs.  

For average daily gain, there was no surprise: if pigs don’t eat, then they don’t grow.
So, we had exactly the same trend here for average daily gain. As we increase leucine
above the requirement, we see the reduction in average daily gain; as we increase
tryptophan above the requirement, we saw some improvement in average daily gain, but it
was not enough to catch up to what we have here with leucine at the requirement. We also
see here that there is no effect of adding tryptophan if we have leucine exactly the 100%
requirement. But in the excess leucine condition, there was clear significant effect of
adding tryptophan in the diet.

What about serotonin? Similar trends as we saw in growth performance data. As we added
more leucine in the diets, there was a pretty dramatic reduction in serotonin. However, if
we added more tryptophan, we could partly ameliorate that leucine effect, but not
completely.

Here we have plasma amino acid data. We saw clear interaction with tryptophan in the
plasma. Of course, if we added more tryptophan in the diet, we see more tryptophan in the
blood; however, there was an interaction, so the increase is greater if we have leucine at
the requirement in the diet.

And leucine in plasma, no surprise here as well. The more leucine we have in the diet, the
more leucine we have in the plasma. But plasma leucine in all cases is reduced by
increased tryptophan in diets, indicating that there is interaction between dietary
tryptophan and leucine.

And when we look at the isoleucine in the plasma, the more leucine we have in the diet,
the less isoleucine we have in the plasma, indicating that excess leucine deaminates some
of that isoleucine; therefore, we get a lower isoleucine concentration in the plasma.

That’s the same thing for valine. The more leucine we have in the diet, the less valine we
have in the plasma, because of reduced availability by excess leucine. However, the effect
of adding tryptophan was almost zero for valine concentration in the plasma.

And we saw one other thing here which we cannot really explain why it happened. Among all
the other indispensable amino acids except tryptophan and branched-chain amino acids,
threonine is also impacted by dietary leucine and dietary tryptophan. This is a pretty new
finding; maybe there is some interaction among tryptophan, leucine, and threonine in terms
of their metabolism. 

Now let’s make an overall conclusion.

Dietary leucine reduced average daily feed intake and average daily gain, but dietary
tryptophan increased average daily feed intake and average daily gain if excess leucine is
added in the diet. For serotonin, dietary leucine reduced hypothalamic serotonin, but
dietary tryptophan increased hypothalamic serotonin. For plasma amino acid profile,
dietary leucine reduced isoleucine and valine concentrations in plasma and dietary
tryptophan changed all 3 branched-chain amino acids concentrations in the plasma.
Interestingly, dietary leucine increased threonine concentration in plasma, but dietary
tryptophan reduced threonine concentration in plasma.

I would like to thank the sponsor, Ajinomoto, for the financial support, and I want to
acknowledge all the members in Stein Monogastric Lab. If you want to learn more about our
research, please visit our website. Thanks for your attention.