Hello, everyone. My name is Joseph Limbach, and I just received my master's degree through
the Stein Monogastric Nutrition Laboratory. The focus of today's podcast is to discuss the
effects of dietary crude protein level on growth performance, blood characteristics, and
indicators of gut functionality in weanling pigs.

I'd like to begin with some background information as to how the weaning process affects
these pigs. So one of the most stressful time points in a pig's life is at weaning, and
this stress can come from a multitude of different areas. There could be nutritional
stress as these pigs transition from a highly digestible milk-based diet to corn- and
soybean-meal based diets that are less digestible, or there could be psychological and
environmental stresses stemming from the commingling of these pigs with pigs from other
litters other nursery rooms and potentially even other nursery facilities. Now any single
or combination of these stresses can lead to what Jayaraman and Nyachoti titled “weaning
anorexia,” which as the name implies is a decrease in feed intake post-weaning. Now that
decrease in feed intake post-weaning then causes a cascade of events that eventually
results in an increase in post-weaning diarrhea. Post-weaning diarrhea is one of the
largest challenges pig producers face because that loss of water and electrolytes can
negatively impact growth performance and potentially even cause mortality, both of which
impact a producer's bottom line.

Historically, antibiotics have been used to try to mitigate the negative effects that may
be associated with weaning. However, due to the increase in legislation and restriction on
the use of sub-therapeutic antibiotics, producers and nutritionists alike have been tasked
with finding alternative ways to try to reduce post-weaning diarrhea. Stein and Kil in
2006 reviewed multiple dietary strategies aimed at achieving this goal. They showed we can
use direct-fed microbials or other types of eubiotics in an attempt to replenish
beneficial bacteria in the guts of these pigs. We could use nucleotides or nucleosides. We
could use alternative cereal grains like cooked rice and barley—so for example, the
fermentation of the fiber from barley seems to have a probiotic type of an effect. We
could supplement these pigs with pharmacological levels of trace minerals like zinc or
copper. Or, we could decrease the crude protein content in the diet. The thought process
behind decreasing the crude protein content in the diet is that weaning pigs are not able
to digest the excessive amounts of protein that are typically found in starter diets, and
so if that protein is not digested and absorbed in the small intestine, then the nitrogen
passage rate into the large intestine will increase and there the nitrogen fermentation
can create an environment in which pathogenic bacteria can proliferate.

And so with that in mind, we designed an experiment with the hypothesis that if we
decrease dietary crude protein level, we will decrease diarrhea and improve intestinal
health in these pigs while having a minimal negative impact on growth performance. In this
experiment, we used 180 weaned pigs that averaged 5.53 kg of body weight, and with five
pigs per pen, we were able to achieve 12 replicate pens per treatment.

We used corn and soybean meal to formulate treatments with three different levels of crude
protein. Both the 22 and the 19% crude protein diets provided amino acids to meet the
requirement established by the NRC. However, the 16% crude protein diet did not, and this
is because amino acid concentration in that originally formulated diet was so low that
even with supplementation of crystalline indispensable amino acids, not all the
requirements were able to be met. We actually fed all of these treatments in a two-phase
feeding program. The phase one diet was fed from day 1–7 post-weaning, and in the 16%
crude protein diet it provided amino acids at approximately 71% of the requirement due to
the limitation in phenylalanine. The phase 2 diet was fed for the balance of this
experiment, and in the 16% crude protein diet, it provided amino acids at approximately
81% of the requirement due to the limitation in histidine.

Next, I'd like to show an experimental timeline for you to better visualize how this
experiment was conducted. So like I just mentioned, that phase one diet was fed from day
1–7 post-weaning, so the phase two diet was fed from day 7 to day 28. Pigs were weighed on
day 1, and a blood serum sample was collected from one pig per pen to establish a baseline
of where these pigs were at pre-weaning. Pigs were then weighed again weekly on day 7, 14,
21, and 28. And then on the day previous to weighing, we collected another blood serum
sample from one pig per pen. And then from those same pigs that we collected all these
blood samples from, we also collected a fresh fecal sample via the grab sampling method on
days 13, 20, and 27. We euthanized one pig per pen on day 12. And fecal scores were
recorded by average per pen every other day for the experiment’s duration. And so
obviously, there was a lot of samples collected within this particular experiment, and
that equates to an overwhelming amount of data to interpret, so for time's sake I’ve only
included results I felt pertinent to making the overall conclusions.

So now that I’ve explained how this experiment is set up, I’d like to move into the
results part of this presentation. And I’ll begin with fecal score by pen, where a lower
fecal score indicates more normal feces and a higher fecal score indicates more severe
diarrhea. I would like to take a second to set up my graph, because these same colors will
be representative of the same diets for the rest of this presentation. The blue bar will
represent pigs consuming the 22% crude protein diet, the orange bar will represent pigs
consuming the 19% crude protein diet, and the brown bar will represent pigs consuming the
16% crude protein diet. So for fecal score, there was a linear reduction in fecal score
for both day 1–7 and day 8–14, so less diarrhea as we decrease crude protein content in
the diet. However, from day 15–21 and day 22–28, the reduction in fecal score was not
significant, but as we consider the entire experimental time period, there was a linear
reduction in fecal score as we decreased crude protein content in the diet. So if you'll
remember, the main goal of feeding this low protein strategy is to reduce post-weaning
diarrhea, and we showed here that we were able to achieve that goal. 

However, the question from literature then becomes, well, does this come at a sacrifice to
growth performance? So for average daily gain, there was a tendency for a linear decrease
from day 1–14 as we decreased crude protein content in the diet. I think it's important to
note that these pigs obviously struggled for the first two weeks post-weaning as indicated
by their low average daily gain, and although there is this tendency for a decrease, the
difference is from 0.1 to 0.09 kg, so not a massive difference. However, from both day
14–28 and from day 1–28, there was a linear decrease in average daily gain as we decreased
crude protein content in the diet. The 16% diet is providing amino acids below the
requirement, so we'd expect a limitation in protein synthesis and thus the decrease in
average daily gain that is observed. As for feed consumption, there is no effect of diet
for either day 1–14 or day 14–28. However, from day 1–28, there was a tendency for a
linear decrease in average daily feed intake as we decreased crude protein content in the
diet.

And then as the gain-to-feed ratio is calculated, from day 1–14 there was a linear
decrease in the gain-to-feed ratio and for day 14–28 and for the entire experimental time
period there were quadratic decreases in the gain-to-feed ratio as we decreased crude
protein content in the diet. And interestingly, it appears that the 16% crude protein diet
is the true driver behind the statistical significances that we are observing.

Next I’d like to move into the presentation of some data that was measured in the blood
samples that were collected weekly. And I’ll begin with blood urea nitrogen or BUN, which
is used as an indicator of amino acid utilization efficiency. There was an effect of crude
protein level, of day, and a diet x day interaction on BUN concentration in the serum. Now
like we've discussed, these pigs struggled in terms of growth for the initial two weeks
post-weaning, so obviously these pigs were not utilizing amino acids efficiently for
protein synthesis and thus the increase in BUN to day 13. However, pigs started growing
better thereafter and so BUN was reduced. But by day 27, we do observe that pigs consuming
the 22% crude protein diet had a greater BUN concentration, indicating that there were
excess amino acids present in that diet. Also, irregardless of day, pigs consuming the 16%
crude protein diet had the lowest BUN concentration.

We also measured albumin, which is a carrier protein in the serum of these pigs. There is
an effect of crude protein level, of day, and a tendency for a diet x day interaction on
albumin concentration in the serum. So whether it's because there was a decrease in
albumin synthesis because of the decrease in protein synthesis, or because there's a
lesser need for albumin to transport nutrients because there's a decrease in protein
synthesis, either way, albumin concentrations were the lowest on day 13. However,
concentrations increased thereafter, and by day 27 the amount of albumin concentration was
reflective of the amount of crude protein content in the diet. And again, regardless of
day, pigs consuming that 16% crude protein diet had the lowest albumin concentration.

We also measured haptoglobin in the serum of these pigs. Haptoglobin is an acute phase
protein synthesized by the liver, and it's increased in response to stress because
haptoglobin functions to control or mitigate oxidative stress. There was an effect of day
and a diet x day interaction on haptoglobin concentration in the serum. We observed that
haptoglobin was the greatest on day 13. We're not sure if this is because these pigs are
still trying to adjust post-weaning or if this increase is associated with the transition
of these pigs from the phase one to the phase two diet there at day 7. However,
haptoglobin concentrations were reduced thereafter, and by day 27, we do observe that pigs
consuming the 16% crude protein diet have the greatest haptoglobin concentration. These
pigs are consuming a diet that's restricted in amino acids, so the pigs are experiencing a
dietary stress, and thus the increase in haptoglobin that is observed.

We also measured immunoglobulin G (or IgG) in the serum of these pigs. IgG is used as an
indicator of the humoral immune response, and it's said that IgG protects the intestinal
gut surface from bacterial damage, so it's also associated with immune protection. There
was an effect of day on IgG concentration, where concentrations were reduced significantly
from day 1–6 post-weaning. These data indicate that these pigs have activated their immune
system as they try to combat the stresses that might be associated with weaning. Now if we
take a step back, I think it's also interesting to note that we've shown that when feeding
these low protein diets, we are able to reduce fecal score in these pigs; however, it
doesn't necessarily seem like we're influencing their immunity.

Now a very similar result was observed in vitamin E concentration, where concentrations
were reduced significantly from day 1–6 post-weaning. Now because vitamin E is used in the
antioxidant system and is used to support the immune system, these data also indicate that
vitamin E is being oxidized to try to support the immune system as these pigs try to
adjust post-weaning.

Now the opposite was actually observed in terms of vitamin A concentration, where
concentrations were increased over the experiment’s duration. Perhaps this increase in
vitamin A is associated with an increased intake of vitamin A and the pig trying to store
more of it in the liver. 

Next, I’d like to change gears and present data from the pigs that were euthanized on day
12, and I’ll begin with morphology measurements in the jejunum and the ileum. So in the
jejunum, there was a quadratic effect on villus height, where pigs consuming the 19% crude
protein diet had the greatest villus height, indicating an increased surface area for
nutrient absorption. However, there was no effect of diet on crypt depth in the jejunum.
So then, as the villus-height-to-crypt-depth ratio is calculated, there was again a
quadratic effect where pigs consuming the 19% crude protein diet had the greatest
villus-height-to-crypt-depth ratio.

As we move into the ileum, there was no effect of diet on villus height in these pigs;
however, there was a quadratic effect on crypt depth, where pigs consuming the 19% crude
protein diet had the lowest or the shallowest crypt depth, and pigs consuming the 22%
crude protein diet had the greatest or the deepest crypts. So deeper crypts, or crypt
hypertrophy or crypt elongation or however you want to describe the term, can be
associated with pathogen colonization and consumption of a feed antigen. So consumption of
that 22% crude protein diet that has an increase in soybean meal inclusion results in pigs
with deeper crypts that also have an increased prevalence of diarrhea. However, then as
the villus-height-to-crypt-depth ratio is calculated, there is no effect of diet in the
ileum.

We also measured pH in the stomach, ileum, and colon of these pigs. So there was a
tendency for a linear increase in pH in the stomach as we decreased crude protein content
in the diet. Although pigs consuming this 16% crude protein diet have a pH that's well
within the normal range, these pigs aren't consuming that extra protein as seen with the
other diets, so the pigs are secreting less hydrochloric acid to aid in protein digestion,
and thus the increase in pH that is observed. Now, the opposite was seen in the ileum,
where there was a linear decrease in pH as we decreased crude protein content in the diet.
Perhaps this is because duct cells in the pancreas of pigs consuming this 22% crude
protein diet are overcompensating in terms of bicarbonate secretion and over buffering the
digesta. As we move into the colon, there was a quadratic effect on pH where pigs
consuming the 19% crude protein diet had the lowest pH. Now this result is difficult to
explain, but here in a few slides, hopefully I’ll present some data that sheds light as to
why we see this result.

We also collected contents from the cecum and the colon for the measurement of products of
fermentation, which would be ammonia and volatile fatty acids (or VFAs). So there was no
effect of diet on either ammonia or total VFA concentration in contents from the cecum.

Now next, I’m going to break down these total VFAs into concentrations of individual VFAs
grouped by those associated with either carbohydrate fermentation or those associated with
protein fermentation. So there was no effect of diet on either acetate or butyrate
concentration in contents collected from the cecum. However, there was a tendency for a
quadratic effect on propionate, where pigs consuming the 19% crude protein diet had the
lowest propionate concentration. There was also no effect of diet on either isobutyrate or
valerate concentration in contents from the cecum; however, there was a tendency for a
linear increase in isovalerate concentration as we decreased crude protein content in the
diet. Now, these data are a bit erratic, and so that makes these results difficult to
explain.

Then as we move into the colon, there was also no effect of diet on either ammonia or
total VFA concentration.

There was also no effect of diet on acetate or propionate concentration; however, there
was a tendency for a quadratic effect on butyrate, where pigs consuming the 19% crude
protein diet had the greatest butyrate concentration. And perhaps this increase in
butyrate concentration is associated with the decrease in pH that was observed in the
colon a few slides ago. There's also no effect of diet on isobutyrate, isovalerate, and
valerate concentrations in contents collected from the colon.

Now, these last few pieces of data that I’d like to present are the relative abundances of
mRNA measured in the ileal mucosa of these pigs. We measured many genes; however, I’m only
going to present the ones that are statistically significant. And we'll begin with
pro-inflammatory genes. There was a quadratic effect on the abundance of interleukin-8, a
linear decrease in the abundance of interferon-γ, a tendency for a quadratic effect in the
abundance of CXCL9, and a linear decrease in the abundance of CXCL10 as we decreased crude
protein content in the diet. Because these pro-inflammatory genes are associated with the
recruitment of other immune cells that result in inflammation, the decrease in the
abundance of these genes that is observed in pigs consuming the 16% crude protein diet
indicates a decrease in inflammation present in the ileum.

So then as we move into anti-inflammatory genes, there was a quadratic effect in the
abundance of interleukin-10 and a tendency for a linear increase in the abundance of
transforming growth factor-β as we decreased crude protein content in the diet. Because
these anti-inflammatory genes are associated with the dissipation of inflammation, an
increase in the abundance of these genes, as observed in pigs consuming the 16% crude
protein diet, indicates a decrease in inflammation present in the ileum of those pigs as
well.

The next group of genes I’d like to present are the ones I’ve coined the gut protective
proteins, and this includes both tight junction proteins and those associated with mucous
secretion. There was a linear decrease in the abundance of occludin and a tendency for a
quadratic effect in the abundance of zonula occludens protein-1 as we decreased crude
protein content in the diet. Now, we assume that an increase in the expression of these
tight junction proteins is associated with less gut leakage; however, as we interpret
these data, perhaps because there's less inflammation—as indicated by the abundance of
those pro-inflammatory genes—there's also a lesser need for the upregulation to increase
expression of these tight junction proteins for protection. There was also a linear
decrease in the abundance of mucin-2, trefoil factor-2 and trefoil factor-3 as we
decreased crude protein content in the diet. Mucin-2 is a major protein found in mucus
which also has a protective function in the gut, and so because there's less inflammation,
there's a lesser need for a mucus to be present for protection and thus the decrease in
MUC-2 that is observed. And because these trefoil factor peptides are produced by
mucus-secreting cells, I think it makes sense that they follow a similar trend in data.

Now, the last group of genes I’d like to present are the glucose transporters. There was a
linear decrease in the abundance of both GLUT2 and GLUT5 as we decreased crude protein
content in the diet. And if you'll remember from the last slide, perhaps because there's a
decrease in the expression of those tight junction proteins, there's more glucose being
absorbed via the paracellular pathway and a decrease in these glucose transporters is then
needed.

So obviously there's an overwhelming amount of data present here within this experiment,
and so I’d like to summarize this data with a few conclusions. So by feeding the low
protein diets, we've shown that we're able to decrease diarrhea score and BUN
concentration in the serum of these pigs. However, we're also reducing albumin in the
process. And by feeding these low protein diets, there's really only a small decrease in
growth performance for the initial two weeks post-weaning. There's also a decrease in the
indicators of inflammation, as shown by the abundance of those inflammatory genes when
pigs are consuming that 16% crude protein diet. And when we're feeding these low protein
diets we are able to change pH in both the stomach and the ileum. Then if we reduce crude
protein level while still providing amino acids at the requirement, we can beneficially
impact morphology as well.

I’d like to wrap up today's presentation with a few take-home messages. So I’ve shown that
by feeding these low-protein diets, that we are able to reduce diarrhea score, which is
the main goal of this nutritional strategy. We're also able to reduce the BUN
concentration, indicating that we're feeding amino acids closer to the requirement in
these pigs. I’ve also shown that when feeding these low protein diets, we're able to
minimize inflammation in the gut as shown by the abundance of those inflammatory genes.
And we're really able to achieve both of these things with only a small decrease in growth
performance for the initial two weeks post-weaning. However, if we are going to feed these
low protein diets for an extended period of time, it then becomes crucial to provide amino
acids to meet the requirement so we can maximize protein synthesis and growth performance
and we can improve gut morphology in the process.

And so with that, I’d like to thank DSM for their financial support and collaboration on
this project. I’d also like to thank all the members of the Stein Monogastric Nutrition
Laboratory, both on the farm and in the lab, for their help in completing this research.
If you have any other questions about this particular project or any inquiries about
nutrition in general, I highly encourage you to visit our website at
nutrition.ansci.illinois.edu. Thank you.