Hello everyone, my name is Vanessa Lagos. I am a first-year Ph. D. student under Dr. Hans Stein, and
today I’ll be talking about the influence of the level of dietary digestible calcium on growth
performance, bone ash, plasma calcium, and abundance of genes involved in calcium absorption in 11-
to 25-kg pigs fed different levels of digestible phosphorus.

To give a little bit of background about the study… unlike requirements for phosphorus that are
expressed as digestible phosphorus, requirements for calcium have been expressed as total calcium
because of a lack of data for the digestibility of calcium in feed ingredients. However, recent work
has generated values for digestible calcium (with and without phytase) in different
calcium-containing feed ingredients, which has allowed for the formulation of diets based on
digestible calcium.

Indeed, research has been conducted to estimate requirements for calcium expressed as a ratio
between standardized total tract digestible calcium (STTD calcium) and standardized total tract
digestible phosphorus (STTD phosphorus) in pigs from 25-50, 50-85, and 100-130 kg. Results from
these studies indicated that if phosphorus is at the requirement, growth performance will be
maximized at a ratio from 1.35:1 for young pigs, to 1.10:1 for finishing pigs. Whereas for bone ash,
the maximum response will be observed at a ratio from 1.80:1, in growing pigs to 2.30:1 in finishing
pigs. So we can clearly observe that requirement for maximizing bone ash are greater than for
maximizing growth performance.  

In terms of 11-25 kg pigs, an attempt to estimate the requirement for digestible calcium was made by
using one level of digestible phosphorus and six levels of digestible calcium. However, because of a
reduction in growth performance as dietary calcium increased, an optimal digestible
calcium:digestible phosphorus ratio could not be estimated. 

Therefore, the objective of this experiment is to test two hypotheses. The first one is that the
calcium requirement to maximize growth performance expressed as a ratio between digestible calcium
and digestible phosphorus is less than 1.40:1. And this number is based on the shown results for
heavier pigs. The second hypothesis is that increased dietary calcium increases plasma calcium
concentration and decreases expression of genes related to transcellular calcium absorption and
tight junction proteins.

The second hypothesis is based on the fact that there are two routes for intestinal calcium
absorption, the paracellular pathway and the transcellular pathway, and the use of each pathway is
dependent on the concentration of calcium in the diet. To set up this slide, here we observe the
enterocytes that line the villi in the intestinal tract. The brush border is facing the luminal side
and in the opposite side it is the basolateral surface. So where there is low calcium in the diet,
calcium is absorbed through the cell. First, it enters the cell through a brush border channel such
as TRPV6. Once in the cell, calcium is bound to calcium-binding proteins such as D9k which move
calcium towards the basolateral side to be finally released by a plasma membrane calcium ATPase.
But, if there is high calcium in the diet, calcium is absorbed through tight junction proteins that
are located in the intercellular space. These proteins could be integral proteins such as claudin-1
and occludin, or peripheral membrane proteins such as ZO1.

Moving on to the materials and methods, 20 corn-soybean meal based diets were formulated using four
levels of digestible phosphorus, from 0.16 to 0.50%, representing from 50 to 150% of the
requirement, and five levels of total calcium, from 0.21 to 1.19% representing from 30 to 170% of
the total calcium requirement. We did not include phytase in these diets, and we used values from
digestible calcium in the feed ingredients included in the diets to calculate the digestible calcium
concentration in each diet. This resulted in 20 different ratios between digestible calcium and
digestible phosphorus that ranged from 0.28:1 to 4.63:1, being 1.33:1 the ratio from the diet where
the estimated requirement were used.  

We fed these diets to 640 pigs. There were four pigs per pen for a total of eight replicate pens per
diet. Diets were fed for 21 days and growth performance parameters were calculated. On day 21, one
pig per pen was euthanized and the right femur, blood samples and duodenal and ileal tissue were
collected to measure bone characteristics, plasma calcium, and gene expression. 

For the statistical analysis, a second-order surface response model was used. The model includes the
lineal effect of calcium and phosphorus, the quadratic effect of calcium and phosphorus, and
interactions between linear and quadratic calcium and phosphorus. 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.

Moving on to the results, I’m going to start with data from growth performance and bone
mineralization. In the model to estimate maximum growth performance, three of the four interactions
between calcium and phosphorus were removed because they were not significant. 

This graph shows the average daily gain of pigs fed the experimental diets. The horizontal axis
represents the five levels of digestible calcium, and the vertical axis represents the predicted
variable. And from now on, the green line represents phosphorus below the requirement, the orange
line represents phosphorus at the requirement, and the purple and red lines represent phosphorus
above the requirement. Being purple, 0.42% and red, 0.50% of digestible phosphorus. So if phosphorus
is below the requirement, increasing dietary calcium reduces average daily gain. This is likely
because excess calcium may bind phosphorus, which exacerbates the already phosphorus deficiency.
Now, if phosphorus is provided at the requirement, from the lowest level of calcium, there is an
improvement in gain, indicating that in these diets phosphorus is limiting gain; but again, as more
calcium is included in the diet, average daily gain is reduced. So there is a detrimental effect of
excess calcium in average daily gain. However, if phosphorus is above the requirement, the negative
effect of increasing concentrations of dietary calcium is ameliorated, meaning that as the
concentration of phosphorus increases, calcium becomes more limiting than phosphorus. The predicted
maximum responses were observed at ratios of 1.93:1 if phosphorus was below the requirement, 1.39:1
if phosphorus was at the requirement, 1.28:1 if phosphorus was at 0.42%, and 1.22:1 if phosphorus
was at 0.50%.

For gain to feed ratio, similar response: decrease in feed efficiency if phosphorus is below the
requirement, improvement if phosphorus is at the requirement, indicating that phosphorus is the
limiting nutrient in the diet. And, by the addition of more phosphorus in the diet, calcium becomes
more limiting than phosphorus. The maximum responses were observed at ratios of 1.51:1 if phosphorus
was below the requirement, 1.25:1 if phosphorus was at the requirement, and 1.20:1 and 1.17:1 to if
phosphorus was at 0.42 and 0.50%, respectively. So here we observe that for gain to feed ratio the
values were smaller than for average daily gain, which needs to be taken into account. But, to
maximize both average daily gain and gain to feed, a ratio between digestible calcium and digestible
phosphorus less than 1.40:1 is needed.

Moving into the results for bone mineralization, for this variable, the full model was used.

In terms of bone mineralization, this graph shows the results from concentration of bone ash in
grams per femur. So here we observe that at the lowest level of calcium, there are small differences
among the four levels of phosphorus, but if we increase the concentration of calcium to the
requirement level, there is an improvement in bone ash if phosphorus is at or above the requirement.
And, if we keep increasing the concentration of calcium in the diet, then we observe differences
among levels of phosphorus. This indicates that if calcium is low in diets, calcium is the limiting
nutrient for bone synthesis. But, if dietary calcium is high, phosphorus becomes the limiting
nutrient, demonstrating that the presence of both calcium and phosphorus is crucial for the
synthesis of bone tissue. The maximum responses were observed at ratios of 1.66:1 if phosphorus was
at the requirement, 1.50:1 if phosphorus was at 0.42%, and 1.30:1 if phosphorus was at 0.50%. Here
we can also observe that if phosphorus was below the requirement, increasing concentrations of
calcium did not affect the concentration of bone ash.

Now let’s move into the results for plasma calcium and gene expression. In the model used for the
concentration of calcium in plasma, all the interactions were removed because were not significant.

Here, we are looking at the results for plasma calcium. We can observe that there is an increase in
the concentration of plasma calcium as more calcium is included in the diet. However, the effect of
calcium is quadratic, meaning that because of hormonal regulation, the increase in plasma calcium as
a result of increasing dietary calcium will plateau. In terms of phosphorus, we observe that the
concentration of plasma calcium decreases as the concentration of dietary phosphorus increases. This
means that if phosphorus is available, more bone tissue will be synthesized, and therefore calcium
will be pulled out from the bloodstream to be stored in bones and less calcium will be present in
plasma.   

Now, in terms of gene expression, the model only contained the linear effect of calcium.

For the transcellular pathway, which was tested in the duodenum, the target genes were TRPV6 for the
brush border channel, s100g for the calcium binding proteins, and ATP2B1 for the calcium membrane
ATPase. However, only TRPV6 and s100g could be predicted by the amount of dietary calcium. 

So here, we have the results. As more calcium is included in the diet, there is a reduction in the
expression of these two genes, so this indicates that if the diet is high in calcium, the
transcellular absorption of calcium will be reduced.

In terms of the paracellular pathway, the target genes were CLDN1 and OCLN for the integral membrane
proteins and ZO1 for the peripheral membrane protein. In the duodenum, only ZO1 and OCLN could be
predicted by the amount of dietary calcium, whereas in the ileum, only ZO1 and CLDN1 could be
predicted by the amount of calcium in the diet.  

Here we have the results for duodenum. Again, we observe a reduction in the expression of these
genes as more calcium is included in the diet. So we believe that less expression of tight junction
proteins means more opportunity for calcium to cross the intracellular space. Therefore the
paracellular calcium absorption will be increased under high calcium diets.   

Similar results were observed for CLDN1 and ZO in the ileum: a reduction in the expression calcium
increases in the diet, indicating that paracellular calcium absorption increases if the amount of
dietary calcium is high.

So in conclusion, increasing dietary calcium decreases growth performance of pigs if phosphorus is
at or below the requirement. The ratio between digestible calcium and digestible phosphorus to
maximize growth performance is less than to maximize bone ash. In pigs of 11- to 25-kg, a ratio of
around 1.35:1 is needed to maximize growth performance if phosphorus is at the requirement. And
increasing dietary calcium increases plasma calcium concentration, decreases transcellular but
increases paracellular calcium absorption.

With this, I would like to acknowledge AB Vista for the financial support, and everybody from Dr.
Stein’s lab. And if you want to learn about the research we are conducting in our lab, please visit
our website at nutrition.ansci.illinois.edu. Thanks for listening.