Hello. I am Hans H. Stein, I'm a professor at the University of Illinois, where I'm working in the Division of Nutritional Sciences and the Department of Animal Sciences. I would like to talk to you today about some work we have conducted in recent years to investigate absorption of calcium and the expression of genes involved in calcium absorption in pigs.

And some of the take-home messages that I would like to leave you with today include the following:

First, I will show some data that demonstrates that microbial phytase added to diets for pigs will increase the digestibility of calcium and, therefore also the absorption of calcium.

Secondly, I will also show you data that indicates that calcium digestibility should be expressed as the standardized total tract digestibility of calcium.

Third, I will also show you some data that indicates that there is minimum regulation of intestinal calcium absorption.

Fourth, the data we have obtained would suggest that the kidney is the main regulatory site for calcium homeostasis, and that's where it is decided if calcium stays in the body or if it is excreted.

Before we start, I would like to acknowledge the large amount of work that has been conducted by two of my previous Ph. D. students: Dr. Caroline González-Vega and Dr. Laura Merriman. And both Caroline and Laura worked in our lab for several years, and they are the ones who conducted most of the research that I will talk about this afternoon, and I want to acknowledge the contributions both of these former students made to our work.

Now, when we started out with this work, we had several questions on calcium digestibility and calcium metabolism.

First, we needed to find out if calcium digestibility should be determined as a duodenal digestibility, ileal digestibility, or total tract digestibility. So that was one question we had.

Another question we had was, we didn't know if there are significant endogenous losses of calcium into the intestinal tract that would influence the values for digestibility that we might obtain. So we needed to determine if there are such endogenous losses.

And third, we needed to determine what the influence of dietary phytate is, what the influence of microbial phytase added to the diets might be, what the influence of fiber is, and what the influence of fat on calcium digestibility might be. So we started out wanting to answer some of these questions.

So the first question we had was if digestibility of calcium should be determined as duodenal digestibility, ileal digestibility, or total tract digestibility. And to answer that question, we inserted two cannulas in growing pigs. One cannula was in the duodenum, and the other cannula was in the ileum. And then we fed different diets to these pigs, and we took samples out from the duodenum and we took samples out from the ileum, and we also collected fecal samples from the pigs to answer this question.

And here are some data from one of the diets we had. This indicates the digestibility of calcium in calcium carbonate. And you can see there's no difference between the duodenal digestibility, the ileal digestibility, and the total tract digestibility. So, this answers the question that it appears that most of the calcium, at least from calcium carbonate, is absorbed very early on in the digestive tract. And there seems to be no further absorption further down in the intestinal tract. However, there are differences among different sources of calcium, but most of the calcium absorption takes place early on in the digestive tract. Based on these results, we concluded that it doesn't really matter where we measure calcium absorption; but because it is easier and less expensive to determine total tract digestibility, we decided that in all future work we conduct we would determine the total tract digestibility of calcium.

The next question we had was whether or not there are significant endogenous losses of calcium into the intestinal tract. So we designed an experiment to answer this question. In this experiment, we had diets that were based on canola meal, and the reason we used canola meal is that canola meal is one of the few plant based ingredients that also contains significant quantities of calcium. So we had canola meal either without microbial phytase or with microbial phytase.

And within each of these two sources of canola meal, we created four diets. And we created the diets in such a way that the diets contained 0.08% calcium, 0.16% calcium, 0.24% calcium, or 0.32% calcium. We made the same diets without phytase and with phytase. And all the calcium in these diets came from canola meal. So the way we designed the diets was simply, we added more and more canola meal to increase the concentration of calcium in the diets.

And here are results from this experiment. We have the orange line indicating the data for the canola meal without phytase, and the blue line indicating the results from the diets with phytase. And a couple of things worth to note here. First, we'll see that the blue line is greater than the orange line. That indicates that when we added phytase to the diet, digestibility increased, and that happened regardless of the level of calcium we had in these diets. Secondly, you can also see that whether we had phytase or not in the diets, the apparent total tract digestibility of calcium increased as we increased the concentration of calcium in the diets. So this indicates that there is an influence of the concentration of calcium in the diet on the apparent total tract digestibility.

And we can see here that both calcium concentration is significant, and also the influence of phytase is significant. Now, this obviously creates a problem if we have to design diets based on digestible calcium, because if the digestibility of calcium is dependent on calcium concentration in the diet, then the question is, which values should we use?

So we had to think about endogenous secretions into the intestinal tract. And in this depiction, the calcium coming from the diet is illustrated by the orange balls, and the endogenous calcium being secreted from the pig into the intestinal tract is illustrated by the blue balls. And here in a second, we will try to feed the pig. And now we will see what happens.

You see the orange balls are coming in, but there are also blue balls being added, indicating endogenous secretions into the intestinal tract. In the small intestine, most of both the endogenous and the diet calcium is absorbed, but you'll notice here that when the calcium exits the pig and is excreted in the feces, we have both blue and orange balls here. That indicates that the calcium we analyze in the feces is a mixture of calcium that was not absorbed from the diet, but also calcium that came from endogenous secretions. So we get both endogenous calcium and diet calcium in the feces.

Because we saw that there was an increase in digestibility as we added more calcium to the diet, we can assume that increase in apparent total tract digestibility was caused by the endogenous losses, because that is a classic response we see if there are endogenous secretions of a nutrient into the intestinal tract of the pigs.

We could take those digestibility values and put a regression coefficient to them and regress back to zero intake, and you will see here that we get a negative intercept; that indicates that there indeed was a significant endogenous secretion into the intestinal tract. And the two values we got for the canola meal without and with phytase was 0.16 mg/kg dry matter intake, and 0.19 mg, and those two values were not significantly different, indicating that phytase does not influence the endogenous losses. However, what is important is we can use those values to correct the values for apparent total tract digestibility to calculate true total tract digestibility.

And if we do that, you'll see here that now the orange line is a straight line, the blue line is a straight line, and that means there's no longer any influence of the concentration of calcium in the diet on the digestibility value that we get.

And you'll see calcium here is no longer significant. The P value is 0.86. So now we have taken the influence of the diet calcium concentration away from the calculated values for digestibility of calcium. However, we still notice that the blue line is greater than the orange line, indicating that if we add phytase to the diet, we get greater digestibility values compared with if we have no phytase in the diet. So, phytase is still significant here. So the conclusion from this work was: one, yes, there is endogenous calcium being secreted into the intestinal tract, and that influences our values for apparent total tract digestibility. And therefore, as the second conclusion, we can say that we should calculate either true digestibility or standardized digestibility to obtain values for calcium digestibility that are additive in mixed diets. In this case, we calculated the true digestibility, but in our further work, we used standardized total tract digestibility of calcium.

Now here is another experiment we conducted. And in this case, we had a corn-soybean meal diet without microbial phytase or with 500 units of microbial phytase, 1000 units of microbial phytase, 2000 units, or 4000 units of microbial phytase. And in this case, we also calculated apparent total tract digestibility of calcium. And you'll see here that there is a significant increase in calcium digestibility as we move from no phytase up to approximately 1100 units of phytase in the diets, and then it plateaus after that. Now, as I said, these diets were corn-soybean meal diets. There's really no calcium being contributed by corn, and the amount of calcium coming from soybean meal is pretty limited. So the majority of the calcium in this diet came from calcium phosphate or from limestone. However, calcium phosphate and limestone do not contain any phytate, so you would not expect that phytase would increase the digestibility of calcium in those feed ingredients. But the only way we can explain results of this graph is that indeed, there must have been some extra release of calcium from either the calcium phosphate or from the calcium carbonate as we fed these diets.

When we add corn and soybean meal to the diet, we also add a significant amount of phytate. At the same time, we have limestone added to the diet, and that is where we get the majority of the calcium from.

So we hypothesized that the calcium from the calcium carbonate or the calcium phosphate may bind to the phytate from corn and soybean meal when it reaches the soluble environment in the stomach.

So based on that, we conducted an experiment in which we determined the digestibility of calcium in calcium supplements without phytase and with phytase.

And in this case, we calculated the standardized total tract digestibility of calcium. And we have here, in the orange bar, the diets without phytase, and in the blue bar, we have the diets with phytase. And the three ingredients we used in this experiment were monocalcium phosphate, dicalcium phosphate, and calcium carbonate. And you'll see here that there was no difference between monocalcium phosphate fed without phytase and with phytase. There was no difference in the digestibility of calcium in dicalcium phosphate without phytase and with phytase. However, for calcium carbonate, there was a significant improvement in digestibility when we added phytase to the diet. So that indicates that it is the calcium from calcium carbonate that may bind to the phytate in corn and soybean meal as it reaches the stomach, and therefore is less digestible because of that phytate. However, if we add phytase to the diet, then we liberate some of that calcium that was bound to phytate from corn and soybean meal, and therefore we increase the digestibility. So data from this experiment really answered the question, yes, there can be binding of calcium from inorganic sources of calcium in the diet to the phytate, and that's why phytase can help us increase the digestibility of calcium in these ingredients.

We conducted another experiment where we pretty much did the same as in the previous experiment but in this case, we used fish meal, which has a high concentration of calcium, primarily in the bones of these fish. And again, we saw the same thing as we did for calcium carbonate, that when we added phytase to fish meal, then we increased the digestibility of calcium. And the reason, again, is that if you add fish meal to a corn based diet, where there's lots of phytate, then some of the calcium from fish meal can bind to the phytate in corn, and therefore we get a low digestibility. But if we add phytase to the diet, then we release that calcium and we increase the digestibility of calcium from fish meal.

We also conducted different experiments where we investigated the effects of zinc oxide on the digestibility of calcium. And the reason we looked at zinc oxide is that zinc and calcium use the same transporter to be absorbed into the enterocyte that lines the intestinal tract. So, our hypothesis was that if we added extra zinc, that might influence calcium digestibility.

So here we have three different types of diets: one with no phytase, one with 1000 units of phytase, and one with 3000 units of phytase. And these, again, are corn-soybean meal type diets, with calcium carbonate as the major calcium source. And then within each of the three diets, we had one diet without extra zinc oxide added, so these diets contained zinc just to cover the requirement (approximately 125 mg/kg). But then we also had a second diet that contained an addition 3000 ppm of zinc oxide, which provided approximately 2500 extra mg/kg of zinc to these diets. So, we had a total of six diets here. The orange diets are the diets without zinc oxide, the blue diets are the diets with zinc oxide. And you'll see here that if you follow just the orange bars, as we increased phytase in the diet, we saw an increase in calcium digestibility. So that is very similar to what we saw in the previous experiments, and again, we can attribute this increase to release of calcium from calcium carbonate that has been bound to phosphate in corn. For the diets that contain zinc oxide, we also see an increase in digestibility as we add phytase to the diet. However, for all three diets containing the zinc oxide, we have a reduced digestibility of calcium compared to the diets without zinc oxide. This tells us that if we add 3000 ppm of zinc oxide to the diets, then we will have a reduced digestibility of calcium, and to some degree a reduced efficiency of the phytase in the diet. So we have to take that into account if we add extra zinc to our diets.

We also conducted an experiment to determine if fat has any influence on digestibility of calcium (in this case it was apparent total tract digestibility). And here we had a corn-soybean meal based diet in light blue—we call that a control diet—and then to that diet we added 7% tallow, 7% choice white grease in yellow, 7% palm oil in purple, 7% corn oil in green, or 7% soybean oil in red. And as you can see here, with the exception of choice white grease, we increased the digestibility of calcium in all cases when we added these fat sources to the diet. So that tells us that at least tallow, palm oil, corn oil, and soybean oil will increase the digestibility of calcium. The reason most likely is that these fat sources will reduce the passage rate in the intestinal tract and therefore there's more time for absorption of the calcium. In the case of choice white grease, we did not see this increase. We're not certain why that is, but it is possible that it has something to do with the quality of that specific source of fat that was used here. But overall, the results from this experiment indicate that fat certainly has no negative effect on calcium absorption, and it appears that for most fat sources, we see an increased digestibility of calcium when we add fat to the diets.

In a different experiment, we wanted to answer the question of whether or not the particle size of calcium carbonate has any influence on the digestibility of calcium.

So here we had four different sources of calcium carbonate. And it was the same basic source we used, but it was ground to different particle sizes, so we ended up with particle sizes of 200 microns, 500 microns, 750 microns, or 1125 microns. And then we included the calcium carbonate from each of these sources in diets fed to pigs as the only calcium source. And you can see here the digestibility values were not changed as we increased the particle size of calcium. So whether it was 200, 500, 750, or 1125 microns, we had exactly the same digestibility of calcium.

We also used these four sources of calcium to feed to pigs to determine growth performance. And we have here in the blue bars average daily feed intake, in the orange bar we have average daily gain. And the diets were fed to pigs from 10-25 kg. And you can see the feed intake was between 728 g and 738 g, no significant difference there. Average daily gain was between 468 g and 475 g, again very very close to each other and no differences among the four groups.

In the case of gain:feed, the values ranged from 0.637 up to 0.649. These values were not significantly different. So we can conclude from this work that the digestibility of calcium is not influenced by the particle size of calcium carbonate, and likewise the growth performance of pigs is not influenced by the particle size of calcium carbonate. So it appears that at least in the range from 200-1125 microns, it doesn't matter what the particle size of calcium carbonate is.

Having conducted these experiments, we had digestibility values for inorganic calcium, we had digestibility values for most of the plant ingredients that contain calcium, we had digestibility values for animal proteins that contain calcium, so based on that, we were able to formulate diets based on digestible calcium. And that was not the case previously.

So, to formulate diets based on digestible calcium, we had to think a little bit about how is calcium regulated in the body. And what happens is that we can have low calcium in plasma, and if that's the case, then there's a hormone called PTH that's secreted from the parathyroid gland.

And that hormone will activate cholecalciferol, which is a form of Vitamin D, and it activates cholecalciferol, into the active form 1.25-hydroxy Vitamin D, and this takes place in the kidneys.

And then, this activated Vitamin D3 called 1.25-hydroxy Vitamin D3, will bind to Vitamin D receptors in the small intestine, and thereby it may increase calcium absorption. So that's what happens when we have low plasma calcium concentrations.

When we have high plasma calcium concentrations, we have another hormone secreted. This hormone is called calcitonin. It's also secreted from the parathyroid gland.

Release of calcitonin will result in reduced calcium release from the bones, meaning we get less calcium pulled out of the bones into the plasma, and therefore we will see a reduction in plasma calcium concentrations.

So based on this, we designed an experiment with 11-25 kg pigs.

And we formulated six different diets, and they had standardized total tract digestible calcium concentrations from 0.32 up to 0.72. If you calculate on a total calcium concentration, it was from 0.40 up to 1.07%.

We fed these six diets to pigs in our research nursery facility, where we have four pigs per pen. And we fed the diets for 22 days. We measured feed intake on daily basis, and we also recorded pig weights at the start of the experiment and on day 10, and at the conclusion of the experiment. In addition, when we terminated the experiment on day 22, we killed one pig per pen and extracted the femurs from this pig. And therefore we were able to determine the bone ash in these different pigs.

We also took these six diets and fed them to pigs that were in metabolism crates. We collected feces and urine, and by doing that we were able to calculate the calcium balance, calcium digestibility, and calcium retention in these pigs.

Results of this experiment include the bone ash. And you'll see here that, if we had very low calcium in the diet, we had low bone ash. But as we increased calcium, we increased bone ash up to a certain point, and then it kind of plateaued and we didn't see a further increase. And we could estimate the maximum point here at 0.48% standardized total tract digestible calcium. So it appeared that we need 0.48% standardized total tract digestible calcium to maximize bone ash in these pigs.

The reason we got the response to bone ash, we think, has to do with changes in the absorption of calcium from the intestinal tract. So let's look at what happens in the intestinal tract. And this is a depiction of the villi in the small intestine, where we have the enterocytes lining the villi, we have  a nucleus in each enterocyte, we have also what we call tight junctions closing the gaps between the different enterocytes. And these tight junctions are the square orange you see here between the cells. On the luminal side of the enterocytes, we have calcium channels, also in orange here, and those calcium channel  responsible for transporting the calcium from the lumen of the intestinal tract into the inside of the enterocytes. Once the calcium is inside the enterocyte, we have calcium binding proteins, and they are blue in this case, and those calcium binding proteins will then transport the calcium from the calcium channels and across the cell to the basolateral side, and on the basolateral side, we have two export mechanisms: one is a calcium-sodium exchanger that can get the calcium out of the cell, and we also have an active transporter we called PMCA1. On the luminal side of the enterocyte, we also have Vitamin D receptors. So the activated Vitamin D that is coming from the kidneys after being released will bind to the Vitamin D receptor, and then the receptor will translocate to the nucleus, and the nucleus will increase expression of both calcium channels and calcium binding proteins. So now we have more calcium channels to absorb calcium, and we have more binding proteins to transport that calcium across the cell.

So when the calcium comes into the intestinal tract here, again at low dietary concentrations, we can see what happens. Get the increased expression of calcium channels, therefore the absorption of calcium through the cell is very efficient, and most of these calciums that are coming in will be absorbed and transported through the enterocyte and released on the basolateral side. So that means that calcium absorption through the cell is very efficient. And this is called the transcellular calcium absorption.

So if we look at the regulation of these calcium transporting proteins, we can see that there is an increased expression of TRPV6, which is the calcium channel, if we have low calcium in the diet. There's also an increase in the calcium binding protein called CABP9K, if we have low calcium in the diet, but there's no effect of calcium diet concentration on the PMCA1 exporter of calcium. So the regulation really is on the absorption of calcium and of the transport of calcium through the cell.

In contrast, if we have high diet calcium, then we do not get the PTH released, that means we do not get Vitamin D activated in the kidneys, that means there's no 1.25-hydroxy Vitamin D3 coming to the intestinal tract to bind to the Vitamin D receptor, and therefore there's no increased expression of those calcium channels or binding proteins. However, because we have more calcium coming in at high diet calcium concentrations, we still get significant calcium absorption. And we can here see what happens when we have high concentrations of calcium in the diet.

And you'll see that calcium in this case will be absorbed not only via the calcium channels but also, and primarily, between the cells. And that is because those tight junctions that sit between the enterocytes, they become selectively permeable if we have high concentrations of calcium in the diet. And because they become selectively permeable, calcium can now be absorbed via those tight junctions, and therefore we still get significant calcium absorption although we don't have the increased expression of the calcium channels and the calcium binding proteins.

So in this case, where we have high diet calcium concentrations, we have a reduced expression of the calcium channels, TRPV6, we have a reduced expression of calcium binding proteins, CABP9K, we still have no change in the expression of the PMCA1, but we have this paracellular absorption of calcium between the cells because the tight junctions, as I said, are selectively permeable now.

So when we look at how much calcium was absorbed when we fed these six diets that contained between 0.32 and up to 0.72% standardized total tract digestible calcium, we will see that there was an a linear increase in calcium absorbed, calculated as g/day as we increased calcium in the diet. And what tells us is that there is really no regulation of total calcium absorption in the intestinal tract—if we feed more calcium, the pigs will absorb more calcium. And it appears to be a linear increase in absorption here, and there's no breakpoint. So, regulation of calcium absorption does not really take place in the intestinal tract. So, although as we showed with the gene expression data that there was a reduced expression of some of the genes needed for transcellular calcium absorption when we fed high concentrations of calcium in the diet, we can see here that that did not result in reduced absorption of calcium. And the reason is that we had an increase in the paracellular absorption of calcium, so the overall absorption of calcium was not really affected by the dietary calcium concentration.

So, as I said before, we also have regulation of calcium in the kidneys. And what happens here—we have a place in the kidneys called the distal convoluted tubules. In these tubules, we have the blood vessels very closely aligned with the urinary tract. And so, when the calcium is being excreted from the kidneys into the urinary tract, there is an opportunity that they can be reabsorbed into these blood vessels here.

And if we look at the cells in the distal tubules in the kidney, you will see they are very similar to the enterocytes we had in the small intestine. We have the same type of cells, epithelial cells; we have the tight junctions between the cells to prevent leakage; and we have calcium channels that eventually can absorb calcium into the cells. They are called TRPV6 as in the intestines, and there's also a second one here called TRPV5. We also have the calcium binding proteins, and there are two of those here: calcium binding protein 9K and also a 28K. And, on the opposite side, we have the calcium-sodium exchanger to transport calcium out of the cell, and we have the PMCA1 active transport, exactly the same way as we had in the enterocytes in the intestines. So in this case, if calcium is absorbed into the epithelial cells from the urinary tract, and it is transported through the cell and released on the other side, it will be released into the bloodstream, and therefore it will be re-used by the animal. We will see here also that we have a Vitamin D receptor sitting on these cells, the same way as we had in the intestinal tract.

So if we have low calcium in the diets, then what happens here is that the calcium that the kidney has excreted into the urinary tract for excretion from the body, it can be reabsorbed here via these calcium channels, and then transported through the epithelial cells and released into the bloodstream. So we get re-usage of the calcium that way, and a very much reduced excretion into the urine of calcium. And, when we looked at the expression of these genes, we saw an increased expression of both TRPV6, TRPV5, and the calcium binding proteins if we had low calcium in the diets.

In contrast, if we had high calcium in the diets, we saw a reduced expression of the proteins, both the calcium binding proteins transporting the calcium inside the cells, and also a reduced expression of the calcium channels, TRPV6 and TRPV5. That means there was very limited absorption of calcium into the cells if we had high calcium in the diet. And one difference between the kidney cells here and the epithelial cells in the intestinal tract is that high calcium in the urinary tract here will not result in paracellular absorption. So there's no absorption through the tight junctions in this case, as we saw in the intestinal tract. And therefore, all this calcium that is coming into the urine will be excreted if we have high calcium concentrations in the diet.

We also measured the output of calcium in the urine from these pigs. And as you can see here, as we had diets with increased calcium concentration, we saw an increase in urinary calcium output. So that tells us that the regulation of the calcium really takes place in the kidneys, and if there is too much calcium in the diet, that calcium will be absorbed, because of the paracellular absorption in the small intestine, but if the pig can't use that calcium, it will be excreted in the urine.

These data confirm some previous observations from our laboratory we had when we fed phosphorus-free diets and we looked at the effect of phosphorus concentration on calcium homeostasis. In this case we had two diets, one in which we had a high concentration of phosphorus, so the pigs were eating 12.21 g of phosphorus every day, and we also had a phosphorus-free diet, so the pigs were eating no phosphorus. And we have the phosphorus-free diet here in the blue bar, and we have the high phosphorus diet in the orange bar. You can see the calcium intake was almost the same in the two diets. The pigs were eating a little bit less of the phosphorus-free diet, but still significant amounts, so they had still significant intake of phosphorus. The amount of phosphorus absorbed was, as a percentage, the same for the two diets. So, the pigs that were fed the phosphorus-free diet absorbed exactly as much calcium as the pigs that were fed the high phosphorus diet. However, because both calcium and phosphorus are needed for bone tissue synthesis, then in for the pigs who were fed the phosphorus-free diet, they couldn't really utilize that calcium that was absorbed because there was no phosphorus to synthesize bone. And therefore, as we can see here, there was much more urine calcium excreted in pigs from the phosphorus-free diet than from pigs from the high phosphorus diet. So this further illustrates that there is no regulation of absorption of calcium in the intestinal tract, even in a case like this where there is no bone tissue synthesis because there's no phosphorus available. Even in that case, pigs will absorb the calcium, as much as they can. But then, because there's no phosphorus for bone synthesis, they will excrete that calcium into the urine.

The overall conclusions here include the following: We have seen that Vitamin D regulates transcellular calcium absorption in the intestinal tract. So that is the calcium that is absorbed via the calcium channels, and they also need the calcium binding proteins. That is regulated by Vitamin D, and indirectly by PTH.

We have also seen that if we have high calcium in the diets, then the paracellular calcium absorption increases. So the overall effect is that we still get the same percentage of calcium absorbed into the enterocyte, whether we have low or high calcium in the diet.

As a consequence, there is very minimal effect of diet calcium concentration on the absorption rate of calcium. We have almost the same digestibility of calcium, regardless of the concentration of calcium in the diet, if we calculate on a percentage basis.

And we've also seen that the regulation of calcium in the body mainly takes place in the kidneys. If we have low calcium in the body, then the kidneys will re-absorb much of the calcium from the urinary tract and direct it back into circulation. Whereas if we have high calcium in the diet, then the kidneys will not re-absorb the calcium and it will instead be excreted in the urine. So, urine calcium concentration will increase as calcium in the diet increases.

So in conclusion, we have seen that the digestibility of calcium has been determined for most ingredients. And we have decided that the most correct way to determine digestibility is to determine the standardized total tract digestibility of calcium because that gives us the same digestibility value regardless of the calcium in the diet. And therefore we get values that are additive in mixed diets.

We've also concluded that phytase and some other factors may affect digestibility of calcium. And phytase affects the digestibility of calcium not only in plant based feed ingredients that contain phytate, but also in some non-phytate feed ingredients such as calcium carbonate, and also in fish meal.

And finally, we have seen that there appears to be no effect of particle size of calcium carbonate on the digestibility of calcium, and there's certainly no negative effect of dietary fat; if anything, there appears to be a positive effect of fat on the digestibility of calcium.

With that, I would like to acknowledge, again, my students who have participated in this work and who have generated all the data that I have talked about here today. Without such great students, this would never have been possible. I also want to acknowledge the funding we have received. The majority of the funding for the work I've talked about here today was provided by AB Vista Feed Ingredients, which we are very grateful for. I also want to mention that if you are interested in more on our research, or if you're interested in more on calcium digestibility, then you can go to our website, nutrition.ansci.illinois.edu. All our research is published on this website; that includes peer-reviewed publications, abstracts, conference proceedings, book chapters, etc. There are press releases, there are newsletters, there are podcasts, and a number of other things. So if you have an interest in swine nutrition, please visit us at nutrition.ansci.illinois.edu.