Hello, everyone, my name is Vanessa Lagos. I am a third year PhD student under the supervision of Dr. Hans Stein, and today I will be talking about requirements for calcium by growing pigs, and I'm going to start talking about limestone.
Limestone is the main source of calcium in diets for pigs, and is an inexpensive feed ingredient that tends to be over supplied in diets, because little attention is paid on the inclusion level of these ingredients in the diets. Also, because it's often used as a carrier in premixes, and as a flow agent in feed ingredients, this last point here, it's very important, because these may result in variability in the concentration of calcium in feed ingredients.
So, I'm going to use this paper published in 2016, as an example, so in this study, 20 sources of soybean meal from the United States were collected, and were analyzed for calcium, and we observe some variability here. But if we cross a line to the point where the value for the average calcium in soybean meal is located 0.33%, we observed that some sources are pretty close to this value, but there are some sources, especially that are quite high, and these may result in a calcium being over supplied in diet.
So, if we look at paper published in 2016, where almost 800 commercial diets from the European Union were collected from both the swine and the poultry industry, we can observe that indeed, there is an excess of 0.22% units in average compared to what is formulated. But why this is important? So this paper was published in 2011, and then here, the increasing levels of calcium from limestone, were used to evaluate the effect of calcium concentration on the apparent total track digestibility of phosphorus. 
From here, we can observe that there is a linear reduction in the digestibility of phosphorus as we increase the concentration of calcium in the diets. So, if we think about the previous slide, and we take some example, this diet here 0.22% units will be around here. So, by doing these, we are decreasing the digestibility of phosphorus by 11% unit, and this is a problem because it has been demonstrated that phosphorus deficiency results in decrease in feeding intake.
So, now let's talk about little bit about requirements. Currently, calcium requirements are expressed as total calcium, and to understand how these values are obtained, first we need to know the content of body protein with this value, we can estimate body phosphorus mass, because there is a linear relationship between body nitrogen content and body phosphorus content. So, with the body phosphorus mass, we can estimate phosphorus requirements expressed as standardized total track digestible phosphorus, and simply by multiplying these number by 2.15, we can obtain the requirement for total calcium, but these value 2.15 is not quite clear where it's coming from and therefore, the NRC stated that a ratio between digestible calcium and digestible phosphorus would be preferred, but because of a lack of data, this was not possible.
So what is the data that is missing the digestibility of calcium in feed ingredients, so we know that we can provide calcium in diets for pigs using plant feed ingredients, animal feed ingredients and inorganic sources. But 10 years ago, this was the only information that we had, the concentration in each individual ingredient, so a number of experiments were conducted in order to obtain these values, and their number of questions came up. So the first question was, whether values for duodenal, ileal, or total tract digestibility should be used. So I'm using this paper published in 2014, where double cannulated pigs were used one cannula was located at the beginning of the duodenum and the second cannula was located at the end of the ileum and feces were also collected. So in this paper, two conclusions were made: First, there is an important amount of calcium being absorbed in the stomach, and second, there are no differences between values for ileal o total tract digestibility. So because of these values for total track digestibility are used in calcium, because this procedure is cheaper and less invasive for pigs. 
The second question was whether we should use apparent total track digestibility ATTD or standardized total track digestibility STTD. So this paper was published in 2013 and in this paper experiment was described, and here increasing levels of calcium, were used and the calcium was coming only from canola meal. So if we look at the effect of the concentration of calcium on ATTD values, we can observe that as calcium increases in the diet, ATTD values also increases. However, this is a result of the presence of endogenous losses of calcium. So if calcium is low in the diets the proportion of calcium that is present in feces that comes from endogenous sources is high, and therefore, the digestibility is small. But if we have diet with high concentration of calcium, this proportion becomes smaller and therefore, it seems that the digestibility is greater. But this is not the case, because if we correct the ATTD values by the endogenous loss of calcium, we can obtain STTD values, and we can observe here that this response is no longer observed, so, because of this, and because STTD values are believed to be more additive in mixed diets, these are the values that are currently used.
Now, the next question is, what about phytase? What is the role of phytase here, so we know that phytase is the …… of phosphorus storage implants, but all their nutrients, including calcium, can bind the phytase molecule. So I'm going to show data from three different papers published in 2015, 2017, and 2019, where the effect of including 500 or 1000 FTU phytase on the STTD of calcium in limestone was observed. Here, we can observe that in the three experiments, there was an increase in the digestibility of calcium in limestone by the inclusion of fiber. And you may wonder why this is the case, even a limestone is an organic source of calcium, where calcium from limestone combined the phytase from other ingredients, let's say corn in the gastrointestinal tract of pigs, and therefore, the presence of phytase, will release calcium will increase digestibility and will increase the amount of calcium available for pigs, so it's also pretty important. 
So currently, we have values for the digestibility of calcium in mineral supplements, plant feed ingredients, and animal feed ingredients, using diets without phytase and with phytase, and if we take these values, and we multiply them by the concentration of calcium in each individual ingredient, we can obtain the concentration of standardized total track digestible calcium or STTD calcium. So with these numbers, we can formulate diets, and we can also estimate requirements for calcium express as STTD calcium. Four number of experiments were conducted in order to determine these requirements. So data from the first experiment was published in 2016, and here, pigs from 11 to 25 kilograms were used. So I'm going to show results for gain to feed ratio when these experiments are fixed level of digestible phosphorus and six levels of digestible calcium were used, and in the horizontal axis, we have the levels of digestible calcium. So what we observe here is that as calcium increases in the diet, there is a reduction in gain to feed. So if we go beyond the requirement, which is this point here, immediately gain to feed is going to be affected. But also because of the nature of this response, it was not possible to obtain a value from digestible calcium that maximizes this response.
Therefore, for the following experiments, not only different levels of digestible calcium, but also different levels of digestible phosphorus were used. So we repeated this experiment, we using the same body weight ranges from 11 to 25 kilograms. But here we use four levels of digestible phosphorus and five levels of digestible calcium, So I'm going to show the results for estimated gain to feed. And I'm calling estimated, because we use a source as response model to obtain the values that I'm going to show in a second, after I set up this slide, so the horizontal axis represents the digestible calcium values, and these values represent 30, 60, 100, 140 and 170% of the calcium requirement. And we also had values for digestible phosphorus that represent 50, 100, 130 and 150% of the requirement for phosphorus, and these values were represented by green, orange, purple, and red lines. So, if we look at the diet with the lowest level of phosphorus. We observed that as we increase the concentration of calcium, there is a decrease in gain to feed. Now, if we increase the concentration of phosphorus add to the requirement level, from the lowest level of calcium, there is an improvement in gain to feed. But if we look here, what happens when we go above the requirement is that we affect growth performance. So excess calcium not only affects phosphorus digestibility, but also performance of pigs. But if we have digestible phosphorus above the requirement, these negative effects of excess calcium is ameliorated, and because we use a model to obtain these values, we obtain the ratio digestible calcium to digestible phosphorus that maximizes each response, and in this case, when phosphorus was at the requirements, this value was 1.39 to 1.
Now we're looking at results for estimated bone ash in grams per femur, and here we observe that at the lowest level of calcium, that are just small differences, among the levels of phosphorus, if we have calcium at the requirement, there is an improvement in bone ash if we have phosphorus at the requirement, or above, but small differences here, and if we keep increasing the concentration of calcium, we observe differences among these three diets, and what we observed is that if there is more phosphorus, then there is more bone ash. So this indicates the need of both calcium and phosphorus for bone synthesis, and the importance of the ratio between calcium and phosphorus. And then the ratio that maximize bone ash when phosphorus was at the requirement was 1.66 to 1. So we can observe that we need more calcium to maximize bone ash than what we need to maximize gain to feed. 
So, the following body weight group that was used was 25 to 50 kilograms. And in this experiment, also 20 diets were used. For here, I'm showing the results for estimated final body weight. And here, we see a similar pattern as in the previous experiment. So if calcium is at the lowest level, there are just small differences among concentrations of phosphorus. But we observe a reduction in body weight as calcium increases on the diet, and these effect is exacerbated if phosphorus is also below the requirements. And again, if we look at the orange line, when phosphorus is at the requirement, if we go above the requirement level for calcium, there is a decrease in final body weight. So the following group that we did was 50 to 85 kilograms, and here, we only use 15 diets. So five levels of digestible calcium and three levels of digestible phosphorus. So we are looking here at estimated average, a daily gain. And again, if we have phosphorus at the lowest level, there is a linear decrease in average daily gain as dietary concentration of calcium increases in phosphorus is the requirement. We observe that if we go above calcium requirement, there is a decrease in average daily gain, but the overall negative response is ameliorated. Now, if we look at the value, where are the diets where phosphorus was above the requirement, we observe a different response. So the implication of this response is that if we are formulating diets for growing finishing pigs that are already high in phosphorus, let's say we're using ingredients such as DDGS, then we may also want to increase the concentration of calcium to maximize this response, as we observe here.

For estimated bone ash but expressed as a percentage, we observe that there is an increase in percentage of bone ash as we increase dietary digestible calcium however, this response again is dependent on the concentration of phosphorus, the more phosphorus, the more percentage of bone ash. So it is important to take into account the ratios not only the individual values, the final group that we analyzed, or we estimated requirements was 100 to 130 kilograms. Here also 15 diets were used and I'm going to show data from estimated average daily feed intake. And first, I would like to point out that this result was the same as in the previous experiment with 50 to 85 kilograms, regardless of the concentration of digestible phosphorus, there was a linear decrease in feed intake, as the concentration in the diet of calcium increased, and the support what I was mentioning at the beginning, that phosphorus deficiency results in decrease in feed intake so here we can observe that.
For estimated average daily gain again there is a negative effect of increasing calcium if we go above requirement, but the response is exacerbated if we have digestible phosphorus also unlimited in diets. So to summarize the results for this experiment, we had four experiments using different body weight groups, and we targeted growth performance and bone ash. So we can conclude that if digestible phosphorus is at the requirement, these values that I'm showing here are the values that will maximize each response. But, I would like to discuss these slides a little bit, if we look in all of the groups, we can observe that there is a greater need for calcium, there is a greater ratio to maximize bone ash than to maximize growth performance. So this is true in all of the groups. So the implication for this is that if we are formulating diets for finishing pigs these values are the ones that we should use. But if we are formulating diets for sows or for pigs that are going to be used in the future for reproduction, we should use these values instead.
Now another observation here is that this ratio decreases as the animal gets heavier, but is the opposite for bone ash. So because these were short term experiments, a follow up experiment was needed to validate these numbers, and to verify what is the effect of using values of maximize growth performance on bone health, especially in this last group, where we can see that the difference is quite high. So we conducted an experiment to validate this data, we use pigs from 11 to 130 kilograms, we use a 5 phase program, and we use the same body weight groups, as the one that are listed in the NRC: 11 to 25, 25 to 50, 50 to 75, 75 to 100 and 100 to 130 kilograms, and because we didn't have an experiment, for 75 to 100, we average the values below and above. In each phase we formulated four diets using a two by two factorial design, we had diet formulated based on the NRC, or using total calcium values and the ratios that I just mentioned in the previous slide. We had also two levels of phytase 0 or 500 FTU and I would like to spend a little bit of time explaining how the diets with phytase were formulated.
So in the diets, with total calcium values, we use matrix values the recommended by the phytase manufacturer, and these values were 0.16% total calcium and 0.11% digestible phosphorus. But for the diet based on digestible calcium and digestible phosphorus ratios, we also use 0.11% digestible phosphorus, but for calcium, we use the digestibility values in the ingredients that we included on these diets, and in these values came from diets with phytase. So that's how we took the phytase effect into account. So here I'm showing the results for growth performance for the overall experimental period, because it was the same response in all of the phases, and for average daily gain an average daily feed intake. So to set up this slide blue colors represent diets without phytase, orange colors represents diets with phytase, lighter colors indicates diet formulated based on total calcium, and darker colors indicate diets formulated based on digestible calcium values. And here we can observe that there was no effect of calcium requirement phytase, and there was no interaction between main effects. So we can conclude here that we can use both values, digestible calcium ratios and total calcium, and we want to obtain the same response as long as we made sure that we are not including calcium in excess.
Now for bone ash we did observe an interaction between calcium requirement and phytase, and here we observed that in the diets without phytase, there was a decrease in bone ash if diets were formulated based on digestible calcium. So these diets contain less calcium, and because they were formulated to maximize growth performance, and not bone ash, so these response was expected. But in diet with phytase, we didn’t observe differences, because the concentration of calcium in these diets was pretty similar. So, we know that we need more calcium to maximize bone mineralization than to maximize growth performance. And we already saw what happens if we use these values that maximize performance on bone ash. 
But also data demonstrated that we also need more calcium to maximize calcium retention and to maximize bone mineralization. So the question was, what happens if we use growth performance values? Are we compromising calcium retention? So to answer this question, we conducted a balance study. So here we use four diets, these diets were the same as in the previous experiment for the body weight range from 50 to 75 kilograms, we had an average body weight of pigs of 60 kilograms, and we did total but separate collection of urine and feces. So looking at the results for digestibility, in terms of calcium, there was no interaction between calcium requirement and phytase, but there was a significant effect of phytase. So we can observe here that regardless of the calcium requirement, estimate, either total calcium or digestible calcium, there is an increase in digestibility of calcium by the inclusion of phytase, and as I mentioned before, calcium also binds a phytase molecule and therefore phytase will release calcium.
In terms of the digestibility of phosphorus, we can observe here that there was an interaction between calcium requirement and phytase. So in the diets without phytase, we can observe that there was an increase in digestibility of phosphorus, if we formulated based on digestible calcium values. So, these diets had less calcium, and therefore we observe greater digestibility of phosphorus. So, as I mentioned, and I showed at the very beginning of the presentation, the more calcium in the diet, the less digestibility of phosphorus and again here in the diets with phytase, the concentrations of calcium were pretty similar, so no differences in phosphorous digestibility were observed. 
In terms of calcium retention, we observed an interaction between calcium requirement and phytase, so here we look at the diets formulated based on total calcium, we observed that there was a decrease in calcium retention if phytase was included in the diet, this was not expected, but if we look the real question here, if there is a difference or negative effect of formulated diets based on digestible calcium, we observe that the answer is no there are no differences and therefore, we are not compromising calcium retention by using digestible calcium values. 
So, in conclusion, calcium is usually over supplied in commercial diets, excess calcium not only results in reduced phosphorus digestibility, but also in reduced growth performance, we have provided ratios between digestible calcium and digestible phosphorus obtained in short term experiments, and these values have already been validated and total calcium values can also be used, we proved that but only if we are sure that calcium is not provided in excess.
So if you take home messages before finishing this presentation, excess calcium must be avoided in diets and in order to these we always have to take into account the phytase effect we can use now, ratios between digestive total calcium and digestible phosphorus in pigs from 11 to 130 kilograms. But if we want to keep using total calcium values or requirements from the NRC, it's totally fine, as long as we understand that these are maximum values if we go beyond these values we are going to compromise growth performance. So now I like to take this opportunity to acknowledge AB Vista for the financial support AB Vista sponsors both my Master's and PhD projects, and also all the trials that I showed before for digestibility and requirements. All the members of the Stein Monogastric Nutrition Laboratory for their support, and all of you for your attention, and if you want to learn more about this topic or other topics related to pig nutrition, please visit our website at nutrition.ansci.illinois.edu. Thank you.