Hi, my name is John K. Mathai, and I am a Ph.D. student in the Hans H. Stein Monogastric Nutrition Laboratory. In today’s podcast, I’m going to talk you about the effects of fiber on the optimum threonine:lysine ratio for 25 to 50 kg growing gilts. Let’s begin with a rather typical outline of the presentation. We’ll start the presentation with the background behind the questions we set out to answer. Then we will move into a discussion of two experiments that we conducted to answer the question of fiber’s effects on the threonine requirement in the pig. And then we’ll finish up with some conclusions and the implications of this research. Now, it’s worth noting that the experiments we are talking about today are actually the last two experiments in a series of experiments. Both the threonine titration and the nitrogen balance study, follow up on a lysine experiment and an amino acid digestibility experiment that we conducted; both of which were presented at the Midwest meetings earlier this year. The lysine titration enabled us to determine the exact lysine requirement for the group of pigs we were using for the subsequent experiments. This was necessary because our efforts were really to determine the effect of fiber on the threonine:lysine ratio. The amino acid digestibility trial allowed us to determine the exact amino acid digestibility of each of the ingredients that would be used in the subsequent trials. All of the ingredients for all of the trials came from single batches, and knowing the exact amino acid digestibility in each of these allowed us to accurately formulate diets in the subsequent studies. So with all that being said, let’s move into the background behind our studies. Overall, more and more fibrous ingredients are making their way into pig diets. And we know this is the case as we see the greater use of DDGS, various types of brans, and canola meals in swine diets. However, we have yet to fully determine the effect of fiber on the digestibility of nutrients in the diet. Most literature has shown that the inclusion of fiber in the diet will reduce both organic matter digestibility and energy digestibility. In terms of lipid digestibility, the effects remain unclear. Some evidence has shown that encapsulation by fiber reduces lipid digestibility, while other evidence indicates that fiber can increase the secretion of pancreatic lipase. But the really important effect that we are most concerned with today is fiber’s tendency to reduce both crude protein and amino acid digestibility. But why do we see this decrease in nitrogen digestibility when we increase fiber concentration in the diets? Well, there are several reasons. For one, like I mentioned with lipids, fiber can encapsulate proteins preventing their hydrolysis and can trap free amino acids preventing their absorption. Additionally, fiber in the gut increases the secretion of endogenous proteins, in the form of mucins and enzymes. High fiber diets have also been shown to result in increased intestinal mass, particularly through the cell-proliferating effects of short chain fatty acids produced via fermentation of fiber in the hindgut. And finally, fiber can increase the endogenous losses of protein, through its abrasive effects as it passes through the gut. When we are talking about endogenous losses, a lot of discussion is focused on mucins. Mucins are glycosylated proteins secreted by the goblet cells. This means that they are composed of sugars bound to a protein backbone. They are secreted to serve as a proteolytic barrier, barrier to pathogens in the gut, and as a buffer against the low pH of digesta. In the case of mucin, it is a 3:1 ratio of polysaccharides to amino acids, the most abundant of which are serine, proline, and threonine. Now, by their very nature, mucins are resistant to hydrolysis, so unlike other endogenously lost protein, they do not get resorbed. So, in the presence of fiber we see a multi-faceted increase in the production and secretion of mucin: 1. In response to fiber in the diet more mucin is secreted to protect the lining from abrasion. 2. The fiber itself pulls mucin from the lining, requiring increased production to compensate. And 3. Fiber can drive the development of larger GI tracts, which naturally will require more mucin to be produced. So, as we see the production and excretion of mucin increasing we really are talking about the utilization and excretion of threonine, because of the high concentration of threonine in mucins. So, with fiber we may see increased endogenous losses of threonine and therefore a potentially increased requirement for threonine for production. It is also worth noting that, although indirectly, fiber may increase the requirement for threonine via immune stimulation. Increased fiber in the diet generally results in a greater concentrations of microbiota in the hind gut. If these microbiota result in immune stimulation, the production of immunoglobulins will increase. And because immunoglobulins have high concentrations of threonine residues, particularly in the case of IgGs, the requirement for threonine may also be increased. So, with all these connections in mind, the question becomes: Does increased fiber in the diet increase the requirement for threonine to optimize protein synthesis? That led us to our objective, to determine the effects of fiber on the optimum SID threonine:lysine ratio in 25-50 kg growing gilts. So with that, we'll move into our first experiment, the threonine titration experiment to determine the effects of fiber on that optimal threonine:lysine ratio. The idea behind the threonine titration was that by formulating diets with low standardized ileal digestible threonine:lysine ratios, and incrementally increasing that ratio in successive diets, the optimum ratio could be determined using simple regression analysis. And then, by creating identical diets with the exception of fiber level, the effect of fiber on that optimal threonine:lysine ratio could be determined. So we went about this experiment with a 28-day growth assay using 192 growing gilts, all of initial body weight of around 26 kg. All pigs were the offspring of G-Performer boars and Fertilis 25 dams of the Pig Improvement Company. The pigs were housed two pigs per pen, and they were allowed ad libitum feed intake. The experiment was set up as a randomized complete block design, with 12 treatments and 8 replications per diet. There were six low fiber diets with 15% cornstarch and six high fiber diets with 15% soybean hulls. Within each level of fiber, there was a basal diet with an SID threonine:lysine ratio of 0.45, and then subsequent diets with ratios of 0.54, 0.63, 0.72, 0.81, and 0.90. We analyzed the data using the UNIVARIATE and MIXED procedures of SAS, with a fixed effect of diet and a random effect of block. We determined our treatment means using the LSMEANS and we completed our regression using the nonlinear procedure of SAS. So we'll jump right in the results here. Keep in mind, these are the low fiber diets. And we'll set up these graphs. On the Y axis, we have our average daily gain in grams, and on our X axis we have the threonine:lysine ratios. Here we determined the requirement not as the plateau point of the broken line or the apex of the curve, but rather the first intersection between the plateau and our quadrilinear curve. So here we see the requirement to optimize ADG at 0.66 SID threonine:lysine. Here, our slides are set up the same way, except here we're looking at the gain:feed. And we see that the optimum SID threonine:lysine ratio here is 0.63. In this slide, we've switched over to the high fiber diets, and again we're looking at average daily gain. We see here to optimize average daily gain, we need a 0.71 SID threonine:lysine ratio. And here in the high fiber diets, for gain:feed we see an optimized SID threonine:lysine ratio of 0.63. So here are all the numbers that we just talked about in a table form that's a little more easy to digest. We see here, between low fiber and high fiber diets, there was no change in the optimum SID threonine:lysine ratio for gain:feed. However, when we look at average daily gain, we see that there's a five percentage unit increase in the high fiber diets. And we see here that there's some evidence that increased fiber in the diet may increase the optimal SID threonine:lysine ratio to optimize growth. So now we're moving to our second experiment. and this experiment was based on an N balance. So unlike our previous experiment, it is not based on optimizing the growth of an animal, but really we're determining the requirement based on the nitrogen balance. So we're using a different metric here to come at the same answer. So like I mentioned, we're not using growth performance here but rather we're using fecal and urine excretion of nitrogen, apparent total tract digestibility of nitrogen, and the retention of nitrogen. So we went about this experiment using 36 growing gilts with an initial body weight of 29 kg. They were from the same genetic line as the previous experiment, and they were housed in metabolism cages with a seven day adaptation and five days of fecal and urine collection. We used the standard marker to marker method. This experiment was also set up like a randomized complete block design, with four treatments and nine replications per diet. The diets here were prepared in a 2x2 factorial arrangement, with two low fiber diets with 15% cornstarch, and two high fiber diets with 15% soybean hulls. Now within each level of fiber, there was one diet with an SID threonine:lysine ratio of 0.45 and another diet with a ratio of 0.60. We chose these numbers based on the results of the previous experiment. We know that 0.45 would definitely be deficient in threonine, and 0.60 is just below the requirement that we saw to optimize gain:feed at 0.63. Now for this experiment, the pigs were fed at 90% of ad libitum intake, to make sure they were eating everything they were provided. It's important to mention that all other amino acids in this diet were provided at 105% of the requirement, and lysine was provided at 90% of our determined requirement. So we really had two hypotheses from this experiment: 1) that increasing the SID threonine:lysine ratio in the diet will increase the nitrogen retention in the diets and 2) that fiber in the diet will reduce nitrogen retention in the animals. So for this experiment, we used the MIXED procedure of SAS to analyze the statistics, with a fixed effect of diet and a random effect of block. Our treatment means were determined using LSMEANS, and we used PDIFF to separate those means and a Fisher's LSD to determine significance. So we'll jump right into the results here. We have our nitrogen excretion in the urine, and keep in mind this is in grams over the total experiment, which was five days. Before I begin, we should set up the slides. On the left half of the slide, we have our low fiber diets, and on the right half of the slide we have our high fiber diets. And we have our two diets, the 0.45 SID threonine:lysine ratio diet, and then our 0.60 SID threonine:lysine ratio diet for each of the fiber levels. So if we look here at our low fiber diets, we see a decrease in the nitrogen excreted in the urine. And we also see that same pattern in the high fiber diets -- a decrease from the 0.45 level to the 0.60 level. Now the reason that we're seeing this decrease is because we're seeing that nitrogen being used in protein synthesis in the animal. Now this is what we'd expect because 0.60 is closer to the animal's requirement for threonine. Here the slides are set up the same way, but we're looking at the nitrogen excreted in the feces, again, in grams over the five days. We see here a decrease in our nitrogen excreted in the feces in our low fiber and our high fiber diets as the threonine level of the diet is increased. Now, you may have noticed here there's a significant fiber by threonine level interaction; and this is what we'd expect. In our previous slide, we didn't see that interaction, and that's because when you add fiber to the diet, you see a shift in the nitrogen excreted from urine to feces. And this is the result of the microbiota in the gut. This slide is also set up like the previous slides. And here were looking at the nitrogen retention, and our unit is percent. This is the capitulation of all the data that we collected in this experiment. And when we see an increase in nitrogen retention, that actually represents an increase in protein synthesis in the animal. So when we look at our low fiber diets, we see an increase in the nitrogen retention in the SID threonine:lysine ratio that is higher – that means closer to the requirement. And we see that same pattern followed when we look at the high fiber diets. What's really interesting to notice here is that pigs fed the high fiber, high threonine diet have nitrogen retention that is statistically equivalent to those of the pigs fed low fiber, low threonine diets. What this means is that pigs fed the high threonine level and the high fiber had the same level of protein synthesis as the pigs fed low threonine, low fiber diets. So what do we conclude from this study? We see that fiber increases total nitrogen output while simultaneously shifting that nitrogen excretion from urine to the feces. And we also see here that fiber reduces the apparent total tract digestibility of nitrogen. And although I did not show that data here, that was indeed the case. Now when we're talking about nitrogen retention, we saw an increase in pigs fed higher threonine. That indicates that these diets were closer to the requirement. And also when we see the difference in nitrogen retention between pigs fed the high threonine diets, that indicates that animals in the high fiber, high threonine diets were not receiving enough threonine, and therefore fiber in the diet may require a greater inclusion level of threonine. So what does this research tell us? Well, combined, the results of these experiments indicate that the optimal SID threonine:lysine ratio is greater in pigs fed high fiber diets than in pigs fed low fiber diets. And therefore, to maximize performance, the concentration of threonine should be increased when fiber level of the diet is increased. And with that, I would like to acknowledge the sponsors of the study, both Ajinomoto and Evonik Industries. And I'd also like to thank my fellow lab members. Without their help, theses experiments would not have been possible. If you enjoyed this presentation and you'd like to hear more podcasts, visit our website at nutrition.ansci.illinois.edu. Thank you for your attention.