- Welcome, everyone, to Wednesday Nite @ the Lab. I'm Tom Zinnen. I work here at the UW-Madison Biotechnology Center. I also work for UW-Extension Cooperative Extension, and on behalf of those folks and our other co-organizers, Wisconsin Public Television, the Wisconsin Alumni Association, and the UW-Madison Science Alliance, thanks again for coming to Wednesday Nite @ the Lab. We do this every Wednesday night, 50 times a year. Tonight, it's my pleasure to introduce to you two researchers in swine here in the Department of Animal Sciences at UW Madison. The first is Laura Amun-, whoops, Laura Amundson. She grew up in Plymouth, Wisconsin, and her husband grew up in Mount Hope...  which if you know the southwestern part of Wisconsin, is over there by Mount Ida. (laughing)  She started her research career as a freshman here at UW-Madison. She majored both in animal sciences and in dairy sciences. She has now finished her PhD in dairy science and,  sorry, in animal science, because she works on monogastrics. That's why she's here talking about swine. And so that's pretty impressive that you came as a freshman, first year, first semester starting in the research career, and now she's a postdoc here. We will-- Our first speaker will be Tom Crenshaw, who's a professor of animal sciences here. He was born in Newbern, Tennessee, got his undergraduate degree at the University of Tennessee-Martin, then did his master's and PhD at the University of Nebraska in Lincoln. And then he came here and has been a professor in the Department of Animal Sciences ever since. This is pretty interesting story tonight, because we're right across the street from the biochemistry building where, back in 1923 in the attic of that building, Harry Steenbock did his famous research on the discovery of UV light as a way to activate precursors of vitamin D. We have another page in this long story of vitamin D here. It's also pretty interesting that we get to talk about swine, which is not something people associate with Wisconsin, with the exception of bratwurst.  - Yeah.  - Yeah, there we go. And also the whole idea that swine might be a very interesting model for human physiology and human health and human diseases. So I'm looking forward to hearing how that all works out. So please join me in welcoming Professor Tom Crenshaw to Wednesday Nite @ the Lab. (applause)  - Thanks, Tom.  Thanks. It's quite a pleasure to be here and to have the opportunity to talk a little bit about some of our research. And it's kind of mentioned being across from the biochemistry building and Steenbock, quite an interesting connection, because I'm going to make a few additional connections with someone else that you might know by the name of Hector DeLuca. So we've had some interactions and discussions with Dr. DeLuca on some of the projects that kind of led us into some vitamin D work that really came about as an accident, and tried to describe some of how this came about. So, as a brief outline of what I would like to do, I'm going to give you some background and foundation with vitamin D from my perspective going through a nutrition program. And animal nutrition certainly would think that we should know a lot about vitamin D, but also establish some of the principles of scientific inquiry that we went through with this kind of accident that was the start of that. And we used that accident as an opportunity, a large one of the products of that opportunity in training the students. So do a brief description of how we have improved our understanding of vitamin D as a result of this, and also share a little bit about how research is an opportunity to train students. I want to point out a little bit about our UW Swine Research and Teaching Center. That's also been a very valuable resource in the establishment of these kind of new opportunities that have come along. We'll kind of lay out a brief timeline of scientific inquiry, trying to take you through a process without maybe boring you with too much of the detail and the data, but we'll talk about some of the traditional traits that we understand and initial observations in this, develop a hypothesis, which has led us to conclude that what we're seeing is this accidental deletion of vitamin D, which is actually a failure in endochondral ossification. I will explain that process. This has led to what we've concluded are lesions of osteochondrosis, which is a joint/cartilage problem rather than a mineralization problem that you might typical think of vitamin D being involved with rickets or poorly mineralized bone. So then some of the inferences from this we actually have, I think, pretty strong evidence that there's a maternal carryover effect. So the diet that mom consumes, or the mother sow consumes, would affect then bone development in the growing pigs. And then we'll look at some of the nutritionally induced signals that might have really lead to some of the better understanding of this chronic problem that we have with pigs. And I'd also mention that dogs have this problem of osteochondrosis. Chickens have a major problem with osteochondrosis. Horses have a problem and humans. All monogastric animals that are involved. And there's really very little that we understand about that. So, to start some of the discussion here, I went back to a presentation that was made by one of my co-graduate students, Dean Boyd. And this was dated, the date is correct, is February 28, 1978, that he and I were in graduate school. Dr. DeLuca had just been to Nebraska and gave a seminar on some of the new discoveries that he'd made on vitamin D. And Dean's presentation included a couple of comments that I pulled out, that there was an explosion of new information regarding vitamin D metabolism, it was difficult for researchers in the field to keep pace with all the discoveries that had been made, and then that was, really had been brought about, because of the realization that vitamin D does not function directly as vitamin D3, but it must be converted into the active hormone 1,25-Dihydroxy D. That was in the presentation of DeLuca. My co-graduate student, Dean Boyd, took advantage of that, or his presentation, and kind of summarized that, got credit for graduate student seminar course, but that was in 1978. And a couple of years later, I found myself in Wisconsin and had the opportunity in the early '80s to work with Dr. DeLuca in using pigs in a natural ingredient diet. We tried to   We have high health status in the herd. And so when something abnormal occurred, it certainly caught our attention, but when it disappeared, that even caught more of the attention. And we did, over about a year time period. I want to do a little bit of a timeline here, but this first appeared as part of an Animal Science 415 Applied Monogastric Nutrition class. We do a class project with the students. We saw these pigs from that experiment, and I accused the students of leaving something out of the diet. They were designing, formulating the diet, mixing the diets, and they convinced me that they had not. So there was something that was missing that we did not expect, but really what led to that started in probably about 2004 when I took the vitamin trace mineral premix that we used to feed all the animals in the herd, and we changed that to intentionally meet the minimum requirement of the animal that's established by the National Research Council. So, there's a guideline that's published for nutrient requirements, and I took the approach if the NRC requirement has set this as an average or an acceptable requirement, then the university, part of our obligation, if there's something wrong, is to identify that and help solve the problem. So, we set all the vitamins and trace minerals at a minimum requirement, and from 2004 until, this was November of 2007 when we saw this problem, we had not seen any problems in our herd. 2007, for a four-month time period, this humpback condition, kyphosis, appeared and disappeared. And so, we set up a series of experiments starting with Laura, then in 2008-2009 when we did the experiment, and we reproduced that condition in the pigs, so it could show under control conditions we'd reproduce that. These are all experiment numbers that we use that have been done since then. The thing that I want to point out that at the same time in the Upper Midwest there was a problem that occurred in the feed industry that was attributed to an accidental deletion or poor source quality of vitamin D that was in the diet, and there was a major feed recall that occurred in 2009-2010, and that feed recall, and these are data from the National Disease Lab in Ames, Iowa, where pigs in the industry in the Upper Midwest, the number of cases of bone diseases that were attributed to a hypovitaminosis D. So the timeline that really, I think, set Laura up for her career was, one, this class project, but also when she had completed an experiment as an undergrad student we had her and one of her classmates on a plane going to a national meeting to present the results of what happened when vitamin D was accidentally left out of the diet. On the plane ride to Denver, Colorado, where the meeting was being held, there was a feed recall that was announced. So the feed industry representatives, the scientists that were at the meeting kind of flocked to this poster, because here we were at the meeting with a poster describing what would happen if vitamin D was left out of the diet. So, it was kind of a fortuitous situation there. So, those two were linked. We spent a lot of time with veterinarians, with nutritionists, on conference calls, two- to four-hour conference calls, going back and forth for a two- to three-year time period, trying to give the benefit of any information that would help to solve the problem. Before we did this experiment to confirm what was out, over that one-year time period when we first saw the problem, it appeared and disappeared. We did about five experiments. We did not immediately jump to vitamin D.  Looking at some of the features of the pigs, we thought there might be a mold mycotoxin problem. We did experiments, even though we could not detect mycotoxins in the corn, we did experiments where we fed diets with no corn in it, trying to prove that there was a mycotoxin contamination in the corn.  We know there was no disease outbreak in our herd. Again, it's a very tightly controlled, monitored herd. We had not introduced any new genetics into the herd. So it must be the diet. Either that or something in the water, right? But if it's something in the water, why did it appear and disappear? So we went to the diet. As a nutritionist, that was an easy conclusion for me to make. And then, actually, working with people over in the medical school, they were interested in doing some vitamin D analysis on the sample. And they were not able to detect any 25-hydroxy D in the serum, in the pig serum. So that took us to the vitamin premix, and we found that there was no detectable level of vitamin D in the premix. And that was actually verified by the company that custom mixes that, that, whoops, it had been accidentally left out. We have a protocol that we turn over the vitamin premix on a regular basis. We identified that it'd been left out. So Laura comes in about that time, and she's ready then to prove if we intentionally leave it out, that we can reproduce the same problem, which is what we did. So we measured the kyphosis incidents using just a visual score at nine weeks of age. At birth it's not visibly evident. At nine weeks of age, in animals that were fed, this should be a red bar here, a diet to the sow that was vitamin D deficient, it was about a 21% incidence of the pigs that were born to the sows that were fed the vitamin D deficient diet that showed evidence of this abnormal spinal curvature.  We continued to watch the pigs another four weeks. And, actually, some of the control pigs begin to show the symptoms again. But the difference in this is during the nursery phase we were not feeding any vitamin D in the diet at that point either. So we'd left vitamin D intentionally out of the nursery diets for the baby pigs. That began to lead us to question the maternal effects. What does the mom's diet do with this?  You have to watch the time for me. Alright, so we initially wanted to characterize some of the traits in the hypovitaminosis kyphotic pig that we had. And there were some relatively dramatic responses that occurred. As you recall, I mentioned in the '80s we did not see deficiencies when we fed diets, pigs over a four- to five-month time period. We were not creating a deficiency. This is the growth rate of pigs over 28 days. The take-home message here, pigs that were fed a diet just during the nursery period, 28 days, with no vitamin D versus pigs that had adequate vitamin D in the diet, there was a 30 percent difference in growth in that response. We measured the bone mineral content using DEXA, that's used actually for humans. The technology, again, was developed here on the Madison campus, but we used DEXA to measure the whole body bone mineral content of pigs, worked out procedures to do that. About a 40 percent reduction in bone mineral content If they grow different, maybe a better measurement to use would be bone mineral density, and a 30 percent reduction in bone mineral density. That corrects for difference in size of the animal. You would expect a bigger pig to have more bone mineral content than a smaller pig. Bone mineral density corrects for the differences in size or area of the bone. So, take-home message is, wow, we're seeing dramatic responses within a 28-day time period. We looked at other classical, traditional responses, serum calcium. These pigs were barely able to maintain normal serum calcium close to the physiological range. They had vitamin D. Homeostatic mechanisms would say they could regulate the serum calcium. Phosphorus, again, if there's extra calcium in phosphorus added, the serum phosphorus is actually dropping. We will not have time to get into all the implications of that. We measured 25-hydroxy in the serum. There was no detectable 25-hydroxy after a four-week time period in the pigs that were fed the deficient diets. In the '80s, we could not create a deficiency. These are corn/soybean mill, very similar diets. And now, for some reason, we're able to create a deficiency. Parathyroid hormone, which is in kind of a counterbalance with serum 25-hydroxy, you would expect the levels, if serum calcium drops, you would expect parathyroid hormone to increase, which it did. We did another study in which we looked at the doses. Maybe there's something, maybe the genetics of the pigs have changed, they have a different requirement. We then designed an experiment to determine the vitamin D requirement of the pig. We went from zero to 1600 international units. The NRC requirement for a pig at this age is 200 international units per kilogram of diet. Our results with our pigs and our research unit, we concluded that the requirement based on a broken stick analysis was 123 international units. So, no evidence that the vitamin D or anything that we're feeding as far as the bone mineral density of the whole had been altered from what maybe the NRC estimates were. Of interest, we also measured the serum 25-hydroxy D concentrations.  There was a lack of fit, if you would, statistically in this in that we could not fit a broken stick to this because, as we increase, the 25-hydroxy continued to increase as we increased the dietary concentrations. The other thing I'd point out, is there seems to be no relationship between the whole body bone ash content, the bone mineral content, and the serum 25-hydroxy. Probably not a very good marker to establish vitamin D status. The requirements of the pigs, when we started these experiments, the NRC requirement that was current at that time was the 1998 version. Those are updated about every 10 years. The updated version for this actually occurred in 2012. They're a little bit behind time. For sows, the requirement was 200 international units. There was a footnote in the NRC requirement that there were no studies that that requirement, that number was based on. In other words, it was kind of a number that was extrapolated from other ages of pigs. The requirement for rats in a 1995 NRC requirement is 1,000 international units. Humans, they express a little bit different international units per day. Recommended daily allowance in 2011 is actually 600 international units. The safe upper limit, 4,000 international units per day.  So with these pigs, now that we had this incidence, and part of the reason that we begin to think about the maternal effects, was this group that was fed the control diet, because we knew that we were feeding right at NRC requirements. Remember, 2004 we went back and fed close to minimum requirements to our animals. Prior to that, we had fed levels that had some type of a safety margin built into the requirements. 

 So, we wanted to further characterize some of the traits that the hypovitaminosis kyphotic pig. We designed an experiment to really explore the maternal diet. And so a little bit of a complicated experiment. Laura was up to the challenge to take this on. We did what you might statistically define as a three by four factorial. There were 12 dietary treatments that we dealt with in a gestation lactation nursery experiment. Over her PhD, undergrad and PhD program, Laura's done three of those experiments. They're quite long experiments to work with. So, we fed the sows diets with no supplemental vitamin D, 325 which is what we routinely had fed in the herd, slightly above what we thought was the requirement, and then the commercial industry might feed as high as 1750, as kind of an average number that they would feed. And then, from breeding all the way through the end of a lactation cycle, they were fed those levels. Then the pigs were weened onto four different nursery diets with different combinations of vitamin D and calcium and phosphorus. The main effects, or actually the maternal effects. This is the incidence at 13 weeks of kyphosis in the pigs. So pigs that were produced by sows that had no supplemental D had a higher incidence of kyphosis at 13 weeks. This middle treatment is what we routinely feed our herds, but if they were fed diets during the nursery period with no supplemental D, we did see about a 10 percent incidence of kyphosis in those pigs. Now, we're really interested in knowing what's going on. What's creating this problem? How is vitamin D creating this bone abnormality? Serum concentrations, as you might expect, increased. If you're familiar with human levels for that, medical doctors might look at the serum 25-hydroxy D concentration and say those animals would be deficient. Okay? Even at the higher concentration. Colostrum concentration right at birth, not much of a reflection of the dietary treatments. A little bit of an increase in day 18, but previous data would show in sows' milk, their vitamin D is not transferred across, so milk is not a very good source of vitamin D.  Tissue concentrations in the pigs, most of the concentrations were in a deficient range or almost non-detectable concentrations.  We looked at the mRNA expression of the genes that are involved in degrading vitamin D and the genes that are involved in activating vitamin D. So we measured the 24-hydroxylase, the mRNA for that. That was really not changed in pigs at birth. So these are concentrations now in the pigs. They were not changed in pigs at birth due to the maternal diets they were fed.  By weening at 25 days of age, there was a dramatic increase in the degradative enzymes if the animals were overfed diets with supplemental D. There was a decrease as you went from diets that were not supplemented with vitamin D to diets that had an excessive amount of vitamin D in the activating enzymes that were involved. So an inverse relationship would occur between the degradation. This simply would say that if you add more vitamin D in the diet, the body has mechanisms to increase the degradation to get rid of that. Okay? The activation at seven weeks, and this is kind of a surprising observation now that occurred. So, at seven weeks of age, these pigs had then been fed nursery diets, in this case with supplemental vitamin D, but there's still a carryover effect of the maternal diet that shows up in these pigs. Degradative enzymes increased if they were fed high levels of D during the maternal diet. They decreased the activation enzymes if they were fed diets that were adequate or excessive during the maternal period. Now, I just wanted to show you a picture of a pig on a DEXA machine. Some of you may have had DEXA scans. And that's the main, we use the DEXA machine in a quite a few different research projects, but we can anesthetize the pig, position the pig on the table. With human patients, you kind of go in and you lay down on the table and you hold real still, and to do that, we have to anesthetize the pigs to get them to lay on the table, but we can use DEXA to measure the whole body bone mineral content in pigs. And we've done quite a big of work in that area. So that was the instrument, now that we used in this experimental design, and if we tease out now 12 dietary treatments is a bit complicated to look at. So, we have evidence then if they were fed no supplemental D during the maternal diet or during the nursery phase, there was really no difference, depending on calcium and phosphorus levels. If anything, if you added the nursery diet, a slight increase here, a decrease if we actually overfed phosphorus in the nursery diet. So excess phosphorus when they have no vitamin D seems to be decreasing the bone mineral content, but it increases bone mineral content if there is vitamin D in the nursery diet if the animals are marginal. An opposite response occurs if there's an excessive amount of vitamin D in the diet. So there seems to be now a critical balance that we've kind of teased out with these diets between the concentration of vitamin D in the maternal diet and what we feed to the pigs during the nursery diets as well.  Now, of interest in this, this is a picture of the vertebral column. This will be the spinal cord here. So a cross-sectional cut of the, a longitudinal cut of the spinal column, but simply showing the kyphosis, that's a wedge in the vertebral body, should be rectangular shaped in this region. They actually grow in a wedge shape. And they fail to grow in the ventral side, and they appear to be growing normally on the dorsal side. So, what would cause a defect in this region in growth? And we see lesions that would occur. This is a cross-section of the distal femur then, and there's a thickened growth plate retained or a cartilage growth plate, cartilage in this region from a pig that was fed a vitamin D deficient diet. If they had an adequate diet, the growth plate would appear more normal and thin. So that led us to thinking about there's a failure in the endochondral ossification of bone, which I'll let Laura here in just a minute explain that in more detail. But we've also used some histology to look at control, the vertebral body we see even stronger histological evidence. In the control animal, this region, this would be the vertebral disk, the growth plate that would be in the vertebral body. This would be a failure in the vertebral disk, the retention of the cartilage in that region. So this would be the region that failed to grow and turn over and replace bone. This side of the vertebral body would appear to be growing normally.  This is a CT scan of a pig that had kyphosis. The obvious curvature that would occur there. The defects are shown. This would be a controlled normal pig. We're in the process now of using some image analysis to go in and look at specific region to look at bone mineral densities in those regions and try to identify differences in mineralization. When Laura came into the lab, she had really more of an interest in cell signaling. This gave us an opportunity to really attack what are the signals and how they begin change in the growth plate region. So I'm going to let Laura take over and explain some of what she's done in looking at some of the cell signals within the bone, trying to identify what is the defect that's occurring within this.  - So endochondral ossification, as Tom was talking earlier, we think that this is the point of failure in our kyphotic pigs, rather than a lack of mineralization, based on some of our DEXA scans and some cell signaling measurements that I'm going to show you. So, just in general, endochondral ossification, what is it? So bones will grow in two different ways, either through endochondral ossification, which I'm gonna talk about now, which is what the long bones, such as the femur, will do. I won't touch on the other way that the flat bones develop, but during endochondral ossification, there's a cartilage matrix, and what happens is there's a progression through chondrocyte, which are cartilage cells, different zones. There will be a mineralization and a degradation of the cartilage matrix, and then an invasion of capillaries will occur that will bring along osteoclasts, which are bone resorping cells, and osteoblasts, which are bone formation cells, and during this process, there's kind of two different forms of bone that get laid down, the first being a primary bone, sometimes referred to as woven bone. It's a weaker bone, not as well structured. Doesn't have as great of strength capacities as what we would consider the secondary, more organized bone to have.  And healthy endochondral ossification or healthy bones is a balance, a coupling between bone resorption and bone formation. And this idea of balance is something that's definitely come up multiple times during my PhD work. And, as Tom alluded to earlier, we don't have time but with the vitamin D and the phosphorus we quickly learned more is not always better. So it's very important to have a balance coupling of bone resorption and formation.  So more of just a schematic of what I just talked about. So this would be a long bone here on the left. And in the growth plate region here, there's the different zones of cartilage cells or chondrocytes, they're green. I'm not sure that it really shows up very well, but the resting zone of chondrocytes kind of disorganized. Just that, it's a resting zone where some progenitor cells kind of hang out and get ready to move through the growth plate. Next is the proliferative zone where the cells become much more organized in columnar shapes. And that columnar organization is pertinent in order for the endochondral ossification process to maintain normalcy.  That's actually one of the signs of a disorganized growth plate is when histologically you look at these growth plates and the cells are not aligned in columns. 

 Next, the cells grow through the hypertrophic zone, which they stay in their columnar organization but increase in size.  And then, right here, at the what's called the chondro-osseous junction, it's where the invading capillaries will come in, shown here in red, with the osteoclasts in purple and osteoblasts in blue. So right at this zone here, at the mature hypertrophic chondrocytes there will be a calcification. And that's where the osteoclasts will come in and remove that calcified matrix and lay down the primary bone. The osteoclasts will come through, chew up the primary bone, and the osteoblasts will lay down the secondary bone. So that's just a general overview, very, as Tom likes to say, 10,000 feet overview of endochondral ossification, but I think it's important to understand that there's so many different areas that things could go wrong. So it's difficult to tease apart what exactly we were dealing with in a kyphotic pig, and 10 years later, here I am. We're still trying to figure it out, but it's a lot of fun. So endochondral ossification, during that process, the modeling and the remodeling of the extracellular matrix, which is what the cartilage cells are laying down, those are the rate limiting steps. So if the cartilage doesn't get laid down, or if the calcified cartilage doesn't get chewed up, that's where things start to break down in the process. So, there are some critical factors during endochondral ossification that are mediated by vitamin D. It's not what we think of classically with vitamin D and the mineralization as Tom spoke earlier about, but after I dug a little bit, I was able to find some things that had more to do with the cartilage matrices. So, these factors called matrix metalloproteinases. They are proteinases that chew up the matrix that the cartilage cells lay down. And just as I showed you through all those different zones those cartilage cells, the different types, the proliferative and the hypertrophic are secreting different types of collagens. So there's a multitude of metalloproteinases that are needed in order to chew up specific aggrecans and collagens, et cetera. So, two that we focused on based on a literature search were the MMP13 and MMP9. And 13 is a collagenase. It will chew up the collagens. And 9 is a gelatinase. So it's going to chew up the gelatin type factors that are within the growth plate. And another factor that's important and related to both of these factors is the vascular endothelial growth factor, or VEGF, which is responsible for angiogenesis which is going to recruit those capillaries to bring in the osteoclasts and osteoblasts. So we thought this is a good starting point to look at some of our kyphotic pigs, tissues from them, to see if these factors were playing a role. So, again, just keep in mind what we eventually wanted to do, and kind of my mindset as a scientist, as Tom's taught me, is we started with a problem at the whole animal level, we're digging a little bit deeper into the molecular side of things what's going on, the nitty gritty, and then bringing it back out. How can we use what we learned to maybe measure something in the live, whole animal, in order to diagnose some of these problems and predict when failures will occur?  So, which signaling pathways are affected? So, again, we looked at those different factors. And within the growth plate, I just want to show you kind of where those factors would come into play. So, as I mentioned earlier, the different zones are going to release different types of collagen. Up here in the proliferative and resting zone, type 2 collagen is prominent. In the hypertrophic zone, type 10 collagen is prominent. And then, as we get down towards the bone, the bony section, type 1 collagen is prominent. And throughout the growth plate, different cells are expressing and making the different MMPs and other metalloproteinases that are responsible for chewing up that matrix and allowing the coupling of the bone resorption. So, MMP13 and VEGF are produced by hypertrophic chondrocytes as well as osteoblasts. And then MMP9 is produced by the osteoclasts, the bone resorbing cell. So just a little schematic of kind of a hypothes  So our initial hypothesis was that because we were saying that retained cartilage, there must have been a decreased expression of the MMPs and the VEGF. We did look at the mRNA. It has been shown on the literature that the production of or the action of those factors are regulated at the mRNA level.  And, as I learned throughout my undergrad and graduate work, your initial hypothesis is not always right, and you have to be very willing to admit that. So what we found was that pigs that were produced, a big message here, pigs that were produced by sows fed excessive levels of vitamin D during gestation and lactation actually had an overall decrease in the femur MMP9 mRNA expression. So, I missed something in the literature or the pig wasn't the same as some of the literature I had read from the human and the dog. But I dug a little bit deeper into some of the articular joint disease literature from the humans, horses, and canines, dogs.  And, actually, in those animals with articular joint diseases, the MMPs are increased. And so what it comes back to, kind of what I said earlier, you know, more is not always better. These animals had increased of these matrix metalloproteinases, which was uncoupling the endochondral ossification process and eventually leading to some pretty detrimental articular joint diseases. TIMPs, or tissues inhibitors of metalloproteinases, are just another example of kind of a check and balance. It's actually the body's own inhibitor of metalloproteinases.  Humans, dogs, we make the tissue inhibitors of the metalloproteinases in order to counteract the MMP actions. So, again, it's a delicate balance, and we're still teasing that part apart. It's kind of taking us maybe a little bit longer on the molecular side of things in the pig than it would have, say, in the mouse or rat. There's not a lot of antibodies for the pig. I spent almost a year trying to get PCR to work in pig bone and getting different primers to work and such, but it was fun. So I threw this up at the end of my PhD, and at first, I didn't have all the other bullet points. I just had it's complicated hoping my committee would be okay with that. (laughing) But it is truly complicated. And, as Tom alluded to earlier, that research is a way to train students, he always said that from the beginning, and as a young, immature student, kind of in one ear and out the other, and he told me he was going to teach me how to think, and I was like what does that even mean? But it's truly been a great process and he has taught me how to think, and even though the research has been a lot of fun, I've learned a lot more than just research results and about vitamin D. But back to our kyphotic pig model, the nutrients, such as vitamin D, act locally, not just systemically, to couple the endochondral ossification that I spoke about.  The nutrient interactions on the molecular signaling within the bone are definitely not clearly established. Bone is a hard tissue to work with. Tom likes to use that joke. It truly is. It's a very heterogeneous tissue made up of a lot of different cells. It's hard to get at, hard to break down in order to do PCR, western blots, things like that, and especially when we're working with such a large animal model. We can't take the whole femur like we could in a mouse. We have to take sections, and then you're introducing even more variability. But we're working on it. One of the biggest things or the biggest, most interesting things, I think, I gathered from my PhD research was the carryover effect of maternal nutrition. It's extremely important in neonatal bone health, and I think sometimes both in the human nutrition world and in the animal nutrition world, we wait 'til we have a problem and then we try to think about what caused the problem, but we don't go back far enough and we're not starting early enough in understanding how the maternal diet is really effecting the offspring. And I read some epigenetic papers that talk about even your grandmother and grandfather's effects on your different outcomes physiologically. So it's something that I think we need to pay attention to. And the kyphotic pig is definitely a model that I think has provided us a lot of new information, both we've interacted with both human and agriculture professionals in understanding more about vitamin D, especially during pregnancy, and offspring outcomes. And, as I said, balance is key. Something definitely was a theme throughout my graduate work. So I'll give it back to Tom.  - So, I wanted to let Laura have the opportunity to kind of go through some of her data, and we've had opportunities over the years to present these results at regional, national, and international meetings. It's really brought quite a bit of attention. But Laura mentioned, you know, the difficulty in techniques that she's worked on, I thought I'd take just a few minutes, if we have some time yet, to talk about some additional things, some additional insights that we've learned from this. And if I can pause my computer a minute, I'm goning to go to one thing that we're working on, we mentioned the osteochondrosis. I've had other students working in the lab, and this is a poster presentation that an undergrad, another undergrad student presented that was a Cargill-Benevenga student. And she won a regional competition with the poster. We use now electronic posters rather than paper posters. And I'm not going to go through the detail on the slides, but we use CT scans of bones and we're trying to develop a technique that we can, rather than histology, which is very tedious, very meticulous work to do, if you watch on the bottom of the screen, the cursor is actually working here, but there's a lesion right in that area. And so, as we take slices through the bone at 0.625-millimeter sections through the bone from a CT scan, we can identify the lesions, and we've worked on. Nicole Gross is the student that worked on this. And I have to give her credit. She put the electronics together here. This shows ... This is the lesion. This is a single slice. If you think of this as a loaf of bread, okay? And that's one slice of bread that she's taken out, but the lesion, the green lesion that you see in this area, we're able to mask and measure the volume, the XYZ coordinates of that lesion, the volume and area of the lesion, the number of lesions that occur across the growth plate. If you were taking a single histological slice of that, you might accidentally miss the lesion, even if you were very systematic in how you collected that slice So I just kind of wanted to show that because we're working on techniques to improve our ability to measure the lesions. With the CT scans, we could actually go in then, again in live animals, and measure the progression of those lesions through-- I've got to get out of this one now. So back to the vitamin D story, this osteochondrosis problem has been around in pigs throughout my career. If you haven't picked it up yet, I've been around for a few years in the research. And osteochondrosis was one of my colleagues in Canada that worked his whole career on osteochondrosis. He told me one time that he had proven everything that did not cause osteochondrosis. (laughter) He tried all kinds of different nutrient supplements, trying to create that, but we typically start, in most of that research we've started with pigs maybe at nursery or at four or eight weeks of age and we look at different nutrient factors that affect that. I think one contribution that Laura has made with her research is to identify that we maybe need to start earlier, during fetal development, looking at the development of these lesions. So we need techniques to do that. Most of my career I've actually focused on mineralization of the bone. Calcium and phosphorus. Some of my colleagues, this is a slide from Cathy Carlson at University of Minnesota, a vet pathologist. She has spent most of her career working with the cartilage in the collagen part of bone. And we've had a lot of fun over the years discussing things and agreeing that we don't agree about some things, but it's been quite an exciting exchange.  With the work that Laura has done, I'm kind of of the opinion that now we can begin to link some of these factors together a little bit. Historical perspective of osteochondrosis in pigs, the prevalence is thought in pigs to be greater than 80%. In other words, most every pig has some osteochondrotic lesion. Is it a lesion that leads to lameness? No. Not that many pigs are lame, but there's evidence, histological evidence, of retained cartilage in the growth plate. There's also problems, as I mentioned earlier, in poultry, horses, dogs and humans. So monogastric animals seem to have a problem with osteochondrotic lesions. For over 45 years, we've kind of studied, have done research on that, and we basically have characterized the lesions, the final stages of the lesions, but we know very little about the factors, the signals that are involved that would initiate the formation of those. And part of that may be that we've not gone back and looked early enough. So if it's been around for 45 years and you think about, okay, I don't know all of your knowledge about the pig industry, but the industry has changed dramatically, even in housing systems, and you could not say that it's a result of modern swine medicine management. Housing, genetics, okay? It's been around and genetics have changed, housing have changed, production management of pigs have changed a lot. So what I think one of the major contributions of this work, other than just training a student, has been that we now have some insights into osteochondrotic lesions, and we may need to begin to look earlier in the development of that. So we will continue. We are continuing to work in those areas, and we've focused down and hopefully with identifying some of the cell signals involved and using a vitamin D deficient model and knowing which signals are changed with that, we can begin to create some differences, understand what's occurring and what to do to prevent those lesions, not only in pigs but also in the other animal species involved, and in humans. So, kind of an overall conclusion, I'd say back to the vitamin D is that the maternal and nursery diet increased the degradation. If there's excess vitamin D fed, degradation went up and there was a decrease in the activation. That's homeostatic mechanism for regulating activation of vitamin D. So just putting more in the diet is probably not going to really solve the problem. Certainly providing a sufficient amount, whatever level that would be, would be good. But the extremes are not necessarily helpful. We were kind of surprised that the maternal carryover effect and actually surprised that we were picking this up in the kidney, the mRNA expression, the maternal carryover effect was even showing up after seven weeks of age. That's the latest that the oldest pig that we've measured this in. What it would do to later ages we don't know. The serum 25-hydroxy, and in defense of people in the medical industry and the animal, veterinary industry that use serum 25-hydroxy as the marker, that's probably about the only thing that we can take a non-invasive sample and try to assess the vitamin D status. But most of our data would point to and say that it's probably not an effective marker to identify some of the lesions, the abnormalities that we've dealt with. So it's not necessarily reflective of the vitamin D status within the body. And this last point, the inadequate vitamin D leading to the osteochondrosis in growing pigs, that gives us, begins to give us a tool in future direction for research. And we've got students that are certainly involved in that. The other thing I'd just like to call your attention to and maybe kind of summarize even, with this slide and an acknowledgment of several things show up here. First, I'd like to say the Swine Research Center, we have a great staff that works there that's been involved with this. Very meticulous in their work, pay careful attention to detail. It was actually Jamie, our unit manager, that suggested, well, let's just continue to watch these pigs from nine weeks to   I've got to go back to the Cargill-Benevenga Laura's set up in the lab. Ben, as I mentioned, comes in still every day. He and I worked together for years, have a lot of respect, and I think we understand each other. Ben is a very, if he listened to this tape, he'll probably agree with this but he'll let me know about it, but he's very kind of an aggressive personality type. He's an Italian, so he ... If any of you know Ben, great guy, and I've really enjoyed working with him over my career. I've learned a lot from him. But sometimes he could actually intimidate certain students. Laura has sat and had lunch with him every day, and he can ask great questions and really help people to begin to think critically about what they're doing, the decisions that they make, and Laura's hung in there really tough with him and learned, I think, a lot, you would admit, from that. But several of these other students, these are all undergraduate students that have worked up in the lab. Some have had Cargill-Benevenga undergrad research stipends, others have worked on different projects in different ways. I'm not goning to go through the whole list of names, but these are only the undergrad students that have kind of worked on the vitamin D project. I think in our research programs in animal sciences, and I'm not the only faculty member in the Department of Animal Science or Dairy Science, we give those guys credit as well, but also in the College of Ag and Life Sciences. I think our faculty, a lot of our faculty will work and really engage with undergraduate students, giving them opportunities in the lab, and I just want to put in a plug. I think it's an important part of our mission as a college is to train to students and to train them how to think. It's not just what they learn in the classroom that's important. If they can learn to think and be engaged and involved in research, that helps a lot with that. (applause)