[Tom Zinnen, Outreach Specialist, Biotechnology Center, University of Wisconsin-Madison]

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.

[laughter]

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 U.V. 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.

[Thomas Crenshaw, Professor, Department of Animal Sciences, University of Wisconsin- Madison]

Yeah.

[Tom Zinnen]

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]

[Thomas Crenshaw]

Great. 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 – its 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 –

[Slide with outline of talk]

– 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 U.W. Swine Research and Teaching Center. That’s also been a very valuable research – 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, and – which has led us to conclude that what we’re seeing is this accidental deletion of vitamin D, was actually a failure in endochondral ossification. I will explain that process. And 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 typically think of vitamin D being involved with rickets or poorly mineralized bone.

[Thomas Crenshaw]

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.

[slide with outline of the Foundations from the early years]

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.

[Thomas Crenshaw]

That was in the presentation of DeLuca. My co-graduate student, Dean Boyd, took advantage of that, and – 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 create a deficiency of vitamin D in pigs, and we were not able to do that.

Now, we did two to three experiments. And Dr. DeLuca had certainly worked a lot in the area of vitamin D, certainly using the rat that you mentioned earlier as a research model, and could create deficiencies in purified diets, very meticulous control over the diets. But we were not able to do that in our research unit with pigs fed a natural ingredient corn/soybean meal type of a diet.

We even kept the pigs in the dark to prevent any light exposure to the pigs. So, I concluded after – after those experiments that vitamin D maybe was not really all that important in pig nutrition. And we understood at that time from what DeLuca had had, and this is a slide –

[slide showing the Traditional Axis for Calcium Homeostasis in the parathyroid, kidney, intestine and bone]

– that I put together to talk about the traditional pathway, and some of you may know about this, but a decrease in serum calcium would be the traditional response that vitamin D is involved in homeostatic mechanisms to regulate the levels. That decrease in serum calcium would stimulate the parathyroid gland to release parathyroid hormone. The parathyroid hormone would act on the kidney, and the 1 alpha-hydroxylase would convert 25-hydroxy D, which is in the serum, into the active form, the 1 alpha,25-Dihydroxy D3. That’s a very classical pathway. Its the 1,25-Dihydroxy D that Dr. DeLuca and his coworkers in the ’70s and ’80s really kind of worked out that pathway and established that.

The active form of vitamin D –

[Thomas Crenshaw]

– then acts with parathyroid hormone on the bone to increase the release of calcium and phosphorus reabsorption from the bone. Works on the intestine to increase activation of certain enzymes and transporters that are there. The net result is an increase in the absorption from the G.I. tract in calcium and phosphorus. But it also acts on the kidney, these hormones do, to increase the reabsorption of calcium –

[return to the Traditional Axis for Calcium Homeostasis slide]

– which results in a decrease in the calcium excretion in the urine, and the phosphorus that would be mobilized from bone or taken up if it’s not needed would be excreted in the urine. So, that’s the traditional pathway that was established, I’m going to say back in the ’70s and ’80s. And I’ve taught that in classes and nutrition classes –

[Thomas Crenshaw]

– and I would say that I had a very good understanding of vitamin D and the traditional pathways as a monogastric nutrition, but for pig nutrition really did not think it was necessarily that important because we had a difficulty creating a deficiency in our animals.

Dr. DeLuca, now he was not maybe totally surprised that we were not able to create deficiencies in the pigs with a natural ingredient diets. And he certainly has continued his career since the ’80s –

[slide of a presentation by Dr. DeLuca of Vitamin D, Bones and Beyond]

– if I can date him to that point anyhow, and he’s gone beyond bones and found certainly a lot of different derivatives of vitamin D and other roles that vitamin D plays within the body.

[Thomas Crenshaw]

So, I don’t want to take anything away from – from that and the contribution that he had.

Another person that I want to mention is a – a guy that I’ve worked with, Ben Benevenga, that was in animal science and nutritional sciences.

[slide with photos of Ben Benevenga]

Ben retired in 2001. He’s continued – he’s still in on campus most every day, but on his retirement, because of Ben’s dedication to students and to student research, the Cargill Nutrena Company actually set up an endowment and Bens encouragement – with Ben’s encouragement. That endowment is used to support undergrad researchers in animal science and nutritional sciences.

So, we have a fund –

[Thomas Crenshaw]

– that we can use to challenge undergrads to get them involved in research. Laura that’s with me today is a result of that. But one of the things, before I talk too much about Laura then, is that I learned from Ben that research is really an opportunity to train students.

And I think that’s sometimes perceived differently in different labs, but I would certainly be one that would encourage that and endorse that, and I want to credit Ben with helping to instill that concept into me. If there’s a problem, research gives you an opportunity to maybe solve problems, but it’s also a great opportunity to train students. And Laura was a result of that. As Tom mentioned earlier, when she came in, she got involved, we got her involved in a project, and it was actually this accidental discovery –

[slide with a photo of Laura Amundson and her discovery of Vitamin D-Induced Kyphosis]

– and she started out with a Cargill-Benevenga undergrad research stipend in, what was that? 2010, 2011 maybe when you did that. Through her PhD program working with me, she was actually awarded an Animal Science Society Young Scholar Award in the last year of her PhD program.

[Thomas Crenshaw]

She’s now, as Tom mentioned earlier, doing a postdoc in the Dairy Science Department with Dr. Laura Hernandez. So, she’s continued in a research credit and I think is one example of what the Cargill-Benevenga Undergrad Research Program has done in our departments to stimulate research interests in our undergraduates.

This is the problem that we accidentally stumbled into, and actually this problem started in one of my undergraduate classes. I teach a class on applied monogastric nutrition.

[slide with photos of swine with Kyphosis or a hump-back both x-ray and normal]

And so, this problem started. These pigs, if you’re not really into pigs, these are not normal pigs, okay? And we in the industry is sometimes described as a hump-back pig or a camelback pig. It’s a sporadic, it appears and disappears. The more technical term is known as kyphosis, which is an abnormal outward curvature of the spinal column in contrast to scoliosis –

[Thomas Crenshaw]

– which would be an abnormal sideward curvature of the spinal column. This condition appeared and disappeared in our research unit over about a four-month time period.

And we knew enough – have enough control over our animals to know that it was not normal. And we did not have a lot of understanding of why it occurred. And we decided to attack the problem, take that on and identify the problem.

[slide with a photo of the Swine Research and Training Center and an overview of its mission]

This is our swine research center that’s up in the Arlington experiment station. And I just want to put a plug in for the College of Ag and the experiment stations that we have. We maintain a breed and herd here. We have very tight control, biosecurity control –

[Thomas Crenshaw]

– of the production of the animals. We know the status of the animal. These are A.A.A.L.A.C. approved facilities. 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 –

[slide with the timeline of the Research Centers work on research with Vitamin D]

– 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 –

[Thomas Crenshaw]

– 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 N.R.C. 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.

[return to the slide of the timeline]

2007, for a four-month time period, this hump-back condition, kyphosis, appeared and disappeared. And so, we set up a series of experiments starting with Laura, then in 2008 is when we – 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 –

[Thomas Crenshaw]

– 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 –

[return to the timeline slide with an emphasis on the graph on the bottom of the page with year on the x axis and number of cases of swine bone disease on the y axis showing a large increase in 2010 and 2011]

– 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 –

[Thomas Crenshaw]

– 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 we 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.

[slide with a decision chart showing the possible causes of Kyphosis including Mycotoxins, Disease, Genetics and Diet]

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 the 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.

[Thomas Crenshaw]

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 –

[slide with Lessons from hypovitaminosis D pigs with graph with age in weeks on the x axis and percentage of kyphosis on the y axis showing an increase over time]

– 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.

[Thomas Crenshaw]

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.

[slide with a graph of Cumulative Growth Rate over a 28 day Trial showing differing growth rates depending on if Vitamin D is in the diet or not]

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.

[slide with two graphs of Skeletal Response to Vitamin D, Calcium and Phosphorus]

We measured the bone mineral content using D.X.A., that’s used actually for in humans. The technology, again, was developed here on the Madison campus, but we used D.X.A. 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.

[Thomas Crenshaw]

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 –

[slide showing two graphs of Dietary D supplements affected serum Calcium and Phosphorus showing increase when Vitamin D is added]

– 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 and phosphorus added, the serum phosphorus is actually dropping. We will not have time to get into all the implications of that.

[slide of Dietary D supplements affected serum hormones]

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.

[Thomas Crenshaw]

And now, for some reason, we’re able to create a deficiency.

Parathyroid hormone, which is in – in – 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.

[slide with a graph of Weanling pig response to Vitamin D]

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 N.R.C. 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 –

[Thomas Crenshaw]

– of the whole pig had been altered from what maybe the N.R.C. estimates were. Of interest, we also measured the serum 25-hydroxy D concentrations.

[slide with a graph of Weanling pig response to Vitamin D ]

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.

[Thomas Crenshaw]

The requirements of the – of the pigs, when we started these experiments, the N.R.C. requirement that was current at that time was the 1998 version.

[slide showing the N.R.C. requirements for Vitamin D for Swine, Rats and Humans]

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 N.R.C. 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 N.R.C. 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.

[return to the slide of Lessons from hypovitaminosis in pigs showing age in weeks and percentage of kyphosis observed]

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 N.R.C. requirements. Remember, 2004 we went back and fed close to minimum requirements to our animals.

[Thomas Crenshaw]

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-

[slide showing graphs of the experiments showing Dietary treatments at each production phase]

– 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, through – 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.

[slide with a graph of the Maternal Diet Carryover Effect of Hypovitaminosis D induced Kyphosis]

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.

[slide with three graphs of Sow Serum and Milk with graphs for Serum, Colostrum and Milk]

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.

[Thomas Crenshaw]

Tissue concentrations in the pigs, most of the concentrations were in a deficient range or almost non-detectable concentrations.

[slide with two graphs of Vitamin D homeostasis at Birth one with a degradative enzyme and one with and activating enzyme]

We looked at the m.R.N.A. 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 m.R.N.A. for that. That was really not changed in pigs at birth. So, these are concentrations now in the – in the pigs. They were not changed in pigs at birth due to the maternal diets they were fed.

[slide of the same two graphs but showing Vitamin D homeostasis during Weaning]

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?

[Thomas Crenshaw]

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 –

[slide with same parameters except of Vitamin D homeostasis at Seven Weeks]

– but there’s still a – 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.

[Thomas Crenshaw]
.
Now, I just wanted to show you a picture of a pig on a D.X.A. machine. Some of you may have had D.X.A scans. And that’s the main, we use the D.X.A. machine in a quite a few different research projects –

[slide of a sow on a D.X.A. machine with the calculations of bone mineral content]

– 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 D.X.A. to measure the whole-body bone mineral content in pigs.

[Thomas Crenshaw]

And we’ve done quite a bit 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.

[slide with three graphs of Whole Body D.X.A. from the End of Nursery showing differences in levels of supplemental Vitamin D]

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 – 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.

[Thomas Crenshaw]

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.

[slide with three photos one of the vertebral column with kyphosis and two cross-sections of a femur one with Vitamin D and one without]

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.

[Thomas Crenshaw]

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.

[slide with histological photos of vertebral endplates showing control versus kyphotic swine]

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.

[slide of two C.T. scans of swine – one with a normal vertebrate and one with kyphosis]

This is a C.T. scan of a pig that had kyphosis. The obvious curvature that would occur there. The defects are – 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 –

[Thomas Crenshaw]

– 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.

[Laura Amundson, Post-Doctoral Fellow, Department of Animal Sciences, University of Wisconsin-Madison]

So endochondral ossification, as Tom was talking earlier, we think that this is the – the point of failure in our kyphotic pigs, rather than a lack of mineralization, based on some of our D.X.A. 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 –

[slide with main points of Endochondral Ossification]

– 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 resorbing cells, and osteoblasts, which are bone formation cells. And during this process there’s kind of two different forms of bone that – 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.

[Laura Amundson]

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 –

[slide with illustration of Endochondral Ossification showing an large bone on the left and a cross-section of the bone growth plate on the right]

– 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 –

[Laura Amundson]

– you look at these growth plates and the cells are not aligned columns – in columns.

Next, the cells grow through the hypertrophic zone, which they stay in their columnar organization –

[return to the Endochondral Ossification illustration of the cross-section of the growth plate]

– but increase in size. And then, right here –

[a large oval is placed on the cross-section illustration between the Hypertrophic zone and the next zone, the Invading capillary]

– 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 – 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.

[Laura Amundson]

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 – its 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.

[slide of Endochondral Ossification showing limiting steps and critical factors mediated by Vitamin D]

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 –

[Laura Amundson]

– 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 V.E.G.F., 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 to keep in mind what we – eventually what 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.

[slide with an illustration of a large bone on the left and a cross-section of the growth plate on the right now with the factors that may affect growth next to their affecting zones]

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 II collagen is prominent. In the hypertrophic zone, Type X collagen is prominent. And then, as we get down towards the bone, the bony section, Type I collagen is prominent.

And throughout the growth plate –

[Laura Amundson]

– 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 V.E.G.F. are produced by hypertrophic chondrocytes as well as osteoblasts.

[return to the slide of the illustration of the cross-section of the growth plate and the factors that affect bone growth next to the zones that they affect]

And then MMP9 is produced by the osteoclasts, the bone resorbing cell.

[slide of a schematic of how the research thinks that Vitamin D affects bone growth in pigs]

So, just a little schematic of kind of a hypothesis of what we think might be going on, at least in terms of the vitamin D model we have.

So, it has been shown that vitamin D acts on the chondrocytes to produce V.E.G.F., which will get released into the extracellular matrix. The osteoblasts can also produce V.E.G.F., stimulated by vitamin D released into the extracellular matrix. At which point the V.E.G.F. in the extracellular matrix produced by the chondrocytes and the osteoblasts is actually in an inactive form because it’s not available. It’s – its within a complex within the extracellular matrix. So, in order for that to be made available and allow for angiogenesis to occur, MMP9 and MMP13 need to come and basically chew up what’s inhibiting the V.E.G.F. from being available.

[Laura Amundson]

So, the MMP9 and 13 produced by the osteoblasts and osteoclasts will come and release this V.E.G.F. which will then cause angiogenesis, bringing in more osteoblasts and osteoclasts for bone resorption and bone formation.

The missing link kind of is how vitamin D would affect MMP9, which I’ll show you it did in our model.

[return to the schematic of how Vitamin D helps in bone growth in pigs]

The reason we think it’s probably not directly through the osteoclasts is because the osteoclast has very few receptors. It does not have a V.D.R., vitamin D receptor. So, a lot of the osteoclast actions actually are mediated through the osteoblast, which does have a V.D.R.

[slide of the Molecular Data of Lauras research]

So, my – our initial hypothesis was that because we were saying that retained cartilage, there must have been a decreased expression of the MMPs and the V.E.G.F. We did look at the mRNA. It has been shown on the literature that the production of –

[Laura Amundson]

– 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 –

[slide with three graphs of Femur Relative MMP9 m.R.N.A. expression accounting for lack of Vitamin D, small supplementation of Vitamin D and large supplementation of Vitamin D]

– 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 –

[return to the slide of Molecular Data from Lauras experiments with a big red X over her initial hypothesis and her findings from Articular joint diseases]

– 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 –

[Laura Amundson]

– which was uncoupling the endochondral ossification process and eventually leading to some pretty detrimental articular joint diseases.

T.I.M.P.s, 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 P.C.R. 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 – 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.

[laughter]

[slide with conclusions of Lauras experiments]

But it is – it is truly complicated. And – and, as Tom alluded to earlier, that, you know, research is a way to train students. He always said that from the beginning, and as a young, immature student, I, 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 signal-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.

[Laura Amundson]

It’s hard to get at, hard to break down in order to do P.C.R., western blots, things like that, and especially with – 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.

[return to the slide of the conclusions of Lauras research]

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 – 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 affecting the offspring. And I read some epigenetic papers that talk about even your grandmother –

[Laura Amundson]

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.

[return to the slide of the conclusions of Lauras research]

Something definitely was a theme throughout my graduate work. So, I’ll give it back to Tom.

[Thomas Crenshaw]

So, I wanted to let Laura have the opportunity to kind of go through some of her data, and – 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 gonna 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.

[slide of a poster of Development of an image technique using clinical C.T. scans to detect osteochondritic-like lesions in femoral growth plates of growing pigs]

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 C.T. 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 –

[Thomas Crenshaw]

– if you watch on the bottom of the screen, the cursor is actually working here, but there’s a lesion right in that area.

[return to the slide of the poster pointing out a lesion in a C.T. scan that is part of the poster]

And so, as we take slices through the bone at 0.625-millimeter sections through the bone from a C.T. scan, we can identify the lesions, and we’ve worked on. Nicole Gross is the student that worked on this.

[the slide of the poster changes pages to show the C.T. scan methods showing images of various C.T. scans]

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 –

[Thomas Crenshaw]

– 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 – I just kind of wanted to show that because we’re working on techniques to improve our ability to measure the lesions.

With the C.T. 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.

[slide showing Skeletal Lameness in Swine with and illustration of a pig with a broken leg and two photos of mineralization of a pig femur and osteochondrosis in a pig bone]

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]

[Thomas Crenshaw]

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.

[return to the Skeletal Lameness in Swine slide]

She has spent most of her career working with the – 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 –

[slide of Historical Perspectives on Osteochondrosis in Swine]

– 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 –

[Thomas Crenshaw]

– 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.

[return to the Historical Perspectives slide]

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.

[Thomas Crenshaw]

So, what I think one of the major contributions of this work, rather – 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 – those areas, and we’ve focused down and hopefully with identifying some of the cell signals involved and using –

[slide of Conclusions of the research – Maternal Diets are Critical]

– 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 –

[Thomas Crenshaw]

– 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 –

[return to the overall Conclusions slide]

– and actually surprised that – that we were picking this up in the – the kidney, the m.R.N.A. 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 – 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.

[Thomas Crenshaw]

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 –

[slide of Acknowledgements]

– an acknowledgment of several things show up here. First, I’d – Id 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 13 weeks. We picked up a different, made a different conclusion by that. So, we have people that are taking care of our animal that are also very much involved and engaged in the research that we’re doing, and I certainly have a lot of respect for them.

Just a full acknowledgment, I’m kind of the faculty supervisor responsible for the unit. And we have great people. So, they – they do a lot to make me look good. I can feel very confident going out and presenting data that we’ve generated from that unit –

[Thomas Crenshaw]

– because, knowing that it’s been accurately recorded, if there’s a mistake, something, the data is not recorded correctly, I have confidence that they would let that be known, not just pretend it didn’t happen. And that’s an important thing to have in a crew that works in a research area.

The other point that I would make now, and we’ve kind of acknowledged Laura and her PhD committee, Laura Hernandez that she’s now doing a postdoc with –

[return to Acknowledgements slide]

– Margaret Dame, Dan Schaefer and Brian Kirkpatrick all have contributed to her training and education. 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.

[Thomas Crenshaw]

Ben is a very, if – 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 –

[return to the Acknowledgements slide]

– undergrad research stipends, others have worked on different projects in different ways. I’m not gonna 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 – in the Department of Animal Science or Dairy Science –

[Thomas Crenshaw]

– 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]