- Welcome, everyone, to Wednes I'm Tom Zinnen. Lab.
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 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 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 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.
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 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 GI tract in calcium and phosphorus,
but it also acts on the kidney, these hormones do,
to increase the reabsorption of calcium, 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,
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,
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.
So I don't want to take anything away from that
and the contribution that he had.
Another person that I want to mention
is a guy that I've worked with, Ben Benevenga,
that was in animal science and nutritional sciences.
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 with Ben's encouragement.
That endowment is used to support undergrad researchers
in animal science and nutritional sciences.
So we have a fund 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,
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.
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.
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 humpback 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,
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.
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
of the production of the animals.
We know the status of the animal.
These are AAALAC 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,
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 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 VEGF,
which will get released into the extracellular matrix.
The osteoblasts can also produce VEGF,
stimulated by vitamin D released
into the extracellular matrix.
At which point the VEGF
in the extracellular matrix produced
by the chondrocytes and the osteoblasts
is actually in an inactive form because it's not available.
It's 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 VEGF from being available.
So the MMP9 and 13 produced by the osteoblasts
and osteoclasts will come and release this VEGF
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.
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 VDR, vitamin D receptor.
So a lot of the osteoclast actions
actually are mediated through the osteoblast,
which does have a VDR.
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 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 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
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,
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.
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)