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The effects of short term high intensity

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presented by

Kristina Marie Hiney

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6/01 c!CIRC/DateDuo.p65-p.15

THE EFFECTS OF SHORT TERM HIGH INTENSITY EXERCISE ON BONE
PARAMETERS OF IMMATURE ANIMALS

By

Kristina Marie Hiney

A DISSERTATION
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY
Department of Animal Science

2003

ABSTRACT

THE EFFECTS OF SHORT TERM HIGH INTENSITY EXERCISE ON BONE
PARAMETERS OF IMMATURE ANIMALS

By

Kristina Marie Hiney

The ability of short duration, high intensity to increase bone formation in
immature animals was investigated in two experiments using two species, the equine and
bovine. In each study, three treatments compared the effects of confinement, voluntary
activity and enforced exercise on bone parameters. Eighteen Holstein calves 8 wk of age
were assigned to one of three treatment groups - group housed (GR), confined with no
exercise (CF), or confined with exercise (EX). Exercise consisted of running 50 m/d, 5
d/wk. Confined calves remained stalled for the 42-d trial. Fused third and fourth
metacarpal bones were scanned using computed tomography for determination of bone
geometry and three point bending tests to failure were performed. Exercise resulted in
increased dorsal cortex thickness, compared to the GR and CF calves (P<0.05). The
exercised calves had a smaller medullary cavity than CF and GR and a larger percentage
of cortical bone than CF (P<0.05). The metacarpi from EX tended to have a higher
fracture force than CF (P<O. l ). Analysis of the changes in concentration of the bone
marker, osteocalcin, indicated greater bone formation in EX than CF (P<0.05).

In the equine study, eighteen Quarter Horses were divided randomly into three treatment
groups similar to the bovine study: group housed (GR), confined with no exercise (CF).

and confined with exercise (EX). The EX group was sprinted 82 m/d. 5 d/wk.

Radiographs of the left third metacarpal bone were taken to estimate changes in bone
mineral content and cortical widths. Dorsal, medial and total RBAE, tended to differ by
a treatrnent*day interaction, with values increasing over time only in the EX group.
Normalized medial and total RBAE tended to differ (P<0. 1) with treatment, with EX
greater than CF. Dorsal-palmar cortical width in EX was greater than GR on d 56 (P =
0.07). The dorsal palmar medullary cavity decreased in EX compared to GR (P<0.05),
while dorsal and medial cortical width increased only in the EX horses (P<O. 1). Both
studies indicate an adaptation in bone geometry indicating that short duration exercise

may be effective in improving bone mass and therefore skeletal strength.

ACKNOWLEDGEMENTS

Okay, this is the part where I get to thank everybody involved until I hear the
music start to play. First, I would like to thank my graduate committee, it is an honor
even being nominated for a Ph.D. Thank you to Dr. Brian Nielsen, who kept the picture
rolling, thank you Dr. Orth for your technical knowledge and editing, Dr. Miriam Weber
for her vision and urging for acceptance of all cultures (even animal rightists and
bovinetologists), and especially to Dr. Diana Rosenstein for her patience and willingness
to help with all the radiographic and computed tomography data. Especially on the day
after Thanksgiving and on all the weekends. Oops, I can’t believe I almost forgot to
thank my chief engineer, Dr. Marks, without whom no legs would have been broken.

Now I would like to give a shout out to all my peeps! To Kristine and David, I
never would have made it out without you two. I bet no one is missing how much noise
we used to make in 1284. Both of you followed your own dreams for the future which I
think is one of the bravest things ever. David you will make an excellent doctor, just try
to cutback on the scab sandwiches. And always remember that you may see some
bicycles, yes you may even see some tricycles, but it is quite doubtfirl you will ever see
any babies.

Kristine, you helped listen to all my grousing about 8000 many things, especially
my namesake. Uggghh! And if you ever need to have a said beverage, just give me a
jingle. You are my ambassador of Qwan. Any time you want to do any sliding and

spirming come on up.

iv

Cara O’Connor. You put up with a lot of craziness in our dysfunctional little
family. But you helped calm my computer craziness. And hey, I got free CD’S and legs
out of the deal. May you and Solo keep jumping higher'fences.

Karen Waite, who apparently I drove right out of the lab tech business. You
know you are the 2"d one I have done that to. But thanks for helping wrestle cows with
me and allowing me access to all of your 4-H children.

Kim, for providing me a place to live and keep my animals, I appreciate
everything you did for me. Remember, we are always better off renting a movie, sitting
on a coach with a dog and having a marguerita than the rest of those miserable saps!

To Troy Prime, who was absolutely instrumental in my project at Merillat, I am
so happy that you and your family are in such a better situation now. You were
completely screwed over by those people, and no one deserved it less.

For those that have helped me improve my horsemanship skills, Jaimie for
alloWing me to start your pleasure horse prospects, Troy for furthering me in pleasure
horse understanding, Pete Kyle for allowing my break from reality and without whom I
probably wouldn’t have my current job, and for Nathan O’Connor and Larry Kasten,
without their assistance, Pico might have been stuck at —1 ‘/2 for forever!

Now for those that have gone before (no, I don’t mean they are dead, except for
Harvey who is buried at Freeman Arena — party on bunny dudel), all my colleagues from
A&M who are still such a huge part of my life. Especially Eric and Erika, my adopted
brother and sister, or are you my guardians? I treasure all of the advice you have given
me over the years, as you two usually see things better than I do. And the

Rammerstorfers who first initiated me into the whole reining game and also taught me

how mean Austrians can be (okay, maybe that is just CR). I fully expect to conquer
Mozart next year, so make the reservations early! And especially to Dr. Potter, whom I
probably respect more than anyone else. As I said in my thesis, I only hope to someday
approach the level of integrity, professionalism and manner in which you treat others that
you exemplified to me.

And last but not least, thank you to my parents, who have never failed to help me
with all of this craziness, especially moving my belongings from place to place without
ever a complaint. Did you think I would ever be out of school? I finally have my new
name — and that is the only one you arwe getting!

Okay, they are dragging me off the stage now, later folks, thanks again .......

vi

TABLE OF CONTENTS

Page

LIST OF TABLES ..................................................................................... ix

LIST OF FIGURES ................................................................................... xi

CHAPTER

I INTRODUCTION ........................................................................ 1

II REVIEW OF LITERATURE ....................................................... 4

Basic bone biology ............................................................... 5

Mechanotransduction ........................................................... 7

Adaptation of bone ............................................................. 10

Extracellular matrix ........................................................... 12

Bone and activity ............................................................... 14

Short duration exercise ...................................................... 15

Disuse ................................................................................. 18

Systemic or hormonal effects ............................................. 20

Bone growth and the young animal ................................... 23

Determinants of bone strength ........................................... 26

Technologies ...................................................................... 29

III BOVINE STUDY ....................................................................... 43

Summary ...... 43

Introduction ........................................................................ 44

Materials and methods ....................................................... 45

Results ................................................................................ 55

Conclusions ........................................................................ 63

IV EQUINE STUDY ....................................................................... 73

Summary ............................................................................ 73

Introduction ........................................................................ 74

Materials and methods ....................................................... 75

Results ................................................................................ 81

Discussion .......................................................................... 87

Conclusions ........................................................................ 95

V SUMMARY AND CONCLUSIONS .......................................... 97

Implications ...................................................................... l 07
APPENDICES

Appendix A ...................................................................... 110

Introduction ...................................................................... 1 10

Materials and Methods ..................................................... 112

Results .............................................................................. 1 14

vii

Discussion ........................................................................ 1 14

Appendix B ...................................................................... 117
Appendix C ...................................................................... 118
Appendix D ...................................................................... 125
LITERATURE CITED ................................................................... 130
VITA ............................................................................................... 150

viii

TABLE

10

11

12

13

14

15

16

LIST OF TABLES

Page
Body weights (kg) and average daily gain (ADG) .................. 55
Cortical areas of MC 111 & IV (cmz) ....................................... 56
Cortical diameters and widths of MC 111 & IV (cm) ............... 56
Bone mineral density as determined by computed
tomography (mg/cc) ................................................................. 57
Mechanical properties of MC III & IV and 9th rib .................. 58
Radiographic bone aluminum equivalence over
time (mm AL) .......................................................................... 59
Ca and P concentrations (mg/g) and Ca:P ratios of
individual cortices within treatment groups ............................. 60
Percent ash of cortical samples on weight and
volume basis ............................................................................. 6O
Bovine serum deoxypyridinoline (ng/ml) over time ................ 62
Bovine serum osteocalcin (ng/ml) over time ........................... 62'
Total duration (%) of behavioral observations averaged
over (I 0, 21, and 42 .................................................................. 63
Frequency of postural shifts summed for each treatment
ond0,21,and 42. .................................................................... 63

Body weight data (kg) and average daily gain (ADG) of horses
separated by treatment ............................................................. 81

Mean cortical RBAE (mm A1) of treatment groups
over time .................................................................................. 82

Mean dorsal-palmar cortical diameters (mm) according to
treatment groups over time ...................................................... 83

Mean medial-lateral cortical diameters (mm) according to
treatment groups over time ...................................................... 85

ix

17 Total duration (%) of behaviors averaged over all days of

recording for each treatment .................................................... 85
18 Total number of occurrences of behaviors surnmed over all

observations and days for each treatment group ...................... 86
19 Equine serum osteocalcin (ng/ml) over time ........................... 87
20 Equine serum deoxypyridinoline (ng/ml) over time ................ 87
Appendix Tables.
1A Regression coefficients and significance levels for

cortical measurements as compared by computed

tomography and radiography ................................................. 116
1B Calf diets for bovine study ..................................................... 1 17
1C Bovine cortical areas of MC III & IV. ................................... 118
2C Bovine cortical widths of MC 111 & IV ................................. 118
3C Bovine computed tomography bone mineral densities .......... l 19
4C Bovine mechanical testing data ............................................. 119
5C Bovine modulus of elasticity and rib mechanical data .......... 120
6C Bovine palmar radiographic bone aluminum

equivalence (mm Al) .............................................................. 120
7C Bovine dorsal radiographic bone aluminum

equivalence (mm Al) .............................................................. 121
8C Bovine lateral radiographic bone aluminum

equivalence (mm Al) .............................................................. 121
9C Bovine medial radiographic bone aluminum

equivalence (mm Al) .............................................................. 122
10C Bovine Ca and P concentrations (mg/g) by cortices .............. 122
11C Bovine serum deoxypyridinoline (ng/ml) .............................. 123
12C Bovine serum osteocalcin (ng/ml) ......................................... 124

1D

2D

3D

4D

5D

Equine radiographic bone aluminum

equivalence (mm Al) .............................................................. 125
Equine cortical widths for d 0 and 28 .................................... 126
Equine cortical widths of MC 111 for d 56 (mm) ................... 127
Equine osteocalcin (ng/ml) .................................................... 128
Equine total deoxypyridinoline (ng/ml) ................................. 129

xi

LIST OF FIGURES

FIGURE Page
1 Schematic illustration of a cross-section of bovine
MC 111 & IV showing cortical measurements .......................... 50
2 Schematic illustration of a cross-section of equine
MC 111 showing bone diameter measurements ....................... 79
3 Schematic illustration of a cross-section of equine
MC 111 showing cortical measurements. .................................. 88
4 Schematic illustrations of cortical measurements
created from Multi-Analyst software ..................................... 112

xii

CHAPTER I
INTRODUCTION

Lameness is a key concern among producers of many species in animal
agriculture, including the swine, poultry, dairy and equine industries. Frequently,
unsound animals must be removed from the herd or flock and euthanized. While this is a
financial concern, these losses also raise issues of animal welfare and well-being. While
the causes for lameness among species may vary, often they can be traced to the
musculoskeletal system. In the equine industry, lameness is of particular concern, as it is
the leading cause of wastage among racehorses (Jeffcott et al., 1982; Rossdale et al.,
1985). The majority of injuries are musculoskeletal in nature, with potentially
catastrophic results. In a survey conducted by Jones (1989), 58.1% of all two-year-old
racehorses experienced an injury. As most young racehorses are placed into training as
long yearlings, the rate of injuries may be due, in part, to the relatively immature skeletal
structure of the animal.

Recently, attention has been focused on the impact of early management of young
animals on skeletal strength. Unfortunately, some management practices of domestic
livestock species may jeopardize skeletal integrity. Within the equine industry,
weanlings and yearlings are often confined in small box stalls for several months to
facilitate preparation for sales or futurities. Stall confinement decreases bone mass in
young horses relative to age-matched horses on pasture (Hoeskstra et al., 1999; Bell et
al., 2001; Firth et al., 1999). Following confinement, these horses are often placed into
rigorous training, which may render them more susceptible to injury than horses with

greater bone strength. Therefore, the goal of much research within the horse industry is

to identify optimal management practices that better prepare the young horse to begin
training.

One approach to decreasing lameness is to strengthen the skeleton prior to the
initiation of traditional “under-saddle” training. Young animals appear to adapt more
readily to exercise (Loitz and Zernicke, 1992; Iwamoto et al., 2000), and increasing bone
mass may be easier if training begins early. Training in young horses increases bone
mass, provided the exercise is of sufficient intensity (McCarthy and Jeffcott, 1991).
Therefore, training at high speeds before “under-saddle” training begins may better
prepare the young horse than waiting until a later age when the skeleton is more mature.

One of the difficulties in performing studies of bone physiology in the equine is
the lack of accurate, non-invasive determinants of bone structure. Computed
tomography, while highly accurate, is difficult to perform due to the expense and risk
involved with anesthetizing large animals. Terminal studies provide the most definitive
information, but again, the expense and use of an animal species considered by some to
be a companion animal limit the option of terminal research. However, the use of a
bovine model is proposed as a suitable alternative. Holstein bull calves are commonly
slaughtered in industry, and are similar in structure and size to young horses. In addition,
bovine skeletal metabolism and adaptation has not been extensively researched. The
hypothesis of the study was that confinement in young animals causes a reduction in bone
mass or strength through a slowing of bone formation, and this reduction in bone growth
can be alleviated or possibly enhanced by providing either a group housing system or
short periods of forces exercise. Therefore the objectives of these studies were to

investigate the usefulness of short periods of intense exercise in improving bone quality

in two different species, as well as to compare bone response to confinement and an

environment which provides ad libidum exercise.

The specific goals of this project were to:

1.

determine if short term, high intensity exercise enhances skeletal mass
and architecture compared to confined or group housed animals.
determine if a reduction in bone mass or bone growth due to
confinement could be alleviated by short term exercise

evaluate the use of the bovine model for studies in basic bone
physiology and comparative studies in the equine.

Determine the influence of voluntary behavior on skeletal parameters

CHAPTER II

REVIEW OF LITERATURE

Bone is a rather unique, highly adaptive tissue that alters its shape and structure in
response to changes in its external environment. The skeletal system responds to patterns
of loading or strain in order to achieve a balance between skeletal strength and skeletal
mass (Rubin, 1984). While a more massive skeleton would be stronger and able to resist
higher forces, it would not be metabolically economical to the animal. Maintaining the
minimal bone mass required to meet the animal’s needs of skeletal strength is an
evolutionary advantage as it reduces the energy expenditure involved with locomotion,
synthesis, and maintenance of extraneous tissue (Loitz and Zernicke, 1992).

This phenomena of the adaptability of bone, first described in the late nineteenth
century, is known as Wolff’s Law (Woo et al., 1981). Numerous studies conducted in
both humans and animals show the positive effect of exercise on bone mass (Heinonen et
al., 1993; Snyder et al., 1992; Biewener and Betram, 1994) while bed rest (Donaldson et
al., 1970; LeBlanc et al., 1990), confinement (Marchant and Broom, 1996) or
weightlessness (Whedon et al., 1977) lead to a reduction of bone mass. The premise of
Wolff’s Law relies on the ability of cells within bone to sense variations in mechanical
strain that fall outside the desired biological window. Cellular processes are then
activated which can alter the extracellular architecture and return strains to a desired
level. For example, in animals subjected to ulnar osteotomy, the radius altered its mass in
response to the sudden increase in strain, and returned recorded strains to pre-operative

levels (Lanyon et al., 1984; Goodship et al., 1979). Similarly, when the mechanical strain

is too low, “excess” bone is removed. Therefore the goal of much current research is to
develop training and management practices which optimize skeletal strength through a

clearer understanding of bone biology.

Basic Bone Biology

In order to prescribe programs to promote skeletal strength, an understanding of
basic bone biology and the mechanisms governing this system is essential. The skeletal
system is a highly specialized form of connective tissue providing numerous essential
functions. While its primary role is that of support, protection, and locomotion, the
skeleton is also the main reservoir of many minerals essential for proper cellular function.
A complex relationship between bone cells and the extra cellular matrix allows the
skeleton to respond to demands between both the need for effective support and the
maintenance of mineral homeostasis.

Bone is comprised of specialized cells within an extensive extracellular organic
matrix strengthened by the deposition of calcium salts, primarily in the form of
hydroxyapatite (Calo(PO4)6(OH)2). Briefly, the four predominant cell types found in
bone tissue include: osteoblasts, osteoclasts, osteocytes and bone-lining cells.
Osteoblasts are the cell type responsible for the formation of bone. Osteoblasts not only
produce bone matrix, but also regulate the mineralization of the organic matrix. As
osteoblasts mature, they either undergo apoptosis, or become completely surrounded by
mineralized tissue and are then referred to as osteocytes (Marks and Ogdren, 2002).
Osteocytes exist within obloid spaces called lacunae, and are believed to be the actual

mechano-sensors of the tissue. Thus, they must communicate with not only other

osteocytes, but other cell types as well. Cellular projections through the canaliculi and
gap junctions allow transfer of electrical signals, as well as intracellular and extracellular
transport of signal molecules between osteocytes and the bone surface. Similar
communication and the passage of the mechanical signal has also been shown in
osteoblasts (Martin, 2000) and osteocytes (Nomura and Takano-Yamamoto, 2000).
Bone surfaces not actively undergoing bone formation or resorption are covered
by fiat, inactive cells, or bone-lining cells. These cells may be the precursors of
osteoblasts, capable of differentiating when needed (Marks and Odgren, 2002). Bone-
lining cells covering the vertebrae of rat tails developed structural features of osteoblasts
following a single loading bout (Chow et al., 1997). Bone-lining cells may help regulate
osteoclastic activity as well. Osteoclasts in resorption pits were found to be in close
contact with bone-lining cells and cytoplasmic extensions of bone-lining cells to
osteoclasts suggested transfer of signals between these cell types (Everts et al., 2002).
Finally, osteoclasts, multi-nucleated cells of hemopoietic origin (versus the
mesenchymal cell origin of those previously discussed) are responsible for the resorption
of mineralized bone. As they orignate from hemopoietic stem cells, they use vascular
channels to migrate to the site of absorption. The mononuclear cells then fuse with other
cells to become the fully active osteoclast. Osteoclasts are distinguished by their ruffled
border, a highly infolded section of the plasma membrane, which rests directly on the
bone surface to be eroded. Osteoclasts also have a clear zone, which surrounds the
ruffled border and serves as the point of attachment to the bone matrix. Osteoclasts are
abundant with lysosomal vesicles containing the proteases and H)r ion concentrations

necessary to solubilize the inorganic matrix (Marks and Ogdren, 2002).

Mechanotransduction

The mechanical stresses placed on the bone must be translated into cellular
signals, a process referred to as mechanotransduction. The appropriate response, either
to increase or decrease bone formation and/or resorption, should be proportional to the
degree of strain experienced. The force on the bone is suggested to be transmitted via a
flow of interstitial fluid, sensed as alterations in streaming potential and shear stresses
(Nomura and Takano-Yamamoto, 2000). Mechanical forces are transformed into
biological irnpedances resulting in specific cellular events. The means by which bone
senses and responds to its loading environment has not been completely defined, but
several cellular pathways and signaling mechanisms are involved. Possibilities for the
exact chemical mediator include changes in ion channel sensitivity altering the flux of
Ca+2 into the cell (Hung et al., 1995), activation of focal adhesion kinases, or
modifications of the cytoskeleton, or integrins and CD44 receptors which anchor bone
cells to the extracellular matrix (Nomura and Takano-Yamamoto, 2000), or perhaps
combination of all of these.

Once the mechanical signal, which can occur after only a brief period of load
application, has been received by the osteocytes, changes in cellular function occur
quickly. Increased RNA production and glucose consumption have been observed in
organ culture between 6 and 24 h post-loading (Markus, 2002) with other reports of
increased RNA production within minutes after application of mechanical strain
(Einhom, 1992). Other factors linked with bone formation such as prostacyclin,

prostaglandin E2 (PGEz) and nitric oxide (N O) are released from bone surface-lining

cells and osteocytes 5 min following loading (F orwood and Turner, 1994) and
transforming growth factor B (TGF-B), insulin-like grth factor — 1 (IGF-l), and c-fos
RNA increase within 2 to 4 h (Raab-Cullen et al., 1994a). In fact, a number of genes
have been discovered which have increased expression levels in response to mechanical
stress including glutamate/aspartate transporter, nitric oxide synthase, c-fos, and cox-2
(N omura and Takano-Yamamoto, 2000) which sustain the release of prostaglandins and
NO. These changes are believed to be mediated by the rapid and transient release of
PGE2, especially as mechanical effects can be abolished by indomethacin, a PGEz
antagonist (Markus, 2002).

Numerous other factors may be involved in the cellular response to loading.
Signals are potentially transmitted through the cytosol through the action of various
hormones as estrogen, prostaglandin, parathyroid hormone (PTH), di-hydroxy vitamin D3
(1,25-(OH)2D3)). Also, glucocorticoid receptors are found on osteocytes. Parathyroid
hormone, previously known only for its role in Ca homeostasis, is now accepted as
modulating the osteocytic response to mechanical strain. (N ijweide et al., 2002; Noble
and Reeve, 2000). Mechanical stimulation via PGE2 activates several classic 2nd
messenger systems, including protein kinase A and protein kinase C which increase
concentrations of cAMP and activate arachidonic acid and PGE2 release (N ijweide et al.,
2002). Elevation of various transcription factors such as AP-l, the product of the c-fos
gene, also result in a cascade of cellular events as it serves as a general activator for a
large number of genes (Yamauchi et al., 1996). Mechanical stimulation increases levels
of growth factors such as IGF-I and -II, which stimulate collagen and DNA synthesis in

osteogenic cells (Damien et al., 2000; Lean et al., 1995). As bone formation following

external loading was 5-fold greater in mice overexpressing IGF-l than wild type mice
(Gross et al., 2002), IGFs may play an important role in mediating bone formation.

While changes in concentrations of 2nd messengers may take only minutes, it does
appear that the actual response of producing osteoid takes 3 to 4 days after activation
(Forwood and Turner, 1994; Pead et al., 1988a). These common cellular events
described above are followed by those more specific to bone tissue, such as the increased
production of bone matrix proteins such as type I collagen, osteopontin, and osteocalcin.
Also, load stimulus may not just increase bone formation, but can reduce bone resorption
as well. Rubin et al. (1999) found a 40% reduction in osteoclast formation in marrow
cultures subjected to strain.

In order for the bone tissue to respond in the appropriate fashion, the osteocytes
that sense strain must coordinate the osteoblasts and osteoclasts responsible for forming
or resorbing bone. Communication can potentially occur through the release of growth
factors or other mediators, or through direct cell-to-cell contact. Bone resorption is
regulated by osteoblasts or cells of the osteoblast lineage, rather than osteoclasts, the cells
ultimately responsible for resorption, as osteoclasts cultured without osteoblasts fail to
form resorption pits (Takashi et al., 2002). In fact, receptors for most osteolytic signals
are found on osteoblasts rather than osteoclasts (Rodan and Martin, 1981) and their
presence may be essential for resorption. While vitamin D, parathyroid hormone,
interleukins, and prostaglandin E2 all stimulate the formation of osteoclasts (Takashi et
al., 2002), the differentiation of immature osteoclasts appears to be mediated through
cell-to-cell contact with osteoblasts and their production of macrophage colony

stimulating factor (M-CSF). Cultured osteoclasts were unable to resorb bone in the

absence of stimulatory factors released by osteoblasts (McSheehy and Chambers, 1986).
Both osteocalcin and osteopontin, proteins synthesized by osteoblasts, may play a role in
the recruitment and attachment of osteoclasts, the first step in the initiation of the bone
remodeling cycle (Rodan and Rodan, 1995; Reinholt et al., 1990).

Bone resorption itself also releases growth factors from the bone matrix that, in
turn, promote the recruitment and activation of osteoblasts, coupling these two processes
tightly together. The release of inorganic phosphate from the demineralization of bone
also may induce apoptosis of osteoblasts in the immediate area, removing the stimulus of
resorption (Meletti et al., 2000). Therefore the function and activity of all bone cell types
are tightly interwoven between the initial mechanical sensing by osteocytes, followed by

biomechanical coupling, cellular communication, and the effector response.

Adaptation of Bone

 

Regardless of the exact pathway by which strain is translated into cellular signals,
the reaction of bone is to preserve its integrity. Bone failure occurs not only from sudden
traumatic failure but also through progressive weakening of bone (Carter et al., 1981).
Indeed, fatigue failure may be one of the most common causes of musculoskeletal
injuries in horses (Estberg et al., 1996). Fatigue damage, due to the repetitive loading of
bone, produces micro-cracks within cortical bone (Burr et al., 1985), decreasing its
stiffness and increasing the strain magnitude (Nunamaker et al., 1990). This initiates the
local repair mechanism of bone remodeling (Mori and Burr, 1993; Burr et al., 1985), a
tightly coupled process where removal of old, weakened bone precedes replacement with

new, undamaged bone. Bone is therefore able to withstand some damage that can be

10

repaired through normal processes before failure occurs. Continuous remodeling also
prevents the accumulation of older, more densely mineralized bone which is more brittle
(Ott, 2002).

Osteocyte apotosis in the region of damaged bone may initiate the activation of
remodeling as well as the increase in strain (Verborgt et al., 2000) while formation alone
is associated with a decrease in osteocyte apotosis (Noble et al., 1997). Bone-lining cells
first remove osteoid covering the bone surface via matrix metalloproteinases which
allows the subsequent attachment of osteoclasts (Everts et al., 2002). Activated
osteoclasts remove a volume of bone during the resorption phase, lasting approximately 3
wk in humans (Parfitt and Chir, 1987). In cortical bone, this results in the removal of a
tunnel or cone of bone. Bone resorption is followed by a reversal phase, normally 1 to 2
wk in duration, during which small mononuclear cells, potentially identified as bone-
lining cells, are attracted to the resorption pit and clean it of any remaining undigested
non-mineralized collagen (Everts et al., 2002). After bone-lining cells deposit a thin
layer of collagen (the cement line), osteoblasts begin to lay down the new organic matrix,
which later will be mineralized to form an osteon (Parfitt and Chir, 1987). The complete
process of bone formation and mineralization for one bone remodeling unit may take as
long as three months. Typically, bone remodeling results in no change in bone mass;
however, with advancing age, the formative phase of remodeling may fail to keep pace
with the resorptive phase and thus senile osteoporosis can occur.

In addition to local repair of damaged bone and an alteration of bone mass or
density, the skeletal system evolved with the ability to alter the geometry of the bone,

sometimes referred to as modeling. Discrepancies in the literature between the terms

11

bone modeling and remodeling can lead to some confusion. Some authors separate the
two processes into separate categories in which bone modeling refers to only addition of
bone mass and never a reduction while remodeling is any change in existing bone (Frost,
1990; Burr et al., 1985). Others refer to bone modeling as a non-localized process by
which bone alters its shape, either by the addition of removal of bone on localized
surfaces (Jee, 1988; Kimmel, 1993). The most commonly accepted definition is a change
in shape of the bone or drift. Drift can occur by either slowing or increasing the relative
rate of formation or resorption of bone. Therefore, a decrease in periosteal bone
formation on a certain aspect of the bone does not always have to be interpreted as a
negative outcome, but merely a corrective geometric adaptation in placing more bone
where needed and away fi'om areas with less strain (Mosely et al., 1997). Several studies
have reported that geometric and biomechanical properties of long bones improve
without an increase in bone mass (Barrengolt et al., 1993; Woo et al., 1981). A
geometric change without a change in mass or density can therefore still be beneficial to

the animal and should not be overlooked.

Extracellular Matrix

 

While most studies examining bone strength and integrity focus on bone mineral
density, the non-mineral, or organic portion of the extracellular matrix also changes with
exercise or disease. Type I collagen, common in many tissues, makes up 95% of the
organic matrix of bone, with the remainder consisting of proteoglycans and
noncollagenous proteins. Type I collagen has a triple helix motif composed of three

polypeptide chains of repeating Gly-X-Y amino acid sequences. Most typically X is

12

proline and Y is hydroxyproline. Hydroxyproline serves to stabilize the triple helix and
is found almost exclusively in collagen. Thus, the concentration of hydroxyproline
within a sample of bone can provide an indication of collagen content which may change
with age and exercise. McDonald et al. (1986) found an increase in hydroxyproline
content of the femur and humerus of 7 mo old rats trained for 12 wk. However, exercise
decreased the hydroxyproline content in the femoral neck (Salem et al., 1993) of
immature rats but concentrations of hydroxyproline did not change with age from 8 to 18
wk of age.

F ibrils of type I collagen are spontaneously formed following the post-
translational modification of N and C terminal propeptides of procollagen. The fibrils
then undergo further modification through the action of lysyl oxidase by cross-linking
lysine or hydroxylysine residues within fibrils. The final mature products are pyridinium
crosslinks, whose metabolism is often monitored as an index of bone activity, which will
be discussed in more detail at a later point. The process of cross-linking confers
additional strength to the extracellular matrix and thus to the whole bone. However, Eyre
et al. (1984) reported that collagen crosslinks, while improving the mechanical properties
of soft tissue, were less important in bone than other tissues. The pattern of collagen
cross-linking may play a role in regulating bone mineralization, as the ratio of
hydroxylysylpyridinoline to lysylpyridinoline was twice as high in bone calluses as
control bone 3 wk after fracturing (Wassen et al., 2000). This ratio returned to normal
levels 21 wk after fracture when collagen was fully mineralized.

Concentrations of pyridinium crosslinks within the bone increase in the first two

_ decades in humans and remain at a constant level thereafter (Eyre et al., 1988). Others

13

have also reported a maturation-related increase in collagen crosslinks (Eyre et al., 1984;
Salem et al., 1989). The constant process of bone remodeling prevents the accumulation
of more crosslinks after skeletal maturity, as the bone matrix is replaced. This is most
likely due to remodeling rather than steric hinderance of mineral formation between
fibrils as low bone turnover in osteopetrotic rats is associated with high concentrations of
pyridinium crosslinks (Wojtowicz et al., 1997). Few studies have examined the effects of
exercise and immobilization however. Crosslink concentrations increased with age and
exercise in the femoral neck of rats (Salem et al., 1993) while Yamauchi et al. (1988)
reported no change in crosslink concentration in primates immobilized for 7 mo. In
roosters exercised at an intensity great enough to temporarily decrease the radial and
longitudinal growth of the tibiotarsus and femur, runners and controls had similar

crosslink concentrations (Maynard et al., 1995).

Bone and Activity

The degree of the bone modeling/remodeling response is dependent on the
magnitude (Robling et al., 2001; Mosley et al., 1997; Rubin and Lanyon, 1985), number,
rate (Mosley and Lanyon, 1998; Turner et al., 1995), distribution (Rubin and Lanyon,
1984) and frequency of cyclic strain (Turner and Pavalko, 1998). The magnitude and
velocity of the fluid flow (and thus the streaming potentials and bioelectric current)
depends on all of these factors which can act independently or additively. The number,
as well as the magnitude, influence osteoregulation independently. High peak strain
magnitudes are more osteogenic than low strain, regardless of cycle number (Rubin and

Lanyon, 1985; Kerr et al., 2001). In addition, bone appears to be more responsive to

14

dynamic loading as opposed to static loading, which either has no effect (Lanyon and
Rubin, 1984) or may depress bone growth (Robling et al., 2001). Presumably this is due
to the absence of stimuli of fluid flow through the canniculi once the static load is in
place. Osteogenic response is positively correlated to strain rate, which refers to the
speed at which a strain is developed, independent of frequency, when all other factors are
held equal (Mosely and Lanyon, 1998; Turner and Pavalko, 1998) and is again related to
the velocity of the fluid flow (Burr et al., 2002). Mosley and Lanyon (1998) showed that
higher strain rates are more osteogenic than increasing strain magnitude. Others have
proposed that the diversity of strain, compared to absolute magnitude, initiated the
adaptive response (Biewener and Bertram, 1993), with more uncommon strains creating a
larger response. For example, large increases in cortical cross sectional area in
experiments involving artificial loading of avian ulnae may reflect the unusual direction
of the strain versus normal wing flapping (Lanyon, 1993). All of these factors should be
considered when implementing a program designed to cause adaptation in bone. The less
dramatic changes observed in traditional physical exercise programs such as running may

be due to the more normal, physiological strains generated by such programs.

Short Duration Exercise

Bone is sensitive to only a few cycles of loading, providing the stimuli is beyond
that normally experienced. Exposure of avian ulnae to a single loading event resulted in
activation of the quiescent periosteurn with flat inactive osteoblasts changing to more
rounded active cells (Pead et al., 1988a). Rat tibia loaded for a single bout of 36 cycles

increased periosteal woven bone formation along 40% of its surface (Forwood and

15

Turner, 1994). Four cycles per day of an externally applied load prevented bone loss in
immobilized limbs, while 36 cycles resulted in an increase in bone mass above controls
(Lanyon, 1984). Lanyon (1984) found no further increase in bone response between 36
or 1800 strains; thus, the response of bone appears to quickly become saturated.
Extended durations of an activity are presumably not important as long as the threshold
level is reached with only a few cycles of unusual strain being significant in a sedentary
animal. Five jumps per day increased bone mass and mechanical strength of rat femora
and tibia over sedentary controls, however an additional response was seen up to 100
jumps per day (Umemura et al., 1997) suggesting saturation had not occurred.

Strain in normal controlled locomotion such as walking may have little effect on
bone architecture while sudden movements of very short duration, such as when an
animal is startled or jumps create strains that may be five times greater than normal
movement (Skerry and Lanyon, 1995). In sheep with an external fixator placed across
the hock joint unloading the calcaneous, daily periods of walking were unable to prevent
bone resorption (Skerry and Lanyon, 1995). The external fixator effectively suppressed
the high-magnitude strains (1,150 microstrains) reported to occur during normal
background loading. With the fixator in place, loading was minimal at only 150
microstrains (Skerry and Lanyon, 1995). Jumping in rats has been reported to cause
loads of 2500 microstrains in comparison to 1200 microstrains due to running (Mosley et
al., 1997) similar to recorded results in roosters as well (Judex and Zernicke, 2000a). In
another study of treadmill-exercised roosters, a defined exercise bout resulted in strains
generally greater than 500 microstrains while background loading (the load created by

normal everyday activity-not training) resulted in intermittent high-magnitude strains

16

 

 

bti

greater than 1,000 microstrains (Konieczynski et al., 1998). Therefore, while brief
experiences of high strains may only account for 1% of the total loading history of an
animal, they may be the largest contributor to the osteogenic response.

Exercise programs which create higher strains in the bone such as jumping or
resistance training, appear to be more effective in causing osteogenesis. In young rats, a
greater increase in femoral and tibia ash weight was observed in jump-trained versus run-
trained rats (Umemura et al., 1995). In addition, while adult rats did not respond to run-
training, an increase in bone mass was observed with jump-training. Jump-training
would provide fewer, but higher, magnitude strains compared to run-training. In roosters
which performed 200 drop jumps/d (roosters were lifted 50 to 60 cm off the ground,
accelerated, and released) for 3 wk, bone formation rates (BF R) increased on both the
periosteal and endosteal surface (J udex and Zernicke, 2000b), particularly in the regions
experiencing the greatest strain rates. Bone formation rates in this study were much
greater than in previous studies by the same laboratory using run-trained roosters. In pre-
pubertal male and female children, after seven months of jump-training consisting solely
of jumping off a 61 cm box 100 times, 3 d/wk , jumpers had a 4.5% and 3.1% increase in
femoral neck and spine bone mineral content (Fuchs et al., 2001). As of yet, the
threshold to elicit a cellular response is unknown and most likely is different for each
system, being highly dependent on the loading history of the bone. The underlying factor
is that those strains must be unusual to those normally experienced.

An alternative to increasing strain magnitude by resistance training or jumping is
to increase the speed, which also will increase strain rate. Strain magnitude and rate has

been reported to increase linearly with velocity (Davies and McCarthy, 1994; Biewener

17

and Betram, 1993). While excessive loading of the skeleton will certainly lead to fatigue
damage or worse, some amount of high-speed exercise may have protective effects.
Thoroughbreds which experienced injuries had less high Speed exercise prior to injury
than non-inj ured controls (Cohen et al., 2000). Horses which had greater cumulative
exercise during the previous 1 to 2 months were at a decreased risk of injury (Cohen et
al., 2000). A lack of high speed exercise may have led to a decreased bone density which
predisposed the animal to injury, although the horses receiving less exercise may have
already been sore and worked less. Therefore the population of horses with greater high

speed exercise were those less predisposed to injury.

Disuse

As mentioned previously, disuse, such as occurs with micro-gravity, bed-rest,
immobilization (Buckingham and Jeffcott, 1991a), or confinement (Marchant and Broom,
1986; Porr et al., 1998), results in unneeded mineral being resorbed in order to maintain
the optimum strain environment and minimal bone mass. Immobilization of a single limb
results in localized demineralization of the bone not observed in the contralateral control
(Inman et al., 1999). This differs to bone resorption caused by hormonal imbalances or
dietary deficiencies which can result in systemic demineralization of the skeleton.

Bone resorption due to immobilization occurs quickly, with decreases in bone
mineral density, stiffness and ultimate load reported after only 6 wk in rats (Inman et al.,
1999). In horses, 4 wk of immobilization of the forelimb increased the medullary cavity
size (Buckingham and Jeffcott, 1991a). While it may take weeks to achieve a detectable

decrease in bone density or altered bone geometry, the cellular resorption response occurs

18

within days. Markers of bone resorption including the cross-linked carboxyterminal
telopeptide of type I collagen (ICTP), hydroxyproline, and deoxypyridinoline (DPD)
increase within two days of bed rest (Lueken et al., 1993). This indicates that skeletal
disuse results in an almost immediate response to begin degradation of “unneeded” bone.
Concentrations of the carboxyterminal propeptide of type I procollagen (PICP) were
found to decrease following three days of bed rest while osteocalcin (OC) values
increased (Pedersen et al., 1995 ).

Confinement rearing of young animals over extended periods of time may lead to
a decrease in the total bone mass able to be achieved at maturity and subsequently result
in an inferior skeleton. The response to immobilization may be very different between
ages of animals, both in degree of bone lost, as well location of resorption. Bone loss in
immature rats was seen after lwk of immobilization, versus 3 wk in mature rats
(Steinberg and Trueta, 1981). Osteoclastic resorption occurred on trabecular and
persiosteal surfaces in young dogs but occurred on the endosteal surface in mature
animals (Uhtoff and Jaworski, 1978). Lack of physical exercise may also lead to
decreased bone formation as opposed to bone resorption due to the over-riding process of
growth. Sciatic neurectomy and tail-suspension in rats resulted in a decrease in bone
mass as a result of decreased bone formation rather than increased resorption as
determined by histomorphometry (Yeh et al., 1993, Kodama et al., 1991).

If confinement is coupled with Ca deficiency, the loss of bone is exacerbated. In
5-mo-old rats, immobilization of the hind-limb concurrent with Ca deficiency resulted in
a 28% loss of BMC in the proximal tibia (Inman et al., 1999) and 32% bone loss in adult

turkey ulnae (Lanyon et al., 1986). Application of a loading stimulus can only partially

l9

compensate for the hormonal effects triggering bone resorption. Loading partially
reduced the bone loss in Ca deficient animals (Lanyon et al., 1986), and attenuated the
decrease in mechanical strength (Inman et al., 1999). Bone loss due to Ca deficiency
becomes even more dramatic if imposed on the immature animal. A dramatic reduction
of 46% of the femoral ash weight was observed after only 72 h of immobilization and Ca
deficiency in 2-mo-old rats (Weinrab et al., 1991). Therefore, if young animals are
confined while being on a diet that provides an insufficient amount of Ca, the owner or

producer may experience greater problems with skeletal injuries.

Systemic or Hormonal Effects

The process of bone modeling/remodeling is not only governed by local laws of
loading events, but also by systemic changes as well. When blood Ca concentrations
drop, the skeleton serves as a large reservoir of available Ca. While bone remodeling
does affect serum levels of minerals, minute-to-minute regulation of serum
concentrations are not dependant on bone remodeling. However, long term changes in
serum concentrations such as Ca defiency can alter bone metabolism.

Receptors for extracellular Ca are found in the main organs involved in Ca
homeostasis: the parathyroid gland, thyroid and kidney. Low serum Ca results in the
release of PTH from the parathyroid gland which increases bone turnover. However, the
rapid increase in serum Ca is not due to bone remodeling, as it takes much longer to
activate osteoclasts. Parathyroid hormone also acts directly on the kidney to increase
reabsorption of Ca and to increase the hydroxylation of 25-OH D3 to l, 25-(OH)2 D3.

The kidney also conserves Ca by altering renal blood flow, glomerular filtration rate, and

20

 

 

Inc

tubular resorption rate to reduce Ca excretion. Vitamin D increases Ca absorption from
the small intestine and has similar effects of PTH on osteoclastic acitivity. Receptors for
PTH and vit D are both found on osteoblasts, despite the fact that their main recognized
role is to increase bone resorption (Takahashi et al., 2002). As discussed previously, the
increased metabolic activity of osteoclasts as well as the increase in their numbers in
response to PTH is believed to be mediated through osteoblasts. When ionized Ca
concentrations increase, calcitonin is released from the thyroid gland which inhibits
osteoclast activity and increases Ca excretion in the kidney (Becker et al., 2002).

Parathyroid hormone has been used as a therapy for bone loss, almost
paradoxically with its known role in bone resorption. Sustained elevations in levels of
PTH can lead to a dramatic decrease in bone mass. However, intermittent doses of PTH
increased bone formation and bone mass (Gunness-Hey and Hock, 1984) and appears to
act synergistically with mechanical loading (Chow et al., 1994; Ma et al., 1999). PTH
administration increases the concentration of 2"d messengers and increased intracellular
Ca similar to effects of strain (Ryder and Duncan, 2001) and increased the sensitivity of
osteoblasts to fluid sheer, perhaps explaining the effect of PTH in increasing bone mass.
Administration of PTH helps regain bone mass following a period of immobilization (Ma
etaL,1999)

Reproduction places large demands on the mineral homeostatic system of the dam
for fetal development and later milk production. Mares (Glade, 1991), rats, monkeys (Ott
et al., 1999) and humans (Heaney and Skillman, 1971) increase Ca absorption during late
gestation in order to counterbalance the increase in Ca demand. Mares fed adequate Ca

increased in metacarpal breaking strength during the last 12 wk of gestation, compared to

21

 

f0;
[11:

Fee

2001

90mm

mares on a deficient diet which showed no change (Glade, 1993), suggesting a
conservation of mineral for upcoming lactation. Adequate Ca stores in the bone are
critical during lactation, as the skeleton is the mineral reserve mobilized during periods of
high demand. For example, bone mass in humans decreased 5 to 20% during lactation,
while monkeys lost 3 % of total body bone mineral between parturition and 3 mo of
lactation (Ott et al., 1999). Metacarpal bone strength decreased in mares during the first
12 wk of lactation and was restored by 24 wk post-parturition in mares on the Ca-
sufficient diet. However, those mares fed the deficient diet had not fully recovered 40 wk
after foaling. More importantly, foals born to the Ca deficient mares had thinner cortical
diameters and weaker third metacarpi (MC III; Glade, 1993). Therefore, during gestation
and lactation, animals not only need to be on a Ca sufficient diet, but also to have an
optimal bone mass for sustaining milk production and maintaining their own skeletal
integrity.

As Ca deficiency results in an increase in systemic factors causing bone
resorption, sufficient Ca in the diet is important for the exercising animal. Exercise could
not prevent bone loss in Ca-deficient rats (Bauer and Griminger, 1983) due to the
overriding hormonal stimulus directed towards bone resorption rather than bone
formation. Porr et al. (2000) found that only horses conditioned while consuming a diet 2
times that of the NRC exhibited a small increase in BMC of the medial aspect of MCIII.
Feeding increased concentrations of Ca above NRC recommendations may also enhance
bone mass of the young racehorse during training (Nielsen et al., 1998; Nolan et al.,
2001), but Ca supplementation of foals through long yearlings did not alter bone mineral

content (Hoffman et al., 2001).

22

 

CS

pr:

dur

Char

apps

Bone Growth and the Young Animal

Much of the work done in the area of bone physiology has been primarily in the
skeletally mature animal. However, most bone modeling occurs very early in life and
mature bone may not be as sensitive to exercise-induced changes as younger, growing
bone (Iwamoto et al., 2000; Loitz and Zernicke, 1992). In immature rats, cortical
thickness (Steinberg and Trueta, 1981) and ash weight (Umemura et al., 1995) increased
in response to running, but not in adult rats. Using the isolated turkey ulna model,
increases in bone area and periosteal mineralizing surface were observed in l-yr-old but
not 3-yr-old turkeys (Rubin et al., 1992). However, at one year of age, turkeys should
have reached skeletal maturity. While not conclusive evidence, thoroughbred racehorses
which received their first start as two-year-olds had more starts and had a longer career in
comparison to those which began racing at an older age (Bailey et al., 1999). However,
this does not eliminate the possibility that these were the stronger horses better able to
tolerate exercise in the first place, and those that did not start were delayed by injury.

Loading of the skeleton during this window of opportunity of active modeling
may therefore be critical for achieving optimal bone mass or structure later in life,
especially for the athletic animal. As human medicine has also focused more on the
prevention rather than the treatment of the age related disease of osteoporosis, exercise
during the growth period has been highly advocated (Bass, 2000; Fuchs et al., 2001). In
human studies with targeted intervention trials, those which have reported the largest
changes in bone mass have been in prepubertal children (Khan et al., 2000) and this

appears to be the critical stage of development. In women recruited to play racquet sports

23

4 to 5 times per wk, those who began training before menarche achieved a 22%
difference in BMC between arms, while this difference was only 10% in those who
started after menarche (Kontulainen et al., 2001). Other studies have shown similar
results in that the BMC of the dominant arm is 2 times greater if playing started before
menarche (Kannus et al., 1995a). Children in the top quartile of physical activity had
significantly higher BMC than their less active peers (Khan et al., 2000). Pre-pubertal
elite female gymnasts were reported to have 30 to 85% greater BMD at the total body,
spine and legs (Bass et al., 1998). However, with human studies, it is harder to separate
effects of predisposition or selection bias in a sport. Bone differences may have existed
prior to beginning the sport or differences in lifestyle (nutrition, genetics) may contribute
as well. However, the arm-to-arm differences in tennis and racquetball players support
mechanical loading effects.

In the young animal, the response of bone to exercise may be more complex due
to the combination of loading along with normal biological growth. Results of similar
loading trials between adult and immature animals cannot be directly compared. Even
where and how new bone is formed in response to exercise may differ between ages and
species of animals. In humans, exercise in the pre- or peripubertal years appears to cause
an increase in periosteal expansion, while exercise late in puberty or after menarche
results in endocortical bone formation (Bass, 2000). However, in an earlier study
reported by Bass et al. (1998), endocortical diameter was reduced in prepubertal
gymnasts. In young animals, the increase in bone mass due to exercise also appears to
occur at the periosteal surface (Yeh and Aloia, 1990; Iwamoto et al., 2000; Notomi et al.,

2001 ). However, others have reported that the dominant effect occurs on the endosteal

24

surface with an accompanying decrease in medullary cavity (Judex and Zernicke, 2000;
Woo et al., 1981, Matsuda et al., 1986).

Training at a young age may alter geometric properties while at a more mature
state volumetric bone density may increase instead. Tennis players who started playing
at a young age had an increase in cross-sectional area of the distal radius, while those
who started playing later increased the trabecular density (Ashizawa et al., 1999). The
strain distribution may differ between adult and immature animals as bone was deposited
in an anteromedial direction in adult roosters, but anterolaterally in growing animals
(Loitz and Zernicke, 1992). Nutritional effects may also have more of an impact on
growing bone, as Ca supplementation in young girls only increased bone mass in pre-
pubescent but not pubescent girls (Slemenda et al., 1997).

Some discretion must be used in exercising the young animal. As the bone of
young animals is more pliable, it may also be susceptible to adverse effects of prolonged
loading or exercise. Conflicting results of positive and negative effects of exercise on
long bone growth have been reported. Intense exercise inhibited longitudinal bone
growth in immature animals (Matsuda et al., 1986; Kiiskinen, 1977; Li et al., 1991) and
increased the amount of trabecular bone in relation to cortical bone in the femoral neck of
rats (Hou et al., 1990). Li et al. (1991) also reported a concurrent decrease in tibial cross-
sectional area and decreased mechanical strength. Artificial dynamic and static
compressive axial loading of rat ulnae retarded longitudinal growth compared to the
contra-lateral control limb (Mosley et al., 1997; Robling et al., 2001) in a dose dependant
manner with strain magnitude, even though periosteal and endosteal bone formation rates

were simultaneously increased in the dynamic loading group (Robling et al., 2001). In

25

humans, elite pre-pubertal gymnasts grew more slowly than controls (Bass et al., 1998).
However, this could be in part due to the intense exercise schedule (15 to 36 h/wk) or the
reduction in body fat (57% less compared to controls). Others have shown an increase in
femoral length in run and jump-trained young rats (Umemura et al., 1995). Many of the
studies showing adverse effects of exercise have used endurance running with immature
rats as a model, an animal which did not evolve for endurance. Therefore, such negative
effects may be more related to the species studied, rather than to just the animal being

young.

Determinants of Bone Strength

While much discussion has been given to the adaptation of bone in response to
loading, some understanding of the physics involved is helpful. When a force is applied
to bone while it is fixed in place, deformation occurs, resulting in internal stress that
resists that force (Einhorn, 1992). This resistance is referred to as stress, which is equal
but opposite to the force applied. Numerically it is expressed as the units of force per

unit area: 0 =force/area. The standard unit is the Pascal (Pa) which is 1 N of force over 1

m2. The measurement of the deformation of the bone is known as the strain. Strain (e) =
the change in length/original length. As it is dimensionless, it can be expressed as a
percentage. The stresses in bone can be described as three types: tension, compression
and shear stresses. Normal loading of bone usually results in bending, which applies both
tensile and compressive forces on opposite sides of the bone. Torsion or twisting
produces shear stresses along the length of the bone, while tension and compression are

also acting to shorten and lengthen the bone in different areas. Most stresses that occur in

26

bone cannot be resolved into one individual component, but are a complex combination
of all three. The predominant stresses in bone as determined by in vivo strain gauge data
are longitudinal normal stresses due to bending, and shear stresses generated from
combined bending and torsion (Einhom, 1992).

Physiological stresses applied to normal healthy bone generally produce small
strains, or little deformation, while poorly mineralized tissue will yield to a greater
degree, producing larger strains. These properties can be tested in a laboratory setting by
applying a known force and measuring the subsequent deformation of the bone. At low
levels of stress, this relationship is linear and is known as Young’s modulus or the
modulus of elasticity. It is a measure of the slope of the force vs. deformation line and
can be calculated by dividing the stress by the strain at any point along the linear portion
of this curve. The linear portion of the curve is known as the elastic region and provides
information about the stiffness of the bone. When the curve becomes nonlinear, this
portion is referred to as the plastic region. The point at which the curve becomes
nonlinear is known as the yield point. Loading beyond this point causes permanent
deformation of the bone. In the elastic region, bone will only deform while the load is
applied and will return to its original shape when the load is removed. Measurement of
the area under the curve up to the point of failure gives the total strain energy stored and
is known as the toughness of the bone (Cullinane and Einhom, 2002).

Bone is a highly anistropic material, meaning that its mechanical properties differ
according to the direction it is loaded (Cullinane and Einhom, 2002). Therefore, when
conducting mechanical testing on bone and comparing data between experiments,

different loading tests may produce different results for the same specimen.

27

Biomechanical properties can be tested at two levels: by either performing standardized
testing on uniform sections, which provide information about intrinsic material
pr0perties, or by testing the bone as a whole with normal geometry in order to obtain its
structural properties (Einhom, 1992).

Bone not only varies in its mechanical properties according to which direction it
is loaded, but also with the rate at which it is loaded. Therefore, strain rate and direction
of load must always be specific when describing material properties of bone. As bone is
a viscoelastic material, material flows internally due to applied stress. An increase in the
strain rates increases the modulus of elasticity and ultimate strength of the bone, but
decreases the ultimate strain (Einhom, 1992). At low strain rates, the bone may show
appreciable deformation, but at high strain rates, it behaves more like a brittle solid,
resisting deformation. Equine cortical bone tested at a range of strain rates showed an
increase in mechanical strength with increasing strain rates (Evans et al., 1992). A linear
relationship exists between the velocity of gait and the strain rate (Rubin and Lanyon,
1982), therefore at higher speeds, bone is more able to resist applied stress. However at
strain rates around 0.1/sec, equine cortical bone showed a lower strain to failure and
energy absorbing capacity as bone acted more like a brittle solid (Evans et al., 1992).
These strain rates were within the rates which would be expected in vivo in the galloping
horse. In addition, at higher strain rates, the increasing cellular stiffness appears to render
the bone cells more insensitive to loading (Jacobs et al., 1998).

Regions of the cortex within a single bone experience less loading than others.
During bending, a region of bone defined as the standard neutral axis experiences no

deformation and thus no strain at the instant of peak loading during running and walking

28

(Carter et al., 1981; Lanyon, 1987; Gross et al., 1992). However, the neutral axis can
shift during different activities such that all parts of the bone will be loaded at some time.
Mosley et al. (1997) showed a non-uniform increase in periosteal expansion in rat ulnae
with more deposition occurring away from the neutral bending axis and towards regions
of greater strain. Greater periosteal apposition rate was observed on the medial aspect of
the rat tibia versus other areas of the bone corresponding with greater recorded strains
during bending (Raab-Cullen et al., 1994b). In jump-trained roosters, both periosteal and
endosteal BF R increased specifically in those sites where strain rates were increased

(J udex and Zernicke, 2000b). Different aspects of a cross-section react differently to the
same stress or loading stimulus, presumably due to the different distribution in strain
magnitude across and along the bone (Rubin, 1984; Biewener et al., 1996). In rats, the
number of osteons and osteocytes increased in the posterior and lateral aspect of the tibia
in responses to exercise, while the posteromedial aspect increased in porosity compared

to controls (Li et al., 1991).

Technologies

 

Numerous markers of bone metabolism have been used in a variety of species to
monitor changes in bone metabolism and the balance between formation and resorption.
Recently markers of bone metabolism have gained in popularity among equine
researchers. Both markers of bone formation and resorption are available based on

metabolism of the bone forming or resorbing cells. Bone resorption markers include

29

pyridinoline crosslinks, carboxy-terminal telopeptide of type I collagen, and tartrate
resistant acid phosphatase, while markers of formation include bone specific serum
alkaline phosphatase, propeptides of type I procollagen and osteocalcin.

Osteoblasts produce many proteins specific to their cell type including bone
sialoprotein, osteopontin, osteonectin, and osteocalcin (OC). Of these bone specific
proteins, 0C is the most frequently used and commonly accepted to be a good marker for
bone formation. Osteocalcin is believed to play a role in matrix mineralization, and is
expressed late in the cell cycle. Also referred to as bone Gla protein due to the presence
of three gamma carboxylic acid residues, 0C is a small non-collagneous protein which is
incorporated into the ECM. It is the most abundant non-collagenous protein in bone,
accounting for 25% of the total of non-collagenous proteins (Azria, 1989). In the
presence of Ca, the GLA residues cause conformational changes which allow CC to bind
to hydroxyapatite and its subsequent accretion into the bone matrix. A portion of the OC
produced is released into the circulation and can be assayed in the serum using
radioimmunoassay (RIA; Delmas, 1990) or enzyme-linked irnmunosorbent assay
(ELISA; Hyldstrup et al., 1989). In a study comparing the effectiveness of these two
techniques on detection of equine OC, recovery of OC in a serial dilution was
underestimated by the ELISA and overestimated by the RIA. In addition, the ELISA
detected greater concentrations and differences between two horses of 82.65 and 77.08
ng/ml while differences between concentrations using the RIA assay on the same samples
was 0.29 and 3.5 ng/ml (Hoyt and Siciliano, 1999). Depending on the assay used,
whether it is a polyclonal or monoclonal antibody, some antibodies may recognize not

only the intact osteocalcin protein, but also fragments of the protein that are released

30

following extracellular matrix (ECM) degradation (Gundberg and Weinstein, 1986). The
RIA uses a polyclonal rabbit anti-bovine-OC antibody while the ELISA uses a
monoclonal mouse anti-bovine-OC antibody.

Equine OC has just recently been isolated and characterized which will lead to
greater specificity and confidence in the results of this assay (Carstanjen et al., 2002).
Osteocalcin concentrations in horses have been shown to be affected by age decreasing
with maturity (Price et al., 1995a; Black et al., 1999; Lepage et al., 1990), growth rate
(Goyal et al., 1981; Petersen et al., 2001), type (Lepage et al., 1998) or breed (higher in
Thoroughbred foals versus Quarter Horses, Reller et al., 2001), circadian rhythms
(Lepage et al., 1991), season (Jackson et al., 1998, Price et al., 1997) and exercise
(Jackson et al., 1998; McCarthy and Jeffcott, 1992; Fletcher et al., 2000). When both
formation and resorption are coupled, 0C is believed to be a good marker of bone
turnover, and when the processes are uncoupled, it is a specific marker. of bone formation
(Delmas, 1993).

Many of the markers of bone resorption are based on the urinary excretion or
serum concentrations of type I collagen degradation products, including hydroxyproline,
and the pyridinium cross links — pyridinoline (PYD) and deoxypyridinoline (DPD).
Urinary hydroxyproline was one of the first bone resorption markers to be developed due
to the high concentration of hydroxyproline in collagen. However, collagen is ubiquitous
and not specific to bone. Bone degradation is assumed to contribute to 50% of the
urinary hydroxyproline, but this percentage may increase with increased bone turnover
due to disease (Robins and Brady, 2002). One of the problems with the use of

hydroxyproline is that its presence is not only due to collagen breakdown, but synthesis

31

as well. As the N- and C- terminal propeptide must be cleaved before incorporation into
the collagen fibril, this presents an additional source of hydroxyproline. Further,
hydroxyproline is extensively metabolized in the liver and dietary sources may further
contribute to urinary concentrations. Therefore, the use of urinary hydroxyproline has
fallen in popularity, especially as short term within-subject variablility is reported
between 17 and 41% with longer term variability of 19 to 53% (Seibel et al., 2002).

Pyridinoline and DPD are two non-reducible pyridinium crosslinks present in the
telopeptide domain of the mature form of collagen. As mentioned previously, they are
created by the posttranslational action of lysyl oxidase linking lysine and hydroxylysine
residues together, a process unique to collagen and elastin molecules. Deoxypyridinoline
is derived from two hydroxylysines and one lysine residue, while PYD is derived from
three hydroxylysine residues. The resulting aldehydes condense with lysyl or
hydroxylysyl residues on adjacent collagen molecules to form the mature pyridinium
crosslink. These interchain bonds strengthen and stabilize collagen molecules within the
extracellular matrix (Robins et al., 1994).

When collagen is degraded during bone resorption, the pyridinoline cross-links
are released into the circulation. These range in size from the free cross-linked amino
acids, to those still bound to segments of N-telopeptide and C-telopeptide sequences.
These small fragments are readily cleared by the kidneys and no evidence exists that they
are themselves metabolically degraded (Calvo et al., 1996). In the human, free
pyridinolines account for about 40% of the total pyridinolines found in urine (Robins et
al., 1994) and most assays only test for the free form unless the samples are hydrolyzed.

Increased bone turnover, as reflected by an overall increase in total DPD, resulted in a

32

preferential increase in the peptide-bound form and a relative decrease in the free form,
although absolute values of both increased (Gamero and Delmas, 1996). Others found
that only the free form of PYD increased with no increase in the peptide bound form in
ovarectomized rats (Black et al., 1989). Free and peptide bound cross-links may be
released from the action of different proteolytic enzymes which are under the regulation
of different aspects of bone resorption. Therefore, as the proportion of cross-links that
appear free vs. bound does not appear to be constant, it may be more useful to determine
total pyridinoline concentrations rather than merely the free form.

In humans, the ratio in which pyridinolines appear in urine reflect their ratio in
bone, thus bone is believed to be the primary contributor to their presence (Robins et al.,
1994). However, in other soft tissues, even though the ratio of PYDzDPD is much
higher, the actual concentration of DPD/mol of collagen is similar to that of bone (Calvo
et al., 1996). Therefore, in certain diseases which may include muscle wasting, the DPD
in urine may originate from non-bone sources (Eyre, 1995). In addition, while the ratio
of PYD to DPD in humans is approximately 3:1, this ratio is variable between species
(Gamero and Delmas, 1996). Data on the ratio of these two crosslinks in equine bone is
currently unavailable and needs to be determined. Different forms of bisphosphonate
therapy in osteoporotic rats resulted in an increase in content of PYD and DPD for some,
while the ratio of PYDzDPD changed in cancellous but not cortical bone (Egger et al.,
1994). The variability in the ratio of PYDzDPD needs to be identified across a large
sampling of the horse population.

The most specific assay used for detection of bone resorption considered to be

the gold standard by most clinicians and researchers is the total urinary excretion of

33

pyridinoline cross-links measured by HPLC (Robey, 1995). However, this method is
tedious and time consuming and therefore simpler assays have been developed using
immunoassays. Urinary free PYD is highly correlated with the. total PYD excretion
measured by HPLC, and increases with menopause (Gamero et al., 1996). The Pyrilinks
(Metra Biosystems, Inc ,Mountainview, CA) assay detects the presence of both PYD and
DPD while Pyrilinks-D (Metra Biosystems, Inc ,Mountainview, CA) is specific for only
deoxypyridinoline, which is believed to be more specific for bone resorption. The
ELISA of free-DPD uses a monoclonal antibody which does not cross react with free
PYD and is also highly correlated with total DPD measured by HPLC (Gamero and
Delmas, 1996). The original ELISA assays measure free pyridinolines unless they are
modified to measure the total pyridinolines. To obtain total pyridinolines, the serum or
urine must first be hydrolyzed to release the peptide bound fragments. However, this is
also a potential source of error as acid hydrolysis can result in destruction of
pyridinolines (Lepage et al., 2001). Within the last few years, an immunoassay was
developed by Metra Biosystems which enables equine serum total DPD to be analyzed
(Weitz et al., 1999).

There are also several assays of bone resorption based on the measurement of
peptides associated with the cross-linked sections of collagen. The NTX assay is based
on a cross-linked peptide from the N-terminal of the telopeptide of type I collagen and
can be used on serum or urine. It is commercially available as an ELISA assay
(Osteomark) from Ostex International (Seattle, WA). Other assays are based on the
metabolism of the C-terminal of type I collagen. Previously known as ICTP, and now

referred to as CTX-MMP, this assay measures the release into the serum of the trivalent

34

crosslink at the C-terminus following matrix metalloproteinase digestion of collagen
(Seibel et al., 2002).

While these markers provide an idea of the balance between bone formation and
resorption at a single time point, they do not provide information about the skeletal mass
or architecture. Circulating concentrations of these markers can also be affected by
factors other than changes in bone turnover, such as their metabolic clearance (liver
uptake, renal excretion, and/or trapping on bone hydroxyapatite), and by the pattern of

immunoreactive moieties recognized by the particular antibodies used. However, since

 

blood or urine can be repeatedly sampled on a single subject and is relatively easy to I
obtain in the equine, changes in these concentrations may provide valuable information
about systemic changes in bone metabolism. Known factors that affect biochemical bone
markers, such as circadian rhythms, diet, age, gender, body and bone mass should be
defined and adjusted when possible. Several studies have shown that with age,
concentrations of bone markers decrease in the horse, reflecting advanced skeletal
maturity (Lepage et al., 1990; Price et al., 1995a). Breed differences also occur in
concentrations of markers as the concentration of osteocalcin and ICTP is higher in draft
horses than in Thoroughbreds which may indicate different rates of bone turnover in
different breeds (Grafenau et al., 2000). Similarly, racial differences exist in human bone
mass, as blacks have higher BMD and lower bone marker concentrations than caucasians
(Kleerekoper et al., 1994; Slemenda et al., 1997). Therefore, data for some breeds of
horses may not necessarily be able to be extrapolated to others. Other factors such as
season, and time of sampling need to also be considered as these influence concentrations

of bone markers (Price et al., 1997; Jackson et al., 1998).

35

 

rep

1her

Bone markers can provide only a somewhat obscure picture of what is happening
at the bone level. In human medicine, markers have been used extensively to identify
patients at risk of osteoporosis as well as to monitor effectiveness of drug therapies.
Single measurements provide only a static picture of a complex process, and higher
concentrations of markers of bone turnover, including markers of both formation and
resorption, have been interpreted by researchers as both a positive (Hiney et al., 2001)
and a negative outcome of exercise (Jackson et al., 1997). Estrogen and bisphosphonate
therapy in osteoporotic women reduces concentrations of bone markers of bone
formation and resorption, indicative of reduced bone turnover (Gamero et al., 1996;
Delmas, 1993). However, both high and low concentrations of OC are found in women
diagnosed with veterbral osteoporosis (Delmas, 1993). Despite decreasing bone
formation, these therapies result in a slowing of bone loss, or a small gain in bone mass.
Lower concentrations of OC were correlated with higher bone mineral density in young
girls (Slemenda et al., 1997). Some see high levels of bone markers such as PICP and
ICTP as an indication that bone turnover is higher and contributing to bone loss while
others proclaim the net result is greater bone deposition. Unless a more complete
understanding of the physiological meaning of fluctuations is determined, and their
relationship is correlated to actual structural changes, their usefulness in a practical, field
situation will be limited. A single measurement will likely never be useful as changes in
blood concentrations must be monitored over time. In addition, seasonal variations in
blood markers may obscure the effects of exercise on these markers. Price et al. (1997)
reported that biochemical markers are at their lowest concentration during the winter and

increase in the spring.

36

The study of bone physiology is very complex due to the composite nature of the
material and the various factors contributing to its strength. Not only is the structural
integrity of bone determined by the state of mineralization and organic matrix
organization, but also by geometry as well. Several non-invasive techniques currently
available to the researcher, physician or veterinarian have been developed to study bone
properties in vivo and predict or diagnose subjects with substandard bone. Several of
these modalities include radiography, radiographic grarnmetry, single and dual photon
absorptiometry, ultrasound velocity (Buckingham and Jeffcott, 1991b), radiographic
absorptiometry (Meakim et al., 1981; Nielsen et al., 1998), dual energy x-ray
absorptiometry (McClure et al., 2001), and quantitative computed tomography. All of
these techniques have their advantages and disadvantages concerning ease of use, cost,
precision, accuracy, and availability and ability to separate cancellous and cortical bone.
These factors must be carefully considered when deciding upon the technique to be used
in either a research setting or for diagnostic evaluation of metabolic bone disease and
treatment effectiveness.

Ultrasonography has been used extensively (Buckingham and Jeffcott, 1991b;
Jeffcott and McCartney, 1985; McCarthy et al., 1990), but is limited in its use as it is
affected by limb temperature and soft tissue swelling, and can only be used in the frontal
plane of the lower part of the limb. Therefore, changes in the dorsal aspect of third
metacarpal bone (MCIII) cannot be assessed. In horses, adaptations in the dorsal cortex
of MCIII are the most common training effect (N unamaker et al., 1989; Stover et al.,
1992; Sherman et al., 1995; Lawrence et al., 1994). Single photon absorptiometry

coupled with ultrasound velocity detected a larger decrease in mineral content in horses

37

 

w
pr
int
0 a
the
Spii
radi

I 994

with immobilized forelimbs in comparison to the control limb, while radiographic
photodensitometry found a larger decrease in the control limb (Buckingham and Jeffcott,
1991a). However, both the fortnightly single photon absorptiometry and weekly
ultrasound measures of the bone showed considerable variation within the same animal,
especially in the 12 wk following cast removal. In addition, single photon
absorptiometry requires a long scan time of up to l min/site and decay of the radiation
source affects long-term accuracy.

Dual energy x-ray absorptiometry (DEXA) offers the advantage of using x-rays of
two different energy levels which are attenuated to a different degree by bone, fat and
muscle. It is the technique most commonly used in human studies and provides an
accurate assessment of site BMC. However, as it is a two dimensional technique, values
of BMD are reported as an areal density rather than a true volumetric density. A DEXA
scan provides areal BMD in units of ycm2 by giving the integral mass of bone mineral
within the region of interest. Few studies examined the accuracy of this technique in
predicting true BMD, as precision has been more the concern in monitoring changes in
individuals. However differences of DEXA values with ashed samples have been within
0 and 15% (Blake et al., 1999). The sensitivity of DEXA is greater than radiographs as
the smallest changes that can be observed in bone density are 4.5% and 7.5% for the
spine and femoral neck respectively versus 10% changes which can be detected by
radiographic bone aluminum equivalence (RBAE; Hodgson and Rose, 1994; Blake et al.,
1999). Dual energy x-ray absorptiometry is used ex vivo in horses and correlates with

bone mineral density as determined by the Archimedes’ principle (McClure et al., 2001;

38

Hanson and Markel, 1994). Correlations improved significantly using a linear regression
which accounted for differences in age, weight and the presence of soft tissue.
Radiogrammetry involves measuring bone diameters, medullary cavities and
cortical widths directly off the radiograph with the aid of a ruler or caliper. It has similar
advantages to standard radiographs in that it is simple to perform and inexpensive.
However, separating cortical from cancellous bone with great accuracy is difficult when
limited to visual inspection. Radiogrammetry can produce a rough estimate of cross
sectional area if the bone is assumed to be symmetrically circular or elliptical. However,
bone will adapt to place more mass in one direction to compensate for compressive forces
(i.e. greater dorsal bone deposition in the equine); therefore, such assumptions should not
automatically be made. Measurements made beyond 1 or 2 mm in definition will
generally not be significant due to the loss of accuracy beyond this level. The sharpness
of the x-ray, or the ability to define an edge, is critical when using this technique. When
measuring cortical widths, the periosteal surfaces are usually easier to distinguish than
the medullary cavity. Therefore, placement of the calipers becomes less certain when
measuring the medullary cavity, especially with the gradual decrease in opacity of the
radiograph due to the presence of the less dense, cancellous bone. In addition, margins of
bone may be clouded by the unavoidable presence of penumbra, or the blurring of the
edges of an image. Penumbra is created by the focal area (rather than a theoretical focal
point) from which the x-rays emanate (Curry etal., 1990). Individual photons hit the
object at slightly different angles, representing the many point sources of the x-rays.
Radiogrammetry has been used previously in the equine, but was poorly correlated with

bone mineral content (Meakim et al., 1981). More recently, radiogrammetry was used to

39

evaluate the effects of exercise and the use of exogenous equine somatotropin on young
racehorses and did appear useful in showing treatment effects (Thomson et al., 2001).
Radiographic absorptiometry improves significantly on the technique of
radiogrammetry as it is much more objective and provides quantitative data. It reduces
the opportunity for manual mistakes and human error involved with visual inspection of
radiographs. Although not as frequently used in human medicine, its ease of use and cost
effectiveness make it very popular for use in animal studies. The optical density of the
bone image is compared with a standard, typically an aluminum step wedge or
penetrometer, which varies by a known thickness for each step (Meakim et al., 1981).
The radiographic image of the stepwedge and the selected site of the bone are
scanned with a densitometer which transfers the information into a digital image with the
aid of a computer software program. To compare data over time or between subjects, a
specific site of the bone should be chosen which can be reliably and repeatedly selected
between different sampling times. Background density due to film base, fog and scatter
are subtracted from the images to provide a baseline of optical density. Logarithms of
the percentage transmittance of the densitometer reading of the stepwedge are plotted
against the known mm of A1 of each step. This produces a linear regression equation
which is used to calculate the percent transmission of the selected point on the bone scan.
The data is now in the form of RBAE. These data are a relationship or an approximation
of bone mineral content, not a direct measure. It is a comparison between the optical
density of the bone and the optical density of the aluminum stepwedge. Radiographic
bone aluminum equivalence has been shown to be a good estimator of bone mineral

content with accuracy within 6% of mineral ash content of bone (Cohn, 1981). In equine

4o

 

 

Pro

61110

bone, RBAE values were highly correlated with BMC expressed as ash per 2 cm section
of bone, but was not highly correlated when expressed on a per weight basis (Meakim et
al., 1981). Radiographic bone aluminum equivalence does not provide information about
bone mineral density which is a volumetric measure.

Total RBAE, a radiographic technique, describes volumetric changes in MCIII
(Nielsen and Potter, 1997). However, scanning the bone at the same site using the
dorsopalmar (d-p) view and lateromedial (l-m) view gives two different values, with the
values from the d-p view being greater. Similarly when using DEXA to assess whole
bone BMD across the width of the bone, the d-p and l~m view provide different numbers,
with the lm view being greater, despite it “seeing” through the same amount of bone
(McClure et al., 2001).

Quantitative computed tomography has the highest sensitivity to disease or age-
related bone loss due to its three-dimensional abilities. Computed tomography (CT)
operates using the same principle of photons being transmitted through the body and
attenuation measurements made by a detecting source. The CT scan shows a value of the
linear attenuation coefficient, u, at each pixel. The p values are given in Hounsfield units
(HU), where air = -1000 HU and water = 0 HU. A linear correlation is determined
between the HU number and BMD by including a hydroxyapetite calibration phantom in
the scan. The data are transferred by computer software to provide tomographic images
that are slices of chosen cross-sections of the body. Quantitative CT provides
information on bone density from cross-sectional rather than projectional images, thus it
provides true physical density rather than proj ectional areal density, as does DEXA. This

allows for areal measurements of the bone and gives true physical density in g/cm’. Total

41

bone area, cortical area and medullary cavity can all be measured accurately with this
method. The use of peripheral CT have revealed differences in cross-sectional area and
trabecular density that DEXA is unable to determine (Adami et al., 1999). Peripheral CT
allows the calculation of biomechanical properties such as the cross-sectional moment of

inertia which is more indicative of bone strength. Computed tomography offers a further

 

advantage as patient positioning does not have to be painstakingly duplicated between I
scans. However, the use of QCT is somewhat limited due to its high installation and I
running costs and the radiation dose is significantly higher with QCT than it is with

DEXA. . E

42

 

WI

“tr
3 big
Were

Key 1

CHAPTER III
BOVINE STUDY
SUMMARY

The ability of short duration, high intensity exercise to increase bone formation in
confined immature Holstein bull calves was investigated. Eighteen bull calves of 8 wk of
age were assigned to one of three treatment groups — group housed (GR, which served as
a control), confined with no exercise (CF), or confined with exercise (EX). The exercise
protocol consisted of rtmning 50 m on a concrete surface once per day, 5 (1 per wk.
Confined calves remained stalled for the 42-d duration of the trial. At the completion of
the trial, calves were euthanized, and both forelegs were collected. The fused third and
fourth metacarpal bones were scanned using computed tomography for determination of
cross sectional geometry and bone mineral density (BMD). Three point bending tests to
failure were performed on metacarpal bones with a universal testing machine. Serum
was analyzed for concentrations of markers of bone metabolism - deoxypyridinoline and
osteocalcin. The exercise protocol resulted in the formation of a rounder bone in EX as
well as increased dorsal cortex thickness, compared to the GR and CF calves (P<0.05).
The exercised calves also had a significantly smaller medullary cavity than CF and GR
and a larger percentage of cortical bone area than CF (P<0.05). Dorsal, palmar, lateral
and total BMD was significantly greater in EX than in CF, and palmar and total BMD
were also greater in EX than in GR (P<0.05). The bones from EX animals tended to have
a higher fracture force than CF (P<0.1). Osteocalcin concentrations normalized from d 0
were higher in EX than CF (P<0.05)

Key words: bone development, confinement, exercise

43

INTRODUCTION
The skeletal system is highly adaptive, able to sense alterations in its loading
environment and respond in order to achieve a dynamic balance between skeletal strength
and skeletal mass. Unfortunately, current management practices of many domestic
livestock species may potentially jeopardize skeletal integrity. Confinement decreases
skeletal strength due to the lack of exercise and loading placed on the bone (Marchant
and Broom, 1996; Knowles and Broom, 1990). In the equine, stall confinement

decreased bone mineral content in both weanlings and yearlings, compared to those left

 

g-
on pasture with access to free exercise (Hoekstra et al., 1999; Bell et al., 2001). This may
be especially detrimental to the young equine if it is then placed into a strenuous training
regime with a skeleton of compromised strength. Injury rates in two-yr-old racehorses
exceed 50% (Rossdale et al., 1985); therefore, the relationship between confinement and
bone strength and its culpability in skeletal injury needs to be explored.

Only a few loading cycles may be needed to stimulate an osteogenic response and
ameliorate the reduction in bone mass observed with confinement. Rubin and Lanyon
(1984) showed that only four cycles per day were needed to maintain bone mineral
density in immobilized turkey ulnae, while 36 cycles prevented disuse osteoporosis in
tibiae of rats (Inman et al., 1999). However, use of an externally applied load in the
above experiments, while usefirl for establishing a very defined strain regime, creates an
unusual, non-physiological strain on the bone. Few studies have examined the
effectiveness of a limited number of physiological loading cycles created by normal

locomotion. Daily periods of walking in sheep were unable to prevent decreased bone

44

mineral density of the calcaneus caused by immobilization with an external fixator placed
across the tarsal joint (Skerry and Lanyon, 1995). Therefore, the stimulus placed on the
bone must be of sufficient magnitude to elicit an adaptive response. Alternatively, the
completely in viva and practical approach of using sprinting exercise as the loading
program is suggested. This would preclude any unusual or artificial effects due to
pressure from the loading apparatus applied to the limb, a previous concern of researchers
(Raab-Cullen et al., 1994b). Additionally, the non-invasive exercise approach subjects
the bone to elevated, but physiologically normal strain patterns, which cannot be
achieved through external mechanical loading. Therefore the goal of this study was to
determine if 6 wk of stall confinement resulted in lowered bone mass compared to pen
housed calves and to determine the potential benefits of short term, high intensity

exercise in increasing bone quality.

MATERIALS AND METHODS

Animals and Management

 

Eighteen Holstein bull calves were obtained from the MSU Dairy Teaching and
Research Center. Calves were age-matched in groups of three, such that each group
began the project at an average age of 8 wk. Individuals from each group were randomly
assigned to one of three treatments, resulting in six calves per treatment group. Calves
were initially weaned from a milk replacer diet at 7 wk of age and thereafter had ad
libidum access to a commercially available pelleted calf grower and allowed free access
to water. Two groups were housed in tie stalls (0.65 m x 1.55 m) that allowed the calves

only to stand and lie down. One group remained in the tie stalls for the 6 wk duration of

45

 

for
113
um
Siren

5042131

the project, with no access to exercise (CF), while 6 calves received controlled exercise
(EX). The exercise regimen consisted of short duration, high intensity running 5 times
per week. Calves were removed from their tie stalls, led to an adjoining barn 32 m from
their stalls and verbally encouraged to run through a 50 m concrete alley once per day.
This approximated 25 strides or cycles per sprint at approximately 4 m/s. They were then
returned to their stalls, with the entire distance traveled being 164 m. The final treatment
(GR) served as a control; these calves were housed in an 8.5 m x 7.3 m pen with free
access to exercise and were allowed to interact with the other calves in this pen. Calves
were weighed on d O, 21 and 42. After 42 d, calves were euthanized, and both forelimbs
were removed above the carpi to allow for collection of the fused third and fourth
metacarpal bone. The left and right 9‘“ ribs were collected as representative of non-

weight bearing bones.

Behavioral Observation

In order to determine the influence of voluntary activity on bone measures,
observations of behavior were made over 24 h on d 0, 21, and 42. Calves were
videotaped in either their tie- stalls or in the group setting with an extended play recorder
which allowed 24 h to be recorded on one tape. Behaviors were observed and recorded
for 4 randomly chosen 15 min periods per 24 h. As the primary objective of this project
was to determine the influence of activity on bone parameters, behavior observations
were limited to those activities which would load the bone, and therefore, impact bone
strength. Therefore, no observations were made of ingestive or eliminative behavior or

social interactions. Behavioral data was recorded with the Observer software (N oldus

46

Information Technologies, Sterling VA). Behaviors for stalled calves were defined as
either standing or lying as they were primarily restricted to these activities. The behavior
of the calves in tie stalls was analyzed for duration of the two-activities as well as the
frequency at which the calves altered from one posture to the next. The frequency of

lying down/standing up was determined as the number of occurrences of the behavior

 

pattern which occurred in the observation period. Duration was recorded as the k
cumulative number of minutes in each of the four 15-min periods for which the animal
stood or was lying. The total duration of the behaviors was recorded as a proportion or
percentage of time for which all occurrences of the behavior lasted over the observation 1

session. Data were averaged over the six calves in each group. Similar observations
were made for the group-housed calves. Voluntary activity of the group-housed animals
was further defined to include bouts of standing, walking, lying, trotting, and jumping.
Walking bouts were defined as four consecutive steps or one stride. If four consecutive
steps were not taken, this behavior was characterized as simply standing. Trotting
consisted of performing a definite two-beat gait and loping as a three-beat gait. Jumping
was also included as a possible behavior, but was defined as an event versus a state, and

thus was not timed.

Measurements and Sample Collection

Blood was collected via jugular venopuncture into non-heparinized vacutainers
for analysis of concentrations of osteocalcin (OC) and deoxypyridinoline (DPD) —
markers of bone metabolism. To determine how quickly bone responds to alterations of

loading regimes, blood was drawn daily at 0800 for the initial 7 (I, followed by once per

47

 

of:

title

wk for the remainder of the project. In order to minimize the effects of diurnal variations
(Black et al., 1999), daily sampling time was not varied. Blood was allowed to coagulate
at 20°C and then centrifuged at 1340 g for 12 min for serum separation. Serum samples
were frozen at —20°C for later analysis. Serum total deoxypyridinoline concentrations
were analyzed using Pyrilinks-D enzyme-linked immunosorbent assay (ELISA). Serum
samples were diluted in a 1:4 ratio with double distilled water. Osteocalcin
concentrations were analyzed using Novacalcin, an ELISA kit obtained from Metra
Biosystems, Inc (Mountainview, CA). Serum samples were diluted in a 1:30 ratio in
order to obtain concentrations within the linear range of the standard curve. All assays

were performed according to manufacturer’s instructions.

Radiographic Bone Aluminum Equivalence

Radiographs were taken by a certified radiologist on d 0, 21, and 42 to determine
radiographic bone aluminum equivalence (RBAE) values, measures of optical density.
Radiographs of the dorsal-palmar and medial-lateral views of the left and right fused
metacarpal bone (MCIII & IV) were taken at a focal length of 30 inches and an exposure
of 60 kVp for .06 sec. An aluminum stepwedge was attached to the radiographic cassette
to standardize readings and calculate RBAE values. Optical density of the bone was
assessed using radiographic bone aluminum equivalence (RBAE) using a BIO-RAD
Model 700 video densitometer (Hoekstra et al., 1999). Radiographs were scanned at 50%
of the bone length of the fused MC 111 & IV. A linear regression was produced from the

thickness of the steps on the aluminum penetrometer. Maximum optical density of each

48

 

cortex was expressed in millimeters of aluminum for both cortices in each view of MC 111

& IV.

Computed Tomography

Following euthanasia, intact limbs with the soft tissue intact were imaged by
computed tomography for determination of cross sectional geometry and bone mineral
density (BMD). Limbs were placed on a QCT phantom pad with hydroxyapetite bone

standards for BMD analysis and scanned with a GE 9800 CT scanner (General Electric

 

Medical Systems, Milwaukee, WI) at 80 KV, 70 mA, with a 2 sec scan time). An initial
scout view was taken to determine the midpoint between the proximal margin of MC 111
& IV and the distal physis. A single IO—mm thick transverse image was then acquired at
this selected location. This location was chosen as it corresponded with the smallest
diameter of the bone and thus the location at which the break was predicted to initiate
during the three-point bending test. This allowed evaluation of both bone density and
cross sectional area at the same location.

The endosteal and periosteal margins of the bone were traced on the CT
computer, which then calculated the area within the region of interest to provide total,
cortical, and medullary cavity cross sectional area. The diameter of the bone was also
measured, including the dorsopalmar (DP) bone diameter (D), DP medullary diameter
(d), lateromedial (LM) bone diameter (B), and LM medullary diameter (b); (see Figure
1). These distances were measured at the line which bisected the bone for the DP
diameter (minor diameter), and at the widest distance across the bone for the LM

diameter (major diameter). Finally, the width of the individual cortices: dorsal (DC),

49

palmar (PC), medial (MC), and lateral (LC) were measured, again in the same plane as

the previous measures were made.

   

Figure 1. Schematic illustration of a cross-section of
__________________ bovine MC 111 & IV showing cortical measurements.
I Measurements included :

d D B = outside major diameter, (lateromedial bone diameter)
n b = inside major diameter, (lateromedial medullary
J diameter)

D = outside minor diameter, (dorsopalmar bone diameter)
d = inside minor diameter, (dorsopalmar medullary
IIIIIIIIIII-I:ullolunl dimeter)

   

 

I
l

B

 

From the cross sectional images, a region of interest (7 mm?) in each cortex was
selected with the cursor in the center of the cortex at approximately the same plane at
which the width of the cortices were measured. Image brightness and contrast were
standardized for the vertebral window. The bone mineral density (BMD; mg/cc
equivalent) was then calculated by a software program which compares the linear
attenuation coefficient of the bone to the BMD phantom for that region. Average BMD
for the entire cross sectional area for each image was also calculated by surrounding the
bone within the cursor as tightly as possible. Each measurement was performed in

triplicate, with the mean value used for statistical analysis.

50

 

165}
and
mm

0.99

Mechanical Testing

 

Ultimate bending strength, modulus of elasticity, and fracture force of the
metacarpi were determined using three-point bending tests conducted on a universal
testing machine (Model 4202, Instron Corp., Canton, MA) according to ASAE Standards
(2000). A cross-head speed of 10 mm/min was used with supports set at 10 cm apart.
Three-point bending to failure was performed on left metacarpi alone, while deformation
to 4 mm was also performed on the right metacarpi. Ultimate bending strength (stress)

was calculated by:

0' = FLC/4I ’
where

c = ultimate bending stress, Pa

F = applied force, N

L = distance between supports, m

C = distance from neutral axis to outer fiber (D/2), m
I = moment of inertia, m4

 

The moment of inertia (MOI) for a hollow ellipse was calculated as:

1 = O.049[(B*D3)-(b*d3)]

Modulus of elasticity was determined by taking the mean of the data from deformation
tests performed on both the right and left limb. The force/deformation curve between 2
and 4 mm deformation was used to calculate the slope of the straight line (F/ 6). This
portion was chosen as it fell in the linear portion of the curve, which provided an r2 =
0.99. Modulus of elasticity (E) was calculated by :

E=FL3/481 5
5 = deformation, m

51

 

Ira
call
the
A-E
b! 11
Prim

their

The modulus of elasticity of the 9‘h rib was determined by three-point bending.
The same cross-head speed and distance between supports was used as for the tests on the
metacarpi. The curved portion of the ribs were cut 18 cm from the costochondral
junction in order to provide a relatively flat sample for bending tests. Modulus of
elasticity (E) was calculated by the same formula as above, with the exception that the
moment of inertia was calculated using the equation for a quadrant of an ellipse:

I = 0.0549(B*D3)

Ash Determination

One cm cross-sections of bone were cut at the midpoint between the proximal
margin of MC 111 & IV and the distal physis in the same location as scanned for
computed tomography. An additional 5 mm cross-section was made proximal to the
carpus and further sectioned into 5 mm cubes to include the same cortical regions (dorsal,
palmar, medial and lateral) analyzed for BMD with the CT. Bone volume was
determined by suspending the intact bone slice within a beaker filled with ionized water.
The volume of the bone was calculated as the difference of the weight of the beaker,
water and sample, and the weight of only the beaker and water. Density of the bone was
calculated using the formula: density = A/(A-B) * P, where P is the density of water, A is
the weight of the bone out of water, B is the weight of the bone submerged in water, and
A-B is the difference in weight, equivalent to the weight of the volume of water displaced
by the bone which is equivalent to the volume of bone according to Archimedes’
Principle. A one cm section from the midpoint of the left rib was removed and ashed as

the metacarpal bone samples. Fat was removed from all bone samples via ether

52

extraction with a Soxhlet apparatus. Samples were then dried at 150° C for 8 h, weighed
to determine the fat-free weight, placed in crucibles and ashed in a muffle furnace at 600°
C for 12 h. Ash was then weighed and either expressed as a percentage of the dry fat-free

weight, or based on the volume of the bone as determined with Archimedes’ Principle.

Biochemical Analysis

After the original 1 cm section of bone was cut from the midsection of the
metacarpal bone for purposes of ash determination, a 5 mm section of bone was sliced
just distal to the midsection. These slices were then sectioned again to be representative
of the dorsal, palmar, medial and lateral cortices of the bone, in the same location as
BMD was determined. Samples were lyopholized for 24 h, weighed and then hydrolyzed
in 1 ml of 6 M HCl for 18 h at 105° C, cooled, and stored at 4° C. For analysis of Ca and
P, 500 ul samples were removed, and dried completely via an ATR Vacuum Concentrator
(Appropriate Technical Resources, Laurel, MD). The dried samples were reconstituted in
1 ml of 6 N HNO3, transferred to acid-washed volumetric flasks, and diluted to 25 ml
with double-distilled water. Samples were diluted ZOO-fold in a 1% lanthanum chloride
matrix. Calcium concentrations were determined by flame atomic absorption
absorptiometry (Unicorn 989 AA spectrophotometer, Thermo Elemental, Franklin, MA).
For analysis of phosphorous concentrations, samples were diluted 10 fold in deionized
water and measured against a standard curve of known phosphorous concentrations
(Beckman Coulter DU 7400 spectrophotometer, Holton, CA).

Of the remaining 500 til, 25 111 was removed for analysis of hydroxyproline
concentrations. For collection of cross-links, 450 ul of the hydrolyzed sample was mixed

with 500 ul of acetic acid, 500 ul of slurry (a cellulose and mobile phase mix), and 2.0 ml

53

of n-butanol. Samples were then vortexed and transferred to gravity-fed columns
containing cellulose in mobile phase. After samples were transferred, 20 ml of mobile
phase was added to elute all proteins except the cross-links. Collection tubes were placed
under the columns following the wash. Cross-links were released from the cellulose with
7 ml double-distilled water followed by 1 ml mobile phase. Samples were dried using an
ATR Vacuum Concentrator, and stored in the dark, at room temperature until analyzed

by HPLC (high performance liquid chromatography).

Statistical Analyses

 

Data were analyzed for the effects of treatment according to the general linear
method of SAS (2001). When treatment effects were significant, means were separated
with the LSD method. A test for homogeneity of variance was performed using a
Bartlett’s F orsythe test. When variance was heterogeneous, the mixed procedure of SAS
was performed on the data. Individual standard errors of the mean (SEM) for each
treatment were then included in the tables. If the variance was homogeneous, pooled
SEMs were provided. Statistical analysis of RBAE and serum data taken throughout the
duration of the project was performed using the mixed procedure of SAS using a
covariance test suitable for repeated measures. The covariance structure was
autoregressive with calf within treatment used as the subject effect. In order to visualize
changes over time in relation to initial values, some data were normalized or subtracted
from d 0 values where appropriate. Correlations between BMD values, moment of
inertia, and results from the mechanical testing were performed using the correlation
procedure of SAS. Duration of behaviors averaged over the three observation periods

were tested for differences between groups using a Fisher’s exact test.

54

RESULTS
Weight Gain

The average age of the calves upon initiation of the project was 56.3 d. The
group-housed calves began the project slightly, but significantly heavier than their stalled
counterparts (Table 1). After 6 wk, while not gaining more weight than the CF calves
(37.2 kg), EX calves tended to gain more weight than the GR calves, 43.9 kg versus 34.9

kg (P<0.1). Average daily gain over the entire period was not different between the

   

 

 

 

 

treatments.
, “be 1- B_Yd _(k_)°“d__ "‘13” dawn” (“mm L
EX CF GR SEM
Body
weight (kg)
Initial 74.7 73 .9 78.2 2.1
Final 118.6 111.1 113.1 2.8
Gain 43.9a 37.2ab 34.9b 1.8
ADG 1.0 0.9 0.8 0.05 ‘
1‘ Treatments lacking a common superscript tend to differ (P<0.1)
Computed Tomography

 

Computed tomography data did not differ between the right and left legs;
therefore mean values were used for statistical analysis. Neither confinement nor
exercise altered the total cross-sectional-area (CSA) of the bone; however, the medullary
cavity of the bone was smaller in the forced-exercised calves versus CF or GR (P<0.01;
Table 2). Absolute cortical bone areas were also not different between groups, but when
the cortical area was expressed as a percentage of the total CSA, the confined animals
had the smallest percentage of cortical bone compared to the GR and EX calves (P<0.01;

Table 2).

55

Table 2. Cortical areas of MC 111 & 1v (cmz).
13x CF GR SEM

 

 

Total bone 4.44 4.38 4.25 0.07
Medullary cavity 1.64b 2.01a 1,33' 0.05
Cortical bone 2.61 2.56 2.37 ' 0.06

%cortical area 0.61b 0.54'I 0.58b 0.01

1 Treatments lacking a common superscript differ (P<.01)

 

Beyond a decrease in the medullary cavity area, the exercise protocol resulted in a
rounder bone versus a more ovoid bone in the other two treatments as evidenced by bone
measurements. Interestingly, the lateromedial bone diameter (B) was the widest in GR
and smallest in EX (P<0.05), while CF animals were not different from either EX or GR
(Table 3). Lateromedial medullary diameter (b) was greatest in CF (due to the overall
larger medullary cavity) with the b of EX again being significantly smaller (P<0.05).
Dorsopalrnar bone diameter (D) did not differ between treatments, but dorsopalmar
medullary diameter (d) was greater in CF versus EX (P<0.1). The group housed calves
were intermediate between both groups and were not different from either group.
Individual cortical diameters did not differ between groups with the exception of the
dorsal cortex, which was greater in EX, 0.41 cm vs 0.31 and 0.35 cm in CF and GR

respectively (P<0.05).

Table 3. Cortical diameters and widths of MC 111 & IV (cm).

 

EX CF GR SEM

 

Bone diameters
(cm) B 2.58b 2.67"b 2.72' 0.03
b 1.59” 1.81' 1.76' 0.04
D 1.94 1.94 1.94 0.03
d 1.13d 128° 1.20“1 0.04
cortical widths
(cm) DC 0.41' 0.31b 0.35b 0.02
PC 0.38 0.34 0.34 0.01
MC 0.50 0.43 0.49 0.02

LC 0.48 0.42 0.47 0.02

 

TTreatments lacking a common superscript differ (P<0.05)
cd Treatments lacking a common superscript tend to differ (P<0. l)

56

Overall, bone density, as determined by CT, was greatest in the exercised calves.
Total BMD and BMD in the palmar cortex of EX was significantly greater than both GR
and CF calves (P< 0.05 respectively) and was greater than CF in the dorsal cortex (Table
4). The EX and GR calves tended to have greater bone mineral density in the lateral

cortex (P<0.l) while only medial BMD did not differ between groups.

Tabe 4- Bnoe M81 We“ Meene Wted tow W _

   

 

 

EX CF GR SEM
dorsal 1234 ‘ 1149 D 1202 ‘5 14
palmar 1144 ' 1072 b 1083 b 13
medial 1225 1190 1226 1 l
lateral 1228‘: 10 1167di25 1225‘: 18 ***
total 664'i3 554%22 597%15 ***

 

 

1"TTreatrnents lacking a common superscript differ (P<0.05)
°" Treatments lacking a common superscript tend to differ (P<0.1)

Mechanical testing

 

Treatment differences seen in bone geometry and density were not reflected in
mechanical properties. Although exercised calves tended to have a higher fracture force
(FF; P<0.1), when the ultimate bending strength (UBS) and apparent modulus of
elasticity (ME) were calculated (which take into account geometric properties) there were
no differences between groups (Table 5). Others have found bone mineral density (Les
et al., 1994) or ash content (El Shorafa et al., 1979; Lawrence et al., 1994) to be
predictive of mechanical properties such as failure stress. In this study, BMD was not
correlated with FF, UBS or ME. However, if bone mineral density values were
compared through regression analysis with the calculated moment of inertia (MOI), there
was a significant positive correlation (R2 = 0.64) between BMD in the medial cortex and

M01 (P<0.05), while there was a weak correlation between total BMD and M01 (P<0. 1;

57

R2 = 0.60). As fracture force had a trend for differences between treatments (P<0.1),
perhaps if the study had been extended beyond 6 wk, or greater numbers of calves were

used, differences in other mechanical properties may have been more readily apparent.

 

 

 

Moment of inertia (mm‘) 8.19 x 10" 7.78 x 109 8.18 x 10'9 2.9 x 10''0

Fracture force (N) 5560’ 4746b 5259ab 152
Ultimate bending strength

(MPa) 170.2 149.9 158.3 4.5
Modulus of elasticity —

metacarpi (MPa) 3819 3286 3572 141
Peak bending force (N) - 243 226 262 14
ribs

Modulus of elast1c1ty — r1bs 4289” 4013b 5074. 214
(MPa)

 

 

.15 Treatments lacking a common superscript tend to differ (P<O. 1)

In order to determine if any treatment effects were present on whole body skeletal
metabolism independent of loading stimuli, the 91" right rib was tested in three-point
bending. Peak bending force sustained by the bones did not differ, but GR calves tended
to have a higher value of ME (Table 5). However, as EX calves were no different from
the CF calves in the MB of ribs, the higher value of FF of the fused third and fourth

metacarpal bone is believed to be due to loading and not differences in management.

Radiographic Bone Aluminum Equivalence

Similar to the CT data, the left and right limb did not differ in RBAE
measurements, thus the mean value for both limbs was used for statistical analysis.
Radiographic bone aluminum equivalence (RBAE) in the lateral, medial, dorsal and

palmar cortices was unaffected by treatment (Table 6). Values did increase significantly

58

Table 6. Radiographic bone aluminum equivalence over time (mm Al).

   

 

Cortice EX CF GR SEM EX CF GR SEM EX CF GR SEM
dorsal 10.3 10.3 10.4 0.2 10.8 10.4 10.6 0.1 ' 11.2 10.7 11.3 0.2
palmar 10.5 10.4 10.2 0.2 10.5 10.2 10.5 0.2 11.0 11.1 11.7 0.2
medial 9.9 9.6 10.0 0.2 10.5 10.0 10.3 0.2 11.3 10.6 11.2 0.2
lateral 10.6 10.4 10.5 0.2 11.4 11.1 11.3 0.2 12.3 12.0 12.1 0.2

'“ Superscripts indicate mean cortical RBAE values across treatments differ by day (P<0.05).

 

over time in all cortices (P<0.001), thus indicating that all calves were increasing in bone
mineral content due to the normal growth process. In order to visualize changes in
RBAE values from initial values, data were also normalized with respect to d 0 values.
No differences existed between treatments in any cortice when data were normalized.
Although bone density, as determined by computed tomography, did show differences in
bone mineral density between treatments, the radiographic technique was not sensitive

enough to detect any such changes.

Ca and P concentrations

Phosphorous or Ca concentrations in the bone sections did not differ with
treatment. While Ca concentrations did not differ according to the region of the bone, P
concentrations tended to differ by cortice (P<0.1), with the medial and lateral cortices
having higher concentrations of P than the palmar region (Table 7). Ratios of Ca to P
were not different between treatments or cortices of the bone. Similarly, bone density of
cross sectional slices when calculated by Archimedes’ Principle, percentage of ash
expressed on a fat-free dry-weight basis, or density of bone calculated from ash weight
and bone volumes did not differ with treatment. Differences between cortices in

percentage ash were seen (P<0.05) as well as a trend for ash weight expressed in relation

59

 

to calculated bone volume (P<0. 1; Table 8). Ash percentage was greater in the dorsal
and medial cortice vs. lateral and palmar ash percentage, with the palmar ash percentage
significantly less than any other aspect of the bone. When ash weight was expressed
relative to bone volume, the dorsal cortical samples were greater in density vs. the palmar

sections.

Table 7. Ca and P concentrastion (mg/g) ad : rastio of indlv1adu o1ticwthii treeatmnt groups.

   

 

dorsal palmar medial Lateral

Ca P ’1 Ca:P Ca P ’ Ca:P Ca P ’ Ca:P Ca P -" Ca:P

 

BX 270 115 2.36 254 108 2.34 264 116.2 2.27 262 119 2.21
CF 266 113 2.36 271 113 2.40 287 119.9 2.39 280 119 2.36
GR 266 116 2.29 255 115 2.21 269 119.5 2.25 277 118 2.35
SEM 6 3 0.03 9 3 0.04 5.8 l 0.05 6 1 0.05

’1 Different superscripts indicate P concentrations tend to differ (P<0. 1) between cortices.
SEMs given are pooled across treatments within day.

Table 8. Percent ash of cortical samples on weight and volume basis.

 

% ash ash weight (g)/bone volume (ml)
dorsal 0.68”l 0.58y
palmar 0.66c 0.51z
medial 0.68' 0.56yz
lateral 0.67b 0.56yz

Cortices lacking common superscripts differ in percent ash (P<0.05)
y“Cortices lacking common superscripts tend to differ in ash weight/bone volume (P<0.1)

Serum Total Deoxypyridinoline

 

Serum DPD has been used as an indicator of bone resorption as it is released into
the bloodstream upon degradation of the organic matrix of bone (Delmas, 1990). As
confinement results in resorption of bone (Hoekstra et al., 1999), stalling calves may
result in higher levels of DPD. However, serum total DPD concentrations did not differ
between treatment groups nor did these values differ over time (Table 9) or when

normalized from d 0 values. Due to the large amount of variation between samples, any

 

biological differences created by the treatments were difficult to determine. The
repeatability between duplicates was low, with duplicates accepted with a 15% error

within the replication.

Serum Osteocalcin
Mean serum osteocalcin concentrations were not affected by treatment. However, 1
if values were normalized from d O, the EX group had higher concentrations of DC in

relation to CF, indicative of greater bone formation as reflected by the changes seen in

 

bone geometry (P<0.05). Even though the EX animals began the project with lower OC
concentrations than CF, they later increased to almost consistently higher values than

those seen in CF.

Behavioral observation

Analysis of behavioral tapes made on d 0, 21 and 42 revealed that the group-
housed calves rarely underwent any high intensity exercise. When observational data
were averaged over the three days, 63.4% of the time budget of the group-housed calves
was spent lying down, 35.0% standing, while actual movement that would significantly
load the bone was limited to 1.58% walking and only 0.02% trotting (Table 11). In
comparison, CF calves spent 35% of the time standing and 65% laying down, with EX
calves standing 27% and lying 73% of the day. The frequency of postural shifts
decreased over time in the confined calves, further reducing the load placed on the bone

(Table 12).

61

  
  

3 .3 -2: 2: .-.....2 n: 6.: 5.212: v.2 mm mi .. «.2 2mm
3”: 2.2 22 3Q 32 we: 3: 3: no: 3: 3: 32 32 mo

fin: _.mm_ mag odm— v.03 Nam— v._m_ AFN— ad: NS: _.NS ade— hd: LU
:62 9mm— véfl v.02 5N2 o6? adv— mém: ad: méi Qum— wdm. as: Xm

NV mm mm 3 3 b e w v m N _ c

}

.08: 53 2:35 528088 838 2:25 .2 2%...

 

v; Nu— N._ h; v; A: 115; w; QN w: ON 1a; 5; 2mm

  
 

Qa— 0.3 Won NE v.2 _.w~ 5cm fiom NAN 9mm «6N Yam 5mm MO
QE 0.2 09 N: Q: ”2 od— adm wdm «.2 5.3 QQ _.mN m0
m.m_ NNN 93 Q: Wm— w.w_ menu QNN v.2 mAN v.3 cdm cAm Xm
Nv mm mm .m E h c n v m N _ c

 

62

Table l 1. Total duration (%) of behavioral observations averaged over (I 0, 21 ,

 

and 42.
—
EX CF GR
standing 35.1 27.5 35.0
lying 64.9 72.6 63.4
walking "* "“ . 1.6

   

Table 12. Frequency of postural shifts summed for each treatment on

 

d 0, 21, and 42.

d0 d2] d42
EX l4 9 6
CF l3 l6 5

GR 7 17 12

   

CONCLUSIONS

Bone resorption due to immobilization occurs quickly, with decreases in bone
mineral density, stiffness and ultimate load after only 6 wk (Inman et al., 1999). In this
study, stall confinement for 6 wk did not appear to adversely affect most properties of the
bone when compared to group-housed calves. While bone geometry and density were the
least favorable in the confined calves, they were not statistically different from the group-
housed calves for most measurements, with the exception of the percentage of cortical
area of the bone. Confinement of these calves probably did not alter the loading pattern
of the bone much beyond that experienced by the calves allowed free exercise. Due to
their own voluntary activity, the control animals (GR) did not differ greatly from the
exercise-restricted animals (CF) in the amount of voluntary activity experienced. Thus
the mechanical loading placed on the confined calves was not decreased greatly beyond
the group-housed animals. While Friend and Dellmeier (1988) found that confined
calves stood more and rested less than group-housed calves due to an increased
motivation for locomotor activity, the calves in the present study did not differ in time

spent lying down or standing. The group-housed calves voluntary activity was quite low,

63

with the majority of their time spent lying down. In fact, CF calves spent almost the same
percentage of time lying down as GR calves, readily explaining the absence of relative
differences in BMD. Even if the stalled calves had stood more, any skeletal effects
likely would not have been observed, as static loading is ineffective in altering bone mass
(Gross et al., 2002; Robling et al., 2001).

Short periods of walking in the GR calves would presumably not load the
skeleton appreciably beyond that of standing in the stalled calves, due to the slow speeds

at which they walked. This is similar to the results seen by Skerry and Lanyon (1995),

 

where periods of walking did not maintain bone mass in otherwise immobilized limbs.
In humans, as much as four hours of walking daily are needed to prevent bone loss
(Anderson and Cohn, 1995). Therefore, in light of the limited activity of GR calves,
conclusive statements in regards to our EX group compared to the control or “normal”
condition are difficult to make. The environment of the GR calves may not have
stimulated enough activity. The author suggests a pasture, rather than a pen setting,
might elicit a greater treatment difference when compared to CF.

While the confinement protocol did not result in any reduction of bone mass,
short duration, high intensity exercise appeared to positively influence both bone mass
and structure even beyond the calves allowed free exercise. Short bouts of running
exercise might improve bone mass beyond confined animals, but the improvement
beyond that of the group-housed calves was unexpected, presumably due to the rather
sedentary pattern of activity in the GR calves. Others have shown that sudden jumps or
movements, while not being an appreciable percentage of daily activity, may be more

important than the influence of standing or walking. Treadmill running in roosters

64

resulted in loads of 500 ustrains while intermittent high-magnitude strains greater than
1,000 ustrains were recorded during normal background activity (Konieczynski etal.,
1998). Similarly in sheep, high-magnitude strains of 1,150 pstrains are recorded during
startling responses (Skerry and Lanyon, 1995). Whalen and Carter (1988) predicted that
bone mass is more dependent on stress magnitude than number of cycles, and as little as a
single loading cycle applied to the foot of a sedentary individual equivalent to that
produced by running could lead to a density change of 61%. Therefore, our hypothesis
that daily running exercise as short as 50 m distance can result in bone hypertrophy
appears to be substantiated.

The greater weight gain in the exercised calves could have contributed to the
differences in bone mass and density as a larger body mass will, of course, produce more
strain on the skeleton. Body weights of calves weakly but significantly correlated with
BMD (P<0.05; R2: 0.28) and % cortical area (P<0.05; R2=O.28). In humans, bone
density in the calcaneus is predicted to be proportional to the square root of the body
weight (Whalen and Carter, 1988). In 5-mo—old foals subjected to confinement or
confinement with sprint training, growth rate had a significant effect on BMD of the
lateral radius, with the faster growing foals having greater BMD (Firth et al., 1999). The
adaptations in EX were not likely due solely to weight gain, as body weight and
medullary cavity were not correlated (P=0.65), and medullary cavity was significantly
smaller in the EX calves. In data from mature cows, metacarpal weight or bone cortex
composition were not different between cows with large differences in age and weight
(Field et al., 1999). We did not anticipate differences in weight gain as calves were

provided ad libidum feed. If similar studies are undertaken, controlling feed intake so

65

that the calves gain weight at a similar rate would be important. This would eliminate the
question of a heavier overall body weight contributing to greater bone mass or density
due to the increased loading.

The exercise protocol led to adaptation in MC 111 & IV, producing a rounder,
more dense bone with less medullary cavity. In this study, the MC III & IV medulla of
the CF and GR calves was larger than EX, yet the total cross-sectional area did not differ.
Other studies have found geometric adaptation of bone to occur while not changing the
cross-sectional area of the bone. In rats, immobilization did not change total cross-
sectional area but did alter cortical width, increasing the marrow cavity and decreasing
the percentage of cortical area (Ma et al., 1999). Such modification occurred through
endocortical bone resorption in the sedentary groups accompanied by a decreased bone
formation rate on both the endosteal as well as the periosteal surface (Ma, 1999). The
normal cyclical process of bone remodeling was not entirely halted, as the increased
resorption on the endosteal surface was accompanied by some new bone formation on the
eroded surfaces. However, as mentioned previously, the confined calves were not very
different from the GR animals, and thus an assumption of bone resorption occurring in
the sedentary animals would be unsubstantiated.

Alternatively, and more likely, the greater cortical mass in EX is due to increased
bone formation. Loitz and Zemicke (1992) found that exercised roosters did lay down
bone endosteally along the anterior-posterior plane, which decreased the a-p endosteal
diameter and increased cortical thickness, similar to our results. However, most studies
have shown that new bone formation occurs on the periosteal surface of the bone rather

than the endocortical surface (Raab-Cullen et al., 1994b). No attempt to resolve whether

66

this adaptation occurred via endocortical bone resorption or by greater bone formation
was made, which would require labeling of the active surfaces or serial CT images.
However, the increase in OC seen in the EX calves indicates greater bone formation, as
mineral apposition rate increases in formerly sedentary sows subjected to exercise (Raab
et al., 1991).

The data suggest that only short periods of high intensity exercise are needed to l'
cause adaptation in the young growing animal. However, the changes in bone structure
and density were not highly reflected by the mechanical testing. Woo et al. (1981) also

found that while exercise caused a reduction of the medullary cavity of the femur of

 

swine and increased ash content, there were no differences in the mechanical properties
of the bone. Loitz and Zemicke (1992) also found that while exercise altered bone
geometry in roosters, elastic modulus did not differ. Thus, exercise changed the quantity,
but not the actual quality of the tissue. Perhaps had the study been continued for a
longer period, it would have resulted in a mechanically stronger bone. Further more bone
strength might have been enhanced in vivo in a manner which could not be measured by
ex vivo testing methods. In addition, as values, while not statistically different,
numerically increased with treatment from CF to GR to EX, a larger sample size may
have lessened the effect of experimental error in the model.

Changing geometry of the bone does change its resistance to bending and thus
alters the strain experienced. While true that bone density is the greatest factor governing
strength of trabecular bone, the primary factor influencing strength of long bones is the
moment of inertia or shape (Whalen et al., 1993). Therefore, changing the shape of bone

may result in a stronger structure than merely an increase in bone density. Bone adapts to

67

decreased usage by reducing bone mass without overly affecting bone strength. The
confined calves overall had a larger total bone area with more medullary area. This
larger, more hollow cylinder appeared to be mechanically similar to the smaller, more
dense bone of the exercised calves. Buckingham et al. (1992), found that although
geldings had lower ultrasound speed velocity through MC III reflecting lower bone
mineral density vs. mares or intact males, the geldings had larger cross-sectional areas.
This adaptation may maintain mechanical integrity in the less dense bone.

Ca concentration as estimated by BMC or BMD using computed tomography,
DEXA, or radiography increases with exercise. Calcium concentrations when analyzed
biochemically have shown an increase in Ca content in all bones in older rats,.and in the
long bones of exercised immature rats (McDonald et al., 1986). Salem et al. (1993)
found Ca concentrations in the femoral neck to be lower in 8-wk-old rats (234 jig/mg) but
were similar after 10 wk between trained and untrained rats (254 and 252 ug/mg,
respectively). Others have also found no differences in Ca concentration following
training in immature rats (Tipton et al., 1972) or swine (Woo et al., 1981). In the present
study, while BMD as predicted by CT differed between treatments, the Ca and P
concentration were unaffected by treatment. This is somewhat difficult to explain as
numerous studies have reported a high correlation between CT and analytical
measurements of mineral concentrations. Phosphorous concentration did tend to differ
between regions of the bone as did the percent ash. Similar to the CT BMD values, ash
% was greatest in the cortex and the least in the palmar cortex. However, no differences
between treatments in physical measurements were observed. Due to the small size of

the cross sectional image of the bone, the region of interested selected in each cortice for

68

 

BMD determination was below the manufacturer’s recommendations and could have
influenced the accuracy of this technique. Therefore, while useful in many studies with
larger animals, with small bones, physical measurements may provide more reliable
assessment of mineral content of bone than CT. Why differences between treatments
existed in CT data is unknown, but may be more related to differences in geometry
between treatment groups.

Adaptations of bone can be limited to geometry with no changes in the
composition of the bone. In mature roosters, exercise redistributed bone mass without
altering total cortical area, or bone density (Loitz and Zernicke, 1992). While cross
sectional area of the middiaphysis of the femur increased in dwarf rats treated with
growth hormone, bone density, calcium and collagen content were smaller in the treated
group (Martinez et al., 1996). Jarvinen et al., (1999) found an increase in failure load in
exercised rats but no difference in BMC determined by DEXA, suggesting a
redistribution of bone. Barengolts et al., (1993) also found an increase in mechanical
properties of femurs of exercised rats despite no change in bone mass, cortical area or
moment of inertia. Changes in collagen fiber orientation confer additional strength
without changing mineral content or architecture of the bone (Turner, 1991). Similarly,
in humans performing exercise intervention targeting the wrist area, BMD as determined
by DEXA did not differ (Adami et al., 1999). However, when the same subjects were
measured using computed tomography, training altered the cross-sectional area of the
bone, and changed BMD, decreasing trabecular and increasing cortical BMD. Therefore
studies focusing solely on changes in BMD may be missing important geometric changes

that have a far greater impact on bending strength and moment of inertia. Thus, despite

69

the disparity in the data between the CT measurements and the direct chemical analysis
of the bone, the observations of the change in bone geometry of the calves indicate that
favorable changes truly were occurring in the EX calves.

While others have shown that stall confinement does result in an increase in DPD
(Hoekstra et al., 1999) and a decrease in OC concentrations (Bell et al., 2000), these
animals were of an older age compared to the 8-wk-old calves in this study. In the very F
young, rapidly growing animal, confinement may not result in an increase in bone

resorption as would be observed by an increase in the bone resorption marker, DPD.

 

Rather a lower bone mass in a sedentary group versus the exercised group is more likely
due to a slower bone gain. Immobilization of the forelimb in young beagle dogs resulted
in the experimental leg developing at a reduced rate compared to the control limb
(Uhthoff and Jaworski, 1978). Immobilized legs had a smaller cross-sectional area than
the normal leg, but both increased in size over time in both total area as well as medullary
area due to normal growth. The total area was greater in the control legs while the
increase in medullary area was greater in the experimental leg. The net result was a
decrease in cortical area in the experimental limb (Uhthoff and Jaworski, 1978).
Additionally, in young, growing rats, a period of deconditioning following exercise
training resulted in bone mass similar to age-matched sedentary controls.
Histomorphometry revealed the decrease in bone mass relative to the increased bone
mass in comparison to the controls at the end of training was due to a decrease in bone
formation, not an increase in resorption (Yeh and Aloia, 1990; Iwamoto et al., 2000).

The change in osteocalcin concentrations seen in the current study also indicate that CC

70

may be more sensitive to increased activity and increased formation rather than an
indicator of a slowing of bone formation.

Some caution may be necessary when implementing an exercise program in very
young animals as some studies have reported an inhibition of bone growth. Dynamic
axial loading suppressed longitudinal growth in rat ulnae (Ohashi et al., 2002). Running
exercise also temporarily stunted longitudinal and radial growth of the tibiotarsus and 1'
femur of roosters (Maynard et al., 1995). In 3-wk-old roosters, moderate exercise caused
the suppression of circumferential growth while at the same time enhancing mid-

diaphyseal cortical thickening (Matsuda et al., 1986). This is similar to results of our

 

bovine study in which 6 wk of exercise did not produce a significantly greater total bone
area in comparison to the other groups but was effective in increasing cortical width.
Similar to our study, they also did not see any change in cross-sectional area moments of
inertia. However, where Matsuda et al. (1986) saw a decrease in bending stiffness,
energy to yield and energy to fracture, our exercise protocol seemed to enhance these
properties. While not significantly different, the EX calves appeared to have a
mechanical advantage versus the other two groups, as evidenced by the trend for a higher
fracture force. Again, perhaps if we had a larger number of calves in each treatment, we
would have seen more statistically significant results.

Whether such an exercise system is useful for other species needs to be tested. In
the equine, interest in promoting skeletal strength in the young animal to lessen the
incidence of injuries is high. Using a short duration high intensity exercise at an early
age may enhance skeletal strength beyond that of normal training regimens typically

implemented in the equine industry. Additionally, one of the difficulties in performing

71

studies of bone physiology in the equine lies in the lack of accurate, non-invasive
determinants of bone structure. While highly accurate, computed tomography is difficult
to perform on the live animal due to the expense and risk involved with the
anesthetization of large animals. Terminal studies would provide the most definitive
information; however, the expense and negative social opinion of using the equine for
terminal research limits this option. However, the usefulness of using a bovine model in
studying the effects of exercise on bone physiology in the immature, rapidly growing
animal appears promising. These animals may provide a better model for some
investigations than rodents, as rodents are quite dissimilar morphologically and have a
very different locomotion pattern. Among domestic ungulates, bull calves especially are
more readily accessible and inexpensive compared to other production animals at many

research institutions.

72

 

CHAPTER IV
EQUINE STUDY
SUMMARY

In order to investigate the hypothesis that short duration exercise may ameliorate
the reduction in bone mass witnessed with confinement, 18 Quarter Horses were weaned
at 4 mo of age and placed into box stalls. After 5 wk, individuals were grouped by age
and weight, and then divided randomly into three treatment groups - group housed (GR),
confined with no exercise (CF), and confined with exercise (EX). The CF and EX groups

were housed in 3.7 m x 3.7 m box stalls for the 56 d duration of the trial. The EX group

 

was sprinted 82 m/d, 5 d/wk in a fenced grass alleyway. The weanlings were led down
the alleyway, turned loose in a small pen, and then released and allowed to run back
down the alley. The GR horses were housed together in 992 m2 drylot with free access to
exercise. On d 0, 28 and 56, dorsopalmar and lateromedial radiographs of the left third
metacarpal bone were taken in order to estimate changes in bone mineral content and
cortical widths. Mean values of medial, lateral, and total RBAE increased over time
(P<0.05), while dorsal and palmar RBAE did not change significantly. Dorsal, medial
and total RBAE, tended to differ by a treatrnent*day interaction, with values increasing
over time only in the EX group. Normalized medial and total RBAE tended to differ
(P<0.1) with treatment, with EX greater than CF. Dorsal-palmar cortical width in EX
(29.3 mm) was greater than GR (27.2 mm) on d 56 (treatment*day; P=0.07). The dorsal
palmar medullary cavity decreased in EX compared to GR (P<0.05), while dorsal and
medial cortical width also increased only in the EX horses (treatment*day; P<0.l).

Although results did not show strong treatment effects, there were indications that such a

73

short duration exercise protocol may be efiective in improving bone mass and therefore

skeletal strength.

INTRODUCTION
Injuries to the skeletal system of the horse are a great concern to the racing
industry. Early training in young horses may be beneficial to the longevity of their
careers. In Australia, horses receiving their first starts as 2-yr-olds had more starts and
raced longer than those which began their racing careers at a later date (Bailey et al.,

1999). The very early training that these horses received in preparation to race as 2-yr-

 

olds may have aided in modeling the skeleton for high speed activity. Most studies show
that the greatest skeletal adaptations occur in very young animals or humans (Iwamoto et
al., 2000; Loitz and Zernicke, 1992; Umemura et al., 1995). Thus, if horses received
exercise at an even earlier age, well before traditional training began, greater benefits to
the skeletal integrity of the horse may be seen.

We have recently shown that very short term sprinting exercise conducted 5 d/wk
enhanced bone geometry in immature calves (Hiney, Chapter III Dissertation).
Therefore, a similar protocol was then implemented for the equine study. However, some
alterations were made to the study after examining results of the bovine study. In the
calves, the RBAE technique did not reveal differences due to treatment that were detected
using more sensitive and accurate computed tomography. However, due to the expense
and risk involved with anesthetizing commercially marketable weanlings, computed
tomography was not an available option for the equine study. Thus, the duration of the

study was lengthened by 2 wk in order to extend the period of skeletal modeling, causing

74

greater differences that could be observed with radiographic absorptiometry. In addition,
as these animals were older than the 8-wk-old-calves which had more readily adaptable
bone, the distance the animals traveled during the exercise protocol was lengthened

slightly.

MATERIALS AND METHODS

Animals and Management

Eighteen Quarter Horses, 9 colts and 9 fillies, were divided into two weaning
groups according to foaling date and were weaned at approximately 4 mo of age. Twelve
weanlings with earlier birth dates were used in the first group and 6 weanlings with later
birth dates were used in the second group. Horses were weaned by removal from their
dams and placed into box stalls (3.7 m x 3.7 m). The foals remained in the stalls for 5 wk
prior to the initiation of the study. The stress due to weaning temporarily reduces feed
intake and thus growth (Knight and Tyznik, 1985). Therefore, this time period allowed
the foals to adjust from the stress of weaning prior to the beginning of the study as well as
to adapt to handling. Individuals from each group were stratified according to age,
weight, and gender as these factors influence growth (Hintz et al., 1979; McKeever et al.,
1981; Pagan et al., 1996). The stratified horses were randomly assigned to one of three
treatments resulting in 6 horses per treatment: confined without exercise (CF), confined
with exercise (EX) and group housed (GR). Mean age of the first weaning group at
commencement of the study was 166 i 3 d and the mean age of the second weaning

group was 172 i 8 d.

75

The GR horses were housed together in 992 m2 drylot with free access to
exercise. The CF horses remained in box stalls for the 8 wk duration of the project with
no access to exercise while the remaining EX horses received forced exercise 5 d per
week. The EX horses were led from their stalls to the end of an 82-m grass alleyway and
turned loose in a small pen. Horses were then released from the pen, and galloped down
the alleyway back towards the barn and were caught in a small pen at the end of the alley.
The average speed of the horses was between 6 to 8 m/s. They were then returned to
their stalls with the entire distance traveled being approximately 264 m, including the

distance walked from the barn to the alleyway.

Behavior Observations

Observations of behavior were made over 24 h on d 0, 28, and 56. For the 24-h
period, horses were kept under constant lighting. Horses were videotaped in either their
box stalls or in the group drylot with an extended play recorder which recorded 24 h on
one tape. For all horses, 4 h of each 24-h period were randomly chosen for observation,
and then 15 min of each hour was analyzed. Behavior observations were again limited to
those activities which would load the bone, and therefore, impact bone strength.
Durations of behaviors were recorded and summed for each 15 min period. The total
duration of the behaviors was recorded as a proportion, or percentage, of time for which
all occurrences of the behavior lasted over the observation session. Total duration of
behaviors were summed for each observation day and over all three days. These
behaviors included bouts of standing, walking, lying down, and trotting. In addition to

the behaviors recorded as cumulative intervals, frequency or number of occurrences of

76

 

shifting of stance from lying to standing, walking bouts, pawing, startling (reaction in
response to a sudden and novel stimulus) and jumping were recorded for each 15 min

period.

Sample Collection

 

Blood was collected via jugular venipuncture into 10-ml non-heparinized
vacutainers at 0800 on d O as well as daily for the initial 7 d in order to monitor any

immediate changes in bone metabolism due to treatment. Blood sampling was then

. RAJ-ll -_-...-

performed once per week for the remaining 7 wk of the project. Blood was allowed to

 

F. '-- __

coagulate at 20° C and then centrifuged at 1340 x g for 12 min for serum separation.
Serum samples were fi'ozen at —20° C for later analysis of osteocalcin (OC) and
deoxypyridinoline (DPD)- markers of bone metabolism. Serum total deoxypyridinoline
concentrations were analyzed using Pyrilinks-D (MetraBiosystems, Inc.) enzyme-linked
immunosorbent assay (ELISA). Serum samples were diluted in a 1:3 ratio with double-
distilled water. Osteocalcin concentrations were analyzed using Novacalcin, an ELISA
kit obtained from Metra Biosystems, Inc (Mountainview, CA). which uses a monoclonal
murine anti-bovine antibody. Serum samples were diluted in a 1:10 ratio in order to
obtain concentrations within the linear range of the standard curve. All assays were
performed according to manufacturer’s instructions.

Radiographs were taken at d 0, 28, and 56 to determine radiographic bone
aluminum equivalence (RBAE) values, measures of optical density and a reflection of
bone mineral content (Meakim et al., 1981). Only the left metacarpal bone (MCIII) was

radiographed for determination of bone mineral content as other studies in mature horses

77

have reported no difference in bone properties between forelimbs (Lawrence et al., 1994;
Glade, 1993). Films of the dorsal-palmar and medial-lateral views of MCIII were taken
at a focal length of 77 cm and an exposure of 65 Kvp for 0.1. s. An aluminum stepwedge
was attached to the radiographic cassette to standardize readings and calculate RBAE

values. Horses were also weighed on a livestock scale on d 0, 28 and 56.

Radiographic Bone Aluminum Equivalence

Optical density of the bone was assessed using radiographic photodensitometry to

 

determine RBAE according to the method of Meakim et al. (1981) using a Bio-Rad
Model GS 700 imaging densitometer (Bell et al., 2001). Radiographs were scanned distal
to the nutrient forarnen of MC 111. A linear regression was produced from the thickness
of the steps on the Al penetrometer. Maximum optical density of each cortex was
expressed in mm of Al for both cortices in each view of MC 111. Total RBAE was
measured by taking the area under the curve of the bone scan and expressing it in relation

to a known volume of Al (Nielsen and Potter, 1997).

Cortical Widths

 

The dorsal-palmar radiographic view was used to measure the width of the
medial (MC) and lateral (LC) cortices, the inner medullary diameter (b) and outer cortical
diameter (B). The beginning of the curve of the bone image as developed by the Multi-
Analyst software to the highest point of the curve was measured for the width of each
individual cortice, and the medullary diameter (or medullary cavity) was measured from

the distance between the two peaks of the curve. The outer cortical diameter was

78

measured as the entire distance of the curve. The similar procedure was used for the
lateral-medial view for determination of dorsal (DC) and palmar (PC) cortical widths,
and the inner medullary diameter (d) and outer cortical diameter across the dorsopalmar
(D) aspect of the bone. The regions of bone measured by this method are depicted in

Figure 2 and 3.

    

Figure 2. Schematic illustration of a cross-section of
equine third metacarpal showing cortical
1 measurements.
D Measurements included :
d B = lateromedial bone diameter
I b = lateromedial medullary diameter
D = dorsopalmar bone diameter
d = dorsopalmar medullary diameter

 
 
 

 

 

\rfi'u/auliu

 

 

 

""‘b

'_—_B

 

Figure 3. Schematic illustration of a cross-section of
equine third metacarpal showing cortical
measurements.

Measurements include:

DC = dorsal cortical width

PC = palmar cortical width

MC = medial cortical width

LC = lateral cortical width

 

Using the digital image potentially limited the human error involved with visual
inspection of the radiographs and caliper placement. An additional study was performed
to examine the validity of this technique, comparing the cortical widths obtained from the
radiographic method to those developed from computed tomography on legs obtained

postmortem. Data from this study is presented in Appendix Chapter A.

79

Statistical analysis

Statistical analysis of RBAE and serum data which were taken throughout the
duration of the project was performed using the mixed procedure of SAS using a
covariance test suitable for repeated measures. When variables were measured at equal
intervals, such as the RBAE data, the covariance structure was first order autoregressive
with horse within treatment used as the subject effect. As serum markers were taken at F
unequal intervals, daily for the first 7 (I followed by weekly sampling, the covariance
structure used was a spatial power. The model tested for treatment, day, and

day*treatment interactions. In addition, because there were two separate groups of horses

 

due to differences in foaling date, a test for period effect was also included in the model
statement. When main effects were significant, post hoc comparisons were used to
separate differences between means. Least square means were separated by the Tukey’s
method. Individual standard errors of the mean (SEM) for each treatment are included in
the tables and figures. To aid in visualizing changes over time within an individual, data
were also normalized by subtracting d O values from all subsequent values. Behavior
data were analde with a multinomial distribution which predicted the probability of
behavior occurrence between groups. Frequency behaviors, such as postural shifts,
walking bouts and pawing, were analyzed using a Poisson distribution. For all analyses, P
values less than 0.05 were considered significant while values less than 0.1 were

discussed as trends.

8O

RESULTS

Weight Gain

 

As horses were balanced and assigned to treatments according to weight, body
weights were similar at the initiation of the study and remained similar throughout (Table
13). However, there was a significant effect of period (P<0.05) on weight gain, with the
foals in the 2"d weaning group gaining more weight over the 56 days than the earlier born
foals, 48.8 kg vs 36.7 kg respectively.

Table 13. Body weight data (kg) and average daily gain
(ADG) of horses separated by treatment.

 

 

EX CF GR SEM
Body
weight (kg)
Initial 226 226 227 4
Final 270 269 263 4
Gain 44 43 36 3
ADG 0.8 0.8 0.6 0. l

 

 

Radiographic Bone Aluminum Equivalence

Dorsal and palmar RBAE values did not differ according to time or treatment,
but there was a trend for a trt*day interaction (P = 0.09) in the dorsal cortex, with values
increasing to d 56 only in the EX group (Table 14). The dorsal cortex of MC 111 in the
CF and GR animals remained essentially unchanged from the initiation of the trial. While
dorsal and palmar values did not increase over time, medial and lateral RBAE values

increased significantly over the duration of the study (P< 0.0005).

81

 

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82

Furthermore, in the medial RBAE, the only increase in RBAE found was in the EX group
(treatment*day interaction; P = 0.05), similar to the dorsal RBAE data. Total RBAE also
increased over time (P<0.005) and similar to both dorsal and medial RBAE data, total
RBAE values tended to increase in only the EX animals (P=0.09). When total RBAE
values were normalized there was a significant effect (P<0.05) of treatment with EX

horses gaining more (137 :t 44 mm2 Al) than either CF (38 :1: 40) or GR horses (8 i: 25). F-

Table 15. Mean dorsal-palmar cortical diameters (mm) according to treatment groups over time.

 

 

 

D d DC PC
dO‘ d28‘ d56h d0 d28 d56 d0 d28 d56 d0 d28 d56"I I
_ I;
EX 27.0" 26.6y 29.32 4.7 4.9 5.9

 
 
 
 
    

4.8 5.1 5.8
5.] SJ 5.0

CF 26.9” 26.4y 28.6'
GR 26.9 27.3 27.2
0.4 0.4 0.5

     

 

Days within a cortical measurement lacking a common superscript differ (P<0.05).
”Indicates a trend for d 56 to be greater than d O and 28 (P<0.1).

’1 Means within a cortical measurement and within a treatment with different superscripts indicate a
trend (P<0.1) for trt‘day interactions.

Cortical Widths

 

Dorsal-palmar cortical diameter (D) increased over time (P<0.05; Table 15), with
values on d 56 greater than either (I 0 or d 28. In addition, EX (29.3 mm) was greater
than GR (27.2 mm) on d 56 (treatment*day; P=0.07). Both CF and EX had greater
dorsal-palmar bone diameters on d 56 in comparison with d 28. When data were
normalized by examining the changes in values from d 0, the treatrnent*day interaction
was significant as EX had increased by 2.3 mm (P=0.07), CF had increased by 1.7 mm
(P=0.02), and GR did not change (P=0.91). Overall, the medullary dorsal-palmar
diameters (d) did not change over the duration of the trial, but treatment*day interactions

were significant (P<0.05). When data were normalized, the medullary cavity decreased

83

in EX (-1.5 mm) compared to GR which gained slightly (0.2 mm; P<0.05). The change
in medullary diameter of CF was not different from either EX or GR. These data seem to
indicate a greater expansion of MC 111 in EX compared to GR as the width of the bone
increased in the dorsal-palmar direction while the medullary cavity was being reduced.
This would suggest the presence of more bone via either a decrease in the normal

expansion of the medullary cavity with growth or by endosteal bone formation.

.. «‘1

Dorsal cortical width (DC) averaged over all treatments did not change over time,
but an increase did occur in the EX horses (treatment*day; P=0.09). When dorsal cortical

width was normalized in relation to d 0, EX was greater than both CF and GR (P<0.05).

 

The average change in the EX was 2.0 mm vs 0.2 mm in CF and —0.3 mm in the GR
foals. Palrnar cortical widths (PC) tended to increase over time (P=0.08), but did not
differ between treatments.

The medial-lateral bone diameter (B) increased over time (P<0.05) but was not
different between treatments (Table 16). Conversely, the medial-lateral medullary
diameter (b) did not change over the duration of the trial but again, the horses in the 2nd
weaning group had a larger medullary cavity diameter measured from the medial to the
lateral aspect of the bone (P=0.07). Mean medial (MC) and lateral (LC) cortical widths
increased over time (P<0.05), and in the normalized medial widths, EX gained more
width in the medial cortex (2.1 mm) compared to GR (0.4 mm) (P=.09). Finally, the
lateral cortex was not different by treatment, but the earlier born foals gained more bone

in the lateral cortex (1.1 mm) vs. the foals weaned later (-0.4 mm; P<0.05).

84

 

13 b ' MC LC

 

dO' d28‘ 056b d0 d28 d56 dO‘ d28” 056b dO‘ r128"b d56b

EX 35.0 35.9 38.3 7.0 8.1 - 9.1 6.8 7.1 7.4
CF 35.2 35.5 38.1 6.9 7.4 7.9 7.2 7.1 8.0
OR 34.7 35.9 36.3 7.5 8.0 7.9 6.5 7.7 7.7
0.3 0.3

Days within a cortical measurement lacking a common superscript differ (P<0.05).

       
 
 

 
 
  

 
 

 

 

1
Table 17. Total duration (%) of behaviors averaged over all days of recording for each treatment.
EX CF GR SEM L
lying 34.2' 20.2b 11.6b 4.5
standing 63.5b 77.9‘ 83.5‘ 4.3
walking 2.3 2.8 7 7 4.9 _. 7 7 1.0 _

 
   

reatments lakrng comonm suprsptcris ffer (P<0.05).

Behavior

Duration of behaviors differed between treatments, but not to the extent expected,
especially in horses allowed free access to exercise (Table 17). Despite their greater
opportunity for movement, the GR horses differed slightly from the box-stalled horses.
The percent of observed time spent walking averaged over all days did not differ between
treatments, 2.3% and 2.8% for EX and CF respectively with the GR horses averaging
slightly more walking at 5% of the observed time. The EX foals spent less time standing
(64% of the observed time), compared to the CF foals (78%) and GR (84%; P<0.05).
The difference in time budget, or how the foals proportioned their activities over time, for
the EX foals was mainly in preference for lying down. The EX foals spent a greater

proportion of time (34%) lying down compared to CF (20%) or GR (12%; P<0.05). The

85

incidence of pawing in the CF foals was greater (Table 18; P<0.05) than in EX or GR,
perhaps showing greater frustration in CF at the lengthy period of confinement. The
frequency in shifting stance between standing and lying was greater in CF foals than GR
foals (P<0.05). The number of walking bouts were similar between treatments. As
animals were never observed startling or jumping during the observation periods, these
behaviors are not reported in the tables. Incidence of trotting bouts were very infrequent. E"
Thus, duration of trotting was very low, did not provide sufficient data for statistical

analysis, and therefore is also not reported in Table 18.

 

Table 18. Total number of occurrences of behaviors summed over all observations and days for each
treatment group.

_
Number of occurrences (#)

 

 

EX CF GR
paw 13" 96‘ 2"
walking bouts 315 410 288
stance change 10ab 15‘ 4b

 

“Treatments lacking common superscripts differ (P<0.05).

 

Osteocalcin

Mean serum OC differed by day (P<0.05; Table 19) and values changed much
the same over time in all three treatments, with an unexplained increase in values on d 7.
Post hoc analysis revealed a trend for the OC concentrations of the CF animals to be
greater than in the GR horses (69.5 vs. 58.2 ng/mL, respectively) while EX was

intermediate (62.8 ng/ml; P = 0.07).

86

Deoxypyridinoline

 

Serum DPD differed by treatment (P<0.05) with mean concentrations in the GR

horses lower than either EX or CF (Table 20). Deoxypyridinoline concentrations tended

to differ by day of trial (P<0.1) but with no discernible pattern of change.

Table 19. Serum osteocalcin (ng/ml) over time.

d

 

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EXFS3.8 49.1 60.4 50.0 55.1 53.3 50.1 80.2 73.2
CF 2 60.3 71.6 65.9 67.2 69.4 54.5 59.9 88.1 79.1
GR y 48.5 49.1 53.9 52.8 62.0 52.0 54.2 84.2 77.7
SEM 4.5 5.1 8.3 5.7 6.4 4.1 4.1 6.4 5.1

_
D

ays lacking common superscripts differ (P<0.05).
’2 Indicates treatments differ (P<0.1).

Table 20. Serum deoxypyridinoline (ng/ml) over time.

71.7
80.7
66.9
4.2

68.0
62.7
64.9
4.8

84.2
66.2
66.5
4.7

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50.1
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492

56"”—

 

Ex‘ 22.6 22.8 25.1 20.2 23.6 20.8 15.9 18.8 23.2
CF' 17.8 23.5 14.6 21.9 21.4 19.0 16.7 22.6 21.6
GR” 21.1 22.6 21.9 14.6 19.1 20.6 12.0 18.4 19.6
SEM 1.3 2.0 2.1 1.8 1.3 1.4 1.4 2.2 1.8

16.6
23.3
16.2
1.8

20.0
20.1
13.3
1.4

22.2

‘21.7

19.9
1.5

19.2
19.0
10.2
1.4

21 .1
23.3
18.6
2.0

18.0
26.1
11.5
2.3

 

1‘72 Days lacking common superscripts differ (P<0.1).
'b Indicates treatments differ (P<0.05).

DISCUSSION

Many studies of weanling and yearling horses have shown an increase in RBAE

values over time, with increasing mineralization of the skeleton occurring with

maturation (Buckingham and Jeffcott, 1987; McCarthy and Jeffcott, 1992; Raub et al.,

1989) with the majority of increase in mineral content of the young horse limited to the

first year and a half of life (Hiney, 1998; Nielsen et al., 1997). In the present study,

87

medial, lateral and total RBAE values increased in all groups over 56 d, but dorsal or
palmar RBAE values did not change.

The increase in RBAE may not have been due solely to increased BMC, but
rather the normal expansion of MC 111 due to growth. While BMD as determined by
ultrasound velocity and single photon absorptiometry did not change, the RBAE of MC
111 increased with growth in weanlings (Buckingham and Jeffcott, 1987). One of the
difficulties of determining changes in mineralization with radiographic
photodensitometry is that it does not specifically measure density. As the animal grows,
bone increases in size, thus the x-rays pass through a thicker, but not necessarily more
dense tissue, appearing more dense on the film.

As the largest recorded strains during galloping occur in the dorsal and medial
cortex of MC 111 (Gross et al., 1992), exercise typically causes greater mineralization to
occur in the dorsal and medial cortex. While overall medial and total RBAE values
increased over time, only the EX group increased in medial, dorsal, and total RBAE
values, as shown by post hoc analysis. Although only trends, the short-term exercise
appeared to be causing more mineral deposition in those areas of the bone experiencing
the most strain during galloping. Again, these changes may have been due to formation
of new bone, rather than increased mineralization of pre-existing bone.

As one of the greatest adaptations created by exercise is in the geometry of bone,
a method of measuring widths from radiographs was attempted. Radiogrammetry is a
method used to measure cortical thickness directly off the film with the aid of a ruler or
digital calipers and this method has been used by others (Meakim et al., 1981; Thomson

et al., 2001). However, while the periosteal surface of the bone may be easier to discern,

88

 

”la-C ._.—- ____- -_ _—_._

 

11E
b0

m1

the gradual reduction in opacity in the medullary cavity makes placement of the calipers
difficult. Greater detail can be observed with radiogrammetry which may aid in cortical
measurements, but must involve the use of either optical or radiographic magnification.
Ordinary medical radiographic film prevents maintenance of fine detail, but finer grain
film can increase the detail able to be seen at greater magnification levels. However, fine
grain film requires longer exposure times (with the patient remaining stationary longer),
and may require hand-processing rather than machine-processing. The processed fine
detail radiograph can then be observed under a stereoscopic microscope (Cohn, 1981).

The higher definition of bone surfaces with this technique allows for more accurate

 

measurement of cortical widths. Therefore rather than using fine grain films, an
alternative strategy of measuring widths from the image scanned into the Multi-Analyst
software was used.

The main effects of growth in just 56 days appear to be more related to the size
and shape of the bone rather than mineral content. While only EX increased in dorsal,
medial, and total RBAE, CF and EX increased in d-p bone diameters and all three
treatments increased in palmar, medial, and lateral cortical widths, as well as the m-l
bone diameter, over the 56 d. However, the tendency of the EX group to increase in
mineral content was reflected in changes in width in the same aspects of the bone.

As changes in shape of the bone may be the dominant loading adaptation
occurring during early life, the changes in the shape of MCIII may be the best indicator
of increased mechanical integrity. The total circumference of the bone (as estimated
from measuring across the medial-lateral and dorsal-palmar widths of the bone) increased

in all groups over the 56 d of the trial, indicating normal periosteal expansion with

89

 

SI

C0

Ire

growth reported previously (Buckingham and Jeffcott, 1992). Periosteal expansion in the
d-p direction was greater in EX in relation to GR and normalized data showed both CF
and EX to be greater than GR. While no mechanical testing was performed in this study,
others have shown that stiffness of equine bone increases with increased bone diameter
(Hanson et al., 1995). As the moment of inertia varies with the 4th power of the outer
diameter of the bone, the EX horses with greater bone diameter may potentially be at a g
mechanical advantage.

The size of the medullary cavity did not change with time, but endosteal

expansion may not occur until later in skeletal development. Even so, in the normalized

 

data, the EX group tended to have less medullary cavity than GR, but whether this was
due to contraction of the endosteal space due to exercise, or an expansion of the endosteal
space in GR is impossible to determine without histomorphometric studies. Mature
roosters strenuously exercised also decreased dorsopalmar medullary diameter, and
increased in dorsal and medial cortical diameters (Loitz and Zernicke, 1992), similar to
the results here. In contrast, in young animals exercise increased periosteal formation
with no change in endosteal space (Kiuchi et al., 1998; Biewener and Betram, 1994).
The location of bone formation, endosteal vs. periosteal, may be due to species or stage
of maturity of the animal at the initiation of exercise, as Woo et al. (1981) found a
significant decrease in endosteal diameter but no change in bone diameter in immature
swine exercised for 1 yr.

Medial and lateral cortical widths increased significantly over time, but palmar
cortical width only tended to increase. Normalized medial cortical width also showed a

trend for an increase in EX vs. the GR horses. While dorsal width didn’t change, the EX

90

group tended to increase. Therefore, the exercise protocol did appear to be causing some
adaptation of MC 111, but usually only in comparison to the GR horses. Why the GR
horses would have shown less periosteal expansion than either of the box-stalled groups,
especially as they underwent no less activity than that performed by the CF horses is
unclear.
While the estimation of cortical widths from radiographic images is not as precise

as data that can be obtained from modalites such as computed tomography, it does at least E-
provide an additional tool of monitoring changes in bone that may not be related to J

density or mineral content changes. In the young animal, adaptation of the architecture of

 

bone is the predominant response to exercise. Exercise increased the dorsal periosteal
apposition rate in young Thoroughbreds (McCarthy and Jeffcott, 1991; McCarthy and
Jeffcott, 1992). Race training also increased dorsal, medial, and lateral cortical diameters
as well as dorsopalmar and mediolateral widths and decreased the medullary cavity,
similar to the results here (Thomson et al. 2001). Thus the data, while only showing
trends for improvement in the EX, corresponds with alterations in bone geometry due to
exercise previously reported in horses. In addition, the foals receiving the short-term
exercise responded in a manner comparable to calves performing a similar exercise
program (Hiney, Chapter II Dissertation).

Stalling the weanlings for 2 mo did not result in bone loss in CF and would not be
expected in such rapidly growing animals. However, the CF horses were not at any
disadvantage when compared to GR horses. The behavioral observations made of the
horses aid in explaining the lack of differences in bone measures between the CF horses

and GR horses allowed freedom of movement. The CF horses did not have lower RBAE

91

or the less favorable bone geometry compared to GR. Rather, the GR horses tended to
have the lowest bone measurements which was unexpected, and most treatment
differences were seen between EX and GR. However, the GR horses did not differ in the
time spent performing activities which would significantly load the bone compared to
CF.

However, the imposed confinement did seem to increase frustration in the CF
horses as they pawed more than either EX or GR. Presumably this is due to the increase
in motivation for locomotor behavior following a period of behavior deprivation, leading
to a variety of abnormal behaviors in horses (Dellmeier, 1989). Even the EX horses,
which were out of the boxed stalls for only a very short time period 5 d per wk, exhibited
less fi'ustrated behaviors. While not a large amount of activity, how much strain on the
bone could result from activities such as pawing is unknown. In addition, while
infrequent, the CF horses were the only animals to ever be observed jumping or startling
in their stalls. Both of these activities have been reported to result in very large strain
magnitudes (Skerry and Lanyon, 1995; Konieczynski et al., 1998). These infrequent
strains may play a significant role in the adaptation of bone and may explain why CF
were not different from GR.

One of the major criticisms of this study is the lengthy stalling period due to
weaning before the initiation of the study. As mentioned previously, weaning does result
in a slowing of the normal growth of the foal and reduced feed intake, due to the stress of
separation from their dam, which the duration of the stalling period would have
eliminated. It would have been of value to have radiographic information from the foals

when they were weaned in comparison to the beginning of the project. However, due to

92

 

W

1161
SU(

06.

8181

any 1

unforeseen management issues, we did not anticipate that the initial stalling period was
going to be 5 wk before the initiation of the project.

In this study, a different question from that originally proposed was raised due to
these unforeseen and unavoidable difficulties. Although not the original design, the study
in part became an examination of the effects of remobilization after a period of
confinement. Remobilization does improve bone parameters but this recovery may be
incomplete, especially if the duration of immobilization is lengthy (Minaire, 1993). The
duration of remobilization may need to exceed that of the initial immobilization period,
as 20 weeks of remobilization of rats immobilized for 18 weeks only partially restored
the minimal cortical width of the tibia (Ma et al., 1999).

In addition, normal rearnbulation is not sufficient to restore bone mass in
previously immobilized animals. In rats with one hindlimb temporarily immobilized for 3
wk, 8 wk of free remobilization only partially restored BMC and BMD compared to both
the control limb and age-matched controls. However, low and high intensity exercise,
while not alleviating the side-to-side differences between limbs, increased BMC such that
the previously mobile limbs were greater than age-matched controls, while the previously
immobilized limbs were equivalent to controls (Kannus et al., 1994). It may therefore be
necessary to load the skeleton to a greater extent than normal voluntary activity to
successfully recover from periods of disuse. Therefore, the GR horses may have needed
greater stimulation to “recover” from confinement, if indeed deleterious effects to the
skeleton had taken place.

As mentioned previously, while body weights did not differ across treatments at

any time point, weaning period had a significant effect on weight gain, with the 2nd group

93

gaining more weight. However, the results of the changes in cortical width would be
opposite of the expected observations if increased weight, and thus greater skeletal
loading, occurred in the more rapidly growing group causing a greater skeletal
adaptation. Unfortunately, additional measurements such as wither and hip height, or
body condition scores were not taken which could have aided in explaining this
phenomena. However, these differences in cortical widths may be merely artifacts of
random individual variation, especially with the unequal animal numbers between the
two periods.

Osteocalcin and DPD did not appear to be useful indictors of differences in bone
modeling between treatments, especially in such young animals undergoing rapid growth.
Similar to this study, Bell et al. (2001) reported no change in stalled and pastured horses
in serum markers of bone formation, other than an age-related decrease in concentration.

The greatest mean OC concentrations in this study were seen in the CF animals.
It would seem unlikely that the greater serum OC in CF horses was due to osteoblastic
activity especially in light of the fact that most changes in bone as determined by
radiography were occurring primarily in the EX group. Serum DPD, a marker of bone
resorption, was lowest in GR horses, but this group was consistently the lowest in terms
of gain in BMC or presumably advantageous geometric changes. The combination of
biochemical and radiographic data seems difficult to resolve, especially without the aid of
more definitive information such as could be gained by histomorphometry.

The imposition of the different treatments did not result in any observable
changes in the initial 7 d of the study. One of the unanswered questions in the use of

makers of bone metabolism in research studies is how soon are changes in exercise,

94

nutrition or other factors reflected in serum markers. In humans, bed rest results in
alterations of bone markers within two days of bed rest (Lueken et al., 1993) indicating
that skeletal disuse results in an almost immediate responseto begin degradation of
“unneeded” bone. Concentrations of PICP were found to decrease following three days
of bed rest while OC values increased (Pedersen et al., 1995). Others have reported that
serum OC changes within only a few hours of moderate-intensity exercise (N ishiyama et
al., 1988). Hiney (1998) reported a decrease in markers of bone turnover in long yearling
horses after 2 d confinement in box stalls. Michael et al. (2001) also reported a similar
effect of stalling as OC and ICTP concentrations were significantly reduced following 4-
d total collection periods. However, the absence of any change in concentrations of
serum markers in this study may be related to the younger age of the animals, and thus
the more rapid growth rate with accompanying higher bone turnover. There was an
unexplained increase in OC concentrations on d 7 of the study but animals were treated
no differently on this day, nor was the sampling protocol altered. As CF animals also
increased on this day, it is doubtful that this is a real physiological effect as they were

already acclimated to their environment for 6 wk prior to that day.

CONCLUSIONS
Results of this study seem to indicate that very short periods of exercise increased
bone mineral content of stalled weanling horses in comparison to those kept in small
paddocks and altered bone geometry Confinement of at least 8 wk duration did not
appear to cause any dramatic effects of disuse osteopenia, however this is difficult to

determine without pre-weaning RBAE values. The exercise protocol was quite easy to

95

implement and was of minimal stress to the young animals. Therefore, similar exercise
protocols might be suggested in order to enhance skeletal strength prior to more

traditional under-saddle training.

96

 

CHAPTER V
SUMMARY AND CONCLUSIONS

The short periods of daily sprinting in both the equine and bovine studies
appeared to positively influence the geometry of the bone. As strain rate is linearly
proportional to strain magnitude and frequency, and as strain rate appears to be the major
factor in controlling osteogenesis, sprinting should be effective exercise for stimulating
the skeleton. Loading at only 1,000 to 1,400 microstrains induces a bone modeling
response in rats, turkeys, dogs and sheep. Strain magnitude experienced by the equine
third metacarpal during galloping may be much greater (3,200 microstrains; Rubin,
1984). Studies of strain magnitude in the immature calf have not yet been performed and
thus no information in this animal is available. It would be interesting to compare the
strains created by the exercise protocol in the forelimbs of both of these species.

Regardless of the exact magnitude of the strains caused by sprinting, the threshold
stimulus was likely reached using that intensity of work. Generally, gains in mechanical
strength are associated with periosteal expansion versus a reduction in medullary cavity
(N otomi et al., 2001). The moment of inertia, and thus weight bearing capacity, varies as
a function of the diameter of the bone raised to the 4th power. Therefore, mathematically,
the easiest way to increase bone strength is to increase the diameter of the bone through
periosteal expansion. In both the equine and the bovine studies, however, the response to
training appeared to be a reduction in the medullary cavity. The reduction in medullary
cavity seen in both calves and horses in response to exercise is difficult to explain.
Altering bone shape in a manner that does not dramatically improve strength would seem

a major disadvantage in energy expenditure to model the bone in that fashion.

97

The suggested endosteal bone formation in the calves and horses may be related
to the age of the animals as the manner in which bone adapts may differ with stage of
maturity. Changes in bone geometry appear to differ between loading and normal
ageing. In humans and roosters, periosteal deposition and endosteal resorption occur
simultaneously with ageing after the cessation of long bone growth (Loitz and Zernicke,
1992). However, when exercised, mature roosters deposited bone endosteally, but F"
showed no increase in persiosteal apposition. Similar changes have also been observed in
swine (Woo et al., 1981).

Contrary to the original hypothesis of the studies, confinement did not appear to

 

adversely affect either CF calves or foals in comparison to GR animals. In the young,
rapidly growing animal, confinement may not result in bone resorption. Suspension of
activity in the young, growing animal may lead to decrease in bone mass via a reduction
of bone formation rather than bone resorption as seen in the adult animal. Unloading
induces resistance in osteoprogenitor cells to such anabolic agents as insulin-like growth
(IGF-l), growth hormone (GH) and parathyroid hormone (PTH; Kostenuik et al., 1999).
In fact, skeletal unloading in the tail suspension rat model increases IGF-l in bone and
receptor expression while at the same time decreasing bone formation (Bilke et al., 1994).
Therefore, the function of osteoblasts may be decreased with unloading or confinement.
Without histomorphometric analysis and labeling of active surfaces of the bone, it is
impossible to ascertain if the observed changes were due to endosteal bone formation in
EX or bone resorption on the endosteal surface in GR or CF.

While no obvious bone loss was observed in the CF calves or weanling horses,

substantial evidence links bone loss with reduced activity. Therefore, confinement of

98

young animals may be a concern when trying to optimize skeletal strength. The more
severe the disuse conditions or the longer the duration, the longer lasting the effects. For
example, in the extreme case of microgravity, Tilton et al. (1980) reported decreased
BMC of the calcaneus of Skylab crew members 5 yr after the flights compared with pre-
flight values. Further, how long a period of immobilization must be before a new steady
state is established and no further bone loss occurs is unknown. Bone loss appeared to
still be occurring 12 wk following stall confinement in previously conditioned mature
horses (Porr etal., 1998). The process of bone resorption must cease or reach a steady
state with bone formation before the skeleton is completely resorbed or structural
integrity is lost, presumably determined by the genetic baseline (Rubin, 1984). Even
within the normal active animal, bone cells close to the neutral axis in long bones
experience minimal strains, yet maintain mineral density.

Whether short-term confinement of growing animals results inlong-term adverse
affects is unclear. The young animal may be more capable of recovery than older
animals placed in confinement. Studies show a greater potential for recovery in young
animals versus mature animals (Jaworski and Uhtoff, 1986; Tuukkanen et al., 1992).
Firth et al. (1999) showed that bone mineral density (BMD) of foals confined to box
stalls for the first 5 mo of life was depressed compared to controls, but were no different
from pasture-reared controls at 11 mo of age, indicating that bone density due to periods
of confinement can be easily compensated for without long term effects. Therefore, the
skeletal system of young animals may recover completely in time, but the time taken to

recover skeletal mass is unknown.

99

 

The time to recover may be longer than the initial immobilization period. In 9 —
11 wk rats subjected to 3 wk of single limb immobilization, followed by 8 wk of free
exercise, BMD, while improved, did not return to values seen in controls (Kannus et al.,
1994). In humans, BMD in casted limbs was still lower than the contralateral limb 10 yr
following immobilization (Kannus et al., 1994). Apparently if immobilization results in
the disappearance of trabecular bone, it cannot be restored. Jaworski and Uhtoff (1986)
also found that while remobilization caused marked improvement in bone tissue, this
restoration was incomplete.

One of the important questions remaining to be answered is how long will
exercise-induced adaptations persist. While bone density was higher in retired athletes
who trained at an early age compared to non-athletes (Bass, 2000), cessation of activity
or a training program may result in an immediate reversal of effect. Studies in young rats
and humans have shown that gains in BMD or BMC due to exercise are subsequently lost
once the exercise protocol was terminated (V uori et al., 1994; LeBlanc et al., 1990; Yeh
et al., 1993). In young, growing rats deconditioning following an exercise protocol
resulted in bone mass similar to age-matched sedentary controls. The reduction in bone
mass back to control levels was due to both a decrease in bone formation and increased
resorption rates compared those recorded during exercise (Yeh and Aloia, 1990; Iwamoto
et al., 2000). Urinary excretion of pre-labeled 3H-tetracycline, a marker of bone
resorption, was significantly higher 4 to 11 d after rats ceased exercise versus those that
continued to train (Yeh and Aloia, 1990), thus the detraining effects may begin rather
rapidly. Six mo of detraining in foals previously receiving sprint exercise for the initial 5

months of life, resulted in bone density lower than pastured foals (Barnevald and van

100

Weeren, 1999). Similarly, Ca supplementation increased bone mass in young girls, but
when discontinued, showed no long-term benefits (Slemenda et al., 1997). Therefore,
whatever measures are taken to increase bone mass must be continued to maintain the
positive effects.

Nevertheless, some encouraging evidence suggests that exercise-induced bone
adaptations persisting after training has ceased. Following an average of 8 yr of
retirement, elite gymnasts maintained a 6 to 16% increased bone mass over controls.
However these former athletes might have continued to lead a slightly more active
lifestyle than controls (Bass et al., 1998). Elite raquet ball players maintained higher
bone mass after 5 yr of a reduced training schedule from 4 to 5 d/wk to only 2 d/wk
(Kontulainen et al., 2001). Male tennis players also maintained the same side—to-side
differences in limbs in a 4 yr follow-up after reducing training frequency (Kontulainen et
al., 1999). Therefore if some amount of equivalent stimulation is performed periodically,
bone mass may be maintained even if training frequency is reduced.

Benefits of exercise lasting decades following training have been suggested in
humans. In postmenopausal women, historical activity between ages of 14 to 21
provided a stronger correlation with bone area than any other activity (Kriska et al.,
1988). In ballet dancers who had ceased training for 26 yr, there was no association
between current activity and hip BMD, but there was a positive correlation between
BMD and hours of ballet training at 10 to 12 yr of age (Khan et al., 1998). Karlsson et al.
(2000) found that soccer players retired for 25 yr had increased bone mass compared to

controls, but these benefits were not maintained beyond that time of retirement.

101

In animal studies of shorter duration, ten wk of exercise training in 4-wk-old rats
increased bone length, cross-sectional area, and BMC and these advantages were not lost
after 10 wk of detraining (Kiuchi et al., 1998). Nelson and Bouxsein (2001) suggested
that alterations in skeletal geometry, similar to those observed in these studies, may be
more resistant to a lessening of training intensity than skeletal benefits that are due only
to increased bone density. Therefore, if exercising the animal at a young age alters bone E
geometry, these advantages may persist longer, especially is the skeleton is still subjected
to periodic stimulation.

An important question for the producer who wishes to implement an exercise

 

program into the management of young animals is how often must such exercise be
conducted. While many studies have indicated a low number of effective cycles
stimulate bone hypertrophy, what the effective frequency at which the stimulus must be
applied in order to maintain the increased bone mass is currently unknown. In mice
subjected to external loading for 5 (I, followed by three wk of no loading, cortical bone
area increased compared to controls (Gross et al., 2002). Apparently just 5 d of loading
was sufficient to maintain a response up to 3 wk later.

The osteogenic response to loading does appear to saturate after a few cycles
(Rubin and Lanyon, 1984) but an increase from five jumps to 100 jumps per day in rats
resulted in greater bone formation in the 100 jump group (Umemura et al., 1997). Others
have suggested that shorter, more frequent bouts with the same total number of cycles
may cause a larger response. When rats were subjected to four separate loading bouts of
90 cycles versus a single bout of 360 cycles, there was a 71% increase in mineral

apposition rate, an 80% increase in bone formation rate, and a 94% increase in

102

mineralizing surface on the endocortical surface of the tibia in the multiple bout animals
(Robling et al., 2001). These changes were observed after only 3 d of loading separated
by 24 h or on d l, 3, and 5 of the study. Raab-Cullen et al. (1,994c) also found an
equivalent response in periosteal mineral apposition in mature rats loaded 3 or 4 d/wk or
daily, indicating exercise or loading every day is probably unwarranted.

Bone cells may become insensitive to repeated cycles at the same strain and thus F
exercise bouts might need to be shortened and performed more frequently. Robling et al.
(2000) assumed a linear association between bone formation and the number of loading

days and suggested that 24-h separation between loading appears a sufficient time span

 

for the bone cells to recover full mechanosensitivity. Robling et al. (2000) suggested that
2 to 3 h separation between 60 loading bouts 6 times/d and 90 bouts 4 times/d appeared
adequate for cells to regain their mechanosensitivity. In a subsequent study of recovery
intervals from 0.5 to 8 h, while an increase in bone formation rate/bone surface was
observed in animals allowed 4 h between loading bouts, the optimal recovery time
appeared to be 8 h intervals when compared to bone formation rates of studies with 24 h
recovery periods (Burr et al., 2002). Raab-Cullen et al. (1994c) suggested that the signal
or intercellular response to loading may continue up to 48 h, while [3H] uridine
incorportation of osteocytes persisted up to 24 h following a single loading bout (Pead et
al., 1988a). Chow et al. (1997) found that maximal gene expression of bone matrix
proteins did not occur until 72 h post-loading of tail vertebrae in rats, while bone-lining
cells stimulated to differentiate to osteoblasts returned to their original state 120 h post-

loading. Others have suggested that the maximum interval between repeated bouts in

103

order to have an additive effect is probably 7 days (Kimmel, 1993), suggesting at least
the need for weekly loading.

Whether these theories apply to physiological versus extemally-applied loading
remains to be tested. This avenue of research does appear promising, however, at some
point, the practicality for implementing such a system does become limiting. For use in
optimizing the skeletal system of domestic animals, the benefits of a possibly slightly
improved skeletal strength or mass must be weighed against the operative time taken to
implement such a system. The studies above suggest that the exercise regimen used here
could be reduced in frequency (perhaps only 3 d/wk) as long as the intensity remained
sufficient and would thus be a valuable line of future research.

Although changes in geometry and bone distribution occurred in response to
exercise in both the equine and bovine studies, actual mechanical properties may not have
changed. It is unknown how large a change in bone geometry is neededto be a
physiological advantage to the animal. In the bovine study, there were definite changes
in cross sectional geometry in the EX calves, yet there was no difference in breaking
strength. In mature roosters, exercise decreased the dorsopalmar endosteal diameter and
increased the diameter of the dorsal and medial cortices of the tarsometatarsus, mirroring
the results of the present equine study, but no differences in mechanical properties were
observed (Loitz and Zernicke, 1992). Woo et al. (1981) also found an increase in femoral
cross-sectional area in exercised swine, but no change in mechanical or material
properties. Alterations in mineral distribution may offer advantages to the animal which
cannot be observed by the ex vivo three-point-bending tests which do not load the bone

in a physiological manner.

104

Single site selection for sampling of BMD or BMC, as used in both the bovine
and equine studies, may have limited the complete picture of changes occurring
throughout the entire bone. In horses, modeling affects different areas of the bone
(Sherman et al., 1995; Warren et al., 1998). In addition, some techniques for measuring
bone density such as dual energy x-ray absorptiometry (DEXA) correlate more with true
BMD in one region of the bone versus another (McClure et al., 2001) and thus may
obscure changes occurring within the bone. While a more thorough investigation along
the length of the entire bone may have yielded a more definitive picture of bone
adaptation, for practicality, the approach taken still provided valuable information.

Furthermore, while most studies tend to focus on changes within a particular long
bone, exercise may affect bones within the same limb differently. Strenuous exercise in
the immature rat resulted in smaller tibial cross-sectional geometry and structural
properties compared to controls, while 2nd metatarsal geometry and structural properties
remained the same (Li et al., 1991). While not equivocal to the equine or bovine due to
the different anatomical site of the bones in rat, it does suggest that different
modifications between bones occur and it may be useful to examine other bones.

Bone response to altered stimuli may be very site specific with differences in
response not only between bones within the skeletal system but also even within the bone
being loaded. In rat ulnae loaded in axial compression, Mosley and Lanyon (1998)
found a reduced rate of periosteal bone formation proximally while an increase in bone
formation occurred distally. Alteration in bone shape can also occur with a simultaneous
increase in periosteal formation and endosteal resorption. Therefore, bone deposition due

to loading may not increase throughout and along the entire bone, but discreet

105

 

populations of cells within a localized region may increase or decrease activity or even
convert from a bone forming population to an area undergoing bone resorption (Mosley
and Lanyon, 1998). Such a localized response would greatly, diminish the likelihood of
observing differences in systemic blood markers.

The results of both studies did not show great promise in the ability of
biochemical markers to detect altered rates of bone turnover due to exercise in the young
rapidly growing animal. Saastamoinen et al. (1994) and Black et al. (1997a) found
considerable inter-individual variation in weanling horses when using a
radioimmunoassay produced by Incstar (Stillwater, MN) due to the amount of bone
activity during growth. Reller et al. (2001) reported similar issues of repeatability in DC
of nursing foals measured with NovoCalcin immunosassays produced by Metra
Biosystems. In the present studies, an enzyme linked immunosorbent assay (ELISA) was
used, which yielded higher OC concentrations in weanlings horses (mean: 64 ng/ml) vs.
traditional radioimmunoassay; (30 to 40 ng/mL; Saastamoinen et al., 1994; Black et al.,
1997a). Differences between assays may be due to the ability of the antibodies to
recognize equine OC, and the possibilities of binding to different OC fragments as both
intact OC and OC fragments appear in the circulation. Development of an assay which
specifically measures intact equine OC may aid in the applicability of this assay.

The ability of serum markers to reflect changes in bone density may be of some
question, especially with young animals. In young adult Oriental males, OC and alkaline
phosphatase increased with resistance training, and DPD was reduced, but no change in
total body, femoral neck, lumber spine, or midradius BMD was observed despite the

elevation in bone formation markers persisting for 4 mo (Fujimura et al., 1997).

106

Conversely in equine studies, OC transiently decreased with exercise (Jackson et al.,
1998; McCarthy and Jeffcott, 1992; F enton et al., 1999, Nielsen et al., 1998). In addition,
no change in serum OC occurred with confinement in mature animals, despite an
observed decrease in bone mineral content (Porr et al., 1998), while others report a
decrease in OC (Hoeskstra, 1999). Here again a considerable range in values was
observed (4 to 31 ng/ml). Without additional information from direct histomorphometry

studies, it is hard interpret alterations in OC concentrations.

IMPLICATIONS

The results of these studies indicate the potential value of implementing an
exercise program in immature animals. By stimulating the skeleton at an early age to
model for intense activity, these animals may be better adapted for training than those
which begin training at later age. Beyond athletic endeavors, increasing bone mass may
produce animals with greater longevity in such production systems as the dairy and swine
industry. Animals with higher bone mass may be better prepared to meet the mineral
demands for multiple cycles of gestation and lactation, as well as reducing their
elimination from the herd due to lameness. Currently, more research needs to be
performed on the long term benefits of these exercise programs as well as the frequency
at which they should be employed.

In order to determine whether such a training protocol was truly useful, it may be
necessary to perform longer longitudinal studies and monitor “performance” of animals
such as incidence of bone trauma during exercise, as well as issues related to production.

If bone mass is truly a concern for the lactating dairy cow, potentially useful information

107

could be gathered concerning bone mass in heifers and if any correlations exist between
duration, total production during lactation, and numbers of lactation before removal from
the herd. Such information concerning the effects of short-term exercise on skeletal
soundness and increasing time spent in production could also be of value for intensively-
managed swine.

More specifically, information on the frequency at which exercise must occur are
most easily addressed using dynamic histomorphometry involving labeling the actively
bone-forming surfaces. This would provide information on site specific changes as well,
and answer the questions concerning whether exercise-induced adaptations in this age of
animal are related to increased periosteal or endosteal bone formation, a reduction to
activation of resorption, or if increased resorption occurred in the medullary cavity of CF
animals. Certainly how frequently exercise must be performed is of critical information
for animal producers as well as those involved in human medicine. Potential studies
could involve graded levels of exercise and monitor bone response using a linear
regression model to determine where or if a plateau in response occurs. The bovine
model should prove the most useful in such studies which facilitate direct tissue analysis.
As the numbers of calves in this study may not have been enough to show significant
differences in mechanical properties, perhaps increasing the number of calves to eight per
treatment might be necessary. If the desired is to pursue responses in horses, a more

sensitive technique of monitoring bone changes should be employed.

108

APPENDICES

109

 

APPENDIX A
INTRODUCTION
The shape of the bone is one of the main determinants of skeletal strength.
Therefore, it is extremely important that the geometry of the bone, in addition to mineral
content, are included in the assessment of bone adaptation to exercise. In the bovine
study, the use of computed tomography (CT) allowed the measurement of cortical and
medullary cross-sectional areas, and cortical widths. Computed tomography provides a
cross sectional image with clearly discernible boundaries between the cortical bone and
the medullary cavity. As significant differences were seen in bone geometry between
treatments in the calves, it was important to attempt measuring bone geometry in the
horses. However, due to the expense and risk involved with anesthesia necessary to use
CT, the only technology available was radiography. The radiographic image of MC 111 as
scanned by the BioRad Densitometer and translated by Multi-Analyst software, produces
a graphic representation of the optical density of the bone. Typically, this curve has two
peaks corresponding to the maximal mineral content of the bone, between which is an
area of lower opacity representing the medullary cavity. The opacity of the object seen
on the radiographic image is not only a function of the density and atomic number of the
object through which the x-rays pass, but also the thickness, with more x-rays attenuated
by a thicker object. Therefore, the longest path of the x-ray through the cortical bone,
and thus potentially the highest point of the peak on the curve, should be just before the
opacity diminishes due to the medullary cavity. Thus measuring cortical diameter from
the initiation of the curve to its highest peak should provide an estimate of an individual

cortical diameter. Similarly, the entire width of the curve should represent the diameter

110

of the bone, with the width of the medullary cavity being the distance between the two
peaks.

Others have used radiogrammetry, the practice of measuring bone widths from
radiographs, in equine studies (Hanson and Markel, 1994; Larkin and Davies, 1996;
Thompson et al., 2001). However, the exact boundaries of cortical bone and medullary
cavity may be difficult to discern with the human eye, and could be a potential source of
measurement errors. The sharpness of an image, and thus its ability to define an edge, is
affected in part by the very object that is filmed. Since MC 111 is a round object, the edge
of the bone will be unsharp, not only due to the presence of penumbra, or the edge
gradient, but simply because the bone does not have sharp edges. Increasing the contrast
of the radiographic film with a lower kVp setting and higher mAs will produce a
radiograph with more visibility of fine detail. However, this also increases the exposure
time, a definite disadvantage when using unsedated animals. Additionally, lower kVp
results in short-scale contrast with most objects appearing in black and white and few
shades of gray. Therefore, the contrast in thickness of A1 of the stepwedge required for
RBAE determination is unable to be discerned, eliminating this possibility if only one
radiograph is to be taken. Others have used a detail screen with the bone placed directly
on the cassette to decrease distortion and penumbra, thereby improving detail and
precision of measurements (Hanson and Markel, 1994). Hanson and Markel used a lower
kVp than that used for the present studies. Larkin and Davies (1996) used a similar
technique of detail screens when assessing radiographic indexes of racehorses. This
equipment was not available for this study. Thomson et al., (2001) measured cortical

widths directly from radiographs that were taken for use in assessing RBAE values, using

111

 

“EL; «1.:

a digital caliper and a magnifying lens. On low contrast films, the ability to detect
cortical margins is diminished, thereby increasing the margin of error. Cortical width
measurements taken from the curve, produced by the Multi-Analyst software, decreases
this human error.

The objective of this preliminary study was to compare the accuracy of cortical

width measurements of the equine MC III on radiographs and CT images.

 

1"
MATERIALS AND METHODS
Thirty forelimbs were obtained post-mortem from the MSU Veterinary Teaching
Hospital and from a commercial slaughter facility. Limbs were radiographed and CT '

scanned with skin and soft tissues in place to simulate field conditions. Dorsopahnar and
lateromedial radiographs were taken (65 kVp for 0.1 s) as typically performed by this

laboratory, necessary to provide enough gray scale when using the aluminum stepwedge.
The dorsal-palmar view was used to measure the width of the medial and lateral cortices,

the inner mediolateral medullary diameter and outer mediolateral cortical diameter.

Figure 4. Schematic illustrations of cortical
measurements created from Multi-Analyst
software.

a a = bone diameter

3, b = medullary diameter

 

c
_L__

c and c’ = individual cortical diameters

 

 

 

 

 

112

The initial rise of the curve above baseline to the highest point was used as the width of
each individual cortice, and the medullary diameter was measured from the distance
between the two peaks of the palmar cortical widths curve. The outer

cortical diameter was measured as the entire distance of the curve. The same procedure

was used for the lateral-medial view for determination of dorsal and palmar cortical

widths. r-

Computed tomography
Limbs were scanned with a GE 9800 CT scanner (General Electric Medical

Systems, Wilwaukee, WI) at 80 kV, 70 mA, 2 sec scan time. An initial scout view was

 

taken to determine the location of the nutrient foramen. A single 10-mm thick transverse
image was then acquired just distal to this location, to correspond with the same site used
for RBAE measurements in this and previous studies. The diameter of the bone was
measured, including the dorsopalmar (dp) bone diameter, dp medullary diameter,
lateromedial (1m) bone diameter and 1m medullary diameter. Finally, the width of the
individual cortices: dorsal, palmar, medial, and lateral were measured in the same plane

as the previous measurements.

Statistical Analysis
The general linear model of SAS with the correlation procedure was used to
estimate the predictability of radiographic measurements in comparison to those obtained

from computed tomography. A P value less than 0.05 was considered significant.

113

RESULTS

All measures from the radiographs were significantly correlated to those obtained
from computed tomography. However, the linearity between the two modalities did vary
considerably between bone measurements (Table 1). It appeared that radiographs more
closely correlated with CT for the dp bone diameter (R2 = 0.66), ml bone diameter (R2 =
0.59), and the dorsal (R2 = 0.77) and medial cortical widths (R2 = 0.76). Measurements
that involved the transition from cortical bone to the medullary cavity were more
variable, and thus the fit of the line was much lower for medullary diameter
measurements. The correlation for palmar cortical widths was also poor, presumably due
to the presence of MC 11 and IV. The difference in repeatability between medial and
lateral cortical widths is more difficult to explain. As most modeling of the equine MC
111 involves an increase of bone in the dorsomedial direction, the greater width of the
medial cortex produces a better fit than measuring the lateral cortex, whose thinner

diameter may produce greater variability.

DISCUSSION

The technique of measuring cortical dimensions from radiographs appears to be a
reasonable alternative to the very costly computed tomography method. The
radiographic technique may detect differences in weanlings caused by short term high
intensity exercise, as previously reported (McCarthy and Jeffcott, 1992; Larkin and
Davies, 1996). The presence of splint bones (MC 11 and IV) in the mediolateral

radiographs increased the error. In the young horses, the splint bones were not yet fused

114

and could be discerned on the radiograph. As the animal ages, this is no longer possible
and increases the error involved, especially in measurements of the palmar cortex.
Piotrowski et al. (1983) found that the fused MC 11 and IV only alter geometry of the
bone in the proximal 20 to 30% of the bone. Measurements taken further distally on MC
111, where MC 11 and IV diminish, have less superimposition and may be more accurate.

Patient positioning can easily effect cortical measurements taken from
radiographs. Slight errors from the true dorsopalmar or lateromedial direction can greatly
change the measurements. In addition, conformation influences the radiographic
appearance of the bones and may require adjustment of film placement. Larkin and
Davies (1996) stated that deviation from a true mediolateral radiograph considerably
altered cortical measurements, but the extent and correction of this effect was not
discussed.

Although the technique described above is not optimal for assessing bone
geometry, it does appear to provide a reasonable compromise between cost, convenience,
and accuracy. Using the digital image ideally minimized the human error component as
compared to visual inspection of the radiographs and caliper placement. In future studies
radiographs may be taken at different kVp exposures, one at higher kVp for RBAE

determination, and one at lower kVp for cortical measurements.

115

 

Table 1A. Regression coefficients and significance levels for cortical measurements as
compared by computed tomography and radiography.

R5 P value
d-p bone 0.66 0.0001
d-p medullary cavity 0.24 0.0048
dorsal 0.77 0.0001
palmar 0.28 0.002
m-l bone 0.59 0.0001
m-l medullary cavity 0.39 0.0001
medial 0.76 0.0001
lateral 0.26 0.0026

116

 

Appendix B
Bovine Data

Calf Diets for Bovine Study _
Appendix Table 1B.

MSU Calf Special
Active drug ingredient
Lasalocid (as lasalocid sodium) ......................................... 30 G/Ton

Guarantee Analysis

Crude Protein ............................................................................ MIN 18.00%
Crude Fat .................................................................................. MIN 1.50%
Crude Fiber ............................................................................. MAX 22.00%
Acid Detergent Fiber .................................................................. MAX 26.00%
Calcium ................................................................................... MAX 1.00%
Calcium ................................................................................... MIN 0.50%
Phosphorus ............................................................................... MIN 0.60%
Selenium (as sodium selenite) ..................................................... MIN 3.00 ppm

Vitamin A ..................................................................... MIN 4,000.00 IU/LB

Ingredients: roughage products, processed grain by-products, plant protein products,
forage products, molasses products, calcium phosphate, salt, bicarbonate of soda, ground
limestone, sodium selenite, zinc sulfate, zinc oxide, manganous oxide, copper sulfate,
ferrous sulfate, cobalt carbonate, calcium iodate, vitamin A acetate, vitamin D-3

supplement, vitamin E supplement

117

 

Appendix Table 1C.
Bovine cortical areas of MC 111 & IV.

 

Calf ID total area (cmz) medullary area (cm’) cortical area (cm’) %medullary cavity %cortical bone

 

 

 

 

 

118

9797 4.84 2.14 2.70 44.2% 55.8%
9801 4.68 2.03 2.65 43.3% 56.7%
9802 4.47 1.84 2.64 41.1% 58.9%
9812 4.37 1.69 2.68 38.7% 61.3%
9817 3.97 1.88 2.09 47.4% 52.6%
9821 4.32 1.72 2.60 39.7% 60.3%
9798 4.58 1.82 2.76 39.7% 60.3%
9799 4.46 1.72 2.74 38.6% 61.4%
9804 3.92 1.44 2.48 36.7% 63.3%
9811 4.54 1.61 2.93 35.5% 64.5%
9819 3.72 1.44 2.29 38.65 61.4%
9820 4.30 1.82 2.48 42.3% 57.7%
9796 4.53 1.88 2.65 41.4% 58.6%
9800 4.37 2.01 2.36 45.9% 54.1%
9805 4.46 2.31 2.15 51.8% 48.2%
9813 4.72 1.88 2.84 39.8% 60.2%
9818 4.03 2.05 1.98 50.6% 49.4%
9822 4.17 1.95 2.22 46.7% 53.3%
Appendix Table 2C. Bovine cortical widths of MC III& IV.
cortical width
Calf lD d-p outer d-p inner m-l outer m-l inner palmar dorsal medial lateral
9796 1.93 1.22 2.73 1.79 0.36 0.335 0.455 0.46
9800 1.99 1.25 2.635 1.765 0.4 0.34 0.415 0.42
9805 2.03 1.435 2.655 1.905 0.315 0.245 0.39 0.355
9813 2.03 1.29 2.755 1.66 0.365 0.335 0.555 0.54
9818 1.78 1.225 2.67 1.935 0.25 0.305 0.375 0.38
9822 1.885 1.24 2.6 1.785 0.335 0.305 0.41 0.39
9798 2.01 1.24 2.68 1.73 0.39 0.4 0.495 0.41
9799 2.035 1.155 2.645 1.635 0.46 0.4 0.53 0.455
9804 1.81 1.06 2.56 1.54 0.34 0.385 0.495 0.48
9811 1.99 1.265 2.615 1.455 0.335 0.36 0.56 0.585
9819 1.83 0.97 2.435 1.555 0.35 0.495 0.455 0.45
9820 1.97 1.095 2.535 1.63 0.375 0.42 0.455 0.47
9797 2.035 1.275 2.815 1.89 0.365 0.365 0.435 0.45
9801 2 1.28 2.815 1.825 0.34 0.31 0.535 0.46
9802 1.925 1.14 2.735 1.76 0.36 0.365 0.51 0.485
9812 1.865 1.14 2.72 1.66 0.35 0.345 0.525 0.505
9817 1.885 1.305 2.525 1.695 0.26 0.31 0.41 0.425
9821 1.905 1.085 2.685 1.72 0.365 0.415 0.51 0.475

Appendix Table 30.
Bovine Computed Tomography Bone Mineral Densities
calf 10 total

9796
9797
9798
9799
9800
9801
9802
9804
9805
9811
9812
9813
9817
9818
9819
9820
9821
9822

61 1.5883
568.925
656.5483
663.5783
564.1 183
61 1.1 1 17
622.965
674.31
491.5017
669.8083
614.2617
608.18
538.03
491.87
654.7233
667.3633
626.9433
557.2467

medial

1252.565
1247.098
1280.127
1239.103
1227.265
1272.947
1217.953
1225.228
1176.795
1212.528
1228.715
1209.793
1174.452
1076.957
1180.305

1210.68
1216.712

1199.07

lateral

1236.51
1254.518
1270.765
1237.888
1206.872
1283.135
1201.713
1220.738
1090.372
1212.177
1237.485
1214.215
1159.865
1109.962
1203.385
1224.045
1212.472
1146.473

dorsal

1230.333
1241.938
1287.445
1248.065

1198.54
1211.093
1156.565
1247.688
1022.058
1213.035
1196.075
1181.537
1187.973
1080.122
1195.965

1209.29
1219.618
1182.673

palmar

1131.252
1111.925
1211.943
1138.485
1100.945
1110.318
1089.847
1087.187
1055.742
1125.813
1061.452
1112.428
1005.707
962.0683
1154.237
1148.687
1118.808
1067.443

Appendix Table 4C. Bovine mechanical testing data.

ultimate bending

calf moment of inertia fracture force strength
9796 8.02412E-09 5098 153.4446
9800 8.48627E-09 441 3 130.5159
9805 8. 123476-09 3986 123.2507
9813 9.566E-09 5915 166.881
9818 5.63762E-09 3887 146.9578
9822 6.86342E-09 5178 178.3223
9798 9.04852E-09 5752 161 .7665
9799 968843509 5293 140.9845
9804 6.53874E-09 5298 188.6756
981 1 8.654368-09 6286 179.1754
9819 6.616795-09 5212 181.9416
9820 858395-09 5520 168.4385
9797 9.71483E-09 5641 142.697
9801 9.15848E—09 4943 136.849
9802 8.28292E-09 5504 162.6589
9812 746038509 5090 172.1 181
9817 6.44595E-09 4554 161 .9687
9821 8.01654E-09 5820 173.5353

119

Appendix Table 50. Bovine modulus of elasticity and rib
mechanical data.

can
9798
9799
9804
9811
9819
9820
9796
9800
9805
9813
9818
9822
9797
9801
9802
9812
9817
9821

modulus of elasticity

3614862666
2989975408
4598095119
3994419697
4608447831
3107639103
3779387253
2590123618
2525662006
3343614207
3529392004
3949949445
3229415858
2907364969
3896793470
3857142622
3988832060
3550515610

rkipeak
rib modulus of elasticity force
5254251245 245
4961875146 252
3949426846 203
2957095789 240
5223934015 248
3388264665 269
3640899441 294
4081136514 131
3573151515 212
4445149303 361
5380507391 161
2960135074 199
5067429911 345
4737066859 252
4247729859 313
5501925379 182
6003893940 222
4886716491 255

Appendix Table 60. Bovine palmar radiographic bone aluminum equivalence

(mm Al).
cafllD
9798
9799
9804
9811
9819
9820
9796
9800
9805
9813
9818
9822
9797
9801
9802
9812
9817
9821

GM

DO

021

8.84770965
9.06720146
8.08378827
12.2883339
9.23295792
8.77995654
1 1 .3199872
1 1 .321 5572
8.41 1 17937
10.187752
9.368861 92
9.86856805
10.6404758
10.7028538
9.2959991 2
1 0.7258232
9.44006035
9.26662477

1 1.2588099
9.57659539
10.8394762
1 1 .4351 889
1 0.201 6501
9.46734795
1 1.3302304
9.7141 5629
8.82526603
10.91 86393
10.8839083
10.6862092
10.165357?
1 0.242771 7
9. 30476308
1 0.0730093
9.4221 3749
12.08431 8

9.13393893
10.6891958
9.81880031
1 1.814378
10.7523485
10.602998
10.3791455
7.85015627
1 1.2695735
1 1.0694055
1 1.0924414
9.710231 16
9.06385784
9.7206352
10.2261078
12.0297439
10.4267218
1 1 .4003613

120

D42
10.2060435
1 1 .9148236
10.4916397

1 1.190622
1 1.1263928
1 1 .1613525
1 1 .7144336
9.53590215
10.4763835
12.1900471
12.0964286

10.432032
10.4906101
120885591
1 1.2260728
13.2967719
10.7519364

12.4429729

 

Appendix Table 7C. Bovine dorsal radiographic bone aluminum equivalence

(mm Al).
CafllD
9798
9799
9804
9811
9819
9820
9796
9800
9805
9813
9818
9822
9797
9801
9802
9812
9817
9821

6wk

9.92128435
9.78227515
8.69559017
1 1.5701 152
9.01959889
9.27536183
10.2174629
10.3036195
8.46616897
1 1 .4802129
9.31 1 15682
10.7037676
10.801712?
9.865741 18
9.36534053

10.864762
9.17054486
9.8675844

00

1 1.3382256

9.139692
9.99885806

1 1.280422
10.3450241
9.95828256
1 1 .0331623
9.64273943
8.52824158
1 1.3382576
9.88998933
1 1.2553545
10.8415264
9.92747902
9.41004929
10.6716791
9.51982988
1 1.7709384

021
10.4013259
10.5971969
10.8500287
1 1.3575526

10.746433
10.7883508
10.7941454
9.24213735
9.51626967
1 1 .0016342
10.9578897
10.6678794
9.86577034
10.0575195
10.151 1368
1 1 .5704013
1 1 .0061628
1 1 .1449957

042
11.5941014
11.5652253
10.0479074
11.6156056
10.6851 194
1 1.5886367
10.941 1228
10.8750378
8.75943504
11.4185295
107012316
1 1.7273204

10.852873
11.4394631
10.6373298
11.6510793
10.5424362
12.8061963

Appendix Table 80. Bovine lateral radiographic bone aluminum equivalence

(mm Al).
CafllD
9798
9799
9804
9811
9819
9820
9796
9800
9805
9813
9818
9822
9797
9801
9802
9812
9817
9821

6wk

9. 5451 8461
9.4961 6895
9.03259498
12.9070837
9.55237546
9. 81 81 7909
9.66390581
1 0.2395088
9.3679233

1 1.8456389
9. 51 827754
10.551 5965
1 0.8949082

10.585776
9. 1 84261 21
1 0.6833097
9.37209956
9.21060822

00
10.8610051
9.071 16472
10.3282131
13.0590031
9.69419445
10.6854874
10.5031364
979876729
9.38812892
12.2779738
9.67030196

10.793034
10.9600226
10.0472443
9.05040678
1 1.4467547
10.2656745
1 1.0022692

021
10.6515231
9.80692719
12.3968631
12.8659712

10.823212
1 1 .5755349
10.5027553
10.8293589
1 1 .2809143
12.1243647
10.5206676
1 1 .1 193825
10.0262978
10.9964976

1 1.809963
1 1.6684461
10.5158806
1 1.0347046

121

042
1 1.9644705
1 1.3745735
12.3733245
13.2970919
1 1 .4629994
13.2041922
1 1.3254503
12.3091944
1 1 .4937132
13.243664
10.621 1006
127734495
12.0880596
12.4651 1 18
12.2192036
1 1.6505355
1 1.5642891
12.6696055

Calf ID
9798
9799
9804
981 1
981 9
9820
9796
9800
9805
981 3
981 8
9822
9797
9801
9802
981 2
981 7
9821

GM
8. 8801 4055
9.54371 1 21
8.32428565
1 1.5986308
8.84839597
8.70126667
9.37199587
9.39435605
8.69353892
1 0.9244661
8.72760147
9. 58954426
1 0.3280208
1 0.4093207
8.3850051 5
9.81 8461 3
8.390781 81
8.58982058

00
10.6493806
9.0430324?
8.992471 12
1 1.0926599

9.1 1586
10.2270701
10.2737995
8.32916512
8.82537302
1 1.2330087

9.2006018
10.01 15517
10.5490027
10.3769919
8.17619497
107022906
9.49452068
10.538921 1

021
10.0918561
9.96033161
10.9014865

1 1 .124595
10.2130017
10.9983254
9.47743691
8.41530879
10.2748374
1 1.3920652
10.3714846
10.2994541

9.352161 1
107688738
10.1239486
10.8375394
9.81693025
10.6993482

Appendix Table QC. Bovine medial radiographic bone aluminum equivalence
(mm Al).

042
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11.5086136
10.6914555
117253027
9.98441201
12.0739268
10.6956646

9.6820386
10.1012423
12.1708455
10.1226485
10.6323256
11.2938104
11.9869141
107743459
10.7531023
10.3396095
11.8495637

 

Appendix Table 100. Bovine Ca and P concentrations (mg/g) by cortices.

dorsal palmar medial lateral
Calf D P Ca P Ca P Ca P Ca

9798 120.50 282.8559 1 17.02 280.2082 1 14.62 247.2494 1 17.36 293.6941
9799 121.18 258.661 115.16 287.3128 117.91 258.278 128.10 246.6087
9804 119.75 280.5267 100.95 217.0426 113.29 235.8008 116.33 232.5234
9811 121.96 282.4392 115.14 266.1921 118.11 281.7288 112.90 254.8084
9819 111.09 283.8298 113.77 283.9798 121.64 294.2654 120.64 250.3715
9820 96.20 234.3303 87.00 190.6979 111.39 266.1654 117.55 294.8814
9796 95.74 221.2418 105.77 247.6323 115.31 258.9095 111.90 261.6117
9800 116.13 276.2619 108.22 251.4645 117.43 333.0842 117.77 274.8709
9805 94.39 227.8165 111.47 247.1362 116.67 268.9172 121.12 249.221

9813 122.60 293.0541 118.39 270.9783 125.27 286.4856 119.88 279.2062
9818 123.96 297.5126 119.11 307.1737 122.76 266.6637 125.63 273.3889
9822 123.93 277.2163 113.55 300.6514 122.12 304.7299 116.73 339.667

9797 119.40 281.849 98.81 217.1594 120.19 249.3683 118.01 258.326

9801 125.11 253.5981 115.78 255.1473 120.46 240.6781 1 17.60 280.7288
9802 112.79 290.874 146.80 340.9481 118.26 274.2467 119.05 274.1463
9812 127.56 286.1493 114.95 260.7644 121.35 270.3678 121.28 288.3396
9817 113.05 237.3885 108.30 227.0087 121.72 281.3749 118.49 299.1609
9821 100.68 243.3787 104.01 226.1912 115.02 297.7698 113.10 258.9109

122

 

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VITA

Kristina Marie Hiney is the daughter of William and Margery Hiney and was born
on October 2, 1973 in Morris, IL.

Following graduation form Morris Community High School, Kristina attended the
University of Illinois, Urbana/Champaign where she received her BS. in Animal Science
in January, 1995 and graduated with University Honors. Kristina entered graduate school
at Texas A&M University in August of 1995 and completed her MS. in December of
1998. Kristina then began her doctoral work at Michigan State University in August of
1999. Throughout her undergraduate and graduate career she has been involved in
research in non-ruminant nutrition, equine exercise physiology and bone physiology and
has been published in the Journal of Animal Science, Nutrition Research Reviews,
Professional Animal Scientist, and the Journal of Equine Veterinary Research. While at
Texas A&M and Michigan State, Kristina served as a research assistant and teaching
assistant and coached several successful collegiate horse judging teams. Kristina remains
active in the equine industry, through showing and judging.

Kristina Marie Hiney’s permanent address currently is 92 Co. Rd. F., River Falls,
WI, 54022.

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