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LIBRARY
Michigan State
University

 

 

 

 

This is to certify that the

thesis entitled

Influence of Housing on the Third Metacarpus and
Markers of Bone and Cartilage Metabolism in

Weanling Horses

presented by

Renee Ann Bell

has been accepted towards fulfillment
of the requirements for

M.S. flegee in Animal Science

 

flafl/fia

Major professor

Date //Z/ //[/9/0?
/

0-7 639 MS U is an Affirmative Action/Equal Opportunity Institution

—" '14- v. 4- _

PLACE IN RETURN BOX to remove this checkout from your record.
TO AVOID FINES return on or before date due.
MAY BE RECALLED with earlier due date if requested.

 

DATE DUE

DATE DUE

DATE DUE

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

1M WMpfiS—p.“

INFLUENCE OF HOUSING ON THE THIRD METACARPUS
AND MARKERS OF BONE AND CARTILAGE METABOLISM IN
WEANLING HORSES
By

Renee Ann Bell

A THESIS
Submitted to
Michigan State University
In partial fulfillment of the requirements for the degree of
MASTER OF SCIENCE
Department of Animal Science

1999

 

Eighte.
Were used to
Expefiment l,

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ABSTRACT
INFLUENCE OF HOUSING ON THE THIRD METACARPUS
AND MARKERS OF BONE AND CARTILAGE METABOLISM IN
WEANLING HORSES
By

Renee Ann Bell

Eighteen Quarter Horse (Experiment 1) and 17 Arabian (Experiment 2) weanlings
were used to determine the influence of housing on third metacarpal bone mass. In
Experiment 1, weanlings were separated into paddock (P1, n = 6), stall (81, n = 6), or
exercised groups (X1, n = 6). Experiment 2 animals were separated into 3 treatment
groups: pasture (P2, 11 = 6), stall (82, n = 5), and partial pasture (PP2, n = 6).
Radiographs of the left third metacarpal were taken every 28 d to determine radiographic
bone aluminum equivalence (RBAE). Serum was collected every 14 d and analde for
osteocalcin (0C), carboxyterminal telopeptide of type I collagen (ICT P) and keratan
sulfate (KS). The P1 group demonstrated an increase in medial and total RBAE in
Experiment 1 while 81 decreased in medial, palmar, and total RBAE. The X1 group
decreased in dorsal and total RBAE. Keratan sulfate decreased in P1, 81, and X1 from d
O to d 56. Group X1 decreased in ICTP from d O to d 42 while P1 and 81 decreased in
ICTP from d 0 to d 56. Osteocalcin decreased in P1 from d 0 to d 56 and in X] from d 0
to d 28 but was unchanged in 81. In Experiment 2, P2 increased in lateral, medial and
total RBAE while PP2 increased in medial and total RBAE. Osteocalcin, ICTP and KS
decreased in all groups. This study concludes that pasture rearing or 12-h daily tum-out

is beneficial to maintaining and increasing bone mineral content in weanling horses.

 

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ACKNOWLEDGMENTS

This project was a great experience for me and helped me understand the horse
industry and how active and exciting weanlings can be. There were a lot of people that
helped and supported me throughout this project that I owe special thanks. Karen Waite
is probably the best research assistant that one could ask for. She was there to help with
assays, statistics, and general support. It all just comes down to Karen being a great
friend!

The animal work for this project would not have been possible without the help
and cooperation of Paula Hitzler and Shorty Parks. Thank you to Paula for letting me
make my mistakes at the barn and letting a novice horse person deal with the weanlings.
Also special thanks to my undergraduate students for assisting me every morning and
night for four months. You made going out to the barn much fun and it was a pleasure
working with you all.

For supporting and listening to me during my stressed out and neurotic moments I
would like to send my gratitude to Ely Rivera, Kristine Lang, and Molly Nicodemus.
Though it does not need to be mentioned I need to thank Lance Garlington, Jada Pierce,
Candace Croney, Marquita Jones, and my parents for being there and giving me the
confidence and faith to finish my degree.

Thank you to my committee, John Shelle, Michael Orth, John Caron, and Camie

Heleski, for guiding me through my program.

iii

Finally, thanks to Dr. Brian Nielsen for taking me at the last minute and
convincing me to stay on with him to finish my degree. I was able to find what I really

enjoy and to truly be happy and proud of my degree.

iv

'3‘" "' warn-“m‘ml 'C' _‘

 

 

LIST OF THE
LIST OF FlC
LYIRODL'C

CHAPTER 1
REVIEW OI
Connectiv
Bone

Cam!
BiOChlei
Bone

Carri
MCaSlIren
Response
Bone

I.

E

h

Cam“

TABLE OF CONTENTS

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

LIST OF TABLES ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, Vii
LIST OF FIGURES .................................. x
INTRODUCTION ........................................................................................................... 1
CHAPTER 1
REVIEW OF LITERATURE ................................................................... 3
Connective Tissue Biology ................................................................ 3
Bone ...................................... 3
Cartilage ................................ 6
Biochemical Markers of Bone and Cartilage Metabolism ,,,,,,,,,,,,,,,, 9
Bone 9
Cartilage 13
Measurements of Bone Mineral Content ............................................ 15
Response of Bone and Cartilage to Limited Physical ActiVity .............................. 17
Bone ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, 17
Housing ....................................................................................................... 18
Exercise ........................................................................................................ l9
Immobilization ..................................... 20
Cartilage ......... 21
Exercise .............................. 21
Immobilization .................................. 22
CHAPTER 2
MATERIALS AND METHODS: EXPERIMENT 1 ..................... 25
Animal Management ..... 25
Bone Mineral Content ............................................................................................... 27
Semm Collection ......................................... 29
Osteocalcin Analysis ................................................................................................. 29
Carboxyterminal Telopeptide of Type I Collagen Analysis .................................. 30
Keratan Sulfate Quantification .............................. 30
Growth Measurement .................................................................. 31
Statistics ..................................................................................... 32
RESULTS: EXPERIMENT l ,,,,,, . ......................... 33
Bone Mineral Content ............................................................................................... 33
Osteocalcin ................... 37
ICTP ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, 38
Keratan Sulfate ....................... 39
Growth Measurements .................................... 39
DISCUSSION: EXPERIMENT l .................... 4 1
Bone Mineral Content 41

 

 

Serum A
Oste
ICTl
Kerr:

Growth 5

CHAPI ER .
MATERLAI
Animal .V
Bone Mir
Serum Cc
Semm Ar
Growth 1V
Statistics"
RESULTS: 1
Bone Min
OSICOC31c

........

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Serum Analysis ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, _ ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, 46
Osteocalcin ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, 46
ICTP ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, 47
Keratan Sulfate ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, 48
Growth Measurements ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, 49
CHAPTER 3
MATERIALS AND METHODS: EXPERIMENT 2 ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, 50
Animal Management ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, , ................................. 50
Bone Mineral Content ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, 51
Serum Collection ................... 51
Serum Analysis ............................................................... 52
Growth Measurement ............................ 52
Statistics .................................................... 52
RESULTS: EXPERIMENT 2 ....................... 53
Bone Mineral Content ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, 53
Osteocalcin ....................................... 55
ICTP ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, 56
Keratan Sulfate ............................................................................... 57
Growth Measurements .............................................................................................. 57
DISCUSSION: EXPERIMENT 2 ................................................................................. 59
Bone Mineral Content__ _ ....................................................................... 59
Serum Analysis .................................... 62
Osteocalcin ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, 62
ICTP ..................................... 6 3
Keratan Sulfate ....................... 6 3
Growth Measurements - ............................... 64
CHAPTER 4
DISCUSSION: EXPERIMENTS l & 2 ............. 66
CHAPTER 5
CONCLUSIONS .............. 70
APPENDIX A
Means Tables ....................................................................................... 75
APPENDIX B
ANOVA Tables ............................... 81
LITERATURE CITED 36

 

vi

ooooooooooooo

 

 

Table l. I
Table 2. (

Table 3. I
Table 4. B
Table 5. 5
Table 6. IV

Table 7. C
01

Table 8, C
or

Table 9, C.

or
Table 10. :
Table 11. 1
Table 12_
Table 1 A.
Table 2A. .
Table 3 A. I
Table 4 A. }
Table 5 A.

Table 6A.

LIST OF TABLES

Table 1. Calculated daily nutrient table for SI and X1 weanlings
on a dry matter basis ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, 26

Table 2. Calculated daily nutrient table for P1 weanlings

on a dry matter basis ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, 26
Table 3. Exercise protocol for Quarter Horse weanlings ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, 27
Table 4. Means table for osteocalcin (ng/ml) in Quarter Horse weanlings ,,,,,,,,,,,,,,, 38
Table 5. Means table for ICTP (ng/ml) in Quarter Horse weanlings ,,,,,,,,,,,,,,,,,,,,,,,, 38

Table 6. Means table for keratan sulfate (ng/ml) in Quarter Horse weanlings ,,,,,,,,, 39

Table 7. Calculated daily nutrient table for S2 weanlings
on a dry matter basis 51

 

Table 8. Calculated daily nutrient table for P2 weanlings
on a dry matter basis 51

Table 9. Calculated daily nutrient table for PP2 weanlings

on a dry matter basis? ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, 3 ,,,,,,,,,,,,,, 51
Table 10. Means table for osteocalcin (ng/ml) in Arabian weanlings ,,,,,,,,,,,,,,,,,,,,,,, 56
Table 11. Means table for ICTP (ng/ml) in Arabian weanlings ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, 56
Table 12. Mean tables for keratan sulfate (ng/ml) in Arabian weanlings ,,,,,,,,,,,,,,,,, 57
Table 1A. Means table for medial RBAE (mm Al) in Experiment 1 ,,,,,,,,,,,,,,,,,,,,,,,, 75
Table 2A. Means table for lateral RBAE (mm Al) in Experiment 1 ,,,,,,,,,,,,,,,,,,,,,,,,, 75
Table 3A. Means table for palmar RBAE (mm Al) in Experiment 1 ,,,,,,,,,,,,,,,,,,,,,,,, 75
Table 4A. Means table for dorsal RBAE (mm Al) in Experiment 1 ,,,,,,,,,,,,,,,,,,,,,,,,, 75

Table 5A. Means table for change in dorsal RBAE (mm Al) in Experiment 1 75

Table 6A. Means table for total (DP) RBAE (mm2 Al) in Experiment 1 ,,,,,,,,,,,,,,,,,, 76

vii

 

 

 

Table 7A.

Table 8A.

I

Table 9A. l1

ii

Table 10A. .

Table 11A.
Table 12A.
Table 13A.

Table 14A.

Table 15A_

Table 16A.

Table 17A.

Table 13 A.

Table 19A,
Table 20A.

Table 21A

N.
M
in
M
M

M

Table 22A. M

In

Table 13. AN

Table 23- ANT

 

 

 

 

Table 7A. Means table for change in total (DP) RBAE (mm2 Al)
in Experiment 1 ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, 76
Table 8A. Means table for total (LM) RBAE (mm2 Al) in Experiment 1 ,,,,,,,,,,,,,,,,, 76
Table 9A. Means table for change in total (LM) RBAE(mm21Al)
in Experiment 1 ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, 76
Table 10A. Means table for change in ICTP (ng/ml) in Experiment 1 _____________________ 76
Table 11A. Means table for height at the withers (cm) in Experiment 1 ,,,,,,,,,,,,,,,,,, 77
Table 12A. Means table for height at the hip (cm) in Experiment 1 ,,,,,,,,,,,,,,,,,,,,,,,,, 77
Table 13A. Means table for weight (kg) in Experiment 1 ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, 77
Table 14A. Means table for third metacarpal circumference (cm)
in Experiment 1 ....................... 77
Table 15A. Means table for medial RBAE (mm Al) in Experiment 2 ,,,,,,,,,,,,,,,,,,,,,, 77
Table 16A. Means table for lateral RBAE (mm Al) in Experiment 2 ,,,,,,,,,,,,,,,,,,,,,,, 78
Table 17A. Means table for tOtal (DP) RBAE (mm2 Al) in Experiment 2 ,,,,,,,,,,,,,,, 78
Table 18A. Means table for change in osteocalcin (ng/ml)
in Experiment 2 .......... 78
Table 19A. Means table for height at the withers (cm) in Experiment 2 ,,,,,,,,,,,,,,,,,, 78
Table 20A. Means table for height at the hip (cm) in Experiment 2 ,,,,,,,,,,,,,,,,,,,,,,,,, 78
Table 21A. Means table for weight (kg) in Experiment 2 ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, 79
Table 22A. Means table for third metacarpal circumference (cm)
In Experiment 2 ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, 79
Table 1B. ANOVA table for medial RBAE (mmAl) in Experiment 1 ,,,,,,,,,,,,,,,,,,,,, 81
Table 2B. ANOVA table for lateral RBAE (mm Al) in Experiment 1 ,,,,,,,,,,,,,,,,,,,,, 81

viii

 

Table 38.
Table 48.
Table SB.
Table 6B.
Table 7B.

Table SB.

Table 9B. .

Table 108.
Table 118.
Table 123.

Table 13B_

Table 14B. ‘
Table 153. ,

Table l6B_ .

Table 173. ‘

Table 18B. ,

Table 198. I

Table 203 I

Table 213 1

Table 223. I

Table 238,

Table 3B.

Table 4B.

Table 5B.

Table 6B.

Table 7B.

Table 88.

Table 9B.

Table 10B.

Table 11B.

Table 12B.

Table 13B.

Table 14B.

Table 15B.

Table 16B.

Table 17B.

Table 18B.

Table 19B.

Table 20B.

Table 21B.

Table 22B.

Table 23B.

ANOVA table for palmar RBAE (mm Al) in Experiment 1 ....................
ANOVA table for dorsal RBAE (mm Al) in Experiment 1 .....................
ANOVA table for total (DP) RBAE (mm2 Al) in Experiment 1 ..............
ANOVA table for total (LM) RBAE (mm2 Al) in Experiment 1 .............
ANOVA table for osteocalcin (ng/ml) in Experiment 1 ...........................
AN OVA table for ICTP (ng/ml) in Experiment 1 .....................................

ANOVA table for keratan sulfate (ng/ml) in Experiment 1 .....................

ANOVA table for height at the withers (cm) in Experiment 1 ..............
ANOVA table for height at the hip (cm) in Experiment 1 .....................
ANOVA table for weight (kg) in Experiment 1 ......................................

ANOVA table for third metacarpal circumference (cm)

 

in Experiment 1

 

ANOVA table for medial RBAE (mm Al) in Experiment 2 ..................
ANOVA table for lateral RBAE (mm Al) in Experiment 2..... ..............
ANOVA table for total (DP) RBAE (mm2 Al) in Experiment 2 ,,,,,,,,,,,
ANOVA table for change in osteocalcin (ng/ml) in Experiment 2 ,,,,,,,,
ANOVA table for ICTP (ng/ml) in Experiment 2 ...................................
ANOVA table for keratan sulfate (ng/ml) in Experiment 2 ...................
ANOVA table for height at the withers (cm) in Experiment 2 ..............
ANOVA table for height at the hip (cm) in Experiment 2 .....................

ANOVA table for weight (kg) in Experiment 2

 

ANOVA table for third metacarpal circumference (cm)

in Experiment 2

 

ix

81

81

81

82

82

82

82

82

82

83

83

83

83

83

83

84

84

84

84

84

84

 

 

Figure 1.

Figure 2. .

Figure 3. I

Figure 4. F

Figure 5. C

Hflm6.C

 

Figure 7. C1
in
Figure 8. M.
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1n

 

LIST OF FIGURES

 

 

 

 

 

 

 

 

Figure l. Scanned image of equine third metacarpal and

aluminum stepwedge penetrometer_,,,,____,_,_,___m_,,,___, ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, 28
Figure 2. Medial RBAE (mm Al) versus day of project

in weanling Quarter Horses ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, 34
Figure 3. Lateral RBAE (mm Al) versus day of project

in weanling Quarter Horses ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, 35
Figure 4. Palmar RBAE (mm Al) versus day of project

in weanling Quarter Horses ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, 35
Figure 5. Change in dorsal RBAE (mm Al) versus day of project

in weanling Quarter Horses ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, 36
Figure 6. Change in total (DP) RBAE (mm2 Al) versus day of project

in weanling Quarter Horses ,,,,,,,,,,,, 36
Figure 7. Change in total (LM) RBAE (mm2 Al) versus day of project

in Quarter Horses ................. 37
Figure 8. Medial RBAE (mm Al) versus day of project in

weanling Arabians ' ........ 54
Figure 9. Lateral RBAE (mm Al) versus day of project in

weanling Arabians ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, 54
Figure 10. Total (DP) RBAE (mm2 Al) versus day of project

in weanling Arabians ................. 55
Figure 11. The Mechanostat Theory (Frost, 1987) ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, 67

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INTRODUCTION

Young horses are often housed in stalls as opposed to being out on pasture with
free access to exercise. Stalling may be detrimental to bone strength due to a lack of
exercise. Exercise allows the bone to respond to changes ensuring bone mass capable of
withstanding loads associated with training later in life (Price eta1., 1995b). Without the
necessary exercise, the extracellular matrix will be resorbed until bone is adapted to
decreased loading. This could result in bone ill-prepared for the rigors of training.
Wolff’s Law states that cortical bone will remodel according to the strains placed on it
(W 00 et al., 1981). Remodeling allows bone to adapt to strains so bending is reduced and
damage will be prevented (Carter, 1984). Numerous Studies have demonstrated that
depriving animals of exercise is detrimental to bone strength. Laying hens housed in
battery cages had 54% weaker bones than those housed in percherys, demonstrating that
bone strength is related to the amount of movement allowed (Knowles and Broom, 1990).
Nielsen et al. (1997) found as bone mineral content of the third metacarpus decreased, the
incidence of bone related injuries increased. McCarthy and Jeffcott (1992) found that
yearling horses exercised on a treadmill demonstrated an increase in bone density.
Hoekstra et al. (1998) found that yearling Arabian horses demonstrated a decrease in the
rate of bone formation, an increase in bone resorption, and a decrease in bone mineral
content when kept in stalls compared to control horses maintained on pasture. The
hypothesis of this study was that Stalling demonstrates a negative effect on bone mass.
The first objective was to determine whether housing weanling horses in stalls is

detrimental to bone and cartilage development compared to that in weanling horses

 

 

maintained

or lZ-h dailj

 

maintained on pasture or in a paddock. The second objective was to determine if exercise

or 12-h daily turnout of weanlings could eliminate a decrease in bone mass.

.fl“

 

 

 

 

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CHAPTER 1

REVIEW OF LITERATURE

Connective Tissue Biology
Bane

Bone is the basic tissue that provides both protection and structure for the body.
It aids in the distribution of forces, provides metabolic regulation of fluids, and acts as the
reservoir for blood cell formation (Jee, 1988; Marieb, 1992). Limb bones, in particular,
are responsible for withstanding repetitive loading and avoiding damage by adapting to
the common loads of normal activity and growth (Lanyon, 1987). Bone is comprised of
cortical (outer wall) and trabecular bone (central region containing marrow). Cortical
bone makes up 80% of total bone (Hassager et al., 1994). Trabecular bone is composed
of soft tissue (75% by volume) including the bone marrow while conical bone is a dense
solid mass (Jee, 1988). Though trabecular bone assists in resisting stresses placed on
bone, cortical bone is primarily responsible for the strength of bone (Marieb, 1992).

Cortical bone contains osteons composed of larnella (Marieb, 1992; Jee, 1988).
The osteons assist in creating structurally sound bone capable of twisting and
withstanding stresses. The Haversian canal runs through each osteon supplying
osteocytes with nutrients. Volkmann’s canals run perpendicular to Haversian’s canals
connecting the vascular and neural fibers to the medullary cavity (marrow) (Marieb,
1992; Jee, 1988). Cortical bone consists of an amorphous ground substance which
contains phosphoproteins, glycoproteins, y-carboxyglutamic acid proteins (e. g.

osteocalcin), proteoglycans, lipids, and peptides (Jee, 1988). Bone collagen fibers are

n .u_--_- ~u‘k:
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embeddec
of hydrox
collagen fi
mineralize.
The
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1988; Mari
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to increase t
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(Tee, 1988). .

embedded within the amorphous ground substance. Bone is made rigid by the presence
of hydroxyapatite (mineral salts) held together by ground substance in between type I
collagen fibrils (Jee, 1988; Marieb, 1992). Approximately 90% of the organic matrix in
mineralized bone consist of type I collagen (Risteli et al., 1993).

The structure of long bones consists of the epiphysis, metaphysis, and diaphysis.
In growing bone, the metaphysis and epiphysis are separated by the growth plate (Jee,
1988; Marieb, 1992). Growth plate cartilage regulates linear growth and the ultimate
length of the bone (Fretz et al., 1984). The outer surface of long bones is covered by the
periosteum. The periosteum is lined with mesenchymal stem cells which can be recruited
to increase bone growth and assist in fracture repair (Jee, 1988).

Osteoblasts and osteoclasts are the cells responsible for bone formation and
degradation, respectively. Osteoblasts synthesize and secrete unmineralized bone matrix
(osteoid), type I collagen, non-collagenous matrix proteins, osteocalcin (0C),
proteoglycans, and other bone forming macromolecules. Osteoblasts are generally
located near bone surfaces. Osteoblasts possess receptors for parathyroid hormone and
1,25-dihydroxy vitamin D3 and contain alkaline phosphatase in its plasma membrane
(Jee, 1933). The life cycle of an osteoblast begins after differentiation of the progenitor
cell and then further matures to participate in matrix formation. The last step is further
maturation into bone-lining cells or osteocytes, participation in calcifying units, or death.
Osteocytes are osteoblasts that have been entrapped in the mineralized matrix and are
responsible for bone modeling and remodeling (Lanyon, 1987). These cells are sensitive
to the distribution of loads, the rate of change, and the magnitude of strain placed on

bone. Osteoclasts are housed in Howship’s lacunae. Osteoclast maturation in the bone

 

 

marrow 1!
environme
acidic em
interstitial
breakdown
Rer
mature bor
remodeling
resorption
maintenanc.
adailm‘fe res
Order to am
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involye any
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ca. '
using dam

marrow is stimulated by 1,25-dihydroxy vitamin D3. Osteoclasts create an acidic
environment and secrete lysosomal enzymes to aid in the degradation of bone. The
acidic environment solubilizes hydroxyapatite allowing the release of calcium into
interstitial fluid (Jee, 1988; Marieb, 1992). Osteoclasts are also responsible for the
breakdown of type I collagen (Allen et al., 1998).

Remodeling is the process in which immature or inferior bone is replaced by
mature bone and old or damaged bone is replaced by new bone (Jee, 1988). During
remodeling in mature bone, formation and resorption are balanced, but as the horse ages,
resorption exceeds formation (Fraher, 1993). This process is necessary for the
maintenance of biomechanically and metabolically competent bone. Remodeling is an
adaptive response to commonly encountered strains to ensure structural soundness in
order to avoid fatigue and failure (Lanyon, 1987). Carter (1984) defines remodeling as
any alteration in the shape, size, and microstructure of bone. These alterations may
involve any change in bone turnover. During training in horses, bones must undergo
adaptive remodeling in order to resist the stress placed on their bones (Smith, 1991).
This adaptation must be gradual to avoid resorption exceeding the rate of formation
causing damage to occur (Carter, 1984). The complete remodeling cycle in which
resorbed bone is replaced in full by new bone takes approximately 4 mo in humans (Jee,
1988). The 4 mo needed for complete remodeling includes 21 d for resorption and 101 d
for formation (Jee, 1988). An increase in remodeling may lead to a loss of trabecular and
cortical-endosteal bone while a decrease in remodeling conserves bone (Frost, 1987). An
increase in remodeling leads to a loss in total bone due to bone being resorbed faster than

it can be repaired.

 

 

 

N
altering t
surfaces .
formation
Any altera
life (Nun:
advantagec
than mature
effects on U
of nutrients
alteration of
formation. 1
Smaller third
analySes indj.

age.

Modeling is the process in which bone develops into its mature adult form by
altering the amount of bone present (Jee, 1998). Modeling occurs at different bone
surfaces and rates during the growth process. In foals and weanlings, the rate of
formation greatly exceeds the rate of resorption (Maenpaa'et al., 1988; Fraher, 1993).
Any alteration to this growing process may have a large impact on bone strength later in
life (Nunamaker et al., 1990). Raub et al. (1989) states that exercise may be
advantageous in growing bone due to its more responsive nature in its adaptation to stress
than mature bone. Weaning may lead to a decrease in growth rate due to its stressful
effects on the young horse (Warren et al., 1998). These stressful effects include the loss
of nutrients from the mare’s milk and a decline in feed intake (Warren et al., 1998). The
alteration of the nutritional patterns causes a decrease in the nutrients required for bone
formation. Though Warren et a1. (1998) found that horses weaned at an earlier age had
smaller third metacarpal bone circumference than those weaned at 6 mo, bone density
analyses indicated no significant changes in bone in horses weaned at 45 mo or 6 mo of

age.

Cannes:
Articular or hyaline cartilage, covers the ends of bones at the joints and serves as
a cushion to limit the stress applied to the bone, providing a smooth gliding surface to
ensure efficient movement (McDevitt, 1973). Synovial fluid lies in between joints
interacting with the cartilage and assists in providing the joint a low coefficient of friction
enhancing the ability of the viscoelastic cartilage to act as a cushion to absorb mechanical

stresses (McDevitt, 197 3).

 

 

composet
proteogly
provides t
II collage
1991; Hau
responsible
1994) and
Cartilage Is
proteOglyca
Prot.
glycosarm'nC
majOr PG fl
provides C01
‘ISROUghley al
aggrecan Prin
up to 100 agg
prom“ (Roug-
the enragenu
appmximatel.‘/
animals (Sandi
replacing ”Te 0-

Mature cartilage is avascular, aneural, and lacks lymphatic vessels. Cartilage is
composed of 70 to 80% water. The remaining dry weight consists of 50% collagen, 40%
proteoglycans, and 10% non-collagenous proteins (Nixon, 1991). Type II collagen
provides the cartilage tensile properties by forming a fibril network (Nixon, 1991). Type
II collagen forms concentric rings around chondrocytes forming a chondron (Nixon,
1991; Hauser et al., 1996). Chondrocytes, making up only 5% of the tissue volume, are
responsible for maintaining organization of the extracellular matrix (Roughley and Lee,
1994) and controlling cartilage turnover (Nixon, 1991). The extracellular matrix of
cartilage is a collagenous matrix containing a ground substance of glycoproteins and
proteoglycans (PG) (Roughley and Lee, 1994).

Proteoglycans are composed of a core protein with covalently bound
glycosaminoglycan (GAG) chains (Hardingham and Fosang, 1992). Aggrecan is the
major PG found in articular cartilage and is a product of chondrocytes. Aggrecan
provides compressive stiffness to cartilage and helps in the distribution of loads
(Roughley and Lee, 1994; Hardingham and Fosang, 1992). In the cartilage matrix
aggrecan primarily exists as a PG aggregate. Proteoglycan aggregates are composed of
up to 100 aggrecan molecules non-covalently bound to hyaluronan and stabilized by link
protein (Roughley and Lee, 1994). Hyaluronan helps maintain the structural integrity of
the extracellular matrix (Haapla et al., 1996). Proteoglycans have a half life of
approximately 300 d, indicating that they do not undergo rapid turn-over in mature
animals (Sandy, 1992). Proteoglycan content remains constant during turnover by

replacing the old aggrecan molecules with newly synthesized molecules (Thonar and

Glant, 1992).

 

 

 

The GAG.

linked by a
sulfated. ir
heparan sul
only glycos
and Fosang
charged. due

which create:

hydrating the

 

 

 

The GAG, attached to PG, are long chains of repeating disaccharide units (hexosamine
linked by a glycosidic bond to a non-nitrogenous sugar). There are two types of GAG:
sulfated, including chondroitin sulfate (CS), keratan sulfate (KS), dermatan sulfate,
heparan sulfate, and heparin and non-sulfated including, hyaluronic acid. Heparin is the
only glycosaminoglycan that is not present in cartilage (McDevitt, 1973; Hardingham
and Fosang, 1992; Roughley and Lee, 1994). Glycosaminoglycans are negatively
charged, due to the presence of carboxyl and sulfate groups, attracting positive ions
which creates an osmotic imbalance thus causing water to be retained in the matrix, thus
hydrating the cartilage (Mankin and Brandt, 1984; White, 1988; Hardingham and
Fosang, 1992).

The lifespan of cartilage consists of two phases: 1) early "developmental" phase
and 2) post-developmental "ageing" phase. The majority of changes cartilage undergoes
occurs in the first phase. During this phase, articular cartilage increases in collagen
content and keratan sulfate. During the second phase, the biochemical composition of
cartilage undergoes less dramatic change (McDevitt, 1973).

During loading, cartilage is compressed causing water to be released by PG.
Once loading is complete and the cartilage bears no weight, water is allowed back in
returning the cartilage back to its original shape. Motion and force facilitates the
diffusion of nutrients and removal of waste from the cartilage (Kincaid and Van Sickle,
1982). Loading allows nutrients to infiltrate the cartilage matrix from the synovial fluid
(Richardson and Clark, 1993).

To maintain cartilage matrix composition, loading of the joint is necessary

(Haapla et al., 1996). The affect of immobilization or lack of exercise on articular

__ai

 

 

cartilage

During th

at repair h
1992). Al'
PG from c:
the early 5
concentrati

increased Pi

cartilage leads to the development of lesions similar to those of osteoarthritis (OA).
During the early stages of OA, chondrocytes increase matrix PG synthesis in an attempt
at repair however, PG depletion is often of greater magnitude (Hardingham and Fosang,
1992). Alwan et al. (1991) observed an increase of GAG in serum indicating a net loss of
PG from cartilage in horses. The rate of PG released from the matrix is very high during
the early stages of OA. Todhunter et a1. (1997) found a tendency for plasma KS
concentration in normal horses to be lower than KS concentrations in OA indicating

increased PG degradation in OA horses.

Biochemical Markers of Bone and Cartilage Metabolism
Biochemical markers are utilized for non-invasive measuring of bone and
cartilage tum-over. Markers are products of tum-over in either bone or cartilage, which
serve particular functions in the matrices of these tissues. Markers can be detected in
either blood or urine and may be used to determine the effects of pasture-rearing versus

stall-rearing on bone growth in young horses.

Bone
Alkaline phosphatase -— Alkaline phosphatase (ALP) is an enzyme found in bone,
liver and kidney (Price et al., 1995a). Bone specific variant of ALP (BALP) is a serum
marker that differs from hepatic ALP by post-translational modifications of the gene
(Delmas et al., 1993). The bone specific variant of ALP is thought to be located on the
cell surface of osteoblasts acting as an osteoinductive cell marker and hence indicative of

bone formation (Allen et al., 1998; Price et al., 1995a, Jee, 1988). Heikkinen et al.

 

 

 

 

(1997) Sta
concentrat
ALP, Delr
analyze du.
by specific
by using re
1998).

Tip
cleaved by
Called (garb.
(PTCP and .
“garliem an
CarbOthem
reUlOdeling
P00rly gene
Sensitive enc

Ostec

binding mat]

(1997) state that BALP is released primarily during matrix maturation and has low serum
concentrations during mineralization. Because BALP is so closely related to hepatic
ALP, Delmas et a1. (1993) reported that BALP lacks sensitivity and is cumbersome to
analyze due to techniques relying on activators, inhibitors, electrophoresis, and separation
by Specific antibodies. However, new methods now exist for analyzing BALP in serum
by using radioimmunoassay or enzyme linked immunosorbent assay kits (Allen et al.,
1998).

Type I collagen propeptides -- Collagen is synthesized from procollagen and then
cleaved by extracellular propeptides releasing the amino- and carboxy-terrninal ends
called carboxy-terrninal propeptide and amino-terminal propeptide of type I collagen
(PICP and PINP respectively). Type I collagen is found in bone, skin, tendon, and
ligament and hence, PICP and PINP are not specific for bone (Price et al., 1995a).
Carboxyterrninal propeptide of type I collagen decreases with increasing age in horses as
remodeling decreases (Price et al., 1995a). Delmas (1993) reports that serum PICP is
poorly correlated with DC and alkaline phosphatase indicating that this marker is not
sensitive enough to be used as a marker of bone formation.

Osteocalcin —- Osteocalcin is a non-collagenous, vitamin K dependent, calcium-
binding matrix protein. Osteocalcin acts as a link between the organic and inorganic
matrices of bone and may account for 3% of bone protein (Fraher, 1993; Hope et al.,
1993). Osteocalcin is synthesized primarily by osteoblasts and incorporated into the
extracellular matrix of bone. Small fractions of 0C are released into circulation during
bone formation which can be detected by radioimmunoassay (RIA) or enzyme linked

immunosorbent assays (ELISA) (Delmas, 1993; Kannus et al., 1996). Osteocalcin

10

 

 

contains th

 

changes in
1990: Lian
upon age a
serum of 5(
lower in Ol
addition tc
concentrati
tsported ti
maximum .
I800 level
directly as
TCSOrpthn
and reserp
Studies ha.

meTRIIOn

H\

v

conagens.
hlv’Ciroywpl
Apprexim
metaboliZe
C02 and u;

the peptide

contains three residues of y—carboxyglutamic acid (gla) which undergo conformational
changes in the presence of Ca promoting 0C binding to hydroxyapatite (Lepage et al.,
1990; Lian and Gundberg, 1988; Price, 1982). Osteocalcin concentrations are dependent
upon age and time of day. Lepage et a1. (1990) determined 0C concentration in the
serum of 50 female Standardbreds of varying ages. Mean serum 0C concentration were
lower in Older horses, indicating a decrease in bone turnover with increasing age. In
addition to age-related changes, 0C undergoes circadian variation so that serum
concentrations are dependent on the time of sample collection. Lepage et a1. (1991)
reported that the lowest serum concentrations occurred between 1900 and 2000 and
maximum concentrations were between 2000 and 0500 in Standardbreds. From 0700 to
1800 levels remained constant. Lepage et a1. (1991) concluded that photoperiod is
directly associated with DC serum levels. During bone tum-over, formation and
resorption occur simultaneously which causes the release of markers of both formation
and resorption. Because formation and resorption are coupled during tum-over, some
studies have indicated the use of DC as a measure of bone tum-over as well as bone
formation (Heikkinen et al., 1997; Kannus et al., 1996).

Hydroxyproline -- Hydroxyproline represents 13% of the amino acid content of
collagens. Since the majority of total body collagen is found within bone,
hydroxyproline is thought to be a marker of bone resorption (Delmas, 1993).
Approximately 90% of the hydroxyproline released during bone resorption is
metabolized, circulates in plasma, is reabsorbed in the kidneys, and is then degraded to
CO2 and urea. This whole process only leaves approximately 10% of hydroxyproline in

the peptide-bound form to be released in the urine (Delmas, 1993). Because only a small

11

 

 

 

percenta
marker c
P
(Dpd) ar
coHagen
creating i
but is 315.
found pm
al., 1996)
markers 0
sensitive I
markers 8)
during the
cOllffcnong
Similar Test
concentratii
Carl
9P6 I Collag

percentage of hydroxyproline is released in the urine, it is not a very sensitive or effective
marker of bone resorption.

Pyridinoline and Deoxypyridinoline -- Pyridinoline (Pyr) and deoxypyridinoline
(Dpd) are cross-links that stabilize collagen. Pyridinoline and Dpd provide stability to
collagen by cross-linking 3 hydroxylysine or lysine residues between chains of collagen
creating a cyclic structure (Valimaki et al., 1994). Pyridinoline is not Specific for bone
but is also found in cartilage and other connective tissues (Delmas, 1993) while Dpd is
found primarily in bone with minute amounts in aorta, dentine, and ligaments (Gomez et
al., 1996). Both Pyr and Dpd can be detected in urine and serum and measured as
markers of bone resorption (Gomez et al., 1996). Both markers are more specific and
sensitive than hydroxyproline making them good markers of bone resorption. Both
markers exhibit diurnal variation with the higher values at night and the lowest values
during the day, making urine collections very cumbersome and often involve 24-h
collections (Valimaki et al., 1994; Delmas, 1993). However, Hoekstra (1998) found very
similar results between 24-h collection analysis and spot samples adjusted for creatinine
concentrations.

Carboxyterminal telopeptide of type I collagen -- Carboxyterminal telopeptide of
type I collagen (ICTP) is released due to the breakdown of pyridinium cross-link of type I
collagen within the extracellular matrix of bone (Allen et al., 1998). Osteoclasts release
proteases that degrade type I collagen into amino acids and peptide fragments releasing
ICTP into serum (Allen et al., 1998; Price et al., 1995a). Carboxyterminal telopeptide of
type I collagen (ICTP) was used in the present study as a marker of bone degradation.

Plebani et al. (1996) and Kylmala et al. (1995) identified ICTP as being the most reliable

12

H‘—
l

 

Ell

 

 

 

and sensi
correlatiOi
This may
cancellous
bone. Val
.667) and
decreases i

had higher

and sensitive marker of bone resorption. However, Hassager et a1. (1994) found no
correlation between serum ICTP and histomorphometric measurements of bone turnover.
This may be a result of only measuring cancellous bone and not cortical bone, Since
cancellous bone is only 20% of total bone and has a different tum-over rate than cortical
bone. Valimaki et a1. (1994) found that ICI‘ P is closely correlated with urinary Pyd (r =
.667) and Dpd (r = .452). As turnover decreases with increasing age, serum ICTP
decreases (Price et al., 1995a). Price et al. (1995a) found that horses less than 1 yr old

had higher concentrations of ICT P than horses older than 1 yr.

Cartilage

Hyaluronan -- Hyaluronan (HA) assists in stabilizing the extracellular matrix of
cartilage and can be used as a marker of cartilage degradation (Thonar et al., 1995b).
Sharif and Dieppe (unpublished data) found that OA patients demonstrating high serum
concentrations of HA progreSsed to more severe joint problems than patients with low
concentrations of HA (Thonar et al., 1995b). Leipold et a1. (1989) studied KS and HA
serum concentrations in dogs with induced 0A and found that KS increased 10 fold while
HA increased only 2 fold. This indicates that HA may be a less sensitive marker of
cartilage metabolism than KS.

Keratan sulfate -- Keratan sulfate is a GAG with repeating disaccharide units of
galactose and N-acetyl glucosamine (Roughley and Lee, 1994). Keratan sulfate is the
second most abundant GAG on aggrecan after chondroitin sulfate. When aggrecan is
proteolytically cleaved, KS is released into the synovial fluid and eventually reaches the

bloodstream. Keratan sulfate is primarily found in cartilage so that serum KS most likely

13

 

 

 

 

reflects i
biochemi
1986). F
decrease
concentra
of age (Tl
(Todhunte
.V'Oung dog
found that
higher thai
Semm [(3
K81
Cartilage \
aggrecan ,
degrading
either Con 1

reflects changes in metabolism of PG, suggesting that serum KS can be used as a
biochemical marker of proteoglycan degradation (Campion et al., 1991; Thonar et al.,
1986). Keratan sulfate concentrations are partly dependent on age and are shown to
decrease with age in humans. During the first 4 yr of "life in children, serum KS
concentrations are high and remain elevated until 12 yr of age and then decrease at 13 yr
of age (Thonar et al., 1988). Plasma KS concentration peaked at 10 wk of age in foals
(Todhunter et al., 1997). Leipold et a1. (1989) concluded that high levels of serum K8 in
young dogs (< 1 yr) are due to the higher rate of synthesis of KS. Okumura et al. (1997)
found that the mean serum KS concentration during the first 3 mo of life were five times
higher than the KS concentration in yearlings or older horses. Between 3 to 5 mo of age,
serum KS concentration decreased rapidly.

Keratan sulfate in synovial fluid indicates the rate of degradation of articular
cartilage within a particular joint while KS in blood measures the average rate of
aggrecan degradation in the body (Thonar et al., 1995a). When cartilage begins
degrading, serum KS concentration increases (Alwan et al., 1990). This increase may
either confirm degradation is occurring or identify a predisposition to osteoarthritis
(Thonar and Giant, 1992). Williams et a1. (1988) injected rabbit knee joints with
chymopapain to induce cartilage degradation and found an increase of serum KS
indicating the acute loss of PG from the joint. Spector et al. (1992) suggested that KS is
not an accurate measure of cartilage degradation or CA due to its high variability
possibly associated with sex, age, and injury. Serum KS concentration may not be an
accurate measure of cartilage degradation because KS reflects both normal and diseased

cartilage PG and that synovial joints account for only 15% of all cartilaginous tissue.

14

 

 

 

Though I

cartilage c

IVC

provide ac
Ult

bone using
horses (Je
ultrasonog]
The disadv
that it is m
boneS(Le.‘
greater Sign
a Weanl in g,
Sing
degree Of a'
1986). Tho
density. it is
et al., 1986)_

Dllal

single phOTOn

Though KS is a variable marker, it is the most sensitive marker found to represent

cartilage degradation (Liepold et al., 1989).

Measurements of Bone Mineral Content

N on-invasive measurements of bone density are used with biochemical markers to
provide accurate measures of bone turnover.

Ultrasonography -- Ultrasonography measures the velocity of sound through a
bone using an ultrasonic beam and two transducers to determine bone mineral content in
horses (Jeffcott and McCartney, 1985). Jeffcott and McCartney (1985) found that
ultrasonography is an accurate, inexpensive method to measure of bone mineral content.
The disadvantages to using ultrasonography, compared to radiographic photometry, are
that it is more labor intensive and requires direct contact with the limb. With smaller
bones (i.e. weanlings) the transducers need to be held on the limb longer in order to have
greater Signal capability of the oscilloscope. It is not feasible to have a horse, specifically
a weanling, to stand perfectly still for more than a few seconds unless tranquilized.

Single photon absorptiometry -- Single photon absorptiometry measures the
degree of attenuation of a low energy photon beam to bone and tissue (Jeffcott et al.,
1986). Though single photon absorptiometry provides a precise measurement of bone
density, it is expensive and requires the animal to stand perfectly still for 90 sec (Jeffcott
et al., 1986). The expense lies in purchasing fluid filled bandages necessary for acting as
a soft tissue conductor, supplying a flat parallel surface, and the machine itself.

Dual photon absorptiometry -- Dual photon absorptiometry is very similar to

single photon absorptiometry except a radiation beam at two separate energy levels are

15

 

 

 

 

used inste
single phO
Ra.
because it
al. (1981)
third meta.
measured
equivalenc:
the RBAE I
content (r =
Changes in
Nielsen an
rmTitil'aliZatj

[Ola] aI-Ca OI

used instead of one. The same limitations exist with dual photon absorptiometry as in
single photon absorptiometry.

Radiographic photometry —- Radiographic photometry is a popular technique
because it provides precise, accurate measurements and is cOnvenient to use. Meakim et
a1. (1981) measured bone mineral content of the dorso-palmar (DP) view of the equine
third metacarpus using radiographic photometry. Radiographs of the DP view were
measured with an aluminum stepwedge to determine radiographic bone aluminum
equivalence (RBAE) expressed in mm aluminum (Al). Meakim et a1. (1981) found that
the RBAE measurement of the third metacarpus was highly correlated with bone mineral
content (r = .88 for the medial side and .94 for the lateral side). This technique indicated
changes in sections of the bone with greatest optical density. Using similar techniques,
Nielsen and Potter (1997) looked at volumetric changes as well as changes in
mineralization by determining total bone mass. This technique uses the optical area and
total area of the A] stepwedge and the optical area of the third metacarpal bone. As in
Meakim et a1. (1981), RBAE was determined but expressed in mm2 Al. Therefore,
RBAE can be used to determine total bone mineral content of the third metacarpus.

Williams et a1. (1991) compared non-invasive techniques of measuring bone
mineral content of the third metacarpal bone in cattle. A correlation coefficient (r)
between bone mineral content and radiographic photometry was .967 (p < .0001). This
was a higher r than photon absorptiometry (.908, P < .0001) and ultrasonography (.565,
P < .0001). Thus, it was concluded that radiographic photometry is more accurate
measurement of bone mineral content then both photon absorptiometry and

ultrasonography.

16

 

 

 

 

Bone
Nu
detrimental
necessary f
stress/strain
(Pratt. 1982
defined as 1;

related to ve}

Response of Bone and Cartilage to Limited Physical Activity
Berle

Numerous studies have demonstrated that depriving animals of exercise is
detrimental to bone strength. Exercise allows the bone to. respond and adapt to loads
necessary for proper bone architecture and mass (Pratt, 1982). An increase in mechanical
stress/strain causes an increase in bone mass while a decrease in loading causes bone loss
(Pratt, 1982). Strain is defined as a deformation created at any point while stress is
defined as internal force intensities (Hayes and Gerhart, 1985). Strain rate is directly
related to velocity:

y = 2500v — 3067
where: y = strain rate in microstrain per sec
v = velocity in feet per sec

indicating that strain increases linearly with increased speeds (Pratt, 1982). In other
words, the faster a horse runs the more force is placed on the leg causing an increase in
strain. Frost (1987) describes'strain as mechanical usage and suggests that the magnitude
of load influences modeling, remodeling, and growth. A lack of exercise in racing horses
will result in bone unable to tolerate high strains which leads to weaker bone potentially
causing injury (Price et al., 1995). This is a major problem in racing horses due to the
high degree of stress put on their legs during training while still skeletally immature
(Price et al., 1995). Larneness is very common among young horses during training (18
to 24 mo of age) possibly due to their bones not reaching a sufficient mass to bear the
necessary load experienced during training (El Shorafa et al., 1979; Nunamaker et al.,
1990). Immature bone is more responsive to alterations in strain than mature bone

(Carter, 1984).

17

 

 

 

 

Housir
Kn'
affects of 1
amount of
restrict ma
percherys i
Specifically
radius and
correlation
breaking Stl
NOtably, m
influences b
Whil
Quarter HO
osteocalcin a
in bone min:
56 to d l 12
“SponsC to
reSOrpthn Ca
the third met,-

Housing

Knowles and Broom (1990) and Fleming et a1. (1994) studied laying hens and the
affects of different housing systems on bone strength. Bone strength was related to the
amount of movement allowed. Both studies demonstrated that battery cages, which
restrict maximal movement, yielded birds with weaker bones than those housed in
percherys did, which allowed for free wing movement. Knowles and Broom (1990)
specifically found that birds in battery cages had only 54% of the bone strength in the
radius and tibia of birds housed in percherys. Fleming et a1. (1994) found a positive
correlation between bone breaking strength and bone mineral density. The increase in
breaking strength was related to changes in cortical bone and not in trabecular bone.
Notably, neither Study discussed the effect of housing on egg production, which
influences bone strength due to Ca utilization for egg formation.

While looking at the effects of mineral balance, Nielsen et a1. (1998) found that
Quarter Horses maintained in stalls during training showed an inerease in serum
osteocalcin as bone density increased. Nielsen et a1. (1998) also found an initial decrease
in bone mineral content at the onset of training (d 0 to d 56) and then an increase from d
56 to d 112. This initial decrease may have been due to the onset of training or a
response to stall confinement. This confinement probably resulted in bone mineral
resorption causing a decrease in bone mineral content, indicating decreased strength of
the third metacarpus. Maenpaa et a1. (1990) found decreased serum alkaline phosphatase
activity and 0C concentration indicating diminished bone formation in foals moved from
pasture to stalls. Hoekstra et a1. (1998) also found that 18.6 mo old Arabian horses

removed from pasture and placed in stalls, with only 1 h/d of walking on a mechanical

18

 

 

 

walker. sh
also showe
and a deer
horses and

compared 1

Exercis
Usii

ulnae of T0(
88% of its (
maintain of
11131 it 100k
CYCles of 102
mineral COn
Showed that
Over [13056 S\
the mechanic
quantity (Cor
Raub

the lefi third 1

bones haVin

walker, showed a decrease in bone mineral content by d 28 of the study. Stalled horses
also Showed increased deoxypyridinoline at d 28 indicating an increase in bone resorption
and a decrease in serum DC at d 14 indicating decreased osteoblastic activity. Stalling
horses and limiting their access to exercise negatively affected normal bone development

compared to horses maintained on pasture.

Exercise

Using photon-absorption densitometry, Rubin and Lanyon (1984) found that the
ulnae of roosters not subjected to loading showed a decrease in bone mineral content to
88% of its original postoperative state. To determine the amount of loading necessary to
maintain or increase bone strength in the rooster ulnae, Rubin and Lanyon (1984) found
that it took four cycles of loading per day to maintain bone mineral content. With 36
cycles of loading per day on the ulnae, bone formation increased with the maximum bone
mineral content being reached at d 28 and stabilizing thereafter. Woo et a1. (1981)
showed that exercised swine had a 35% increase in maximum load strength of the femur
over those swine not undergoing exercise. Woo et a1. (1981) also determined that though
the mechanical properties of bone remained unchanged following prolonged exercise, the
quantity (cortical thickness and volume) of bone increased.

Raub et a1. (1989) noticed an increase in medial bone mineral content/density of
the left third metacarpus in exercised weanling horses. This increase was associated with
bones having greater bone strength, hence, being able to withstand the forces associated
with increased loading. McCarthy and Jeffcott (1992) studied the effects of exercise and

inactivity on 12 Thoroughbred yearlings. They found that the exercised group had lower

19

 

 

 

 

cortical b0
lower seru
porosity in'
These Chan
structure mt
metacarpal 1
increase was
the most ef
activities. P
Short activi
exceeding th
that growing

bones and jO;

cortical bone porosity, higher bone mineral content, higher bone mineral density, and
lower serum OC concentrations than those receiving no exercise. The decrease in
porosity indicates a decrease in bone remodeling causing an increase in bone density.
These changes demonstrated the ability of cortical bone to adapt to exercise due to a
structure more capable of resisting injury. Sherman et al. (1995) found that the third
metacarpal bone of horses increased in peripheral thickness in response to exercise. This
increase was concluded to be associated with an increase in strength. Pratt (1982) found
the most efficient way to develop bone mass is to participate in short but vigorous
activities. Prolonged vigorous activities may lead to fatigue fractures of the bone. These
short activities provides bone the loading necessary for optimal growth without
exceeding the delicate balance between formation and resorption. Jeffcott (1991) states
that growing horses should remain on pasture 12 h/d for the normal development of

bones and joints.

Immobilization

Biewener and Bertram (1994) immobilized the wings of 2-wk—old chickens and
found that cortical thickness was greater in exercised animals than in the
sedentary/immobilized or denervation (excised sciatic nerve) animals. Kannus et a1.
(1996) immobilized patellas of rats to identify 0C concentrations. In the groups in which
limbs were immobilized and then remobilized and exercised, OC increased indicating
increased mineralization of newly formed bone during exercise. Yeh et a1. (1993) found
similar results in the limbs of immobilized rats. The resorption of the immobilized limb,

analyzed by radioactive ”Ca retention, was 21% greater than that Of the control rats.

20

 

 

 

   

Immobiliz

formation.

effects on
loading.

increased we

Exercise

The 11
when Comp;
demonstrates
Palmer et a1.
Cartilage of e
iOUnd to be C:
antibody 1C6
did a Similar S
horses pOSSibll
find that the ex
"latn‘x than the

ability f0r fluid

Immobilization decreased bone modeling while exercise caused more active bone

formation.

Cartilage
Similar to studies in bone, lack of exercise has been shown to have negative
effects on cartilage. A decrease in PG content has been associated with decreased
loading. Decreased PG content of the cartilage causes a decrease in elasticity and

increased water content (Palmoski et al., 1980).

Exercise

The incidence of osteochondrosis in foals subjected to forced exercise decreases
when compared to foals provided with limited exercise (Jeffcott, 1991). This
demonstrates the importance of exercise on cartilage metabolism in young horses.
Palmer et al. (1995a) found. a significant increase of newly synthesized PG in the
cartilage of exercised horses compared to non-exercised, stalled horses. The PG was
found to be composed of both large and small monomers that reacted with monoclonal
antibody 1C6 indicating the potential to form aggregates with HA. Palmer et al. (1995b)
did a similar study but found no difference in PG content between exercised and stalled
horses possibly due to the short time period of the study (6 wk). Palmer et al. (1995b) did
find that the exercised group demonstrated a greater permeability constant of the cartilage
matrix than the non-exercised group. This suggests that the exercised group had a greater

ability for fluid movement through the articular cartilage.

21

Little et al. (1997) compared strenuous and moderate exercise in horses and found
that strenuous exercise causes an increase in PG metabolism in regions of high contact
stress. Thus, too much exercise can damage cartilage. Saamamen et al. (1994) found
that dogs running 20 km/d for 15 wk had a higher water Content and lower collagen
content in the cartilage than did control animals. These changes in cartilage were thought
to be a result of damaged collagen matrix due to over-exertion of the cartilage. Vasan
(1983) also demonstrated cartilage degradation in dogs running 6 d/wk for l h over an 8
mo period at 9.7 to 12.9 mm with a 20° incline. In order for cartilage to attempt repair
caused by this excessive stress, an increase in synthesis activity was seen. However, this

increase in PG synthesis remained less than PG loss.

Immobilization

Numerous immobilization studies have been performed to identify the effects of
immobilization and increased weight bearing on both PG content and GAG content
within the joints. Richardson and Clark (1993) describe the effects of immobilization to
be complete erosion of the affected points of contact and soft tissue infiltration in non-
contact areas. Comparing PG and GAG content in exercised, immobilized, and control
rabbit joints, Tammi et a1. (1983) found an increase in PG concentration in the cartilage
of the exercised group. An increase in CS in the immobilized knee and an increase of KS
in the weight bearing knee were observed when compared to controls. Immobilization
yields PG richer in CS making aggregation more difficult but normal joint loading will
counter-act this effect (Tammi et al., 1983). A decrease in GAG synthesis was observed

by Eronen et a1. (1978) when rabbit legs were immobilized in extension. Immobilization

22

caused a rapid induction of osteoarthritic characteristics in the weight bearing cartilage.
By immobilizing a leg in extension, there is constant compression of the cartilage causing
a disruption of chondrocyte function leading to a decrease in GAG synthesis. This
decrease continues until the degradation becomes so extenSive that an increase in GAG
synthesis begins to compensate for the vast amount of degradation (Eronen et al., 1978)
but by this time the damage may be greater than the synthesis causing a net loss of
cartilage. A similar decrease of GAG in immobilized rabbit joints was Observed as well
as an increase in the KS/CS ratio (Videman et al., 1981). The increase KS/CS ratio
differs from what is found in OA joints. Videman et al. (1981) could not explain this
difference. Caterson and Lowther (1978) found that sheep with an immobilized foreleg
demonstrated a decrease in PG content while the supporting leg had an increase in PG.
Increased loadbearing on, and movement of, the joint stimulated metabolism of
chondrocytes, causing an increase in CS and an increase resistance to deformation.

In a study of dogs, ankle cartilage was transected and then immObilized (Palmoski
et al., 1980). The immobilized cartilage was 14 to 29% thinner than the contralateral
cartilage. There was also a decrease in PG content and synthesis, an increase in water
content, and defective PG aggregation. Haapala et al. (1996) found a 23 to 31% lower
hyaluronan concentration in immobilized dogs than in the contralateral limb. This lower
concentration of hyaluronan is thought to cause deleterious affects on cartilage matrix
organization.

Palmoski et al. (1980) also adds decreased elasticity and compressive stiffness to
the adverse effects of immobilized cartilage. While looking at horses immobilized with a

cast, Richardson and Clark (1993) found a decrease in PG content of cartilage in the

23

immobilized joint due to the mechanical stimuli being below a level necessary to
maintain normal cartilage. This lack of loading and motion causes a decrease in the
movement of synovial fluid nutrients into cartilage causing a decrease in PG synthesis

and content.

24

CHAPTER 2

MATERIALS AND METHODS: EXPERIMENT 1

Animal Management

Eighteen Quarter Horse weanlings from the MSU Merillat Equine Center were
pair-matched by age and randomly assigned to three treatment groups of Six horses each.
Horses remained on the study for 56 d. Prior to the start of the Study, weanlings were
kept outside during the day and stalled each night with their dams. Horses were weaned
in two separate groups consisting of nine horses each (three horses in each treatment
group). The first weaning group began the study on June 9, 1998 while the second
weaning group began on August 29, 1998. The paddock group (P1, n = 6) was
maintained on a 62.6 m x 15.9 m dry lot with free access to exercise. The stall group (S 1,
n = 6) was housed in 3.7 m x 3.7 m stalls with no free access to exercise. The exercised
group (X1, n = 6) was housed in 3.7 m x 3.7 m for the first 28 d Of the study with no free
access to exercise and followed an exercise protocol from d 28 to d 56 described later.
One horse in X1, from the second weaning group, died after d 28 from respiratory
problems. The data for this horse was still included in the results excluding d 42 and 56.
The average ages, followed by the range of ages in parentheses, of each group were P1 -
143 d (115 d to 187 d), 81 - 143.2 d (116 d to 193 d), and X1 - 141.2 d (108 d to 154 d).
Groups S1 and X1 were individually fed 1.5 kg of Strategy" (Purina Feeds, St. Louis,
MO) twice daily (0730 and 1530) and approximately 1.2 kg of alfalfa-grass mixed hay
every morning at (0730) (Table 1). If hay was eaten in the morning, a second feeding of

1.2 kg was fed at night at (1530). Group P1 was group fed 3.8 kg of Strategym (1.3

25

kg/horse) twice daily (0730 and 1530) and followed the same regimen of hay as groups
81 and X1 (Table 2). Strongid® C Daily Deworrner (Pfizer, Inc., New York, NY ) was
fed every morning according to manufacturer recommendations. Water was available at

all times.

Table 1. Calculated daily nutrient table for SI and X1 weanlings on a dry matter basis

 

 

Nutrient Calculated
Nutrient Content Total Daily Intake
DE 2.9 Meal/kg 12.4 Mcal
CP 15.0% 645.0 g
Ca 0.81% 35.0 g
P 0.46% 20.4 g

 

Table 2. Calculated daily nutrient table for P1 weanlings on a dry matter basis

 

 

Nutrient Calculated
Nutrient Content Total Daily Intake
DE 2.8 Meal/kg 11.2 Mcal
CP 15.1% 595.0 g
Ca 0.81% 32.1 g
P 0.46% ' 18.0 g

 

Before longe training began, each weanling in the exercised group was introduced
to longeing by walking 5 min daily, 5 d/wk on a mechanical walker (15.2 m diameter
circle) in both directions. The longeing procedure began on d 28, 1 wk after introduction
to the mechanical walker. Horses were longed on a 6.1 In to 9.1 m diameter circle once a
day for 5 d/wk. They were longed in an indoor arena on a dirt surface. Horses were
trotted 10 circles, cantered 20 circles, reversed and the circles repeated in the opposite

direction. Longeing lasted for approximately 15 min daily.

26

The exercised group, in the first weaning group, were longed with the preceding
procedure from d 28 to d 42. From d 42 to d 56 they were walked for 15 min, 5 d/wk on
the mechanical walker. The stalled and exercised weanlings in the second weaning group
were “halter broke” by walking on the mechanical walker fOr the first 14 d of the study.

On (1 28, the exercised group began the longeing procedure as stated above (Table 3).

Table 3. Exercise protocol for Quarter Horse weanlings

 

 

 

 

Day
Treatment 0-14 15-28 29-42 43-56
Exercise L MW
(W1)
Exercise I-IB L L
(W2)
stall (W 1)
stall (W2) HB
L - longeing procedure
MW - walking on the mechanical
walker

HB - halter broke on mechanical walker
W1 - weaning group 1
W2 - weaning group 2

Bone Mineral Content
On d 0, 28, and 56 radiographs of the left third metacarpal were taken to
determine radiographic bone aluminum equivalence (RBAE) (Meakim et al., 1981).
Dorsal-palmar and lateral-medial radiographs were taken at 17 mA at 71.1 cm focal film
distance for .08 sec at 70 kV, using mobile x-ray equipment and medical x-ray film. The
radiograph cassette was positioned against the palmar and medial points of the leg
allowing the beam to be parallel to the ground centered at the midpoint of the metacarpal

region. An aluminum stepwedge penetrometer was used as a reference standard for each

27

radiograph. Radiographs were scanned at the nutrient foramen, to determine RBAE.
using the Bio-Rad GS-700 Imaging Densitometer (Bio-Rad Laboratories, Hercules, CA).
Logarithmic regression was used to determine the dorsal, palmar, lateral, and medial
RBAE (mm Al) using the thickness of the stepwedge and the maximum optical density
readings of these cortices (Meakim et al., 1981). Total RBAE (mmZAl) was used to
determine density and volumetric changes using the total area of bone divided by the total
area of the aluminum stepwedge (Figure l). The total area under the stepwedge
corresponding to the steps with thickness of 14, 17, 20, 23, and 26 mm Al and the total
area of the third metacarpal bone curve were used. The area of the stepwedge was 1270
mm’. The total RBAE of the third metacarpal bone was determined by multiplying the
area of the third metacarpus (mm*0ptical Density) by 1270 mm2 and dividing by the

scanned area of the stepwedge (mm*Optical Density) (Nielsen and Potter, 1997).

(O.D.)

.l—l

 

 

 

-0 " r I
0 50 100 150
(mm

Figure l. Scanned image of equine third metacarpal and aluminum stepwedge
penetrometer (area of the stepwedge used for calculation is between the two
arrows) (Nielsen and Potter, 1997)

28

Serum Collection

Every two wk (d 0, 14, 28, 42, 56), blood samples were taken via jugular
venipuncture using a 20 gauge Vacutainer" needle and a sterile Vacutainer“D tube (Becton
Dickinson, Franklin Lakes, NJ) with no additives. BloOd was allowed to clot for
approximately 1 to 2 h and was subsequently centrifuged at 1340 g for 15 min at room
temperature. Serum was collected using a disposable Pasteur pipet (VWR Scientific,
West Chester, PA) and placed in 1.5 ml microcentrifuge tubes. Each sample was then
stored in a -20°C freezer until analysis. Serum was analyzed for DC, ICTP, and KS.
Sampling times for d 0, 14, 28, 42, and 56 for the first weaning group were 1615, 1030,
1535, 0935, and 0930 respectively. Sample collection for the second weaning group

were 0930, 0930, 0945, 0930, and 0930 respectively.

Osteocalcin Analysis

Osteocalcin was quantified using Novocalcin" ELISA kits (Metra Biosystems,
Inc. Mountain View, CA) according to manufacturer’s instructions. Serum was diluted
between 1:10 and 1:15 dependent on the sample. Dilutions were determined by running
the OC assay on a few samples at dilutions of 1:2, 1:4, 1:6, and 1:8. The values were
then compared to the standard curve graph in the manufacturer’s instructions. When the
values fell on the linear portion of the graph (between .4 and 1.5 optical density) the
corresponding dilution was used. Each sample was run in duplicate. Standards and
controls were reconstituted using .5 III] 1X wash buffer. Twenty-five til of standard,
sample, or control was added to each well with 125 pl of anti-0C antibody. This mix

was incubated at room temperature for 2 h and then each well washed with 300 pl 1X

29

wash buffer three times. Enzyme conjugate (150 1.11) was added to each well and
incubated for 1 h at room temperature. Each well was then washed with 300 [.11 1X wash
buffer. The last step was the 40 min incubation of 150 it] of working substrate solution.
Each plate was then read at 405 nm optical density on a spectra Max 340 plate reader

(Molecular Devices Corp, Sunnyvale, CA).

Carboxyterminal Telopeptide of Type I Collagen Analysis

Carboxyterminal telopeptide of type I collagen was quantified using ICTP 1251
RIA KitO (Diasorin Corp. Stillwater, MN) according to manufacturer’s instructions.
Each sample, standard, control, and nonspecific binding tubes were run in duplicate. The
tubes were set up as follows: nonspecific binding tubes - 100 1.11 sample and 200 111
distilled water, standards (0 and A-F) - 100 111 standard and 200 It] ICI‘ P antiserum,
controls and samples - 100 til serum and 200 111 ICTP antiserum. Each tube was
incubated for 2 h at 37° C with 200 pl of 125ICTP. Separation reagent (500 iii) was added
to each tube and incubated for 30 min at 25 ° C. Tubes were then centrifuged for 30 min
at 2000 X g at 10° C. The supernatant was decanted and placed in the 1290 GammaTrac

Gamma Counting System (Tm Analytic, Elk Grove Village, IL).

Keratan Sulfate Quantification
Keratan sulfate was quantified by an ELISA with an inhibition Step previously
described by Thonar et al. (1985) and Williams et al. (1988). The standard was diluted at
1:24 and serial diluted onto the uncoated plate. Samples were diluted to 1:4 (70 |J.l sample

+ 210 til PBS-Tween 20:1% BSA buffer) and serial diluted to 1:32 in the uncoated plate.

30

Both the standard and the samples were diluted with PBS-Tween 20:1% BSA, pH 5.3.
For the inhibition step, a 1212,000 dilution of the anti-KS monoclonal antibody (ICN
Pharmaceuticals Inc., Costa Mesa, CA) was made and 140 111 was added to each well.
Each plate was covered and incubated, with shaking, for l h at room temperature. The
plates were then stored at 4° C overnight. Coating buffer was removed from the coated
plates with three washes of PBS-Tween 20, pH 5.3 for 5 min. Two hundred til from each
well from the inhibition step were transferred to the washed coated plates. The coated
plate was covered and incubated, with shaking, for 1 h at room temperature. Ten min
prior to use, the horseradish peroxidase-conjugated anti-mouse IgG antibody (second
antibody) (ICN Pharmaceuticals Inc., Costa Mesa, CA) was diluted in PBS-Tween 20: 1%
BSA, pH 5.3, at a 1: 1,000 dilution. After 1 h incubation, the inhibition mixture in the
coated plates was removed and 5 min washes were performed three times. The second
antibody (200 It] ) was added to each well and incubated, with shaking, for 1 h at room
temperature. After 1 h of incubation, the inhibition mixture in the cOated plates was
removed and 5 min washes were performed three times. O-phenylenediamine was added
(200 til/well ) and incubated at room temperature for 15 min (Sigma Chemical, St. Louis,
MO). To stop the substrate-enzyme color development, 50 til/well of 2M H2804 was
added. The plates were then read with the Spectra Max 340 plate reader (Molecular

Devices Corp).
Growth Measurement

Cannon circumference, hip height, wither height and weight were taken every 28

(1. Weight was determined using a weight tape placed around the girth just below the

31

withers. Cannon circumference was measured around the mid-section of the third
metacarpus. Hip and wither heights were measured using a measuring stick from the

ground to the point of the hip or the withers.

Statistics

Differences between treatments, day of study and day*treatment interactions were
determined using a two-factor ANOVA (PROC MIXED) in SAS 6.12 (1997). The
blocking effect of weaning in two groups was included in our model. This was added to
account for any differences that may have existed due to weaning time. The LSMEAN S
statement was included in the analysis to obtain treatment means, differences between
means and standard errors. Data were normalized (d 0 values subtracted from other days
to examine changes from d 0) when treatment differences existed at d 0. A P-value of
less than .05 was considered significant while a trend was investigated at a P-value of less

than.l.

32

RESULTS: EXPERIMENT 1

Bone Mineral Content

The medial RBAE of the paddocked weanlings (Figiire 2) increased from 16.6 _+_
.7 mm Al on d 28 to 18.3 i .7 mm Al on d 56 (P = .03). Stalled weanlings demonstrated
a decrease in medial bone mineral content from 17.1 i .7 mm Al on d 0 to 15.6 :1; .7 mm
Al on d 28 (P = .05). On (1 56, P1 tended to have a greater mineral content of 18.3 i .7
mm Al than the stalled horses at 16.4 i .7 mm Al (P = .06) in medial RBAE. There were
no differences seen in the exercised weanlings or between X1 and P1 or S1 weanlings.

There were no time or treatment differences in lateral RBAE (Figure 3).

No treatment differences were seen in palmar bone mineral content. However,
the palmar RBAE of 81 (Figure 4) tended to decrease from 15.2 _-i_- .7 mm Al on d 0 to
13.6 1; .7 mm Al on d 56 (P s .1). No differences were seen in paddocked or exercised
weanlings.

Due to differences on d 0, dorsal, total (LM) and total (DP) views were
normalized by subtracting all values from d 0. The exercised group tended to decrease in
dorsal bone mineral content from d 0 to d 28 (P = .06) but increased from d 28 to d 56 (P
= .04) (Figure 5). On (I 28, the paddocked group had greater dorsal mineral content than
X1 (P = .02) while 81 tended to have greater mineral content than X1 on d 28 (P = .1).

Total (DP) RBAE demonstrated a tendency to decrease in X1 from d 28 to d 56 (P
= .09) (Figure 6). On d 56, paddocked weanlings had greater total (DP) mineral content

than exercised weanlings (P = .03). Total (DP) RBAE did not change in the stalled

weanlings.

33

The exercised group decreased in total (LM) RBAE from d 0 to d 56 (P = .002)
(Figure 7). An increase in the paddock group was seen from d 0 to d 28 (P = .04). The
total (LM) RBAE of P1 was greater than that of X1 on d 28 (P = .02) and on d 56 (P =
.01). Stalled weanlings tended to decrease from d 0 to d 56 (P g .1) in the total (LM)

view. On d 28, the stalled group tended to be greater than the exercised group (P = .1).

—a— Paddock (P1)
...-o--- Exercised (XI)
- -o - - Stall (81) x

1.9-i

 

Medial RBAE (mm Al)

 

 

14" | I

28 56
Day

:—

Figure 2. Medial RBAE (mm Al) versus day of project in weanling Quarter Horses
2differences between a 28 and d 56 (P _<_ .05)
I"different from d 0 (P 5 .05)
x”treatments with different superscripts at a given day differ (P _<_ .1)

34

—D— Paddock (P1)

 

 

 

 

 

 

18.5-
....o.... Exercised (X1)
18.0-
--.“ Stall (SI)
3‘?
17.5-
E
5
II: c:
5 17.0
'5 16.5-
0
‘5
t-I
16.0-1
15.5‘ I
0 28 56
Day

Figure 3. Lateral RBAE (mm Al) versus day of project in weanling Quarter Horses

16- + Paddock (Pl)

 
  

 

 

 

"“"'° Exercised (X1)
"0" Stall (51)
C? l 5- :‘:-?.:.:.~ ~
«e an“...
E ......................
5 ......
‘31 14-
: 1’
B
E
a. 13-
12- I I I
0 28 56
Day

Figure 4. Palmar RBAE (mm Al) versus day of project of weanling Quarter Horses
Tdifferent from d o (P s .1)

35

2_ —u— Paddock (P1)
----o-- Exercised (X1)

- -O - Stall (Sl)

 

 

 

 

 

2"
E
E
m
<
a:
a
Z .
c: I
<1 -2- ,,,, t
‘I b
'3‘ I l I
0 28 56
Day

Figure 5. Change in dorsal RBAE (mm Al) versus day of project in weanling Quarter Horses
1different from d 0 (P 5 .l)
2differences from d 28 to d 56 (P 5 .05)
a9l’treatments with different superscripts at a given day differ (P 5 .05)

+ Paddock (Pl)
.--o--- Exercised (X1)

 

 

 

 

 

9 100~ --O-- Stall (81) I la
<
"a l
a:
5
E:
e
3
E-
q -100- l
-150-‘ [ ‘ 1
0 28 56
Day

Figure 6. Change in Total (DP) RBAE (mm2 Al) versus day of project in weanling Quarter Horses
1differences from d 28 to d 56 (P 5 .1)
"btreatments with different superscripts at a given day differ (P 5 .05)

36

—D— Paddock (Pl)

 

 

 

 

 

100-
..-o--- Exercised (X1)
".- suu (51) l;
2 50-
"a
a: 0‘
5
g .50- .
e. 1 “““““““
3 """"" ll
6 ...... a
:- -100-4 "°b
<1 l
'150" [ I I
0 23 56

Day

Figure 7. Change in total (LM) RBAE (mm2 Al) versus day of project in weanling Quarter Horses
‘different from d 0 (P 5 .05)
ldifferent from d o (r s .1)
"btreatments with different superscripts at a given day differ (P 5 .05)

Osteocalcin
The serum DC of the paddocked group tended to decrease from 152.3 ng/ml on d
0 to 108.3 ng/ml on d 28 (P = .06) and continued to decrease to 101.5 ng/ml on d 42 (P =
.03) (Table 4). The final P1 serum 0C concentration was 98.4 ng/ml (P = .02). The
serum 0C concentration of the exercised weanlings decreased from 154.7 ng/ml on d 0 to
99.3 ng/ml on d 14 (P = .01) and continued to decrease to 91.9 ng/ml on d 28 (P = .007).
Serum DC of the exercised group tended to increase from 91.9 ng/ml on d 28 to 134.2

ng/ml on d 56 (P = .06). Stalled weanlings had greater 0C concentration than X1 on d 14

37

(P = .05) and tended to be greater than X1 on d 28 (P = .1). The serum DC of the stalled
group also tended to be greater than the paddocked weanlings on d 42 (P = .1).

Table 4. Means table for osteocalcin (ng/ml) in Quarter Horse weanlings

 

 

 

Day
Treatment 0 14 28 42 ' 56 SEM
Paddock 152.3' 123.4"my 108.3“ 101.5” 98.4" 16.4
Exercise 154.7' 99.3bx 91.9” 1 17.1“” 1342”” 16.4
Stall 147.6'I 145 .8‘y 129.4' 139.0‘ 1 13.8‘ 16.4

 

“days with different superscripts within rows differ (P 5 .05)
“treatments with different superscripts within columns differ (P 5 .05)
‘SEM = 17.9
"SEM = 18
ICTP

Due to differences in ICTP on d 0 (Table 5), ICTP data was normalized by
subtracting all values from d 0 (Table 10A). Paddocked weanlings decreased from d 0 to
d 42 (P = .05) with a continued decrease of 3 ng/rnl to d 56 (P = .05). The exercised
weanlings demonstrated a decrease in ICTP concentration of 2.8 ng/ml from d 14 to d 28
(P = .05) and increased by 3.0 ng/ml from d 28 to d 56 (P = .05). The stalled weanlings
decreased by 6.7 ng/ml from d 0 to d 28. An increase of S1 serum ICTP of 4 ng/ml was
seen from d 28 to d 42 (P = .007). On (1 28, paddocked weanlings had greater ICTP

concentration than the stalled group (P = .02).

Table 5. Means table for ICTP (ng/ml) in Quarter Horse weanlings

 

 

 

Day
Treatment 0 14 28 42 56 SEM
Paddock 15.7"" 13.1‘” 14.3‘bx 12.6” 12.6" 1.1
Exercise 16.8"l 14.9‘I 12.1'my 13.9“” 15.1" 1.1
Stall 20.0" 14.7be 13.3by 17.3‘c 15.7c 1.1

 

“days with different superscripts within rows differ (P 5 .05)
"treatments with different superscripts within columns differ (P 5.05)
'SEM = 1.2

38

Keratan Sulfate

All treatment groups demonstrated a decrease in KS concentration from d O to d
56 (Table 6). Initial KS concentrations of P1, X1, and S] were 618.6, 548.5, and 623.8
ng/ml respectively. All treatment groups decreased to d 14 (P 5 .02), d 28 (P 5 .003), and
d 42 (P 5 .03). The final concentrations were 473.2 ng/ml for P1 (P 5 .001), 476.1 ng/ml
for X1 (P = .08), and 535.9 ng/ml for S] (P = .02). On (I 14, the paddocked group
demonstrated greater KS concentration than the exercised group (P = .02). Serum KS
concentration of the stalled group tended to be greater than the exercised group on d 28

(P = .06).

Table 6. Means table for keratan sulfate (ng/ml) in Quarter Horse weanlings

 

 

 

Day
Treatment 0 14 28 42 56 SEM
Paddock 618.6' 565.2bx 488.5Cd 524.7“ 473.2c 36
Exercise 548.5‘ 440.6” 435.0” 464. 1'” 476. 1“" 36
Stall 623.8' 5140""y 533.8” 522.9b 535.9"~ 36

 

“”‘days with different superscripts within rows differ (P 5.05)
“treatments with different superscripts within columns differ (P 5 .05)
'SEM = 37.1
”SEM = 37.9
Growth Measurements

All groups demonstrated an increase from d 0 to d 56 in height at the withers (P 5
.01). Initial wither heights were 123.5 cm for P1, 125.3 cm for X1, and 123.3 cm for 81.
Final height at the withers were 128.5 cm for P1, 128.5 cm for X1, and 128.5 cm for 81.
There were no treatment differences seen in wither height (Table 11A). All groups

increased from d 0 to d 56 (P 5 .05) in hip height with no treatment differences observed.

Initial and final hip heights were 129.3 cm to 133.6 cm for P1, 131.4 cm to 135.0 cm for

39

X1, and 130.1 cm to 135.3 cm for 81 (Table 12A). All treatment groups increased from
d 0 to d 56 (P 5 .02) in weight with no treatment differences seen. Initial weights were
233 kg for P1, 232 kg for X1, and 221 kg for 81. Final weights for P1, X1, and 81 were
265 kg, 250 kg, and 257 kg respectively (Table 13A). Both paddocked weanlings and
stalled weanlings demonstrated increases in cannon circumference from d 0 to d 56
(Table 14A). The cannon circumferences increased from 15.7 cm on d 0 to 16.4 cm on d
56 (P = .0003) for P1 and 15.5 cm on d 0 to 15.9 cm on d 56 (P = .03) for S1. The
exercised group decreased from 16.0 cm on d 0 to 15.7 cm (P = .02) on d 28. The cannon
circumference of exercised weanlings then increased from d 28 to 16.2 cm on d 56 (P =

.002).

40

DISCUSSION: EXPERIMENT 1

Bone Mineral Content

As expected, the paddocked group increased in both medial and total (LM) RBAE
and demonstrated greater bone mineral content than the stalled group in medial RBAE.
The stalled weanlings demonstrated a decrease in medial, palmar, and total (LM) bone
mineral content. The greater bone mineral content of the paddocked weanlings,
demonstrated in the present study, parallel those of Hoekstra (1998) where paddocked
yearlings demonstrated greater bone mineral content than the stalled horses on d 28 and d
56 in both the lateral and medial cortices. The results are also similar to Knowles and
Broom (1990) and Fleming et al. (1994) that demonstrated that allowing an animal room
to move (i.e. hens in perchery’s or weanlings in paddocks) causes an increase in bone
mineral content and, hence, in bone strength. The increase in space to move allows for
free access to exercise. McCarthy and Jeffcott (1992) found that eXercised yearlings
demonstrated greater bone mineral content than those horses not receiving exercise. The
lack of exercise received by the stalled group would account for the decreased bone
mineral content.

Along with the present study, the influence of housing on behavior was examined
using the stalled and paddocked treatment groups (Heleski et al., 1999). It was found that
stalled weanlings spent more time lying down than paddocked weanlings (P < .001). The
stalled animals also spent less time standing, interacting with other horses, and moving

around than the paddocked weanlings. The increase in lying down and lack of movement

41

from the stalled horses would account for the decreases in bone mineral content observed
in the present study.

The lack of changes seen in the palmar and lateral cortex of the paddocked group
and in the lateral cortex of the stalled group is dependent on where the most compressive
loads were experienced. Where compression occurs may relate to the conformation of
the animal, the ground surface, the architecture of the bone, and other factors. During
“normal everyday activities”, the dorsomedial quadrant of bone receives the most
concussion (Rooney, 1978). Because the dorsolateral quadrant does not receive as much
concussion as the dorsomedial quadrant the risk of injury to that area increases (Rooney,
1978). As cortical bone thickens in response to training, the medial, lateral, and dorsal
cortices change while the palmar cortex remains unchanged (Nielsen et al., 1997). This
results in the dorsal, medial, and lateral cortices increasing in strength and size while the
palmar does not. This lack of change in the palmar cortex may be due to the lack of
compression the palmar cortex receives (Pratt, 1982). The palmar cortex has been shown
to primarily undergo tension and not compression (Pratt, 1982).

Exercised weanlings demonstrated an initial decrease in dorsal RBAE followed
by an increase. The exercised group also decreased in both total (DP) and (LM) bone
mineral content. The paddocked and stalled weanlings demonstrated greater dorsal and
total (LM) bone mineral content then the exercised weanlings. The sharp increase in the
dorsal view, of the exercised group, corresponds with the onset of longe training, which
began on d 28. Raub et al. (1989) found that weanlings worked on the mechanical walker
at the trot 5 d/wk demonstrated an increase in medial bone mineral content with no

change in the lateral cortex. The differences of the cortices was attributed to the media]

42

cortex having greater bending properties than the lateral cortex. Though an increase was
seen in dorsal RBAE, the increase of bone mineral content was not great enough to detect
an increase in the total bone mineral content. The initial decrease observed in dorsal
RBAE, of the exercised group, may be due to the lack of exercise the weanlings were
receiving for the first 28 d of the study.

Paddocked weanlings may have demonstrated greater bone mineral content than
the exercised weanlings because the longeing procedure and the work on the mechanical
walker did not supply enough concussion on the bone to stimulate an increase greater
than that seen in the paddocked group. There seems to be no explanation for the greater
bone mineral content of the stalled horses, in dorsal and total (LM) RBAE, when
compared to the exercised weanlings.

When looking at the weaning groups separately, we find that in weaning group 1
(data not reported), the stalled weanlings never demonstrate greater bone mineral content
than the exercised weanlings. In fact, the stalled weanlings decreaSe in total (LM),
palmar, and medial RBAE. The stalled weanlings tended to increase in lateral RBAE
from d 28 to d 56. This increase in the stalled weanlings does not correspond with any
increase in activity as seen by Heleski et al. (1999) and does not seem to have an
explanation. The exercised weanlings in weaning group 1, tended to decrease in total
(DP) RBAE from d 28 to d 56 and increase in medial RBAE from d 28 to d 56. The
exercised group also decreased from d 0 to d 28 in medial RBAE. The decrease
demonstrated in the exercised group in medial RBAE may be due to being stalled from d
0 to d 28. The increase in medial RBAE is likely due to the longeing procedure

counteracting the affects of stalling this group for the first month of the study. Because

43

the bone received little concussion during the first month of the study, due to stalling, one
would expect to see a decrease or no change in the bone mineral content (Hoekstra et al.,
1998; Fleming et al., 1994; Knowles and Broom, 1990). Once longe training began
however, an increase in bone mineral content is expected because of the increase in
concussion the bone would then be receiving (Hoekstra et al., 1998; McCarthy and
Jeffcott, 1992). In the exercised group, the bone went from little concussion on d 0 to d
28 to loads associated with cantering and trotting on the longe line from d 28 to d 56. In
both lateral and total (DP) RBAE, exercised weanlings demonstrated greater bone
mineral content than the stalled weanlings on d 28. This response of the exercised
weanlings corresponds with the start of longe training following the 1 wk of walking on
the mechanical walker. Though walking does not result in much change in bone mineral
content (strain is related to speed which is lacking in the walk), as weanlings are
introduced to a mechanical walker they usually jump, rear, or bolt forward to escape the
pressure on their halters or because they are startled. These actions may cause enough
strain on the bone to induce increases in bone mineral content, which would result in
greater bone mineral content than the stalled weanlings.

The total (LM) or (DP), lateral, and medial RBAE in the paddocked group in the
first weaning group is always greater than the stalled or the exercised weanlings. An
increase in medial RBAE from d 28 to d 56 was seen in the paddocked group. This
correlates with other studies indicating that confinement rearing is detrimental to bone
strength (Hoekstra et al., 1997; Knowles and Broom, 1990; Fleming, 1994). There were
no differences seen in the normalized data of dorsal RBAE. This lack of change may

have been due to the lack of compression the dorsal cortex was receiving. Though the

everyday activity causes compression on the dorsomedial quadrant of the bone, there may
not have been enough concussion surpassing this “normal activity” to elicit an increase in
the dorsal cortex of the paddocked weanlings. In racing horses, it is necessary to have
compression of the dorsal cortex in order to increase bone Strength of the dorsal cortex.
The dorsal cortex is the area that is injured the most due to increases in strain
(Nunamaker et al., 1990).

In the second weaning group, the stalled weanlings increased from d 0 to d 56 in
both media] and lateral RBAE. The exercised group increased in lateral RBAE from d 0
to d 56 and tended to be greater than stalled weanlings on d 28 in medial RBAE. The
paddocked group decreased from d 28 to d 56 in total (LM) RBAE but increased from d
28 to d 56 in medial RBAE. The increase demonstrated by the stalled weanlings does not
have an explanation. The increase seen in the exercised group is likely due to the
introduction to the mechanical walker and the longeing procedure. The decrease seen in
the paddocked group may be due to the weanlings lack of activity in the paddock
(Heleski et al., 1999).

Since the exercised group was treated so differently between weaning groups, this
may explain why the data of the exercised group demonstrate both increases and
decreases in RBAE when both weaning groups are combined. No blocking effect was
seen because only six horses (three stalled and three exercised from the second weaning
group) out of the 18 weanlings used in the study were treated differently. However, the
weaning groups were quite different and yielded different responses in all groups
although significant power in the design was not present to detect a difference by

weaning groups. Due to the relative inactivity of the exercise group from d 0 to d 28, one

45

would expect the bone mineral content to either decrease or remain unchanged. But as

the longeing procedure began on d 28 an increase in bone mineral content was expected.

Serum Analysis
Osteocalcin

Paddocked weanlings decreased in serum OC concentration. The stalled
weanlings remain unchanged throughout the study and resulted in the stalled weanlings
demonstrating greater OC concentration than both the exercised and paddocked
weanlings. Osteocalcin decreases with age in growing horses (Hope et al., 1993; Lepage
et al., 1991; Lepage et al., 1990). This decrease can be seen in the paddocked weanlings.
The exercised weanlings demonstrated an initial decrease followed by an increase in
serum OC. This increase may represent the increase in bone formation or bone tum-over
once the longeing procedure began. Kannus et al. (1996) found similar results as in the
present study. Kannus et al. (1996) immobilized the hind legs of rats for 3 wk, similar to
the inactivity of the exercised group, and found a decrease in OC concentration. After 8
wk of remobilization and forced exercise, similar to the exercised group working on the
longe line, the OC concentration increased to baseline. Though immobilization is more
severe than the lack of activity the exercised group underwent, both the present study and
Kannus et al. (1996) demonstrated similar results indicating that decreased activity
followed by an increase in activity may result in an increase in OC.

Osteocalcin has been shown to fluctuate with time of day. Lepage et al. (1991)

found that OC serum concentration levels remained constant from 0700 to 1900. The

46

serum analyzed in the present study was collected between 0700 and 1900. Hence, this

should not have contributed greatly to any variation.

ICTP

All treatment groups had decreases in serum ICTP concentration from d O to d 42
or d 56. This corresponds with numerous studies indicating that serum ICTP
concentration decreases with age in equine (Price et al., 1995a; 1995b), canine (Allen et
al., 1998), and humans (Crofton et al., 1997; Zanze et al., 1997). Price et al. (1995a)
reported ICT P serum concentrations of 15 female Thoroughbred horses less than 1 yr to
be 13.7 to 26.7 llg/l. In the present study our ICTP serum concentrations on d 0 ranged
from 15.7 i 1.0 ng/ml to 20.0 i 1.0 ng/ml, thus falling within the range Price at al.
(1995a) reported. Price et al. (1995a) reported the ICTP serum concentrations to
decrease to 7.9 to 22.8 jig/l by 1 to 2 yr of age. The final concentrations in the present
study ranged from 12.6 :1: 1.0-ng/ml to 15.7 i 1.0 ng/ml, also falling Within the range of
Price et al. (1995a). The stalled weanlings increase in serum ICTP concentration from d
28 to d 42 is likely due to the lack of exercise received by this group. The increase in the
exercised group from d 28 to d 56 may be due to the increase in bone tum-over from
being longed returning serum ICTP concentrations to baseline. The increase of ICTP and
OC serum concentrations represents an increase in bone tum-over due to remodeling and
modeling. There is no explanation as to why differences existed on d 0, randomization

should have accounted for this difference.

47

W

All treatment groups decreased in KS serum concentration from d 0 to d 56. This
decrease parallels other studies that demonstrate decreases in KS concentration with
increasing age (Todhunter et al., 1997; Okumura et al., 1997; Roughley and Lee 1994;
Spector et al., 1992; Nixon, 1991; Sweet et al., 1990; Leipold et al., 1989). Okumura et
al. (1997) reported KS concentrations in 15 foals to be approximately 4000 ng/ml at 3 mo
of age and decreasing to 500 ng/ml at 6 mo of age. Todhunter et a1. (1997) reported KS
values in ten 3-mo-old foals to be approximately 300 ng/ml. The differences between
these two studies may be due to Okumura et al. (1997) analyzing serum while Todhunter
et a1. (1997) analyzed plasma levels. The present study reports KS concentration values
to range from 435.0 3; 37.5 ng/ml to 623.8 i 37.5 ng/ml, in the range of Okumura et al.
(1997). Okumura et al. (1997) found KS serum concentrations to decrease rapidly after 3
mo of age. To have an average concentration at 4 mo of age of 597.0 :1; 37.5 ng/ml would
seem to agree with Okumura et al. (1997) study. The paddocked grOup demonstrated
greater KS concentrations than the exercised weanlings on d 14 while the stalled
weanlings demonstrated greater KS concentrations than the exercised weanlings on d 28.
These differences may be due to the paddocked and stalled groups having greater (though
not significant) starting values. The paddock group demonstrated a smaller decrease to d
14 than the exercised group resulting in the paddocked group having greater serum KS
concentration. Similarly, the stalled group demonstrated a smaller decrease to d 28 than

the exercised group resulting in the stalled group having greater serum KS concentration.

48

Growth Measurements

There were no treatment differences in height at the withers, hip, or weight of the
weanling horses. All groups increased which follows the normal pattern of growth. The
third metacarpus circumference increased from d 0 to d 56in the paddocked and stalled
groups. The exercised group demonstrated an initial decrease from d 0 to d 28 and then
an increase from d 28 to d 56. The increases in the paddocked and stalled groups may be
due to normal growth resulting in larger bones. The decrease initially observed in the
exercised weanlings may be due to experimental error. One would expect to see either an
increase related to normal growth or no change in circumference at all due to lack of
activity. The increase seen from d 28 to d 56 is related to grth of the animal and the
onset of longe training which may have caused thickening of the bone and of the tendons
and ligaments (Raub et al., 1989). An increase in all growth measurements was expected
due to normal growth of the weanlings. The initial decrease of the cannon bone in the

exercised group was unexpected but was likely due to experimental errOr.

49

CHAPTER 3

MATERIALS AND METHODS: EXPERIMENT 2

Animal Management

Eighteen Arabian weanlings from the Michigan State University Horse Teaching
and Research Center were pair-matched by age and randomly assigned to three treatment
groups of six horses each. Horses remained on the study for 56 d. Before the start of the
study, weanlings were kept outside with their dams. Horses were weaned in three
separate groups of six horses each with two horses in each treatment group. The first
weaning group began the study on August 25, 1998, the second group on September 21,
1998, and the third group on October 19, 1998. The pasture group (P2, n = 6) was
maintained on a 432.8 m x 53.6 m pasture allowing free access to exercise. The stall
group (S2, n = 5) was housed in 3.1 m x 3.1 m stalls with no free access to exercise. The
partial pasture group (PP2, n: 6) was maintained with the pasture group allowing free
access to exercise for 12 h and housed in 3.1 m x 3.1 m stalls for 12 h. One horse in the S
group died and resulted in a smaller treatment group. The average ages of each group,
followed by the range of ages in parentheses, were P2 - 136 d (119 d to 151 d), S2 — 134
d (127 d to 143 d), and PP2 — 135 d (128 d to 149 (1). Each group was individually fed .9
kg com, .85 kg oats, and .05 kg 40% dairy protein pellets (Kent Feeds Inc., Muscatine,
IA) twice daily (0700 and 1900). Stalled horses had ad libitum access to alfalfa and grass
mixed hay (Tables 7 and 8). Horses on pasture had ad libitum access to a mixed grass
pasture (brome, orchard, timothy and Kentucky blue grasses). Water and mineral blocks

were available at all times.

50

Table 7. Calculated daily nutrient table for 82 weanlings on a dry matter basis

 

 

Nutrient Calculated
Nutrient Content Total Daily Intake
DE 3.06 Meal/kg 14.7 Mcal
CF 14.0% 670.0 g
Ca 0.27% 12.8 g
P 0.33% 15.9 g

 

Table 8. Calculated daily nutrient table for P2 weanlings on a dry matter basis

 

 

Nutrient Calculated
Nutrient Content Total Daily Intake
DE 3.0 Meal/kg 14.3 Mcal
CP 11.9% 570.0 g
Ca 0.16% 7.8 g
P 0.33% 16.0 g

 

Table 9. Calculated daily nutrient table for PP2 weanlings on a dry matter basis

 

 

Nutrient Calculated
Nutrient Content Total Daily Intake
DE 3.02 Mcal/kg 14.5 Meal
CP 12.9% 620.0 g
Ca 0.21% 10.3 g
P 0.33% - 15.9 g

 

Bone Mineral Content
Bone mineral content was measured as reported in Experiment 1 with the
exception of no lateral-medial radiographs were taken due to an insufficient number of

available radiographic cassettes.

Serum Collection
Serum was collected as reported in Experiment 1. The times of sample collection

for d 0, 14, 28, 42, and 56 for the first weaning group were 1700, 0730, 1430, 1300, and

51

1300 respectively. Sample collection for the second and third weaning groups were

between 1300 and 1400.

Serum Analysis

Serum was analyzed for OC. ICT P and KS as reported in Experiment 1.

Growth Measurement
Cannon circumference, hip height, wither height and weight were taken every 28

d as described in Experiment 1.

Statistics
Data was analyzed as reported in Experiment 1. The blocking effect of weaning
in three groups was included in our model. This was added to account for any

differences that may have existed due to weaning time.

52

RESULTS: EXPERIMENT 2

Bone Mineral Content

The pastured group tended to increase from 17.4 i .4 mm Al on d 0 to 18.4 i .4
mm Al on d 28 in medial RBAE (P = .06) (Figure 8). The partial-pastured group
demonstrated an increase in medial RBAE from 17.2 :t .4 mm Al on d 0 to 18.5 i .4 mm
Al on d 56 (P = .02). In medial RBAE, the stalled group was lower than P2 (P = .003)
and PP2 (P = .05) on d 28. On d 56, S2 was lower than PP2 in medial RBAE (P = .01).

The lateral RBAE of pastured weanlings increased from 15.7 :1: .4 mm Al on d 0
to 16.8 i .4 mm Al on d 28 (P = .05) and continued to increase to 17.8 i .4 mm Al on d
56 (P = .001) (Figure 9). The lateral RBAE of the partial-pastured group was lower than
the pastured group on d 56 (P = .02). Partial-pastured weanlings tended to have greater
lateral RBAE than the stalled weanlings on d 28 (P = .08). Stalled weanlings had a lower
lateral RBAE than pastured weanlings on d 28 (P = .005) and d 56 (P =‘ .007).

In total (DP) RBAE (Figure 10), pastured weanlings increased from 150 i 20
mm2 Al on d 0 to 208 i 20 mm2 Al on d 56 (P = .05) and tended to be greater than the
stalled weanlings on d 28 (P = .08) (Figure 10). The partial-pastured weanlings tended to
increase in total (DP) RBAE from 145 i 20 mm2 Al on d 0 to 196 j: 20 mm2 Al on d 56
(P = .08) and was greater than the stalled weanlings on d 28 (P = .05). The stalled group
increased from 136 j; 22 mm2 Al on d 0 to 204 j; 22 mm2 Al on d 56 in total (DP) RBAE

(P = .04).

53

19.0‘ -D— Pasture (P2)
--o---- Partial Pasture (PP2)

18.5- - -O- - Stall (82) 1' ,-

18.0-

17.5-

Medial RBAE (mm Al)

 

 

 

Day

Figure 8. Medial RBAE (mm Al) versus day of project in weanling Arabians
*different from a o (P 5 .05)
Idifferent from d 0 (P 5 .1)
“’btreatments with different superscripts at a given day differ (P 5 .05)

19_ —fl-'— Pasture (P2)
---o---- Partial Pasture (PP2)

I I I * t
1 8-1 '. Stall (82) T

17-

16‘

Lateral RBAE (mm Al)

15-1

 

 

 

14-

G-
N
on
U!
a

Da
Figure 9. Lateral RBAE (mm Al) versusyday of project of weanling Arabians
.dlfferent from d o (r 5 .05)
.Idlfferent from d 0 (P 5 .01)
a’I’treatmeuts with different superscripts at a given day differ (P 5 .05)

54

-D- Pasture (P2)

 

 

 

2504 "-0"- Partial Pasture (PP2)
--O-- Stall (S2)

2
N
E
5 200.
In)
5
E:
e
_ 150-
a
‘5
I-I

100- fee I I

0 28 56
Day

Figure 10. Total (DP) RBAE (mm2 Al) versus day of project in weanling Arabians
1"different from d 0 (P 5 .05)
Idifferent from d 0 (P 5 .1)

“’btreaments with different superscripts at a given day differ (P s 45)

Osteocalcin

Due to differences in serum QC on d 0 (Table 10), the data were normalized
(Table 18A) by subtracting all days from d 0. Pastured weanlings decreased by 46.6
ng/ml from d 0 to d 42 (P = .006) and continued to decrease to d 56 (P = .06). Partial-
pastured weanlings also demonstrated a decrease of 66.0 ng/ml from d 0 to d 56 (P = .01).
The stalled group tended to decrease by 34.7 ng/ml from d 0 to d 56 (P = .06). On d 42,
stalled weanlings had a greater serum OC concentration than partial-pastured weanlings

(P = .05).

55

Table 10. Means table for osteocalcin (ng/ml) in Arabian weanlings

 

 

 

Day
Treatment 0 14 28 42 56 SEM
Pasture 120.6“ 1 14.7‘c 108.6ac 74.1'“ 88.5ab‘ 11.6
Partial Pasture 154.5" 113.7b 106.0” 894"" 112.7b 11.6
Stall 144.5‘” 124.1‘ 132.5‘ 126.4"y 109.48 12.8

 

abcdays with different superscripts within rows differ (P 5 .05)
“treatments with different superscripts within columns differ (P 5 .05)

ICTP

All treatment groups decreased in serum ICT P from d 0 to d 56 with no treatment
differences shown (Table 11). The pastured group demonstrated a decrease in serum
ICT P from 20 ng/ml on d 0 to 17 ng/ml on d 14 (P = .006) remained low until (1 28 (P =
.04) and experienced a further decrease to 14 ng/ml on d 42. On d 56, the ICTP
concentration of the pastured group was 16 ng/rnl (P = .003). Partial-pastured weanlings
had an initial ICTP concentration of 19 ng/ml and decreased to 16 ng/ml on d 14 (P =
.006). Concentrations continued to decrease to 16 ng/ml on d 42 with final ICT P
concentrations of 14 ng/ml (P = .003). The stalled weanlings demonstrated a similar
decrease in ICTP concentrations from 19 ng/ml on d 0 to 14 ng/ml on d 42 (P = .0001)

and 15 ng/ml on d 56 (P = .002).

Table 11. Means table for ICTP (ng/ml) in Arabian weanlings

 

 

 

Day
Treatment 0 14 28 42 56 SEM
Pasture 19.9‘ 17.0" 17.5b 14.4° 16.1'Dc 0.9
Partial Pasture 19.1' 16.4" 16.0” 16.3” 14.8” 0.8
Stall 19.0‘ 17.1"‘I 17.9'” 14.4c 15.4Cd 0.8

 

“days with different superscripts within rows differ (P 5 .05)

56

Keratan Sulfate
There were no treatment differences in KS concentration (Table 12). The serum
KS concentration of the pastured group decreased from 537 ng/ml on d 0 to 465 ng/ml on
d 28 (P = .04), with a continued decrease to 460 ng/ml' on d 42 (P = .05). Stalled
weanlings demonstrated a decrease from 497 ng/ml on d 14 to 434 ng/ml on d 56 (P =

.05). There was no change in the serum concentration of the partial-pastured weanlings.

Table 12. Means table for keratan sulfate (ng/ml) in Arabian weanlings

 

 

 

Day
Treatment 0 14 28 42 56 SEM
Pasture 536.6‘ 537.2‘ 465.2” 460.4” 491. 1‘” 25.9
Partial Pasture 495.6‘ 507.8‘l 492.9‘ 501.5‘ 474.8‘ 23.6
Stall 486.0'” 497.4' 490. 1‘” 460.2‘” 434.2” 23.6

 

“days with different superscripts within rows differ (P 5 .05)

Growth Measurements

Initial height at the withers for P2, PP2 and S2 were 119.9 1 1.2 cm, 119.7 :1; 1.2
cm and 119.2 :1: 1.4 cm respectively. All groups increased from d 0 to d 56 (P 5 .01) with
no differences between groups. Final wither heights were 125.1 1; 1.3 cm for P2, 124.9 i
1.2 cm for PP2, and 125.0 j: 1.4 cm for S2 (Table 19A). On d 0, pastured weanlings
(127.0 1; 1.1 cm) tended to be taller at the hip than stalled weanlings (123.8 i 1.2 cm ) (P
= .06). The partial-pastured group had an initial hip height of 124.7 i 1.1 cm. All groups
increased from d 0 to d 56 (P 5 .001). Final heights at the hip were 131.8 i 1.2 cm for
P2, 132.0 1 1.1 cm for PP2 and 129.5 3; 1.2 for S2 (Table 20A). All treatments increased
in weight from d 0 to d 56 (P 5.01) and demonstrated no differences between groups.
Initial weights were 187 j; 7 kg for P2, 195 -_1-_ 7 kg for PP2, and 188 j; 8 kg for S2. Final

weights for P2, PP2, and S2 were 235 i 8 kg, 228 j; 7 kg, and 238 :1: 8 kg respectively

57

(Table 21A). Partial-pastured weanlings were greater than stalled weanlings in cannon
circumference (P = .03). Pastured weanlings tended to have greater cannon
circumference than stalled weanlings (P 5 .07). In cannon circumference, both P2 and
PP2 increased from d 0 to d 56 (P = .0001) with no change in 82. The initial and final
circumference of P2 were 15.2 i- .2 cm on d 0 to 16.6 i .2 cm on d 56. The cannon
circumference for PP2 on d 0 was 15.5 :1; .2 cm and 16.5 :1: .2 cm on d 56. The stalled
group had an initial circumference of 15.3 i: .2 cm with a final circumference of 15 .6 i .2
cm. Both P2 and PP2 had larger cannon circumferences than S2 on d 28 (P 5 .05) and d

56 (P = .004) (Table 22A).

58

DISCUSSION: EXPERIMENT 2

Bone Mineral Content

In the present study, the pastured group increased in bone mineral content in both
lateral and total (DP) RBAE and tended to increase in medial RBAE. The stalled group
remained lower than the pastured group in the medial and lateral views and tended to be
lower in total (DP) RBAE. These data correspond with a study looking at the influence
of housing in yearling horses (Hoekstra et al., 1998). Hoekstra et al. (1998) normalized
the lateral and medial RBAE data due to differences on d 0 but found that the, pastured
horses had greater bone mineral content than the stalled horses on d 28 and d 56 in both
cortices. Hoekstra et al. (1998) found no change in total RBAE.

A partial pasture group was used to begin to elicit how much time on pasture was
necessary to prevent the disuse osteoporosis seen by Hoekstra et a1. (1998). The present
study indicates that the partial-pasture horses increased in medial and total RBAE. The
stalled group had lower medial RBAE and total RBAE than the partial-pastured group
and tended to be lower in lateral RBAE. This demonstrates the benefits of 12-h daily
tum-out on bone strength. The results seen in the partial-pasture group are similar to the
pastured group.

The data in the present study concur with studies indicating that allowing an
animal room to move (i.e. placing an animal in a larger cage or area) causes an increase
in bone strength. Knowles and Broom (1990) found that restricting the amount of
movement in. laying hens was detrimental to bone breaking strength. When the hens

were housed in percherys versus battery cages or the Elson terrace system, more

59

movement was allowed and hence those birds housed in percherys had stronger bones. In
fact, birds housed in battery cages demonstrated 54% less strength than their counterparts
in percherys. A similar study by Fleming et al. (1994) found that laying hens housed in
percherys or Naturel housing systems allowed more wing movement, which in turn
caused an increase in bone strength.

The increases in the pastured and partial-pastured group and the differences found
between both groups and the stalled group in this study are supported by numerous
studies looking at the effects of exercise or immobilization on the equine third
metacarpus. McCarthy and Jeffcott (1992) found an increase in bone mineral content of
exercised Thoroughbred yearlings. By wk 12, exercised horses had greater bone mineral
content than the non-exercised horses. This increase in the exercised horses was
determined to be due to the horse exercising to its maximum potential (galloped for up to
1.2 km or until exhaustion). Both groups in the McCarthy and Jeffcott (1992) study were
relatively inactive for 2 mo before the start of the study. This was suggested to be
enough time for the horses to reach a steady state of bone remodeling so all horses started
the study at the same baseline. In the present study, when the partial-pastured horses
were brought out to the pasture every morning, it was very rare that they did not sprint off
to join the other weanlings and once all the horses were together they would often run
around chasing one another. This was probably enough loading on the bone to initiate
increases in bone mineral content. This parallels Jeffcott’s (1991) suggestion that
growing horses should be pastured for 12 h/d to develop normal bones and joints. Pratt
(1982) states that the most efficient way to increase bone strength is to run short vigorous

sprints, hence it is likely that even less than 12-h of tum-out per day may be adequate.

60

The stalled group became very active in their stalls after (1 28 of the study. Their
activity included jumping, bucking, and rearing as well as running around in small
circles. This increase in activity may explain the increase of the stalled group from d 0 to
d 56 in total (DP) RBAE. This activity may have provided the third metacarpus enough
strain to increase the mineral content. The increase in bone mineral content may not have
been great enough to see in the lateral and medial views separately, but combined were
great enough to detect in the total (DP) RBAE. In the medial and lateral cortices, the
stalled group demonstrated no changes with time though remained lower than the pasture
group.

The only concern as to whether 12-h of tum-out is as good as continuous tum-out
was that partial-pastured weanlings demonstrated lower lateral RBAE than pastured
weanlings on d 56. The partial-pastured weanlings demonstrated no increase in lateral
RBAE which may account for the difference with pastured weanlings in lateral RBAE on
d 56. No increase may have been seen based on the amount of compression the lateral
cortex received. Each cortex will receive differing amounts of compression depending
on how the animal moves and where most of the force is placed, as previously discussed
in Experiment 1. The increases seen in total RBAE are consistent with other studies
indicating an increase in RBAE in growing horses (Nielsen et al., 1997; Frey et al., 1992;
Meakim et al., 1981). Nielsen et al. (1997) found an initial decrease in lateral and medial
RBAE in 18 mo old horses entering race training. This decrease may be due to only
working the animals at slower gaits in order to get them broke to ride. But, after speed
was placed on theses horses, lateral and medial RBAE increased. This is consistent with

the present study indicating that the lateral and medial cortices were greater in the

61

 

 

pastured or partial-pastured weanlings versus the stalled weanlings. This was probably
due to the applied forces on the animals legs when out in the pasture running around and

playing with each other.

Serum Analysis
st i

All treatment groups decreased in OC serum concentration. But, on d 42 the
stalled weanlings demonstrated a greater OC concentration than the partial-pastured
weanlings. The greater concentration may be explained by the increase in activity of the
stalled weanlings after d 28. The decrease with age seen in this study corresponds with
other studies demonstrating the OC serum concentrations decrease with age in equine
(Hope et al., 1993; Lepage et al., 1991; Lepage et al., 1990). Lepage et al. (1990)
reported the OC serum concentration of eight horses, under the age of 1 yr old (4 mo to 1
yr), to be 47.3 :1; 10.1 ng/ml and found that it decreased in nine horses, between 1.5 and
2.5 yr of age, to 35.7 :1: 14.2 ng/ml. These values are clearly lower than the values
reported in the present study of 74.1 3; 11.2 ng/ml to 154.5 i 11.2 ng/ml. The lower
concentrations reported in the present study were from horses ranging in age from 119 d
to 151 d (4 mo to 5 mo) and ended with them being 179 d to 211 d old (6 mo to 7 mo).
This range of ages is smaller than the range in the Lepage et al. (1990) study. This
decrease in bone formation corresponds with the decrease in modeling and remodeling
with increasing age (Fraher, 1993; Maenpaa et al., 1988). Osteocalcin has been shown

to fluctuate with time of day. Our samples were collected around 1400 on all sample

62

 

days (with exception to the first two days of the first weaning group). Lepage et al.

(1991) found that OC serum concentration levels remained constant from 0700 to 1900.

1212

All treatment groups decreased in ICT P serum concentration. This corresponds
with numerous studies indicating that ICT P concentration decreases with age in equine
(Price et al., 1995a, 1995b), canine (Allen et al., 1998), and humans (Crofton et al., 1997;
Zanze et al., 1997). Price et al. (1995a) reported ICI'P serum concentrations of 15 female
Thoroughbred horses less than 1 yr to be 13.7 to 26.7 ug/l. In the present study our ICT P
serum concentrations of 14.4 ng/ml to 19.9 ng/ml fall within the range Price at al. (1995a)
reported. Price et al. (1995a) reported the ICTP serum concentrations decreased between

7.9 to 22.8 jig/l in 15 horses ranging in age from 1 to 2 yr of age.

W

Stalled and pastured weanlings decreased in KS serum concentration. This
decrease parallels other studies that demonstrate decreases in KS concentration with
increasing age (Todhunter et al., 1997; Okumura et al., 1997 ; Roughley and Lee, 1994;
Spector et al., 1992; Nixon, 1991; Sweet et al., 1990; Leipold et al., 1989). Okumura et
al. (1997) reported KS concentrations, in 15 foals, to be approximately 4000 ng/ml at 3
mo of age and decreasing to 500 ng/ml at 6 mo of age. The present study reports KS
concentration values to range from 434.2 :1: 24.4 ng/ml to 537.2 :1: 27.0 ng/ml, in the range
of Okumura et al. (1997). Okumura et al. (1997) found KS serum concentrations to

decrease rapidly after 3 mo of age. The average concentration at 4 mo of age of 506.1 :

63

 

25, in the present study, is in the correct range when compared to the Okumura et a1.
(1997) study. The failure of the partial-pastured group to decrease with age seems to

have no explanation .

Growth Measurements

Height at the withers, hip, and weight increased in all treatment groups from d 0
to d 56. There was an overall treatment difference between the pastured weanlings and
the stalled weanlings on d 0 in hip height. For cannon bone circumference, the pastured
and partial-pastured weanlings increased from d 0 to d 56 with no change in the stalled
weanlings. The pastured and partial-pastured horses also demonstrated larger cannon
circumferences than the stalled horses on d 28. Sherman et al. (1995) measured the
breaking strength and cortical cross-sectional area of the third metacarpal bone in 24
Thoroughbreds (2 to 4 yr of age) with various training backgrounds. Horses with the
more extensive training background had greater peripheral thickening and greater area
moment of inertia. By looking at the correlation between area moment of inertia and
failure load, Sherman et al. (1995) found that this increase in size corresponds with an
increase in strength. Woo et al. (1981) agrees with Sherman et al. (1995) that the
increase in femoral cross-sectional area of the exercised swine compared to the non-
exercised group is directly related to bone strength. The increase of the pastured and
partial-pastured weanlings in the present study may correspond with the increase in bone
mineral content seen in the radiographs and hence is associated with an increase in bone

strength (Fleming et al., 1994; El Shorafa et al., 1979).

There were nutritional differences in the present study where the stalled
weanlings had access to hay while the pastured weanlings had access to the pasture
grasses and the partial pastured weanlings had access to both hay and pasture grasses.
The nutrition tables indicated inverse Ca to P ratio but ”no detrimental effects of this
inverted ratio were observed. It is not believed that nutrition had a part in the results
observed. Hoekstra (1998) performed a similar study to the present study, but the study
took place during the winter months while Experiment 2 took place from late summer to
early winter. During the winter months, there was no grass in the pasture so all horses
had ad libitum access to alfalfa-grass hay and were fed the same amount of concentrate
(Hoekstra, 1998). The results observed in Hoekstra’s (1998) study were very similar to

those in the present study.

65

 

CHAPTER 4

DISCUSSION: EXPERIMENTS l & 2

There were no breed differences demonstrated in the present study. Numerous
studies have identified the effects of exercise or lack of exercise on bone mineral content
or bone strength in different breeds of horses (McCarthy and Jeffcott, 1992; Hoekstra et
al., 1998; Nielsen et al., 1998). Though the methods of exercise differed in these studies
the results remained the same -- increase in availability to exercise through pasturing
causes an increase in bone mineral content. In the present study, the paddocked group of
Experiment 1 and the pastured group of Experiment 2 both demonstrated increases in
bone mineral content. The exercised group and the partial-pastured group are not able to
be compared because of the vast number of differences between the two.

Though not scientifically proven, the increase in total RBAE of the stalled group
of Experiment 2 is related to” the amount of activity of the stalled weanlings. Though
these weanlings were not exercised, they were quite active in their stalls. In Experiment
1, stalled weanlings had no significant increase in bone mineral content. Differences in
housing conditions may explain why S2 were more active than S1. In Experiment 1, the
stalls had walls that allowed no interaction with other horses. The walls were from the
floor to the ceiling with only a small window looking outside and a small window
allowing the horses to interact vocally across the barn aisle. This design does not allow
for much interaction while the stalls of Experiment 2 were open on three sides allowing
both vocal and physical interactions. The barn of Experiment 2 was a very busy barn

with a lot of activity. Numerous classes were held in the barn and during feeding times

66

 

 

students would groom the weanlings. All of the activity in the barn caused the weanlings
to become excited and run or jump around. The barn in Experiment 1 was not as active
besides the running of the broom vacuum during the mornings and cleaning of the stalls.
Yeh et al. (1993) found that rats whose legs were immobilized demonstrated a
decrease in bone formation for the first 31 d of the study and then stabilized until the end
of the study on d 41. This stabilization may have occurred from the rats adapting to the
decrease in load. In contrast, the exercised rats demonstrated an increase in bone
formation of the cortical bone. Yeh et a1. (1993) related his results to be in agreement
with Frost’s (1987) mechanostat theory (Figure 11). This theory is based on the
mechanical usage of the bone determining its bone mass. Frost explains the mechanostat
as acting like a home thermostat that activates in response to an error and turns “off” in
absence of an error. The mechanostat can be growth, modeling, or remodeling. As these
properties increase or decrease the mass of the bone is effected which then loops around

and affects how the bone adjusts in growth, modeling, and remodeling.

Mechanical Usage -> bone amechanostat —> bone mass effect

l

Figure 11. The Mechanostat Theory (Frost, 1987)
The present study corresponds with the mechanostat theory in that horses
maintained on pasture or partial—pasture demonstrated increases in bone mineral content

and hence bone strength given the correlation between bone breaking strength and the

amount of structural bone present (Fleming et al., 1994). E1 Shorafa et a1. (1979) found

67

 

_,’-‘1 '~ 'D

a positive correlation between bone breaking strength and ash content of the third
metacarpal (r2 = .58; P< .01). The mechanical usage of the stalled weanlings has
changed which caused a decrease in bone formation which decreased bone mass.
Hoekstra (1998) hypothesized that once the bone has maintained a bone mass that has
adapted to the optimal strain placed on it, the bone will stop increasing in bone mass and
reach a steady state.

The bone and cartilage serum markers indicate changes of the entire skeleton
which may have masked any specific changes that were occurring in the left third
metacarpal bone and its joints. Any treatment differences that were seen may have been
due to changes occurring in other parts of the body and not necessarily changes in the
third metacarpus as a response to housing. During the growth phase of young horses,
modeling and remodeling occur at a very high rate allowing for appositional growth and
strengthening of long bones with the rate of formation exceeding the rate of resorption
(Maenpaa et al., 1988; Fraher, 1993). This increased bone turn-over is occurring
throughout the skeleton and is not known how it is affected by environmental changes
such as stalling. Nunamaker et al. (1990) believes that any alterations to this growth
phase may have a large impact on bone strength later in life. Cartilage turnover is also
occurring at a faster rate in young animals than in older animals (Leipold et al., 1989). In
8 to 10 mo old dogs predisposed to OA (dogs from a genetic line with a high incidence of
hip dysplasia), no change in KS or HA serum concentrations were seen (Leipold et al.,
1989). That study concluded that KS and HA were poor markers of indicating
predisposition to CA but it may have been due to the age of the dogs masking any

changes that may have been occurring. The serum markers were found to be

68

 

 

inconclusive but mirror the results of a previous study indicating that bone turnover
decreases with age (Price et al., 1995). This was indicated by the decrease in both OC
and ICTP. The decrease in KS is also similar to a previous study that found KS decreases
very rapidly between 3 to 5 mo of age in horses (Okumura et al., 1997). Synovial fluid
KS is more specific for the fetlock joint and the carpal joint of the third metacarpus but is
an invasive technique (Campion et al., 1991).

As we did not know how variable the serum measurements would be in the
weanling horse, we can use this study to determine the relative power. As power of a test
determines the probability of correctly rejecting the null hypothesis when the hypothesis
is not correct, a low power study often fails to find differences between treatments when
one actually exists. After completing this study and having a better indication of the
variability of the serum measurements in weanlings, we found that our power ranges
from a high of 91% to a low of 5%. Hence, the failure to find treatment differences, for
instance in some of the serum markers, is due to a relatively low number of animals.
Thus, we conclude that because of large overall changes in the entire skeletal system of
the weanling during growth, the likelihood of detecting a difference due to our imposed

treatments would require an unrealistic number of animals for an equine study.

69

 

CHAPTER 5

CONCLUSIONS

An increase in mineral content of the third metacarpal of the pastured and
paddocked groups in both experiments suggests an increased strengthening of bone. A
decrease in bone mineral content of the third metacarpal bone or a lack of change in the
stalled weanlings in both experiments suggests a weakening of the third metacarpal bone
or prevention of maximal bone mineral deposition, respectively (Fleming et al., 1994; El
Shorafa et al., 1979). The increase in RBAE is likely due to exercise while the decrease
in RBAE is most likely due to the lack of exercise and loading on the bones of the stalled
horses. These studies are a practical example of Wolff’s Law which states that as the
function of a bone changes, the architecture of that bone must change to adapt to this new
function (Woo et al., 1981).

Maintaining weanlings on pasture for 12 h/d in Experiment 2 proved to be just as
effective in increasing bone mineral content in horses pastured continuously. Pasture-
rearing may not be very feasible for many horse owners because of feeding and aesthetic
reasons. Differences between the handling of the longeing groups in Experiment 1,
prevent us from drawing conclusions about the positive or negative effects of longeing at
the trot and canter or walking on the mechanical walker. However, Raub et a1. (1989)
found that weanlings worked on the mechanical walker at the trot 5 d/wk demonstrated
an increase in medial bone mineral content. Longe training at the trot or canter or
working on the mechanical walker at the trot would likely be more advantageous to bone

strength than stalling with no exercise. Though the exercise received on the longe line or

70

'.'-.‘."1*" t. 1
i.
F

 

mechanical walker may not be sufficient to increase bone mineral content it may be
enough to prevent disuse osteoporosis and maintain bone mineral content.

Thoroughbreds do not train at racing speeds which predispose the horse to an
increase risk of injury once racing begins due to the animals legs not being sufficient to
withstand the increase in strain (Nunamaker et al., 1990). Maintaining a horse in stalls
preventing strains necessary for racing will lead to an increase of injury once speed is
placed on the leg (Nielsen et al., 1997). In order to prevent this, a training regimen that
includes a slow progression of speed work may be beneficial to increase the strength of
the bones so eventually they will be capable of withstanding strains associated with
racing.

Photometric radiography is an accurate measure of bone mineral content which
correlates with bone strength (Fleming et al., 1994; El Shorafa et al., 1979). The
measurements were specific for the third metacarpal and allowed conclusions to be made
regarding bone mineral content without dependency on the serum markers. We are
confident that pasture- or paddock-rearing horses is beneficial for increasing bone
mineral content based on similar results in previous studies. Though pasture- and
paddock-rearing horses responded similarly in bone mineral content, pasture-rearing
elicited more consistent responses in increases in bone mineral content. The larger
pasture allows for more area to run at fast sprints while a smaller paddock may be too
small to really allow the ability to pick up a fast sprint. When raising horses in a paddock
or a pasture, the size must be considered. Enough room must be allowed for the horses to

run at fast sprints.

71

 

We conclude that stalling weanlings without exercise prevents maximal mineral
deposition in the third metacarpal. This may prove detrimental to some athletic horses
when they enter training later in life. Stalling horses while “sale prepping” is a common
occurrence in the horse industry. Horses are often stalled for 3 to 4 mo before a sale to
improve hair coat and prevent injuries. This “prepping” likely yields horses with weaker
bones that are more prone to injury once training commences. The results of the present
study indicate that if “sale prepping” is necessary, 12-h daily tum-out or perhaps only a
few hours outside allowing for the horse to sprint are enough to prevent decreases in bone
mineral content. Exercise is particularly important during the skeletal maturation stage
so that once skeletal maturation is complete, the bone is strong enough to withstand
increases in strain. For example, if a horse is raised with only access to walking (no fast
sprints), the bone will mature capable of withstanding the strains of standing and
walking. Once speed work is introduced, the bone will not be strong enough to withstand
the increase in strain associated with training and injury will occur. -

The next research to be performed should be to determine how much time is
necessary outside to prevent disuse osteoporosis. The present study indicates 12 h is
plenty but this may not be feasible for many horse producers. Because this study
determined the influence of housing on bone mass and cartilage markers, it has not been
determined what exactly causes the increase in bone strength seen in the
pastured/paddocked horses. Though it is thought to be growth and exercise that facilitate
this increase and a lack of exercise that causes a decrease, a more controlled study would

need to be done to determine why a decrease or lack of growth is seen in stalled horses.

72

 

A more controlled study would be one that allowed the stalling of horses outside so that
all horses experience the same daylight, weather conditions, and feeding conditions.

Pratt (1982) states that the most efficient way to develop bone mass and hence
bone strength is to participate in short vigorous activities (i.e. sprints). The combination
of this study with the results indicated in the present study implies that it should only take
a minimal amount of strides at a sprint to prevent disuse osteoporosis in the horse.
Therefore, we have concluded that disuse osteoporosis can be prevented by 12-h daily

tum-out and quite possibly less.

73

 

 

.1"

APPENDIX A

74

APPENDIX A

Means Tables

Table 1A. Means table for medial RBAE (mm Al) in Quarter Horse weanlings

 

 

 

Day
Treatment 0 28 56 SEM
Paddock 17.2 16.6 18.3 0.6 “-1
Exercise 17.6 16.9 17.8‘ 0.6 ':
Stall 17.1 15.6 16.4 0.6

 

'SEM = 0.7

Table 2A. Means table for lateral RBAE (mm Al) in Quarter Horse weanlings

 

 

 

 

Day r-
Treatment 0 28 56 SEM E "
Paddock 16.6 16.4 17.2 0.7
Exercise 16.5 16.6 16.9 0.7
Stall 17.4 16.2 16.9 0.7

 

Table 3A. Means table for palmar RBAE (mm Al) in Quarter Horse weanlings

 

 

 

Day
Treatment 0 28 56 SEM
Paddock 14.5 - 14.7 14.1 0.7
Exercise 15.1 14.4 14.6‘ 0.7
Stall 15.2 14.4 13.6 0.7

 

'SEM = 0.8

Table 4A. Means table for dorsal RBAE (mm Al) in Quarter Horse weanlings

 

 

 

 

 

 

 

Day
Treatment 0 28 56 SEM
Paddock 16.1 16.5 15.9 0.6
Exercise 17.5 16.2 16.7 0.6
Stall 16.5 16.0 15.3 0.6
Table 5A. Means table for change in dorsal RBAE (mm Al) in Quarter Horse

weanlin 5

Day
Treatment 28 56 SEM
Paddock 0.4 -0.2 0.7
Exercise -2. l -0.7 0.7
Stall -0.5 -1.2 0.7

 

75

Table 6A. Means table for total (DP) RBAE (mm2 Al) in Quarter Horse

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

weanlings
Day
Treatment 0 28 56 SEM
Paddock 168 229 255 42
Exercise 287 270 221' . 42
Stall 232 220 21 1 42
'SEM = 45
Table 7A. Means table for change in total (DP) RBAE (mm2 Al) in Quarter Horse
weanlings
Day
Treatment 28 56 SEM
Paddock 60.6 79.8 43.8
Exercise —16.8 -68.7' 43.8
Stall -31.3 -20.6 43.8
‘SEM = 45.3
Table 8A. Means table for total (LM) RBAE (mm2 Al) in Quarter Horse
weanlian
Day
Treatment 0 28 56 SEM
Paddock 84 140 99 21
Exercise 188 133 83' 21
Stall 117 . 104 67 21
'SEM = 23
Table 9A. Means table for change in total (LM) RBAE (mm2 A1) in Quarter Horse
weanlirgs
Day
Treatment 28 56 SEM
Paddock 56.6 18.8 28.7
Exercise -54.6 -103.9' 28.7
Stall 16.5 -49.5 28.7
'SEM = 30.7

Table 10A. Means table for change in ICTP (ng/ml) in Quarter Horse weanlings

 

 

 

 

Day
Treatment 14 28 42 56 SEM
Paddock -2.6 -l.3 -3.0 -3.1 1.5
Exercise -1.9 -4.7 -2.9' —4.3' 1.5
Stall -5.3 -6.6 -2.6 -1.5 1.5
.SEM = 1 .6

76

 

Table 11A. Means table for height at the withers (cm) in Quarter Horse

 

 

 

 

weanlifls
Day
Treatment 0 28 56 SEM
Paddock 123.5 125.3 128.5 1.4
Exercise 125.3 125.2 128.5' _ 1.4
Stall 123.3 125.2 128.5 1.4
'SEM = 1.5

3 Table 12A. Means table for height at the hips (cm) in Quarter Horse weanlings

 

 

 

Day
Treatment 0 28 56 SEM
Paddock 129.3 132.1 133.6 1.7
Exercise 131.4 132.1 135.0' 1.7
Stall 130.1 131.4 135.3 1.7

 

'SEM = 1.8

 

Table 13A. Means table for weight (kg) in Quarter Horse weanlings

 

 

 

 

 

 

 

 

 

 

 

Day
Treatment 0 28 56 SEM
Paddock 233 236 265 14
Exercise 237 232 250 14
Stall 221 224 257 14
Table 14A. Means table for third metacarpal circumference (cm) in Quarter Horse
weanm
Day
Treatment 0 28 56 SEM
Paddock 15.7 15.5 16.4 0.3
Exercise 16.0 15.7 16.2 0.3
Stall 15.5 15.4 15.9 0.3
Table 15A. Means table for medial RBAE (mm Al) in Arabian
weanlin s
Day
Treatment 0 28 56 SEM
Pasture 17.4 18.4 17.8 0.4
Partial Pasture 17.2 17.7 18.5 0.4
Stall 16.4 16.6 17.1 0.4

 

77

Table 16A. Means table for lateral RBAE (mm Al) in Arabian

 

 

 

weanlifis
Day
Treatment 0 28 56 SEM
Pasture 15.7 16.8 17.8 0.4
Partial Pasture 15.6 16.1 16.4 0.4
Stall 15.9 15 15.6 ' 0.4

 

Table 17A. Means table for total (DP) RBAE (mm2 Al) in Arabian weanlings

 

 

 

 

 

 

 

Day
Treatment 0 28 56 SEM
Pasture 149 186 208 20
Partial Pasture 145 193 196 20
Stall 136 133 204 22
Table 18A. Means table for change in OC (ng/ml) of Arabian
weanlings
Day
Treatment 14 28 42 56 SEM
Pasture -6.0 -12.1 -46.6 -32.1 19.2
Partial Pasture -40.8 -48.5 -65.1 -41.7 19.2
Stall -13.1 -5.8 -18.0 -30.1 22.1

 

Table 19A. Means table for height at the withers (cm) in Arabian weanlings

 

 

 

 

 

 

 

 

- Day
Treatment 0 28 56 SEM
Pasture 119.9 122.1 1251' 1.2
Partial Pasture 119.7 124.4 124.9 1.2
Stall 119.2 122.9 125.1 1.4
‘SEM = 1.3
Table 20A. Means table for height at the hip (cm) in Arabian

weanliggs
Day

Treatment 0 28 56 SEM
Pasture 127.0 129.7 131.8‘ 1.1
Partial Pasture 124.7 127.5 132.0 1.1
Stall 123.8 127.1 129.5 1.2
'SEM = 1.2

78

 

Table 21A. Means table for weight (kg) in Arabian weanlings

 

 

 

 

 

 

 

Day
Treatment ' 0 28 56 SEM
Pasture 186 205 235' 7
Partial Pasture 195 209 228 7
Stall 188 208 238 8
’SEM = 8
Table 22A. Means table for third metacarpal circumference (cm) in Arabian

weanlian

Day
Treatment 0 28 56 SEM
Pasture 15.2 15.9 16.6 0.2
Partial Pasture 15.5 15.9 16.5 0.2
Stall 15.3 15.2 15.6 0.2

 

79

 

APPENDIX B

80

 

1W?
1
'1

 

APPENDIX B

AN OVA Tables

Table 1B. ANOVA table for media] RBAE (mm Al) in Quarter Horse weanlings

 

 

NDF DDF F-Value P-Value
Treatment 2 l4 1 .62 0.2326
Day 2 29 4.72 0.0169 9.
Day*Treatment 4 29 0.56 0.6917
Block 1 14 0.14 0.7098

 

Table 2B. ANOVA table for lateral RBAE (mm Al) in Quarter Horse weanlings

 

 

 

NDF DDF F-Value P-Value
Treatment 2 14 0.04 0.9637
Day 2 29 0.55 0.5354 1 ‘-
Day*Treatment 4 29 0.30 0.8752
Block 1 14 0.08 0.7768

 

Table 3B. ANOVA table for palmar RBAE (mm Al) in Quarter Horse weanlings

 

 

NDF DDF F-Value P-Value
Treatment 2 14 0.10 0.9091
Day 2 29 1.20 0.3170
Day *Treatment 4 29 0.46 0.7612
Block 1 14 0.55 0.4695 '

 

Table 4B. ANOVA table for dorsal RBAE (mm Al) in Quarter Horse weanling

 

 

NDF DDF F-Value P-Value
Treatment 2 14 1.10 0.3596
Day 2 29 1 .42 0.2588
Day *Treatment 4 29 1 .06 0.3955
Block 1 14 1.68 0.2160

 

Table 5B. ANOVA table for total (DP) RBAE (mm2 Al) in Quarter Horse weanlings

 

 

NDF DDF F-Value P-Value
Treatment 2 14 0.53 0.6002
Day 2 29 0.14 0.873 1
Day *Treatment 4 29 1. 1 1 0.3717
Block 1 14 0.18 0.6810

 

81

Table 6B. ANOVA table for total (LM) RBAE (mm2 Al) in Quarter Horse weanlings

 

 

NDF DDF F-Value P-Value
Treatment 2 14 2.09 0.161 1
Day 2 29 4.83 0.0154
Day*Treatment 4 29 2.86 0.0412
Block 1 14 0.52 0.4843

 

Table 7B. ANOVA table for Osteocalcin (ng/ml) in Quarter Horse weanlings

 

 

NDF DDF F-Value P-Value
Treatment 2 12 1 .53 0.2555
Day 4 54 3. 12 0.022
Day*Treatment 8 54 1.04 0.4163
Block 1 12 0.18 0.6791

 

Table 8B. ANOVA table for ICTP (ng/ml) in Quarter Horse weanlings

 

 

 

NDF DDF F-Value P-Value
Treatment 2 12 9.02 0.0041
Day 4 54 8. 19 0.0001
Day*Treatment 8 54 2.05 0.0579
Block 1 12 38.69 0.0001

 

Table 9B. ANOVA table for Keratan Sulfate (ng/ml) in Quarter Horse weanlings

 

 

NDF DDF F-Value P-Value
Treatment 2 12 1.42 0.2803
Day 4 ' 54 14.90 0.0001 .
Day*Treatment 8 54 2.78 0.01 18
Block 1 12 0.26 0.6224

 

Table 108. ANOVA table for height at the withers (cm) in Quarter Horse weanlings

 

 

NDF DDF F-Value P-Value
Treatment 2 14 0.08 0.9260
Day 2 29 25.92 0.0001
Day*Treatment 4 29 0.99 0.4307
Block 1 14 4.75 0.0468

 

Table 11B. ANOVA table for height at the hip (cm) in Quarter Horse weanlings

 

 

NDF DDF F-Value P-Value
Treatment 2 14 0.16 0.8555
Day 2 29 10.71 0.0003
Day*Treatment 4 29 0.77 0.5529
Block 1 14 5.67 0.0320

 

82

 

Table 12B. ANOVA table for weight (kg) in Quarter Horse weanlings

 

 

 

 

 

NDF DDF F-Value P-Value
Treatment 2 14 0. 16 0.8500
Day 2 29 21.92 0.0001
Day*Treatrnent 4 29 0.80 0.5334
Block 1 14 2.38 0.1454
Table 13B. ANOVA table for third metacarpal circumference (cm) in Quarter Horse

weanlings

NDF DDF F-Value P-Value
Treatment 2 14 0.42 0.6621
Day 2 29 37.24 0.0001
Day *Treatment 4 29 l .84 0. 1477
Block 1 14 4.26 0.0580

 

Table 14B. ANOVA table for medial RBAE (mm Al) in Arabian weanlings

 

 

NDF DDF F-Value - P-Value
Treatment 2 12 7.90 0.0065
Day 2 28 3.55 0.0423
Day*Treatrnent 4 28 1. 14 0.3576
Block 2 12 2.98 0.0888

 

Table 15B. AN OVA table for lateral RBAE (mm Al) in Arabian weanlings

 

 

 

NDF DDF F-Value P-Value
Treatment 2 12 7.29 0.0085 _
Day 2 28 3.85 0.0332
Day*Treatment 4 28 2.55 0.0613
Block 2 12 0.35 0.7103

 

Table 16B. ANOVA table for total (DP) RBAE (mm2 Al) in Arabian weanlings

 

 

NDF DDF F-Value P-Value
Treatment 2 12 1.23 0.3256
Day 2 28 6.26 0.0057
Day*Treatment 4 28 0.68 0.6125
Block 2 12 0.02 0.9772

 

Table 17B. ANOVA table for osteocalcin (ng/ml) in Arabian weanlings

 

 

NDF DDF F-Value P-Value
Treatment 2 8 4.5 1 0.0488
Day 4 48 6.22 0.0004
Day*Treatment 8 48 1. 1 1 0.3722
Block 2 8 3. 14 0.0987

 

83

Table 18B. ANOVA table for ICTP (ng/ml) in Arabian weanlings

 

 

NDF DDF F-Value P-Value
Treatment 2 12 0.19 0.8332
Day 4 56 13.58 0.0001
Day *Treatment 8 56 1 .74 0. 1087
Block 2 12 0.47 0.6358

 

Table 19B. ANOVA table for keratan sulfate (ng/ml) in Arabian weanlings

 

 

NDF DDF F-Value P—Value
Treatment 2 l 1 0.63 0.5494
Day 4 52 1.89 0.1267
Day*Treatment 8 52 0.99 0.4571
Block 1 1 1 0.55 0.4757

 

Table 20B. ANOVA table for height at the withers (cm) in Arabian weanlings

 

 

NDF DDF F-Value P-Value
Treatment 2 12 0.13 0.8818
Day 2 25 17.40 0.0001
Day*Treatment 4 25 0.87 0.4973
Block 2 12 7.52 0.0076

 

Table 21B. ANOVA table for height at the hip (cm) in Arabian weanlings

 

 

NDF DDF F-Value P-Value
Treatment 2 12 1.71 0.2218
Day 2 25 36.84 0.0001 .
Day *Treatment 4 25 1 .3 1 0.2929
Block 2 12 5.20 0.0236

 

Table 22B. ANOVA table for weight (kg) in Arabian weanlings

 

 

 

 

 

NDF DDF F-Value P-Value
Treatment 2 12 0.06 0.9457
Day 2 25 25.72 0.0001
Day*Treatment 4 25 0.48 0.7466
Block 2 12 0.89 0.4360
Table 23B. ANOVA table for third metacarpal circumference (cm) in Arabian

weanling

NDF DDF F-Value P-Value
Treatment 2 12 2.92 0.0928
Day 2 25 34.0 0.0001
Day*Treatment 4 25 4.70 0.0057
Block 2 12 1.37 0.2923

 

84

 

 

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