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Copper Nutrition in Pasture-Fed 
New Zealand Thoroughbreds, and 
its Role in Developmental 
Orthopaedic Disease 
A thesis presented in partial fulfilment of the 
requirements for the degree of Doctor of Philosophy at 
Massey University, Palmerston North, New Zealand 
Simon George Pearce 
1997 
ii 
ABSTRACT 
This thesis reports studies to test the hypothesis that "dietary supplementation of pasture­fed New Zealand Thoroughbreds with copper will reduce the incidence and severity of 
developmental orthopaedic disease". Thoroughbred foals were raised based on New Zealand 
Thoroughbred industry standards at AgResearch's Equine Research Stud. Thus the first stage 
required the development of methods to define indices of copper status and growth plate 
development. Techniques were developed and evaluated for in vivo sampling of liver from the 
mare and neonatal foal and the distal radial physis of the neonatal foal. 
Pregnant Thoroughbred mares (n=24) were divided into either copper supplemented or control 
groups. Live foals born to each group of mares were also divided into copper supplemented or 
control groups. Supplementation was administered orally with aqueous copper sulphate at a 
rate of 0.5 mg Cu/kg liveweight (LW), and controls received a placebo of equivalent fluid 
volume. Mares were supplemented for the final 13 to 25 weeks of gestation until parturition. 
Foals were supplemented from 21 days of age with 0.2 mg Culkg LW increasing to 0.5 mg 
Culkg LW by 49 days remaining at that level until euthanasia at approximately 150 days. All 
animals grazed tall fescue pasture containing 4.4 to 8.6 mg Culkg dry matter (OM) for the 
duration of the experiment. This experimental design allowed independent investigation of 
both the effect of mare treatment during gestation and foal treatment on the evidence of 
developmental orthopaedic disease in the foals. Randomisation was stratified based on mare 
age, sire of the foal, last date of service and in the case of the foals only, sex of the foal. 
Indices of copper status were measured in both the mares and foals throughout the experiment, 
and at postmortem. The foals were examined regularly for evidence of developmental 
orthopaedic disease (DOD) which included clinical, conformational and radiological 
examinations. At euthanasia, an exhaustive postmortem examination was performed which 
included investigation of all limb and cervical spine articulations, and examination of the 
physes from the proximal humerus, proximal and distal radius and tibia, and distal femur, 
third metacarpus and third metatarsus. Physes were examined after cutting bone ends into 
slabs of approximately 6 mm widths using a bone saw. All abnormalities in cartilage or bone 
found at postmortem were submitted for histological examination. A histomorphometric study 
was also performed on the growth plate and metaphyseal primary spongiosa of standard 
sections cut through the distal radius. 
iii 
Plasma copper concentration of mares declined throughout the final trimester, and was not 
affected by copper supplementation. There was a trend toward increased copper concentration 
in the livers of supplemented mares, and a significant increase in the liver copper 
concentration of foals born to supplemented mares as determined by liver biopsies of mares 
and foals shortly after parturition,. Plasma and blood cell copper concentration, and plasma 
caeruloplasmin oxidase activity in the foals were not affected by copper supplementation, but 
liver copper concentration was significantly greater in copper-supplemented foals compared 
with controls. 
There was no effect of copper supplementation on the evidence of DOD in vivo, in the physes 
examined at postmortem, or on the histomorphometry in the distal radius. However there was 
an effect of mare supplementation during gestation on indices of physitis in the foals assessed 
from postmortem radiographs and on mild abnormalities found in the articulations of foal 
limbs. Copper supplementation of the foal had no effect on these indices. The low incidence 
and severity of lesions found in foals grazing a pasture based diet containing 4.4 to 8.6 mg 
Cu/kg OM, combined with the failure to demonstrate an effect of foal copper supplementation 
on the evidence of DOD is contrary to published and anecdotal reports within New Zealand 
and internationally. This prompted the investigation of a possible dietary constituents which 
may affect the requirement for copper. 
Molybdenum was identified as a possible antagonist which under certain circumstances may 
be found in high concentrations in a pasture diet. A pasture was prepared containing 8 to 15 
mg Mo/kg OM. Weanlings were grazed on this pasture for 70 days and then supplemented 
with oral copper sulphate at a rate of 1.0 mg Cu/kg OM for a further 14 days (84 days total). 
There was no effect of high dietary molybdenum on plasma copper, trichloroacetic acid 
insoluble plasma copper, or blood cell copper concentration, or on the activities of 
caeruloplasmin oxidase or red blood cell superoxide dismutase. There was also no effect of 
dietary molybdenum on the liver copper concentration after 70 or 84 days. It was therefore 
concluded that at concentrations that might be expected in a pasture diet molybdenum is 
unlikely to increase copper requirements of horses. 
The possible implications of this work to the New Zealand Thoroughbred industry, and 
suggestions for further research are discussed. 
iv 
Acknowledgements 
I am greatly in debt to the outstanding commitment of time and energy provided by my 
supervisors Elwyn Firth and Peter Fennessy. I am particularly thankful for the time Elwyn 
spent performing many integral functions related to the research and the preparation of 
manuscripts. Peter's rigorous, logical and creative approach to experimental design, data 
analysis and manuscript preparation also merits special mention. I would also like to thank my 
other supervisors, Jeff Wichtel, whose assistance with statistical analysis and manuscript 
preparation was particularly appreciated, and Sabina Holle. 
I am particularly grateful to AgResearch's Neville Grace who taught me the basics of 
nutrition, was a major co-investigator in the studies described in this thesis, and was 
invaluable in the preparation of manuscripts including this thesis. Also at AgResearch I thank 
Warren Hunt who managed the research stud at Flock House, Bob Fletcher and Peter 
Johnstone, who provided advice on experimental design and statistical analysis and Willi 
Stiefel, who provided technical support for these studies. 
In the Department of Veterinary Clinical Sciences at Massey University, I am thankful for the 
technical assistance of Simone Holmes, Michelle Hayes, Tracey Smith and Guy Hessell. 
Maureen Wichtel and Roz Machon anaesthetised foals for the technique development studies. 
Merv Birtles provided advice on histological techniques and took the photomicrographs in this 
thesis. Grant Guilford and Keith Thompson provided advice on different sections of this 
thesis, and Keith also assisted in the interpretation of histological sections. I would also like to 
thank the many other people in the department of Veterinary Clinical Science who offered 
advice, support and encouragement throughout. 
The work in these studies was supported financially by AgResearch, the Foundation for 
Research, Science and Technology, the New Zealand Equine Research Foundation and 
Massey University. The New Zealand Equine Research Foundation also provided a travel 
scholarship which allowed me to present results of this work in North America. 
Simon Pearce 
Department of Veterinary Clinical Science 
Massey University 
April 1997 
I dedicate this thesis to my parents, Pam and Martin, 
with my love and respect. 
v 
vi 
Table of Contents 
page 
Abstract 11 
Acknowledgements IV 
Table of Contents VI 
List of Figures XlI 
List of Tables xv 
Chapter 1 Introduction 1 
1.1 Experimental hypothesis 2 
1.2 The New Zealand Thoroughbred 2 
1 .2.1 Brief history of the Thoroughbred racing horse 2 
1.2.2 The Thoroughbred in New Zealand 4 
1 .2.3 The Thoroughbred management system in New Zealand 4 
1 .2.4 Wastage 5 
1 .3 Equine skeletal anatomy 6 
1 .3.1 Introduction 6 
1.3.2 Anatomy of the growing long-bone 6 
1.3.3 Physis 6 
1.4 Developmental orthopaedic disease 7 
1.4.1 Introduction 7 
1.4.2 Osteochondrosis 8 
1.4.3 Physitis 10 
1.4.4 Cervical vertebral malformation 11 
1.4.5 Limb deformities 12 
1.4.5.1 Angular limb deformity 13 
1 .4.5.2 Flexural limb deformity 13 
1.4.6 Proposed aetiologies for DOD 13 
1.4.6.1 Growth rate 14 
1.4.6.2 Trauma 15 
1.4.6.3 Ischaemia 16 
1.4.6.4 Genetics 17 
1 .4.6.5 Nutrition 19 
----------------------------- -
vii 
1.4.6.6 Dexamethasone 26 
1.4.6.7 Hypothyroidism 26 
1.4.6.8 Infection 26 
1.4.7 Conclusions from the review on DOD 27 
1.5 Review on copper nutrition 28 
1.5.1 Introduction 28 
1.5.2 Copper content and distribution in the body 29 
1.5.3 Copper metabolism 30 
1.5.3.1 Absorption 30 
1.5.3.2 Transport and storage 3 1  
1.5.3.3 Excretion of copper 32 
1.5.3.4 Effect of pregnancy on copper metabolism 33 
1.5.3.5 Copper metabolism for the neonate 33 
1.5.4 Cuproproteins 34 
1.5.5 Measurement of copper status 38 
1.5.6 Dietary copper sources and bioavailability 42 
1.5.6.1 Copper content and availability in pasture 42 
1.5.6.2 Copper supplements (bioavailability) 43 
1.5.7 Copper dietary interactions 43 
1.5.8 Copper metabolism studies and copper requirements in horses 46 
1.5.9 Copper deficiency 48 
1.5.10 Copper toxicity 49 
1.5.11 Therapeutic applications for copper 5 1  
1.6 Introduction to the objectives of the thesis 52 
Chapter 2 Liver Biopsy Techniques for Adult Horses and Neonatal 53 
foals to Assess Copper Status 
2.1 Abstract 54 
2.2 Introduction 54 
2.3 Materials and methods 56 
2.3.1 Liver biopsy general details 56 
2.3.2 Mare liver biopsy procedure 56 
viii 
2.3.3 Foal liver biopsy procedure 57 
2.3.4 Liver biopsy sample preparation and copper analysis 58 
2.3.5 Site variation in liver copper concentration 59 
2.4 Results 60 
2.4.1 Mare liver biopsy procedure 60 
2.4.2 F oal liver biopsy procedure 60 
2.4.3 Liver biopsy copper concentration 61 
2.4.4 Site variation in liver copper concentration 61 
2.5 Discussion 63 
Chapter 3 Biopsy of the Distal Radial Physis in Neonatal Foals 66 
3.1 Abstract 67 
3.2 Introduction 67 
3.3 Materials and Methods 68 
3.3.1 Animals 68 
3.3.2 Biopsy technique 68 
3.3.3 Conformation 70 
3.3.4 Postmortem examination 70 
3.4 Results 71 
3.4.1 Surgery 71 
3.4.2 Conformation 71 
3.4.3 Postmortem 73 
3.5 Discussion 76 
Chapter 4 The Effect of Copper Supplementation on the Copper 79 
Status of Pregnant Thoroughbred Mares and their 
Neonates 
4.1 Abstract 80 
4.2 Introduction 80 
4.3 Materials and methods 81 
4.3.1 Animals 81 
4.3.2 Sampling 82 
4.3.3 
4.3.4 
4.4 
4.4.1 
4.4.2 
4.5 
Chapter 5 
5.1 
5.2 
5.3 
5.3.1 
5.3.2 
5.3.3 
5.3.4 
5.3.5 
5.4 
5.4.1 
5.4.2 
5.4.3 
5.4.4 
5.4.5 
5.4.6 
5.5 
Chapter 6 
6.1 
6.2 
Analytical 
Statistical analysis 
Results 
Plasma copper concentration 
Liver copper concentration 
Discussion 
The Effect of Copper Supplementation on the Copper Status of 
Pasture-Fed Young Thoroughbreds 
Abstract 
Introduction 
Materials and methods 
Animals 
Diet 
Samples 
Analytical 
Statistical analysis 
Results 
Animals 
Pasture mineral composition 
Mare milk copper concentration 
Foal plasma copper concentration, caeruloplasmin activity and 
blood cell copper concentration 
Foal soft tissue copper concentration 
Foal bone copper concentration 
Discussion 
The Effect of Copper Supplementation on the Evidence of 
Developmental Orthopaedic Disease in Pasture-Fed 
Thoroughbreds 
Abstract 
Introduction 
ix 
82 
83 
83 
83 
84 
86 
90 
91 
91 
93 
93 
94 
94 
95 
97 
98 
98 
98 
99 
99 
103 
103 
104 
109 
110 
110  
x 
6.3 Materials and methods 112  
6.3.1 Animals and diet 112 
6.3.2 Clinical examination 112 
6.3.3 Radiographic examination 112  
6.3.4 Postmortem 114  
6.3.5 Bone physical properties 114  
6.3.6 Gross pathology 115  
6.3.7 Histopathology 115  
6.3.8 Statistical analysis 116  
6.4 Results 116  
6.4.1 Growth rate 116  
6.4.2 Clinical examination 117 
6.4.3 Radiographs 117  
6.4.4 Bone physical properties 118  
6.4.5 Pathology 119  
6.5 Discussion 127 
Chapter 7 The Effect of High Pasture Molybdenum Concentrations 130 
on the Copper Status of Grazing Horses 
7.1 Abstract 131 
7.2 Introduction 1 31 
7.3 Materials and methods 133 
7.3.1 Experimental design 133 
7.3.2 Animals 133 
7.3.3 Diets 133 
7.3.4 Part 1 :  Molybdenum copper interaction 133 
7.3.5 Part 2: Copper challenge 134 
7.3.6 Analytical 134 
7.3.7 Statistical analysis 135 
7.4 Results 136 
7.4.1 Pasture minerals 136 
7.4.2 Part 1: Molybdenum copper interaction 136 
7.4.3 
7.5 
Chapter 8 
8.1 
8.2 
8.3 
8.3.1 
8.3.2 
8.4 
8.4.1 
8.4.2 
8.4.3 
8.5 
8.6 
8.6.1 
8.6.2 
8.6.3 
8.7 
8.8 
8.9 
Appendices 
Appendix A 
References 
Part 2. Copper challenge 
Discussion 
General Discussion 
Introduction 
Experimental design 
Development of surgical techniques 
Liver biopsy technique 
Physeal biopsy technique 
The concept and measurement of adequate copper status 
Plasma indices of copper status 
Blood cell copper concentration and RBC Cu-Zn SOD as indices 
of copper status 
Liver copper concentration as an index of copper status 
The effect of copper supplementation of the mare and foal on indices 
of copper status 
The effect of copper supplementation of the mare and foal on indices 
of bone and cartilage development 
The influence of energy in the copper response experiments 
The theory of an association between dietary copper requirements 
and energy sourcellevel 
The theory of copper as a therapeutic agent 
Molybdenum as a copper antagonist for pasture-fed horses 
Recommendations to the New Zealand Thoroughbred Industry 
Further Research 
Pilot Studies on Circulating Copper Indices 
xi 
139 
140 
143 
144 
144 
145 
145 
146 
146 
146 
147 
147 
148 
149 
152 
152 
153 
154 
154 
155 
158 
159 
170 
Figure 2.1 
Figure 3.1 
Figure 3.2 
Figure 3.3 
Figure 3.4 
Figure 4.1 
Figure 4.2 
Figure 4.3 
Figure 5.1 
Figure 5.2 
List of Figures 
Mean difference (mg/kg dry weight) between liver copper 
concentration at each site (30 to 60 mg fresh weight) and the total 
(homogenised) liver copper concentration for 3 mature Thoroughbred 
horses. 
Stainless steel trephine, 9mm internal diameter, 11 mm external 
diameter, 24 mm trephine depth. 
The difference (right subtracted from left) in carpus (valgus) angle as 
viewed from in front: 
a) 21 foals with time from the physeal biopsy procedure 
b) 6 age-matched, unoperated control foals with time from the median 
day of physeal biopsy (day 7) in operated foals; 
Angles were calculated using video image analysis 
Thin section cabinet radiograph of a frontal section through the distal 
radius showing the effect of the biopsy on the physis laterally. 
Low power view of the histological features of an epiphyseodesis at 
the biopsy site showing the trabeculae aligned perpendicular to the 
physis, extending into the physis. 
The relationship between plasma copper concentration and time 
prepartum in the 21 mares. 
The relationship between mare and foal liver copper concentration 
(mg Cu/kg DM) at 4 to 10 days postpartum from copper control and 
supplemented mares. 
The relationship between mare age and foal liver copper 
concentration at birth for foals from control and copper supplemented 
mares. 
Mean milk copper concentration (mgll) of all lactating mares from 
birth to 150 days (n=21). 
Mean plasma copper concentration (mgll) and blood cell copper 
concentration (mg/kg fresh weight) of all foals (n=21) from birth to 
150 days. 
xii 
Page 
62 
69 
72 
74 
75 
84 
85 
86 
99 
100 
Figure 5.3 
Figure 5.4 
Figure 6.1 
Figure 6.2 
Figure 6.3 
Figure 6.4 
Figure 6.5 
Mean plasma caeruloplasmin activity (UIl, 37°C) of all foals (n=21) 
from birth to 150 days. 
Relationship between plasma copper concentration and 
caeruloplasmin activity of all foals (n=2 1) 
a) from birth to 2 1  days 
b) from 28 to 150 days. 
Radiograph of distal third metatarsal bone showing metaphyseal 
shouldering, metaphyseal sclerosis, and widening of the physis. 
An invaginated type lesion showing: 
a) the gross appearance in the articular and epiphyseal cartilage of 
the proximal humerus and distal scapula and, 
b) the histological appearance of cartilage invagination in the 
proximal radius, showing fibrocartilage adherent to the articular 
cartilage, chondrones, subjacent myelofibrosis, and a mature 
subchondral microfracture. 
A retained cartilage lesion showing: 
a) the gross appearance in the lateral trochlear ridge of the distal 
femur and, 
b) the histological appearance in the distal third metacarpal bone 
showing a widened zone of hypertrophic cartilage, a decrease in 
matrix staining, subjacent myelofibrosis, and a subchondral 
microfracture associated with fibrin and necrotic cartilage. 
Low power micrograph of a retained cartilage lesion in a "trough" of 
the distal radial physis, showing retention of physeal hypertrophic 
chondrocytes protruding into the metaphysis. 
Percentage trabecular covered area of the metaphyseal spongiosa 10 
rnm proximal to the physis, and width of the distal radial physis in 
control and copper supplemented foals, measured from medial to 
lateral in a standard section of the distal radii in 13 foals ( 150 days of 
age) 
xiii 
101 
102 
113 
12 1-
122 
123 
125 
126 
Figure 7.1 
Figure 7.2 
Figure Al 
Figure A2 
Figure A3 
Figure A4 
FigureA5 
Figure A6 
Copper indices in weanling Thoroughbred fillies grazing pasture with 
low (n=6) or high (n=6) molybdenum concentration. 
a) Plasma copper concentration. 
b) Plasma caeruloplasmin oxidase activity. 
c) Blood cell copper concentration. 
d) Red blood cell superoxide dismutase activity. 
Mean liver copper concentration from weanling Thoroughbred fillies 
on high molybdenum (n=6) and low molybdenum (n=6) pastures. 
Mean plasma copper concentration and caeruloplasmin activity of 
mares receiving a single dose of copper sulphate (500 mg) on day 0, 
and controls. 
Plasma copper concentration of individual mares receiving a single 
dose of copper sulphate (500 mg) on day 0, and controls. 
Caeruloplasmin activity of individual mares receiving a single dose of 
copper sulphate (500 mg) on day 0, and controls. 
Plasma copper concentration of pasture-fed pregnant mares receiving 
different levels of copper supplementation. 
Caeruloplasmin activity of pasture-fed pregnant mares receiving 
different levels of copper supplementation. 
Plasma copper concentration of two foals following laparoscopic 
liver biopsy and growth plate biopsy performed under general 
anaesthesia. 
xiv 
138 
139 
161 
162 
162 
165 
166 
168 
Table 1.1 
Table 2.1 
Table 2.2 
Table 4.1 
Table 5.1 
Table 5.2 
Table 5.3 
Table 6.1 
Table 6.2 
Table 6.3 
Table 6.4 
Table 6.5 
Table 7.1 
List of Tables 
Studies on the influence and heritability of osteochondrosis (OC) 
from Scandinavian countries. 
Copper concentration (mglkg dry weight) at six sites across the liver 
of six feral horses, with mean, standard deviation (sd) and coefficient 
of variation (cv). 
Liver copper concentration (mg Cu/kg dry weight) with standard 
deviation (sd) and coefficient of variation (cv) of 30-60 mg (fresh 
weight) samples taken at 12 sites within each liver, and the total liver 
(homogenised) copper concentration, from three mature horses. 
Effect of copper supplementation of mares on mare (n= 16) and foal 
(n=2 1) liver copper concentration (mg Cu/kg DM) at 4 to 10 days 
postpartum. 
The mineral composition of the tall fescue pasture from May 1994 to 
March 1995 and tall fescue hay fed to the mares to supplement the 
pasture diet from May to July 1994. 
Effect of copper supplementation of the mare prepartum, and the foal 
from 2 1  to 150 days on the mean copper concentration (mg Cu/kg 
DM) of foal organs at 150 days. 
The mean ash, copper, zinc, iron, calcium and phosphorus 
concentration of the atlas and ilium from all foals (n=2 1). 
Mean physitis scores from radiographs of the distal metatarsus of all 
foals at 150 days of age. 
Pooled mean values (n=2 1) for physical properties of the foal bones 
at 150 days of age. 
Number of foal articular cartilage lesions by classification at 150 
days of age according to mare and foal treatment. 
Number of foal physeal cartilage lesions at 150 days of age according 
to bone and mare and foal treatment. 
Location of classified foal articular cartilage lesions at 150 days of 
age. 
Concentrations of sulphur, molybdenum and copper from the 
molybdenum treated and control paddocks. 
xv 
Page 
18 
6 1  
62 
85 
98 
103 
104 
1 18 
1 18 
1 19 
1 19 
120 
136 
Chapter 1 
Introduction 
2 
1.1 Experimental hypothesis 
D evelopmental orthopaedic disease (DOD) causes unacceptable wastage in the rearing of foals throughout the world. Dietary copper supplementation above .National 
Research Counci l  (NRC) ( 1 989) recommendations ( 1 0  mg Cu/kg OM) in a small 
number of experiments resulted in a decrease in the incidence and severity of DOD \0 
horses raised under North American management systems. 
In  the New Zealand Thoroughbred industry, pasture is the major part of the diet of pregnant 
mares and their offspring. However pasture provides dietary copper concentrations that are 
often below NRC ( 1 989) recommendations. The dietary copper requirements of pasture-fed 
horses to provide adequate copper for healthy development of bone and cartilage, have not 
been studied. Therefore the work in this thesis is centred on the hypothesis that 
"Dietary supplementation of pasture-fed New Zealand Thoroughbreds with 
copper reduces the incidence and severity of developmental orthopaedic disease." 
1.2 The New Zealand Thoroughbred 
1.2.1 Brief history of the Thoroughbred racing horse 
During the period of Imperial Rome, horse rac ing (usually in chariots) held great prestige. It 
was considered that in order to win races, it was necessary to have an Oriental horse. These 
horses were imported to Rome from North Africa (the Barbs), Turkey, Egypt and the 
Arabian Peninsula. The Romans contributed to the spread of these Oriental horses by using 
them in their invading armies and by breeding and trading them throughout Europe. By the 
time of the English invasion (55 BC) some Oriental blood was already noticeable in the 
Britons' horses, presumably having been traded across the Channel by the Belgians (Tesio 
1958). 
Under Roman rule came the inception of organised races on the British Isles (Tesio 1 958 ;  
Edwards 1980). These races were very popular with the Britons, and they continued after 
the fall of the Roman Empire. Movements of the Crusaders ( l 1 th - 1 3th centuries), provided 
3 
great opportunity for further importation of the superior Oriental horses to the British Isles 
(Tesio 1 958 ;  Anonymous 1 978). 
Rac ing thrived during the periods of the Stuart and Tudor kings. In 1 533 King Henry VIII, a 
great horse racing enthusiast, prohibited stall ions from grazing on publ ic lands. This was to 
improve the general quality of horses by encouraging judicious breeding with wel l bred 
stallions (Borton 1 977; Tesio 1 958) .  On two occasions, he was gifted "pure bred" oriental 
broodmares which figure prominently in the ancestry of Thoroughbreds (Tesio 1 958) .  
In 1 575, an eminent Italian horse breeder, Signor Prospero D'Osma was invited to the court 
of Queen Elizabeth, to rectify what was considered to be a deterioration in the quality of the 
royal rac ing stock. D'Osma was a firm believer in keeping blood lines pure, and in his 
records he comments that" It is not advisable to mix two breeds of horses lest bastards be 
produced" (Tesio 1 958) .  It is believed that during this period the foundation stock of 
oriental mares gifted to King Henry VIII were bred almost exclusively to Oriental sires 
(Tesio 1 958) .  
The Puritans prohibited horse rac ing and betting, the royal studs were dispersed, and many 
horses were requisitioned by the state (Edwards 1 980; Borton 1 977). However, Cromwel l  
himself was a keen horseman and there are records of h im importing horses of pure oriental 
blood from Naples and the Orient (Tesio. 1 958) .  With the Restoration ( 1 660) came the 
reopening of the racetracks, and racing thrived again (Edwards 1 980). From the late 1 600s 
until 1 750, a number of Oriental sires were imported to the British Isles for the purpose of 
increasing speed in race-horses. Male lines survive in only three of these horses, namely the 
Byerly Turk, the Godolphin Barb (also called the Godolphin Arabian) and the Darley 
Arabian, from which all modem Thoroughbreds are said to be descended (Napier 1 973; 
Borton 1 977; Edwards 1 980). The success of each stallion can be attributed to one stallion 
offspring, Godolphin Barb's grandson Matchem ( 1 748), Darley Arabian' s  great great 
grandson Ecl ipse ( 1 764), and Byerly Turk's great great grandson Herod ( 1 758), which were 
all outstandingly successful as racehorses and as sires. 
The first stud book ( 1 793), contained approximately one hundred broodmares and a simi lar 
number of sires (Tesio 1 958) .  All were proven producers of winners and nearly all could be 
traced back to Oriental origins . From these horses came the term "Thoroughbred", meaning 
carefully bred. This is an appropriate name for these horses which developed from a nuc leus 
4 
of King Henry VIII purebred oriental stock, as they were carefully bred by D'Osma to 
imported Oriental stall ions, and after many generations were selected on racing performance 
for entry into the studbook. 
The Thoroughbred was bred primarily for speed at intermediate distances. The modem 
Thoroughbred differs from its antecedents which were nearly two hands (8 inches) smaller 
(Borton 1 977) and were said to race with their heads and tails elevated in the fashion of 
Arabians (Napier 1 973). Charles Darwin wrote about Thoroughbreds that, "By a process of 
careful selection and by careful training, the whole body of English racehorses has come to 
surpass in fleetness and size the parent stock." (Anonymous 1 978). 
1.2.2 The Thoroughbred in New Zealand 
Initially New Zealand relied on Australia for the importation of Thoroughbreds. The first 
Thoroughbred to be imported to Australia was the English-bred stal lion Rockingham in 
1 799 (Edwards 1 980) and the first Thoroughbred mare arrived in 1 825 .  The yearling colt 
Figaro, which arrived in Wellington in 1 840, was the first Thoroughbred to be imported to 
New Zealand (Redwood 1 979). 
The first New Zealand studbook, compiled in 1 862, contained the pedigrees of 1 45 mares 
and 5 8  stallions (Redwood 1 979). Since then New Zealand has become renowned as one of 
the world's great Thoroughbred nurseries. An American horse buyer observed that "New 
Zealand enjoys the sunshine of Cal ifornia, the grass of Kentucky, and the rainfall of Ireland 
and there is not a more natural place to raise Thoroughbreds" (Bridewell 1 993).  
1.2.3 The Thoroughbred management system in New Zealand 
The Thoroughbred industry in New Zealand is predominantly pasture based (Hunt 1 994). 
High average rainfall and fertile soils along with a temperate climate allows for horses to 
graze pastures all year. The cl imate also allows for a longer growing season compared with 
many other countries (Hunt 1 994), and minimises the requirements for supplementation. 
Due to the imposition of the Thoroughbred horse's birthday in the Southern Hemisphere on 
August 1 ,  most foals are born in late August, September and October to give them the 
greatest opportunity to mature and therefore successfully compete in the sales ring and on 
5 
the race track with other horses of the same "age". This imposed foaling season coincides 
with Spring pastures, which in general provide adequate feed for the late pregnant and 
lactating mare, and the neonatal foal . It is also during Spring that yearlings are being 
prepared for sale. To maximise growth rates, and body condition, yearlings may be fed 
supplements of high energy concentrates in addition to the highly digestible pasture feed 
available at that time. 
Yearling value is of prime importance to horse studs (Bridewell 1 993), because the majority 
of Thoroughbreds are intended for sale as yearlings. However, sale of wean lings produces a 
significant part of many studs' revenue. Value at sale is detennined jointly by pedigree and 
an individual ' s  type. Type consists of conformation, size, and the presence or absence of 
defects. No matter how good the pedigree of a particular individual, a confonnational defect 
or the presence of a defect in bone and cartilage development is likely to significantly 
reduce sale value, or prevent sale. 
1.2.4 Wastage 
Yearlings and weanlings must be presented to sales in the best condition possible. 
Withdrawal from sales is considered a better option than presenting an animal to the sales in 
poor or an unsatisfactory condition. One significant cause of late withdrawal is DOD, which 
may manifest as having unacceptable conformation or an unsound gait with corresponding 
clinical abnonnalities. 
O'Donoghue et al. ( 1 992) found that 70 % of foals born in Ireland exhibited some fonn of 
DOD. Of these, approximately 1 0  % required treatment and half of those foals receiving 
treatment fai led to reach expected sale value. Alvarado et al. ( 1 989) found that only 25 % of 
yearl ing standardbreds on one fann had no radiological evidence of DOD. However they 
also found that radiographic evidence of DOD had no relationship with future athletic 
perfonnance based on prize winnings. 
Nutrition of the horse in its first year of l ife is likely to affect its immediate future in the sale 
ring, but it may also affect its future racing career. There is no epidemiological infonnation 
linking the occurrence of DOD with future lameness in racehorses, but several researchers 
have proposed that there is a significant relationship (Krook and Maylin 1 988, Stover et al. 
1 992, Kaneko et al. 1 993). 
6 
1.3 Equine skeletal anatomy 
1.3.1 Introduction 
The skeleton of the horse consists of 205 bones. There are 20 bones in each forelimb and 
hindlimb, forming the appendicular skeleton. The other bones are found in the axial skeleton 
and consist of the skull ,  ribs, vertebrae and sternum (Getty 1 975). Equine bones can also be 
divided into long, cuboidal, irregular and flat bones. The long-bones of the limbs function as 
weight bearing bones, and as levers in the process of locomotion. The cuboidal bones are in 
complex joints such as the carpus and tarsus, and dissipate concussive forces (Deane and 
Davies 1 995). In other animals they also al low for a greater range of motion such as 
pronation in carnivores, but th is greater mobility has been sacrificed in the horse in favour 
of greater stabi lity and a resultant increase in speed. The flat bones (skull , rib and scapula) 
provide large areas for the attachment of connective tissues, and protect vital organs. The 
irregular bones are in the vertebral column, and provide the semi-rigid framework of the 
body, and protect the central nervous system. The formation of bone requires a connective 
tissue model, which in the case of long-bones is hyaline cartilage. 
1.3.2 Anatomy of the growing long-bone 
The growing long-bone consists of a diaphyseal shaft which is formed from the primary 
ossification centre of the original carti laginous model. Secondary ossification centres are 
present at the ends of the long-bone, in either compression or traction epiphyses. 
Endochondral ossification is the process whereby bone is formed from the physis and from 
the epiphyseal cartilage. The region of newly formed bone on the side toward the primary 
ossification centre is called the metaphysis. Remodel l ing of this metaphyseal bone forms the 
diaphysis and medul lary cavity. The joint surfaces of bone are covered by articular carti lage 
which is continuous with growth cartilage of epiphyseal origin. This carti lage is sometimes 
referred to as the articular-epiphyseal (A-E) complex. Endochondral ossification of the 
cartilage surrounding the epiphysis ceases first, and bone trabeculae form a subchondral 
bone plate adjacent to articular and physeal cartilage. 
1.3.3 Physis 
7 
There are various classifications of histological zones in the physis. This thesis refers to the 
three main zones described by Brighton ( 1 978), namely the reserve, prol iferative and 
hypertrophic cell zones. The reserve cell zone (also called the resting cell zone) consists of 
slowly dividing cells arranged in a haphazard array of collagen fibres. The proliferative or 
stacking cell layer consists of rapidly dividing chondrocytes in columns arranged parallel to 
the longitudinal axis of the bone. The hypertrophic zone adjoins the proliferative zone and 
consists of cells of increasing size. The hypertrophic zone can be subdivided into the 
hypertrophic zone closest to the proliferative zone, and the zone of calcifying cartilage 
adjacent to the metaphysis (Olsson 1 982; Brighton 1 978). 
Endochondral ossification consists of chondrocyte differentiation, cartilage calcification and 
chondrocyte death, followed by blood vessel invasion, cartilage matrix removal and bone 
formation. This process occurs in an ordered fashion in normal bone and cartilage 
development. Disruption of part of this process results in a retention of cartilage. This can 
occur both on the metaphyseal side of the physis, and also in the cartilage of the A-E 
complex at either subarticular or 'subphyseal' sites. Disturbance of endochondral 
ossification, the resultant carti lage retention and the sequelae in this tissue result in a group 
of diseases in growing foals called developmental orthopaedic diseases (DOD). 
1.4 Developmental orthopaedic disease (DOD) 
1.4.1 Introduction 
The l iteral definition of developmental orthopaedic disease is any orthopaedic disease 
occurring during skeletal development. However the term, first coined at a symposium in 
1 986 on growth disorders in horses, has become associated spec ifically with diseases caused 
by a fai lure of the normal conversion of the cartilaginous model to bone. Therefore a list of 
diseases was made to comprise this syndrome. These include osteochondrosis, cuboidal 
bone malformation, physitis (and primary spongiosa microfracture), wobbler syndrome 
(cervical vertebral malformation), and angular and flexural l imb deformities (McIlwraith 
1 986). There has been some debate as to whether other diseases such as juvenile arthritis 
should be included (Watrous 1 99 1 ). Gabel ( 1 988) proposed that the term "metabolic bone 
disease" be used because it implies a common pathogenesis namely the disorder in the 
conversion of cartilage to bone. This has not achieved much support because of its usage in 
8 
orthopaedic diseases after development such as post menopausal osteoporosis, and because 
it implies a disease of the bone and not associated cartilage (Jeffcott 1 99 1 ) . 
Further confusion arises because Palmer et al. ( 1 993) considers that DOD represents the 
clinical manifestations and sequelae of osteochondrosis, namely the diseases l isted above, 
and yet, Hurtig and Poole ( 1 996) make a greater distinction between OC and DOD in a 
discussion of these diseases. In this thesis, DOD is used as a collective term to describe 
diseases, with or without clinical signs, which have been associated with a disorder in the 
normal progression of endochondral ossification. 
1.4.2 Osteochondrosis (OC) 
Osteochondrosis is defined as a disorder in endochondral ossification. I nitial descriptions of 
the disease were based on radiological and postmortem examination of clinical cases and 
incidental findings. Rooney ( l 975a, 1 975b) described osteochondrosis (OC) in horses based 
on gross and histological observations of postmortem specimens. Predilection sites were 
identified, at which lesions were often present bi laterally. Lesions were divided into those 
occurring at the margin of joint surfaces, and those in non-marginal areas. Grossly, marginal 
lesions had a pedunculated mass of cartilage at the site of the lesion, whereas the non­
marginal lesion was characterised by a groove or infolding of the cartilage, often continuous 
with a cystic structure in the subchondral bone. The lesion types had similar histological 
changes including retention of cartilage, fai lure of vascular invasion of the hypertrophic 
cartilage, and carti lage necrosis in the hypertrophic zone. The relationship between the 
marginal lesions of OC and the non-marginal lesions which develop into subchondral bone 
cysts has been debated and is reviewed by Baxter ( 1 996). 
In their extensive radiological, clin ical and pathological investigation of OC in the femoro­
patel lar and tarso-crural joints of the horse, Stromberg and Rejno ( 1 978) and Rejno and 
Stromberg ( 1 978) also found that OC was often bi laterally symmetrical, and occurred in 
predilection sites. Predilection sites were observed to be located near insertion of tendons 
and joint capsules. The morphologic description of articular lesions was simi lar to that of 
Rooney ( l975a, 1 975b), and to lesions in pigs (Reiland 1 978) and cattle (Reiland et al. 
1 978; Jensen et al. 1 98 1 ), with the exception that there was a greater tendency for articular 
osteochondral fragments to be mineralised. The four histological hal lmarks for OC were 
thickening of cartilage, disturbance of endochondral ossification, cartilage degeneration and 
---------------------
9 
carti lage necrosis. Stromberg ( 1 979) re-iterated the c lassifications as described by Rooney 
( 1 975a, 1 975b) for articular cartilage lesions and also included physeal lesions in the 
syndrome of equine Oc. 
Pool ( 1 993) has recommended that OC be divided into an idiopathic form resulting from a 
constitutional defect in the cartilage model, and an acquired form. The idiopathic form 
causes lesions, commonly bilateraI Iy, in recognised predilection sites, and it is proposed that 
excessive biomechanical forces on a normal structure, or normal forces on a weakened 
structure, may be responsible for these lesions. The acquired form is proposed to cause 
generalised lesions without bilateral symmetry in a range of sites simultaneously, and may 
be caused by a variety of factors including nutritional excess and defic iency. 
OC is clearly intimately related to DOD, but whereas DOD was previously defined as a 
collective term for diseases associated with a failure of endochondral ossification, OC is 
spec ifically characterised by a subsequent retention of cartilage. 
Retained cartilage has been investigated histologically and biochemically to better define 
the lesion morphology in osteochondrosis. Brown and MacCallum (1976) identified retained 
carti lage in the distal third metacarpal and metatarsal physes of foals in a survey of normal 
foals. The authors considered that these may be within normal variation, or may have been 
evidence of the presence of subclinical physitis .  Similarly, Hill et al. ( 1 985) found retention 
of hypertrophic chondrocytes in a high percentage of one day old p igs examined. It was 
proposed that these retentions may have been part of the normal growth pattern for these 
p igs. 
Retained cartilage lesions in the distal radial growth plate of foals at 3 to 70 days of age 
contained one of three abnormal ities: retention of carti lage, retention of cartilage with 
discontinuity of the primary spongiosa (with or without identification of microfracture), and 
microfracture without retained cartilage (Firth and Poulos 1 984). It was proposed that 
microfracture may be the fore-runner to carti lage retention with discontinuity of primary 
spongiosa, as unl ike primary retention, the retained cartilage had regular borders. However, 
primary retention followed by microfracture could not be exc luded. The possibil ity of the 
different types of OC having different aetiologies was raised. In a study of calves reared on 
concrete and c lay flooring, mild retention of carti lage with no obvious discontinuity in the 
primary spongiosa was not considered to be a lesion (White et al. 1 984). 
1 0  
Biochemical abnormalities include decreased concentration and aggregations In pigs 
(Nakano et al. 1 979, 1 985;  Nakano and Aherne 1 995) and horses (Lill ich et al. 1 997) and a 
decrease in the amount, crosslinking and relative distribution of col lagen types (Wardale 
and Duance 1 994). Farnum and Wilsman ( 1 986) identified a pericellular matrix 
macromolecular material which supposedly stabilised the interface between chondrocytes 
and their matrix, which was disturbed in osteochondrotic cartilage. Other abnormalities 
include increased intracellular lipid inclusions in osteochondrotic articular cartilage of dogs, 
particularly in the superficial chondrocytes (Kincaid et al. 1 985b; Carlson et al. 1 986) and 
decreased carbonic anhydrase activity in osteochondrotic cartilage of pigs (Ekman and 
Ridderstrale 1 992). 
Investigation of the pathogenesis of avian tibial dyschondroplasia has indicated that retained 
chondrocytes are not a result of increased chondrocyte proliferation or diminished activity 
of osteo/chondroclasts, and that oxygen is not limiting to these cells. Instead, a fai lure of 
chondrocyte differentiation, such that there is an accumulation of transitional chondrocytes, 
has been impl icated (Thorp et al. 1 993 ). Unl ike the findings of Wardale and Duance ( 1 994), 
Thorp et al. ( 1 993 ) found no difference in collagen type I I  distribution, and therefore 
concluded that the failure of chondrocyte differentiation could be localised to the 
transitional chondrocytes. 
1.4.3 Physitis 
Physitis (also referred to as epiphysitis, physosis, physeal dysplasia and physeal 
dyschondroplasia) is a disease associated with rapidly growing well muscled animals on a 
high plane of nutrition (Thompson et al. 1 988). 
The physes of the distal third metacarpal/metatarsal bone, distal radius and distal tibia are 
the most commonly affected (Turner 1 987), but other physes, including those in the 
vertebrae (Mayhew et al. 1 978; Powers et al. 1 986), may also be involved. Physitis is 
considered to be a self limiting disease occurring when growth plates are open (Turner 
1 987; Firth 1 990). There are two peaks of incidence in growing horses, the first is between 4 
and 8 months of age, when the distal third metacarpal/metatarsal bone is most affected, and 
the second is between one and two years, in the distal radius and tibia (Turner 1987). The 
onset of physitis has also been linked with the period of closure of growth plates, and it has 
1 1  
been p roposed that this may be due to an increased susceptibil ity of the growth p lates at this 
time to mechanical compression (Rooney 1 963). 
Mildly affected horses exhibit subtle pain free swelling of the physes, and appear otherwise 
normal. Severely affected horses have painful warm swel l ings at the level of the physes, and 
secondary problems such as contracted tendons (flexural deformities) and stress fractures 
may supervene (Fretz 1 980; Auer 1 983 ; Firth 1 990). 
Radiographic changes associated with physitis include angular limb deformities, 
metaphyseal flaring, broadening and asymmetry of the metaphysis, metaphyseal sclerosis, 
asymmetrical cortical thickness, regional flaring of the epiphysis, and a scal lop ing and 
wedging of the epiphysis (Turner 1 987). leffcott ( 1 99 1 )  considers that physitis is the cl inical 
manifestation of physeal osteochondrosis, whereas McIlwraith ( 1 996) disagrees and 
considers them separate entities. A distinction between physeal histological changes for 
physitis and the physeal form of osteochondrosis are not reported, but any differences are 
likely to be small (Firth 1 990). 
1.4.4 Cervical vertebral malformation (wobbler syndrome) 
The two most common syndromes in cervical vertebral malformation (CVM) are cranial 
cervical vertebral instability most commonly present at the articulation between second and 
third cervical vertebrae (C2-3) to C4-5 ,  and caudal cervical stenosis at C5-6 and C6-7 (Pool 
1 993). Cranial cervical instability occurs in young horses usually in their first year and is 
known as a dynamic instabil ity, in which malformation of the dorsal facets results in 
stenosis that is exaggerated by flexion. Caudal cervical stenosis is a static stenosis, caused 
by enlargement of the articular process and/or soft tissue hypertrophy in horses usually 
between one and four years of age. 
Mayhew et al. ( 1 978) performed an extensive investigation of sp inal cord disease and 
possible aetiologies. The authors found an association between CVM (which they called 
cervical stenotic myelopathy) and well grown horses. It was proposed that the risk of 
develop ing CVM was enhanced by performance based selection of horses for rap id growth 
and large size, and excessive nutrition to ensure that genetic potential for growth was met. 
Donawick et al. ( 1 989) also made the association between CVM and overfeeding and 
included vigorous exercise. These authors found that feed and exercise restriction of 
1 2  
affected young horses could amel iorate the clinical signs. Powers et al. ( 1 986) examined 25 
horses with cervical static stenosis, and in one horse, found evidence of osteochondrosis in 
the articular facet. Based on the extensive examinations at postmortem, the authors 
concluded that biomechanical forces and osteochondrosis were the most likely factors 
involved. 
Mayhew et al. ( 1 978) found increased osteochondrotic lesions in the costochondral junction 
in animals with CVM, although an increase in OC lesions in other joints was not found. 
However Stewart et al. ( 1 99 1 )  examined 1 1  Thoroughbreds with CVM and found an 
increase in the frequency and severity of OC lesions in the limbs and an increase in the 
severity but not frequency of lesions in the cervical vertebrae compared with seven age and 
breed matched controls. These authors came to the conclusion that they could not implicate 
OC as the cause of the CVM but that the aetiological factors responsible for the CVM were 
likely to also predispose horses to OC. This  was supported by the finding that in some areas 
compression sites were found without any evidence of physeal or articular Oc. 
Wagner et al. ( 1 985a) bred two stal lions with cervical vertebral instabi l ity to 1 2  mares with 
the same condition. This study was unable to demonstrate an increased incidence of CVM in 
offspring. However, the offspring suffered a high level of osteochondrosis, physitis and 
flexural l imb deformities, but the numbers of animals were too small to test the significance 
of this association. 
Pool ( 1 993) considers that the pathological changes seen in cases of cervical vertebral 
malformation are not consistent with osteochondrosis, but instead are manifestations of 
hyperflexion in the case of cervical vertebral instabil ity and hyperextension in cervical 
vertebral stenosis. He believes that the articular cartilage lesions are consistent with a 
secondary degenerative joint disease as a result of biomechanically induced remodelling. 
1.4.5 Limb deformities 
Limb deformities or "bent legs" are a common problem in growing foals. Limbs can be 
deformed such that there is a deviation in the normal conformation when viewed from along 
the long axis of the horse (angular l imb deformity) or from the lateral perspective (flexural 
l imb deformity). 
1 3  
1.4.5.1 Angular limb deformity (ALD) 
Angular limb deformities most often occur at the carpus and less frequently in the tarsus and 
distal third metacarpal/metatarsal bone. Fretz ( 1 980) describes five classifications for ALD 
which include imbalanced ep iphyseal growth and defects in endochondral ossification of 
the cuboidal bones of the carpus and tarsus.  With the former condition growth p late lesions 
can have the same radiographic and histologic appearances as found in physitis (Hurtig and 
Pool 1 996), and OC may be associated with the latter (Fretz 1 980; Auer et ai. 1983) In a 
study of six foals examined for ALD, four had OCD in either the third or fourth carpal 
bones (McLaughlin et ai. 1 98 1 ). Most foals are born with a mild angular l imb deformity (5 
to 1 0  degrees) which resolves within about four weeks (Auer et ai. 1 982). 
1.4.5.2 Flexural limb deformity 
A number of factors have been implicated in the aetiology of flexural limb deformities 
including malposition in utero (Mason 1 98 1  ), rap id growth (Hintz 1 996) and 
musculoskeletal lesions that may cause pain (Wagner et ai. 1 985b) such as osteochondrosis 
(Moore and McI lwraith 1976). Foals receiving diets deficient in copper ( 1 .7 mg Cu/kg OM) 
showed clinical signs of musculoskeletal pain and developed flexural l imb deformities 
(Bridges and Harris 1 988). Rooney ( 1 966) carried out postmortems on approximately 50 
affected foetuses and foals with flexural deformities and found no evidence of abnormalities 
in endochondral ossification, but all foals had malformed distal th ird metacarpal/metatarsal 
bones. Based on these findings, Rooney ( 1 966) proposed that flexural deformity is 
secondary to joint instability and subsequent innervation of the musculature due to "random 
uncoordinated proprioceptive impulses". The impulses cause the contraction of all muscles, 
but joint flexion results because the flexor muscles are stronger than the extensors. Szczurek 
et al. ( 1 988) attempted to investigate the effect of confinement, level of nutrition and foot 
conformation on flexural limb deformities, but the condition could not be induced. 
1.4.6 Proposed aetiologies for DOD 
As a result of the limited abil ity of bone and cartilage to respond to injury, different 
aetiologies of DOD may cause morphologically similar clinical signs and/or postmortem 
lesions, despite having different mechanisms. This situation has been recognised with tibial 
dyschondrop lasia in birds (Orth and Cook 1 994) in which analysis of dyschondroplastic 
cartilage caused by different aetiologies appears similar but has different ultrastructural 
1 4  
abnormalities. A s  a result of this, information gained regarding a particular model for DOD 
may not pertain to other forms. 
1.4.6.1 Growth rate 
Mature skeletal dimensions are genetically predetermined, with the foal being 
approximately 60% of mature skeletal size at birth . However, nutrition can have a marked 
effect on growth rates, which is primari ly due to an increase in body weight rather than 
skeletal growth (Green 1969). Even with undernutrition, height is less affected than body 
weight (Green 1 96 1 ). 
There is a remarkable simi larity in growth curves for different species of horses in different 
climates and under different management regimes (Green 1 96 1 ). Growth rates are maximal 
in neonates, and decrease with age (Green 1 96 1 ). However growth rates in yearlings are 
considered to be more a function of season than age (Pagan et al. 1 996), and yearlings in 
training stables can exhibit irregular growth rates (Green 1 976). Thoroughbreds retain the 
capabi l ity for rapid growth (increase in weight) until 1 5  to 1 6  months of age, and in the next 
4 -5 months will increase their weight by only around 1 0  % (Jelan et al. 1 996). However, 
dogs fed restricted diets grew slower and had significantly decreased skeletal dimensions 
(Dammrich 1 99 1 ) . ,  whi le dogs fed ad libitum, had more OC lesions, and thinner 
metaphyseal spongiosa. Weakened subchondral bone support combined with extra skeletal 
requirements for weight bearing were strongly impl icated as the causes for the OC lesions. 
In a study on overnutrition in Great Danes, puppies were fed diets rich in energy, protein, 
calcium and phosphorus from birth to weaning. Severe abnormal ities in endochondral 
ossification were found in the costochondral junction, articular cartilage of the humeral head 
and the cervical vertebrae. Other abnormalities in bone were found, and the lesions were 
associated with excessive apposition of bone and retardation in osteocytic osteolysis and 
chondrolysis (Hedhammer et al. 1 974). High calcium may also have had a significant role in 
the induction of lesions and will be discussed below. 
Reiland ( 1 978) found that restricting growth rates in domestic pigs greatly decreased the 
incidence of Oc. Rapid growth rate up to 4 months of age fol lowed by restricted growth 
rates did not greatly reduce the incidence of lesions, indicating that the growth rate of pigs 
in the first 4 months of life may be of great importance to the incidence of lesions in older 
pigs. Progeny of pigs bred to wild hogs grew slower than pure-bred domestic pigs and had 
1 5  
no lesions of osteochondrosis compared with domestic pigs, of which approximately 90 % 
were affected. The domestic pigs commonly had a marked inferior prognathism which is a 
common finding in humans with acromegaly, and thus growth hormone may have a 
significant role in the induction of Oc. 
Similar effects of rapid growth rates have been found with poultry (Poulos et al. 1 978), and 
bulls (Reiland et al. 1 978). The bulls were slaughtered at simi lar weights thus the rapidly 
growing animals were examined at a younger age than the bulls on the lower energy 
intensity diet. This may have influenced this study if lesions tended to resolve in older 
animals (Reiland et al. 1 978). 
Pagan and Jackson ( 1 996) found a pattern of onset of osteochondrosis lesions in foals on a 
Thoroughbred farm in Kentucky which may impl icate high growth rates at particular times 
in development. For example hock and stifle lesions were commonly diagnosed between 
304 and 335  days in heavy foals that were growing rapidly, and so it was considered that 
rapid growth in heavy foals following weaning may predispose to the clin ical expression of 
these lesions. However in a recent study no significant differences were found in body­
weight or height between normal and DOD-affected horses (Jelan et al. 1 996). 
1.4.6.2 Trauma 
Internal trauma, or trauma due to ground reaction forces in response to weight bearing has 
become a popular theory for the aetiology of DOD. 
Stromberg and Rejno ( 1 978) considered that the bilateral and symmetrical nature of lesions 
indicated that the aetiology for DOD probably included an underlying cause other than 
simply trauma such as a metabolic derangement. However Pool ( 1 993 ) has argued that the 
location of predilection sites in the horse coincide with areas of high biomechanical loads, 
and the bi lateral nature of the disease may be due to the effect of excessive loading in a 
particular window of vulnerabil ity. McIlwraith ( 1 993) found that while lesions were often 
symmetrical, it was rare for more than one set of joints to be affected, supporting the 
biomechanical aetiology operating in a window of vulnerability. Firth et al. ( 1 988) proposed 
that altering hoof balance using wedges on the weight bearing surface of the hoof may 
predispose horses to OC, but this did not provide a model for OC and alterations to 
compressive forces and strain in the limb returned to normal over 1 0  days without any 
lasting side-effects. 
1 6  
Experimental evidence for the biomechanical aetiology was provided in pigs by inducing 
OC in a front leg by tying up the contralateral leg (Grondalen and Grondalen 1 974). Olsson 
and Reiland ( 1 978) proposed that the location of naturally occurring lesions in pigs at sites 
subjected to greater than normal biomechanical forces, suggests that weight-bearing or 
ground reaction forces may be an important triggering factor for OC, but not sufficient on 
its own. Nakano and Aherne ( 1 988) found that the prevalence of OC dramatically increased 
after loading and unloading pigs on a transport vehicle, compared with control pigs. Trauma 
was considered to be an important factor in increasing the severity and prevalence of lesions 
found in calves raised on concrete floors compared with controls on clay (White et al. 
1 984). Pool ( 1 993) has recognised the difficulty in differentiating between lesions caused by 
normal biomechanical activity on weakened connective tissue versus excessive 
biomechanical activity on normal connective tissue structures. 
The presence of OC lesions in the costochondral junction of pigs (Reiland 1 978), horses, 
(Rejno and Stromberg 1 978) and calves (White et al. 1 984) was considered to mitigate 
against biomechanical trauma being the primary cause. However, the costochondral junction 
probably experiences continual traction and compression forces due to respiratory 
movements, support of the vertebral column and support of thoracic and abdominal 
contents. 
1.4.6.3 Ischaemia 
A fai lure of vascular penetration of the zone of hypertrophic chondrocytes has been 
recognised as a consistent histological feature of osteochondrosis (Jeffcott 1 99 1 ). 
Experimental disruption of the metaphyseal blood supply at the ossification front of physes 
will reproduce retained cartilage lesions (Trueta and Amato 1 960). 
The role of cartilage canals in the pathogenesis of OC has been investigated in swine 
(Kincaid and Lidvall 1 982; Kincaid et al. 1 985a; Woodard et al. 1 987b; Visco et al. 1 99 1 ), 
and more recently in horses (Carlson et al. 1 995). Carti lage canals provide conduits for a 
capillary network in the "growth" cartilages, have been associated with the nutrition of these 
cartilages (Stockwel l  1 97 1 ), and are found in epiphyseal, but not articular cartilage (Carlson 
et al. 1 989; Firth and Poulos 1 983). Disruption of these canals may cause an ischaemic 
necrosis of the cartilage consistent with the earliest lesions of osteochondrosis (Kincaid et 
al. 1 985a; Woodard et al. 1 987b; Carlson et al. 1 989). It has also been shown that large 
1 7  
forces appl ied to articular cartilages of dogs, that were insufficient t o  cause long bone 
fracture, caused chondrocyte necrosis (Donohue et al. 1 983) .  These large in vivo forces may 
cause degeneration of carti lage canals, especially in anatomical locations where immature 
carti lage is excessively thick. Degeneration of cartilage canals m ight also be part of the 
normal process of cartilage maturation with age, but Woodard et al. ( 1 987b) proposed that 
in OC, the associated cartilage necrosis and chondrolysis is excessive. 
In a recent study, Carlson et al. ( 1 995) found that cartilage canals in medial femoral 
condyles, lateral femoral trochlear ridge and distal ends of the tibia and proximal phalanx 
were evident only in horses less than 7 months of age, and that lesions of osteochondrosis 
found in horses between 3 weeks and 5 months of age were always associated with necrotic 
cartilage canal blood vessels. This strongly supports the role of carti lage canals in the early 
lesions of OC and suggests that the formation of lesions has a remarkably narrow time 
frame. This narrow "window" is supported by a radiographic survey performed on 77 
standardbreds in Sweden (Carlsten et al. 1 993), in which 1 9  (25 %) horses had radiographic 
lesions consistent with osteochondrosis of the tarsocrural or fetlock joints at 1 2  months of 
age. Four horses showed radiographic changes before 1 month of age, four before 3 months, 
and 1 1  before 5 months. Eighteen horses had early radiographic changes which had resolved 
before 8 months. No new lesions developed after 8 months of age. The conclusion was that 
osteochondrosis of the fetlock and tarsocrural joint develops early, and can be considered 
permanent in cases which persist until 1 2  months of age (Carlsten et al. 1 993). 
1.4.6.4 Genetics 
In 9 1  cases of equine OC diagnosed at the Royal Veterinary College in Stockholm, 
Stromberg and Rejno ( 1 978) found an over-representation of the progeny of two sires. One 
was an English Thoroughbred stall ion renowned for having foals that became large horses, 
and the other a Swedish Thoroughbred stallion. The authors noted that, in general, animals 
affected with OC grew rapidly and were offspring of large parents. 
Further studies have been performed in Scandinavia, using larger numbers of standardbred 
and warmblood horses. The detection of osteochondrosis was based on radiographic studies 
of certain predilection sites, and the true incidence of OC was probably greater than reported 
because of the difficulty in detecting mild cartilage lesions. The incidence varied from 1 0  to 
26 % in different populations, with al l studies showing a difference in the incidence of OC 
in the progeny from different stal lions. The variable difference between stall ions suggests 
1 8  
that the mode of inheritance is polygenic rather than due to a single gene pair (Philipsson et 
al. 1 993 ; Phil ipsson 1 996). Schougaard et al. ( 1 990) examined the progeny from two 
cohorts of two standardbred stall ions and found little difference in the incidence of OC in 
the progeny, between years, compared with the (large) difference in incidence between the 
two stallions. Therefore it was concluded that genetics appeared to be more important than 
environment in the incidence of OC in that population. Heritabil ity estimates from these 
studies are summarised in Table 1 . 1 .  The heritability of OC in these populations appears to 
be quite high, suggesting that breeding policies to reduce the incidence of osteochondrosis 
would be successful .  The observation that some sires are asymptomatic but produce progeny 
with a high incidence of OC suggests that progeny testing would be the most successful 
technique for selecting stallions. 
Table 1.1 Studies on the incidence and heritability of osteochondrosis (OC) from Scandinavian 
countries 
Author Breed Horse Numbers 
Sire Progeny 
Hoppe and Standardbreds 
Philipsson 1 985 Sweden 69 1 30 
Gottland 60 208 
Swedish 
Wannbloods 39 99 
Schougaard et al. Standardbreds 9 325 
1 990 
Grondahl and Standardbreds 39 753' 
Dolvnik 1 993 
Philipsson et al. Standardbreds 
1 993 Random 24 674 
With OC 4 1 1 9 
. . * Only 644 horses were used for hentablllty calculations . 
Area Incidence Heritability 
examined 
Hock 26.2 Not Done 
Hock 1 0 . 1  Not Done 
Hock 1 5 .2 Not Done 
Hock 1 4.2 0.26 
Hock 1 4.3 0.52 
Fetlocks 1 1 .8 0.2 1 
Hock 1 0.5 0.34 
Hock 1 6.8  (all horses) 
Pool ( 1 993 ) considers that the identification of DOD caused by nutritional or other 
managemental causes is necessary before animals can be culled from the genetic pool. This 
assumes that animals showing DOD as a consequence of nutritional or managemental 
causes are not manifesting the disease because of a genetic predisposition. This assumption 
may not be val id. 
----------------------------------------
1 9  
There are l ikely to be differences in the incidence and heritabil ity of OC in different 
populations of horses, and also on the effect of environment on the incidence of DOD. The 
incidence and heritabil ity of OC in Australian and New Zealand Thoroughbreds have not 
been studied 
1.4.6.5 Nutrition 
Energy 
Glade and Belling ( 1 984) found that diets high in energy and protein caused a high, early 
peak in insulin and glucose, and relatively low T4 levels postprandially. It was proposed 
that the high, early insulin peak stimulated the conversion of T4 to T3, preventing the 
"normal", transient, postprandial hyperthyroxemia. It was postulated that this 
hyperthyroxemia was important in the differentiation of hypertrophic chondrocytes. In a 
further experiment Glade and Bel ling ( 1 986) found that high energy/protein diets fed to 
weanlings were associated with decreased hexosamine and hydroxyprol ine content and 
increased DNA content of physeal cartilage harvested in vivo from the distal radial growth 
plate. These findings indicated a decreased production of cartilage matrix by chondrocytes, 
and an increase in chondrocyte cellularity. It was proposed that these changes to the matrix 
may have been responsible for a failure of metaphyseal capillary invasion. Further support 
for the role of aberrations in endogenous hormones was provided by Ralston ( 1 996) who 
found that postprandial glucose and insulin in four horses with OCD was significantly 
higher than in 1 5  controls. 
Savage et al. ( 1 993a) fed foals 1 29 % NRC ( 1 989) energy recommendations for 1 6  to 1 8  
weeks, and found a significantly greater incidence of dyschondroplasia (OC) than in 
controls fed 1 00 % NRC ( 1 989) energy recommendations. No mechanism could be 
elucidated from this trial, and glucose, insulin and thyroid hormones were not measured. 
There was no s ignificant effect of dietary energy level on average daily live weight gain, 
which would preclude a biomechanical aetiology unless high energy diets resulted in a 
greater degree of activity in their restricted pens. The source of supplemental energy was 
maize oil and maize starch (fat and complex carbohydrates) which was expected to have 
less effect on insulin and glucose than the soluble carbohydrates fed in the work of Glade 
and Bell ing ( 1 986). However in the work of Savage et al. ( 1993a), the fai lure of an 
increased energy intake to increase live weight gain is surprising. 
20 
Variable results have been obtained from a number of experiments with pigs, investigating 
the relationship between dietary energy, growth rate and OC (Nakano et al. 1 987). Carlson 
et al. ( 1 988) recognised the difficulty in differentiating the effect of increased bodyweight 
from increased growth rate, and therefore they slaughtered gilts receiving different levels of 
dietary energy at the same weight, and found that at the same weight, the high dietary 
energy group had a higher incidence of Oc. 
Protein 
Savage ( l 993a) fed foals on two different levels of dietary protein ( 1 00 and 1 29% NRC 
( 1 989) recommendations. None of the foals receiving the high protein diet had clinical or 
radiographic evidence of dyschondroplasia (OC), and at postmortem the incidence of 
lesions was not significantly different between the two diets. Schryver et al. ( 1 987) found 
that protein diets at approximately 1 40 % of NRC recommendations had no skeletal effects 
in growing horses including calcium and phosphorus metabol ism. Several investigators 
found that the dietary level of protein in pigs had no effect on the occurrence of skeletal 
lesions (Reiland 1 978); Woodard et al. 1987a; Jorgensen 1 995) .  
Calcium/Phosphorus/Vitamin D 
Excessive dietary calcium can affect bone by increasing growth rate, increasing calcitonin 
production, decreasing trace mineral absorption and decreasing iodine uptake by the thyroid 
gland (Kronfeld et al. 1 990). Krook and Maylin ( 1 988) considered that high dietary calcium 
was a major factor in the presence of lesions associated with catastrophic racetrack 
fractures. Osteochondrotic lesions were often associated with osteoporosis, and the lesions 
were consistent with changes found with hypercalcitonism . However, Savage et al. ( l 993b) 
found no effect of feeding 1 .95 % calcium to weanl ing foals which is greater than 3 times 
NRC ( 1 989) recommended levels on the incidence or severity of OC lesions. Knight et al. 
( 1 985)  performed an epidemiological study of the influence of dietary components on DOD 
on several farms in Kentucky and Ohio, and found that farms with the highest incidence of 
DOD had the lowest dietary calcium levels (other dietary associations were also reported) .  
Slater et al. ( 1 992) performed a matched case control study in dogs and found that high 
dietary calcium was associated with an increased risk of having Oc. In another study 
(Hazewinkel et al. 1 985 ;  Goedegebuure and Hazewinkel 1 986), Great Dane puppies fed 
three times the recommended levels of calcium had an increase in lesions consistent with 
OC, including retention of articular and physeal carti lage, disturbances in endochondral 
21  
ossification, and retarded maturation and remodell ing of trabecular and cortical bone. The 
authors postulated that hypercalcitoninism and hypoparathyroidism may have been 
responsible for the lesions although these changes were not supported by circulating levels 
of these hormones. High levels of dietary calcium have caused parafollicular cell 
hyperplasia in beagles (Stephens et al. 1985) and also in foetuses of ewes fed supplementary 
calcium (Corbellini et al. 1 99 1 ). The high concentrations of calcitonin impair the normal 
process of osteoclastic and chondroclastic resorption resulting in osteopetrosis and impaired 
endochondral ossification. The dogs receiving high levels of dietary calcium had lower 
growth rates, and so it was concluded that the effect of calcium on lesions was independent 
of growth rate. 
Diets h igh m phosphorus and low in calcium result m an increased production of 
parathyroid hormone which increases bone calcium resorption, causing nutritional 
secondary hyperparathyroidism. Diets high in phosphorus can also interfere with calcium 
absorption due to the formation of complexes which bind calcium. This dietary abnormality 
can be caused by grain based diets high in phosphorus and low in calcium, inadequate 
vitamin D, and pastures high in oxalates. Savage et al. ( 1 993b) found that feeding foals diets 
containing 1 .7 % phosphorus (388 % NRC 1 989 recommendations) for 1 6  to 1 8  weeks 
significantly increased incidence of dyschondroplasia (osteochondrosis) compared with 
controls. The mechanism was not elucidated, but in poultry, high phosphorus diet are 
considered to cause an acidosis, by exhausting cationic buffering, which interferes with 
provisional calcification (Lilburn et al. 1 989). However sodium bicarbonate fed to pigs did 
not reduce the incidence of skeletal disease (Van der Wal et al. 1 986). 
Vitamin D metabolites affect calcium and phosphorus metabolism by increasing their 
intestinal absorption, decreasing their urinary excretion, as wel l  as increasing osteoclastic 
bone resorption. Vitamin D can also regulate the expression of osteoblast phenotype, 
demonstrating an abi l ity to influence osteoblast differentiation and matrix competence 
(Owen et al. 1 99 1 ). Inadequate vitamin D is unl ikely to occur in horses at pasture because 
their dietary vitamin D requirement is low, as they obtain ample exposure to sunlight 
allowing the synthesis of Vitamin D in the skin. 
Copper/Zinc/Molybdenum 
22 
There is a growing weight of evidence to support the role of copper deficiency in the 
aetiology of DOD, and in particular but not limited to physitis. 
Copper deficiency in horses was first considered in 1 949 when it was investigated whether 
supplementary copper fed to horses would decrease the prevalence of cartilage erosions 
found incidentally at necropsy (Cupps and Howell 1 949). Numbers of animals were 
insufficient to test the significance of the relationship between copper nutrition and 
abnormalities in bone and cartilage development, but results from this study have formed 
the basis for the NRC copper requirements. 
More recently copper deficiency has been induced experimentally in foals, by feeding diets 
containing 1 .7 mg Cu/kg DM (Bridges and Harris 1 988) .  The clinical signs that developed 
were predominantly related to disorders in endochondral ossification. The foals first 
developed intermittent diarrhoea which coincided with decreasing serum copper 
concentrations . Lameness, stilted gait and tendon contracture developed within 6 weeks of 
the decrease in serum copper concentrations, but there was no apparent change in common 
indices of copper deficiency in other species such as packed cell volume, white blood cell 
count, coat colour and growth rates. At necropsy, there were lesions in the articular and 
growth plate cartilages consisting of retention of viable carti lage beyond the ossification 
front, areas of necrotic cartilage with intracartilaginous fractures in the hypertrophic zone of 
articular-epiphyseal and physeal carti lage, and eosinophilic streaks. Cartilage flaps, and free 
cartilage separated from joint surfaces were also present within the joint. Col lagen solubility 
was 3 to 6 fold greater in deficient than control foals. It was presumed that a reduction in 
lysyl oxidase activity was likely to be responsible for the clinical signs, although the 
enzyme was not assayed. A sim ilar increase in collagen solubil ity, and also a decrease in 
Iysyl oxidase (amine oxidase) activity has been found in the bones of chickens raised on 
copper-deficient diets (Rucker et al. 1969). 
In a study of 1 9  farms in Ohio and Kentucky, Knight et al. ( 1 985)  found that there was a 
significant negative correlation between dietary copper concentration and the prevalence of 
DOD. After adjustment of dietary rations, there was a significant decrease in the prevalence 
of DOD (Gabel et al. 1 987), and copper was the nutrient intake which changed most 
significantly. Epidemiological studies in Japan on pasture based weanlings also found a 
significant negative correlation between pasture copper concentrations and the cl inical 
23 
evidence of physitis of the fetlock (Fuj ikawa et al. 1 993 ; Asai et al. 1 993), but not the distal 
radius (Asai et al. 1 993). 
There are reports of favourable clinical responses of fetlock physitis to copper 
supplementation in Ireland (Egan and Murrin 1 973a), New Zealand (Carberry 1 978) and 
North America (Hildebran and Hunt 1 986). However these trials were poorly controlled 
with several factors changing simultaneously. 
Copper supplementation trials have been performed on mixed breed foals in North America 
(Knight et al. 1 990; Hurtig et al. 1 990, 1 993 ).  Knight et al. ( 1 990) fed mares and foals with 
32 and 5 5  mg Cu/kg DM respectively compared with mare and foals receiving 1 3  and 1 5  
mg Cu/kg DM respectively. Foals were euthanased at either 90 or 1 80 days. There was a 
significant effect of copper supplementation on decreasing the incidence of lesions at 1 80 
days. Lesions consisted of thickening of articular and physeal carti lage, associated with a 
fai lure of vascular invasion, often with eosinophi lic streaks, necrosis in the hypertrophic 
chondrocytes and subchondral fibrosis. Thinned carti lage and osteophytes were also scored 
as lesions. The effect of mare supplementation could not be separated from that of foal 
supplementation, and there was little information on the copper status of the animals. Hurtig 
. et al. ( 1 990, 1 993) studied Quarter horse and Quarter horse x Thoroughbred foals, a number 
of which had pre-existing OC (in particular femoropatellar OCD). Copper supplemented 
foals received 25 mg Cu/kg OM versus 8 mg Cu/kg OM for controls. Both groups received 
1 20 % NRC recommendations for energy and protein as this was considered to be normal 
husbandry practice. The 3 to 4 month old foals received the experimental diets for 5 months, 
after which time all foals were euthanased. Copper supplementation significantly decreased 
the incidence of DOD, and produced histomorphometric evidence of an increase in 
subchondral bone volume and more trabeculae in the primary spongiosa. However 
histomorphometry was performed on a subset of the foals, the selection of which may have 
been biased, being based on the presence of lesions in the low copper group. The lesions 
were categorised as cartilaginous flaps, osteochondral fragments that were undermined or 
free in the joint space, cartilage thinning with erosion and eburnation, and carti lage defects 
with subchondral cavitation. No foal in the copper treated group had clinically significant 
DOD, and it was considered that including foals with pre-existing disease in both groups 
demonstrated a palliative effect of high copper diets on OC. In this experiment l iver copper 
concentration in the low copper group declined and was considerably lower than in the 
supplemented animals, but there was no difference in the serum copper concentration or 
24 
caeruloplasmin activity between groups. Biochemical studies of the articular cartilage of 
severely affected foals demonstrated a decrease in collagen crosslinking, and it was 
proposed that the effect of copper supplementation was to improve the biomechanical 
structure of the cartilage and subchondral bone matrix. Both of the above trials found a large 
number of lesions in the cervical vertebrae, and it was suggested (Hurtig et al. 1 990) that 
this site may be a good location for occult joint lesions in foals suspected of having Oc. It 
was further considered that the lesions created in the low dietary copper group occurred in 
multiple sites and the disease more resembled that found in copper deficient cattle than 
naturally occurring OC in horses. 
A wel l-reported syndrome of lameness in cattle attributed to copper deficiency and often 
l inked to high molybdenum intake (Suttle and Angus 1 978), includes osteoporosis, beading 
of the ribs, spontaneous fractures and enlargement of the fetlock joints. The predominant 
histopathological and radiological changes include an irregular widening of the growth 
plates, with tongues of retained cartilage (Irwin et al. 1 974; Mills et al. 1 976; Suttle and 
Angus 1 978). Calves raised on copper-deficient diets ( 1 .5 mg Cu/kg OM) exhibit angular 
l imb deformities, lameness, and swol len joints. The physis is focally thickened, with 
overgrown disorganised cartilage columns and some metaphyseal cartilage sequestration. 
Osteoblast numbers are depleted, but osteoclasts are numerous and mineralisation appears to 
be unaffected. Subchondral bone becomes rarefied with some cross trabeculation (Suttle and 
Angus 1 978). Irwin et al. ( 1 974) distinguished the changes seen with a molybdenum­
induced copper deficiency, which caused a "rickets- like" physeal widening, from the focal 
retentions seen in primary copper deficiency. 
Lambs raised on copper-deficient diets developed metaphyseal osteoporosis without 
widening of the growth plate, and was considered to be secondary to an inhibition of 
osteoblastic activity (Suttle et al. 1 972). Copper deficient lambs also exhibit decreased 
growth rates, spontaneous fractures, periosteal and muscle insertion l ifting and haemorrhage 
(Hogan et al. 1 97 1 ). 
OC associated with copper deficiency has been described in red deer in New Zealand 
(Thompson et al. 1 994). The lesions which were simi lar to those found in the experimental 
copper deficiency of foals (Bridges and Harris 1 988), included intracartilaginous fractures 
and flap formation in articular cartilage surfaces, as wel l  as epiphyseolysis in severe cases. 
25 
Lesions occurred commonly in carpal, tarsal, femoropatellar and coxofemoral joints, and 
were usually bilateral. 
The role of copper in DOD has been presumed to be due to a reduction in the activity of the 
cuproenzyme lysyl oxidase (Bridges et al. 1 984; Bridges and Harris 1 988). This is 
supported by the similarities of growth plate lesions seen with osteolathyrism and those 
induced experimentally in copper deficient rats fed thiomolybdates (Spence et al. 1 980) in 
which the growth plate abnormalities included cartilage dysplasia resulting from a decrease 
in osteogenesis, which progressed to the cessation of osteogenesis. However the activity of 
lysyl oxidase on copper deficient diets has not been measured in horses. 
There are a number of reports of skeletal abnormalities in horses fed excessive amounts of 
dietary zinc e ither experimentally (Wil loughby et al. 1 972; Bridges and Moffitt 1 990) or by 
ingestion of polluted feed (Gunson et al. 1 982; Eamans et al. 1 984; Campbell-Beggs 1 994). 
Cl inical signs and pathological changes (severe generalised OC) were consistent with the 
changes found by Bridges and Harris ( 1 988) in foals fed a copper deficient diet, and a zinc 
induced secondary copper deficiency was the most l ikely cause for the skeletal changes. 
Bridges and Moffitt ( 1 990) found that foals fed over 1 000 mg Zn/kg OM had significantly 
lower serum copper concentration, and at euthanasia after 9 to 22 weeks on the high zinc 
diets the serum copper concentration was less than 0.2 mg Cull. 
Manganese 
Manganese is important for the integrity of connective tissue matrices, through its role in 
the production of chondroitin sulphate, and also by its activation of alkaline phosphatase 
(Georgievskii et al. 1 982; Lewis 1 995). Manganese deficiency may have been responsible 
for a case of weak, brittle bones and lameness with joint pain in a neonatal foal on pasture 
polluted with smelter effluent which had been treated with high concentrations of lime to 
decrease soil pH, and consequently with decreased manganese avai labil ity (Cowgill et al. 
1 980). 
26 
Vitamin A 
Vitamin A is a l ipid soluble vitamin that induces the production of osteocalcin (Schule et al. 
1 990), and thereby regulates osteoblast activity. Reiland ( 1 978) found no effect of 
supplemented vitamin A (3500 to 1 5000 IV/kg bodyweightlweek) in a group of pigs with a 
high incidence of osteochondrosis. Vitamin A supplementation at very high levels ( 1 00 to 
1 000 times NRC ( 1 989) recommendations) caused physitis and contracted tendons in a 
small number of fill ies (Kronfeld et al. 1 990). Supplementation with vitamin A was also 
associated with an increased incidence of DOD on farms in Kentucky and Ohio (Knight et 
at. 1 985). Vitamin A may interact with trace element metabolism, and should therefore not 
be supplemented excessively (Kronfeld et al. 1 990). 
1.4.6.6 Dexamethasone 
Glade et al. 1 983 found macroscopic histological changes consistent with OC in the 
proximal femoral physis and articular/epiphyseal cartilage of pony foals treated with 0.5 
mg dexamethasone/ 1 00 kg bodyweightl day for 3 ,  8 or 1 1  months, and horse foals treated 
with 5 .0  mg! 1 00 kg bodyweight / day for 20 weeks. The changes consistently showed a 
decrease in chondrocyte activity, and a failure of epiphyseal or metaphyseal vessels to 
penetrate hypertrophic cartilage. 
1.4.6.7 Hypothyroidism 
Hypothyroidism has been investigated as a cause of DOD in Canada. A syndrome has been 
defined in animals diagnosed with hyperplastic goitre, secondary to excessive nitrate intake. 
The skeletal abnormalities include delayed ossification, stunted stature, cuboidal bone 
hypoplasia, angular and flexural l imb deformities and mandibular prognathism (McLaughlin 
and Doige 1 98 1 ). This condition is congenital, and without disruption of endochondral 
ossification, and it is not considered to be a DOD. However another foal was diagnosed with 
hypothyroid of unknown aetiology, and had severe OCD evident clinically and at 
postmortem, as well as physeal retained cartilage and transverse trabeculation in the 
metaphyses (Vivrette et al. 1 984). This may indicate that hypothyroidism should be 
included as a possible cause of DOD. 
1.4.6.8 Infection 
The proposal that ischaemic necrosis of carti lage is important in the pathogenesis of DOD 
indicates that thrombogenic vascular occlusion due to haematogenous bacteria may be 
associated with osteochondrotic lesions. Firth and Goedegebuure ( 1 988) have shown that 
27 
predilection sites for septic arthritis include locations in the epiphysis with the thickest 
carti lage. Growth plate lesions were found predominantly in sites of greatest inclination. 
These sites are also commonly affected with DOD. This association has not yet been 
investigated. 
1.4.7 Conclusions from the review on DOD 
The exact relationship between the different diseases comprising DOD remains contentious, 
and the nomenclature is potentially confusing due to different interpretations of definitions. 
For example these may refer to aetiological factors of a subtle intrinsic nature (for example 
organelle based abnormality), or to gross, non quantifiable extrinsic factors such as 
biomechanical forces acting on tissue. This is further confused because the term DOD is 
used to refer to abnormal ities present, unseen and possibly undetectable by conventional 
pathological examination, as well as abnormalities that are present and identifiable but 
clinically s ilent, or lesion present and clinically evident. 
The pathogenesis of a disruption in endochondral ossification is difficult to study due to the 
uniformity of the response of bone to a range of insults, the lack of pathognomonic changes 
in affected bone and cartilage, and the overriding effects of continued weight bearing on 
damaged tissue. Distribution of lesions has been suggested as a useful method of 
differentiating aetiologies, but this is based mainly on opinion and observations. 
Tissue from retained cartilage lesions has been investigated in several species, including the 
horse, to define the nature of lesions. These are useful approaches to investigate 
abnormalities present in affected cartilage, and may be helpfu l  in identifying potential 
aetiologies. However, it is impossible to differentiate between changes to tissue that cause 
lesions, or changes occurring due to the presence of lesions. There remains a need for 
control led experimental studies to investigate possible aetiologies. 
Several aetiologies are closely related. Excessive energy intake results in increased growth 
rate, which in tum increases trauma due to weight bearing, which may cause ischaemia by 
damage to cartilage canals. Other aetiologies such as excessive intakes of calcium, zinc and 
excessive dexamethasone administration all have the potential to influence copper 
avai lability, and further study may reveal a unified nutritional aetiology. 
28 
Excessive energy intake appears to be a reliable model for the induction of DOD, but the 
mechanism is stil l  unresolved, especially issues regarding the effect of different energy 
sources. Copper deficiency is also a reliable model for the induction of DOD. Presently the 
dietary copper intakes that have been recommended by some authors are considerably 
higher than pasture diets can provide. Therefore copper may be an important factor in the 
high prevalence of DOD found in pasture-based horse raising systems, as uti l ised in New 
Zealand. It is evident that more information is required on copper metabol ism in horses. 
1.5 Review on copper nutrition 
1.5.1 Introduction 
Copper, discovered between 3000 and 6000 BC, was named after the Island of Cyprus, the 
location of the ancient copper mines (Wertime 1 964). S ince its discovery, it has had a great 
impact on humanity. Copper was valued as an ornamental metal, but it was also the first 
metal to be used for practical purposes in alloys with tin (bronze), zinc (brass) and nickel 
and zinc (nickel si lver). 
Copper was recognised as an essential nutrient in the 1 920s, when rats fed copper-deficient 
diets developed an anaemia which would not respond to iron supplementation (McHargue 
1 926; Hart et al. 1 928). Early work on copper requirements in horses was performed in 
1 949, when Cupps and Howell ( 1 949) proposed that copper supplementation may prevent 
erosions found in articular cartilages. The horse numbers were too small to be conclusive, 
and the role of copper in bone and cartilage development of foals sti l l  remains a research 
priority. 
Copper is a transitional metal with atomic number 29 and a mass of 63 .5 .  There are two 
naturally occurring stable isotopes, 63CU and 65CU, in relative abundance of approximately 
69 and 3 1  % respectively. There are 3 radioisotopes 6 ICU, 64Cu, and 67CU, with half l ives of 
approximately 3, 1 2  and 60 hours respectively. Copper ions exist as either the monovalent 
(cuprous) ion which forms tetrahedral complexes or the divalent (cupric) ion that forms 
square planar complexes and is a potent oxidising agent. In biological systems, copper is 
predominantly in the form of the cupric ion (Cu++). However the cuprous ion (Cu+) also 
exists, and the ready exchange of valencies accounts for copper's activity in oxidation-
\ 
29 
reduction reactions and the scavenging of free radicals. At physiological pH, copper is 
almost completely bound, forming insoluble complexes. In acidic environments there are 
higher concentrations of free and chelated (protein-bound) copper. 
1 .5.2 Copper content and distribution in the body 
There are l ittle data on the distribution of copper in the body of horses, although Schryver et 
al. ( 1 974) calculated the copper content of horses less than one year of age to be about 4 to 5 
mg Cu/kg l iveweight (L W) and Meyer and Ahlswede ( 1 978) found the body copper 
concentration of neonates to be 5 mg Cu/kg LW. The high copper concentration in the 
neonate is largely due to the high l iver copper store which accounted for approximately 60 
% of the total body copper. The copper concentration in the neonatal liver varies widely, 
and in one survey I iver copper concentration was found to vary from 1 8 1  to 874 mg Cu/kg 
DM at 1 to 2 weeks postpartum (Meyer and Tiegs 1 995). Simi lar widely variable liver 
copper concentrations have been reported in horses up to eight months of age (Egan and 
Murrin 1 973b). Schryver et al. ( 1 974) found that l iver copper accounted for approximately 
2.5 to 5 percent of total body copper in horses from 1 2  to 24 months of age. This is very low 
compared with ruminants (Grace 1 983a), and is contributed to by both a relatively low liver 
copper concentration (23 and 14 mg Cu/kg fat free DM respectively) and a high total body 
copper concentration. Cymbal uk and Christensen ( 1 986) found a similar liver copper 
concentration in yearlings (approximately 20 mg Cu/kg DM), and slightly higher kidney 
copper concentrations (20 to 30 mg Cu/kg DM). Liver copper concentration is responsive to 
dietary copper intake, and was approximately 4300 mg Cu/kg DM in ponies fed a diet 
containing 790 mg Cu/kg DM (Smith et al. 1 975). This abi lity of the liver copper 
concentration to vary widely according to dietary copper intakes is likely to cause large 
differences between studies on the copper distribution in horses, and any such study should 
include information on the dietary copper intake. It may also be helpful when presenting 
total body copper concentrations to separate values for the liver due to this wide variation, 
as is done in ruminants (Suttle 1 979; Grace 1 983a). 
Schryver et al. ( 1 974) found the total body copper concentration (excluding the liver) of 
more mature horses (24 months), to be approximately 3 mg Cu/kg LW. This is considerably 
greater than estimations of the copper content of man (Sass-Kortsak 1 965) (with l iver) and 
lambs and calves (Suttle 1 979; Grace 1 983a) (without liver), which are both approximately 
1 mg Cu/kg DM. 
30 
1 .5.3 Copper metabolism 
There are few data on copper metabolism in horses and much information is extrapolated 
from studies on other species. 
1 .5.3.1 Absorption 
The major site of copper absorption is the stomach and small intestine based on studies in 
which humans fed radioactive isotope (64CU) had rapid uptake of the isotope into systemic 
circulation (Beam and Kunkel 1 955) .  However it is believed that all segments of the 
intestine are able to absorb copper in ruminants (Grace 1 975) .  The acid environment of the 
stomach is important in the dissociation of copper complexes, and constituents in the bile 
may improve absorption suggesting the importance of the jejunum and more distal intestine 
for copper absorption (Solomons 1 988). One study on extensive large colon resection in 
horses found no decrease in copper absorption, although this may have been due to a wide 
variation in faecal excretion (Bertone et al. 1 989). 
Copper absorption is thought to require two distinct mechanisms. Copper in the intestinal 
lumen enters the mucosal cells by passive diffusion across an ionic gradient. Transport 
across the basolateral border of mucosal cells into interstitial fluids requires an energy 
dependent (ATPase) mechanism, which is saturable (Linder 1 99 1 ). At high dietary copper 
intakes, additional transport mechanisms across the basolateral membrane are used. These 
additional mechanisms are the site for the competitive antagonism of other transitional 
metal ions (Linder 1 99 1 ). The activity and abundance of the transport mechanisms are 
increased in cases of copper deficiency, pregnancy and cancer, and in humans they are 
decreased in the case of Menke's disease, a genetic disorder of copper absorption (Cohen et 
al. 1 979; Linder 1 99 1 ). Ionic copper in the mucosal cells is bound to a cytosolic protein 
called metallothionein. Metallothionein is likely to play an important regulatory role in the 
absorption of copper across the mucosal cells, and it is thought that with very high dietary 
copper, sloughing of mucosal cells laden with copper bound to metal lothionein may be 
important in preventing toxicity (Bremner 1 987). 
Copper excretion via the bile results in large differences between apparent absorption and 
true absorption. Re-absorption of copper is negligible (Evans 1 973) .  Studies with stable and 
radio-isotopes in humans estimate a copper absorption of between 40 and 70 % with wide 
31  
individual variation (King et al. 1978). Horses, ruminants, rats and humans demonstrate an 
ability to alter copper absorption relative to requirements with absorption increasing with 
low intakes, and decreasing with high intakes (Cymbal uk et al. 1 98 1 a; Woolliams et al. 
1 983;  Turnlund et al. 1 989; Linder 1 99 1 ). 
1.5.3.2 Transport and storage 
After absorption, copper has two recognised distribution patterns. Absorbed copper, bound 
to plasma proteins, is taken to the l iver which is the first phase of systemic copper 
distribution. I n  the liver, the copper is bound to caeruloplasmin and released into the 
circulation as the second phase of copper distribution. These distributions have been studied 
in vivo in rats and humans using radio and stable isotopes (Lee et al. 1 993; Scott and 
Turnlund 1 994) . 
Copper in the portal circulation is loosely bound to two plasma proteins, albumin and 
transcuprein. Copper is readily transferred between the two proteins in vitro (Linder et al. 
1 987). Albumin is abundant in plasma and can accommodate far more copper than is 
required for b inding to absorbed copper in the portal circulation . Transcuprein is a larger 
protein than albumin, and the affinity of copper for transcuprein is considered to be greater 
than albumin as both proteins bind a simi lar amount of copper despite a much greater 
concentration of albumin in p lasma (Weiss and Linder 1 985 ;  Tsai et al. 1 992). A very small 
amount (0.5%) of absorbed copper is bound to small peptides and amino acids (Linder 
1 99 1 ). As the portal circulation delivers the copper complexes to the liver, they are 
transferred to metallothionein in the cytosol of hepatocytes. A small percentage of the 
copper complexes transported from the gut are not transferred to the l iver, and enter the 
systemic circulation, and constitute the non-caeruloplasmin bound plasma copper (Linder 
1 99 1  ). 
Metallothionein-bound copper in the liver is either stored bound to the metallothionein, 
excreted via the bile, combined with the transport protein caeruloplasmin or combined with 
other cuproproteins in the liver. Caeruloplasmin-bound copper enters the circulation and 
comprises the majority of circulating plasma copper. 
32 
1.5.3.3 Excretion of copper 
Copper excretion is important In maintaining copper homeostasis. Copper is excreted 
predominantly in the bile, which has the highest copper concentration of all body fluids 
(Linder 1 99 1 ). Copper in bile forms complexes that are poorly reabsorbed minimising 
enterohepatic recirculation. Copper is also excreted in salivary, gastric and intestinal 
secretions. Therefore fluids entering the digestive tract from bile and other secretions in 
humans contain 4 to 5 times as much copper as copper from dietary sources, indicating that 
there is significant reabsorption. However most of the copper which is reabsorbed comes 
from the non-biliary component of the secretions (Linder 1 99 1 ). Cymbaluk et al. ( 1 98 1  a) 
recovered 70 % of parenterally administered dose of 64CU in the bile of bile duct cannulated 
ponies and only 3 .6 % in the faeces, thus demonstrating the much greater contribution of 
biliary copper than gastric and intestinal secretions to copper excretion . 
It is unclear how copper from other tissues is transported to the l iver for biliary excretion 
(Linder and Hazegh-Azam 1 996). It is possible that caeruloplasmin may return copper to the 
liver from other organs (a third phase of systemic copper distribution), and it has been 
proposed that, copper bound to caeruloplasmin destined for excretion is absorbed by 
hepatocytes and excreted in the bile. Caeruloplasmin is not readily degraded by digestive 
enzymes and may account for the lack of absorption of copper associated with biliary 
excretions (Linder and Hazegh-Azam 1 996). 
Urine is a minor route of copper excretion . This is probably due to the negl igible 
concentration of free copper in the plasma, and the efficient reabsorption of low molecular 
weight copper complexes which do get filtered by the glomerulus (Linder and Hazegh­
Azam 1996). Urinary copper excretion is not influenced by dietary copper intake, and has 
only a l imited ability to increase in cases of bil iary obstruction (Solomons 1 988) .  However 
chelation therapy, as used with copper toxicity and more specifically in Wilson' s  disease, a 
disease in humans associated with increased copper retention due to an inherited 
abnormality in caeruloplasmin production, increases the plasma concentration of copper 
associated with low molecular weight ligands resulting in increased unnary copper 
excretion. This increase in urinary copper excretion is also found with horses that are 
injected subcutaneously with copper D-penicillamine (Auer 1 989b). 
33 
1 .5.3.4 Effect of pregnancy on copper metabolism 
The pregnant mare transfers copper to the foetus trans-placentally, the mechanism of copper 
transfer is complex, and it is bel ieved to be via di-histidine bound (transferable) copper 
(McArdle and Van den Berg 1 99 1 ). The foetal l iver accumulates copper bound to 
metallothionein which is located in the lysosomes (Ryden and Deutsch 1 978). The foetal 
horse total body copper concentration is high (20 mg Culkg DM) with the hepatic copper 
containing 50 to 60 % of the total body copper. More than half of the total term copper is 
deposited in the foetus in the final two months of gestation (Meyer and Ahlswede 1978). In 
this respect, human prematurity is recognised as a risk factor for copper deficiency since the 
majority of foetal copper transfer and hepatic storage occurs in the last trimester (Lonnerdal 
1 996). A greater requirement for copper would be expected in pregnant mares, but has not 
yet been demonstrated. In rats, pregnancy increases copper absorption enabling them to 
retain up to 30  % more copper than non-pregnant rats in the final trimester (Davies and 
Williams 1 976). 
1.5.3.5 Copper metabolism for the neonate 
Mare colostrum contains 0.8 to 1 .0 mg Cull which decreases to approximately OJ mg Cull 
in mare mi lk (Ullrey et al. 1 974; Schryver et al. 1 986). It is thought that mi lk alone cannot 
provide adequate copper to meet neonatal needs, and that the l iver copper store maintains 
the neonate in a copper-sufficient state until such time that it consumes diets richer in 
copper (Meyer and Tiegs 1 995). Human infants may suffer from copper deficiency if they 
are weaned onto cow's  milk at an early age. This is thought to be due to a lower copper 
concentration in bovine milk compared with human milk. Therefore, in humans at least, it is 
considered that lactational copper is an important source of dietary copper (Lonnerdal 
1 996). 
Copper absorption in neonatal foals has not been investigated. In neonatal rats copper 
absorption is very high (about 75 %), and decreases with age (Misti lis and Mearrick 1 969; 
Lonnerdal et al. 1 985). This decrease in copper absorption can be induced by steroid 
administration (Mistil is and Mearrick 1 969). A large proportion of the absorbed copper in 
neonates is retained in the gastro- intestinal mucosa. However as the induction of intestinal 
metallothioneins also increases with age, this suggests that metallothioneins are not 
responsible for the high copper retention in the neonatal intestine (Lonnerdal 1 996). It 
appears that the absorption of copper in neonates is related in a l inear manner to copper 
intake and is considered to be non-saturable (Varada et al. 1 993 ). Balance data from human 
34 
infants have shown that despite efficient intestinal absorption, these individuals had a 
negative copper balance (Cavell and Widdowson 1 964; Dauncey et al. 1 977; Tyrala 1 986). 
Copper absorption declines in lambs as they age, and after weaning. The decline is 
associated with the maturation of the intestine, as well as the development of a functional 
rumen (Suttle 1 975) .  
In cattle, a critical foetal liver concentration of 300 mg Cu/kg OM has been proposed as the 
amount of stored liver copper that a calf requires to maintain it in a copper-adequate state 
over lactation until it ingests sufficient copper from non-milk sources to meet requirements. 
Calves born to cows receiving more than 1 0  mg Cu/kg OM are recommended as having a 
liver copper concentration above the critical level, but it is recommended that maternal 
copper liver levels should be 25 mg Cu/kg OM to ensure optimum function of copper­
dependent processes (Gooneratne et al. 1 989). No such data are available for horses. 
1 .5.4 Cuproproteins 
Copper in mammals is associated with numerous intracellular and extracel lu lar enzymes 
and proteins. These cuproproteins function predominantly as cofactors for enzymes and 
electron transport proteins in energy or antioxidant metabol ism (Linder and Hazegh-Azam 
1 996). Cuproproteins contain copper as a part of their molecular structure, which does not 
dissociate on isolation of the protein. The numerous and varied functions of cuproproteins 
highlight the essentiality of copper in animals. 
Caeruloplasmin 
Caeruloplasmin in humans, is a l S I  kDa 0.2 glycoprotein (0.34 % Cu), which binds 8 
copper atoms per molecule. In horses, unl ike other species, caeruloplasmin is an 0. 1  
glycoprotein (Okumura et al. 1 99 1 ), and has an activity peak at a higher pH (6.6 - 6 .8) than 
the 0.2 glycoprotein (5 .8  - 5 .9) (Bingley and Dick 1 969; Smith et al. 1 983). Caeruloplasmin 
is synthesised in and secreted almost entirely from the liver, but lung and lymphocytes may 
also contribute (Linder 199 1 ). Caeruloplasm in synthesis is regulated by hormones operating 
at the transcription level .  These include hormones associated with inflammatory processes 
(short term regulation) and oestrogen which is mainly responsible for long term regulation 
probably by stabil isation of Cp-mRNA (Middleton and Linder 1 993). Caeruloplasmin, in 
addition to being the main protein associated with copper transport, is also involved in free 
radical scavenging as well as an abi lity to catalyse the oxidation of ferrous (Fe++) ion to the 
35 
ferric (Fe+++) ion. Fe +++ is able to bind to the iron transport protein transferrin. The 
mechanism by which extracellu lar caeruloplasmin is able to act on the Fe+++ bound to the 
intracellular storage protein ferritin is unknown. A deficiency in caeruloplasmin results in 
the accumulation of stored iron in the liver and other organs including the brain (Linder and 
Hazegh-Azam 1 996). The oxidase activity of caeruloplasmin is dependent on its copper 
content and copper is important in regulating the amount of caeruloplasmin produced as 
hepatic copper binds with the caeruloplasmin regulator gene and increases transcription. 
However copper is not required for caeruloplasmin synthesis and the copper atoms are 
added post-translationally (Linder et al. 1 979). Copper antagonists are able to decrease 
caeruloplasmin activity in rats with silver being the strongest antagonist fol lowed by 
cadmium, molybdenum, ZInC and then sulphate. Copper deficiency decreases 
caeruloplasmin activity which can be restored by supplementary copper (Whanger and 
Weswig 1 970). However toxic doses of copper injected into monkeys markedly decreased 
their serum caeruloplasmin concentration (Neifakh et al. 1 969). Caeruloplasmin has both 
pro- and anti-inflammatory properties, as well as oxidase activities. Its antioxidant activities 
are important in preventing auto-oxidation of lipids and combined with its free radical 
scavenging properties are important in preventing host tissue from damage at a site of local 
tissue injury (Gutteridge 1 978; Linder 1 99 1 ). In the horse, plasma caeruloplasmin activity 
increased fol lowing intramuscular injection with Freund's  adjuvant, peaking at 24 days, and 
was stil l  significantly elevated 50 days after injection. Plasma copper and caeruloplasmin 
concentration were also elevated, but declined more rapidly (Auer 1 989a). 
Cytochrome c oxidase 
Cytochrome c oxidase has a molecular weight of approximately 200 kDa in mammals and 
consists of 1 2  or more subunits, all of which are located in the mitochondrial wall .  
Cytochrome c oxidase functions as the terminal enzyme of the electron transport chain in 
m itochondria, and catalyses the production of two electrons from the Kreb's cycle, required 
for the reduction of respired oxygen (2H+ + O2 � H20). Copper deficiency decreases 
cytochrome c oxidase activity, but due to the excess of respiratory enzymes in most cel ls, 
impaired mitochondrial function occurs only in severe copper deficiency. The mitochondria 
of the myocardial and hepatic cells appears to be more susceptible to copper deficiency than 
renal cel ls (Linder 199 1 ). Cytochrome c oxidase is thought to play a role in axonal and 
neuronal integrity. CNS lesions including those of enzootic ataxia of sheep (swayback) are 
thought to be due to a lack of this enzyme in deficiency states (Fell et al. 1 965) .  However 
36 
this is a contentious issue and the exact origin of the lesions seen in this condition has not 
been agreed upon. 
Lysyl oxidase 
Lysyl oxidase is an extracel lular enzyme, and is widely distributed through body tissues and 
is particularly abundant in structures containing large amounts of connective tissue (skin, 
blood vessels, cartilage etc .). Lysyl oxidase catalyses the inter-molecular cross-linking of 
newly formed col lagen and tropoelastin fibres by catalysing the deamination of lysine side­
chains. The cross-links are an important factor for the tensi le strength of these proteins. A 
decrease in lysyl oxidase activity is associated with skeletal abnormalities in young animals, 
and defective sheathing of blood vessels with elastin (Linder 1 99 1 ). Bone formation may be 
retarded due to a decreased abil ity to mineralise less organised collagen fibres (Suttle and 
Angus 1 978). 
Superoxide dismutase (SOD) 
SOD, also known as erythrocuprein, is a homodimer of approximately 32 kDa. Each dimer 
contains one copper and one zinc atom per molecule. Manganese and iron forms of the 
enzyme also exist in mitochondria. Cu-Zn SOD is found in the cytosol of most (if not all) 
cells, and it is particularly abundant in the liver, kidney, adrenal, and red blood cells and 
neurones (Paynter et al. 1 979; Linder and Hazegh-Azam 1 996). In the cytosol it catalyses 
the dismutation of superoxide radicals to hydrogen peroxide and oxygen (2H02- � H202 + 
O2). The peroxide is then further disposed of by other enzymes including the se1enoprotein 
glutathione peroxidase. Copper but not zinc appears to regulate the functional activity of 
Cu-Zn SOD. Dietary copper deficiencies reduces the enzyme's activity, which may be 
restored by copper supplementation (Paynter et al. 1 979; Harris 1 992). 
Free radical damage has been associated with cellular senescence and therefore aging. The 
ratio of Cu-Zn SOD activity to glutathione peroxidase activity has been investigated, and an 
increase in this ratio results in increased production of H202, and cellular senescence in vitro 
(de Haan et al. 1 996). Transgenic mice bred with greater than normal concentrations of Cu­
Zn SOD have an increased resistance to reperfusion injury (Yang et al. 1 994). Decreased 
Cu-Zn SOD in endothelial cells and hepatocytes decreases the susceptibility of these cells to 
oxidant-induced damage (Beckman et al. 1 988; Markey et al. 1 990), however a similar 
decrease in susceptibi l ity was not demonstrated in cardiac myocytes (Sarvazyan et al. 
1 995). The fami l ial form of amyotrophic lateral sclerosis (ALS), a genetic CNS 
37 
degenerative disease has been associated with mutations of the Cu-ln SOD gene (Rosen et 
al. 1 993 ) .  It is considered that the mutation causes a gain of an adverse function of the Cu­
In SOD enzyme rather than a decrease in enzyme activity (Rowland 1 995) .  The relatively 
high proportion of Cu-ln SOD in motor neurones are therefore hypothesised to put these 
neurones selectively at risk (Pardo et al. 1 995). A comparison has been made between 
equine motor neuron disease (EMND) and ALS, although there is no evidence that EMND 
is heritable, and is therefore more likely to have simi larities to the sporadic form of ALS 
than the familial form (Mayhew 1 994). 
Tyrosinase 
Tyrosinase is found in the melanosomes of melanocytes and is involved in melanin 
pigmentation which protects against ultraviolet exposure and provides hair, skin and eye 
colour. Tyrosinase catalyses the conversion of tyrosine to dopaquinone, which readi ly 
polymerises to form melanin (Linder 1 99 1 ). A lack of tyrosinase results in a decrease in the 
conversion of tyrosine to melanin, and a lighter than normal coat colour. This is a well 
recognised manifestation of copper deficiency in cattle (Poole 1 982; Gooneratne et al. 
1 989). 
Dopamine �-monooxygenase (Dopamine �-hydroxylase) 
Dopamine \3-monooxygenase is a tetrameric homoprotein found in noradrenergic neurones 
within norepinephrine storage granules near synapses and also in chromaffin granules of the 
adrenal glands. The nor-adrenergic neurones are concentrated in the locus ceruleus, brain 
stem and posterior hypothalamus (Linder and Hazegh-Azam 1996). Dopamine f3-
monooxygenase catalyses the hydroxylation of dihydroxyphenylamine (dopamine) to 
norepinephrine. This enzyme is therefore integral to nerve transmission in the central 
nervous system as wel l  as the stress response mediated by the adrenal medulla. Deficiency 
of this enzyme activity impairs the function of the CNS and results in an accumulation of 
the substrate dopamine in the brain (Linder 1 99 1 ). 
Metallothioneins 
Metallothioneins are small cysteine-rich polypeptides that are expressed in all cells exposed 
to divalent transitional metal ions (except Fe++). The cysteines confer metallothionein with 
metal binding properties, which are important in the storage and detoxification of these 
metal ions. As well as the ions themselves, metallothionein expression is regulated by stress 
(glucocorticoids), inflammatory hormones, and liver glucagon concentration (Linder 1 99 1 ). 
38 
Of the metal ions commonly present in animals, copper has the greatest affinity for 
metallothioneins, and can displace other elements from metallothionein binding sites. 
Generally the amount of copper bound to metallothioneins is small because of the ready 
excretion of excess copper in the bile, preventing significant storage of copper in tissues 
(Linder 1 99 1 ). As wel l  as metallothionein' s role in metal storage and detoxification, 
metallothionein is able to donate copper (and zinc) to apo forms of enzymes, and therefore 
may also play a role in copper deficiency, or when caeruloplasmin is lacking. There has also 
been recent interest in the free radical scavenging properties of copper-bound 
metallothioneins, and this activity may account for the regulation of metallothionein by 
inflammatory hormones (Linder and Hazegh-Azam 1 996). 
Others 
Other cuproproteins are l ikely to perform numerous biological functions. In  studies on 
severely copper deficient animals, a role has been ascribed for copper in the amidation of 
neuropeptides, dopamine synthesis, myelination, and activation of enkephalin proteins 
responsible for binding opiate receptors in response to pain (Linder and Hazegh-Azam 
1 996). There are also several amine oxidases, in addition to Iysyl oxidase, in connective 
tissue, which probably serve to deaminate various cytokines (Mason 1 979). 
1 .5.5 Measurement of copper status 
Horses do not exhibit the typical signs of copper deficiency exhibited by ruminants grazing 
copper deficient pastures, and so it has been assumed that their dietary copper requirements 
are low. This may not be true, and elucidation of the role of copper dependent enzymes in 
immune function, connective tissue structure and function and in providing protection from 
oxidant induced injury may result in the identification of more copper-responsive diseases 
in horses. As a result, being able to accurately measure the copper status of a horses is l ikely 
to become clinically important. 
An adequate copper status exists when tissue copper concentrations, and more particularly 
l iver copper stores, are sufficient to ensure that copper is not l imiting biochemical (eg 
enzyme activity) and physiological function (eg connective tissue integrity) and that animal 
performance and health are not improved by copper supplementation . 
39 
Liver copper concentration 
The l iver is important in copper homeostasis, being the major site for copper storage and 
excretion, as well as caeruloplasmin production. Hepatic copper concentration is used to 
determine copper status in cattle, with reference values established (Smart et al. 1 992).  In 
horses, hepatic copper concentration has been shown to reflect dietary copper intake (Cupps 
and Howell  1 949; Smith et al. 1 975;  Hurtig et al. 1 990). Suttle et al. ( 1 996) have proposed 
liver copper reference values for horses based on combinations of horse and ruminant data, 
and Meyer and Tiegs ( 1 995) have proposed recommended minimum l iver copper 
concentrations for new born foals. However reference ranges for adult horses, based on 
amounts required for proper function of copper dependent processes, have not been 
developed. 
Hepatic copper concentration varies with age, being very high in the foetus and the neonate 
(Egan and Murrin 1 973b; Meyer and Tiegs 1 995). Due to the varied functions of the liver in 
copper metabolism, including metal ion storage and detoxification, the availability of 
hepatic copper may not be certain, making interpretation hazardous. For example in zinc 
toxicity, Bridges and Moffitt ( 1 990) found clinical copper deficiency including 
hypocupraemia and lameness with articular and growth plate cartilage fractures, without a 
decrease in l iver copper concentrations. Therefore the interpretation of copper status from 
l iver copper concentrations may be improved by determination of the subcellular 
compartmentalisation of the copper (Bremner 1 99 1 ;  Cym baluk and Smart 1 993).  The 
relationship between plasma copper and liver copper concentration has been investigated in 
sheep (MacPherson et al. 1 964), cattle (Claypool et al. 1 975; Vermunt and West 1 994) deer 
(Mackintosh et al. 1 986) and horses (Cymbal uk and Christensen 1 986; Suttle et al. 1 996). In 
cattle and deer, a curvilinear relationship best fits the model (Vermunt and West 1 994), but 
in horses, Suttle et al. ( 1 996) found a linear relationship whereas Cymbaluk and Christensen 
( 1 986) found no relationship. No useful relationship was found in sheep in one study 
(MacPherson et al. 1 964). 
Blood copper status indices 
Circu lating copper is located in caeruloplasmin bound and non-caeruloplasmin (protein) 
bound fractions in the plasma and is predominantly bound to superoxide dismutase in red 
blood cel ls. The amount of circulating copper incorporated in caeruloplasmin is species 
specific, and in horses it is about 70 % of total plasma copper (Auer et al. 1 988b). Plasma or 
serum copper concentration is often considered to be a useful indicator of copper status, as it 
40 
declines reliably in moderate and severe copper deficiency (Solomons 1 979). However at 
higher dietary copper intakes, plasma copper does not follow changes in dietary copper 
intake. P lasma, serum and red blood cell copper concentrations are regarded as being 
roughly equivalent (Solomons 1 979), and therefore haemolysis is not considered to greatly 
alter the interpretation of serum copper levels (this is not the case with zinc or iron). A 
diurnal variation has been reported in serum copper in humans, with the highest 
concentration in the mornings (Lifschitz and Henkin 1 979). Serum copper concentrations 
and caeruloplasmin activity are affected by age, sex and pregnancy, and are increased in 
inflammatory or infectious conditions, with cancer and with the administration of 
anticonvulsant therapy or oestrogens. Conversely, copper concentrations are decreased by 
administration of corticosteroids and adrenocorticotropic hormones (Fisher et al. 1 990; 
Milne and Johnson 1 993). 
Seasonal variations in copper status of horses have been reported in horses in the UK and 
Australia (Stubley et al. 1 983;  Auer et al. 1 988b), which the authors suggested were 
probably due to differences in dietary copper availability. Changes with age and pregnancy 
have also been reported, with serum or plasma copper concentrations increasing with 
pregnancy and decreasing with age in mature animals, but great individual variation reduced 
the significance of these relationships (Stowe 1 968; Auer et al. 1 988a; Auer et al. 1 988b). 
At birth, foals have very low circulating copper concentrations and caeruloplasmin activity, 
which increases to adult levels by 3 to 4 weeks of age (Bell et al. 1 987). 
Cuproenzyme activity 
Cuproenzymes may provide a more sensitive measure of copper status in mi ld copper 
deficiencies and are l ikely to reflect more closely the relationship between an animal's 
copper status and its clinical significance. For instance Glade ( 1 986) suggested that 
caeruloplasmin is  the most reliable indicator of copper status.  Danks ( 1 98 1 )  proposed a 
simple non-invasive technique for determining the adequacy of a diet with respect to copper 
by measuring the plasma cuproenzyme level, then adding copper to the diet. He proposed 
that the cuproenzyme activity should increase only if the diet was deficient. However a 
major problem with measuring caeruloplasmin activity is its variabil ity which is apparently 
not related to dietary copper intake. For example caeruloplasmin activity may vary as a 
result of inflammatory states (Firth et al. 1 987; Smith and Cipriano 1 987; Auer 1 989a). It is 
also increased by chronic infection and inflammation, and is decreased by malnutrition, 
nephrosis and chronic hepatitis .  In humans, plasma copper concentration is h igher in women 
41 
than men, and higher in pregnant women and in individuals on oral contraceptives (Milne 
1 994; Solomons 1 979). Milne and Johnson ( 1 993) also showed that in some cases of copper 
deficiency in humans, the concentration of caeruloplasmin is not decreased despite a 
reduction in enzyme activity due to the presence of the apo-enzyme (not bound with 
copper) . It has therefore been proposed that the ratio of the enzymatic activity to 
caeruloplasmin concentration may be a better indicator of copper status, as this ratio is not 
greatly affected by other influences including hormones and sex. 
Red blood cell copper-zinc superoxide dismutase (RBC Cu-Zn SOD) activity in humans and 
animals has been shown to decrease with copper deficiency, with the reduction in activity 
proportional to the magnitude of the deficiency (Bettger et al. 1 978; Uauy et al. 1 985).  RBC 
Cu-Zn SOD appears not to be affected by age, sex or hormonal therapy (Saik et al. 1 982; 
Fisher et ai. 1 990). In one study performed on copper-deficient humans, the activity of 
cytochrome c oxidase in platelets and leukocytes responded more quickly to copper 
deficiency than RBC Cu-Zn SOD activity (Milne et al. 1 988). Milne ( 1 994) also reported 
that platelet cytochrome c oxidase activity correlates well with liver copper concentration. 
Barry et al. ( 1 983) found that sheep grazing kale (Brassica oleracea) depleted their liver 
copper stores, and reduced their serum copper concentrations and RBC Cu-Zn SOD activity. 
Copper supplementation of these sheep increased their heart cytochrome oxidase activity, 
and diminished the reduction in their RBC Cu-Zn SOD activity. Thus the available data 
suggest that parameters of cuproenzyme activity may provide valuable indicators of copper 
status in a variety of situations and hence merit further investigation. 
Hair copper concentration 
Hair copper has been found to reflect liver copper in rats (Jacob et al. 1 978) and cattle 
(Kel laway et al. 1 978), but doubt has been expressed as to its usefulness in humans 
(Ol ivares and Uauy 1 996) because hair copper concentration is reduced only after prolonged 
copper deficiency and is subject to contamination from external sources (Solomons 1 979; 
Danks 1 988). In horses, variation of hair copper due to several factors including season, age 
and dexamethasone administration (Cape and Hintz 1 982), as well as the possibil ity of 
contamination with environmental copper, may limit the usefulness of this measurement. 
42 
Urinary copper concentration 
Urinary copper in horses is independent of intake at adequate dietary copper concentrations 
(Cymbaluk et al. 1 98 1  a). In humans, urinary copper has been reported to be normal in 
copper deficient states and elevated in disease states such as fol lowing bums (Carr and 
Wilkinson 1 975) .  Consequently, urinary copper is not regarded as an accurate measure of 
copper status. 
1 .5.6 Dietary copper sources and bioavailability 
The oral route is by far the most significant route of entry for copper, although it is possible 
that small amounts of copper may be absorbed dermally from applications of ointments rich 
in copper. Small amounts of copper have been reported to enter through the skin in humans 
wearing copper bracelets through the formation of glycine-copper complexes dissolved in 
sweat (Walker et aI 1 982). 
1 .5.6. 1 Copper content and availability in pasture 
Factors influencing the copper composition of pasture plants include: pasture type, 
vegetation stage, fertilisers, soil type and pollution (Georgievskii et al. 1 982). 
Legumes and mixed herbs have higher copper concentrations than grasses, with the 
vegetative parts of the plants containing more copper than the reproductive parts. Therefore 
pasture copper concentration is at a maximum in young legume dominant pastures, and 
decreases as the plants age and flower (Georgievskii et al. 1 982). The addition of copper in 
fertil isers increases copper content of pasture plants (Cunningham and Perrin 1 946). 
Ferti lisers containing zinc may reduce the absorption of copper as described above. 
Increased pH will decrease the absorption of copper by plants, and it increases molybdenum 
concentration of pasture plants (Grace 1 994). Pasture copper concentrations vary 
considerably on different soil types (Cunningham 1 956; Wells 1 957). Low pasture copper 
concentrations are found on coastal sandy soils as well as marine and river silts and peat 
soils (Wells 1 957; Grace 1 994). Within New Zealand, topsoil copper concentrations can 
vary from 2 to over 1 20 mg Cu/kg OM, However topsoil copper concentration is poorly 
correlated with pasture copper concentration (Wells 1 957; Grace 1 994). 
Copper is less likely to be a cause of pasture pollution from industry than some other trace 
elements, such as zinc which wil l  adversely effect copper absorption. 
43 
1 .5.6.2 Copper supplements (bioavailability) 
Supplemental copper is available as inorganic salts (eg. cupric chloride, acetate, oxide, 
sulphate and carbonate), and as copper proteinates (or chelates). The bioavai labi l ity of 
supplemental copper can be assessed by various methods which are reviewed by Baker and 
Ammermann ( 1 995). Suttle ( 1 974) describes a method of assessing copper bioavailability 
by depleting ewes on a copper deficient diet and then repleting them with the copper 
supplement and using the increase in plasma copper concentration as a relative measure of 
bioavailabi l ity. Changes in liver copper concentration following supplementation has also 
been commonly used. Molybdenum supplementation has been used to ensure a low liver 
copper concentration prior to supplementation. More recently cupro-enzyme activities have 
also been used (Baker and Ammermann 1 995). 
Copper sulphate is often used as a reference to compare the bioavailabi lity of other copper­
compounds. In vivo studies in ruminants have found copper sulphate to have the greatest 
bioavailabi l ity of the metal salts (Ledoux et al. 1 995) and l ittle difference between the 
bioavailabi l ity of copper sulphate and the copper proteinate copper lysine (Kegley and 
Spears 1 994). Copper histidine complexes have been used successfully for the treatment of 
copper deficiency in humans and has been given subcutaneously to treat patients suffering 
from Menke's  disease (Sherwood et al. 1 989), an inherited disorder of copper absorption. 
1 .5.7 Copper dietary interactions 
Dietary components may affect copper absorption from the digestive tract. For example, 
dietary phytates, complexes with amino acids, reducing agents (eg ascorbic acid), the form 
of dietary carbohydrates, and other minerals influence bioavailability. 
Zinc 
Evidence regarding the role of zinc, as a dietary antagonist for copper is conflicting. Zinc, 
having the same electron configuration as copper, interacts with dietary copper at the site of 
absorption (Van Campen and Scaife 1 967; Hall et al. 1 979). Zinc is a potent inducer of 
metallothioneins, which have a greater affinity for binding with copper than zinc. High zinc 
intake by horses, both experimentally and due to ingestion of pasture with high zinc 
concentrations, has been reported to cause skeletal abnormalities consistent with copper 
deficiency, and decreases in blood copper concentrations (Wil loughby et al. 1 972; Gunson 
44 
et al. 1 982; Eamens et al. 1 984; Bridges and Moffitt 1 990; Campbell Beggs et al. 1 994). 
However the opposite was found in a study with pony foals, where a high zinc intake ( 1 200 
mg/kg DM) did not produce skeletal disease or reduce liver copper concentration (Coger et 
al. 1 987). The inhibitory effects of zinc have been shown to increase in the presence of 
excess histidine (Wapnir and Lee 1 993) and the balance between the chelating amino acids, 
and other trace elements and copper may be very important (Lonnerdal 1 996). 
iron 
Iron is also considered to decrease copper absorption, which, similar to ascorbic acid, may 
be due to the reduction of Cu++ to Cu+ in the intestine. High iron intakes in calves 
(Humphries et al. 1 983), and goats (Schonewille et al. 1 995), reduced plasma copper 
concentration, caeruloplasmin activity and liver copper concentration, and also markedly 
reduced the liver copper concentration in lambs (Grace and Lee 1 990). Increased iron intake 
occurs often in winter/early spring when herbage iron intake increases due to soil 
contamination. High dietary iron intakes in human infants has also been associated with 
induced copper deficiency (Barclay et al. 1 99 1 ;  Lonnerdal 1 996), and the combination of 
iron and ascorbate which is commonly supplemented in combination is thought to be 
synergistic on decreasing copper availability (Lonnerdal 1 996). 
Molybdenum/sulphur 
Elevated d ietary molybdenum and sulphur concentrations are the most important factors in 
secondary copper deficiency in ruminants (Dick et al. 1 975;  Gooneratne et al. 1989). 
Thiomolybdates appear to induce copper deficiency by forming insoluble complexes with 
copper in the digestive tract, and therefore limiting copper absorption. Absorbed 
thiomolybdates also alter copper ligand binding to proteins (ie albumin) decreasing copper 
bioavailabi lity, and increasing copper biliary excretion (Gooneratne et al. 1 989). However 
the effect of molybdenum in horses is considered to be less important than in the case of 
ruminants, and there have been no reports of an induced copper deficiency secondary to 
excess molybdenum intake in the horse (Cymbaluk and Smart 1 993), although recently 
there has been a report of a case of molybdenum toxicity in horses grazing pastures 
contaminated with fly ash, with necropsy evidence of decreased liver copper stores 
(Ladefoged and Sturup 1 995). A study on three ponies aged between 1 and 2 years 
demonstrated that increasing d ietary molybdenum decreased the amount of copper 
absorption and increased excretion. There was no change in serum copper concentration, but 
there was a decrease in caeruloplasmin activity, and an increase in TCA insoluble copper 
45 
concentration (Cymbal uk et al. 1 98 1  b). Another study on six horses of mixed age, breed and 
pregnancy status, found that molybdenum supplementation had no effect on plasma copper 
levels, and there was no increase in TCA insoluble copper, suggesting that there was no 
significant absorption of thiomolybdenates (Strickland et al. 1 987). 
Cadmiumlsilverlnickelltin 
Cadmium and si lver, like zinc, share the same three dimensional configuration as copper 
and therefore compete for binding sites in the intestine and on caeruloplasmin (Whanger and 
Weswig 1 969), and increase metal lothionein induction. High dietary intakes of nickel and 
tin reduce copper absorption and probably act either by antagonism of intestinal transport 
mechanisms, or by the reduction ofCu++ to Cu+ (Yu and Beynen 1 995;  Gunson et al. 1 982). 
Selenium 
Copper pretreatment of ponies attenuated the clinical signs of experimentally induced 
selenium toxicity. The copper did not appear to inhibit selenium absorption, but increased 
removal of selenium from the serum (Stowe 1 980). 
Phytates 
Phytates have been shown to reduce the bioavailability of copper and other trace elements in 
rats (Davies and Nightingale 1 975). However studies in humans have fai led to show this 
effect (Turn lund et al. 1 985)  despite a decrease in zinc absorption (Turn lund et al. 1 984). 
Unlike zinc-phytate complexes, copper-phytate complexes do not precipitate at intestinal pH 
(Vohra et al. 1 965), and the phytate in soluble copper-phytate complexes is easily displaced 
by amino acid and polypeptide chelators, and therefore should not significantly decrease 
copper absorption (Jacobsen and Slotfeldt-Ellingsen 1 983). Phytates could indirectly affect 
copper availabi l ity by this action on zinc. 
Amino acids 
Complexing of copper with amino acids affects intestinal copper absorption, and it is likely 
that the copper uptake is influenced by the absorptive properties of the bound amino acid 
(Linder and Hazegh-Azam 1 996). Histidine forms complexes with copper with much greater 
affinity than with zinc (Ashmead et al. 1 985)  and enhances copper absorption (Wapnir and 
Balkman 1 99 1 ). Cysteine has an inhibitory effect on copper absorption in chickens (Robbins 
and Baker 1 980; Baker and Czarnecki-Maulden 1 987) by increasing zinc antagonism at 
intestinal binding sites (Aoyagi and Baker 1 994). However, complexes with other amino 
46 
acids, such as methionine and lysine may increase copper absorption in the presence of 
copper antagonists, as the copper in these complexes could be absorbed via the amino acid 
intestinal transport mechanism (Aoyagi and Baker 1 994), bypassing the need to be absorbed 
at the antagonised sites. 
Ascorbic acid 
Ascorbic acid decreases copper absorption by reducing the cuprous ion to cupric ion, which 
is less wel l  absorbed (Hill and Starcher 1 965;  van Campen and Gross 1 968). Other reducing 
agents also depress copper absorption by the same mechanism (Hill and Starcher 1 965) .  
Ascorbic acid also affects copper metabol ism post-absorption, and was shown to decrease 
copper uti l isation in chicks injected with copper intraperitoneally (Disilvestro and Harris 
1 98 1 ). Ascorbic acid (or I-ascorbate) may also play an important role in the reduction of 
caeruloplasmin-bound copper to make it available intracellularly at target organs (Lonnerdal 
1 996), and it may also be required for the transport of copper from mucosal cells. 
Carbohydrates 
Dietary fructose (as compared with starch) decreases copper balance in copper-deficient rats 
(Fields et al. 1 984). This effect may be specific to ma:le rats. In humans, fructose appears to 
increase copper intake/retention compared with starch, but it decreases Cu-Zn SOD activity 
(Reiser et al. 1 985)  which returns to normal following copper supplementation. The effect 
of fructose on mineral balance is not specific to copper as similar changes were seen with 
Ca, Mg, Fe and Zn (Holbrook et al. 1 989). 
1 .5.8 Copper metabolism studies and copper requirements in horses 
There is a dearth of information on copper metabolism and requirements for horses. 
Cymbaluk et al. ( 1 98 1 a) performed a series of metabolism experiments on ponies using 
radio- and stable- copper isotope techniques as well as bile duct cannulation to measure 
copper absorption and endogenous losses. In the bile duct cannulated ponies it was observed 
that the apparent copper absorption coefficient was 0.27, and the true copper absorption 
coefficient was 0.46. Endogenous (obligatory) losses were calculated from the intercept of a 
regression between copper retention and dietary copper intake. Endogenous losses were also 
determined using isotope dilution techniques, and there was good agreement between the 
two methods. The copper requirement for maintenance was determined by dividing the daily 
endogenous loss by the true absorption coefficient (0.66). The values obtained were 3 . 5  mg 
47 
CullOO kg LW/day for endogenous loss and 5 .3 mg Cu/ 1 00 kg LW/day as a minimum 
copper intake for maintenance. The true absorption coefficient obtained by the regression 
approach was 0.66 which is considerably higher than the true absorption coefficient of 0.47 
determined using bile duct cannulated ponies. 
A regression approach was also used by Pagan ( 1 994) using 500 to 600 kg mixed breed 
horses on diets containing copper ranging from 7 to 38 mg Cu/kg OM. In this study it was 
determined that the true absorption coefficient was 0.40, endogenous losses were 7 mg 
Cul l 00 kg L W /day and minimum dietary copper intake for maintenance was 1 7 .5  mg 
Cu/ I OO kg LW/day. 
A balance study performed on miniature ponies in metabolism stalls, calculated the apparent 
absorption coefficient of copper to be from 0.4 to 0.5 (Hoyt et al. 1 995). 
Clearly there is considerable variation in the measurement of absorption and endogenous 
losses of copper because: 
1 .  small number of animals were used in these trials, 
2. trace element balance studies can suffer from significant errors because they involve the 
measurement of smal l amounts of mineral element in large volumes of ingesta and excreta 
and 
3 .  in horses copper absorption decreases with increasing dietary copper intakes (Cymbal uk 
et al. 1 98 1 a) indicating that the relationship is not linear. 
Estimates of dietary copper requirements for maintenance, based on the studies of 
Cymbaluk et al. ( 198 1 a) and Pagan ( \ 994) are 3 . 5  mg Cu/kg OM and 7 mg Cu/kg OM 
respectively. Both these dietary copper requirements are less than current NRC ( 1 989) 
recommendations of 1 0  mg Cu/kg OM. However, the above maintenance requirements do 
not account for extra dietary copper that is required during growth, exercise, pregnancy or 
lactation. 
48 
1 .5.9 Copper deficiency 
Two fonns of copper deficiency are normally described, namely primary deficiency due to 
an insufficient amount of copper present in the diet or secondary (or induced) deficiency 
due to a decrease in absorption or abil ity of tissues to uti lise absorbed copper. Some causes 
of secondary copper deficiency include the presence of antagonistic ions, copper chelating 
agents and conditions causing malabsorption including diarrhoea. Menke's disease, a 
genetic disorders of copper metabolism causing copper deficiency has been recognised in 
humans (Danks 1 995), but not in horses. Clinical signs of copper deficiency can be 
attributed to a reduction in the activity of copper enzymes, as well as decreasing amounts of 
free copper. 
Copper deficiency in growing horses is primarily manifest as abnonnalities in bone and 
cartilage development and has been discussed previously. Stowe ( 1 968) made an association 
between copper deficiency and rupture of the uterine artery in foaling mares, which may be 
due to the reduction in the activity of lysyl oxidase in adult horses, and a consequent 
weakening of the connective tissue (elastin) which ensheaths major blood vessels. 
Breed and species susceptibil ities to copper deficiency have been reported in ruminants. 
Cattle are more susceptible to copper deficiency than sheep, and also are more susceptible to 
molybdenosis. Simmental cattle are more susceptible to copper deficiency than other breeds 
(Smart and Gudmudson 1 980). This is probably because they excrete relatively more copper 
in the bile than other cattle (Gooneratne et al. 1 989). The association between ruminant 
copper deficiency and abnormalities in bone development has been discussed previously. 
Other clinical signs of copper deficiency in cattle include anorexia, diarrhoea, depressed 
growth rates, decreased reproductive perfonnance, microcytic hypochromic anaemia, 
achromotrichia, poor keratinisation of hair and hoof, nervous disorders, cardiovascular 
disorders, depressed immune status and death (Poole 1 982; Gooneratne et al. 1 989). Copper 
deficiency in pregnant sheep is associated with a nervous disorder of lambs (called enzootic 
ataxia or sway back) and is characterised by ataxia. It is considered to be caused by 
degeneration of neurones and a fai lure in myelination of neurones due to decreased 
cytochrome c oxidase activity (Fell et al. 1 965 ;  Jubb and Huxtable 1 993). A similar 
condition affects deer on copper deficient diets, but unl ike sheep, older animals rather than 
neonates are susceptible (Peet and Hepworth 1 993) .  
49 
Copper deficiency is not common in humans, but premature infants and individuals on total 
parenteral nutrition are at a higher risk. The most common clinical manifestations of copper 
deficiency in humans are anaemia, neutropaenia and bone abnormalities (Heller et al. 1 978; 
Williams 1 983;  Danks 1 988; Shaw 1 992). The anaemia is usually a hypochromic, 
normocytic or macrocytic anaemia, but microcytic anaemias are also found (Will iams 1 983 ; 
Shaw 1 992). The anaemias are considered to be due to defective iron mobi lisation from 
tissues secondary to reduced caeruloplasmin activity, and the anaemias respond to copper 
but not iron supplementation . Abnormalities in bone include osteoporosis, fractures, 
epiphyseal separation, widening of growth plates with spur formation and the formation of 
sub-periosteal new bone (Danks 1 988; Shaw 1 992). Less frequent manifestations of copper 
deficiency include hypopigmentation of hair, reduced growth rates, and reduced resistance 
to infections (Danks 1 988; Shaw 1 992). Also abnormalities to glucose and cholesterol 
metabol ism have been described, resulting in copper deficiency (as well as excessively high 
levels of copper) being considered to be a risk factor for atherosclerosis (Klevay 1 990; 
Kenneth et al. 1 994). 
The role of copper deficiency in depressed immune function has been reported in humans 
(Kelley et al. 1 995), ruminants (Jones and Suttle 198 1 ;  Woolliams et al. 1 986; Xin et al. 
1 99 1  Stabel et al. 1 993 ; Niederman et al. 1 994) and rats (Mulhern and Koller 1 988; 
Hopkins and Fai lla 1 995). Typical findings include decreased resistance to infections, and 
decreased lymphocyte numbers and phagocytic abil ity. Copper deficiency decreases 
humoral and cell mediated immunity, as wel l as affecting non-specific immunity by 
decreasing the phagocytic activity of macro phages and neutrophils. Woolliams et al. ( 1 986) 
found that lambs born from rams genetically selected for high copper status had a decreased 
mortality due to sway back and infection compared with lambs born to rams bred for low 
copper status. Mechanisms for the changes in immunity are sti l l  not clear, although it 
appears that there may be a failure of cellular differentiation, or a decreased cellular 
viability (Percival 1 995).  
1 .5.10 Copper toxicity 
The mechanism of copper toxicity is yet to be definitively establ ished, but several theories 
have been proposed (Luza and Speisky 1 996). For example, free copper may bind to 
proteins, changing their structure and preventing them from performing their normal 
enzymatic functions which are essential for cell metabolism (Nakamura and Yagasaki 
50 
1 972). Alternatively free copper may interact with hydrogen peroxide and superoxide anions 
to form highly toxic hydroxyl radicals (Samuni et al. 1 98 1 ;  Goldstein and Csapsky 1 986). 
These hydroxyl radicals cause oxidative degradation of proteins and lipid peroxidation of 
cell and organelle membranes. 
Metallothioneins are likely to be important in the protection of cells from copper toxicity by 
efficiently binding and therefore sequestering any free ionic copper (Luza and Speisky 
1 996). Metallothionein may also have a significant cytoprotective role via its antioxidant 
activities, and it has the highest known reactivity towards free radicals of any protein 
(Thomalley and Vasak 1 985). In addition, zinc may be displaced from metallothionein by 
copper and free zinc can also perform antioxidant and cytoprotective functions (McMillan 
and Schnell 1 984). 
Glutathione affects the abil ity of metallothionein to bind copper. Glutathione has a good 
affinity for Cu+ and forms stable complexes which are then incorporated into 
apometallothionein (Freedman and Peisach 1 989; Da Costa Ferreira et at. 1 993) .  
Glutathione also plays an important role in oxygen scavenging and as a cofactor for the 
seleno-protein glutathione peroxidase. Deficiency of glutathione may cause individuals to 
be more susceptible to copper cytotoxicity (Luza and Speisky 1 996) 
In grazing animals there is a species difference in susceptibil ity to copper toxicity with 
sheep being more susceptible than cattle which are more susceptible than horses. It is 
suggested that sheep may be more susceptible to toxicity because of their impaired abil ity to 
excrete excessive copper in bile (Gooneratne et al. 1 989). In sheep a breed difference in 
susceptibility to copper toxicity has also been recognised, with British breeds of sheep more 
susceptible to copper toxicity than merinos. Cl inical signs of copper toxicity include 
inappetence, increased respiratory rate and bil irubinaemia (Georgievskii et al. 1 982). 
Copper toxicity is also common in Bedl ington terrier dogs due to a genetic disorder of 
copper excretion, while Wilson 's  disease in humans is also a genetic disorder which results 
in impaired copper transport and excretion. 
Horses are very resistant to excessive copper intakes, and a trial with horses fed up to 800 
mg Culkg DM copper in the diet showed no adverse effects to the horse (Smith et al. 1 975); 
one animal that had been on this highest level subsequently foaled normally 3 to 4 months 
after completion of the trial . However chronic copper toxicity was induced in horses fed a 
51 
diet containing 2800 mg Cu/kg DM for two months. The horses developed hepatic and renal 
damage and died four months after finishing the trial (Bauer 1 975). A case of acute copper 
toxicity was reported in a standardbred injected subcutaneously with 4 mg Cu/kg L W of 
copper D-penicillamine. The horse became agitated and ataxic initially, and then later 
developed signs of abdominal pain. The horse made an apparent recovery after 36  hours 
(Auer 1 989b). 
1 .5.1 1  Therapeutic applications for copper 
Historically, copper has been used to treat a great variety of diseases with varying successes 
(Mason 1 979). More recently the anti-inflammatory and anti-oxidant qualities of copper 
have been investigated, and therapeutic formulations containing copper have been 
manufactured. Sorenson ( 1 982) found that copper complexed with anti inflammatory 
compounds were more effective at reducing inflammation than the compound alone. It has 
further been proposed that the anti-inflammatory activity of some therapeutic agents 
(including nonsteroidal anti-inflammatory drugs (NSAIDs) may rely on the drugs' abi lity to 
bind copper in vivo (Sorenson 1 985). NSAIDs are commonly used to treat inflammatory 
conditions of the musculoskeletal system in the horse, and copper bound NSAIDs have been 
evaluated in horses, and found to be effective at inhibiting inflammatory oedema. A parallel 
experiment on rats found the copper-bound drugs had a greater efficacy than the NSAIDs 
alone (Auer 1 990). Copper supplementation has also been investigated with respect to its 
ability to provide gastroprotection from NSAID induced lesions (Franco and Velo 1 995), 
and research in neurosurgery has found that copper inhibitors (D-penicillamine) prevent 
tumour angiogenesis, and are therefore useful as adjunctive therapy in cancer patients 
(Yoshida et al. 1 995). 
The wide range of biological activities of copper containing compounds, as well as 
advancing research technology, especially in the field of molecular biology, is l ikely to 
result in an increase in the therapeutic applications for copper. 
52 
1 .6 Introduction to the objectives 
The fol lowing chapters describe experimental work performed to test the hypothesis. 
Objectives for the experimental work include: 
2. Investigate and evaluate in vivo liver biopsy techniques for mares and neonatal foals 
(Chapter 2). 
1 .  Develop an in vivo technique for col lecting physeal cartilage for histomorphometric 
analysis (Chapter 3 ). 
3 .  Assess the effect of copper supplementation of mares on: 
i .  measurable indices of their copper status (Chapter 4), 
ii. the copper status of their offspring (Chapters 4 and 5) and 
i i i .  the incidence and severity of DOD in their offspring. (Chapter 6). 
4 .  Assess the effect of copper supplementation of growing foals on: 
i .  measurable indices of their copper status (Chapter 5)  and 
i i .  the incidence and severity of DOD (Chapter 6). 
5. Investigate the effect of high pasture molybdenum concentrations on the copper status of 
young horses grazing pasture (Chapter 7). 
Conclusions from this work, including recommendations for industry and areas requiring 
further research, are presented in the general discussion (Chapter 8). Pilot trial studies are 
included in the appendices. 
Chapter 2 
Liver Biopsy Techniques for Adult 
Horses and Neonatal Foals to Assess 
Copper Status ! 
I Published as: SG Pearce, EC Firth, ND Grace and PF Fennessy ( 1 997) Australian 
Veterinary Journal 73, 1 94- 1 98. 
53 
54 
2.1 Abstract 
The objective of this study was to evaluate standing, percutaneous, ultrasound-guided, transthoracic l iver biopsy in mares, and transabdominal, laparoscopically-guided, liver 
biopsy under general anaesthesia in foals, as techniques for obtaining tissue for 
assessment of copper status. The techniques were evaluated with respect to ease of use and 
effect on the animal. 
Twenty of 24 Thoroughbred mares and 21 of their foals were biopsied. The animals were 
part of a larger study of the effect of copper supplementation on copper status and the 
prevalence of developmental orthopaedic disease. Livers were also col lected from unrelated 
horses and sampled to investigate the variability in the distribution of copper in l iver tissue. 
The biopsy techn ique caused no lasting effect on the mares, but there was an increased risk 
of viscus penetration associated with taking multiple biopsy cores. The use of 
ultrasonography to scan the target area for the liver, identified four cases that were not 
appropriate candidates for liver biopsy, because of large intestine being located in the 
biopsy area. In the foals there were no serious postoperative adverse effects, nor was there 
any evidence of problems caused by the procedure when the abdomen was examined 
postmortem at five months of age. In l ivers collected to investigate the variabi lity of copper 
concentration, copper appeared to be relatively evenly distributed through the l iver. 
Standing, percutaneous, ultrasound-guided, transthoracic liver biopsy in mares, and 
transabdominal, laparoscopically-guided, liver biopsy under general anaesthesia in foals are 
convenient procedures for obtaining l iver tissue for assessing copper status in horses. The 
use of ultrasound to identify l iver tissue is recommended, especially in older mares. 
2.2 Introduction 
The liver is a major storage organ for copper in domestic animals and therefore l iver biopsy 
is routinely used in cattle and sheep to determine copper status (Chapman et al. 1 963). In 
the horse, liver biopsy is less commonly used for the determination of mineral status, and 
instead is used mainly as an aid to diagnosis and prognosis in suspected hepatic disease. 
55 
Recently, the assessment of copper status in horses has become more important because of 
the suggestion that some diets for growing horses do not provide adequate dietary copper 
intakes for normal bone development. In particular, it has been proposed that inadequate 
dietary copper intakes may result in a disease characterised by flexural limb deformities, 
epiphysitis, joint swel ling and radiographic and postmortem evidence of osteochondrosis 
(Knight et al. ! 990; Hurtig et al. 1 990). 
For the adult horse there are several methods for liver biopsy (Gibbons 1 964; Tennant et al. 
1 973 ; Pearson and Craig 1 980; McIIwraith 1 984; Milne 1 992), which vary with respect to 
the site of insertion of the biopsy needle (between the 1 2th to 1 4th intercostal spaces) and 
the angle at which the needle should be inserted (90 degrees to skin or oriented cranio­
ventrally). Traditionally, a l ine drawn between the tuber coxae and the point of the shoulder 
identified the target area for insertion of the biopsy needle (Gibbons 1 964; Milne 1 992). A 
modification of this  technique is to replace this line by a triangle which includes the above 
two points and the proximal olecranon (McIIwraith 1 984). 
Standing laparoscopic procedures have been described in the horse for the purposes of 
investigating the reproductive tract (Wilson 1 986; Fischer 1 99 1 ), as a diagnostic aid in colic 
cases (Fio 1 995) and other abdominal disorders (Fulton et al. 1 990), and for guided biopsy 
of abdominal organs, including the liver (Fischer 1 99 1 ). Laparoscopically-guided liver 
biopsy in anaesthetised sheep has been reported to cause no adverse effects (Hidiroglou and 
Ivan 1 993 ).  There is less information on foal liver biopsy techniques. However, Hurtig et al. 
( 1 990) reported on serial, blind, percutaneous liver biopsies performed on sedated foals. 
There is a concern about the val idity of liver biopsy to determine l iver copper stores because 
of the variation of copper concentration between sites in the liver (O'Cuill et al. 1 970), 
possibly as a result of differences in portal blood flow to different parts of the liver, as 
observed in ruminants (Heath and Perkins 1 985). Therefore, some biopsy procedures 
involve sampling at specific site(s) to reduce variation associated with such differences 
(Cymbaluk and Christensen 1 986). 
In a study to assess the effect of copper supplementation in pregnant mares and their foals, a 
safe technique was required that would permit sampling of the liver for copper 
concentration in young foals. Therefore, a laparoscopic technique was used to ensure the 
safe passage of the biopsy instrument across the abdomen and into the l iver, and to assess 
56 
the effect of the biopsy procedure on the liver. When the foals were euthanased at five 
months of age the long term effect of the biopsy was also assessed. In the mares, standing, 
percutaneous, ultrasound-guided, transthoracic l iver biopsy was used. This paper reports 
these techniques and also describes the variabil ity of copper concentration with respect to 
site in the l iver of 1 2  other horses obtained at necropsy or slaughter. 
2.3 Materials and methods 
2.3.1 Liver biopsy general details 
Thoroughbred mares aged between 4 and 20 years (mean age 1 0  years) were subjects in a 
study to determine the effect of increasing copper intake (7 versus 30 mg Cu/kg dry matter 
(DM)) on the copper status of the young horse and the possible role of copper in 
developmental orthopaedic disease in pasture fed horses. Liver biopsy was attempted on 24 
thoroughbred mares and on 2 1  of their foals. The biopsies of a mare and its foal were 
performed on the same day, 4 to 1 0  days after foal ing. 
The mare l ivers were biopsied using a 1 4  gauge 1 50 mm trucut needle (Baxter Healthcare 
Corporation, Deerfield, IL, USA), after scanning the liver with a 3 . 5  MHz l inear ultrasound 
probe. The foal l ivers were biopsied using either a 1 4  gauge trucut biopsy needle (n=1 8) or 
the cannula of a Rhone Merieux l iver biopsy needle (Rhone Merieux, Wellington, New 
Zealand 240 mm long, 4 mm diameter) attached to a 5 ml syringe (n = 3) .  The laparoscopy 
was performed using a CO2 insufflator (Wisap®, Sauerlach, Germany), 1 2  mm by 1 00 mm 
disposable trochar and cannula (Ethicon Endo Surgery, Cincinati, Ohio, USA), Verres 
needle (Ethicon Endo Surgery, C incinati, Ohio, USA) and a 1 0  mm rigid laparoscope 
(Olympus Winter & Ibe, Hamburg, Germany) attached to a 250 watt l ight source (Karl Storz 
GmbH and Co, Tuttl ingen, Germany). The foals were anaesthetised using a small animal 
circle system with an out-of-circuit precision vapouriser. 
2.3.2 Mare liver biopsy procedure 
The mares were restrained without a twitch during this procedure in an open sheltered area. 
They were sedated with 200 mg of 1 0  % xylazine hydrochloride intravenously and 1 0  mg of 
butorphanol tartrate. Fractious mares received a further 1 00 mg of xylazine hydrochloride 
intravenously. A target site on the horse's  right side was identified by considering a triangle 
57 
formed by joining the tuber coxae to the point of the olecranon and the scapulo-humeral 
joint, and then counting back from the last rib to identify the 1 2th to the 1 4th intercostal 
spaces. A 20 cm by 20 cm area was then cl ipped and washed, and a 3 .5 MHz ultrasound 
probe used to identify liver parenchyma. The area with the greatest depth of liver 
parenchyma and a lack of large hepatic blood vessels was chosen as the site for the biopsy. 
Two ml of 2 % lignocaine hydrochloride was injected intradermally to mark the site, which 
was prepared aseptically. A further 5 to 8 ml of lignocaine was injected into subcutaneous 
tissue and intercostal muscles. A 5 mm stab incision was made with a No. 2 1  scalpel blade, 
and the biopsy needle was inserted, oriented cranio-ventrally towards the contralateral 
shoulder, and advanced. When the biopsy needle penetrated the diaphragm, which was 
evident by the oscillations of the needle corresponding to respiratory frequency, it was 
advanced a further 5 cm into the liver, and a biopsy was taken. The cannula was left in place 
while the trochar was removed and the biopsy core harvested. The trochar was then re­
inserted and another biopsy taken. This procedure was repeated two or three times with the 
trucut needle to obtain 30  to 60 mg of l iver tissue. Any mare showing signs of colic after 
biopsy was treated with flunixin meglumine intravenously. The mares and their foals were 
stabled and examined twice daily for 3 to 4 days, turned out into a small yard for 1 to 2 
weeks and then returned to the herd. 
2.3.3 Foal liver biopsy procedure 
After sedation with xylazine hydrochloride (0.5 to 0.75 mg/kg), a naso-tracheal tube was 
inserted. Anaesthesia was induced, and then maintained via an endotracheal tube, using 
isoflurane and oxygen. A 1 6  gauge catheter was placed in the right jugular vein through 
which lactated Ringer's solution with I % dextrose was infused at 5 mllkgih. Blood pressure 
was monitored using a Doppler probe on the tai l. The foal was placed in left lateral 
recumbency, rotated dorsally to about 20° from true lateral, so that the ventral midl ine was 
easily accessible, and the right lateral and ventral surfaces of the caudal abdomen prepared 
aseptically. The table was ti lted about 1 5°, with the head raised, so that the intestines moved 
more caudally in the abdomen. A Verres needle was inserted into the abdomen through the 
ventral midline, attached to the insufflator, and the abdomen inflated with carbon dioxide. A 
stab incision was made through the skin, 5 cm caudal to the costochondral arch, at the level 
of the end of the 1 4th rib. The trochar and cannula were introduced through this incision 
into the abdomen. The trochar was removed, the laparoscope inserted through the cannula, 
the carbon dioxide insufflator removed from the Verres needle and attached to the cannula, 
58 
and the Verres needle removed from the ventral midline. Distension pressure was 
maintained at less than 1 6  mmHg. 
The right ventral liver lobe was visualised, and a biopsy needle was inserted through a small 
skin incision (5mm) made with a No. 2 1  scalpel, and then into the right lateral body wall a 
few centimetres from the ventral midline, midway between the xiphoid and the umbil icus. 
The exact s ite was determined by digital pressure on the abdominal wall being visualised, 
along with the right liver lobe and other structures, through the laparoscope. The biopsy 
needle was then advanced across the abdomen, under visualisation by the laparoscope and 
passed into the right liver lobe. The biopsy needle approached the liver lobe almost parallel 
to the diaphragmatic surface of the lobe, and was inserted into the liver 4 to 5 cm from the 
l iver margin. When the trucut needle was used repeat biopsies were required to obtain 30 to 
60 mg of l iver tissue. However, only one biopsy was required to obtain sufficient liver ( 1 50 
mg) when the larger bore Rhone Merieux biopsy cannula was used. The biopsy instrument 
was removed and the abdomen was explored with the laparoscope. The liver was observed 
several times to check for haemorrhage at the biopsy site. After removing the laparoscope 
and cannula, the abdominal musculature and skin at these two sites were sutured with 2-0 
PDS in three separate layers. The total anaesthesia time was 1 .5 to 2 h, because a bone 
biopsy and a radiographic skeletal survey were also conducted. Procaine penicillin ( l .5 x 
1 06 IU, intramuscularly) and phenylbutazone ( 1 00 mg, intravenously) were administered 
intraoperatively. The foals were returned to their dams, stabled for 3 to 4 days, turned out 
into a small yard for I to 2 weeks and then returned to the herd. 
At necropsy of the foals, at 5 months of age, the abdomen, with the l iver in situ, was 
examined for adhesions. The liver was removed and examined for evidence of scarring and 
other lesions that might be associated with the biopsy. 
2.3.4 Liver biopsy sample preparation and copper analysis 
Acid washed glassware was used to collect and store the liver samples. All care was taken to 
prevent any contamination of the sample. The tissue samples (30 to 60 mg) were handled 
with stainless steel forceps, washed in deionised water to remove any blood and patted dry 
with a paper towel, sealed in a small plastic vial and stored at -20°C for analyses.  The liver 
samples were freeze-dried, subsamples (6 to 1 0  mg) taken and 0.5 ml concentrated HN03 
added before being wet-ashed in a closed system microwave furnace. The sample was made 
59 
up to 5 ml  with deion ised water and the copper determined by Zeeman graphite furnace 
atomic absorption . Samples were run with a l iver standard produced by the National Bureau 
of Standards (USA) of known copper concentration (NBS bovine l iver 1 577-B). The l imit of 
detection is 2 flg Cull . 
2.3.5 Site variation in liver copper concentration 
S ix l ivers were col lected at s laughter from feral horses that grazed pasture in the central 
plateau of New Zealand 's  North Island. Six sites were sampled (50 g each) :  the peripheral, 
lateral border of the left and right lobes, the peripheral, ventral border of the quadrate lobe 
and the centre of the right, left and quadrate lobes. L iver subsamples were homogenised, 
freeze-dried and 0 .25 g of the subsample wet-ashed in 1 0  ml concentrated HN03, the residue 
taken up in 1 0  ml 2M HCI and the copper determined by inductively coupled p lasma 
emission spectrophotometry (ICP) (Lee 1 983) .  Analysis of variance was performed to test 
for a difference between sites with in the l iver. 
Livers were also harvested from three mature horses that were euthanased at the Massey 
University Veterinary cl inic for reasons unrelated to the present investigation . No l iver 
disease was detected in these horses, and the d ietary h istory of the horses was unknown. 
Twelve sites were identified, three evenly spaced across the diaphragmatic surface of each 
of the dorsal right lobe, ventral right lobe and quadrate lobe (from right to left), and the left 
lobe (from dorsal to ventral) .  The sites were biopsied with a trucut needle w ith 30  to 60 mg 
of fresh tissue being harvested. The l ivers were then homogenised and 5 subsamples of 
approximately 1 g of fresh tissue were col lected from each homogenate. Liver copper 
concentrations were determined by ICP as described above. The mean concentration of the 5 
subsamples of the homogenised l iver was used to define the total l iver copper concentration . 
The difference between the copper concentration at each of the 1 2  b iopsy sites and the mean 
copper concentration of the homogenised I iver was analysed using paired (-tests to 
determine if there was a systematic difference in copper concentration across the l iver. 
60 
2.4 Results 
2.4.1 Mare liver biopsy procedure 
The mare procedure took approximately 1 5  m inutes from sedation of the mare until 
completion. Of the 24 mares, four ( 1 7  %) were not biopsied, because l iver tissue could not 
be v isualised with the ultrasound scan over the target site. In  al l  of these cases intestine was 
identified. I n  another two horses (8 %) the l iver was identified, but on repeated biopsy, gut 
content was obtained and the biopsy was term inated before adequate l iver was harvested for 
analysis.  I n  both these cases the mares immediately received 30 000 IU/kg of procaine 
penic i l l in .  Within 1 to 2 h, both of these mares showed signs of colic and became restless 
with an increase in temperature and heart rate and a decrease in gut sounds. One other mare 
also showed signs of colic after the biopsy. These three mares were treated with 1 0  ml  of 
flunixin meglumine i ntravenously, and responded immediately; al l  recovered uneventfu l ly .  
The mares not biopsied because of the u ltrasonic identification of intestine over the target 
site were aged 1 1 , 1 3 , 1 3  and 1 8  years of age. F ive other mares aged 1 1  or older were 
biopsied successful ly. 
2.4.2 Foal liver biopsy procedure 
There was a small amount of bleeding from the l iver after b iopsy with the trucut needle, but 
seepage of blood from the needle track ceased within 3 to 4 min. There was more 
haemorrhage fol lowing b iopsy with the Rhone Merieux cannula, which also stopped after 3 
to 4 min in al l  cases. The foals showed no signs of i l l  effects attributable to the b iopsy 
(including no disruption to appetite or body weight gain), except for one foal,  which had 
m i ld subcutaneous emphysema due to a cannula being partly misplaced during insufflation. 
Two other foals became hypotensive during the anaesthesia ( systol ic pressure less than 90 
mm Hg), but they responded to an increase in the flow rate of intravenous fluids and a 
decrease in the concentration of the inhalant anaesthetic.  
At necropsy, l iver scarring attributable to the biopsy could not be d iscerned, but there were 
occasional small ,  irregular pale white scars in the hepatic capsule. The distribution of these 
scars and their  irregular shape were considered to be consistent with parasitic damage. 
There were no adhesions in the abdomen of any of the foals. 
- - -- � ----��- �---
2.4.3 Liver biopsy copper concentrations 
61  
The l iver biopsy concentrations in  the mares ranged from 1 2 .3 to  23 .5  mg Cu/kg OM (mean 
1 7 . 8  mg Cu/kg OM). The l iver biopsy concentrations in the foals ranged from 1 34 to 805 
mg Cu/kg OM (mean 3 5 1  Ilg Cu/g OM). 
2.4.4 Site variation in liver copper concentration 
The l iver copper concentrations (mg Cu/kg DM) at the six sites in the six feral horses are 
presented in Table 2 . 1 .  The coefficient of variation for the six feral horses ranged 2 .9 to 9 .0  
% .  There was no s ignificant difference (P > 0 .05)  between sites within the l iver for the six 
feral horses, but there was a s ignificant difference (P < 0.05)  between horses. 
Table 2.1 Copper concentration (mg/kg d ry weight) at  six sites across the liver of six feral 
horses, with mean, standard deviation (sd) and coefficient of variation (cv). 
Horse site 1 site 2 site 3 site 4 site 5 site 6 mean sd cv (%) 
23 .6 20.4 1 9.6 23 .2 24.4 20.8 22 .0 1 .98 9 .0 
2 20.0 20.4 1 9.6 22.4 2 1 . 6 2 1 .2 20.9 1 .06 5 . 1  
3 20A 20.4 20.0 20.0 20.4 2 1 . 6 20.5 0.59 2 .9 
4 29.6 32.4 30.4 27.6 3 0.4 29.6 30.0 1 .5 6  5 .2 
5 22.4 22.4 2 1 .2 20.4 23.2 23.2 22. 1 1 .2 1  S . l 
6 22.6 24.4 20.0 24.4 24.0 22.4 23.0 1 .70 7 .4 
Lobe Right Right Quadrate Quadrate Left Left 
location periphery centre periphery centre periphery centre 
The coeffic ients of variation for the mean copper concentration for the 1 2  biopsy sized (30 
to 60 mg fresh weight) samples were 1 2 .3 ,  1 5 .2 and 20.9 % .  No site had a significantly 
different copper concentration from the mean of the homogenised l iver Table 2 .2 .  
However there was a significant (p < 0.05), though m inor b ias, in that the concentration 
recorded in the total l iver homogenate was higher ( 1 .2 +/- 0 .05,  n = 36, t =: 2 . 1 2) than that of 
the sUbsample.  The mean difference between the copper concentration at each site and the 
copper concentration of the homogenised l iver (total l iver copper concentration) is 
presented in F igure 2 . 1 .  
62 
Table 2.2 Liver copper concentration (mg/kg dry weight) with standard deviation (sd) and 
coefficient of variation (cv) of 30-60 mg (fresh weight) samples taken at 12 sites within each 
liver, and the total liver (homogenised) copper concentration, from three mature horses. 
S ite Lobe Location Horse 1 Horse 2 Horse 3 
Right dorsal left 1 2.4 2 1 .2 24.9 
2 Right dorsal middle 1 2 .4 20. 1 2 3 . 1  
3 Right dorsal right 1 1 .6 2 1 .3 2 5 . 5  
4 Right ventral left 1 2 .8 20.9 1 9.3  
5 Right ventral m iddle 1 1 .4 20.9 2 3 .4 
6 Right ventral right 1 2 .4 29.8 2 3 . 8  
7 Quadrate left 1 7 .6 1 5 .0 24.9 
8 Quadrate m iddle 1 0. 7  1 9. 8  2 1 . 8 
9 Quadrate right 1 2.8 3 0. 9  1 9.7 
1 0  Left dorsal 1 5 .2 2 1 . I  2 1 . 8 
1 1  Left m iddle 1 2 .4 1 8. 5  1 6.6 
1 2  Left ventral 1 5 .5 1 8.7 20 . 1  
mean 1 3 . 1 2 1 .5 22. 1 
sd 1 .99 4.49 2 . 7 1 
cv (%) 1 5 .2 20.9 1 2. 3  
Homogenate of total liver 1  (sd) 1 5 .0 +/- 0 . 3 8  2 1 .0 +/- 0 .45 24.3 +/- 0.29 
1. mean of 5 samples from the whole liver homogenate 
Figure 2 . 1 .  Mean difference (mg/kg dry weight) between liver copper concentration at each site 
(30 to 60 mg fresh weight) and the total (homogenised) liver copper concentration for 3 mature 
Thoroughbred horses (refer to Table 2.2 for identification of sites). Differences were not 
statistically significant. 
5 -. 
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Liver site 
63 
2.5 Discussion 
In the mares, the l iver biopsy technique was a short procedure and was performed with l ittle 
difficulty. Occasionally, the mares reacted when the b iopsy instrument penetrated the deep 
layers of the thoracic wal l .  Deep infi ltration of l ignocaine at the biopsy site helped diminish 
this response. The sedated mares required minimal restraint, and the b iopsy procedure could 
eas i ly be performed in the field .  The mares were not fasted, because it is  considered that 
fasting is not usefu l  in large herbivores, as the gut content helps to hold the l iver against the 
diaphragm (Pearson and Craig 1 980).  
The use of the ultrasound before b iopsy identified situations when the right l iver lobe was 
not present, u itrasonographical ly, in the target area. In these mares the presence of intestine 
in the b iopsy area rendered them inappropriate candidates for performing standing, 
transthoracic, l iver b iopsies; they were re-scanned 1 to 2 weeks later and the situation had 
not changed. One mare (aged 1 8  years) was later necropsied for unrelated reasons and was 
found to have an atrophied right l iver lobe. Atrophy of the l iver is common in older horses 
and occurs more frequently on the right side because of pressure of the right dorsal colon 
and the caecal base (Dyce et al. 1 987). The left side is not suitable for transthoracic biopsy 
in the horse, as the l iver is asymmetrical with two thirds of the l iver to the right of the 
midline, and the l iver on the left does not extend beyond the caudal margin of the lungs. 
The necessity of performing mUltiple passes with the trucut needle to obtain sufficient l iver 
tissue (30 to 60 mg wet weight) for copper determination was a consequence of the low 
concentration of copper in mature equine l ivers (5 to 20 times less than rum inant l ivers). 
One pass with the trucut needle harvested 20 to 30 mg, hence the need for two to three 
passes. The trucut needles were re-used after steri l isation, but the more times they were 
resteril i sed, the less satisfactory was their performance. It was considered that the biopsy 
needles should only be re-used once or twice at best. Resharpening of the b iopsy needles 
was not attempted, but may increase their performance when re-using them. It was helpful 
to make a bold stab i nc ision with the scalpel through the skin, as th is enabled the b iopsy 
trochar to be manoeuvred more easi ly. 
In this study, gut content was obtained on two occasions, and it was thought that in  the 
course of doing repeated passes with the stylet, the b iopsy needle exited the l iver and 
penetrated a v iscus. Whi le the gut penetration was presumably the cause of the subsequent 
-_ . .  __ . - _.  ---------- - - -
64 
mild to moderate col ic, the horses recovered ful ly after simple symptomatic anti­
inflammatory treatment. The most successful location for performing the l iver biopsy was 
the 1 3th intercostal space, at about the level of the l ine between the tuber coxae and the 
point of the elbow, with the needle d irected towards the contralateral elbow. 
In  the l iterature, standing, percutaneous l iver biopsies are reported to be safe, and one study 
reported more than 200 l iver biopsies from horses, cattle, sheep and goats without adverse 
effects (Pearson and Craig 1 980) .  One adverse response to a l iver biopsy in a horse was 
associated w ith a delayed prothrombin time, and therefore delayed coagulation is considered 
to be a contraindication for th is techn ique (Tennant et aI. 1 973).  Gibbons ( 1 964) claimed 
that a branch of the hepatic or portal circulation is frequently encountered, but is rarely of 
consequence as hepatic b lood clots very rapidly. 
The foal b iopsy procedure requires dedicated equipment, and is best performed where 
faci l ities and ski l led staff are available .  The procedure is costly and time consuming. 
Furthermore, foals in their  first week of l ife may, in  some situations, be an anaesthetic risk. 
The procedure, however, was straightforward and no major complication was encountered. 
At this young age, and with sufficient insufflation, v isual i sation of the organs in the 
abdomen was excellent, and there was no requirement to fast the foals, which could increase 
anaesthetic  risk. Because of the importance of minimising morbidity with these animals, we 
used this laparoscopic technique to ensure that the b iopsy was accurate and so that we could 
assess the amount of haemorrhage and other effects of the biopsy. Haemorrhage was m inor, 
and the effects of the biopsy on the foals was minimal, which would suggest that an 
u ltrasound-guided technique may be used successful ly in the future on these animals. 
The aftercare of the mares and foals described in this paper was more rigorous than required 
for the above procedures, but was necessary because of concurrent bone b iopsies performed 
on the foals. The procedure appeared to be innocuous, since there were no post-operative 
complications attributable to the l iver b iopsy, nor was there any evidence of scarring or 
adhesions in the l iver on gross examination at necropsy at five months of age. There were 
no adverse effects reported from blind, serial, percutaneous, l iver biopsy in the work of 
Hurtig et al. ( 1 990), and this technique is l ikely to be cheaper and avoids the anaesthetic risk 
associated with neonatal foals. 
65 
O'Cui l l  et al. ( 1 970) indicated that the variabi l ity of copper concentration across the l iver of 
horses (coefficient of variation up to 47 %) meant that an assessment of the l iver copper 
stores from a l iver biopsy could be subject to large error. However, our own investigation on 
l iver copper concentration showed much less variab i l ity (coefficient of variation < 2 1%). In 
the case of the feral horses, the same number of sites per l iver were taken as in the study of 
O'Cui l l  et al. ( 1 970), and the coefficient of variation was considerably less « 9 %).  While it 
is  d ifficult to explain the difference between studies, dietary and physiological factors may 
influence the distribution of copper in the l iver. The study of O'Cui l l  et al. ( 1 970), analysed 
1 g samples from each l iver site whereas we took 50 g samples; this may have influenced 
the variabi l ity detected, and both these samples are considerably greater than the amount 
harvested by the biopsy procedures (30 to 60 mg fresh weight). Therefore the study 
performed on the l ivers from the 3 mature horses was designed to determine the variabi l ity 
across the l iver that may be expected when taking s imi lar sized samples to the samples 
harvested by the described l iver biopsy technique. S ites 1 to 6 were located in the right l iver 
lobe which is the biopsy site in the standing (mare) technique. There was no systematic 
difference in copper concentration across the l iver determined, and it can be expected that a 
l iver biopsy of 30  to 60 mg (fresh weight) wi l l  give an estimation of total l iver copper 
concentration with a coefficient of variation of 1 0  to 20 %. Whether th is is accurate enough 
wil l  be determined by the magnitude of difference from a reference value, or other animals, 
that needs to be establ ished. 
This study has shown l iver biopsy to be an easy and reasonably accurate method for 
determining l iver copper concentrations in neonatal and adult horses. The interpretation of 
l iver copper concentration as a measure of dietary copper intake and relationsh ips to plasma 
copper and caeruloplasmin oxidase activity are the subjects of continuing research . 
Chapter 3 
Biopsy of the Distal Radial Physis in 
Neonatal F oals2 
2 Submitted as :  SG Pearce, EC Firth and WF Hunt ( 1 997) Veterinary Surgery. 
66 
67 
3.1  Abstract 
A technique was devised for taking biopsies from the distal radial growth plate in neonatal foals for the purpose of studying physeal carti lage morphology. The b iopsy 
of 2 1  Thoroughbred foals, 4 to 1 0  days of age, was performed under inhalant 
anaesthesia, using a trephine on a multi speed dri l l .  Five foals (24%) developed excessive 
incisional granulation tissue, due to their exercise not being adequately restricted 
postoperatively. Nine foals (43%) developed some flexion of the carpus (over at the knee) 
and 1 5  (7 1 %) developed outward rotation of the left l imb. There was s ignificantly greater 
carpal valgus in the left leg compared with the right (p < 0.005), which was not present in 
six age-matched, unoperated, control foals. There were no abnormalities in the congruency 
of the radiocarpal joint detected grossly at postmortem at 5 months of age. However there 
were gross and h istological abnormal ities of the distal radial growth plate. Postoperative 
morbidity, s ign ificant changes to conformation, and the unknown long term consequences 
of the d isruption to the physis  are l ikely to make th is  procedure inappropriate for c l in ical 
usage. However, the b iopsy procedure was easi ly carried out and provided a suitable sample 
for h i stological and histomorphometric examination of the growth plate carti lage. Therefore 
th is  technique provides a useful research method for study of growth p late morphology in 
neonates. 
3.2 Introduction 
Abnormal ities in the development of bone and carti lage present a s ignificant problem to the 
equine industry worldwide, and there is some evidence that the incidence of the resulting 
d iseases i s  on the increase (Jeffcott 1 99 1 ). Developmental abnormalities of the growth plate, 
such as "physitis", are a s ignificant cause of morbidity in young foals (O'Donohue et al. 
1 992), and may result in undesirable conformational abnormalities (Fretz 1 980;  Auer et al. 
1 983) .  
Examination of growth plate morphology in vivo may help define early pathological 
changes. Pathological changes to carti lage canals, present in  the early l ife of a foal, have 
recently been implicated in the development of articular osteochondrosis (Carlson et al. 
1 995) .  Biochemical abnormal ities have also been reported in retained cartilage from cases 
of osteochondrosis of the intermediate ridge of the distal tibia (Li l l ich et al. 1 997), and 
s imilar changes m ight also be present in retained carti lage of physes. Early changes to the 
68 
anatomical and biochemical structure of the growth plate may be associated with subsequent 
developmental abnormal ities of the physis.  
B iopsies from the wing of the i l ium (Savage et a1 1 99 1 )  have been investigated for the early 
d iagnosis of bone abnormal ities. However, the h istomorphometric parameters of biopsies 
from the wing of the i l ium had l ittle association with the incidence of dyschondroplastic 
lesions in a group of wean l ings fed different levels of phosphorus and digestible energy 
(Savage et al 1 993c).  Misheff and Stover ( 1 992) found that biopsies from the 1 2th rib, for 
the same purpose, were not suitable for histological or h i stomorphometric analysis .  A 
technique for standing surgical growth plate (cartilage) b iopsy has been described for the 
study of growth plate d isorders in weanl ings (Bel l ing and Glade 1 984). We required a 
techn ique for obtaining tissue for immunocytochemical studies of neonatal foal growth 
plates (Hol le  and F i rth unpubl ished data). The purpose of this study is to describe and 
evaluate the b iopsy technique we used and to indicate its usefu lness for c l in ical and research 
purposes. 
3.3 Materials and methods 
3.3.1 Animals 
A b iopsy was performed on 2 1  m ixed sex, healthy, 4 to 1 0  day o ld Thoroughbred foals 
which were subjects in  a study of the effect of oral copper supplementation on copper status, 
and its possible role in developmental orthopaedic d isease. 
3.3.2 Biopsy technique 
The foal was sedated with 0 .5  to 0.75 mg/kg of xylazine, and anaesthesia was induced with 
isoflurane delivered through a nasotracheal tube. Anaesthesia was maintained with 
isoflurane using a smal l animal c ircle system with an out of c ircuit precision vapouriser. 
Lactated R inger 's  solution with 1 % dextrose was infused at 1 0  ml/kg/h throughout the 
procedure. The foal was placed in left lateral recumbency, and the right carpus was prepared 
for steri le surgery and draped conventionally. 
A 25 mm proximodistal curvi l inear incision was made over the lateral aspect of the distal 
radial physis, and the skin and subcutis retracted. An 8 x 8 mm square of tissue from the 
------- -
69 
periosteal-perichondrial complex was either excised or hinged proximally, to reveal 
metaphyseal and epiphyseal bone. A trephine (Figure 3 . 1 )  (9 mm internal diameter, 1 1  mm 
external diameter), on a battery powered slow speed drill, was placed over the physis such that 
epiphyseal and metaphyseal tissue would be included in the core to be removed. The drill was 
directed slightly distally since the physis inclines in this direction. The burr penetrated 1 5  mm . 
When the burr was withdrawn, either it contained the excised core (to be removed with small 
needles) or the core remained attached at its base, and was removed by gentle leverage with a 
20 gauge needle bent at right angles 3 mm from its end. Haemorrhage was controlled with 
surgical swabs using digital pressure. The periosteum was reap posed with simple interrupted 
sutures using 2-0 PDS, and the skin and subcutis were closed in two layers . A pressure 
bandage was placed on the limb, and the foal was hand recovered. The foals received 1 .5 x 
1 06 ill of procaine penicillin (im) and 1 00 mg of phenylbutazone (iv) intraoperatively. 
Total anaesthetic time was 1 .5 to 2 hours and included laparoscopically guided liver biopsy 
and radiological survey of the limbs. The recovered foals were returned to their dams, stabled 
and observed three times a day for three days and then placed in a small yard for 1 to 2 weeks 
before returning to the herd. The bandages were replaced every 3 to 4 days for 1 0  to 1 2  days. 
Figure 3. 1 Stainless steel trel)hine, 9 mm internal d iameter, 1 1  mm external diameter, 24 mm 
trephine depth. . 
70 
3.3.3 Conformation 
The foals were assessed for conformational changes every one or two weeks unti l  they were 
euthanased at 5 months. Conformation was recorded on a standard sheet, subjectively 
scoring the d istal l imb joints for angulation from in front, and for excessIve 
flexion/extension from the side. Limb rotation, and joint distension were also scored. A 
scoring system from 0 to 3 was used ranging from normal to severe abnonnalities. A m i ld 
abnormality (score = 1 )  was considered significant if it was present on two consecutive 
scoring occasions. 
At the same time as the foals were scored, they were videoed, from in front and the right 
hand side, and the angulation of their carpus and front fetlocks was measured in both 
dimensions. The use of v ideo- image analysis for conformation examination is described 
elsewhere (Hunt et af. in press). 
The angulation of the biopsied (right) leg measured by video image analysis was subtracted 
from the left leg at each sampl ing occasion. The resulting values were analysed with time 
from biopsy by l inear regression . Six age-matched, unoperated, c l inical ly normal control 
foals were also v ideoed, and the difference between the angulation in their left and right legs 
were also calculated and analysed by l inear regression. Linear regression of control foals 
was taken from the average day of b iopsy in operated animals (day 6). A comparison of 
l inear regressions for operated and unoperated foals was performed. Analyses were 
performed with Statistical Analysis System (SAS) version 6 . 1 1  (SAS Institute Inc. Cary, 
N .C . ,  USA) using procedures for the general l inear model .  
3.3.4 Postmortem examination 
Intravenous pentobarbitone injection was used to euthanase the foals at 5 months of age. 
The biopsy site was examined for gross abnormalities of the distal radius and the 
radiocarpal joint. The left and right radius from each foal was weighed, its length and mid 
d iaphyseal circumference measured, and its volume estimated by water d isplacement. 
Differences were tested for significance by paired-t tests (ex = 0 .05) .  In 20 of the 2 1  foals, 
the distal radius was sawn frontally into approximately 6 slabs 6 mm wide. The frontal slabs 
were examined grossly, and cabinet radiographs taken .  A section through the biopsy site 
71  
was decalcified in  formic ac id/formate, paraffin embedded, sectioned at 6).lm, and stained 
with haematoxyl in,  eosin and alcian blue. 
3.4 Results 
3.4.1 Surgery 
Removal of a core of the distal radial physis  was readi ly achieved. Al l  foals recovered from 
anaesthesia uneventfully. Lameness was not evident when the foal was returned to the dam, 
nor during observations over the fol lowing 1 -2 weeks.  In a few cases, after-care instructions 
were not compl ied with and the foals  were returned to the herd early. In one case, the 
surgical wound dehisced, developed excessive granulation tissue and lameness persi sted for 
several weeks. In  another 4 cases, the wound partially dehisced, and healed uneventfu l ly by 
secondary i ntention after granulation tissue exc ision, bandaging, and confinement. Of these 
4 cases, complete heal ing occurred with in 4 weeks for 3 foals, and 1 2  weeks for 1 foal, with 
no residual lameness. 
3.4.2 Conformation 
Nine foals (43 %) developed m i ld flexion of the carpi fol lowing b iopsy. In all cases the 
carpal flexion was bilateral .  In 6 foals, the flexion had resolved after 5 to 6 weeks post 
surgery, whi le 2 took 8 weeks, and the foal that developed chronic lameness had a m ild 
b i lateral carpal flexion for the duration of the trial (5 months) .  Fifteen foals (7 1 %) had mild 
outward rotation of the left forel imb that became evident from 2 to 9 weeks fol lowing 
surgery. Two foals ( l 0  %) developed outward rotation of the right forel imb at 8 and 1 6  
weeks postoperatively. 
There was a s ignificant relationship between the difference in carpus angle ( left - right) in 
the operated foals (p < 0.005) with time (Figure 3 .2a), but no such relationship existed for 
the control foals (Figure 3 .2b) .  Separate l inear regressions for both sets of data provided a 
s ignificantly better model (p < 0.05)  than a common regression relationship.  
72 
Figure 3.2 The difference (right subtracted from left) in carpus (valgus) angle as viewed from in 
front of: 
a) 2 1  foals with time from the physeal biopsy procedure. 
b) 6 age-matched, unoperated control foals with time from the median day of growth plate 
biopsy (day 7) in operated foals. 
Angles were calculated using video image analysis. 
Figure 3.2a 
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Time post day 7 (d) 
• 
• • • • . . . .
, 
• • • . + • • • 
• 
1 00 1 20 1 40 • 
.
+ 
• 
• • 
1 0Q ·  1 20 • 1 40 • 
· 
• 
• 
73 
3 .4.3 Postmortem 
There was no s ignificant difference (p > 0.05) between weights, lengths or densities of the 
left and right radi i .  The external morphology of the right distal radius did not appear grossly 
different to that of the left s ide, and there were no abnormalities detected in  the radiocarpal 
joints. On frontal sections (6 mm thick) of the distal radius, the physea\ disruption 
associated with the biopsy was apparent in all cases. The normal path of the lateral aspect of 
the physis  was disrupted, with struts of bone pass ing through the physis and carti lage or 
fibrocartilage present in the metaphysis .  
Radiographs of the s labs revealed 3 different morphological abnormalities i n  the area of the 
biopsy. These were c lassified A, B and C (Figure 3 .3 ). "A" consisted of a curved, roughly 
parallel  radiolucency in the epiphysis, approximately 3 mm from the physis on the lateral 
margin of the s lab and curving distal ly toward the articular surface. "B" consisted of an 
i rregular radiolucency, roughly triangular or rectangular shaped, continuous with the physis  
and extending into the metaphysis. "C" consisted of a tal l ,  thin, roughly rectangular area of 
radiolucency on the lateral border of the frontal s lab, at the level of the growth plate 
extending into both the epiphysis and metaphysis .  
Changes A, B and C were present in the radiographs of 1 5  foals (75%). Of the 5 remaining 
foals only B was seen in 2 foals, only C in 1 foal, A and B were seen in I foal, and in 1 foal, 
obvious epiphyseodesis, and periosteal reaction proximal to the growth plate on the lateral 
cortex could be seen, but the changes were not typical of A, B or C. In most cases the 
radiographic changes were evident on two or three of the 6 mm frontal s labs through the 
distal radius. In only 8 of the 1 5  foal s  with changes A, B and C, were these changes present 
on the radiograph of the same s lab. 
74 
Figure 3.3 Thin section cabinet radiograph of a frontal section through the distal radius 
showing the effect of the biopsy on the physis laterally. Morphological changes A, B, and C as 
described in the text, are depicted. A small ossicle (0) is seen in the m iddle of change C on the 
lateral border of the radiograph, at the level of the physis. 
Histological examination of the distal radial growth plate revealed no abnonnalities medial to 
the site of the biopsy. Changes began abruptly about 1 5  mm from the lateral margin of the 
radius .  A defect in the epiphysis, consistent with change A seen on cabinet radiographs, 
contained no trabecular bone and was filled with mature collagen, fat, and vascular tissue. 
The radiographic change B was consistent with an epiphyseodesis which consisted of areas of 
trabecular bone, and in places, bundles of col lagen and rows of fibroblasts arranged parallel in 
the direction of longitudinal growth (Figure 3 .4). The collagen bundles merged with trabecular 
bone surrounded by numerous active osteoblasts at both ends of the epiphyseodesis. Some 
fibrocartilage was also often seen continuous with the physis at the point of disruption, and 
was also seen occasionally at the periphery of the epiphyseal defect. 
75 
Figure 3.4 Low power view of the histological features of an epiphyseodesis (E) at the biopsy 
site showing the trabeculae alligned perpendicular to the physis (P), edending into the 
metaphysis (M). Stain :  Alcian Blue, Haematoxylin and Eosin. Magnification: Bar = 500 IJ.m. 
Histologically change C was evident as a thickening of the periosteal-perichondrial complex 
encroaching on the lateral margin of the distal radius at the level of the physis. The foal with 
the atypical radiographic appearance of the biopsied slab, appeared histologically to have a 
more mature epiphyseodesis containing less fibrous tissue, and more trabecular bone. 
76 
3.5 Discussion 
The lateral aspect of the d istal radius was chosen as a s ite for b iopsy due to easy surgical 
access, and its predisposition for carti lage retention and possible role in physitis and angular 
l imb  deformities (Firth and Poulos 1 984). 
The surgical procedure was eas i ly performed. The only problem encountered was the 
possibi l ity offa i l ing to obtain the complete thickness of physi s  in the biopsy core, due to the 
distal incli nation of the growth plate from the lateral margin. Also, occas iona l ly the biopsy 
core broke off i ns ide the trephine, and removal of the core was difficult and could result in  
fracture of the b iopsy core. These problems were min im ised by preventing change in 
d irection of the trephine. An adaptation to the burr such that the biopsied core could be 
expelled would be advantageous for th is procedure. 
The internal diameter of the trephine was chosen such that the b iopsied core would contain 
ful l  thickness of the physis  connected to bone of the metaphysis  and epiphysis .  In a smal l 
number of cases, this objective was not achieved, and so a larger diameter burr may have 
been more suitable. However a larger diameter burr would result in a greater degree of 
physeal d isruption, and greater chances of s ignificant effects of epiphyseodesis .  Prevention 
of epiphyseodesis ,  for instance by use of fat grafts, might then become important. 
Autogenous fat grafts, harvested from the flank, have been reported for the prevention of 
bony bridging after osteotomy in dogs (Craig 1 98 1 ) . 
Postoperatively, 5 of the foals suffered some degree of wound dehiscence at 1 0  to 20 days 
after surgery. Thi s  may have been prevented if the after care of the foals involved a longer 
period of confinement. However, the foals were part of an experiment which required their 
consuming a pasture diet, and thus they were returned to pasture prematurely. 
Most of the foals developed outward rotation of their non-operated ( left) forel imb. Two 
foals had a m ild right forel imb outward rotation. Outward rotation is described as being 
secondary to angular limb deviation which results in uneven weight bearing on the affected 
l imb. The rotation decreased in severity as the foals aged, and there was no apparent need 
for intervention. 
-------------------------
77 
There was a significantly greater carpal valgus in the left l imb than in the right. Data from 
control foals were analysed to document that the difference was abnormal in growing foals. 
Contralateral l imb  deformities have been produced in the h ind l imb of immature lambs by 
excisional femoral arthroplasty (Duff 1 986) .  These animals developed an inward rotation of 
the l imb and a varus deviation at the level of the distal tibia. It was also noted that a mi lder 
inward rotation of the tibia was a normal ageing process for control animals. Most foals in  
th is  study developed an outward rotation and valgus deformity in the unoperated forel imb. 
Mi ld valgus deformity was commonly found in  al l  carpi including those of control foals. 
Therefore it i s  possible that extra weight bearing in  the contralateral l imb, as a result of the 
biopsy, exacerbated th is normal conformation and caused an associated outward rotation . 
However there were no joint incongruities obvious, nor any gross morphological differences 
between the biopsied radius and the contralateral l imb.  There is l ittle known about the 
normal age related changes to conformation at different sites in quadrupeds and the role of 
weight bearing on this (Duff 1 986). 
The cabinet radiographs of the frontal sections through the radius and the h i stology of the 
b iopsy site demonstrated that the cavity remaining after biopsy was retained in the 
epiphysis, and the physis was d isrupted by trabecular bone .  There was also considerable 
bone remodell ing into the metaphysis. The morphological change A was consistent w ith the 
s ite of the biopsy core. It had a curved appearance, presumably due to the uneven pattern of 
growth in  the epiphysis from biopsy until postmortem . The radiolucency extending into the 
metaphysi s  (B) was consistent with the collagenous fibres laid down during the reparative 
process fol lowing biopsy .  These fibres were oriented in  the direction of longitudinal growth 
and merged with the trabecular bone in a simi lar manner to Sharpey fibres; the trabecular 
bone appeared to contain osteoblasts actively producing osteoid, and with time, the collagen 
fibres may have been replaced by trabecular bone. 
Bel l ing and Glade ( 1 984) used a hammer and a curved 10 mm bone gouge to remove 
growth plate tissue in standing sedated yearling horses and claimed that after 4 weeks the 
site was healed sufficiently to repeat the b iopsy in a neighbouring site. They also c laimed 
that postmortem examination of 6 yearl ings 4 weeks postoperatively revealed "no evidence 
of mechanical damage or persistent intracarti laginous or osseous wounds in any of the 
specimens". This differs from our postmortem results, and m ight reflect the fact that we 
b iopsied neonatal foals rather than yearl ings, and we performed necropsy much longer after 
surgery. There may also have been differences in the methods of postmortem examination. 
78 
In thi s  study, the foals exhibited s ignificant conformational changes, some postoperative 
morbidity, and obvious postmortem physeal changes. These effects m ight preclude this  
technique from cl in ical usage. However, better adherence to a stricter postoperative regime, 
the use of anti-inflammatory medication, and feet trimming to m inimise morbidity and 
conformational abnormal ities are expected to s ignificantly decrease the adverse effects of 
this  procedure which was conducted in a research environment which could not be 
completely regulated for investigation of the surgical technique. Thus the technique might 
be usefu l  in c l in ical s ituations. At present the technique certainly provides a useful research 
tool for further studies on physeal morphology and the characterisation of early 
developmental lesions. 
79 
Chapter 4 
The Effect of Copper Supplementation 
on the Copper Status of Pregnant 
Thoroughbred Mares and their 
Neonates3 
3 Submitted as: SG Pearce, ND Grace, JJ Wichtel, EC Firth and PF Fennessy ( 1 997) Equine 
Veterinary Journal. 
80 
4.1 Abstract 
P regnant Thoroughbred mares (n = 2 1 )  were grazed on tal l  fescue pasture contain ing 4.4 to 8 .6 mg Cu/kg Dry matter (DM). Twelve mares were supplemented with 
approximately 0 . 5  mg Cu/kg l iveweight (L W)/day as copper sulphate for the final 1 3  
to 2 5  weeks o f  gestation . P lasma copper concentration declined during the third trimester 
for all mares and was not affected by copper supplementation . Liver copper concentration of 
mares and foals was determined when the foals were 4 to 1 0  days old.  Copper 
supplementation of the mares tended to increase mare l iver copper concentration (p < 0 .08), 
and increased foal l iver copper concentration (p < 0 .0 1 ). There was a s ignificant relationship 
between mare and foal l iver copper concentration (p < 0 .00 1 ,  r2 = 0.7). Increasing the copper 
intake of mares is effective in enhancing the copper status of foals. 
4.2 Introduction 
There has been considerable interest in the copper requirements of growing horses, with the 
d ietary copper requirements recommended by the NRC ( 1 989) being chal lenged (Hurtig et 
al. 1 993 ;  Lewi s  1 995) .  Maternal and foetal copper metabol ism are i ntimately l i nked (Mason 
1 979, Hidiroglou and Knipfel 1 98 1 ), although l ittle is known about this relationship in 
horses. A greater understanding would al low more informed recommendations of copper 
requirements of the dam during pregnancy, and offer the possibi l ity of influencing the 
copper status of the neonate for either treatment of congenital copper deficiencies, or as a 
prophylaxis for putative copper-responsive conditions of the growing foal . 
D ietary copper requirements for pregnant and lactating mares are considered to be the same 
as for other c lasses of horses (NRC 1 989). However copper metabol ism in pregnant mares 
m ight be expected to be different to non-pregnant horses, because of changes in appetite and 
intake in the mare, and the extra nutritional requirements of the foetus. In thi s  respect, peak 
daily deposition of copper in the foetus ( l Oth month of gestation) has been estimated at 
around 4 mg  Cu (Meyer and Ahlswede 1 978). In rum inants, foetal demand for copper 
increases throughout gestation, with up to 50 % of the newborn lamb's total copper being 
stored in the l iver (Pryor 1 964; Grace et al. 1 985) .  
Copper deficiency in calves i s  considered to be evident postnatal ly, whereas symptoms of 
copper deficiency in sheep and goats may be evident in utero (Hidiroglou and Knipfel 
8 1  
1 98 1 ) . The relatively lower foetal copper accumulation in  sheep compared w ith cattle may 
account for the h igher incidence of congenital copper deficiency syndromes in sheep 
(Gooneratne et al. 1 989). In sheep and goats, copper deficiency late in gestation may result 
in an inadequate supply of copper to the foetal central nervous system, resulting in a d isease 
characterised by motor disturbances (Jubb and Huxtable  1 993) .  Some ewes are relatively 
res istant to the effects of low copper diets and may produce normal lambs because of 
increased copper transfer across the placenta in these animals (Hidiroglou and Knipfel 
1 98 1 ) . Transplacental copper transport in the final part of the third trimester i s  important to 
provide adequate copper for human neonates, and premature b irth i s  cons idered a risk factor 
for copper deficiency in the infant (Tyrala 1 986; Solomons 1 988).  This  can appear c l inical ly 
as osteoporosis, oedema, ataxia, anaemia, neutropenia, and apnoea in the neonate (Sutton et 
al. 1 985 ;  Solomons 1 988) .  The effect of copper deficiency on foals in utero has not been 
described. 
In mares, i ncreased maternal copper intake may also be important as low copper status 
during pregnancy has been associated with fatal rupture of the uterine artery during foal ing 
(Stowe 1 968) .  
The objectives of this study were to determine the effect of two leve l s  of d ietary copper 
intake of pregnant pasture fed mares on their plasma copper concentration in the final 
trimester, and on mare and foal l iver copper concentration shortly after parturition. 
4.3 Materials and methods 
4.3 .1  Animals 
Pregnant Thoroughbred mares (n = 24, 4 to 20 years of age) were purchased, and grazed on 
tal l  fescue pasture from May 1 994 for the duration of the experiment at AgResearch'  s F lock 
House Research Centre near Bul ls  on the North I sland of New Zealand. When pasture 
avail ab i lity was inadequate to meet requirements from May to July ,  the mares received 
supplementary tal l  fescue hay, produced from the same property. The copper concentration 
of the pasture ranged from 4.4 to 8.6 mg Cu/kg DM, and the hay contained 1 1  mg Cu/kg 
DM. 
82 
The mares were randomly allocated to either a control or copper supplemented group, 
matched for mare age, date of last service and the s ire of the pregnancy. Copper 
supplementation began in June and continued until parturition. Mares foaled from 30th 
August to 24th November, so that at the start of supplementation they were 1 3  to 25 weeks 
from foal ing. The copper supplemented group received a drench three times a week which 
contained copper sulphate (equivalent to 0.5 mg Cu/kg L W/day), as wel l  as zinc sulphate 
(0.5 mg Zn/kg LW/day) and sodium selenate (0.02 mg Se/kg LW/day). The control animals 
received zinc and selenium only. Zinc and selenium were supplemented to ensure that all 
trace elements except copper were above NRC ( 1 989) recommendations throughout the 
trial . Three foals d ied at birth leaving 2 1  mare-foal pairs (9 control and 1 2  copper­
supplemented mares) 
4.3.2 Sampling 
Jugular venous b lood was collected into trace element, sodium heparin vacutainers (Becton 
Dickinson, #367735) .  Samples were taken monthly during the final trimester and analysed 
for p lasma copper concentration. 
A laparoscopically-guided, transabdominal, l iver b iopsy (Chapter 2) was performed on each 
foal, at 4 to 1 0  days postpartum.  On the same day, a standing, ultrasound-guided, 
transthoracic, l iver biopsy was attempted on each mare on the same day. However only 1 6  
of the 2 1  mares were successfu l ly b iopsied ( 6  control and 9 supplemented); fai lure was due 
to the presence of large intestine in the biopsy target s ite (Chapter 2). 
4.3.3 Chemical analysis 
Plasma copper concentration 
P lasma ( 1  ml )  and concentrated HN03 ( 1  ml)  were placed in a 1 5  m l  tube and left overnight. 
The m ixture was incubated in a water bath at 70°C for 3 hours, 1 ml of 3 0  % H202 was 
added and then the mixture was incubated again at 70°C for a further 2 hours. The sample 
was made up to a volume of 6.5 ml with 2M HCl and the copper concentration determined 
using an i nductively coupled plasma emission spectrometer ClCP) (Lee \ 983) .  
83 
Liver copper concentration: 
The b iopsy sample was prepared and analysed as described in Chapter 2. The copper 
concentration was determined using a Zeeman graphite furnace atomic absorption 
spectrometer. Concentrations were expressed on a dry matter basis .  
4.3.4 Statistical analysis 
All  analyses were performed on the 2 1  mare-foal pairs . The effect of copper 
supplementation on mare prepartum plasma copper concentration and mare and foal l iver 
copper concentration at 4 to 1 0  days postpartum was analysed by analysis of variance 
(ANOVA). Regression analyses were performed to investigate the relationship between 
plasma copper concentration and stage of gestation and also between mare and foal l iver 
copper concentration. 
The regression models for ANOV A included mare age and foaling date as covariates, whi le  
the model for foal l iver copper concentration also inc luded gestational length as a covariate. 
Al l  analyses were performed with Statistical Analysi s  System (SAS) version 6. 1 1  for 
Windows (SAS Institute Inc. ,  Cary, N .C. ,  USA). 
4.4 Results 
4.4.1 Plasma copper concentration 
Changes in p lasma copper concentrations for the 1 50 days prior to parturition are presented 
in F igure 4 . 1 .  There was a s ignificant (p < 0.00 1 )  decrease in the p lasma copper 
concentration during this period, with foaling date significant (p < 0 .00 1 )  as a covariate. 
These two factors accounted for approximately 20% of the variation . There was no 
significant difference in the relationship between control and copper supplemented mares 
and the stage of gestation. 
84 
Figure 4. 1 The relationship between plasma copper concentration and time prepartum in the 2 1  
mares. 
1 .8 
1 .6 -
• 
0.2 _ 
o 
- 1 60 
• 
• 
• • • 
• 
- 1 40 
• 
• 
• 
- 1 20 
. . 
. . 
• 
• 
+. 
• •• 
- 1 00 
y -0.002x + 0.9 1 
y2 = 0. 1 9  
• 
• •  • •• • 
. . . . 
+. 
• 
• • 
. • 
n = 88 
• 
••  • 
. .. .. .. 
• 
• • • 
• 
• • • • 
• • . . 
• 
• 
• • 
• 
-80 -60 -40 -20 
Days prepartum 
4.4.2 Liver copper concentration 
Mare 
• 
o 
The mean (+/- standard error of the mean (SEM» copper concentration for control and 
copper supplemented mares was 1 6.6 +/- 1 .0 1  and 1 8 . 5  +/- 1 .28 mg eu/kg DM respectively 
(Table 4 . 1 ) . A lthough copper supplementation did not have a significant effect on mare l iver 
copper concentration there was a trend toward increased l iver copper concentration in the 
supplemented mares (p < 0 .08).  
Foal 
The mean (+/- SEM) copper concentration for foals, at 4 to 1 0  days of age, from control and 
copper supplemented mares was 253 .9 +/- 20.2 and 423 . 8  +/- 7 1 .3 mg eu/kg DM 
respectively (Table 4 . 1 ), and the foal l iver copper concentrations ranged from 1 34 to 805 
mg eu/kg DM. Mare supplementation had a s ign ificant effect on l iver copper concentration 
of the foals (p < 0.05) .  
85 
Table 4.1  Effect of copper supplementation of mares on mare (n = 1 6) and foal (n= 21) liver 
copper concentration (mg Cu/kg DM +/- SEM) at 4 to 1 0  days postpartu m .  
Control Mare 
Supplemented Mare 
Mare Liver 
1 6.6 ( 1 .0 1 )  
1 8.5  ( 1 .28) 
Foal Liver 
2 5 3 .9 (20.2) 
423 . 8  (7 1 .3 )  
There was a s ignificant (p < 0 .005) positive l inear relationship between mare and foal l iver 
copper concentration (Figure 4.2). The quadratic term for the relationship was investigated, 
but was not s ignificant. Age of the mare (p < 0.0 1 ), but not parity, was s ignificant as a 
covariate. L iver copper concentration of foals from supplemented mares increased with the 
age of their dam (Figure 4 .3) .  
Figure 4.2 The relationship between mare and foal liver copper concentration (mg Cu/kg DM) 
at 4 to 10 days postpartum from copper control (D) and supplemented (_) mares. 
900 
800 
c: 700 .-0 
.� 600 
B �  c: " 500 . 0 -u ClJ '- ..>e  <l.) � 0.. := 400 o.. u 2; ClJ 
o S  300 :E: 
ca 0 200 -"-
1 00 
0 
0 
y 0= 43.2x - 428 
r 0= 0.55 
n 0= 1 6  
5 1 0  
o 
• 0 • 
1 5  
• • 
• o 
o 
o • 
20 
Mare liver copper concentration (mg Cu/kg DM) 
• 
• 
• 
25 
-- - --- -
86 
Figure 4.3 The relationship between mare age and foal  liver copper concentration at birth for 
foals from control (D) and copper supplemented (_) mares. 
900 -
800 - • 
• 
c 700 -
.2 
g 600 � • 
� �  
0 0  500 u CJJ .... -'< <U �  0.. ::l 400 g- u  
u CJJ • • 
ts s  300 .2: • 
c;; 0 200 r..:... 
0 0 0 0 0 0 
• 0 • 
0 • • 
1 00 
0 
0 5 1 0  1 5  20 
Mare age (years) 
4.5 Discussion 
The decrease in plasma copper concentration of the pregnant mares in their final trimester 
suggested possible negative copper balance during this period. However, supplementation of 
the pasture diet with copper su lphate did not increase the copper status of the mare during 
this period. This suggests a possible physiological cause for the decrease in the plasma 
copper concentration. There was a trend, however, toward increased l iver copper 
concentration in the copper supplemented mares (p < 0.08).  Neonatal foal l iver copper 
concentration was significantly increased by mare dietary copper supplementation, and there 
was also a strong relationship between mare and foal l iver copper concentration measured 
soon after parturition. 
The New Zealand Thoroughbred industry is predominantly pasture based, and the copper 
concentration for most pasture diets is s imi lar to our control d iet (4.4 to 8 . 6  mg Cu/kg DM). 
The dietary copper intake of the supplemented mares was approximately 3 to 4 times that of 
the control animals (8 vs 30 mg Cu/kg DM). 
87 
The decreasing plasma copper concentration in the final trimester is contrary to the study of 
Stowe ( 1 968), who found a smal l increase in serum copper concentrations during gestation . 
Auer et al. ( 1 988a) found that pregnant mares had s imi lar p lasma copper to their non­
pregnant pasture mates with no apparent effects of stage of gestation, but a second study 
(Auer et al. 1 988b) found a significantly h igher p lasma copper concentration in pregnant 
mares than in lactating mares. Our results are not in agreement with these studies, and may 
reflect differences in genetic composition, or in management regimes. Foaling date was 
used as a covariate in our analyses to account for the l ikely confounding effect of season . 
Increasing plasma copper concentration with increasing gestation occurs in women (Mason 
1 979) where plasma copper concentration can increase to up to 2 .5  times that in non­
pregnant women (Widdowson et al. 1 974). Some theories explain ing the increase in  plasma 
copper in women (reviewed by Mason 1 979), include: alterations in  the competitive binding 
of copper and zinc with metalloth ioneins, a response to the continual inflammatory stimulus 
of the foetus, and an association between plasma copper concentration and oestrogen, which 
may also exp lain the h igher plasma copper concentration in  women compared w ith men. 
Increased d ietary copper intakes in pregnant women (from 2 .5  to 4 .6 mg Cu/day) does not 
alter serum copper concentration or caeruloplasmin oxidase activity (King and Wright 
1 985) .  
For horses, ruminants m ight be a more appropriate companson species than humans  in  
respect to their dietary and management conditions. In one experiment (Butler 1 963), 
housed pregnant ewes were fed either half estimated dietary copper requirements (2 
mg/day) or a h igh copper d iet (approximately 1 5  mg/day). P lasma copper i ndices including 
whole b lood, plasma, erythrocyte, and caeruloplasmin-bound copper concentrations all 
declined throughout gestation in  both treatment groups. The blood copper concentration 
continued to dec l ine for another month postpartum, and then increased to pre-mating levels  
shortly afterwards .  This  pattern of change occurred in both dietary groups, but d id not occur 
in a group of barren ewes used as controls. The decl ine in b lood copper indices was not as 
marked in the h igh dietary copper group, but was sti l l  s ignificant. 
D ietary copper intake has been shown to be reflected in the l iver copper concentration of 
young horses receiving 8 to 1 08 mg Cu/kg DM and mature ponies receiving 8 to 790 mg 
Cu/kg DM (Cupps and Howel l  1 949, Smith e t  al. 1 975), but thi s  relationship was not so 
obvious in  the pregnant mares of this  study (8 or 30 mg Cu/kg DM). This may be a result of 
88 
differences in copper metabolism associated with pregnancy. The l iver copper concentration 
of our neonatal foals was influenced by the dietary copper intake of the mares suggesting 
preferential storage of the supplementary dietary copper in the foetal l iver. A s imilar 
relationship between dietary copper intake and foetal l iver copper concentration has been 
observed in cattle (Smart et al. 1 992). In a region with a h igh prevalence of hypocuprosis, 
the copper status of calves was related to the copper status of their dams, and maternal l iver 
copper concentration and foetal age were determined to be good predictors of foetal l iver 
copper concentration (Gooneratne and Christensen 1 989). 
There was cons iderable individual variation III l iver copper concentration of foal s  from 
supplemented mares, and the distribution of the data was b imodal .  Of these 1 2  foals from 
supplemented mares, 4 had l iver copper concentrations greater than 600 mg Cu/kg DM, and 
the remaining 8 were less than 400 mg Cu/kg DM. The higher l iver copper concentrations 
m ight indicate the abi l ity of some foals to accumulate copper in utero to a greater extent 
than others. The variation in the l iver copper concentrations of foals from control mares was 
relatively smal l and the sample d istribution was normal. The effect of mare age on foal l iver 
copper stores at parturition is of interest, as a l l  foals with l iver copper concentrations greater 
than 600 mg Cu/kg DM came from supplemented mares aged 1 2  or greater. Thi s  i s  possibly 
due to an age related difference in the copper metabol ism of pregnant mares, which does not 
seem to be related to parity. However the numbers were too small to make definitive 
conclusions. 
The l iver b iopsy, as a techn ique for estimating l iver copper concentration has been criticised 
due to the variation of the copper concentration with in the l iver (O'Cuil l  et al. 1 970). 
However, much smaller variation was found in a recent study of New Zealand horses 
(coefficient of variation of 1 0  to 20 %) (Chapter 2). The variation and magnitude of the l iver 
copper concentration from our neonatal foals was consistent with two other reports of 
neonatal l iver copper concentrations from mares of unknown d ietary h istory (Egan and 
Murrin 1 973b; Meyer and Tiegs 1 995) .  
The lack of a copper transport protein (caeruloplasmin) i n  utero enables h igh levels of 
copper to accumulate in the foetal l iver (Chang et al. 1 975 ;  Widdowson 1 974). The role of 
these l iver copper stores at birth is uncertain. Copper absorption of young rum inants (Suttle 
1 975)  and neonatal rats during ingestion of m i lk diets is very h igh due to the p inocytosis  of 
copper complexes, which enables the resorption of copper excreted in the bi le (Mist i l i s  and 
89 
Mearrick 1 969). B i l iary excretion is also reduced in these animals, thereby enhancing 
copper conservation. This decreases d ietary copper requirements while on m i lk, a diet 
contain ing low copper concentrations (0.2 to 0.3 mg Cull). Therefore stored l iver copper at 
birth may reflect the inabi l ity of the foetal l iver to excrete copper rather than foetal supply 
of copper for early l ife (Bremner 1 99 1 ; Cymbaluk and Smart 1 993) .  However Meyer and 
Tiegs ( 1 995)  claim that despite this  h igh copper absorption, there is a shortfall  between 
daily copper requirements and absorbed copper, which can be met by mobi l i sation of copper 
from the l iver copper stores accumulated in utero. 
Increased copper intake of the mare provided increased copper storage in the foetal l iver, 
which may be of importance for the growing foal. However, there was considerable 
variabi l ity in foal l iver copper concentration in foals from supplemented mares, and the 
relationship between mare dietary copper intake and foetal copper stores appears to be 
complex. The effect of mare age on foetal copper stores needs further investigation . 
- - - - - ------------- - - -
90 
Chapter 5 
The Effect of Copper Supplementation 
on the Copper Status of Pasture-Fed 
Young Thoroughbreds4 
4 Submitted as :  SG Pearce, ND Grace, EC Firth, JJ WichteI ,  SA Holle 
and PF Fennessy ( 1 997) Equine Veterinary Journal. 
91  
5.1 Abstract 
The effect of copper supplementation of pasture fed mares and foals on the copper status of the foals ,  in terms of plasma, soft tissue and bone copper concentrations and 
caeruloplasmin activity, was investigated. Twenty one Thoroughbred foals from either 
control mares (n = 9), or copper-supplemented mares (n = 1 2) were randomly divided into 
control (pasture only, n = 1 0) or supplemented (pasture and oral copper sulphate, n = 1 1 ) 
groups. The pasture diet was grazed by al l  animals, and contained 4.4 to 8 . 6  mg Cu/kg dry 
matter (DM). The copper supplement for the mares contained copper sulphate equivalent to 
0 . 5  mg CU/kg l iveweight (L W)/day, and was administered three times a week for 1 3  to 2 5  
weeks prior to foal ing. The supplemented foals, also dosed three t imes a week, received 0.2 
mg Cu/kg LW/day at 2 1  days of age, which was increased to 0 . 5  mg Cu/kg LW/day at 49 
days and was continued at th is level unti l  euthanasia at 1 5 0 days. Foal p lasma copper 
concentration and caeruloplasmin activity increased from birth to 2 1  days postpartum and 
then plateaued at a concentration simi lar to the mare, but the rise in these indices was not 
affected by copper supplementation of the mare or foal .  Copper supplementation of the foal 
increased foal l iver copper concentration at 1 50 days (p < 0 . 0 3 ) .  Copper intake of diets 
containing approximately 8 to 30 mg Cu/kg DM is well reflected by l iver copper 
concentration, but is poorly reflected by bone, other soft tissue copper concentrations and 
circulating copper status indices. 
5.2 Introduction 
Copper is an essential trace element, associated with a variety of metal loenzymes 
responsible for many biological ly important functions, and there is growing interest in the 
role  of copper deficiency in certain c l inical conditions. Recent work has suggested a 
relationship between dietary copper intake and abnormalities of bone and cartilage 
development in the young growing horse (Knight et al. 1 98 5 ;  Gabel et al. 1 98 7 ;  Asai et al. 
1 993), and has resulted in some concern regarding the adequacy of dietary copper 
requirements recommended by the NRC ( 1 989). 
Many factors influence the effect of d ietary copper intake on copper status in  animals. Much 
of the work has involved ruminants and includes factors such as the post-weaning 
development of the rumen (Suttle 1 97 5 )  and mineral antagonists such as molybdenum, 
sulphur, iron and zinc, that affect copper absorption and metabol ism (Gooneratne et al. 
92 
1 994; Suttle 1 994). Less information is avai lable for the horse, and differences in copper 
requirements for horses of different age, sex, breed and pregnancy status have not been well 
defined. Cymbal uk et al. ( 1 98 1  a) studied copper metabolism in a smal l number of mature 
ponies using isotope techniques, and from calculations of obl igatory (endogenous) losses, 
estimated dietary copper requirements to be 3 . 5  mg Cu/kg DM. Isotope kinetic studies 
provide accurate i nformation on mineral metabol i sm, but generally involve only small 
numbers, and breed differences may l imit extrapolation . For instance, breed differences in 
copper absorption and metabol ism on various copper d iets have been demonstrated in  sheep 
(Wool l iams et al. 1 983)  and cattle (Ward et al. 1 995) .  Two other commonly used 
approaches for determining m ineral requirements are factorial model l ing techniques and 
dose response trials. 
The factorial model (Grace and Clark 1 99 1 )  i s  a mathematical approach which determines 
mineral requirements from information obtained from isotope kinetic studies, nutrition 
balance trials and s laughter experiments. This approach is l imited by the lack of information 
on copper metabol ism in the horse. For example, there are very wide ranges reported for the 
apparent absorption of copper from various feeds (Cymbaluk and Smart 1 993) and for 
faecal endogenous losses (Cymbaluk et al. 1 98 1 a; Pagan 1 994). Changing growth rates, 
interactions with other minerals, and substitution of mi lk  with sol id feed further compl icate 
the model. 
Cupps and Howel l  ( 1 949) performed a dose response trial (8, 1 8  and 1 08 mg Cu/kg DM) in 
Percheron foals (aged 1 03 to 1 85 days), to determine d ietary copper requirements. No 
differences in  growth rate, plasma copper, packed cell volume, blood cell count and 
haemoglobin concentration were detected, and so it was concluded that copper requirements 
were less than 8 mg Cu/kg DM. It was also noted that the role of h igher d ietary levels of 
copper on the incidence of carti lage erosions needed to be further investigated. This study 
was influential in establ i shing the NRC ( 1 989) dietary copper requirements for horses. 
The relationship between dietary copper intake and bone disease in  growing horses has 
recently been reviewed (Cymbaluk and Smart 1 993).  Knight et al. ( 1 990) and Hurtig e t  al. 
( 1 993) performed dose response trials specifical ly investigating abnormalities of bone and 
cartilage development. Both groups found that increases in d ietary copper above the NRC 
( 1989) requirements ( 1 0  mg Cu/kg DM) resulted in a lower incidence of developmental 
orthopaedic disease (DOD). The increased dietary copper levels did not abol ish DOD, as 
93 
might be expected with a multifactorial disease. These studies resulted in recommendations 
to increase dietary copper intake. For instance Hurtig et al. ( 1 993 ) suggests a diet containing 
20 to 2 5  mg Cu/kg DM for all growing horses, and Lewis ( 1 99 5 )  suggests 5 0  mg Cu/kg DM 
for creep feeds, and 2 5  mg Cu/kg DM for weanl ings. These copper concentrations are 
considerably greater than can be suppl ied by pasture alone. 
Despite the proposed beneficial effect of increased copper intake on bone and carti lage 
development, the relationship between dietary copper intake, copper status and bone and 
carti lage development in foals is sti l l  unclear. The work described here was part of a larger 
study investigating this relationsh ip.  The objective of th is  study was to assess the effect on 
foals of oral copper supplementation of their pregnant dams and, of oral copper 
supplementation of the foals from 2 1  to 1 50 days of age, in terms of changes in copper 
concentration of plasma, soft tissue and bone, and caeruloplasmin activity. 
5.3 Materials and methods 
5.3.1  Animals 
Mares 
Pregnant Thoroughbred mares (n = 24, 4 to 20 years of age) were purchased, and grazed on 
tal l  fescue pasture from May 1 994 for the duration of the experiment at AgResearch ' s F lock 
House Research Centre near Bul ls  on the North Is land of New Zealand. The mares were 
randomly al located to either a control or copper supplemented group, matched for mare age, 
date of last service and the s ire of the pregnancy. Copper supplementation began in June 
when the mares were 1 3  to 2 5  weeks prior to foal ing. 
Foals 
Nine foals from the control mares and 1 2  foals from the copper supplemented mares were 
then randomly divided again, into control and copper supplemented groups. Three of the 24 
foals died in the perinatal period. Copper supplementation of the foals began 2 1  days 
postpartum. Foals were weighed weekly unti l January 1 995 (foals aged 2 to 5 months), and 
thereafter were weighed fortn ightly. 
94 
5.3.2 Diet 
Pasture 
The mares were grazed tal l fescue pasture, and when pasture avai lab i l ity was inadequate to 
meet requirements (from May to July), they received supplementary tall fescue hay 
produced from the same property. Hay constituted less than 25 % of the total dai ly dry 
matter intake of the mares during peak hay consumption. 
Copper supplementation 
The mares and foals were supplemented orally, three times a week, with an aqueous solution 
of copper sulphate administered using a feeding syringe or a drench gun. The control groups 
received a placebo consisting of an equivalent amount of water and the basic m ineral 
supplement of zinc and selen ium. Supplementation of the mares ceased at term. The mares 
were dosed at a rate of 0.5 mg Cu/kg LW/day (equivalent to approximately 30 mg Cu/kg 
DM of the diet). At 2 1  days the foals were dosed at 0.2 mg Cu/kg L W /day which was 
increased to 0 .5  mg Cu/kg L W /day by 49 days. The foals  were then dosed at that rate until 
euthanasia. Dosages were adjusted weekly according to l iveweight gain.  Both the p lacebo 
and the copper sulphate supplement contained the equivalent of 0.02 mg/kg L W /day Se and 
0.5 mg/kg L W /day Zn to ensure that these trace elements remained above NRC ( 1 989) 
recommended levels throughout the trial .  
5.3.3 Samples 
Pasture and hay 
Pasture was col lected at 1 0  to 1 4  day intervals and bulked to give monthly samples. 
Subsamples of hay fed over winter were also col lected. Samples were stored at -20°C prior 
to mineral determination. 
Blood 
Jugular venous blood from the foal s  was collected into trace e lement, sodium heparin 
vacutainers (Becton Dickinson, #36773 5), on days 0, 3, 7, 1 4, 2 1  and 28 postpartum, for 
determination of p lasma and b lood cel l  copper concentrations and p lasma caeruloplasmin 
activity. The animals were then p laced in s imilar aged cohorts and sampled at 
approximately 60, 90 and 1 50 days. The vacutainers were placed on ice for transport to the 
laboratory, and then the plasma and blood cells were separated by centrifugation, and stored 
95 
at -2oDe until analysis. P lasma samples for determ ination of caeruloplasm in activity were 
stored at 4°e and analyses were performed w ithin 2 to 3 days from sampling. P lasma copper 
concentration and caeruloplasmin activity were used in preference to serum because of the 
possibi l ity of caeruloplasmin bound copper being incorporated into the b lood clot resulting 
in erroneously low values (Paynter 1 982). 
Milk 
Milk samples (20 to 5 0  m!) were collected at the same time periods as the b lood samples, by 
manual expression, after exc luding the foal for a short period. 
Soft tissues 
The foals were euthanased at 1 46 to 1 6 1  days (20 foals) while one foal was euthanased 
earlier ( 1 1 2  days) as a result of a traumatic injury sustained in the paddock. Fol lowing 
sedation with xylazine, the foals were euthanased with a lethal injection of barbiturate and 
placed on a postmortem table. The brachial vessels  were opened and the blood collected. 
The internal organs were excised, weighed and subsampled. The gastrointestinal tract was 
removed, emptied of ingesta, and tissue subsamples were taken from the stomach, small 
intestine and large intestine and homogeni sed.  The carcass was skinned and the integument 
(not including the hooves) subsampled. A muscle subsample was taken from the m. 
semitendinosus, m. semimembranosus and m. biceps jemoris . 
Bones 
The atlas was d issected and defleshed, and a 1 5  mm wide by 80 mm long section was cut, in  
a frontal p lane, through the wing of the i l ium, midway between the tuber coxae and the 
tuber sacrale. 
5.3.4 Analytical 
Al l  g lassware used was acid washed, and all care was taken to prevent contamination from 
external sources. 
96 
Pasture and hay 
Samples were dried in an oven at 60°C for 1 6  h, and then ground unti l  the sample could pass 
through a I mm sieve. The pasture was weighed out accurately (0.5 g) and wet d igested. Five 
ml  of concentrated HN03 was added to the sample and left overnight. The mixture was then 
refluxed at I 20°C for 1 hour, heated at 200°C for 3 to 4 hours to evaporate the ac id, and the 
residue was reconstituted in 1 0  ml of 2M HC} and the copper concentration determined 
using inductively coupled plasma emission spectrometry (lCP) (Lee 1 983) .  
Plasma copper concentration 
Analyses were performed by ICP, as presented in Chapter 4 .  
Blood cell copper concentration 
The blood cel ls were separated by centrifugation, washed twice with i sotonic saline and then 
after centrifugation at 2000 rpm for 1 0  m inutes, approximately 1 g of the blood cells was 
weighed out accurately and wet digested as for the pasture samples, and the copper 
concentration determined using ICP. The concentration was expressed as mg Cu/kg b lood 
cells wet weight. 
Caeruloplasmin activity 
Plasma caeruloplasmin  oxidase activity was measured using a colorimetric method (Ravin  
196 1 ). CaerulopJasmin catalyses the oxidation of P-phenylenediami ne-dihydrochloride 
(PPD), a white crystal (Holmberg and Laurell 1 95 1 ). A spectrophotometer was used to 
measure the optical density after incubation of PPD with caeru loplasmin at a pH of 5 .4 to 
5 . 5  and a temperature of 3 JOC. The results are expressed as absorbance units at 530  nm 
(37°C) per l itre of p lasma. 
Milk 
Three m l  of concentrated HN03 and I .S ml 3 0  % H202 were added to 3 m l  of m i lk, from a 
thoroughly m ixed whole mi lk sample, in a 25 m l  conical flask. Wet d igestion was 
performed, evaporating the acid and H202• The digest was made up to 5 ml  with 2 M HCl 
and the mineral e lement concentration was determined by Iep. 
Soft tissue 
----- - �- �------
97 
A sample of fresh tissue (approximately 1 .0 g) was weighed accurately into a 5 0  ml  conical 
flask and desiccated in a dry matter oven at 1 00°C for 1 6  hours, and the dry matter 
determ ined . Concentrated HN03 ( 1 0  ml)  was added and left overnight. The sample was 
refluxed for 4 to 5 hours and the acid evaporated. The sample was made up to 1 0  m l  with 
2M HCI and the concentration of m ineral e lements determined using the ICP. 
Bone 
Subsamples of bone ( 1 00 to 250g), dried at 60°C for 24 h, were ashed at 480°C for 1 6  h to 
determine dry matter and ash content. The ashed bone was then ground to a fine powder and 
a 0.2 g sample was dissolved in 1 0  ml of 1 .6 M HN03. The copper was determined using 
Zeeman flameless atomic absorption spectrometer by the method of additions. Standards 
were made up in a synthetic bone solution prepared from calcium phosphate. 
5.3.5 Statistical analysis 
The p lasma copper concentration, b lood cel l  copper concentration, caeruloplasmin activity 
and m i lk copper concentration were analysed by analysis of variance (ANOVA) with 
repeated measures over time, according to mare treatment up unti l  2 1  days postpartum and 
then (except mi lk  copper concentration data), as four groups according to foal and mare 
treatment. Soft tissue and bone data were analysed by ANOV A according to treatments. The 
bone copper concentrations were log transformed prior to analysi s  to standardise the 
varIance. 
The regression relationship between plasma copper parameters and time were generated for 
each foal over the 5 sampl ings from day 0 to 2 1 .  A separate pooled regression was 
establ ished for the foals from control mares and copper-supplemented mares and compared. 
The relationship between caeruloplasmin activity and plasma copper concentration was 
determined for the whole time period and for the periods from day 0 to 2 1  and day 28 to 
1 50 .  
The standard model used included sex of the foal, foal ing date, post-conceptional age and 
age at euthanasia (postmortem samples), as covariates. Analyses were performed with 
Statistical Analysis System (SAS) version 6 . 1 1  (SAS Institute Inc. Cary, N .C . ,  USA) using 
proc glm for ANOVAs, and proc mixed for ANOV As with repeated measures over time. 
98 
5.4 Results 
5.4. 1 Animals 
The mares remained healthy throughout the experiment and were maintained at a condition 
score above 5 on a 1 to 9 scale (Henneke et al. 1 983). Likewise, all foals remained healthy 
throughout the trial, except for one foal that developed complications fol lowing a biopsy of the 
distal radial growth plate, but continued to grow well, and at euthanasia at 1 4 2  days it 
weighed 200 kg with an average growth rate of 1 . 09 kg/day. Another foal developed an acute 
non-weight bearing left hind limb lameness of traumatic origin, and was euthanased 
prematurely at 1 1 2 days. Its growth rate to that stage was 1 .3 9  kg/day. The remainder of the 
foals grew well and at euthanasia their growth rates from birth ranged from 0.99 to 1 .2 2  
kg/day. There was no effect of either mare or foal copper supplementation on growth rate. 
5.4.2 Pasture mineral composition 
The tall fescue copper concentrations ranged from 4.4 to 8 .6  mg Culkg DM. The 
concentrations of the pasture minerals, and mineral analysis of the hay which supplemented 
the pasture diet of the mares from May to July 1 994 are presented in Table 5 . 1 .  
Table 5.1 Mineral composition of tall fescue pasture from May 1994 to March 1995 and tall 
fescue hay fed to the mares to supplement the pasture diet from May to July 1 994. 
Na K Ca P Mg S Cu Fe Zn Mn Mo 
g/kg OM mg/kg OM 
May 1 .9 29 2 .9 3.6 1 .7 2 .8  5 .6 1 1 0  21  55 1 .0 
June 1 .7 47 3 . 1  4.3 1 .6 4.4 8.6 74 25 40 2.8 
July 1 .2 44 3.2 3.4 1 .5 4.4 6 .5  83 29 37 1 .5 
Aug 1 .5 46 2.8 3 .1  1 .5 3 .6 7 .9  95 20 53 0.7 
Sept 1 .7 69 2 .8 3.6 1 .5 4.4 7.7 69 25 53 1 .5 
Oct 1 .7 28 3 .8  2 .7  1 .2 3.4 5 .5  48 20 56 1 .2 
Nov 1 .3 35 2.5 2.6 1 .5 4.0 6 . 1  1 06 20 50 1 . 1 
Dec 1 .8 34 2.8 2.8 1 .3 2.8 8 . 1  1 32 20 57 0 .9  
Jan 1 .4 30 2.7 2 .3 1 .3 3.2 4.5 1 06 1 9  40 1 .5 
Feb 0.7 3 1  3 . 1  2.4 2.2 3 . 1  4.4 290 1 9  68 0.9 
Mar 1 .2 33 3.6 2 .8 2 .1  3 .2 4 .8  340 22 95 1 . 3 
Hay 1 . 5 30 4.8 3 .9  2 .4 3 . 1  1 0.9  239 33 66 1 .7 
99 
5.4.3 Mare milk copper concentration 
The mare's milk copper concentration was highest on the first day postpartum, ranging from 
0 . 3 2  to 1 .7 mg Cull milk. The milk copper concentrations decreased, plateauing after the 1 4  
day sample at approximately 0 . 2  mg Cull (Figure 5 . 1 ) .  There was a significant effect of tirne 
on copper concentration but there was no effect of prepartum mare copper supplementation on 
the milk copper concentration. 
Figure 5.1 Mean milk copper concentration (mg!l, +/- SEM) of all lactating mares from birth to 
150 days (n "" 21). 
0 .9  
0 .8  
� 0.7 
5 
>:: 0 .6  
'E 
8 
1:: 0.5 'l) u 
§ u 0.4 
t; p.. 
g. 0.3 u 
= 
� 0.2 
0. 1 
0.0 +------+--------'----_-+-____ --1-____ --., 
o 30 60 90 1 20 1 5 0  
Days postpartum 
5.4.4 Foal plasma copper concentration, caeruloplasmin activity and blood cell 
copper concentration 
There was no effect of copper supplementation of the prepartum mare on foal plasma copper 
concentration and caeruloplasmin activity, nor was there an effect of copper supplementation 
of the foal on these plasma indices from days 2 8  to 1 5 0 .  
1 00 
The foal  plasma copper concentration was initially low compared to adult values, ranging 
from 0 . 1 5  to 0.29 mg Cull, and increased over the first 2 1  days and plateaued at a level of 
1 .0 to 1 .4 mg Cull (Figure 5 .2).  Plasma caeruloplasmin activity was also low on the first day 
after b irth (4 to 1 1  U/I) and increased, reaching a plateau at 2 1  to 28 days. There was 
considerable indiv idual and sample variation in the caeruloplasmin activity with values 
ranging from 36 to 1 0 8 Ull in the period from 28 to 1 5 0 days (Figure 5 .3) .  From day 0 to 2 1 ,  
there was a s ignificant effect of time (p < 0.00 1 )  on both plasma copper concentration and 
caeruloplasmin activity. Analysis  of regression equations for individual foals for both 
plasma copper concentration and caeruloplasmin activity from day 0 to 2 I found no 
significant difference due to mare copper supplementation on the rate of increase of these 
indices over this  period. 
Figure 5.2 Mean plasma copper concentration (B, mg/I, +/-SEM) and blood cell copper 
concentration (+, mg/kg fresh weight, +I-SEM) of all foa ls (n= 2 1 )  from birth to 1 50 days. 
1 .4 
1 .2 
1 .0 
0.8 
0.6 
0.4 
0.2 
0.0+---------�-------+--------�--------�------_4 
o 30 60 90 1 20 1 50 
Days postpartum 
1 01 
Figure 5.3 Mean p lasma caeruloplasmin activity (U/I, 37°C, +/- SEM) of al l  foa ls (n = 21)  from 
birth to 150 days. 
80 
U 70 0 t--M 60 
2- 50 
.q > 
tl 40 '" 
c 
E '" 30  '" 
"C.. 
..s 20 
2 v OJ 1 0  U 
0 
0 30  60 90 1 20 1 50 
Days postpartum 
Overall there was a significant relationship between plasma copper concentration and 
caeruloplasmin activity (p < 0.00 1 ,  r = 0 .69). From 0 to 2 1  days, plasma copper 
concentration and caeruloplasmin activity were highly correlated (r2 == 0.73)  (Figure 5 .4a), 
whereas in the period from day 28 these two indices were less wel l  correlated (r == 0.23) 
(Figure S .4b). 
The mean foal blood cel l  copper concentration ranged from 0.63 to 0 .82 mg Cull (Figure 
5 .2), and there was no effect of time or of copper supplementation of either mare or foal on 
blood cel l  copper concentration in  either time period (0 to 2 1  or 28 to 1 50 days). 
1 02 
Figure 5.4a Relationship between plasma copper concentration and caeruloplasmin activity of 
all foals (n = 2 1 )  from :  
a )  birth to 2 1  days, and 
b) 28 to 1 50 days. 
Figure 5.4a. 
1 20 
G 
1 00 
0 t-M 
� 80 
i::' 
:2: 
u 60 « 
c: 
"2 
</l 
� 40 0. 0 
::; 
V '" u 20 
0 
Figure 5.4b. 
1 20 
G 1 00 0 t-M 
::::: 80 ::J '-' 
i::' 
.;::: 
U 60 '" c 
.§ 
Vl 
40 0:: 0. 
.5: ::l .... <l) '" 20 u 
0 
0 - 2 1  days 
y = 47.2x + 3 .0  
[2 = 0.73 
n =  1 05 
• 
• 
• 
• 
a .  
• • 
.. '. �. . 
r1' . • •• 
• • .. . . . • 
28 - 1 50 days 
y = 32.4x + 23 . 1  
r =  0.23 
n = 84 
• 
• • • • • I • • • • 
.- • • -• • • 
• • • B. • 
• • • • • • • • 
• 
• • 
• 
• • • 
0 0 .2 0 .4 0.6 0.8 1 .2 1 .4 
Plasma copper concentration (mg/l) 
• 
• 
• 
• 
• 
• 
• • • 
• 
• • 
• • • 
1 .6 1 .8 2 
1 03 
5.4.5 Foal soft tissue copper concentration 
The mean organ copper concentrations for the 4 treatment groups, at 5 months of age are 
presented in Table 5 .2 .  Mare supplementation, foal supplementation and the interaction of 
the two treatments had no effect on any organ except l iver. Foal treatment had a significant 
effect (p < 0.03) on l iver copper concentration. The l iver copper concentration at 5 months 
(mean +/- SEM) was 2 5 . 1 +/- 2 .4 and 6 8 . 8  +/- 1 5 .3 mg Cufkg DM for control and copper 
supplemented foals respectively. 
Table 5.2 Effect of copper supplementation of the mare prepartum, and the foa l  from 21 to 1 50 
days on the mean (+!- standard error of the difference (SED)) copper concentration (mg Cu/kg 
DM) of foa l  organs at 1 50 days. 
Mare treatment Foal treatment SED Effect of copper 
(MT) (FT) supplementation I 
Organ control + Cu control + Cu MT FT 
mg Cu/kg OM 
Brain 9.3 9 . 7  9.2 9.8 0.8 NS 0.079 
Gut 7 . 8  7 . 1 7 . 2  7 . 7  1 .2 NS NS 
Heart 1 7. 2  1 8 . 1  1 7. 6  1 7. 8  1 .2 NS NS 
Kidney 3 7 . 8  4 5 .3 43 . 2  3 9 . 8  8 . 3  0 . 0 5 7  NS 
Liver 4 5 . 8  4 8 . 1 2 5 . 1  6 8 . 8  3 9.2 NS 0.022 
Lung 7 . 6  8 . 7  8 . 2  8.0 1 . 5 NS NS 
Muscle 7 . 4  7 . 1 7 . 2  7.3 2 . 1 NS NS 
Skin 1 .4 1 .4 1 .3 1 . 5 0 . 4  NS NS 
Spleen 4.0 4.0 4 . 0  4 . 0  0 . 5  NS NS 
I NS, P > O. I O  
5.4.6 Foal bone copper concentration 
There was no effect of copper supplementation of the mare or foal on the percentage of ash, 
copper, iron, zinc, calcium or phosphorus concentration of the atlas and i l ium. The mean 
(+/- SEM) bone copper concentration for the atlas and i l ium were 0.63 +/- 0 . 024 mg Cu/kg 
OM and 1 .00 +/- 0 . 0 8 8  mg eu/kg OM (Table 5 .3 ) .  
1 04 
Table 5.3 The m ean (+1- SEM) ash, copper, zinc, iron, calcium and phosphorus concentration of 
the atlas and ilium from all foals (n = 2 1 )  at 1 50 days of age. 
Ash Cu Zn Fe Ca P 
g/ 1 00g OM mg/kg OM g/kg O M  
Atlas 42 (0.4) 0.6 (0.02) 53 (2.6) 1 29 (7.7) 1 53 (7.2) 75 (3.2) 
I lium 35 (0.8) 1 .0 (0.09) 37 (2.8) 1 1 5 ( 1 3 .0) 89 (6.8) 45 (3 .5) 
5.5 Discussion 
Increasing the foal ' s  average dai ly copper intake 3 to 4 fold, by oral copper supplementation 
(8  vs 30 mg Cu/kg DM), increased the l iver copper stores of the foal (measured at 
euthanasia at approximately 1 50 days). However there was no effect of mare or foal 
treatment on plasma copper concentration, blood cell copper concentration and plasma 
caeruloplasmin activity or copper concentration of bone and organs, other than the l iver. 
The observed changes in p lasma copper concentration and caeruloplasmin activity with time 
are consistent w ith the study of Bell et al. ( 1 987), and rose more slowly than in  sheep, which 
reach adult level s  by 3 to 4 days postpartum (McCosker 1 968). 
Oral copper sulphate was chosen as the source of supplementary copper. An oral 
formulation was required because our primary interest was to determine the effect of an 
increase in d ietary copper on copper status. Injectable copper formulations are available, 
and have been reported to al leviate apparent copper deficiency syndromes in  horses (Egan 
and Murrin 1 973a). However they can cause considerable local tissue reaction in some 
individuals (SG Pearce and EC F irth, unpubl ished data), and there has been one report of 
induced copper toxicity in a mature horse after inj ection with copper D-penici l lamine (4 
mg/kg) (Auer et al. 1 989b). For these reasons, some authors do not recommend the use of 
copper i njections in horses (Cymbaluk and Smart 1 993).  
An investigation on the b ioavailab i lity of cupric chloride, acetate, oxide, sulphate and 
carbonate, by measuring the hepatic copper uptake in sheep, showed that copper sulphate 
had the h ighest biolog ical avai labil ity (Ledoux et al. 1 995) .  In  another experiment in cattle 
with experimental ly-induced copper deficiency, the bioavailabi l ity of copper sulphate and 
copper lysine was s imi lar and superior to copper oxide, as assessed by plasma copper 
concentration and caeruloplasmin  activity (Kegley and Spears 1 994). Recent work suggests 
that copper amino acid complexes may be absorbed via a different route or pathway than 
1 05 
inorganic copper (Ou et al. 1 996). There may also be species and breed differences In 
copper absorption and avai labi l ity from different sources of supplemental copper. 
Mi lk and plasma copper concentrations recorded in the present experiment were simi lar to 
those in mares fed diets with copper concentrations of 7 and 1 2  mg Cu/kg OM/day 
(Breedveld et al. 1 988). However, the mi lk  copper concentrations reported by Ul lrey et al. 
( 1 974) are a l ittle h igher (0.99 mg/l at parturition, plateauing at 0 .29 mg/l after 3 weeks), 
and those of Schryver et al. ( 1 986) are considerably h igher (0 .85 ,  0 . 5 5  and plataeuing at 
0.29 mg/l at I ,  4 and 8 weeks respectively). Al l  analyses were performed using atomic 
absorption spectrometry and we are unable to explain the differences in these results, but 
they may be due to unknown interactions in with other d ietary constituents, and, although 
unlikely, differences in laboratory technique. There was no relationship between mare l iver 
copper stores (determined in Chapter 4) and mi lk  copper concentration, and there was no 
residual effect of copper supplementation of the prepartum mares on the milk copper 
concentration. Several other studies have also found that copper supplementation of mares 
does not alter their mi lk copper concentration (Breedveld et al. 1 988, Baucus et al. 1 989, 
Asai et al. 1 995) .  
Oietary copper intake i s  thought to account for some of the variabi l ity in p lasma and serum 
copper concentration in horses (Auer et al. 1 988a; Stub ley et at. 1 98 3 ;  Mee and McLaughl in  
1 995), but th is  was not evident in  this study. In another study, horses fed diets containing 
either 7 or 30 mg Cu/kg OM did not exhibit detectable differences in serum copper 
concentration despite large increases in l iver copper and fewer bone and carti lage lesions in  
the h igh copper intake group (Hurtig e t  al. 1 990). In a further study, h igh levels of dietary 
copper (79 1 mg Cu/kg OM) fed for 6 months, did not affect whole b lood or p lasma copper 
concentration in ponies (Smith et al. 1 975) .  In rum inants, if l iver copper stores are adequate, 
increasing copper intakes wi l l  not usually alter blood copper concentration but wi l l  continue 
to increase copper accumulation in the l iver (Wool liams et al. 1 983) .  Thi s  appears also to be 
true with horses in th is study. 
Caeruloplasmin being acute phase protein (Gutteridge 1 978), is sensitive to many factors 
other than dietary copper intake. It is increased by chronic infection and inflammation, and 
is decreased by malnutrition, nephrosis and chronic hepatitis. In humans, p lasma copper 
concentration is h igher in women than men, and h igher in pregnant women and in  
individuals on oral contraceptives (Mi lne 1 994; Solomons 1 979). There i s  some evidence 
1 06 
that procedures that provoke an inflammatory response may affect circulating copper 
indices in horses. For example, Smith and Cipriano ( 1 987) found that caeruloplasmin 
activity increased fol lowing intramuscular injection of ponies with turpentine oi l ,  Firth et al. 
( 1 987) found an increase in serum copper fol lowing intra-articular l ipopolysaccharide 
administration III ponies, and Auer et al. ( 1 989a) found an increase in plasma copper 
concentration, caeruloplasm in concentration and caeruloplasmin activity fol lowing 
intramuscular i njection of Freund' s  adjuvant into horses. In  the present experiment, foals 
underwent laparoscopic l iver biopsy and a biopsy of the distal radial growth plate in  one 
surgical procedure performed 4 to 1 0  days after parturition. The surgical procedures 
performed on our foals m ight have been a sufficiently inflammatory stimulus to alter the 
pattern of change of p lasma copper concentration and caeruloplasmin activity. However in a 
separate experiment, foals of the same age and under s imi lar conditions, but without 
surgery, showed simi lar changes in copper concentration and caeruloplasmin activity over 
days 0 to 28 as presented here (SG Pearce and EC Firth, unpubl ished data). 
Caeruloplasmin is also a transport enzyme (Gutteridge 1 978) for p lasma copper and 
accounts for approximately 70 % of circulating copper in horses (Auer et al. 1 988b). Shortly 
after birth there is a very low concentration of caeruloplasmin due to an inab i l ity of the 
foetal l iver to produce the active enzyme (Chang et al. 1 975 ;  Mi lne 1 994). Thus at very low 
but increasing levels of caeruloplasmin activity soon after birth, it is expected that the 
caeruloplasmin and plasma copper concentration wil l  be h ighly correlated . After 28 days, by 
which time the caerulop lasmin activity had reached adult levels and was probably no longer 
l im iting plasma copper concentration, there was a poorer correlation. A s imi lar situation has 
been demonstrated in rats, whereby supplementation of copper deficient rats with copper 
wi l l  increase caeruloplasmin activ ity, but in rats with adequate copper stores, copper 
supplementation does not alter caeruloplasmin activity (Linder et al. 1 979). 
B lood cel l copper concentration was not affected by age of the foal nor by copper 
supplementation of the mare or foal. Intracel lular copper concentration has been suggested 
to be a better indicator of copper status than p lasma copper concentration as it is less lab ile, 
and therefore less responsive to non-dietary factors (Milne 1 994). 
There was no sign ificant relationship between neonatal l iver copper concentration 
(determined III Chapter 4) and l iver copper concentration at euthanasia ( 1 50 days). 
However, copper supplementation of the foal significantly attenuated the decl ine in l iver 
1 07 
copper concentration over the 1 50 days. There was a decl ine of approximately 92% and 
82% of l iver copper concentration, from birth (ie at 4 to 1 0  days of age), of control and 
copper supp lemented foals respectively. Therefore at euthanasia, the l iver copper 
concentration from copper supplemented foals was nearly 3 fold h igher than control foals. 
This suggests that l iver copper concentration equi l ibrates to levels that reflect the dietary 
copper intake in sucking foals.  Liver copper concentration has been shown to not reflect 
copper availab i l ity in situations of zinc-induced hypocuprosis (Bridges and Moffitt 1 990), 
and therefore other information regarding the d iet and other indices of copper status must be 
taken into account along with l iver copper concentration when assessing an animal ' s  copper 
status (Cymbaluk and Smart 1 993) .  
Copper supplementation of the mare, foal, or both mare and foal had no significant effect on 
the copper concentration in any organ except the l iver. However in  other studies, brain 
copper concentration has been reported to be sensitive to marginal copper deficiency in rats 
(Hopkins and Fai l la 1 995), and kidney copper concentration has been reported to vary with 
different d iets in yearling horses (Cymbaluk and Chri stensen 1 986). There was some 
evidence of such effects in th is study with a trend toward increased brain copper 
concentration in supplemented foals (p < 0 .08), and increased kidney copper concentration 
in foals born to copper supplemented mares (p < 0.06). 
Two predominantly cancellous bones ( i l ium and atlas) were chosen to examine the effect of 
copper supplementation on bone percent ash content and mineral concentration. In sheep, 
increased copper intake (8 vs 1 7  mg Cu/day) of pasture fed sheep resulted in a sign ificant 
increase in calc ium and phosphorus content of the skeleton (Grace and Lee 1 988). Another 
study found an increase in bone ash copper concentration in sheep receiving copper 
supplementation (Hogan et at. 1 97 1 ). In cattle, copper supplementation did not increase 
bone copper concentration, but copper depletion s ignificantly reduced the organic matter 
content of bones (Suttle and Angus 1 978). Copper-depleted rats had increased bone zinc 
concentration and decreased iron concentration (Tsai and Lei 1 979). In the present study, 
there was no effect of copper supplementation of the mare or foal on bone mineral 
concentration or bone ash content. This might be due to the copper intake levels of the 
animals in thi s  trial not being extreme enough to detect a difference, although the 
difficulties of accurate analysis may also be important. For example, a sample from the 
i l ium was relatively easy to col lect from a fixed, standard site, and the whole the atlas was 
taken, both of which m inim ised any bias due to variations in the sampling s ite. However the 
1 08 
detennination of bone copper concentration was difficult as there was only a small amount 
of copper in the presence of large amounts of calcium and phosphorus. The variation 
between animals in the bone copper concentrations was also substantial .  These factors 
would seem to m itigate against the use of bone copper concentrations as useful indices of 
copper status. 
Oral copper supplementation of mares in late gestation and nursing foals altered the copper 
balance in these horses, resulting in an increase in the foal ' s  l iver copper stores at birth and 
at 5 months of age respectively. The l iver copper concentration of weanl ing foals appears to 
be a good indicator of their dietary copper intake. Increased l iver copper stores of the 
neonate may be important for ensuring healthy development of the skeleton during the 
period of maximum post-natal growth. 
1 09 
Chapter 6 
The Effect of Copper Supplementation 
on the Evidence of 
Developmental Orthopaedic Disease 
in Pasture-Fed Thoroughbreds5 
5 Submitted as : SG  Pearce, EC Firth, ND Grace and PF  Fennessy ( 1 997) Equine Veterinary 
Journal. 
1 1 0 
6.1  Abstract 
The effect of copper status on the evidence of bone and cartilage lesions was investigated in 2 1  Thoroughbred foals .  The foals, and their dams, were grazed on 
pasture containing 4 .4 to 8 .6  mg Cu/kg dry matter (OM). Four treatment groups were 
created by randomly allocating mares and their foals to either copper supplemented (0.5 mg 
Cu/kg l iveweight (LW)/day), or control (pasture only) groups. This experimental design 
allowed the effect of copper supplementation of mare and foal to be examined 
independently. Parameters of bone and cartilage development were assessed in the foals 
both in vivo, and at postmortem at approximately 1 50 days of age. Mare copper 
supplementation significantly (p < 0.0 1 )  decreased radiographic indices of physitis in the 
d istal third metatarsal bone of the foals at 1 50 days, and the prevalence of articular cartilage 
lesions (p < 0.05) .  Articular carti lage lesions were minor in all foals, with no evidence of 
cl inical developmental orthopaedic disease (DOD) in vivo, with the exception of m inor 
radiographic changes assessed at postmortem.  Copper supplementation of the foal had no 
effect on any of the bone and carti lage parameters. Copper supplementation of the mares did 
not abolish DOD in the growing foals, emphasising the l ikely multifactorial nature of this 
condition. However, mare supplementation may be a useful treatment regime on a farm 
where the incidence and severity of DOD are of concern. 
6.2 Introduction 
Developmental orthopaedic d isease (DOD) is a term used for a group of diseases in growing 
foals that are caused by abnormalities in  the development and maturation of carti lage and 
bone. This group includes: osteochondrosis, subchondral bone cysts, physitis, acquired 
angular l imb deformities, flexural l imb deformities and cuboidal bone malformation 
(Beeman and Mcllwraith 1 986).  Cervical vertebral malformation and juveni le arthritis have 
also been proposed to be included in thi s  group because of their putative relationship with 
osteochondrosis (Reed et al. 1 987; Watrous et al. 1 99 1 ) .  The aetiology for these d iseases is 
l ikely to be multifactorial, and one particular aetiology may only be associated with a subset 
of the group of diseases (Palmer 1 993 ; Pool 1 993 ; Hurtig and Pool 1 996). 
DOD i s  considered to contribute to major economic wastage In the horse i ndustries 
worldwide. In New Zealand, a relationship between copper status and DOD was recognised 
cl in ically over 20 years ago, when exostoses above and below the fetlock responded to 
1 1 1  
copper supplementation (Carberry 1 978). Other simi lar c l inical observations of copper 
responsive DOD have been reported in America (Hildebran and Hunt 1 986) and Ireland 
(Egan and Murrin 1 973a) .  S imi larly, in New Zealand, deer grazing copper-deficient pasture 
developed lameness, angular l imb deformities and swol len joints (Thompson et al. 1 994). 
Lesions described were simi lar to those found in copper-deficient foals (Bridges and Harris 
1 988) .  
Bridges and Harris ( 1 988) fed diets containing very low concentrations of copper ( 1  to 2 mg 
Cu/kg DM) to foals and found that the outstanding manifestations of copper deficiency were 
abnormal ities of bone and carti lage development. Al l  foals became lame, and developed 
flexural l imb deformities. At postmortem, there was a generalised distribution of articular 
and physeal carti lage lesions. Lesions consisted of intracarti laginous fractures, areas of focal 
chondrocyte retention and chondrocyte necrosis. The suggested underlying mechanism was 
a decrease in the activity of the copper metalloenzyme Iysyl oxidase, which catalyses the 
formation of inter-molecular bonds of collagen fibri ls .  This  was supported by the greatly 
increased collagen solubi l ity of a sample of the aorta and selected articular cartilages 
compared with control foals. S imi lar pathological changes have been found in foals after 
long term dexamethasone administration (Glade et al. 1 983), zinc toxicosis (Gunson et al. 
1 982; Eamens et al. 1 984) and in early cases of osteochondritis dissecans of unknown cause 
(Bridges et al. 1 984). 
Studies performed on farms in America (Knight et al. 1 985 ;  Gabel et al. 1 987) and Japan 
(Fuj ikawa et al. 1 993; Asai et al. 1 993) have found an inverse relationship between dietary 
copper concentration and the prevalence of DOD. Two North American dose-response trials 
examining the effect of increased d ietary copper intakes on bone and carti lage abnormalities 
(Knight et al. 1 990; Hurtig et al. 1 993) found that the incidence of DOD lesions was 
decreased by increasing the copper content of the d iet above NRC ( 1 989) recommendations. 
D ietary copper intake at NRC ( 1 989) recommendations ( 1 0  mg Cuikg DM) is considered to 
be associated with mi ld  copper deficiency resulting in an increase in  tissue fragi l ity (Hurtig 
and Pool 1 996), predisposing subchondral bone and the hypertrophic chondrocyte zone to 
m icro-fractures. As a result, dietary levels of 50 mg Cu/kg dry matter (DM) for creep feeds 
and 30 mg Cu/kg DM for wean lings have now been recommended (Lewis 1 995).  
The New Zealand horse industry is essential ly pasture-based, in that pasture constitutes the 
major part of the d iet of mares and foals. New Zealand pastures contain a dietary copper 
1 1 2 
concentration ranging from 3 . 5  to 1 8  mg Cu/kg DM (Grace 1 994), and therefore, a shortfall  
in  copper i ntake might be expected in foals raised under this management system . This 
paper describes the effect of increased copper intake on the evidence of developmental 
orthopaedic d iseases of pasture-fed young Thoroughbreds. 
6.3 Materials and methods 
6.3.1 Animals and diet 
Mixed sex Thoroughbred foals (n = 2 1 )  were used. For the 1 3  to 25 weeks prior to foal ing, 
their dams had been treated, with e ither oral copper sulphate at a rate of 0 .5  mg Cu/kg 
l iveweight (L W)/day, or a placebo of equivalent fluid volume. The foals at 2 1  days of age 
were randomly allocated to either a copper-control or supplemented group, taking into 
account sex, s ire foaling date and mare treatment. The supplemented foals received oral 
copper sulphate at a rate increasing from 0.2 to 0 .5  mg Cu/kg LW/day from 2 1  to 49 days of 
age, and then 0.5 mg Cu/kg L W /day from 49 days of age until euthanasia at approximately 
1 50 days of age. Control foals received a placebo. Al l  animals received supplementary zinc 
(0 .5  mg Zn/kg L W/day) and selenium ( 0.02 mg Se/kg L W/day). Detai ls  of the mineral 
content of the pasture diet, and of the mares' mi lk  are presented in Chapter 5 .  
Laparoscopically-guided l iver biopsy and uni lateral distal radial growth plate biopsy, 
described in Chapters 2 and 3, were conducted at 4 to 1 0  days of age. 
6.3.2 Clinical examination 
Foals were weighed and examined at one or two week intervals throughout the experiment. 
Al l  l imbs were scored for the presence of joint effusion and angularities as v iewed from 
lateral and frontal aspects. All parameters were scored on a scale from 0 to 3 ,  where 0 was 
normal and 1 ,  2 and 3 indicated the presence of mi ld, moderate and severe abnormalities. 
Physitis was not scored cl in ical ly, but was assessed on radiographic examination of 
postmortem samples. 
6.3.3 Radiographic examination 
Survey radiographs of al l  foals were performed from 4 to 1 0  days of age, under general 
anaesthesia, and on the legs of foal s  fol lowing euthanasia at 1 50 days.  Views included 
1 1 3 
dorso-palmar/plantar (dp) and lateral v iews of the metacarpo/tarso-phalangeal joints and 
carpi, and dorso-plantar and dorso-Iateral plantero-medial obl ique v iews of the tarsi and 
dorso-medial plantero-lateral obl ique views of the stifles. 
Al l  radiographs were examined for abnormal ities by two investigators. The postmortem dp 
radiograph of the metatarso-phalangeal joint of both hind legs, taken at 5 months, was also 
scored by four observers for morphological abnormalities consistent with physitis of the 
distal metatarsal bone. The scores were summed to give a "physitis" score for each hind leg. 
The radiographs were scored in random (shuffled) order with the observer bl inded to 
treatment groups. Radiographs were scored for the fol lowing categories: presence of 
beaking, physeal flaring, sclerosis medially or laterally, metaphyseal shouldering medially 
or lateral ly, epiphyseal scal loping medial ly or lateral ly, and epiphyseal wedging (Figure 
6. 1 ) . 
Figure 6.1  Radiograph of distal third metatarsal bone showing metaphyseal shouldering (S), 
metaphyseal sclerosis (arrow heads), and widening of the physis. 
1 14 
Beaking and physeal flaring were scored as 1 or 0, if present or absent respectively, 
regardless  of whether medial or lateral or both . A beak was defined as a concavity in the 
contour of both the metaphysis and epiphysis adjacent to the physis, such that the physis 
appeared to protrude outward. Small beaks that could be clearly v iewed only with a hot l ight 
were scored as 0. Physeal flaring was defined as a local widening of the physi s  at the medial 
or lateral extremities, that was inconsistent with the width of the rest of the physis .  
Metaphyseal sclerosis, shouldering and epiphyseal scalloping were scored from ° to  3 ,  
scores of 1 ,  2 and 3 representing m i ld, moderate and severe abnormalities respectively. 
Medial and lateral aspects were scored independently. Sclerosis was defined as an increase 
in metaphyseal radio�density proximal to the physis. Metaphyseal shouldering was defined 
as an abnormality of the normal contour of the metaphyseal cortex, resulting in a convex 
metaphyseal "shoulder" proximal to the physis .  Epiphyseal scalloping was defined as an 
increase in the concavity of the medial or lateral contour of the epiphysis .  
Epiphyseal wedging was scored from 0 to 3 ,  a s ingle score covering both medial and lateral 
aspects. Wedging was defined as being present when a l ine joining medial and lateral 
margins of the physis was not parallel to the joint surface .  
6.3.4 Postmortem 
Twenty of the foals were euthanased at 1 46 to 1 6 1  days and one at 1 1 2 days fol lowing a 
traumatic injury sustained in the paddock. Foals were sedated with 250 mg xylazine and 
euthanased with intravenous barbiturate. The legs and cervical spine were removed, 
defleshed and d isarticulated. 
6.3.5 Bone physical properties 
The left and right humerus, femur, radius, tibia, third metacarpal and third metatarsal bone 
were weighed, and their length, and m id-shaft circumference measured. The volume of the 
bones was measured using a water-displacement technique, and the dens ity calculated. The 
bones were subsamp\ed and ash content determined as described in Chapter 5 .  
1 1 5  
6.3.6 Gross pathology 
All  cervical vertebral and l imb joint surfaces were examined, after disarticulation, for the 
presence of lesions. Lesions were counted but not scored for severity. 
After examination of the articular surfaces, the ends of the long bones were sawn into slabs 
about 6 mm thick for examination of the articular-epiphyseal complex and the physeal 
cartilage. The proximal humerus, distal femur and proximal radius were slabbed in sagittal 
section. The distal tibia and talus were s labbed in the plane of the intermediate ridge and the 
talar ridges respectively. The distal radius and 3rd metacarpal/metatarsal bone were slabbed 
frontally. Lesions were characterised, described, and their location given by slab number 
(counting from medial or dorsal), bone-end, foal and leg. All bone slabs containing lesions 
were radiographed and formalin fixed. 
6.3.7 Histopathology 
S labs submitted for h istology were decalcified in formic acid/formate, fol lowed by vacuum 
embedding in paraffin wax. Sections (6 f..tm th ick) were prepared and stained with a\cian 
b lue, haematoxyl in and eosin, and selected sections were stained with Lendrum' s  acid Picric 
Mallory. 
Categorisation of lesions 
Lesions were categorised as occurring in e ither articular or physeal carti lage. Articular 
carti lage lesions were divided into three categories: invaginated carti lage (Figure 6.2), 
thickened carti lage (Figure 6 .3), and lesions on the medial trochlear ridge of the talus. 
Articular carti lage wear l ines were categorised separately. Physeal cartilage lesions were 
categorised as either retained carti lage (Figure 6 .4), or interruptions to the physis.  
Histomorphology 
The left and right distal radi i  from 7 supplemented foals and 6 control foals ( 5  of the foals 
were from control mares and 8 from supplemented mares) were examined 
h istomorpho logically. A standard frontal cross-section of the distal radius was located by 
measuring the m idpoint between dorsal and palmar aspects of the lateral styloid process, 
and extending a l ine from lateral to medial, paral lel  to the palmar surface of the interprocess 
area (Firth and Hartman 1 983) .  Histological sections were prepared, and a l ine was drawn 
1 16 
from the medial to lateral border of the physis. Beginning medial ly, a series of parallel 
perpendicular intersections of this l ine 5 mm apart, defined histomorphological 
measurement sites along the physis .  The sl ides were projected at a known magnification, 
and at each measurement site, the width of the total physis and the combined hypertrophic 
and pro l iferative cel l  layer were measured. Also at each measurement site, 1 mm proximal 
to the physis, the metaphyseal trabecular area in a 1 mm by 1 mm square was determined by 
tracing the trabeculae of the projected image. The tracings were then digitised (sigma 
scanner), and the percentage trabecular area for each site determined. 
6.3.8 Statistical analysis 
All  data were analysed for the effect of mare and foal treatment using analysis of variance 
(ANOVA) procedures. They included growth rate, weight at euthanasia, (adjusted to 1 50 
days of age), radiographic distal radial "physitis" scores (the mean for the four observers), 
and articular and cartilage lesions by ranks of the scores. The scores were also analysed for 
inter-observer variation. The histomorphometric data were analysed as a repeated measures 
analysis according to site. Analyses were performed with Statistical Analysis System (SAS) 
version 6 . 1 1  (SAS Institute Inc. Cary, N.C., USA) using proc glm for ANOVAs, and proc 
m ixed for repeated measures analyses. 
6.4 Results 
6.4. 1 Growth rate 
There was no effect of foal or mare copper supplementation on growth rates, birth weights 
or final weights (adjusted to 1 50 days), of the foals. The growth rates for the individual foals 
from birth to euthanasia ranged from 0.9 to 1 .4 kg/day, with foal weights at euthanasia 
ranging from 1 84 to 234 kg l iveweight. The mean growth rate (+/- standard error of means) 
for al l  foals was 1 . 1  +/- 0.03 kg/day. 
1 1 7 
6.4.2 Clinical examinations 
All  but one of the foals remained healthy, the one foal became lame fol lowing laparoscopic 
l iver b iopsy and b iopsy of the distal radial growth plate performed under general anaesthesia 
on day 5. The foal appeared lethargic and was often recumbent. However this foal continued 
to grow wel l  and was 200 kg when euthanased at 1 42 days (growth rate 1 .4 kg/day). She had 
a m ild flexural deformity of the right carpus that persisted throughout, and at euthanasia had 
evidence of degenerative joint disease of the right radio-carpal joint, severe physitis in both 
d istal metatarsi  and calcification of the suspensory l igament distal to the sesamoids. It was 
considered that the degenerative joint disease and other c l in ical signs were associated with 
complications fol lowing the growth plate biopsy. Inclusion of this foal did not alter the 
statistical significance of the parameters examined. 
Conformational abnormal ities caused by the biopsy of the distal radial growth plate, are 
described in Chapter 3 .  F ifteen (7 1 %) of the foals developed an outward rotation of the left 
(unoperated) forel imb and a mi ld valgus conformation of the left leg, centred at the carpus. 
There was no evidence of joint effusion in  the foals, nor lameness in any of the foals 
excluding the one previously described. Conformation was not different in  copper­
supplemented foals compared with control foals. 
6.4.3 Radiographs 
Smal l carti lage retentions were evident on the survey radiographs in the lateral trochlear 
ridge of the distal femur in one foal, and in the distal physis of the 3rd metacarpal bone in  
another foal. 
Copper supplementation of mares was associated with a s ignificant reduction in  the physitis 
scores (p < 0.0 1 ), assessed radiographically, of the foals at 1 50 days of age (Table 6 . 1 ) . A 
relationsh ip between the foal or mare l iver copper concentration (from Chapter 4) and the 
radiographic scores was also investigated, but was not s ignificant. There was no s ign ificant 
effect of copper supplementation of the foals on radiographic scores. There were no 
sign ificant differences in radiographic scores between observers (p > 0.05) .  
1 1 8 
Table 6.1 Mean physitis scores from radiographs of the d istal metatarsus of a l l  foa ls at 1 50 days 
of age (C = control, S = copper supplemented). 
Mare Treatment Foal Treatment F value 
C S C S SED Mare Foal 
Number of foals 9 1 2  1 0  1 1  
Observer 
6.7 4.8 5 . 8  5 . 7  2.0 9 .4" 0 
2 5 .5 3 . 3  4 . 3  4.6 2.6 7 .4*' 0.2 
3 6.6 4.4 5 . 5  5 . 6  2 . 6  7.2' 0 
4 5 .8 3 .4 4 .6  4.6 2 .8  7 .5 ** 0 
mean 6.0 3 . 7  4 .8  4 .9  2 .4 9. 1 *** 0 
p < 0.05 
P < 0.01 
...  P < 0.005 
6.4.4 Bone physical properties 
There was no effect of mare or foal copper supplementation on any of the bone physical 
parameters. Pooled mean values for the foal bone physical properties are presented in Table 
6.2.  
Table 6.2 Pooled m ean values (+1- SEM, n = 21) for physical properties of the foa l  bones at 1 50 
days of age. 
Bone Weight Volume Length Circumference Density Ash 
(g) (m!) (mm) (mm) (g/m) (% fresh wt) 
Humerus 1 029 (33 .0) 794 ( 1 7 .0) 2 80 ( 1 .8) 1 2.3 (0. 1 3) 1 .3 (0.03) 39.4 (0.99) 
Radius 927 ( 1 7.8) 664 ( 1 4.3) 406 (2.8) 1 1 .3 (0. 1 2) 1 .4 (0.02) 42.3 (0.7 1 )  
Metacarpus 3 9 1  (8.0) 243 (5 .8) 268 ( 1 .7) 1 0.0 (0.06) 1 .6 (0.04) 40.4 (0.3 1 )  
Femur 1 585 (32.2) 1 226 (29.8) 364 (2.0) 1 4.3 (0. 1 6) 1 .3 (0.0 1 )  38 .2 (0.52) 
Tibia 999 ( 1 6.7) 7 1 2  ( 1 5 . 7) 348 ( 1 .9) 1 1 .7 (0. 1 2) 1 .4 (0.02) 42.7 (0.54) 
Metatarsus 499 (35 .9) 308 (2 1 .7)  3 1 3  (2.5) 1 0. 5  (0.74) 1 .6 (0. 1 2) 40.2 (2.88) 
1 1 9 
6.4.5 Pathology 
The prevalence and severity of lesions at postmortem was considered to be minor. There 
was a significantly lower (p < 0.05) incidence of articular carti lage lesions in foals from 
supplemented mares (Table 6.3) .  However there was no s ignificant effect of copper 
supplementation of the foals on articular and physeal carti lage (Table 6.4) lesions or wear 
l ines. 
Table 6.3 Num ber of foa l  articu lar cartilage lesions by classification at 1 50 days of age 
according to mare and foal  treatment (c == control, s = copper supplemented). 
Mare treatmentIFoal treatment 
clc cis sic sis 
Number of foals 4 5 6 6 
Lesion classification 
Invaginated 6 6 2 4 
Thickened 4 5 2 
Talus 3 5 4 6 
Mean (Iesions/foal) 3 .3 3 .2 1 .2 2 .0 
Mean (mare treatment)' 3 . 3  l .6 
• Values significantly different (p < 0.05). 
Table 6.4 Number of foal physeal cartilage lesions at 1 50 days of age according to bone and 
mare and foa l  treatment (c = control, s == copper supplemented). 
Number of foals 
Location 
Proximal humerus 
Proximal radius 
Distal radius 
Distal m etacarpus 
Distal femur 
Proximal tibia 
Distal tibia 
Distal m etatarsus 
mean (lesions/foal) 
Mare treatment/Foal treatment 
clc 
4 
7 
2 
o 
4 
4.3 
cis 
5 
o 
o 
o 
o 
o 
2 
5 
1 .6 
sic 
6 
o 
3 
o 
3 
o 
2 
2 
1 .8 
sis 
6 
2 
2 
4 
2 
o 
3 
2 
2 . 7  
Table 6.5 Location of classified foa l  articular cartilage lesions at 1 50 days of age. 
Lesion Classification S�mmetr� 
Location Invaginated Th ick Uni lateral B ilateral Total 
Proximal humerus 
D istal humerus 
Proximal radius 8 4 2 8 
3 rd Carpal bone 2 2 
Distal metacarpus . 5 3 
Front phalanges 4 2 4 
Distal femur 3 3 0 3 
Distal metatarsus 3 3 
Hind phalanges 2 2 2 
Talust 8 5 1 8  
* Mu ltiple lesions at  one s ite 
t Talus  lesion type classified separately 
Site 
middle articulating surface (head) 
lateral condyle 
medial/lateral articulating facet 
junction articu lation of radial and intermediate carpal bones 
medialllateral condyle 
median sagittal groove/dorsal em inence (P 1 )  
medial condyle/lateral trochlear ridge 
median sagittal ridge 
median sagittal groove/dorsal eminence (P I )  
medial trochlear ridge 
1 2 1  
Articular cartilage 
Invaginated cartilage lesions were found on the proximal humerus (Figure 6.2a), proximal 
radius, distal femur, front and hind proximal and middle phalanges and 3rd carpal bone (Table 
6.5) .  Histologically, these lesions appeared as an inward folding or cleft of the articular 
cartilage into the subchondral bone, often with some retention of predominantly hypertrophic 
chondrocytes. There was usually disruption to the organisation of the tangential cell layer, 
which was occasionally missing altogether, and a decrease in matrix staining intensity. 
Chondrones, subchondral fibrosis and subchondral sclerosis surrounding these areas of fibrosis 
were commonly present. Occasionally there were areas of necrotic articular cartilage at the 
chondro-osseous junction and fibrocartilage adherent to the surface of the invaginated articular 
cartilage. In one lesion there a cleft in the subchondral trabeculae entered the region of 
hypertrophic chondrocytes in the invaginated cartilage (Figure 6.2b). The cleft was associated 
with necrotic chondrocytes and numerous chondrones, its contents stained positive for fibrin by 
Lendrum 's acid Picric Mallory method, and was interpreted as being a mature m icro-fracture. 
Figure 6.2 An invaginated type lesion showing: 
a) the gross appea rance in the articular and epiphyseal cartilage (a rrow heads) of the proximal 
humerus (h) and d istal sca pula (s), and 
b) the histological appearance of cartilage invagination in the proximal radius, showing 
fibrocartilage (I) adherent to the articular cartilage, chondrones (s mal l  arrow heads), subjacent 
myelofibrosis (m), and a matu re subchondral microfracture (large a rrow heads). Stain : Alcian 
blue, Haematoxylin and Eosin. Magnification bar: 500 /-lm. 
a 
1 22 
m 
Thickened articular cartilage lesions were most clearly evident on examination of slabbed sections 
and on cabinet radiographs of slabs, and were found on the distal femur (Figure 6 . 3 a), humerus, 
metacarpus (Figure 6 . 3b) and metatarsus. The histological appearance of these lesions was the 
same as for invaginated cartilage except that the cartilage was more obviously thickened, with 
most of the thickening in the hypertrophic cell layer. Also the degree of depression of the articular 
cartilage was usually mild. One such lesion had a fissure extending to the articular surface, which 
was evident on gross examination, and histologically there was a subchondral trabecular 
microfracture at the chondro-osseous junction (Figure 6 .3b) .  It was not apparent histologically 
whether or not the microfracture communicated with the dissecting fissure. 
1 23 
Thirteen (62 %) foals had lesions on the medial trochlear ridge of the talus. These lesions were 
bilateral in 5 (24 %) cases. Lesions appeared grossly as a depression in the contour of the 
articular cartilage, with occasionally a linear invagination of cartilage. Lesions were located 
midway along the axis of the medial trochlear ridge, either on the apex of the ridge, or 2 to 4 mm 
medial to the apex. A variation of the lesion was a string of small punctate depressions in the 
cartilage, running parallel to the long axis of the ridge. Histologically these lesions showed one or 
more of: thickened cartilage, deep retention of cartilage, chondronecrosis, eosinophilic streaks, 
myelofibrosis, and surrounding osteosclerosis. Most had chondrones in or adjacent to the 
cartilage abnormalities. There was no significant effect of copper supplementation of mares or 
foals on the incidence of these lesions. 
Figure 6.3 A " etained cartilage lesion showing: 
a) the gross appearance in the lateral trochlear ridge of the distal femur (arrow heads) and, 
b) the histological appearance in the distal third metacarpal bone showing a widened zone of 
hYI)el1rophic cartilage, a decrease in matrix staining, subjacent myelofib rosis (m), and a 
subchondral microfracture (arrow heads) associated with fibrin and necrotic cartilage. Stain:  
Alcian blue, Haematoxylin and Eosin. Magnification bar:  500 f..Lm. 
a )  
1 24 
b)  
Wear lines 
Wear lines were present in the humero-radial joint of 1 1  (52 %) foals and the femoro-tibial joint 
of 3 ( 1 4 %) foals . Histologically they appeared as a disruption in the tangential layer, along with 
a decrease in the number of chondrocytes superficially, and a decrease in the matrix staining 
intensity. There were no abnormalities evident in the subchondral bone associated with the wear 
lines . 
Physeal cartilage 
Lesions were categorised grossly as either physeal disruption or retention of cartilage. Disruption 
was most commonly present on steeply inclined segments of the physis. Grossly these disruptions 
appeared as small (2 to 5 mm wide) epiphyseodeses, which histologically were either complete or 
partial (probably due to the plane of the section), consisting of trabeculae aligned perpendicular 
to the physis, and often accompanied by some thickening and disorganisation of the adjacent 
physis and fibrosis on the metaphyseal side of the epiphyseodesis. Disruptions to the physis in the 
distal metacarpus and metatarsus were considered part of the normal process of closure when 
there were other indications that the physis was beginning to close, such as considerable 
narrowing of the more central physeal cartilage, especially the reserve cell zone, and complete 
closu re beginning centrally and extending to the lateral and medial margins . 
1 25 
Retained cartilage lesions, most common in the distal troughs of the distal radial (Figure 6.4) and 
third metatarsal physes, were 4 to 10 mm high, and consisted primarily of retained hypertrophic 
cells and usually were associated with eosinophilic streaks. Usually the retentions were cone­
shaped extensions into the metaphysis with disruption to the endochondral ossification on one or 
both sides of the cone. When endochondral ossification was occurring, the primary spongiosa in 
the area of the retained cartilage was shortened and often not aligned parallel to the direction of 
growth. Occasionally there appeared to be active endochondral ossification on the epiphyseal side 
of retained cartilage lesions, despite the presence of a subchondral bone plate in other areas . 
Figure 6.4 Low power microgral)h of a retained cartilage lesion in a "trough" of the distal radial 
physis, showing retention of physeal hYI)ertrOI)hic chondrocytes protruding into the metaphysis 
(m). Stain:  Aldan blue, Haematoxylin and Eosin. Magnification bar: 2.0 mm. 
1 26 
Histomorphology 
Copper supplementation of the mare or foal had no significant effect on the histomorphometric 
parameters measured from the distal radii of foals (Figures 6.5) .  The physeal depth was greatest 
at the periphery and significantly greater medially than laterally (p < 0.00 1) .  The physis consisted 
predominantly of reserve cells at the periphery, but approximately 80 % hypertrophic and 
proliferative cells in the central region. The percentage trabecular covered area was significantly 
different (p < 0.00 1 )  at different sites along the physis, being low at the medial edge, and in the 
centre of the physis, and having a peak close to the medial edge and being greatest at the lateral 
edge. 
Figure 6.5 Percentage trabecular covered area of the metaphyseal spongiosa 10 mm proximal to 
the physis in control (0) and copper supplemented (_) foals, and width of the distal radial physis 
(mm) in control (0) and copper supplemented (e) foals measured from medial to lateral in a 
standard section of the distal radii in 13 foals (150 days of age). 
60 T 
I 
'" 55 + 
� i '" 
50 + � 
� .., .. 
45 0 u ... '" 
:; u 40 .., I .D 
35 + '" .b ...... 0 
"E I .., 30 + u ... I .., I p... 
25 + 
20 I I 
2 3 4 5 6 7 8 9 
Site 
T 
I 1 
I I 
10  1 1  12  13  14  
3 .0  
2 .5  
! � 
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. -= � 
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. . . . t 1 .5 til OJ en ;>.. ..: p... 
i I 
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1 27 
6.5 Discussion 
The dietary copper intake of control foals in the present study was within the range considered 
by Hurtig and Pool ( 1 996) to be mildly deficient. However, there was no histomorphometric 
evidence of tissue fragi l ity in the primary spongiosa of the distal radius of these foals compared 
with supplemented foals. The articular carti lage lesions were simi lar to the lesions found in the 
studies of Knight et al. ( 1 990) and Hurtig et al. ( 1 993). Pool ( 1 993) has argued that bone and 
cartilage lesions occurring as a result of nutritional excesses or deficiencies are l ikely to be a 
different entity to the commonly recognised form of equine osteochondrosis. He considers that 
the distribution of lesions may differentiate the two forms, as the nutritional form tends to be 
generalised, often not bilateral, nor in predilection sites. It was also suggested that 
interpretation of nutritional trials can be complicated by the occurrence of both forms of 
osteochondrosis simultaneously. 
Physeal lesions commonly consisted of an increase in the width of the zone of hypertrophic 
cel ls, and disruptions to the physis.  Lesions occurred commonly in wel l  defined areas 
particularly in the distal incl inations of the distal radius medially, and the distal 3rd metacarpal 
and metatarsal bones medially and laterally. These lesions were not cl inically apparent, except 
for radiographic evidence of physitis and retained cartilage seen on survey radiographs in two 
foals. The histological morphology and gross distribution of these lesions were similar to the 
physeal abnormalities described by Brown and MacCallum ( 1 976) in clinically normal foals. 
The low incidence and severity of lesions in our foals differs from the findings of Knight et al. 
( 1 990) and Hurtig et al. ( 1 993) who found significant DOD lesions in foals fed on diets 
containing 1 5  and 8 mg Cu/kg OM respectively, and an inverse relationship between copper 
supplementation and bone and cartilage lesions. It is possible that foals under North American 
management systems, commonly receiving high energy diets ( 1 30 % NRC ( 1 989) 
recommendations) (Hurtig et al. 1 990), may have a greater dietary copper requirement than 
pasture-fed foals. Glade et al. ( 1 984) and Glade and Belling ( 1 984) proposed that high energy 
diets interfere with the normal process of chondrocyte differentiation and maturation. Savage et 
al. ( l 993a) found an increase in the incidence of osteochondrosis lesions in foals fed 1 29 % 
NRC ( 1 989) energy recommendations, compared with controls fed 1 00 % NRC ( 1 989) energy 
recommendations. The high energy diets were achieved by adding maize oil to the ration which 
increased starch and fat intake. The copper concentration in the diets of both groups was 1 1  mg 
Cu/kg DM, and it was considered that due to the low incidence of lesions in the control 
1 28 
animals, high energy diets may be a more significant cause of osteochondrosis than copper at 
that dietary copper concentration . However, the effect of copper supplementation of the high 
energy diets on the incidence of lesions was not tested. An association between energy intake 
and copper requirements has been proposed by Glade ( 1 986) who suggested that it may be 
mediated by insulin like growth factor. It is not known what the energy intake of our foals was, 
but they were well grown compared to NRC ( 1 989) estimations for liveweights at 5 months, 
and compared with the average daily gains and final live weights of the foals in the study of 
Hurtig et al. ( 1 993) as reported by Burton and Hurtig ( 1 99 1 )  suggesting that our foals also 
received a high energy diet. Savage et al. ( 1 993a) found no effect of increased energy intake on 
average daily gain, which is perplexing. 
The source of carbohydrates may be important in the induction of DOD, and in particular 
soluble carbohydrates have been impl icated (NRC 1 989). A relationship between dietary 
carbohydrate source and copper bioavailability in male rats was described in a review by 
O'Del l ( 1 990). However this effect described in male rats has not been demonstrated in other 
species; in addition, simple sugars decreased copper bioavailabi lity relative to the effect of 
starch. In  this respect , the effect in rats was due to fructose and sucrose, whereas starch was the 
major carbohydrate supplemented to foals receiving high energy diets in the work of Savage et 
al. ( l 993a). 
The experiments of Knight et al. ( 1 990) and Hurtig et al. ( 1 993) found a high incidence of 
cervical vertebral lesions, suggesting that this may be a predisposition site for copper 
responsive articular cartilage lesions. However, we examined all vertebral facets and found no 
evidence of articular cartilage lesions in any of the foals. This is consistent with the low overall 
incidence and severity of DOD in our foals. 
In our study, copper supplementation of the mare, and not the foal, decreased articular cartilage 
lesions and the radiographic indices of physitis in foals at 1 50 days. However there was no 
relationship between neonatal liver copper stores and either radiographic physitis scores or 
articular carti lage lesions. Therefore mare supplementation probably had an effect on the foal 
skeletal development in utero rather than through the provision of greater copper stores in the 
liver of neonates. This possible in utero effect needs to be investigated further. Osteoblasts in 
lambs are thought to be particularly sensitive to copper deficiency in utero and in early life, as 
the effects of reduced osteoblastic activity have been observed before other clinical or 
pathological signs of copper deficiency (Suttle et al. 1 972). Treatment of the dam presents a 
1 29 
convenient approach for increasing copper status of foals in utero, and of new born foals 
(Chapter 4). In this respect, treating hinds with copper has been used to decrease the incidence 
of articular cartilage lesions in growing deer grazing New Zealand pastures (Thompson et al. 
1 994). 
There are few data concernmg the prevalence of DOD in pasture-fed New Zealand 
Thoroughbreds, but there are anecdotal reports of farms with very high incidence of clinical 
signs which in some years have considerable economic consequences. There is a great variety 
of supplementary nutritional regimes on pasture based production systems, as wel l  as 
differences in climate, pasture and soil types throughout New Zealand. Thoroughbred farms 
with higher prevalence and severity of DOD than found in our trial may use greater amounts of 
supplementary feeding. Unknown constituents of these feeds may interact with copper 
metabol ism, or increased foal growth rates may increase copper requirements of horses on 
these farms. There also may be a variation in copper supply between seasons. In our study, all 
horses were supplemented with zinc and selenium to ensure that they were not l imiting, and it 
is uncertain whether deficiencies of these minerals may contribute to the nutritional aetiology 
of DOD on some farms. Animals in this study had low calcium and phosphorus intakes (2.5 to 
3 .8 g Calkg DM and 2.3 to 4.3 g P/kg DM ) as wel l  as a low calcium to phosphorus ratio 
(ranging from 0.72 to 1 .4, and below l .0 for 6 of the 1 1  months of the trial), compared with 
NRC ( 1 989) recommendations. Low calcium to phosphorus ratios have been proposed as an 
important cause of DOD (Savage et al. 1 993b), but this is not supported by our findings. 
In conclusion, copper intake of foals grazing pastures containing 4.4 to 8.6 mg Cu/kg OM, is 
adequate for healthy development of bone and cartilage. However copper supplementation of 
mares in the final half of gestation did reduce the evidence of abnormalities in bone and 
cartilage development of their foals at 1 50 days of age, although the clin ical significance of this  
evidence was minor. Copper supplementation of mares may provide a convenient treatment 
regime on farms experiencing a greater incidence and severity of developmental orthopaedic 
diseases. 
Chapter 7 
The Effect of High Pasture 
Molybdenum Concentrations on the 
Copper Status of Grazing Horses6 
1 30 
6 Submitted as : SG Pearce, EC Firth, ND Grace and PF Fennessy ( 1997) American Journal of 
Veterinary Research. 
1 31 
7.1 Abstract 
Inconsistencies exist in the recommendations on the copper requirements of horses. The objective of this study was to determine whether molybdenum could act as an antagonist 
causing secondary copper deficiency in pasture-fed horses. Twelve Thoroughbred 
weanl ing fil l ies were divided into two groups and fed either on a pasture containing 8 to 1 5  mg 
Mo/kg OM, or on a control pasture containing 0.6 to 1 .2 mg Mo/kg OM for 84 days. Both 
pastures contained 6 to 9 mg Cu/kg OM. The animals were chal lenged with copper over the last 
1 4  days to detennine their response to supplemental copper ( 1 .0 mglkg liveweight) given orally 
three times a week. Copper status indices included liver, plasma, blood cell and plasma 
trichloroacetic acid insoluble copper concentration as wel l  as the enzyme activities of 
caeruloplasmin oxidase and red blood cel l  superoxide dismutase. None of these indices were 
affected by the level of molybdenum intake. Horses grazing pasture high in molybdenum did 
not respond differently to a copper challenge when compared to controls. The results of this 
study suggest that molybdenum is unlikely to increase copper requirements by causing a 
secondary copper deficiency at copper and molybdenum concentrations nonnally found in the 
diet of New Zealand pasture-fed Thoroughbreds. 
7.2 Introduction 
It is considered that copper (Cu) deficiency may contribute to abnonnal skeletal development 
of foals (Lewis 1 995; Hurtig and Poole 1 996). There are reported cases of copper-responsive 
orthopaedic disease in foals on pasture (Egan and Murrin 1 973a, Carberry 1 978), with evidence 
of a high dietary molybdenum (Mo) intake in one case (Egan and Murrin 1 973a). Similarly, 
studies in North America have found beneficial effects of copper supplementation of foals on 
diets of 8 to 1 5  mg Cu/kg OM prior to supplementation (Knight et al. 1 990; Hurtig et al. 1 993) .  
However, a supplementation trial performed on horses grazing pasture containing 4 .4 to 8.6 mg 
Cu/kg DM showed only minor benefits of foal copper supplementation (0.5 mg Cu/kg 
Liveweight) on indices of bone and cartilage development (Chapter 6). Therefore we propose 
that a dietary copper antagonist such as molybdenum might explain these apparent 
inconsistencies. 
Walsh and O'Moore ( 1 953)  suggested that molybdenum might be a cause of osteodystrophy in 
horses. Two studies in which horses were fed molybdenum had contrasting results. In one 
study, aqueous ammonium molybdate was fed at increasing concentrations ( 1 ,  27 and 1 07 mg 
1 32 
Mo/kg DM), for 2 to 3 weeks at each level, to bile duct cannulated ponies. The ponies had 
increased bi l iary copper excretion, decreased caeruloplasmin activity and increased 
trichloroacetic acid (TCA) insoluble copper which was suggestive of the presence of systemic 
thiomolybdates (Cymbal uk et al. 1 98 1  b). In the other study, aqueous ammonium molybdate 
was fed at a rate of 20 mg Mo/kg DM for 4.5 months to horses of m ixed breeding, and there 
was no apparent change in plasma copper or TCA insoluble copper concentrations (Strickland 
et al. 1 987). In addition, excess molybdenum due to industrial pollution has been reported to 
cause death in horses associated with decreased liver copper stores (Ladefoged and Sturup 
1 995). 
In ruminants, molybdenum (associated with sulphur) interferes with copper metabolism by the 
formation of thiomolybdates in the reductive environment of the rumen (Dick et al. 1 975, Price 
et al. 1 987). Thiomolybdates interfere with copper absorption in the smal l intestine, and alter 
the protein l igand binding of absorbed copper. This increases the concentration of copper in the 
TCA insoluble fraction of the blood, which is less readily available. There is also an increase in 
bil iary excretion of copper as demonstrated in horses (Cymbaluk et al. 1 98 1  b) and cattle 
(Gooneratne et al. 1 994). In ruminants, diets high in molybdenum cause a depletion of l iver 
copper stores (Van Ryssen 1 994), and are used as models for copper deficiency in ruminants 
(Ledoux et al. 1 995). Intravenous administration of thiomolybdates has also been 
recommended for the treatment of copper poisoning in sheep (Gooneratne et al. 1 98 1 ;  
Humphries et al. 1 988). 
In the absence of a rumen, the hindgut might be a site in horses suitable for the formation of 
thiomolybdates.  S ince significant copper absorption occurs in the small intestine, any effect of 
hindgut thiomolybdate formation on copper absorption is unl ikely, and any influence of the Cu­
Mo interaction would therefore be associated with systemic thiomolybdates. However, 
Strickland et al. ( 1 987) concluded that insufficient molybdenum entered the large intestine to 
form significant amounts of thiomolybdates because of the rapid absorption of molybdenum 
from the small intestine and its subsequent urinary excretion. 
In the experiments of both Cymbaluk et al. ( 1 981  b) and Strickland et al. ( 1 987) aqueous 
solutions of molybdenum were used as a supplement. This form of molybdenum may be more 
readily available than molybdenum contained in pasture plants, and therefore grazing pasture 
plants with high molybdenum concentrations may allow sufficient molybdenum to reach the 
large intestine leading to the formation of sufficient amounts of thiomolybdates to antagonise 
1 33 
copper metabolism systemically. This study reports on the influence of grazing pasture 
containing high molybdenum concentrations on the copper status of horses 
7.3 Materials and methods 
7.3.1 Experimental design 
There were two treatments (low and high molybdenum pasture), with six horses per treatment. 
The horses grazed the experimental paddocks for a total of 84 days from the 1 3th May to the 
5th August 1 996 (Winter). The experiment was run in two parts. In part 1 (day 0 to 70), the 
horses grazed the pasture without any supplementation, and in part 2 (day 70 to day 84), all 
horses received a thrice weekly supplement of copper sulphate orally at a rate of 1 .0 mg Culkg 
liveweight (L W)/day. 
7.3.2 Animals 
The 1 2  weanling Thoroughbred fil lies were aged 1 70 to 260 days and were assigned to either a 
treatment or a control group by stratified randomisation. The fill ies were allocated to groups 
based on their sire, age and caeruloplasmin oxidase activity, which was determined 7 days prior 
to the start of the experiment. The two groups were grazed on low endophyte ryegrass (Lolium 
perenne L.) in adjacent paddocks. 
7.3.3 Diets 
The pasture for the treatment group was sprayed with an aqueous solution ofNa2Mo04 at a rate 
of 500g Molha, 5 weeks prior to day 0, while the pasture for the control group was not treated. 
The pasture for both groups was not grazed for a period of 5 weeks fol lowing appl ication of the 
fertilisers, to allow uptake of the molybdenum by the plants and to provide adequate pasture for 
the duration of the experiment. Pasture samples were collected for mineral analysis every 2 
weeks after topdressing for the duration of the trial. 
7.3.4 Part 1 :  Molybdenum copper interaction 
A standing, ultrasound-guided liver biopsy (Chapter 2) was performed on all animals on day -4 
and 67, for the assessment of liver copper concentration. The biopsied l iver tissue was frozen 
1 34 
at -20°C until analysis. Animals were examined cl inically at the same time as the l iver biopsies 
were taken. Plasma copper and 5% TCA insoluble plasma copper concentrations and the 
plasma caeruloplasmin activity, were measured on days -7, -4, 0, 7, 1 4, 28, 42, 56, and 67. 
B lood cel l  copper concentration was also analysed on days 42, 56 and 67. Red blood cel l  
copper-zinc superoxide dismutase (RBC Cu-Zn SOD) activity was determined on day 67. 
7.3.5 Part 2 :  Copper challenge 
In the event of molybdenum depleting liver copper stores, and influencing blood copper 
indices, the copper challenge was designed to measure the rate of liver copper repletion, and 
the influence on circulating copper indices. 
On day 70, all animals were weighed and given oral copper sulphate supplementation at a rate 
of 1 .0 mg/kg LW/day. The supplementation was given orally as 3 doses per week. Plasma and 
blood cell copper concentration and caeruloplasmin and RBC Cu-Zn SOD activity were 
monitored on days 77, 84 and 87 after the onset of copper supplementation . A third liver biopsy 
was performed on day 84. 
7.3.6 Analytical 
Plasma and blood cell copper were measured using inductively coupled plasma emISSIon 
spectrometry (lCP) (Lee 1 983). TCA insoluble copper was measured by calculating TCA 
soluble copper and subtracting from total plasma copper concentration . TCA soluble copper 
was measured by mixing plasma with an equal volume of 1 0  % trichloroacetic acid (w/v) for 30 
s and then centrifuged at 2400 g for 1 5  min. The supernatant was harvested, and the precipitate 
was reconstituted in 5 % TCA, and the procedure repeated (Mason et af. 1 979). The 
supernatants were pooled and the copper concentration determined via ICP. 
Plasma caeruloplasmin activity was measured using a colorimetric method (Ravin 1 96 1 )  to 
measure the oxidation of the substrate p-phenylenediamine-dihydrochloride (PPD). The results 
are expressed as absorbance units at 530 nm (37°C) per l itre of plasma. 
The Cu-Zn SOD activity of lysed red blood cells was determined spectrophotometrically using 
a kit (Cruml in, Northern Ireland) based on the method ofPodczasy and Wei ( 1 988). 
1 35 
Analysis of caeruloplasmin oxidase activity was performed within 3 days after each sampling 
occasion. Analysis of RBC Cu-Zn SOD activity and pasture copper concentrations were 
performed in one batch at the end of the trial . All other samples were analysed in four batches 
fol lowing the day 7, 28, 67 and 84 day samples. 
Liver copper concentration was analysed by Zeeman graphite furnace atomic absorption. 
Samples were run with a liver standard of known copper concentration produced by the 
National Bureau of Standards, USA (NBS, bovine liver 1 577-B). All  l iver samples were 
analysed in one batch after completion of the experiment. 
7.3.7 Statistical analysis 
Blood copper status indices were compared between groups by analysis of variance (ANOV A) 
with repeated measures on time. The effect of molybdenum intake on liver copper 
concentration and the response of the l iver copper concentration to the copper challenge, was 
analysed by ANOV A, using the initial l iver copper concentration as a covariate. Analyses were 
performed with Statistical Analysis System (SAS) version 6. 1 1  (SAS Institute Inc. Cary, N.C., 
USA) using proc glm for ANOVAs, and proc mixed for ANOVAs with repeated measures over 
time. 
1 36 
7.4 Results 
7.4.1 Pasture minerals 
Mean pasture mineral concentrations for the molybdenum treated and control paddocks were: 
4.7 and 3 .9 g calcium/kg OM, 3 .6 and 3 . 5  g phosphorus/kg OM, 1 .9 and 1 .9 g magnesiumlkg 
OM, 32 and 32 g potassium/kg OM, 2.0 and 2. 1 g sodium/kg OM, 577 and 438 mg iron/kg 
OM, 76 and 72 mg manganese/kg OM, and 24 and 25 mg zinclkg OM. The pasture 
concentrations of sulphur, molybdenum and copper for the control and molybdenum treated 
paddocks, over the period of the experiment, are presented in Table 7 . 1 
Table 7. 1 Concentrations of sulphur, molybdenum and copper from the molybdenum treated and 
control paddocks. 
Molybdenum treated paddock Control paddock 
S Mo Cu S Mo Cu 
Trial day g/kg OM mglkg OM g/kg OM mg Cu/kg OM 
0 4.2 1 6 . 1  8 .5  3 .5 0.8 8.2 
28 3 .9 1 3 .2 8.9 3 .6 1 .0 8 .4 
42 3 .3 9.9 7.5 3 .5 1 .2 7.4 
56 2 .8  9 .5  6 .8  3 .8 0.7 8.0 
77 2.4 8 .6 6.9 2.4 2.0 7.0 
84 3 .2 7.2 7 . 1  3 .0 2.6 6.4 
7.4.2 Part 1 :  Molybdenum copper interaction 
Clinical examinations 
Clinical examinations revealed no differences in conformation or clinically evident musculo­
skeletal disease between high molybdenum and control groups. 
Blood copper indices 
There were no significant differences between high molybdenum and control groups, for any of 
the blood copper status indices, but there was a significant effect of time for all indices. The 
plasma copper concentration of both groups declined until day 56  (Figure 7 . 1 a), so that the 
level was significantly lower than the initial plasma copper concentration (p < 0.005). The 
caeruloplasmin activity did not show a simi lar decrease in the day 56  sample (Figure 7 . 1  b). 
1 37 
There was no difference between treatment groups in TCA insoluble copper concentration, which 
remained below 0. 1 mg Culkg DM for both groups. 
Liver copper 
There was a large variation in the initial liver copper concentrations with the high molybdenum 
group having a mean liver copper concentration (6 1 . 1  mg Culkg DM) approximately twice that 
of the low molybdenum animals (34.6 mg Culkg DM) prior to the start of the triaL However the 
differences were not significant due to the wide variation among individuals. The variation 
between groups was greatly diminished at the 67 day liver biopsy, with mean liver copper 
concentrations of 20.9 and 1 4.9 mg Culkg DM for high molybdenum and control groups 
respectively; the difference was not significant (Figure 7.2). 
Figure 7.1 (on following page) Copper indices in weanling Thoroughbred fillies grazing pasture 
with low (0, n=6) or high (., n=6) molybdenum concentration. Error bars represent standard 
error of means, arrow heads indicate the start of the copper challenge. 
a) Plasma copper concentration. 
b) Plasma caeruloplasmin oxidase activity. 
c) Blood cell copper concentration. 
d) Red blood cell superoxide dismutase activity. 
Figure 7.1a  
t: 1 .6 -.:2 
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en 0.4 -'" 
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Figure 7.1 b  
.q 90 _ > 80 .;-t) ro � 70 � t: �  .- r-E M en ro _" 
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Figure 7.1 c  
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-0 1 .0 _ 0 
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Figure 7.1d  
2500 T 2000 T 
1 500 _ 
1 000 _ 
--5.0.0.- -
- 1 0  o 
1 0  
1 0  
1 0  
1 0  
20 30 
20 30 
20 30 
20 30 
. .
.
t . . . . 
40 50 60 70 80 
. . . . . . . . . .  
+ . . · · · · · ·l · · 
40 50 
40 50 
40 50 
Time (d) 
60 
60 
60 
70 80 
70 80 
A �. I 
70 80 
1 38 
90 1 00 
90 1 00 
90 1 00 
90 1 00 
1 39 
Figure 7.2 Mean liver copper concentration from weanling Thoroughbred fillies on high 
molybdenum (II, n=6) or control (0, n=6) pastures. Error bars represent standard error of 
means. 
80 T , 
0 
� 
. .§ ea .b � ,-.  
13 ;:E  
S Ci  <.) 01) 
� �  8:: 5 o '-' <.) ... Q) > 
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I e I 
- 1 0  0 1 0  20 30 40 SO 60 70 80 90 
Time (d) 
7.4.3 Part 2: Copper challenge 
Blood copper indices 
There was no significant difference between high and low molybdenum groups of any of the 
blood copper status indices measured. The mean plasma copper concentration peaked on day 77 
(7 days after the onset of the copper challenge) and then declined on the day 84 and 87 samples. 
Following onset of the copper challenge, blood cell copper concentration decreased to a minimum 
at day 77 and then increased, with the day 84 and 87 samples being greater than the day 77 
sample (Figure 7. l c). The RBC Cu-Zn SOD activity increased significantly between day 77 and 
a peak at day 84 (Figure 7. 1d). 
Liver copper 
The liver copper concentration after the copper challenge (84 days) was significantly greater than 
at day 67 in both groups, but there was no significant difference between treatment groups in the 
rate of change of the liver copper concentration between the two samples. 
140 
7.5 Discussion 
There was no effect of the high molybdenum pasture diet on blood copper indices, or on the 
liver copper concentration of the horses, including TCA insoluble copper which is considered 
to be an early, sensitive indicator of mi ld molybdenosis in ruminants (Lamand et al. 1 980). 
This suggests that there was no significant absorption of thiomolybdates in the horses, and is in 
agreement with the study of Strickland et al. ( 1 987) but contrasts with the study of Cymbal uk 
et al. ( 1 98 1  b). Changes to blood copper indices in ruminants on high molybdenum intakes 
include an increase in plasma copper concentration, and a decline in caeruloplasmin activity. 
Simi lar changes were described in horses by Cymbaluk et al. ( 1 98 1 b). 
The liver copper concentrations of the horses at the beginning of the experiment varied widely, 
with the animals in the high molybdenum group having a mean liver copper concentration 
almost twice that of the control animals. The differences in liver copper concentration between 
groups was not significant because of the wide variation. The liver copper concentrations of the 
horses after 67 days on the experimental pasture had decreased, and using the initial liver 
copper concentrations as a covariate, the difference between treatment groups was not 
significant. It was unfortunate and unexpected that the initial mean liver copper concentrations 
of the two groups was so different, and the reason for this was not known. All liver biopsies 
were analysed together after completion of the experiment. 
There is no ready explanation for the changes in the plasma copper concentration which 
declined to a minimum at 56 days and then increased. Venipuncture was taken at 
approximately the same time each day to minimise any diurnal variation, as has been reported 
in human plasma copper concentrations (Lifschitz and Henkin 1 97 1 ). The increase in plasma 
copper concentration could not be attributed to the copper challenge because the increase began 
before the challenge. Previous work with copper supplementation has also suggested no 
relationship between dietary copper intake and plasma copper concentration at mean pasture 
dietary copper intakes of 6 to 7 mg Cu/kg DM) and greater (Smith et al. 1 975;  Chapter 5) .  The 
caeruloplasmin activity increased significantly from day 67 to 77, mimicking the increase in 
the plasma copper concentration. The changes to the blood cell copper concentration also 
mimicked the changes to the plasma copper concentration, but they occurred 3 weeks later. 
RBC Cu-Zn SOD activity also increased when the blood cel l  copper concentration increased. 
The ready response of the RBC Cu-Zn SOD may indicate reconstitution of an apoenzyme in 
14 1  
circulating blood cells. There were less data for RBC Cu-Zn SOD than the other indices, as the 
assay was being developed during the trial. 
The plasma copper concentration showed two distinct patterns of change. There was a 
downward trend to day 56 followed by a rise as well as transient changes between sampl ing 
days which were consistent for all foals. Possible causes for the general trends include changes 
in diet, excluding copper and molybdenum, or seasonal changes such as temperature or 
photoperiod. There was a very high concentration of iron in the pasture analyses, which was 
most likely due to soil contamination as the pastures became quite muddy during the 
experiment. This is likely to reflect an increase in iron intake by the horses, which may have 
affected copper status. The effects of photoperiod on homeostatic regulation have been well 
described with reproductive and growth hormones in a number of domestic species, and it is 
possible that this may have influenced copper status in this trial as the shortest day of the year 
was on day 39.  Seasonal effects on copper status in horses have been reported by Auer et al. 
( 1 988a), and were considered to be most likely due to changes in dietary copper intake or 
availabil ity. 
Causes for the transient changes in plasma copper concentration may include daily cl imatic 
changes such as temperature or rainfall as well  as inflammatory episodes such as viral 
respiratory infection as plasma copper and caeruloplasmin activity are known to be responsive 
to inflammation (Auer 1 989a). However, there was no evidence of disease in the fi ll ies during 
or preceding the trial. The liver biopsy procedure had no apparent effect on plasma copper 
status following the first liver biopsy (day -4), and was not considered to be a significant cause 
of the variabil ity of the indices. 
The inabi lity of pasture containing 7 to 1 8  mg Mo/kg DM to deplete copper stores in 
Thoroughbred weanlings compared with control animals, is consistent with prevIOUS 
observations that horses are able to graze "teart" pasture that cause secondary copper 
deficiency in cattle, without cl inical signs of disease (Underwood 1 977). Presumably 
differences between horses and ruminants in molybdenum metabol ism relate to the greater 
abi lity of the rumen compared with the hindgut of horses, to produce thiomolybdates, and the 
location of the rumen, preceding the site of copper oxidation and absorption . Further research is 
required to determine the manner in which horses are "protected" from the effects of high 
molybdenum intakes. Whether molybdenum is prevented from reaching the large intestine in 
sufficient amounts, or the large intestine is not a suitable environment for the production and 
1 42 
subsequent absorption of thiomolybdates, or whether absorbed thiomolybdates are prevented 
from interacting with tissue copper is not known. 
It is possible that a greater amount of molybdenum than fed to the horses in this trial may result 
in greater formation and absorption of thiomolybdates. However since the pasture molybdenum 
concentration in this trial was very high compared with New Zealand pastures which can 
contain 0.2 to 1 0  mg Mo/kg DM (Grace 1 983b), this would preclude molybdenum as a likely 
cause of secondary copper deficiency responsible for copper responsive developmental 
orthopaedic disease in foals. 
1 43 
Chapter 8 
General Discussion 
144 
8.1 Introduction 
The original hypothesis for the studies described in this thesis was to determine whether dietary 
supplementation of pasture-fed New Zealand Thoroughbreds with copper would reduce the 
incidence and severity of DOD, and to identify suitable indices related to copper status and 
features of bone and carti lage development in order to better define the relationship between 
dietary copper, copper status and DOD. This was achieved by a copper supplementation trial 
and investigation of changes in copper status and bone and cartilage development. These 
studies found that mare copper supplementation increased neonatal foal liver copper 
concentrations and was associated with a reduction in the (mi ld) evidence of DOD in foals up 
to 5 months of age. Foal copper supplementation increased l iver copper concentrations but had 
no effect on any index of bone or carti lage development. 
8.2 Experimental design 
The rationale for this investigation was based on the concerns of the New Zealand 
Thoroughbred industry regarding the amount of wastage, largely in terms of decreased sale 
value, of animals with DOD. These concerns were documented in a survey produced by the 
New Zealand Equine Research Foundation for the Racing Industry Board. Many aetiologies 
have been proposed for DOD but copper seemed the most appropriate factor for immediate 
study because recent work had suggested that the copper requirements for growing horses (20 
to 30 mg Cu/kg OM) were greater than can be provided by a pasture diet (5 to 1 0  mg Cu/kg 
OM) (Knight et al. 1 990; Hurtig et al. 1 993) which has already lead to a widespread practice of 
copper supplementation. 
The studies were performed at the AgResearch Flock House equine research faci l ity, a new 
faci l ity which had not run horses previously. Therefore the level of DOD that might be 
expected was not known. The pasture copper concentration (4.4 to 8.6 mg Cu/kg OM) was 
below NRC ( 1 989) recommended levels ( 1 0  mg Cu/kg OM) and at similar levels to the foals 
fed low copper diets in the studies of Knight et al. ( 1 990) and Hurtig et al. ( 1 993), in which 
copper supplementation was found to reduce the incidence of DOD. Our supplemented animals 
received 0 .5  mg Cu/kg LW/day, a similar dietary copper concentration to the high copper 
treatments in the previous studies (30 mg Cu/kg OM). However, the literature on factors 
affecting copper bioavailability in horses is sparse, which makes comparison between 
1 45 
measured dietary copper levels in different diets problematic as unknown factors may influence 
copper absorption . 
Copper sulphate is often used as the benchmark for comparing the bioavai labil ity of other 
copper containing compounds (Baker and Ammermann 1 995). Copper-containing compounds, 
other than copper sulphate, and in particular copper proteinates, might have a greater 
bioavailabil ity in horses, but this has not yet been documented. In horses satisfactory methods 
of long term copper supplementation have not been reported, and so we resorted to frequent 
oral dosing. An alternative could be the feeding of a small quantity of copper "spiked" 
supplement, a method which may sti ll not be satisfactory for herds of horses on pasture where 
the intake of supplements by individuals cannot be regulated. Therefore there is a need for the 
development of depot formulations. In our work, copper sulphate doses were administered 
three times per week. Whether more or less frequent dosing at the same dose rate would have 
had a different effect on copper bioavailabi l ity is not known. 
8.3 Development of surgical techniques 
8.3.1 Liver biopsy technique 
The l iver biopsy technique described in this thesis was developed to obtain samples to 
determine liver copper concentration. The morbidity and adverse effects of the techniques were 
also investigated so that their usefulness for further studies could be assessed. The 
transabdominal, laparoscopical ly-guided technique performed on the neonatal foals under 
anaesthesia was considerably more involved than the standing, percutaneous, ultrasound­
guided, transthoracic technique which was easily performed. The latter procedure was 
performed on the mares in the main study, and on the weanlings in the investigation of high 
dietary molybdenum. In the case of 4 mares the liver could not be visualised 
ultrasonographically, presumably due to atrophy of the liver in these horses. Liver biopsies 
performed by these techniques provide an estimate of the liver copper concentration with a 
coefficient of variation of 1 0  to 20 percent. Both procedures had few 
adverse effects on the horses. 
1 46 
8.3.2 Physeal biopsy technique 
The physeal biopsy technique was developed to obtain tissue for the investigation of bone and 
cartilage development in neonatal foals. The tissue sample was adequate for histological 
examination, and was used in a separate study on immunohistochemistry of the growth plate 
(SA Holle and EC Firth, unpublished data). There were some problems with the foals following 
this procedure including wound breakdown, excessive granulation tissue formation and 
conformational abnormalities. These problems might preclude the clinical usefulness of this 
technique, but more rigorous aftercare and the use of anti-inflammatory medication may 
attenuate the adverse effects. The time the foals spent confined postoperatively was minimised 
because the main study required them to be grazing a pasture diet, and anti-inflammatories 
were not administered lest they interfere with copper metabolism and status. The physeal 
biopsy is a useful technique as it provides adequate sample for examination of physeal 
composition and structure, and morbidity need not be significant. 
8.4 The concept and measurement of adequate copper status 
Adequate copper status was defined (Chapter I )  as existing when tissue copper concentrations, 
and more particularly liver copper stores, are sufficient to ensure that copper is not limiting 
biochemical and physiological function and that animal performance and health are not 
improved by copper supplementation. When copper intake is inadequate we would first expect 
a depletion of tissue copper stores, which would progress to a state of deficiency whereby there 
is a decrease in plasma copper concentration and insufficient copper for the copper containing 
enzymes. The resultant decline in cupro-enzyme activity will result in a dysfunction in their 
b iological activity which will then progress to copper deficient disease which is well defined in 
ruminants, but is not commonly recognised in horses. 
8.4.1 Plasma indices of copper status 
Plasma and serum copper concentrations consistently fal l  below normal in cases of 
experimental copper deficiency (Bridges and Harris 1 988) in which case a threshold is 
presumably reached whereby tissue reserves are depleted and normal blood copper 
concentrations can no longer be maintained. 
1 47 
The measurement of plasma copper and caeruloplasmin oxidase activity can be influenced by 
various factors and conditions discussed in Chapter 1 .  The data in this  thesis also present the 
normal postpartum changes in these parameters. Pasture-fed Thoroughbred foals are initially 
hypocupraemic with low activities of caeruloplasmin, and these parameters increase to adult 
levels by approximately 3 weeks of age. This corroborates the data of Bell et al. ( 1 987). 
If a dietary copper threshold for maintenance of plasma indices exists, then the pasture diet of 
both mares and foals in our experimental work was above this level, as there was no effect of 
copper supplementation on plasma and blood cel l  copper concentration and plasma 
caeruloplasmin activity. However, according to the above definition, copper supplementation 
of the mare reduced the evidence of DOD in their foals indicating that these animals were 
responsive to copper supplementation, and therefore not of an adequate copper status . This 
apparent contradiction is considered later and indicates that indices may differ in sensitivity. 
8.4.2 Blood cell copper concentration and RBC Cu-Zn SOD as indices of copper 
status 
Blood cell copper represents a tissue copper pool that can be readily easily sampled. A large 
proportion of blood cell copper is associated with the cupro-enzyme superoxide dismutase 
although there is no apparent correlation between total blood cell copper concentration and 
RBC Cu-Zn SOD activity (Scudder et al. 1 976). B lood cell copper in these studies was not 
affected by dietary copper levels and showed little change postpartum .  The activity of Cu-Zn 
SOD may be a more useful  index of copper status than plasma copper concentration as it is less 
responsive to inflammation and other such factors (Milne 1 994). 
8.4.3 Liver copper concentration as an index of copper status 
The l iver is considered a copper storage organ in ruminants, because increased copper intake 
results in increased liver copper concentration (Woolliams et al. 1 983 ) .  A simi lar relationship 
between copper intake and l iver copper concentration was found in our horses, and has also 
been reported in other studies (Cupps and Howell 1 949; Smith et al. 1 975;  Hurtig et al. 1 990). 
This contrasts with the situation in humans, in which it is considered that copper is not stored in 
the body to any great extent as excess copper is readily excreted via the bile (Linder and 
Hazegh-Azam 1 996). 
148 
At present the lim itation of liver copper concentration as a measure of copper status in horses is 
that the l iver copper concentration at which copper inadequacy occurs, that is decreases in 
plasma copper and retardation of copper-dependent processes is not known. Therefore the l iver 
copper concentration may be used to compare different individuals or treatments, but there are 
no data to define whether a horse has an adequate copper status based on its liver copper 
concentration. In this respect, plasma copper concentration and cuproenzyme activities are 
likely to be more useful as they reflect the avai lability of copper to copper-dependent 
processes. 
8.S The effect of copper supplementation of the mare and foal on indices of copper 
status 
Copper supplementation of pregnant mares did not influence their plasma copper 
concentration, which decreased over the final stages of gestation (Chapter 4). This decline was 
presumably physiological and was not responsive to dietary intake. However the relationship 
with stage of pregnancy was low (r = 0.2) indicating that the effect of pregnancy explained 
only a minor part of the total variation. Large general trends and smaller transient changes in 
plasma copper concentration and caeruloplasmin activity were often detected and could not be 
explained, but these indices never showed a response to copper supplementation. Changes in 
copper indices are discussed in more detai l in Chapter 7 .  
Copper supplementation of pregnant mares increased the liver copper concentration of their 
foals, and there was a trend toward increased liver copper concentrations in the mare. It is 
considered that the high liver copper concentration of new born foals is required to provide 
copper during nursing when dietary copper intake (from milk) is low (Meyer and Tiegs 1 995) .  
The apparent preferential storage of copper in the foetal liver rather than the mare may support 
the theory that the copper stored in the neonatal liver performs an important function in the 
neonatal period. However, there was no relationship between l iver copper concentration at 4 to 
1 0  days of age and any of the blood copper status indices. Other suggestions to explain the high 
neonatal l iver copper concentrations include storage of copper for chromosomal function 
(Leighton et al. 1 990) and a consequence of the failure of foetal l iver to excrete accumulated 
copper (Cymbaluk and Smart 1 993) .  More information is required on copper requirements of 
the foal as well as the increased absorption of dietary copper by neonates, as described in sheep 
(Suttle 1 975) and rats (Mistilis and Mearrick 1 969), which may compensate for low dietary 
copper concentrations during nursing. 
149 
Copper supplementation of pasture-fed foals from birth to weanmg at 5 months of age 
significantly increased their liver copper concentration measured at postmortem at 5 months of 
age. There was no residual effect of mare copper supplementation on the foal 's  liver copper 
concentration at 5 months of age. The rate at which the high neonatal liver copper 
concentration fal ls, and the effect of dietary copper intake on this decline, would help 
determine the importance of the neonatal l iver copper stores. None of the other organs appeared 
to store significant amounts of copper although there was a trend toward increased brain copper 
concentration in copper-supplemented foals. 
8.6 The effect of copper supplementation of the mare and foal on indices of bone 
and cartilage development 
No study has yet identified the mechanism by which supplementary copper may reduce 
abnormalities in bone and carti lage development in horses. A decrease in the cupro-enzyme 
lysyl oxidase activity was strongly impl icated in copper-depleted foals because of the 
concurrent increase in collagen solubil ity (Bridges and Harris 1 988) .  A decrease in lysyl 
oxidase (amine oxidase) activity was accompanied by increased collagen solubil ity in chickens 
raised on copper deficient diets (Rucker et al. 1 969). 
It may be expected that lysyl oxidase activity in our animals, and also with the animals in the 
study of Hurtig et al. ( 1 993) were adequate because plasma and blood cell copper 
concentrations and plasma caeruloplasmin activity were not influenced by copper 
supplementation. However lysyl oxidase levels were not measured in either study, and it is 
unlikely, that a decrease in lysyl oxidase activity preceded a depletion of caeruloplasmin 
activity or a decline in plasma copper concentration. Plasma and tissue copper concentrations 
or cupro-enzyme activity were not measured in the study of Knight et al. ( 1 990). Connective 
tissue was col lected and stored for the determination of lysyl oxidase in our study, but due to 
the lack of significant bone and cartilage lesions, it was decided not to perform these assays. 
However, the situation where horses responded to copper supplementation despite being above 
the threshold required for the maintenance of normal blood copper indices was reported in the 
experiment of Hurtig et al. ( 1 993) where copper decreased the incidence of bone and cartilage 
abnormalities, but had no effect on blood copper concentrations or caeruloplasmin activity. 
1 50 
Copper supplementation of our foals had no effect on indices of bone and cartilage 
development, but supplementation of the mare resulted in a decrease in the radiographic indices 
of physitis and a decrease in abnormalities detected at postmortem (at 5 months of age) in the 
cartilage and subchondral bone of limb joints. 
The bone and cartilage abnormalities present were minor. In the articular cartilage they 
consisted of either small retentions of cartilage, usually in commonly described locations such 
as the lateral and medial condyles of the distal third metacarpal/metatarsal bone and lateral 
trochlear ridge of the d istal femur, or small invaginations of cartilage in locations where 
subchondral bone cysts have been described such as the medial condyle of the distal femur, the 
proximal radius, the proximal humerus and the proximal and distal articular surfaces of the 
proximal phalanges. 
A number of mi ld lesions consisting of retained cartilage with irregular pitting of the surface of 
the articular cartilage were found on the medial trochlear ridge of the talus which is not 
considered a predilection site for OC lesions, nor other cl inically evident skeletal disease. Other 
sites in the hock commonly associated with OC (intermediate ridge of distal tibia and lateral 
trochlear ridge of the talus) were not affected, and therefore, while the significance of the 
lesions on the medial trochlear ridge was not known they were considered to be minor. 
There was no effect of mare copper supplementation on retentions of cartilage in the physis, 
but there was an effect on radiographic indices of physitis in the distal third metatarsal bone. 
This might be because only the distal third metacarpal/metatarsal physes were affected at the 
time of euthanasia, and as the growth plates were closing, it was not possible to evaluate them 
accurately by gross or histological examination, whereas the radiographic features of physitis 
were sti l l  evident. Radiographs might be better for assessing features of physitis, as the plane of 
section is not important as for gross and histological examinations, and it is easier to assess 
bone remodel l ing radiographically. The technique used for assessing features of physitis 
described in Chapter 6 had a very good repeatabi l ity between observers and might be a very 
useful clinical tool, but there would be obvious advantages to an automated image analysis 
procedure. The presence of physitis in only the metacarpal/tarsal growth plates is an i l lustration 
of the proposed "window of vulnerabil ity" (Pool 1 993) .  
1 51 
No lesions appeared to be clinically significant at the time of euthanasia and only two cases of 
retained cartilage (distal third metacarpal bone and lateral trochlear ridge of the distal femur) 
were seen on the radiographs taken at postmortem at 5 months of age. 
The fact that mare but not foal copper supplementation affected bone and cartilage 
development of foals may either reflect a greater requirement for copper in utero and/or a 
greater susceptibi l ity of the skeleton in utero to lesions which may be attenuated by copper 
supplementation of the mare. In particular it suggests that articular and physeal cartilage 
abnormalities may originate in utero, which can be influenced post-natally by environmental 
and managemental factors. The in utero effect which we observed at 5 months of age might 
have been caused by abnormalities in conformation at birth which would suggest a role of 
copper on skeletal conformation in utero. In this case, the abnormalities that we scored would 
haver been a result of normal weight bearing on an abnormal conformation. Treatment effects 
on conformation were not detected on clinical examinations of the foals, but they might not 
have been sufficiently sensitive. Alternately copper supplementation of the mares might have 
affected the integrity of the foetal connective tissue matrices and therefore the abnormalities 
detected might have been due to normal weight bearing forces on structural ly weakened tissue. 
These two possibilities might be associated. Further research is required to determine a 
mechanism for the effect of supplemental copper on developing connective tissue in the 
absence of other indices of copper deficiency. 
Whether high l iver copper stores in utero and during the neonatal period play a role in cartilage 
and bone development is not known. The liver copper concentrations at birth were highly 
variable but there was no relationship between l iver copper concentrations at birth and any 
evidence of DOD in this study. 
Differences between our results and those of Knight et al. ( 1 990) and Hurtig et al. ( 1 993) are 
curious. Both these North American studies found considerable differences in lesion severity 
and frequency between copper-supplemented and control foals. Hurtig et al. ( 1 993) even 
considered that the copper may have been pall iative, as a number of the foals were affected 
with femoropatel lar OCD when they started the trial at 3 months of age, but after completion of 
the trial there was no clinically evident sign of OCD in supplemented animals. Differences 
between the North American studies and our results may be explained if the North American 
foals have a greater frequency and severity of DOD due to managemental causes or genetic 
constitution, which is attenuated by supplemental copper. Alternately, if the level of DOD seen 
1 52 
in the untreated animals is a reflection of inadequate dietary copper, then other dietary factors 
must either increase the requirement of those foals for copper, or must decrease the 
bioavailabil ity of copper compared to pasture-fed foals, because our foals with confirmed low 
dietary copper levels had scant evidence of DOD lesions. 
8.6.1 The influence of energy in the copper response experiments 
The foals in the study of Hurtig et al. ( 1 993) were fed 1 20 % of NRC recommendations as this 
was considered to be standard North American management practice at the time (Hurtig et al. 
1 990). These animals were fed control led diets on dry-lots. The energy intake of our foals on an 
ad libitum pasture-diet was not known. However as discussed in Chapter 6, the liveweight 
gains of our foals were greater than those of Hurtig et al. ( 1 993) as reported by Burton and 
Hurtig ( 1 99 1 ). This may indicate that the energy intake of our foals was at least as high as the 
North American foals, unless breed differences accounted for large differences in body weight. 
The source of energy, rather than just the level of energy intake, may be of importance, and 
Glade et al. ( 1 984) has proposed that soluble carbohydrates may be primarily responsible via a 
postprandial hyperinsulinaemia, and a consequent transient hypothyroxemia. For example, 
Glade and Belling ( 1 986) studied the effect of increased intakes of soluble carbohydrates in 
horses and found histologically evident abnormalities in endochondral ossification and 
biochemical changes in growth plate cartilage. The issue regarding energy intake/source of 
energy is largely unresolved as Savage et al. ( 1 993a) found an effect of energy (using maize 
oil) on the incidence of DOD, scored as cl inically evident disease as wel l  as gross and 
histological abnormalities present at postmortem, but not on liveweight or l iveweight gain. The 
increased energy was contributed by complex carbohydrate (maize starch) and l ipids (maize 
oi l), which may not have had the same effect on endogenous postprandial hormones, but these 
were not measured. 
8.6.2 The theory of an association between dietary copper requirements and energy 
sourcellevel 
If energy source can explain differences between the incidence and severity of lesions between 
North American studies and those found in our foals, then it fol lows. that there is a l ink between 
dietary copper and energy source/level. The proposal has been studied in humans and rodents 
because of the concern that Western diets, and especially fast foods, are h igh in soluble 
1 53 
carbohydrates and low in dietary copper. In addition low copper diets are being investigated as 
a possible risk factor for human cardiovascular disease (Wapnir and Devas 1 995) .  However the 
results have been confusing; an effect of soluble carbohydrates versus starch on copper 
avai lability has been identified, but seems to be limited to male rats (O'Dell 1 990), while other 
studies have found that in humans, soluble carbohydrates increase copper balance but decrease 
the activity of the tissue cupro-enzyme Cu-Zn SOD, compared with starch (Lonnerdal 1 996). 
As wel l  high fat diets aggravate the effect of soluble carbohydrates (fructose) on the expression 
of copper deficiency in rats (Wapnir and Devas 1 995). 
The practice of meal feedings as opposed to continual grazing may be important in increasing 
the incidence and severity of DOD, and may support Glade's observations of the involvement 
of aberrations in normal endogenous hormones associated with feeding. Ralston ( 1996) has 
given further support to this by showing that horses affected with OCD had significantly higher 
postprandial glucose and insulin peaks compared with controls. 
8.6.3 The theory of copper as a therapeutic agent 
An alternative scenario is that dietary copper might reduce DOD caused by other aetiological 
factors . This is supported by the normal plasma copper concentration and caeruloplasmin 
activity in unsupplemented foals with a high incidence of DOD (Hurtig et al. 1 990). An effect 
of copper supplementation on tibial dyschondroplasia in birds, in the absence of copper 
deficiency has been reported (Orth and Cook 1 994), and may be due to the complexing of other 
dietary aetiological agents, reducing their absorption. Similarly in horses and other species in 
which there are anecdotal or reported observations of cl inical responses of DOD in some 
animals to supplementary copper, a mechanism other than a primary copper deficiency cannot 
be excluded. For example Davies et al. ( 1 996) found a direct effect of copper on the connective 
tissue matrix in vitro, including increased proteoglycan production and decreased production of 
cartilage degrading enzymes. 
A response in foals to copper supplementation without being copper deficient would be 
analogous to the situation where zinc is fed to sheep for the prevention of facial eczema despite 
no nutritional deficiency of zinc existing in these animals. The zinc ameliorates the effect of the 
mycotoxin sporidesmin. Based on these examples, the definition of adequate copper status may 
need to take into account a possible pharmacological effect of supplementation on animal 
1 54 
perfonnance and health especially ill cases where other indices of deficiency can not be 
demonstrated. 
8.7 Molybdenum as a copper antagonist for pasture-fed horses 
Molybdenum has been largely discarded as a possible copper antagonist in equine nutrition 
following the study of Strickland et al. ( 1987) in which it was found that oral aqueous sodium 
molybdate was rapidly absorbed from the small intestine and excreted in the urine. There was no 
effect on the measured copper status indices and it was concluded that there was little opportunity 
for the fonnation of thiomolybdates in the hindgut, and that any thiomolybdates fonned were not 
interfering significantly with copper absorption. Another study found that molybdenum 
significantly affected indices of copper status, including a decrease in copper balance in ponies 
fed molybdenum for short periods (Cymbaluk et al. 1981 b). 
We hypothesised that molybdenum incorporated in forage plants might be less readily absorbed 
because of its association with plant constituents, allowing greater quantities of molybdenum to 
reach the hindgut for the formation of thiomolybdates which once absorbed may interfere with 
tissue copper metabolism. The treated pasture contained 8 to 15 mg Molkg OM, and was 
considered very high but within possible pasture concentrations in certain situations (Grace 
1983b). At these concentrations we were unable to observe any effect on indices of copper status 
including plasma copper concentration, blood cell copper concentration, TCA insoluble plasma 
copper concentration, caeruloplasmin activity, superoxide dismutase activity and liver copper 
concentration. This does not preclude higher concentrations of pasture molybdenum from 
inducing a copper deficiency, but it rules out molybdenum as being a likely cause for increased 
copper requirements of horses raised on New Zealand pasture. 
8.8 Recommendations to the New Zealand Thoroughbred Industry 
At present we have little evidence to support recommendations, based on North American studies, 
to supplement pasture-based diets with copper for nursing animals (up to 5 months of age). 
However, the effect of copper supplementation of the mare on DOD lesions in the foal may 
reflect an increased requirement of copper in mares during gestation. Although the bone and 
cartilage lesions found may be considered minor, the possiblity of their becoming clinically 
significant could not be excluded, and thus supplementation of the mare is justified. 
1 55 
Our postmortem findings did not reflect the degree of industry concern regarding DOD. This is 
likely to be due to the stage at which the lesions were examined, and to unknown factors that 
control the expression of the disease, which resulted in evidence of very mild DOD lesions on our 
research farm yet severe, clinically evident DOD elsewhere. The incorporation of zinc and 
selenium in the supplement used in the main experimental work may have contributed to the low 
incidence encountered. However it is clear from these studies, that a dietary copper intake 
between 4.4 to 8.6 mg Cu/kg OM, low by most standards, for growing foals up to 5 months of 
age, is not an important factor in the expression of DOD. This may be due to the absence of 
dietary constituents that antagonise copper bioavailability on this farm. 
From our findings of the influence of copper supplementation of mares on the radiological and 
postmortem evidence of mild DOD in foals, copper supplementation can be recommended, 
especially on farms suffering significant incidence and severity of DOD. In this respect, 
supplementation of mares appears to be an appropriate way of increasing the copper status of the 
foetus and the neonate. 
8.9 Further research 
These studies raised a number of questions including the need to study further aspects of copper 
metabolism and its role in the aetiology of DOD. 
1 .  Measure indices of copper status in copper depleted animals to determine the relationship of 
changes in dietary copper intake and blood and liver copper concentration at which various 
cupro-enzymes (especially lysyl oxidase) become limiting. 
2 .  Determine the size, fate, and rate of depletion of liver copper stores in neonates, and the impact 
of these copper stores on copper metabolism of the neonate. 
3 .  Determine factors affecting copper bioavailability in horses (iron, energy source, others not yet 
identified) . 
4. Develop a long term remedy for copper deficiency based on depot copper administration. 
5. Determine the prevalence of DOD in New Zealand and develop detailed case descriptions. 
1 56 
6. Evaluate the effect of copper supplementation on farms suffering a high incidence and severity 
of DOD and develop a model of copper responsive DOD (identify a farm). 
7. Investigate the mechanism of action of copper supplementation in cases of copper responsive 
DOD. 
---- --
1 58 
Appendices 
1 59 
Appendix A: Pilot Studies on Circulating Copper Indices. 
General introduction 
Prior to the commencement of the main project, a small pi lot project was carried out to develop 
techniques and evaluate procedures. These covered the measurement of plasma copper 
concentration and caeruloplasmin activity, and the development of techniques for sampling 
liver tissue and growth plate tissue in vivo. The liver and growth plate biopsy techniques are 
described in Chapter 2 and 3 .  This chapter describes three pilot trials: a spike trial, a copper 
dose response trial and a trial investigating the effect of surgery on plasma copper 
concentrations. 
Spike Trial 
Introduction 
The l iterature is ambiguous as to whether dietary copper intake influences blood indices of 
copper status. Changes in dietary copper levels had no influence on serum copper concentration 
in some trials (Smith et al. 1 975; Hurtig et al. 1 990), while some authors consider that 
variations in serum and plasma copper concentrations between horses from different stables, 
and in different seasons are most l ikely due to changes in dietary copper intake (Stubley et al. 
1 983; Auer et al. 1 988a). 
Therefore, for the present study, it was important to accurately characterise the copper status of 
both control horses and horses receiving oral copper supplementation . If oral copper 
supplementation resulted in a transient increase in plasma copper concentration and 
caeruloplasmin oxidase activity, then the time of sampling fol lowing supplementation could 
influence the value of these indices. Thus this study investigated the nature of any rise in 
circulating copper indices following a single large dose of oral copper sulphate. 
Materials and methods 
Eight mature mares in the second trimester of gestation were randomly divided into copper 
supplemented and control groups based on age and date of last service. All  animals were kept 
in a smal l paddock and fed pasture for the duration of the trial . 
160 
Copper supplemented animals received 500 mg of copper sulphate given per os using a drench 
gun. Control groups received a water placebo of equivalent fluid volume. 
Animals were sampled by jugular venepuncture, and the blood was col lected into blue top 
sodium heparin trace element tubes (Becton Dickinson # 367735) .  Tubes were placed on ice 
for transport to the laboratory. Plasma was then separated and the sample split for analysis of 
plasma copper concentration and caeruloplasmin oxidase activity. Plasma copper concentration 
was analysed by inductively coupled plasma emission spectrometry (Lee 1 983), and 
caeruloplasmin oxidase activity was analysed by the colorimetric measurement of the oxidation 
of the substrate p-phenylenediamine (Ravin 1 96 1 ). 
Samples were collected at -24, 0, 2, 4, 6, 8, 1 2, 1 6, 25,  32, 48, 72 and 80 hours after oral 
supplementation. All samples were analysed as one batch. 
The results were analysed by analysis of variation with repeated measures on time to test for a 
significant effect of treatment, and changes with time. Analyses were perfonned using 
Statistical Analysis System (SAS) version 6 . 1 1 for windows (SAS Institute Inc., Cary, N.C., 
USA). 
Results and discussion 
There was no significant effect of time or copper supplementation on the plasma copper 
concentration or caeruloplasmin activity (Figure A I ). 
1 61 
Figure At Mean plasma copper concentration (boxes) and caeruloplasmin activity (diamonds) of 
mares receiving a single dose of copper sulphate (500 mg) on day 0 (complete lines), and controls 
(incomplete lines). 
� 
� 
5 
c:: o 
.� 
C .., u c:: o u .... .., c.. c.. o u ro 
E CIl ro 
0:::: 
1 .6 _ 
. . . . . . . . . . .  · · ·(J
.
S · 
0.6 + 
0.4 ..:. 
0.2 _ 
---------,QD--- -- -
-30 -20 - 1 0  o 1 0  20 30 
Time (h) 
. 50 
_ 45 
2-. .  30 .£ 
· · · · · · · · ·f · · · · · · · · · · · · · · · · · · · t · · · · ·+ 25 .� 
40 
j c:: 
.::.. 20 .§ CIl ro 
_ 1 5  g. 
"2 
1 0  � u 
.). 5 i 
____ ._.+ _____  1 0 
50 60 70 80 
There was a remarkable similarity in the trends in the plasma copper concentration and 
caeruloplasmin activity of both control and supplemented horses. 
Figure A2 and A3 shows the pattern of change of the individual animals plasma copper 
concentration and caeruloplasmin activity. All animals had a peak in caeruloplasmin at the 2 
hour sample and a trough at the 25 hour sample, with another smaller peak at the 32  hour 
sample. 
Figure A2 Plasma copper concentration of individual mares receiving a single dose of copper 
sulphate (500 mg) on day 0 (complete lines, open boxes), and controls (incomplete lines, closed 
boxes). 
� 
� 
g 
c o 
� 
C Q) u c o u 
lii a. a. o u 
co 
E rJ) co 
a:: 
. . . . . . . . . . . .  
2 __ 
• 
1 . 8  - !: : : " 
1 .6 -I :: 
, : 
. . . . . . . . . . . . . . . . . . .. 
. . . . .
. . . . · · ·o:; ( � 
• • 0 ' 
0.2 _ 
----.- -- .-0-_ 
-30 -20 - 1 0  o 
: ........ . . 
• 
. .• . . . . . . . . . . . . . . . . . . . . . • . . . . . . • 
1 0  20 30 40 50 60 70 80 
Time (h) 
162 
Figure A3 Caeruloplasmin activity of individual mares receiving a single dose of copper sulphate 
(500 mg) on day 0 (complete lines, open boxes), and controls (incomplete lines, closed 
boxes). 
G o r­C""') 
25 -
_-�' .---_, -20- _ 
-30 -20 - 1 0  0 1 0  
. .• . . . . .  - .  
'.' 
20 30 40 50 60 70 80 
Time (h) 
1 63 
Plasma copper concentration shows a simi lar pattern of change, but with greater variability 
between individuals. This pattern of change is unlikely to be explained by a diurnal effect as 
the peak at 2 hours is only one hour different when corrected for the time of day with the 
trough at the 25 hour sample. It is also clearly not related to dietary copper intake as both 
controls and supplemented mares showed the same pattern of change, with the greatest peak 
occurring in the control mares. It is possible that the peak at the 2 hour sample was related to 
the stress involved with yarding, drenching and bleeding the mares. Copper and caeruloplasmin 
activity has been shown to increase in horses in response to inflammatory stimuli (Firth et al. 
1 987; Smith and Cipriano 1 987). However, Auer et al. ( 1  989a) found that caeruloplasmin 
activity peaked after 24 days and was sti l l  significantly elevated after 50 days, but the 
inflammatory stimuli used in that study (intramuscular injection of Freund's  adjuvant) was far 
more severe than the stimuli in the present project. These transient changes in circulating 
indices, which are consistent between animals, are discussed further in Chapter 7. 
The ranking of the individuals with respect to their plasma copper indices, stayed reasonably 
consistent throughout the trial. This indicates that individuals may have different homeostatic 
levels, possibly under strong genetic control. 
There also appeared to be a good relationship between plasma copper concentration and 
caeruloplasmin activity. This relationship is further explored in Chapter 5 .  
Time of sampling after oral copper supplementation is unl ikely to bias plasma copper 
concentration and caeruloplasmin activity data collected to determine copper status of horses. 
There was no effect of supplementation, and despite significant changes between sampling 
occasions, all individuals showed simi lar changes. 
1 64 
Copper Dose Response 
Introduction 
Danks ( 1 98 1 )  proposed that a useful way of diagnosing dietary copper adequacy was to 
measure caeruloplasmin activity, then supplement the diet with copper and remeasure the 
caeruloplasmin activity. If the caeruloplasmin activity increased, the initial diet was inadequate 
in dietary copper. According to Matusda et al. ( 1 974), the resultant increase in caeruloplasmin 
activity after copper supplementation of copper-deficient humans begins after 4 hours and is at 
a maximum 2 days following supplementation. In copper deficient rats, restoration of 
caeruloplasmin activity occurred 6 to 8 hours after intubation and 1 4  to 1 6  hours after feeding 
supplementary copper sulphate (Linder et al. 1 979). 
Neifakh et al. ( 1 969) found that excessive supplementation of copper (parenterally) in monkeys 
resulted in an inhibition of caeruloplasmin activity. 
It has been suggested that dietary copper levels of 8 to 1 5  mg Culkg DM, which is in the high 
range of pasture copper concentrations, is not adequate for growing horses (Knight et al. 1 990; 
Hurtig et al. 1 993).  Burton et al. ( 1 990) found that caeruloplasmin activity reflected copper 
intake in pregnant and barren mares at dietary copper concentrations of 4, 1 0, 20 and 30 mg 
Culkg. Caeruloplasmin activity was suppressed at the higher dietary copper concentrations. 
The objective of this trial was to feed horses on different planes of dietary copper and to 
measure any changes in plasma copper concentration and caeruloplasmin activity, to ensure 
that the copper supplemented horses in the experimental part of this thesis received adequate 
copper supplementation for maximum cuproenzyme activity, but not an excessive amount. 
Materials and methods 
Nineteen mature Thoroughbred mares in the second trimester of gestation were randomly 
assigned to one of ten dietary copper supplementation levels (0, 50, 1 00, 1 50, 200, 250, 300, 
350, 400, and 500 mg Cu/day). Two horses received each dose, except only one horse received 
350 mg Cu/day. 
1 65 
Horses were grazing pasture containing approximately 7 mg Cu/kg DM throughout the trial. 
Assuming a dietary dry matter intake of 14 kg/day, the dietary copper intake from the pasture 
would be about 1 00 mg/day. 
Horses were supplemented daily for 4 days by oral drenching. B lood samples were collected on 
day 0, before the first supplementation and then dai ly for a further 4 days. The samples were 
collected and analysed as described for the previous trial. Samples were taken at approximately 
the same time each day, and before copper supplementation on that day. 
Samples were analysed by analysis of variance with repeated measures over time. The effect of 
treatment level, and of time were investigated. Analyses were performed using SAS as above. 
Results and discussion 
A family of curves for the plasma copper concentration and caeruloplasmin activity were 
created for each level oftreatment group and are presented as 3 dimensional surfaces in Figures 
A4 and AS.  Mean values were taken for each treatment level. Standard errors are not presented. 
Figure A4 Plasma copper concentration of pasture-fed pregnant mares receiving different levels of 
copper supplementation. 
1 .4 
I: 1 .2 .!2 
'" .... I i E l .., '-' 0.8 � I: 0 :::::::-'-' "Ob t E I c. '--' 0.6 l c. Copper dose 0 '-' 0 .4 (mg/day) C<l 
E en 0.2 C<l 0:::: 
2 3 4 
Time (day) 
Figure AS Caeruloplasmin activity of pasture-fed pregnant mares receiving different levels of 
copper supplementation. 
60 
.2 50 500 
.� 40 400 � G  350 .:: 0 300 
e t- 30 250 ", M  '" -" 
200 Copper dose Q.. :J � �  20 (mglday) 
2 ., '" 1 0  u 
2 3 4 
Time (day) 
There was no significant effect of the level of copper supplementation, or on the time after 
copper supplementation on either plasma copper concentration or caeruloplasmin activity. 
1 66 
This indicates that the mares in their second month of gestation and grazing pasture are 
receiving adequate copper for optimal caeruloplasmin activity. In  addition, assuming the 
proposal of Danks ( 1 98 1 )  is valid for pregnant mares, pasture provides adequate dietary copper 
for mares in their second trimester of gestation . However, the requirements for copper may 
increase in the third trimester and also for foals experiencing high growth rates. 
This trial did not corroborate the findings of Burton et al. ( 1 990), in that caeruloplasmin 
activity did not reflect dietary copper intake and we failed to demonstrate any adverse effect of 
excessive copper supplementation on caeru\op\asmin activity. This supports previous work 
which found a resistance of horses to copper toxicity on very high dietary copper intakes 
(Smith et al. 1 975) .  
1 67 
The Effect of Surgery 
Introduction 
Neonatal foals in the experimental work of this thesis underwent growth plate and laparoscopic 
liver biopsies under general anaesthesia as described in Chapters 2 and 3 .  
Inflammatory procedures increase plasma copper concentration (Firth et al. 1 987; Smith and 
Cipriano 1 987; Auer et al. 1 989a). The objective of this trial was to define the effects of the 
surgical procedures (described in Chapters 2 and 3) in terms of post-operative changes to 
plasma copper concentration. 
Materials and methods 
Two nursing foals aged approximately 3 months of age underwent growth plate and 
laparoscopic l iver biopsies under general anaesthesia for the purpose of technique 
development. The foals and their dams were confined to stalls following the surgery for the 
duration of this trial. Blood was col lected and analysed for plasma copper concentration as 
described in the pilot trials above. 
No statistical analyses were performed due to the small number of animals and lack of controls. 
Results and discussion 
There were no control foals, and the two foals were operated on different occasions. Therefore 
there was no way of confirming that any changes to blood copper concentration were due to the 
surgical procedures. Therefore the results were interpreted cautiously, and were used only to 
describe the changes observed fol lowing the surgical procedures. 
The plasma copper concentration of one foal increased for 5 days following the surgical 
procedures, and then declined. The plasma copper concentration of the second foals showed a 
smaller increase in plasma copper which plateaued for approximately 1 0  days following 
surgery (Figure A6). 
1 68 
Figure A6 Plasma copper concentration of two foals following laparoscopic liver biopsy and growth 
plate biopsy performed under general anaesthesia. 
� 
en 
.s 
c o 
� 
C (!) t) c o t) 
'-(!) a. a. o t) 
ro 
E 
2.5 _ 
2 _ 
gJ 0.5 ;-
0: 
. ___ ____ . ---CL- __ _ _ _ . 
-� 0 � 100 1� 
Time (h) 
200 2� 300 3� 
There was a considerable difference between the two individuals in the response of their 
plasma copper concentrations fol lowing the surgical procedures. This may reflect a difference 
in the foals response to inflammation, their previous copper status, or differences in the amount 
of surgical inflammation between the two procedures. Auer ( 1 989a) reported that changes to 
plasma copper concentrations were a good measure of the degree of inflammation, and that the 
decrease in the elevated plasma copper concentration may be useful in monitoring the 
resolution of an inflammatory processes. 
Four mature horses in the study of Auer et al. ( 1 989a) received intramuscular injections with 
Freund's  adjuvant. Their mean plasma copper concentration rose from approximately 1 .2 mg 
Cull to peak after 24 days at approximately 2.3 mg Cull. The mean plasma copper 
concentration was sti l l  significantly elevated after 36 days. 
The resolution of the increase in plasma copper over a relatively short period indicates that the 
surgical procedures performed were not highly inflammatory stimul i .  However the age of the 
foals in this pi lot trial were older than the age of the foals undergoing these surgical procedures 
in the experimental work of this  thesis. 
1 69 
General conclusion of pilot trials 
Plasma copper concentration and caeruloplasmin activity have a wide variation between 
samples which is not able to be explained by dietary copper intake, and is consistent between 
animals. There appear to be similarities in the changes in plasma copper and caeruloplasmin 
activity most l ikely due to the large percentage (73 %) of circulating copper that is bound to 
caeruloplasmin (Auer 1 988b). 
There is no evidence from circulating plasma copper status indices of an inadequacy of dietary 
copper in pasture-fed mares in their second semester of gestation . Hurtig et al. ( 1 990, 1 993) 
also found no difference between copper supplemented and control foals in their serum copper 
concentrations or caeruloplasmin activity despite increased bone and cartilage lesions in 
control foals. 
Surgery and other inflammatory procedures are likely to influence circulating copper status 
indices. However in the main experimental work of this thesis, all foals were supplemented, 
and therefore surgery should not affect the comparison in copper status between copper­
supplemented and control foals. 
1 70 
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