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This is to certify that the

dissertation entitled

The Relationship Between Selenium and Vitamin E
Nutrition and Exercise in Horses

presented by

John Edward Sheiie

has been accepted towards fulfillment
of the requirements for

 

 

 

Ph.D. degree in Animal Science

 

mam

Major professor

Dr. Duane E. Ullrey

[Mme 6/15/84

 

.‘WSU 13 an Affirmative Action/Equal Opportunity Institution 0- 12771

 

 

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THE RELATIONSHIP BETWEEN SELENIUM AND
VITAMIN E NUTRITION AND EXERCISE IN HORSES

By
John Edward Shelle

A DISSERTATION

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

DOCTOR OF PHILOSOPHY

Department of Animal Science

1984

1 4.3K--.) V'

J10

ABSTRACT

THE RELATIONSHIP BETWEEN SELENIUM AND VITAMIN E
NUTRITION AND EXERCISE IN HORSES

By

John Edward Shelle

The similarities between exercise—induced my0pathies in
horses and white muscle disease have led to supplementation
of both selenium (Se) and vitamin E to heavily exercised
horses. Exercise increases 02 delivery and metabolism in
muscle tissue resulting in.zn1 increased generation of
reactive (k3 byproducts. The Se—containing enzyme
glutathione peroxidase (GSpr) and vitamin.E help protect
the cell from these reactive 02 species.

To establish Se status of horses at the MSU Horse
Teaching and Research Center (HTRC), plasma and milk Se
concentrations of 6 mares and their foals were analyzed and
found 'within. normal ranges despite classification. of
Michigan as a Se-deficient state. Previous studies at the
HTRC have offered no explanation for these normal plasma Se

concentrations. Analyses of forage, soil and cinder-surfaced

roads support the conclusion that high forage Se was a
consequence of soil contamination with Se-rich cinder dust.

Eight Arabian mares were used in a 2x2 double split-plot
design to determine the effects of conditioning, exercise
and daily supplements onLB mg Se and/or 750 IU vitamin E
on blood constituents of the GSpr system and on muscle and
blood enzymes. Three levels of treadmill exercise
conditioning were provided: 1) non-conditioned, 2)
conditioned for 45 days and 3) conditioned and allowed 2
days of stall rest before sampling. Blood samples were
obtained before, during and after exercise. Conditioning
mares for 45 days reduced blood malondialdehyde (MDA) and
lactate concentrations, indicating improved animal fitness.
High blood MDA concentrations in non-conditioned mares, and
increased plasma creatine phosphokinase activities after 2
days of rest in conditioned mares, suggest that sudden
changes in physical activity should be approached
cautiously.

Blood reduced glutathione concentration and erythrocyte
g1ucose-6-phosphate dehydrogenase activity decreased with
conditioning. Eighteen weeks of vitamin E supplementation
increased plasma alpha-tocopherol concentrations but did not
significantly affect any other parameter measured.
Erythrocyte GSpr activities were significantly higher as a
result of conditioning and Se supplementation. Mares in this
study appeared to have adequate plasma Se levels. However,

the observed increases in GSpr activity associated with

conditioning may indicate a need for supplemental Se when

diets of exercising horses are low in this element.

DEDICATION

To my wife Pam, whose faith in me

made this work possible.

ii

ACKNOWLEDGEMENTS

There so many people who helped and supported me
during my education it would be impossible to thank them
all, so let me start with a blanket thank you to those
people whose names are not mentioned here but were
instrumental in the completion of this works Data gathering
during this project took many hands. I would like to thank
Dr. Joe Rook, Dave Anderson, Chris Johnson, Dave Baer, Dave
Cross, Marilyn Loudenslager and especially Mike Yoder, for
helping to make things run smoothly. A special thanks to
Dr. Wayne Van Huss from the Department of Health and
Physical Education for his assistance in monitoring heart
rates. I would like to thank Phyllis Whetter and Dr. Pao Ku
for their patience and willingness to share their experience
with laboratory procedures.

I would like to express a special thank you to Norm
Oswald from the University Farms Department for his
assistance in the design and fabrication of the treadmill
and the many modifications it took to make it Operable. His
friendship and support will always be remembered.

The*willingness of my graduate committee, Dr. Harold
Henneman, Dr. Elwyn Miller and Dr. Howard Stowe, to help in

giving direction and guidance in my course of study was

iii

greatly appreciated. Most importantly I would like to thank
my committee chairman Dr. Duane Ullrey for his constant
support and friendship. I feel fortunate to have been able
to work with a person whom I so greatly respect and admire.
Lastly I would like to thank my wife, Pam for the many
sacrifices she made during our education. Her love and
support makes the completion of this work that much more
meaningful. I would like to thank my kids Michelle, Kevin
and Aaron for understanding that dad couldn’t always be

there. This degree is indeed a family accomplishment.

iv

TABLE OF CONTENTS
Page
LIST OF TABLES..........................................iv
LIST OF FIGURES.........................................vi
LIST OF PLATES.........................................vii
INTRODUCTION.............................................1
REVIEW OF THE LITERATURE.................................7
Selenium and Vitamin E in Nutrition of the Horse.......7
Selenium Toxicity....................................7
Selenium Deficiency..................................8
Selenium Requirement.................................9
Vitamin E...........................................11
Vitamin E and the Immune System.....................12
Vitamin E and Exercise..............................13
Myopathies............................................14
Exertional Myopathy.................................14
Muscle Disorders in Horses..........................15
MATERIALS AND METHODS...................................19
Selenium in Plasma and Milk...........................19
Exercise Trial........................................21
Animals.............................................21

DietSOOOO0.000000000000000000000000.00.00.00.000000022

conditioning.OOOOOOOOOOOOOOOOO0.0.00.00000000000000025

Blood Sampling......................................25
Heart Rate..........................................28
Blood Analyses........................................28
Statistical Analyses..................................30
Treadmill Design and Construction.....................32
RESULTS AND DISCUSSION..................................4O

The Testing, Use and Modification of
an Equine Exercise Treadmill..........................4O

conCluSionOIOOOOOOOOOOOOOOO....00....00000000000000046

Plasma and Milk Selenium Levels from
Maresand Foals........... ............. .. ....... ......47

conCIuSionOOOCOOOIOOOOOI0.0.0.0....0.0.0.000... ..... 52

Changes in Blood Parameters During
Conditioning andExercise in Horses..n..uu.un..u..53

summaryl.0..OOOOOOOOOOOOOOOO0....0.0.0.000000000000058

Selenium and Vitamin E Supplementation During
Exercise euui Conditioning in Horses.n.u.u.n.n.u.59

summary. 0 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O .76
CONCLUSION. O O O O O O I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O I O O O O O .77
LITERATURE CITED 0 O O 0 O O O O O O O O O O O O O O O O O O O O O O O C O O O O O O O 0 O O O .79

vi

LIST OF TABLES

Table Page

1 Vitamin E and selenium concentrations in air
dry matter Of feed.OOOOOOOOOOOOOOOOOOOOOO..000.00.23

Horse weights and air dry feed intakes............23
Supplemental pellet formula.......................24
Conditioning routines.............................26
Blood sampling times..............................27
Key for figures 2-6...............................34

Se concentration of feeds, bedding and soils......48

CDx'lmU'I-PVJN

Mean Se concentration of mare's milk
and plasma and foal plasma........................51

9 The effects of conditioning on heart rate and
blood lactate levels..............................54

10 Changes in blood lactate as a result of
conditioning and exercise.........................55

11 Changes in blood hemoglobin and hematocrit
and plasma hemoglobin as a result of exercise.....57

12 The effects of vitamin E supplementation on
plasma alpha-tocopherol concentrations............63

13 The effects of conditioning and Se treatment
on erythrocyte GSpr activity.....................64

14 The effects of conditioning and time of sampling
on blood malondialdehyde concentration............66

15 The effects of level of conditioning and time
of sampling on plasma protein.....................68

16 The effects of conditioning and time of sampling
on plasma creatine phosphokinase activity.........70

17 The effects of conditioning and time of sampling
on blood reduced glutathione levels...............73

vii

18 The effects of conditioning and vitamin E
supplementationcnl erythrocyte glucose-6- .
phosphate dehydrogenase activity..................75

viii

Figure

QmU‘I-P-W

11

LIST OF FIGURES

Systems for cellular defense against 02
induced damage.O...OOOOOOOOOOCOOOOOOOOOOO0....0.0.04

Treadmill, side elevation, with
motor and walkway removed............. ......... ...35

Treadmill, front elevation.. ..... ........ ......... 36
Treadmill, rear perspective.......................36
Treadmill, safety latch............. ..... .........39
Treadmill, motor and drive, top view .............. 39

Changes in Se concentration in mare's
milk and mare and foal plasma over time...........49

The effects of Se treatment on plasma
Se levelsIOOO0.0.0....OOOOOOOOOOOOOOOOOO000.......60

Changes in plasma Se during exercise.. ..... .......61

Changes in plasma glutathione-S-transferase
activity as a result of exercise..................69

Changes in blood reduced glutathione levels
With exerCiseOOOOOOOOOOOOOOO ...... .0. 0000000000000 72

ix

LIST OF PLATES

Plate Page
1 Treadmill, side view...............................41
Treadmill, loading gate and ramp...................41
Treadmill, front view..............................42

2
3
4 Treadmill, rear view...............................42
5 Treadmill, motor and drive.........................43
6

Treadmill belt, lacings and s1ide..................43

INTRODUCTION

Selenium (Se) and vitamin E have not routinely been
added to equine rations as with diets of other species.
This may be due primarily to the lack of conclusive evidence
of Se-vitamin E deficiency diseases in the horse.
Nutritional muscular dystrophy (NMD) does occur in foals and
is responsive to vitamin E and Se supplementation (Caple et
al., 1978; Gabbedy and Richards, 1970; Schougaard et al.,
1972; Wilson et al., 1976). However, the frequency of NMD
has been low, even in areas with low soil Se concentrations,
and horsemen have not routinely provided supplementation.

Evidence of Se and vitamin E deficiencies in adult
horses has been even more elusive. Heimann et a1. (1981)
demonstrated improved reproductive efficiency in pony mares
grazing fescue pasture by providing Se or fertilizing
pastures with nitrogen which increased Se content of the
forage. Reproductive problems involved perinatal foal
losses due to thickening of the placenta or postnatal losses
from agalactia. Owen et al. (1977) hypothesized that
maxillary myositis and dystrophic myodegeneration in adult
horses may be the same disease entity caused by a deficiency
of vitamin E and Se. However, conclusive evidence supporting

this thesis has not been forthcoming. Dewes (1981) reported

improved athletic performance in horses with parenteral
administration of vitamin E. Improvement was seen in
attitude and disposition and was thought to be the result of
prevention of a mild subclinical myositis, manifested in
progressively deteriorating performance and behavior.

There are no less than seven types of myopathies of
horses described in the literature. The etiology of most is
unknown.(Hansen, 1970). Azoturia.(Monday morning disease)
and tying-up are among the most common and are thought to be
the same disease state, varying only in the severity of
signs (Farrow et al., 1976). Signs normally appear in well-
fit individuals on the first day of exercise after a period
of rest; hence, the common name "Monday morning disease".
Elevated serum activities of glutamic oxalacetic
transaminase and creatine phosphokinase and, at necropsy,
lesions of skeletal and cardiac muscle are also described
(Cardinet et al., 1967; Lindholm et al., 1974). Affected
animals exhibit myoglobinuria and resist further muscular
activity. The similarities between NMD in lambs and foals,
and exercise-induced myopathies have led veterinarians to
prescribe supplementation of Se euui vitamin E both
prophylactically and therapeutically in the treatment of
azoturia and tying-up with favorable results (Farrow et al.,
1976; Hill, 1962; Stewart, 1960).

Azoturia and tying-up may occur when conditioning
horses for show, race or endurance competition. Because

they occur more frequently in some breeds and families than

in others, there is concern that there may be a genetic
predisposition in some horses. There is also a tendency for
reoccurrence of signs once an animal has had a problem.
All of this amounts to losses in both training time and
medical expenses which are of concern to a great many
horsemen.

The cell has evolved a variety of defense mechanisms
to protect against peroxidative membrane damage. Super-
oxide dismutase, often called the first line of cellular
defense, acts on oxygen radicals reducing them to less toxic
hydrogen peroxides. The seleno-enzyme, glutathione
peroxidase (GSpr) and vitamin E protect the cell from
peroxidative damage as follows. GSpr, working together
with catalase, destroys hydrogen peroxide and organic
peroxides. Reducing equivalents for these reactions are
provided by the enzymes of the hexose monophosphate pathway
(Figure 1). Vitamin E exerts its protective effect by.
preventing perpetuation of peroxidative chain reactions in
unsaturated fatty acids within membranes (Diplock, 1981).

The effects of exercise on this system were first
alluded to by Young and Keeler (1962). Immobilizing one
limb of Se-deficient lambs significantly reduced muscular
lesions in that limb, indicating that exercise exacerbates
the effects of Se deficiency. Exercise increases oxidative
metabolism at the cellular level which may lead to an

increased generation of potentially damaging byproducts. If

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this is indeed the case, then perhaps rigorous exercise
programs may increase the need for vitamin E and Se above
the minimum requirements.

Thoroughbred horses which had been described as
unsatisfactory performers were found to have lower serum Se
levels than horses whose performance was as expected
(Blackmore et al., 1979). Brady et al. (1978) reported
increased peroxidative damage in exercised horses but could
not show an increase in GSpr activity with Se
supplementation" IHowever, this study was conducted using
non—conditioned horses during a single exercise bout. The
need for Se supplementation may increase with conditioning.
A constant challenge to the GSpr system through frequent
exercise may stimulate synthesis of GSpr and hence increase
the Se need.

The effect of mega-doses of vitamin E on performance of
human and equine athletes has been tested by Sharmon et al.
(1971) and Lawrence and Slade (1979), respectively. In both
cases, athletes were (n1 conditioning programs and
performance was not significantly altered by
supplementation. However, the effect of supplementation on
limiting peroxidative damage was not assessed.

This study was undertaken to:1) reproduce as nearly as
possible the factors which result in equine exercise
myopathies, by varying days of rest and exercise, 2)
determine the effect of Se and vitamin E supplementation on

changes in serum enzyme activities resulting from exercise-

induced stress, 3) evaluate changes in the erythrocyte
GSpr system due to supplementation and conditioning, 4)
design and build an equine exercise treadmill, with
adjustable speed and slope of incline, to accurately produce
controlled exercise stress for use in studies of this
nature, and 5) investigate further the unusually high plasma
Se levels found in horses at the MSU Horse Teaching and

Research Center.

REVIEW OF THE LITERATURE
Selenium and Vitamin E in Nutrition of the Horse

Selenium Toxicity

Toxicity signs were first reported in horses in the
United States by Madison (1860). In the 1930s the causative
agent was identified as Se, occurring in high levels in
plants that concentrate the element naturally (Franke,
1934). The acute form of toxicity is called blind staggers
with the chronic form known as alkali disease. Chronic
signs include hair loss (especially of the mane and tail),
laminitis and hoof sloughing. Lack of pathologic lesions of
the visceral organs has been a feature of poisoned horses at
necropsy (Knott and McCray, 1959). Crinion and O'Connor
(1978) saw elevated sorbitol dehydrogenase activity,
indicative of liver damage, in the serum of horses consuming
plants high in Se. However, death in most instances has at
least partially been attributed to an inability to forage
for food due to lameness. The acute form of toxicity is
evidenced by elevated temperature, labored respiration, a
bloody froth from the mouth and nose, respiratory failure
and death. Acute toxicity normally follows consumption of

very high levels of Se (Gerken, 1982).

Selenium Deficiency

The essentiality of Se has over-shadowed its toxic
properties in recent years. The occurrence of nutritional
muscular dystrophy (NMD) has been correlated to areas of the
United State where Se content or availability in soils is
low (Muth and Allaway, 1963L.It has further been shown that
the signs of NMD can be eliminated with the addition of Se
to livestock diets (Ewan et al., 1968). INMD is also seen in
horses and is responsive to Se and vitamin E supplementation
(Caple et al., 1978; Gabbedy and Richards, 1970; Jones &
Reed, 1948; Schougaard et al.,l972; Wilson et al.,l976).
Foals with NMD show increased serum activities of creatine
phosphokinase (CPKL. glutamic oxaloacetic transaminase
(SGOT) and glutamic pyruvic transaminase (SGPT) which would
indicate muscle damage and the subsequent release of these
enzymes into the circulation. Lesions of cardiac and
skeletal muscle, typical of NMD in‘livestock; are seen at
necropsy. .Affected foals commonly are nursing mares that
are consuming diets low in Se or low in both Se and vitamin
E and are seen to improve with supplementation.

Perinatal foal losses in mares grazing fescue pasture
were found to respond to Se supplementation (Heimann et al.,
1981a). Death of the foals was attributed to suffocation
due to a thickened placenta which failed to rupture at
parturition or to agalactia. All foals fromSe—supplemented
mares survived while 50% of the foals from the non-

supplemented group died at birth. In a concurrent study, the

Se status of mares grazing fescue was shown to improve when
pastures were fertilized with nitrogen, which increased the

Se concentration in the forage (Heimann et al., 1981b).

Selenium Requirement

Serum Se levels in surveys of normal horses conducted
by Stowe (1967) and Maylin et al.(l970) ranged from .070 to
.124 ppm and .077 to .156 ppm, respectively. Se levels
were found to be dependent on whether feed was home grown or
commercially obtained, and on quantity and type of bedding
used. Stowe (1967) and Bergsten.et al. (1970) found lower
serum Se levels in suckling foals than in their dams. In a
depletion—repletion study, Stowe used orphaned foals on a
Torula yeast diet to determine the Se requirement. Minumum
serum Se levels reached..057 ppm, and foals on the deficient
diet showed a tendency for decreased weight gain when
compared to their normally reared contemporaries. Optimal
physiological effect from parenteral supplementation was
reached at approximately 45 days and gave an estimated
requirement of 2.4 ug Se/kg body weight per day.

Bergsten et al. (1970) orally supplemented adult horses
with low serum Se levels with sodium selenite at doses of
1.5, 5, 6, 18, or 30 mg Se per week. Serum Se levels
responded linearly from 1.5 to 6 mg of supplementation with
only a slight increase with higher dose levels. Bergsten et
al. (1970) concluded that 6 mg Se per week was the

requirement of the horse.

10

These values can be used to calculate dietary Se
intake, approximating a minimum requirement of .09 ppm and
.10 ppm daily for the studies of Bergsten et a1. and Stowe,
respectively. These studies have led the National Research
Council to suggest that Se is required at the .10 ppm level

in equine diets (NRC, 1978).

11

Vitamin E

Rations for horses normally contain sun-cured green
hays which are high in vitamin E. This fact, in combination
with the interrelationship of Se and vitamin E function in
maintenance of cellular integrity, has made it difficult to
produce substantial evidence of a vitamin.E deficiency in
horses. Nutritional muscular dystrOphy in foals has'been
shown to respond to Se and vitamin E supplementation (Dodd
et al., 1960). However, when supplementing vitamin E only
to dystrophic foals, Dodd saw no response and concludedthat
Se supplementation alone was of prophylactic benefit.

In a survey of New York breeding farms, plasma vitamin
E concentrations varied by horse and season of the year
(Maylin et al., 1980). The highest concentrations were
seen during Spring and summer sampling periods, when intake
of fresh rather than stored forage was greatest. It was
suggested that during the months of peak reproductive
performance, vitamin E content of feeds was increased and
this may be useful in maintenance of normal reproductive
function. Vitamin E deficiency has been implicated as a
possible cause of male sterility and in failure to maintain
pregnancies in females of other species. Icheve , Rich et
a1. (1985) could not improve semen quality in stallions with
supplementation of 5,000 or 10,000 IU of vitamin E daily,
and concluded that the current NRC recommended minimum
requirement 'was adequate ‘h: maintain reproductive

performance.

12

Stowe~(1968):fed orphaned foals a vitamin E-deficient
diet. Erythrocyte stability, as determined by layering
hemolysis, was used to evaluate vitamin E status. Foals
were fed the deficient diet for 200 days. Rations were then
supplemented with alpha—tOCOpherol, and the return of
erythrocyte stability was monitored. A value of 233 ug
alpha-tocopherol per kg of body weight produced normal
erythrocyte stability and is currently the recommended

minium daily requirement (NRC, 1978).

Vitamin E and the Immune System

Recent deve10pments in immunology have spurred new
interest in vitamin E supplementation. Polymorphonuclear'
leukocytes (PMN) engulf and destroy invading bacteria. The
destructive mechanism is closely related to an increased
oxygen uptake. The reduced byproducts of this oxygen surge,
especially H202, account for much of the microbicidal
activity of the PMN (Baehner et al., 1980; Iyer et al.,
1961). The ability of vitamin E to protect erythrocytes
from oxidative cellular damage, and because erythrocytes and
lymphocytes are of similar cellular origin, interest has
been stimulated in the effect of vitamin E supplementation
on the immune system (Sheffy and Shultz, 1979). Megadoses
of vitamin E have been shown.to enhance immune response by
increasing phagocytic activity as well as T—cell lymphocyte
mitogenesis (Linn et al., 1981; Watson and Petro, 1982;

Nockels,1979).

13

Vitamin E and Exercise

Most of the effects of vitamin E on cellular mechanisms
can be directly attributed to its antioxidant properties.
When oxygen metabolism is increased, the potential need for
additional supplementation is apparent. It was this logic
which led to the widespread use of megadoses of vitamin E to
enhance performance of human athletes (Cooper, 1972;
Shepard, 1980). However, Sharman et a1. (1971, 1975) in two
separate studies with untrained and highly trained swimmers
could show no benefit from vitamin E supplementation when
athletic performance was evaluated. Lawrence and Slade
(1979) did see increased packed cell. volumes (PCV) in
exercising horses when supplemented with vitamin E. A
decrease in red celJ. lysis due to supplementation was
considered a possible explanation for the increase in PCV.

Vitamin E supplementation of exercising horses is
thought to be of benefit in preventing muscle disorders
which result from strenuous exercise (McLean ,1973; Geiser,
1975; Hill ,1962). Improvement in performance has not been
a consideration except in terms of decreased incidence of
muscle damage and the resulting decrease in periods of lay-

off for recovery from these disorders.

14

Myopathies

Disorders which result in muscle cell necrosis are
numerous and occur in many species, including man. Most
cases in humans are idiopathic and are accompanied by
varying degrees of myoglobinuria and elevated serum enzymes
(Afifi et al., 1968; Savage et al., 1971; Schutta et al.,
1969; Scarpelli et al., 1963). Occurrence of signs is
normally associated with some insult, physical exertion,
drugs, trauma or mild infections. Kontos et al. (1963)
described one such exertional related case study.
Myoglobinuria accompanied by muscle soreness and atrophy
were reported. Administration of glucose or glycogen
produced myoglobinuria in this subject, and it was suggested
that an uncoupling of oxidative phosphorylation in skeletal

muscle during activity was the cause.

Exertional Myopathy

In most domestic species, myopathies have been shown to
be of nutritional origin rather than activity related.
White muscle disease in sheep and cattle has been shown to
respond to Se and vitamin E suppIementation. However, Young
and Keeler (1962) demonstrated that muscular activity
exacerbates the effect of Se deficiency.

Exertional myopathies in wild animals resulting from the
stress of chase and capture occur in various species of

ungulates. A profound acidemia has been described and

favorable results have been obtained with bicarbonate

15

infusion immediately post-capture (Harthoorn and Young,
1974; Harthoorn, 1976). Serum creatine phosphokinase (CPK)
and SGOT activities as well as plasma lactate concentrations
were elevated (Hofmeyr, 1973).

Harthoorn (1976) expressed concern that lactating animals
exhibited the lowest plasma Se levels of the age groups
studied and were most frequently lost as a result of the
stress of chase and capture. However, injecting the animals
with a commercially available Se and vitamin E preparation
(Bo-Se; 2.19 mg sodium selenite, 68 IU vitamin E) did not
significantly change blood chemistry at capture. Similarly,
Robbins and Parish (personal communication) could not alter
the effects of capture by supplementing the diet of mountain
goats with both Se and vitamin E. Harthoorn (1976) concluded
that conditioning animals to the capture procedure could
reduce the stress on the animal and, subsequently, the

severity of capture myopathy.

Muscle Disorders in Horses

Hansen (1970) studied 35 cases of myopathy in horses and
classified them into 9 categories. Dark colored urine and
elevated activity of SGOT and serum glutamic pyruvate
transaminase (SGPT)were commonly seen in many of these
syndromes. The relative increase in serum enzymes and the
rate of decline during treatment were shown to be useful
indicators of prognosis.

Muscle degeneration (Gabbedy and Richards, 1970; Jones

16

and Reed, 1948; Schougaard et al., 1972; Wilson et al.,
1976) or yellow fat disease (Dodd et al., 1960; Platt and
Whitwell, 1971) in foals have, at least in part, been
attributable to diets low in Se and vitamin E. Lesions of
skeletal and cardiac muscle, typical of white muscle disease
in lambs were evident at necrOpsy. In some cases,a
generalized fat necrosis and fat discoloration were also
present. Owen et al. (1977) reported similar histological
muscle findings in adult horses. Low dietary vitamin E
intakes were associated with ataxia, muscular incoordination
and skeletal muscle lesions in adult and juvenile Mongolian
horses (Liu et all, 1983L. chever, skeletal muscle lesions
are not pathognomonic of Se and vitamin E deficiency, and
direct evidence supporting this thesis has been limited.

Bowen (1942) and Pope and Heslop (1960) reported
myoglobinuria in adult horses. Muscles were flaccid. Horses
were extremely weak and resisted movement. In both cases,
animals were in outside drylots and fed poor quality diets.
Se and vitamin E levels of plasma or feeds were not
determined.

Azoturia and tying—up are the most common muscular
disorders in horses. Azoturia occurs during the early
stages of exercise and is characterized by extreme muscle
tenseness and myoglobinuria. Muscles which are most
drastically affected are those of the loin and croup.
Horses are normally in good physical condition and on high

concentrate rations. Azoturia most frequently occurs on the

17

first day of exercise following a 1 to 2 day period of rest.
Tying-up has been associated with excessively nervous
animals and is similar to muscle cramps experienced by human
athletes after a post—exercise rest period when movement is
resumed (Farrow et al., 1976).

Cardinet et a1. (1963), in an effort to associate high
carbohydrate consumption with the occurence of azoturia and
tying-up, evaluated SGOT activity in exercise and at rest in
horses fed high grain rations. Activities were elevated in
exercise. When exercise was restricted and feed intake was
maintained, SGOT activities also rose.

In a later study, Cardinet et al. (1967) compared SGOT
and CPK activities in exercising horses and horses with
azoturia. SGOT activities rose appreciably in both exercise
and azoturia” However, CPK activities were 10-100 times
greater in azoturia than in exercise and was preferable to
SGOT in diagnostic use. The disappearance rates of both
enzymes were useful in predicting rate of animal recovery.

Lindholm et alt (1974) evaluated serum enzyme and
‘muscular changes in 59 Standardbred trotters showing
clinical signs of tying-up. SGOT and CPK levels were
elevated in all horses. Muscle biopsies showed myofibrillar
degeneration and necrosis with invasion of inflammatory
cells. Fast twitch fibers were most frequently affected.
Muscle samples were low in glycogen, ATP and CPK and had

high concentrations of glucose and lactate. It was

18

suggested that muscular alterations may be caused by a
derangement of carbohydrate metabolism resulting from a
local hypoxia.

Historically, treatment has consisted of restricting
concentrate intake, administration of muscle relaxants and
rest. Prophylactic and therapeutic administration of vitamin
iE and Se has met with widespread acceptance by equine
practicioners and appear to produce favorable results

(Geiser,1975; Hill, 1962; McLean, 1973; Stewart, 1960).

MATERIALS AND METHODS

Selenium in Plasma and Milk

Four Arabian and two Quarter Horse mares weighing
approximately 500 kg and their foals were used to determine
plasma and milk Se levels for horses at the MSU Horse
Teaching and Research Center. Mares were fed a corn-oats
concentrate and alfalfa-bromegrass hay which.was grown on
the same farm. No supplemental Se was provided. First
cutting hay was fed during gestation, and second cutting hay
was fed post-parturition. During gestation a 1:1 mix of
cats and corn was fed at approximately 25% of total dry
matter intake. Concentrate intake was doubled after
foaling. Mares were allowed access to grass pastures during
the daylight hours, both pre- and post-partum.

Blood samples were taken at approximately 7 and 14 days
prior to foaling, at foaling and 1, 4, 7, 14 and 21 days
after foaling. Milk samples were taken by allowing the foal
to suckle one teat while a 50 ml sample was collected from
the other. Colostrum and milk samples were taken on blood
sampling days post-partum. Blood samples were centrifuged
at 4°C and 2000 x g for 15 minutes to separate plasma. The

plasma, colostrum andlnilk samples were stored in plastic

19

2O

containers and frozen until analyzed. Feedstuffs were
analyzed for Se content as were additional potential Se
sources such as soil and cinders which arearmajor portion

of the roadways at the MSU horse unit.

21

Exercise Trial
Animals

Eight Arabian mares ranging in age from 5 to 14;years
were used inzi2x2 double split-plot design with repeated
measures. Two levels of Se and of vitamin E supplementation
were provided at 3 levels of conditioning, with repeated

samples taken during each collection period, illustrated as

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

follows:
DOUBLE SPLIT—PLOT DESIGN
Vitamin E (IU/day)
O 750 O 750 O 750
Se 0 ] l
(mg/day)
I 1
1 2 _ 3

Conditioning Level

The three conditioning levels were 1) non-conditioned
(prior to sampling, horses were acclimated to the treadmill
then housed in 3.0m x 3.6m box stalls and allowed one hour
of free exercise daily); 2) conditioned (horses were
conditioned 6 days per week for 45 days); 3) conditioned
with rest (horses were conditioned for 3 days beyond
conditioning level 2 and allowed 2 days of stall rest

immediately prior to sampling).

22

Diets

Two horses were assigned to each of 4 experimental
diets with the concentrate portion of the ration consisting
of crimped oats with supplemental energy provided in the
form of cracked corn to some horses. Second-cutting
alfalfa-bromegrass hay was also fed. Vitamin E and Se
concentrations for each of the major components of the
ration are given in Table 1. All diets were fed for 2 months
prior to blood samples being taken at conditioning level 1.

Mares were weighed at the beginning of the conditioning
period and at 2-week intervals during conditioning. A final
weight was taken at the completion of the trial. All horses
were fed over the entire trial to achieve a desired weight
change or to maintain weight, depending upon the animal's
physical appearance at the initiation of the feeding period.
Mares were in good physical condition at the beginning of
the feeding trial. Howevewg limiting exercise caused
increased fattening in some mares; after exercise began,
other mares lost weight rapidly. An effort was made to
minimize fat differences. Therefore, animal weights and
feed intakes differed in some cases during the conditioning
stage of the trial (Table 2).

Supplemental vitamin E and Se were provided in pelleted
form (Table 3) and fed twice daily with the concentrate
portion of the ration. Mares were bedded on wood Shavings
and supplied fresh water and trace mineralized salt ad

libitum. Mean daily Se and vitamin E intakes, respectively,

23

Table 1. Vitamin Ilznni selenium concentrations in air dry
matter of feeds.

 

Vitamin E

 

Feed Se(ppm)a (mg alpha-tocopherol/kg)b
Crimped oats .022 834
Cracked corn .033 10v4
Alfalfa-bromegrass .408 6043

hay

 

a Analyzed values.
b Hoffman.-IaRoche Inc. ,NutleyjNJ (personal communication).

Table 2. Horse weights and air dry feed intake.

 

Weight (kg) Ave. daily Intake
Initial Final intake(kg) (% BW/day)

Animal
number Trt.

 

DF Control 465 477 £112 1.72
51 diet 365 387 7.49 1-99
32 +Se 414 416 7.17 1.73
37 387 398 7.59 1-93
21 +Vit E 448 448 6575 1.51
25 488 477 6.75 1.40
18 +Se +Vit E 475 482 6.75 1.41
27 450 446 6.75 1.51

 

24

Table 3. Supplement pellet formulation (%).

 

Supplementpellet

 

 

Ingredient Placebo +Se +Vit E
Ground corn 65.6 64.5 65.14
Wheat middlings 30.0 30.0 30.00
Se premixa —- 5.5 _-
Vit E premixb -- -— .66
Ca carbonate 4.2 -- 4.20

 

a 200 mg/kg. Sodium selenite in calcium carbonate
carrier.

b 500 IU vitamin E/g. Vitamin E activity in the form of
dl-alpha tocopheryl acetate.

25

for each treatment group were: control, 1.99 mg, 327 IU;
plus Se, 4.47 mg, 321 IU; plus vitamin E, 1.93 mg, 1057 IU;
plus Se plus vitamin E, 4.43 mg, 1057 IU.

Conditioning

Horses were conditioned on an equine exercise
treadmill. During each exercise bout, animals were started
slowly, and speed was then increased in two stages. Daily
distance traveled by each horse was recorded as well as
environmental temperature and total time on the treadmill
(Table 4). After each bout, horses were rinsed with warm
water and tied until cool. The 22% grade of the treadmill
was held constant during the conditioning and sampling
periods. One day of rest was provided after every 6 days of
conditioning. Time on the treadmild. was increased as
animals showed visual improvement in signs of fatigue and
time required to recover from exercise, normally after 5 to

6 days of conditioning at the preceding level.

Blood Sampling

Blood samples were taken before, during and after
exercise (Table 5). Mares were fitted with indwelling
jugular venous catheters at 8:00 arm. on sampling days.
These remained in place until the 1 hr post—exercise sample
was taken. The 24 hr post-exercise sample was taken by
venous punctureu Thirty ml of blood were obtained at each
sampling, placed in 2 heparinized 15 ml test tubes and mixed

by repeated inversion. Subsamples were taken immediately

 

26

Table 4. Conditioning routinesa.

 

 

Week 323:7323) Distance(m) (373:2) Eaiiézgé gpmp.
1 6.3 1211.4 192.9 6.8
2 6.7 1437.4 213.9 6.2
3 7.6 1312.4 199.0 8.3
4 8.7 1798.3 206.7 8.3
5 9.6 1946.9 202.8 3.3
6 10.0 ‘1980.0 198~0 1.7

 

a Daily, except Sunday.

27

Table 5. Blood sampling times.

 

Sample no.

Time of sampling

 

1 hr
24 hr

Pre-exercise

1 minute of exercise

Exercise heart rate of
200 bpm

1 hr post-exercise

24 hr post-exercise

 

28

for glutathione and malondialdehyde determinations. A 20 ul
sample of whole, non-heparinized blood was taken for lactate

analysis.

Heart Rate

Areas on either side of the sternum, approximately 7.5
cm behind the elbow; and in the center of the'back,ELO cm
behind the withers, were clipped and shaven to expose the
skin surface. The skin was cleaned with 70% ethanol and
surface electrodes were attached. Lead wires were connected
to the electrodes with alligator clips and wrapped with an
elastic bandage to maintain good contact during the exercise
bout. Horses were placed on the treadmill, and an initial
heart rate was taken with a model 30-30 Cambridge Electro-
Cardiogram recorder (Cambridge Co" Inn», Ossaning,lNYL
Exercise did not begin until a steady resting heart rate was
achieved. Heart rates were monitored during the entire
exercise period and for approximately 1 min into the

recovery phase.

Blood Analyses

Blood samples were taken to the laboratory immediately
after the 1 hr post-exercise samples wereobtained.
Heparinized whole blood samples were analyzed for packed
cell volume (PCV) and hemoglobin concentration as soon as
possible. The blood was then centrifuged at 2000 x g for 15
min at 4°C, the resultant plasma was pipetted into plastic

containers, residual air was displaced with nitrogen and the

29

plasma was stored at -20°C until analyzed. The remaining
cells were washed three times with physiological saline and
refrigerated at 4°C until analyzed for GSpr and G-6-PDH
activity.

Fluorometric Se analyses were conducted on all plasma
samples and diet components as described by Whetter and
Ullrey (1978). Plasma vitamin E levels were determined by
high pressure liquid chromatography (HPLC) using the
procedure of Bieri et al. (1979). Glutathione peroxidase
activity of erythrocytes and plasma was determined by a
modification (Lawrence et al., 1974) of the procedure of
Paglia and Valentine (1967). Plasma glutathione-S-
transferase activities were measured as described by Habig
et al. (1974).

Whole blood was precipitated in 5% metaphosphoric acid,
centrifuged at 39,100 x g for 15 min at 4°C, and the
resulting supernatant was analyzed for reduced glutathione
levels (Beutler et alt, 1963). Similarly, 1 m1 of whole
blood was precipitated in 10% trichloroacetic acid,
centrifuged at 1500 x g for 20 minutes at 4°C, and the
supernatant analyzed:finrmalonyldialdehyde concentration
(Mengel 1967; Placer 1966). Plasma creatine phosphokinase
and erythrocyte G-6-PDH activities were evaluated using
commercially prepared diagnostic kits (Sigma, l983a,b).

Lactate levels were determined in the Laboratory for

the Study of Human Performance of the Department of Health

30

and Physical Education. A Roche Lactate Analyzer 640
(Hoffmann—LaRoche Ltd., Bio-Electronics Div., Basle,
Switzerland) was used. Ihi this procedure, lactate is
oxidized to pyruvate by hexocyanoferrate in the presence of
cytochrome b2; hexocyanoferrate then liberates two electrons
to the reaction medium, and they are collected on a platinum
electrode. The resulting change in current is measured and
used to determine lactate concentration based on lactate

standards.

Statistical Analyses

Selenium concentrations in plasma and milk from mares
and foals were analyzed as a completely randomized block
design with repeated measures. ZHorses were used as blocks
and time as treatment. Statistical analyses were performed
on the negative log y scale for all values to minimize
heterogeneity of variance. Missing cells were generated by
least squares estimation and used to calculate sums of
squares. This procedure produces values which, by
definition, minimize variance and bias the test statistic
upward. Therefore, treatment sums of squares, for
marginally significant omnibus test statistics, were
adjusted to eliminate biases. Comparisons between selected
means were made using Scheffe's test (Gill, 1978).

A multivariate analysis of variance for a double split-
plot design with repeated measures was conducted for all

exercise values. Similarly,a split-plot analysis was

31

performed for samples collected over the entire feeding
period to evaluate changes due to time on experimental diets
(Statistical Packages for the Social Sciences). Scheffe's
test was applied to selected comparisons of means as

described by Gill (1978).

32

Treadmill Design and Construction

Providing a constant and repeatable work load for
exercise research is difficult, if not impossible, when
traditional training and exercising methods are used.
Sampling subjects during exercise poses additional problems,
as exercise must be interrupted to facilitate blood
sampling. The introduction of exercise treadmills in both
human and animal exercise studies has greatly increased
the accuracy and ease with which these studies may be
conducted.

The cost of commercially-constructed equine exercise
treadmills, which are variable in both speed and incline of
walking surface, has made them difficult to purchase on
limited research budgets. Less expensive mills lack much of
the versatility’ needed 'ho conduct extensive) exercise
research. Most commercially-built treadmills are not
designed to take the type of abuse they would be subjected
to in a research situation. For these reasons, an equine
exercise treadmill was designed and built for use in this
and subsequent animal studies.

To produce maximum animal stress, the belt, which
provides a non-slip moving walking surface, must travel in
excess of 200 m/min. This speed results in a fast trot for
pleasure horses. Commercial mills which are designed to
travel at this speed use a system of conveyor—type
cylindrical rollers beneath the walking surface to decrease

friction and facilitate the use of smaller, less expensive

33

motors to drive the belt.

Traveling at high speed, these rollers are subject to a
great deal of stress and, consequently, wear out quickly.
Providing an even walking surface with this type of system
is also difficult. 'Rollers must be placed as close together
as possible, and a thick, less pliable belting is used to
help lessen the vibration which occurs as the horse's hooves
are carried over these rollers. The large number of rollers
required results in considerable noise and presents some
difficulty in acclimating horses to exercise on the
treadmill.

Because of the problems resulting from the use of
rollers, the treadmill designed for this study used a flat,
stainless steel skid sheet as the surface over which the
belt was driven. This provided a level walking surface and
greatly decreased operating noise. However, eliminating the
rollers increased both friction and heat generation, and
means of overcoming these problems will be discussed later.

Detailed drawings of all elevations are given in
Figures 2 through 4. Measurements are given in the English
units currently used for structural materials. The frame-
work was constructed with 4 inch channel iron, and 1 1/2 by
1/4 inch angle iron provided the support for the walking
surface. Side panels were 3/4 inch plywood attached to 1
1/2 inch black pipe. Plywood provided a durable, rigid
sidewall, which was also helpful in decreasing operating

noise.

34

Table 6. Key for figures 2—6.

 

Figure 2. Treadmill, side elevation, with motor and walkway

*IdthOtfiIP

removed.

Adjustable front support.
Conveyor belt.

Rear pillow block bearing.
Loading ramp.

Loading gate.

Safety latch, rump rope.

Figure 3. Treadmill, front elevation.

thdeIP

Curved chest gate.

Speed adjustment, to variable speed
Walkway, handler.

Right angle gear box, ratio 1:1.
Adjustable front support.

Figure 4. Treadmill, rear perspective.

thOtflIP

Rump rope.
Conveyor belt.
Walkway, handler.
Motor.

Loading ramp.

Figure 5. Treadmill, safety latch.

A
B

Chain, to rump rope.
Mounting bracket.

sheave.

Figure 6. Treadmill, motor and drive, top view.

A

B
C
D
E
F

Motor.

Chain drive, 60 chain, 6.9:1 sprocket ratio.

Driver conveyor pulley.

Variable speed adjustment, leading to operator.

Variable speed sheaves, 10 in., 6:1
Conveyor belt.

ratio.

 

 

Figure 2. Treadmill, side elevation, with motor and walkway removed.

36

.m>fluommmumm
Hmmn .HHHEUmmMB .v ousmflm

 

 

T :mm 1

 

A

  
  

 

”Tim
“.5112 _

:Nv

 

 

.coflum>oam uconm

.Hawscmmua

.m magmas

 

 

37

Front supports were constructed of 2 inch black pipe
and were adjusted by sliding through a 2 1/2 inch sleeve, 6
inches long. A 1/2 inch steel pin was used to secure the
sliding support at any of 4 desired heights. Rear supports
were 1/2 by 2 inch flat iron curved to maintain a constant 1
1/2 inch clearance for the rear drive conveyor drum,
regardless of how the incline was changed.

The loading ramp was made from 1 1/2 inch pipe and
supported with 2 by 1/4 inch angle iron. It was covered
with 3/4 inch plywood and carpeted to provide good footing.
The ramp was wide enough to accomodate both horse and
handler and made loading animals the first time easier and
safer. A loading gate also was designed to simplify loading
young or balky horses. It was constructed from 1 1/2 inch
pipe and hinged to swing away from the mill and then back to
provide a narrow walkway to help force animals to load. The
angle at which the gate was hung could be adjusted as the
incline of the treadmill was changed.

Once horses were on the mill they were restrained in
front by a chest gate and in the rear by a cotton rump rope.
The chest gate was curved forward from top to bottom so that
the stride of the animal was not obstructed when the mill
was in operation. The rump rope was secured, after the
animal was loaded, with a safety latch (Figure 5) which
could be released quickly and easily in all situations.
This was particularly useful if a horse should fall or

resist exercise.

38

A walkway for the handler was constructed of metal
grating supported by 1 1/2 by 1/4 inch angle iron. This
also served as a protective housing for the motor and drive
train assembly which were mounted on 4 inch channel iron.

A 5 HJ%, 220 volt single phase motor was used.
Treadmill speed was adjusted with variable speed sheaves
which drove a jackshaft. The conveyor drum was driven from
this jackshaft with size 60 chain. The driver sprocket had
thirteen teeth while the driven sprocket had 90 teeth,
giving a 6.9 to 1 speed reduction (Figure 6). The speed
range of the mill was from 10 meters per minute (mpm) to in
excess of 500 mpm. Normal operating speed was from 100 to
250 mpm, producing a slow walk to a very fast trot for most

horses.

 

 

 

 

 

 

 

 

 

 

39

Side
View

 

 

 

 

 

[\1 03::
[/‘\\*\;——i

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

7\\;m_
T913 ) B
View
I , ~
Figure 5. Treadmill, safety latch.
I n j
Pm
D
I E .
__L-/ I: B»
'— __J
£;=FT==‘ ‘—t:::T'
'(———-z___.___A
Figure 6. Treadmill, motor and drive, top View.

RESULTS AND DISCUSSION

The Testing, Use and Modification of an

Equine Exercise Treadmill

Plates 1 through 4 show “Mme completed treadmill.
During the exercise trial, the treadmill was used intensely
six days per week. Minor breakdowns did occur, and were
corrected as quickly as possiblth so that no exercise days
were missed” IHowevery some major changes still need to be
made to improve its durability and ease of Operation.

Most of the early changes dealt with the motor and
drive train. To reduce cost, a 3'HJL, 110 volt electric
motor was installed when the treadmill was constructed.
Treadmills with rollers under the walking surface are
equipped with 1 1/2 to 2 HQP. motors. Replacing the rollers
with a slide sheet increased the drag;and the power needed
to drive the belt. The 5 HJ%, 220 volt motor (Plate 5),
which was installrxi later, had sufficient power to operate
the mill without difficulty.

Finding a durable conveyor belt, which provided good
footing;for the horses and also helped to reduce friction,
was difficult. Grip-TOp rubber belting (B. F. Goodrich,

Columbus, OH), fulfilled these criteria better than any

40

41

 

 

Plate 1. Treadmill, side view.

 

' - 2.. . ' .1 »‘ 1
., ‘ ~ .~ ' . . ' .
' . " .. ~ ’ ‘ “‘ ,
a \a. - . ' “use; . -222, .v

Plate 2. Treadmill, loading gate and ramp.

 

42

.Bmfl> wmmu .Haflecmmus .v mumam .Bmfl> ucoum .Haflscmmna .m mumam

a

    

o

L

a

i

,. v
._,I i.
u-

5
u...
a

 

v

10.1“

L in
c .
_. _ .
1...,
.

 

 

 

 

43

 

Plate 5. Treadmill, motor and drive.

 

 

 

Plate 6. Treadmill, belt, lacings and slide.

44

other observed commercial conveyor belting (Plate 6%.The
underside was nylon fiber, as compared to rubber backing
found on other belts.

A polypropylene sheet (Cadillac Plastics Co., Detroit,
MI) was initially used as the slide. This posed several
problems. As the nylon underside of the belt moved over the
plastic sheet, static electricity was generated. The
electricity was transferred to the horse on the treadmill,
and resulted in static shock when contact was made with the
pipe supports on the sides of the treadmill. Covering the
pipes with rubber pipe insulation solved this problem.
However, if the operator touched the horse, both would
recieve a shock. An antistatic spray was applied to the
underside of the belt and helped to reduce the generation of
static electricity.

Belt lacings (Lovejoy Co., Downers Grove, IL) were
required to allow for easy conveyor belt installation.
Lacings were bolted to both end of the belt, and were
connected by sliding a flexible metal cable through the
lacings (Plate 6). These lacings cut into the polypropylene
sheet and wore it out quickly. Polypropylene is a soft
plastic with a low melting point (110°(IL When.the mill
was in operation, the heat generated from friction between
the belt and slide warmed the plastic. This caused it to
warp and become softer, which made it more susceptible to
damage from the belt lacings.

The plastic slide was replaced with a stainless-steel

45

sheet (Plater6), which was more durable. Stainless-steel
increased friction between the belt and the slide, and also
warped when heated. One inch holes were drilled in the
plywood, under the slide, to help dissipate some Of the
heat. This did reduce heat build up. However, when the
treadmill was used for an extended period of time, the steel
could not cool sufficiently, and would warp and become
noisy. Liquid soap was applied to lubricate the underside
of the belt. This was effective; however, it was messy and
required an additional person for its application.

Tetrafluoroethylene (Teflon; Dupont, Midland, Michigan)
is material which fulfills many of the necessary
requirements for use as a slide. It is a smooth, heat
resistant material (operating temperature, 230° C) that is
very durable. Teflon vdJLL be used in future tests to
replace the stainless-steel slide.

When the incline of the treadmill was maximized, and
friction reduced as much as possible, horses could move the
belt without the motor running. Some horses, when
exercising, were difficult to stop after the motor was
turned Off. A temporary brake was applied to the driver
variable speed pulley. A brake which can be Operated by the
handler, and applied to the driven conveyor pulley will be

installed in the future.

46

Conclusion

Constructing the treadmill provided a less expensive,
reproducible method to stress horses through exercise. Cost
of construction was about one-half that of commercial
treadmills made similarly. Speeds which were used to
exercise horses during this trial were comparable to those
attained with other treadmills. However, the maximum speed
possible greatly exceeds that of treadmills which can be
purchased currently. Having the ability to change the slope
of the incline, and exercise horses at various speeds
(including the canter), allows for more flexibility when

designing future exercise research.

47

Plasma and Milk Selenium

Levels from Mares and Foals

Selenium concentrations Of feeds are shown in Table 7.
Using these feed values, and assuming a daily intake Of 8575
kg Of dry matter (1.75% Of body weight), an estimate of
dietary Se intake was made. Mares were fed approximately
4.7 and 1.9 mg Se daily from natural feed sources during
gestation and lactation, respectively.

The Se concentrations Of cinders and soil are also
given in Table 7. Cinders from the Michigan State
University Power Plant were used to establish and maintain
driveways at the Horse Teaching and Research Center for many
years. Many of the pastures contain one of these driveways,
or large areas where cinders were used as fill to eliminate
moisture problems. As a result, pastures are bathed in
cinder dust during the summer months. This may explain the
unusually high Se concentrations found in hays grown on
these fields, and the resulting high plasma Se values
observed in horses grazing them (Brady, 1978). Furr et al.
(1978) and Mandisodza et al. (1979) found high Se levels in
sweet clover grown on fly ash, which is a residue from
burning soft coal in electrical power-generating plants.
When this sweet clover was fed to livestock, Se
concentrations were increased in all tissues studied.

Changes in Se concentration Of plasma from mares and

foals and mare milk are compared graphically in Figure 7.

48

Table 7. Se concentration of feeds, bedding, soils, and
driveway surfaces.

 

 

Item Se (ppm dry basis)
Crimped oats .022
Cracked corn .033
Alfalfa-bromegrass hay

1st cutting .717

2nd cutting .408
Wood shavings (bedding) .048
Soil (pasture) .242

Driveway surfaces
Cinders 2.418

Gravel .040

 

Lm>o mammPa Pace can mess was xpme m.mtme cw coppmtucmocou

mxma

va 5 v
p _

mm cw mmmcmgu .u mesmwu

 

 

4. _

 

49

l'

3
”"“‘|’

I
/

 

mEmde Hood
3H“:

mEmem mumzl_l.l.1.l

 

 

qqflufluqqudu

140.

Imo.

-8.

ldo.

lmo.

loo.

lHo.

Imo.

lmo.

ch.

lfla.

 

lNa.

(Iw/fin)as

5O

Foal plasma Se levels remained constant throughout the
collection period. Mare plasma Se levels tended to rise
prepartum and fall Off after parturition; however, these
means were run: significantly different. lhean Se
concentrations of milk and plasma from individual mares and
their respective foals are given in Table 8. In general,
mares had higher plasma Se levels than their respective
foals.

Colostrum Se concentrations were higher than those
found in milk (P<.001). McConnell. and Roth (1964)
demonstrated that nearly all of the Se found in milk is
protein bound. The decline seen in Se levels, between
colostrum and milk, is coincident with the rapid decline in
immunoproteins during the first 24 hr Of lactation. The
average Se concentration Of milk samples (.029 ug/ml)
compares favorably with Se concentrations found in milk
from Hereford cows fed diets with similar Se levels (Perry

et al., 1977).

51

Table 8. Mean Se concentration of mare's milk and plasma
and foal plasma.

 

Se concentration (ug/ml)

 

 

Mare Plasmaa Colostrumb MilkC Foal plasmad
DC .098:.023 .053 .026:.OO3 .051:.03
EB .097i.o1 .075 .028_+_.003 .055:.01
FR .093:.02 .119 .018i.006 .080:.O3
rs . 1 2515.02 .084 .0393002 ~093i-02
R .097:.o1 .070 .037:.011 .071:.o1
z .129:.01 .131 .O30:.OO6 .088:.O2

 

a Values represent the mean Of 8 samples per mare, Obtain-
ed 7 and 14 days prepartum, at parturition, and 1, 4, 7,
14 and 21 days after foaling.

b Values represent 1 colostrum sample per mare, Obtained
at the time of foaling.

0 Values represent the mean of 5 samples per mare, Obtain—
ed at 1, 4, 7, 14 and 21 days after foaling.

d Values represent the mean of 6 samples per foal, Obtain-
ed at the time of foaling and 1, 4, 7, 14 and 21 days
after foaling.

e ‘:SEM

52

Conclusions

Plasma Se concentrations of mares, at the MSU Horse
Teaching and Research Center, were found to be within normal
ranges in an area known to have low soil Se levels.
Apparently adequate Se intakes could be provided at this
facility by feeding hays which were grown on soils
contaminated with Se-rich cinder dust. Plasma Se
concentrations Of foals in this study were low—normal and
may ‘warrant further investigation ‘ha ensure that
prophylactic administration Of Se, to nursing foals, is not

needed.

53

Changes in Blood Parameters During

Conditioning and Exercise in Horses

The exercise time required to reach a maximum heart rate
of 200 bpm increased, while blood lactate concentrations
decreased, with conditioning (Table 9). These changes, due
to conditioning, have been used as indicators Of improved
physical performance in horses (Milne et al., 1976; Milne et
al., 1977; Rodiek et al., 1983; Sigler et al., 1979). The
two days of rest prior to exercise, provided in conditioning
level 3, resulted in a decrease in the time required to
reach maximum heart rate, when compared to conditioning
level 2. Whether this is a result of an increase in
excitability Of the horses or an actual decrease in animal
fitness is not kmcwn. However, Foreman et a1. (1983)
reported that a one month rest period did not significantly
change cardiopulmonary fitness in well-trained thoroughbred
horses.

Changes in blood lactate concentrations as a result of
conditioning and exercise are given in Table 10.
Significantly higher lactate concentrations were seen for
all conditioning periods at one minute of exercise and when
a heart rate Of 200 bpm was reached as compared to initial
values. Blood lactate concentrations remained elevated 1 hr
after exercise for non-conditioned horses, again indicating
improved animal fitness for conditioning levels 2 and 3.

Rodiek et al. (1983) demonstrated the increased

54

Table 9. The effects of conditioning on heart rate and

blood lactate levels.

 

Conditioning level

 

 

Item 1 2 3 'Y
Speed (m/min) 220 210 219 216
Time (min:sec)a 1:50 4:15 2:30 2:52
Lactate (mmoles/l)b 9.7 5.3 5.5 6.9

 

a Exercise time required to reach a heart rate Of 200 bpm.

b Lactate concentration when heart rate was 200 bpm.

Table 10.

55

Changes in blood lactate (mmoles/l) as a result
of conditioning and exercise.

 

Conditioning level

 

 

Time of sampling 1 2 3 P value
I 1.68 1.0a 1.3a NS9
1 min 5.2b0 3.1bd 3.5bd .05
r 9.8bC 5.4bd 5.5bd .001
1 hr 3.830 1.5ad 1.5ad .01
24 hr 1.3a 1.0a 1.5a NS
P value .001 .001 .001

 

a,b Values in the same column with different superscripts
differ significantly.
c,d Values in the same row with different superscripts
differ significantly.

e Values are not significantly different (P>.10).

56

physical stress associated with increasing Huaincline Of
the treadmill. Increases in both heart rate and blood
lactate levels were seen when the grade was changed from 3%
to 9%. Webb et al. (1979) indicated the same type of
response when the incline (9% grade) was held constant and
treadmill speed was increased from 123 m/min to 172 m/min.
In both studies, maximum heart rate achieved, after 30
minutes of exercise, was approximately 150 bpm.

The treadmill, used in the present study, was Operated
at a 22% grade and an average speed Of 216 m/min.
Maximizing physical stress, as measured by blood lactate and
heart rate, was accomplished in a minimum amount of
exercise time. These values exceed those Obtained on
conventional treadmills and are similar to values seen in
horses under simulated racing conditions (Lindholm and
Saltin, 1974).

Blood hemoglobin concentration and hematocrit increased
with exercise (Table 11), consistent with the results Of
Torten and Schalm (1964) who reported splenic release of
erythrocytes in horses during exercise. This phenomenon is
well documented (Boucher et al., 1981; Kitchen et al., 1965;
Parks and Monohar, 1983; Rose et al,, 1983) andaallows for
rapid changes in the oxygen carrying capacity of the blood
of horses under stress. It is interesting to note that
initial samples had significantly higher hematocrit levels
when compared with the 1 and 24 hr post-exercise samples.

This may be attributable to splenic release Of red cells as

57

 

 

 

Table 11. Changes in blood hemoglobin and hematocrit and
plasma hemoglobin as a result Of exercise.
Time Of Hematocrit Hemoglobin (g/dl)

sampling (%) Blood Plasma
I 42.3a 15.2ab .156de

1 min 47.8b 18.8a .192d

r 51.9b 20.4a .226d

1 hr 34.7c 13.5b .1198

24 hr 38.9C 15.6ab .146de

 

a,b,c Values in the same column with different
superscripts differ significantly (P<.001).

d,e Values in the same column with different
superscripts differ significantly (P<.O1).

58

a result of nervous anticipation of exercise.
Centrifugation of blood samples, which were mixed by
inversionznuiused to determine hematocrit and hemoglobin
values, produced plasma with noticeably more hemolysis than
blood centrifuged immediately after collection. Hemoglobin
values for these plasma samples are shown in Table 11.
Hemolysis was significantly higher in samples obtained
during exercise, when compared to the 1 hr post-exercise
samples. The extensive reticulO-endothelial system Of the
spleen functions to remove damaged erythrocytes from blood
(Wintrobe, 1980). Strenuous physical activity increases red
blood cell hemolysis (Buskirk, 1980). The increased
hemolysis seen in exercise samples may be a result of two
factors, the release of these damaged cells back into the

circulation and exercise-induced hemolysis.
Summary

Exercise bouts conducted in this study produced
hematological and blood lactate changes similar to those
reported elsewhere in iflua literature. The ability to
increase per cent grade and treadmill speed provided an
effective method of producing maximum stress in_a minimum
amount Of time. Hematocrit values were shown to increase
with exercise and level Of excitement. This would indicate
that care should be taken to minimize animal excitement when
blood samples are Obtained for baseline hematocrit or

hemoglobin determinations in the horse.

59

Selenium and Vitamin E Supplementation

During Exercise and Conditioning in Horses

Changes in plasma Se levels over the 18-week feeding
period are shown graphically in Figure 8. Plasma Se
concentration increased with Se supplementation above pre-
feeding levels(P<JXH). The basal ration would appear to
provide dietary Se intakes high enough to maintain plasma Se
concentration. However, feeding this ration produced a
significant decline in plasma Se over the length Of the
trial (P<.001). Before mares were fed the experimental
diets, they were maintained in outside lots and fed only
first cutting alfalfa-grass hay. This hay had an analyzed
Se content of .711 mg/kg dry matter (DM). Therefore,
feeding the basal ration reduced daily dietary Se intake 3
fold and resulted in the Observed decrease in plasma Se
levels for non-supplemented mares.

Plasma Se concentrations were elevated (P<.05) during
exercise, when compared to the 1 hr post-exercise sample
(Figure 9). These differences are thought to have resulted
from changes in plasma volume rather than mobilization of Se
from some body store. During severe exercise in humans,
hemoconcentration and reduction in plasma volume have been
described (Kaltreider and Meneely, 1940; Johnson and
Buskirk, 1980). This decrease is thought to result, at least
in part,from increased circulatory hydrostatic pressure and

loss Of water from the vascular compartment. Astrand and

60

.mHo>oH mm mammam co ucmEummnu mm HO muommum one .m mucosa
5x38 onwademm mo mafia

ha Ha

 

-1-.°.:

v o
_ T _
_ fl.

 

Lr 2.

mm + Hmmmmlllll

Hmmmm

 

 

61

H: vN

.Omfiouoxm magnum mm mammam cw mmmcmzo

GmHUHOXQ NO mafia

a:

CHE a

.m magmas

 

 

_

—_

l mma.

1 and.

l ova.

l med.

11hva.

.lmva.

 

62

Saltin (1964) found an increase in plasma volume, above pre—
exercise levels, 1 hr after a strenuous exercise bout. This
initial hemoconcentration and subsequent hemodilution, may
explain observed changes in plasma Se concentration during,
and 1 hr after strenuous exercise.

Changes 1J1 plasma alpha-tocopherol concentrations
resulting from vitamin E supplementation are shown in Table
12. Concentrations tended to be higher with supplementa-
tion; however, these differences were not significant.
Exercise and conditioning did not affect plasma alpha-
tocopherol levels.

Mean plasma GSpr activities were 5.3 and 7.7 EU/g of
plasma protein.(PK501), for non-supplemented and supplemen—
ted mares, respectively. Erythrocyte GSpr activities
increased as a result Of conditioning (Table 13). Selenium
supplementation augmented the effect Of conditioning and
resulted in a significant treatment/conditioning interaction
(P<.01).

Brady et al. (1978) and Shellow et al. (1983) saw no
effect of Se supplementation on GSpr activities in
exercised and non-exercised horses, respectively. However,
plasma Se levels were within normal ranges in both studies
prior to supplementation, and in neither study were horses
conditioned. Exercise Inna been shown ‘hl increase
peroxidative damage, as determined by changes in lipid
peroxide levels, in horses and rats (Brady et al., 1978;

Brady et al., 1979). This increase in peroxidative damage

63

Table 12. The effect of vitamin E supplementation on
plasma alpha-tocopherol concentrations (ug/ml).

 

Weeks on feed

 

 

Diet 0 4 11 17 18 P value
Basal 3.6 3.1 3.5 2.5 3.3 NS3
Basal+vit E 3.4b 4.5bc 4.6bc 4.9bC 5.80 .01

 

a Values are not significantly different (P>.10).
b,c Values with different superscripts in the same row
differ significantly.

64

Table 13 The effects Of conditioning and Se treatment on
erythrocyte GSpr activity (EU/g Hb)a.

 

Conditioning level

 

 

Selenium treatment 1 2 3
NO added Se 42bd 108Cd 104cd
2.5 mg added Se/day 54bd 14908 15408

 

1 EU (enzyme unit): 1 umole NADPH oxidized x min‘1.
Values in the same row with different superscripts
differ significantly (P<.001).

,e Values in the same column with different superscripts
differ significantly (P<.05).

0- C7113
0

65

is thought to be a result Of an increase in oxygen delivery
and metabolism which occurs during exercise. Therefore,
exercise may increase the need for mechanisms to protect
against cellular oxidative damage, and in so doing,
stimulates GSpr synthesis. Godwin (1972) did demonstrate a
moderating effect, due in) conditioning, (n1 plasma CPK
activities in lambs fed a Se—deficient diet.

The lifespan of the mature red blood cell in horses is
151 days (Carter et al. 1974), and, being a non-nucleated
cell, it lacks the genetic machinery to alter its protein
make-up. The magnitude of change in erythrocyte GSpr
activity, seen in this study, and the length of time
required for this change to occur, are difficult to explain
within these restrictions. However, extensive destruction
of red cells occurs with the onset of intensive conditioning
(Davidson, 1964; Johnson and Buskirk, 1980). As
conditioning continues, erythropoiesis increases to restore
red cell count to normal levels and then decreases to a new
level of production commensurate with the needs of
conditioning (Johnson and Buskirk, 1980). In effect, the
onset Of intensive physical conditioning reduces the
lifespan Of the red cell and may account for the Observed
rapid changes in erythrocyte GSpr activity.

Blood malondialdehyde (MDA) levels at 24 hr after
exercise were significantly lower than levels during
exercise, for non-conditioned mares (Table 14).

Conditioning lowered MDA levels, efljuflnated the effect Of

66

Table 14. The effects Of conditioning and time Of sampling
on blood malondialdehyde concentration (U/ml)a.

 

Conditioning level

 

 

 

Sampling
time 1 2 3 value
I 13.7bd 3.6C 3.2C .001
1 min 8.7bd 3.6C 3.2C .001
r 11.5bd 3.9C 3.8C .001
1 hr 9.2bd 3.0C 3.10 .001
24 hr 5.6be 2.2C 2.9C .05
P value .01 NSf NS
a U (unit) is defined as 10 x absorbance at 535 nm.
b,c Values in the same row with different superscripts
differ significantly.
d,e Values in the same column with different superscripts
differ significantly.
f Values are not significantly different (P>.10).

67

exercise, and resulted in a significant conditioning/time of
sampling treatment interaction(PK.O5%. Blood MDA levels
are a measure of cellular lipid peroxidative damage and, as
has been discussed earlier, increase during exercise in non-
conditioned horses (Brady et al, 1978). Daily conditioning
of rats by swimming for several weeks has been shown to
decrease lipid peroxides in both blood and liver (Tani and
Aoki, 1981). This is in agreement with the present study
and may indicate the importance Of conditioning in
stimulating cellular protective mechanisms.

Changes in total plasma protein resulting from exercise
and conditioning are given in Table 15. Increased plasma
protein concentrations during exercise have been described
elsewhere (Poso et al., 1983; Rose et al., 1983). Strenuous
exercise causes a transient hemoconcentration and a
subsequent hemodilution after exercise has stopped. These
changes in plasma volume are thought to be responsible for
the Observed fluctuations in plasma protein concentrations.
Plasma glutathione-S-transferase activity tended to rise
after exercise (Figure 10). However, there were no
significant differences.

Plasma CPK activity was significantly lower at all
sample times in non-conditioned horses (Table 16). Cardinet
et al. (1967) and Poso et al. (1983) saw elevated plasma CPK
activities with exercise in strenuously exercised horses.
Increases in CPK activities are thought to indicate muscle

damage as a result of extreme exertion. The length of time

68

Table 15. The effects of level of conditioning and time of
sampling on plasma protein (mg protein/ml plasma).

 

Conditioning level

 

 

Time of
sampling 1 2 3 P value
I 640 660d 64 NS9
1 min 65ac 69bc 69b .10
r 65ac 69bc 69b .10
1 hr 57ad 63bd 66b .01
24 hr 68ac 64bod 64b .10
P value .05 .05 NS

 

a,b Values with different superscripts in the same row
differ significantly.

c,d Values with different superscripts in the same column
differ significantly.

e Values are not significantly different (P>.10).

69

1
Wm

oe_e~

CHE x crummsflcoo mzo meaoe:

.2

_

H

as .2m x coca u a aaaas a. omaouoxo
uazmmu m we >uw>fiuom mmmuwmmamuu1m1mc0wnumusam mammam cw momcmnu

unwoumxo no cede

m
_

.oH magmas

 

A
_
_

1 1
I 1
U1 1v

1 1
r* 1
I" \O
"raogd 6oat/orttrmlt-s-Hso

l 1
1 1
c: «a

1

1
u-O
,4

 

70

Table 16. The effect Of conditioning and time of sampling

on plasma creatine phosphokinase activity

(IU/1OO g protein).

 

Conditioning level

 

 

Time Of
sampling 1 2 3 value
I 2.3a 4.8b 4.6bd .01
1 min 2.48 4.7b 5.5bd .001
F 2.7a 5.2b 4.6bd .01
1 hr 3.0a 6.3b 5.7bd .001
24 hr 2.5a 4.8b 9.0ce .001
P value NSf NS .001

 

a,b,c Values

differ
d,e Values
differ
f Values

in the same row with different superscripts
significantly.
in the same column with different superscripts
significantly.
are not significantly different (P>.10).

71

mares were on the treadmill at conditioning level 1 was
shorter than for conditioning levels 2 and 3 (refer to Table
9), and may not have been Of sufficient length to produce a
level of muscular activity resulting in the leakage Of
muscle enzymes into the circulation.

The highest CPK activities were seen in conditioning
level 3, 24 hr after exercise. This mean was calculated on
seven Observations. Mare number 37 had a 24 hr CPK activity
Of 191.5 IU/1OO g plasma protein. This value was calculated
as an outlyer and removed for statistical analysis. Mare 37
showed no visible signs of lameness or plysical fatigue
after this exercise bout. She was, however, an excitable
animal and always appeared to be under more emotional stress
than other mares. The elevated CPK activities after
exercise in conditioning level 3 may indicate the importance
Of gradually increasing work intensity after days of rest to
minimize muscle damage.

Reduced glutathione (GSH) levels in whole blood were
elevated (P<KXM) during exercise (Figure 11). The highest
concentration of GSH in blood is within the red cell. As
discussed earlier, red cell numbers are increased with
exercise in the horse, asziresuld;of splenic contraction.
Changes in GSH concentrations may be the result Of this rise
and subsequent decline in erythrocyte numbers during
exercise. The effects of conditioning on blood GSH
concentrations are given in Table 17. Concentrations were

significantly higher at all sample times for conditioning

72

u; vm

.mmaoumxm nua3 mHO>OH OCOwcumusam cocoons cooan :H mmmcmnu

on a

omfiouoxo no mass

.m
_

.Ha oaamae

 

 

——

11 on

11om

1110?

[low

110m

1.... on

(tr/saromn) Hss

110m

Liced

119:

 

73

 

 

 

Table 17. The effects of conditioning and time of sampling
on blood reduced glutathione levels (umoles/dl).
Conditioning level
Time Of
sampling 1 2 3 P value
I 76ac 62bC 56be .01
1 min 93ad 77bd 80bd .01
r 100ad 83bd 82bd .01
1 hr 65ac 52be 49b0 .01
24 hr 75ac 52bC 58bc .001
P value .001 .001 .001

 

a,b Values
differ
c,d Values
differ

in the same row with different superscripts

significantly.

in the same column with different superscripts

significantly.

74

level 1. A significant interaction between vitamin E
supplementation and level of conditioning occurred for
erythrocyte glucose-6-phosphate dehydrogenase (G-6-PDH)
activity (Table 18).

Erythrocyte G-6—PDH activities and.CKHi levels were
affected similarly by conditioning. This result is
reasonable, as the reducing equivalents required to maintain
GSH levels in the erythrocyte are supplied exclusively from
the hexose monOphosphate pathway. G-6—PDH is the regulatory
enzyme in this pathway, and is responsive to cellular NADPH
levels. Glutathione reductase, in the presence of
riboflavin, uses NADPH to reduce oxidized glutathione to
GSH. Heavy exercise in the rat has been shown.to increase
liver G-6—PDH activity (Mayanskaya,1982). Maximum
activities were reached at 6 and 16 hr after exercise.
However, the effects of conditioning were not studied.

The increase in erythrocyte GSpr activity seen in this
study would indicate an increased demand for reducing
equivalents provided.by the hexose monophosphate pathway.
However, the reverse may be true. A reduction in G-6-PDH
activity and the resultant assumed rise in oxidized
glutathione levels, may stimulate synthesis Of GSpr. This
may, in part, account for the magnitude of change seen in
red cell GSpr activity as a result of conditioning.
Additional work is needed to determine the effects Of
exercise and conditioning on these enzymes and further

elucidate the reasons for these changes in enzyme activity.

75

 

 

 

Table 18. The effects Of conditioning and vitamin E
supplementation on erythrocyte glucose-6-phosphate
dehydrogenase activity (IU/g hemoglobin).

Conditioning level
Dietary
treatment 1 2 3 P value
Basal (B) 9.2a 7.3b 7.1bC .001
B + vitamin E 9.1a 7.2b 7.9bd .001
P value NS6 NS .01

 

a,b Values

differ
c,d Values
differ
e Values

in the same row with different superscripts
significantly.

in the same column with different superscripts
significantly.

are not significantly different (P>.10).

76

Summary

Strenuous conditioning programs appear to increase
GSpr activities in equine erythrocytes. This may indicate
an increased need for mechanisms to protect against
peroxidative cellular damage resulting from the increased
production of oxygen byproducts during exercise. Decreases
in G-6-PDH activities seen as a result of conditioning were
not expected. The reason for this decline is not
understood, and additional research is needed to define the
mechanisms responsible. The Observations 1xf horsemen
indicating an increase in the incidence Of muscular
disorders as a result of exercise, after periods of rest, is
supported by the prolonged increase in CPK activity seen in

conditioning level 3.

CONCLUSION

Supplementation Of vitamin E, above the levels found in
the basal ration, increased plasma alpha—tocopherol
concentrations, However, the addition of vitamin E did not
significantly affect any other parameter measured. When
good quality hays were provided, supplementation with
vitamin E appeared to be Of no additional benefit to
exercising horses.

Hematocrit, hemoglobin annl blood lactate levels
increased with exercise. These changes were similar to
published values for heavily exercised horses. Conditioning
mares for 45 days reduced the magnitude Of blood MDA and
lactate concentration increases resulting from exercise,
indicating an improvement in animal fitness.

Erythrocyte GSpr activity increased as a result of
conditioning and Se supplementation. Conditioning had the
most profound effect, increasing baseline activities two-
fold. Selenium supplementation did not improve animal
performance or reduce the potentially damaging effects Of
exercise, as measured by blood MDA levels and plasma CPK
activities. The effects of Se supplementation upon Se-
deficient horses during exercise were not studied. However,

the conditioning-induced increase in erythrocyte GSpr

77

78

activity may indicate that providing adequate dietary Se
intake becomes increasingly more important as the level Of
physical activity increases.

Blood GSH levels decreased with conditioning. This may
have been a result Of an increase in oxygen metabolism with
exercise, and a decrease in reducing equivalents provided
from the hexose monophosphate pathway. Conditioning
decreased erythrocyte G-6—PDH activity. The reason for this
decrease is unknown and warrants further investigation.

Plasma CPK activities, during and after exercise, were
highest in conditioned mares after 2 days Of rest. Blood
malondialdehyde levels, during exercise, were highest in
non-conditioned mares. These results indicate that care
should be taken when exercise intensity is increased or
when exercise is resumed after periods of rest. Rapid
changes in the level of physical activity may cause
increased lipid peroxidation and muscle damage and may

result in impaired animal performance.

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