HISTOCHEMICAL PROFILES OF RAT TRICEPS SURAE
AND PLANTARIS AFTER SEVEN EXERCISE 'REGIMENS

Thesis for the Degree of Ph. D.
MICHIGAN STATE UNIVERSITY
JAMES FRANCIS TAYLOR
1971

 

 

vi

”-12.5 1

 

This is to certify that the
thesis entitled

HISTOCHEHICAL PROFILES OF RAT TRICEPS SURAE
AND PLANTARIS. AFTER SEVEN EXERCISE REGIMENS

presented by
James Francis Taylor

has been accepted towards fulfillment
of the requirements for

_P_h_.£.__ degree in w

MW

Major professor

Datg November, 1971 - ‘

 

gm

ABSTRACT

HISTOCHEMICAL PROFILES OF RAT TRICEPS SURAE
AND PLANTARIS AFTER SEVEN EXERCISE REGIMENS

BY

James Francis Taylor

This study was undertaken to determine the effects
of seven different exercise regimens on the histochemical
characteristics and distribution of selected muscle fibers
of the triceps surae and plantaris muscles of albino rats.

One hundred and seventy-six 72-day—old, normal,
male, albino rats (Sprague-Dawley Strain) were randomly
assigned to one of the seven treatments. Treatments be-
gan after a 12 day adjustment period when all animals were
85 days of age. The treatment groups were sedentary con-
trol (CON); voluntary running (VOL); short-duration, high-
speed endurance running (SHT); medium-duration, moderate-
speed endurance running (MED); long-duration, low-speed
endurance running (LON); electric stimulus control (ESC);
and long-duration, low-intensity swimming (SWM). Treat-
ments were administered once a day, Monday through Friday.
All animals had access to food and water aleibitum.

Seven animals, limited to the same duration, were weighed

 

and sa

of tra

peritor
was inj
muscle

taris m
iSOpent
proxima
USing a
Procedu.

glycolyd

fiber 1::

Cinate C
(adenOsl-l
each Spe
plantari

inteIISit

 

chemiCal

were rEC

James Francis Taylor

and sacrificed after zero, four, eight, and twelve weeks
of training. The final sample consisted of 94 animals.

Animals were sacrificed under anesthesia by intra-
peritoneal injection of pentobarbital sodium. Pelikan ink
was injected into the vascular system for capillary per
muscle fiber calculations. The triceps surae and plan—
taris muscles were removed as a unit, and frozen in an
isopentane-liquid nitrogen system. Fresh-frozen, distal-
proximal serial cross sections, were cut at 10 microns
using a rotary microtome-cryostat. Four histochemical
procedures were utilized for identification of relative
fiber type intensities of glycogen (periodic acid-Schiff),
glycolytic enzyme (phosphorylase), oxidative enzyme (suc-
cinate dehydrogenase), and energy producing systems
(adenosine triphosphatase). Fifty muscle fibers, from
each specific intramuscular area of medial gastrocnemius,
plantaris, and soleus muscles, were graded according to
intensity and distribution patterns of the various histo-
chemical procedures. The percentages of intensity ratings
were recorded for individual animals.

The prominence of duration, as well as treatment,
effects suggested that the seven different chronic physi-
cal activities had specific effects upon the alteration of
fiber characteristics, but the effects were highly time
dependent.

The results indicated there were diverse regional

responses and patterns of change over time, to the same

exerci
variou
in pro

planta

Schiff
four an
Produce
intermeI
Stimulu.
nificam
tWelve y
Ir
PAS inte
and Swim
control
Creases
twelIIe w
result a:
Specific

The
gaStrOCnEI

TH

 

James Francis Taylor

exercise stimulus. Four, eight, and twelve weeks of
various activity programs produced metabolic alterations
in proportions of fiber types in the medial gastrocnemius,
plantaris, and soleus muscles.

In the soleus muscle area, similar periodic acid~
Schiff (PAS) changes occurred for the voluntary group at
four and eight weeks. The voluntary (VOL) group activity
produced a significant increase in the percentage of
intermediate ATP fibers at eight weeks, while the electric
stimulus control (ESC) treatment produced a similar sig-
nificant increase at four weeks, which was reversed at
twelve weeks (p < .20).

In the plantaris muscle area increases in similar
PAS intensities were found at four weeks for long (LON)
and swimming (SWM) groups (p < .20). The electric stimulus
control (ESC) and SWM programs produced significant in-
creases in the percentage low intensity ATP fibers at
twelve weeks, while the VOL treatment produced the same
result at four weeks. The short (SHT) group showed a
specific pattern of increasing intermediate ATP fibers
from four to twelve weeks (p < .20).

The general adaptive patterns for the medial
gastrocnemius muscle area showed that anaerobic fibers
were able to acquire aerobic fiber characteristics and
supported the hypothesis of specificity of alteration.

The greatest relative rise in ATP occurred in the medium

(MED) I
observq

and bet

 

 

James Francis Taylor

(MED) and long (LON) running groups. This change was
observed between four and twelve weeks for the MED group

and between zero and eight weeks for the LON group.

HISTOCHEMICAL PROFILES OF RAT TRICEPS SURAE

AND PLANTARIS AFTER SEVEN EXERCISE REGIMENS

BY

James Francis Taylor

A THESIS

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

DOCTOR OF PHILOSOPHY

Department of Anatomy

1971

(Chairn
Van Hug
throngh

to Dr.

 

 

ACKNOWLEDGMENTS

I wish to thank my committee, Dr. R. E. Carrow
(Chairman), Dr. R. Echt, Dr. M. H. Ratzlaff and Dr. W. D.
Van Huss for their guidance, consideration and criticism
throughout the study. Sincere appreciation is extended
to Dr. R. E. Carrow, who served as chairman and advisor,
for his painstaking guidance, interest, patience and en-
couragement to develop my tentative ideas into a practical
research project.

I am grateful to Dr. R. O. Ruhling, and A. T. Reed
for their involvement in other phases of this research
project, which yielded many hours of intellectual dis—
cussion.

It is a pleasure to thank Mrs. Barbara Wheaton and
Miss Patricia L. Lamb for contributing their technical
assistance and Laboratory experience.

Special thanks should be given to Dr. W. W. Heusner
for the care and training of the animals in the Human
Energy Research Laboratory, Department of Health, Physical

Education and Recreation.

ii

I deeply extend appreciation to my wife, Jan and
son Matthew for their unending patience and forebearance
during this most often neglectful period.

This study was supported by National Institutes of

Health Grant HD 03918.

iii

INTROE

REVIEW

MATERII:

RESULTS

 

TABLE OF CONTENTS

INTRODUCTION . . . . . . .

General Comments . . .
Statement of the Problem
Rationale . . . . .

Significance of the Problem .

REVIEW OF PERTINENT LITERATURE .

Red, Intermediate, and White

Muscle Fibers . . .

Skeletal Muscle Fiber Response to

Skeletal

Experimentally Induced Conditions.

Skeletal Muscle Fiber Response to
Exercise Biochemical and Histochemi-

cal Studies . . . .
MATERIALS AND METHODS . . . .

Experimental Animals. .
Treatment Groups . . .
Duration Groups . . .
Treatment Procedures. .
Animal Care . . . .
Sacrifice Procedures.

Histochemical Procedures
Histological Procedure .
Tissue Analysis . . .
Statistical Procedures .

RESULTS 0 C C O O O O O 0

Training Results . . .
Histochemical Results .

DISCUSSION. . . . . . . .

Limitations of this Study

0 O O O O
O O 0 0 O

and

Suggestions for Future Study

iv

Page

kWh-IF

OS

20

25
34

34
34
37
38
40
40
44
45
45
54

56

56
58

71

82

SUMMAI

LIST

APPENZ

 

Appen ‘

 

SUMMARY

LIST OF
APPENDIC

Appendix

AND CONCLUSIONS . .

Summary. . . . .
Conclusions . . .

REFERENCES. . . .
ES
Training Programs .

Training Performance
Conditions . . .

and

Environmental

Histochemical Fiber Type Raw Data .

Statistical Tables for Medial Gastroc-
nemius, Plantaris, and Soleus Muscles.

Histochemical Fiber Type Mean Data.

Page
84

84
87

88

107

111
117

123

132

Table

 

‘~m.v .F!.. W.» . r
.

Table

1.

10.

LIST OF TABLES

Nomenclatural schemes for classifying fiber
types 0 O O O I O O I I O O I O I

Comparison of training programs used by
various investigators. . . . . . . . .

Final cell frequencies by treatment and
duration of treatment. . . . . . . . .

Localization and reaction intensity of end-
product in red, intermediate, and white
skeletal muscle fibers (cross section) . . .

Summary of Scheffé contrasts for area/histo—
chemical Sin’l percent ratings within
duration . . . . . . . . . . . . .

Summary of Scheffé contrasts for histochemical
Sin-1 percent ratings for statistically

significant treatments at four weeks in medial
gastrocnemius muscle . . . . . . . . .

Summary of Scheffé contrasts for histochemical
Sin'l percent ratings for statistically
significant treatments at eight weeks in
medial gastrocnemius muscle. . . . . . .

Summary of Scheffé contrasts for histochemical
Sin“1 percent ratings for statistically
significant treatments at twelve weeks in
medial gastrocnemius muscle. . . . . . .

Summary of Scheffé contrasts for area/histo-
chemical Sin"1 percent ratings within
duration of treatment. . . . . . . . .

Summary of Scheffé contrasts for histochemical
Sin"1 percent ratings for statistically
significant duration of treatment in medial
gastrocnemius muscle . . . . . . . . .

vi

Page

19

26

42

52

60

61

62

63

64

65

Table

11.

12.

A-l.

C~2

c~3

.- V Hnt de mnpa

Table

11.

12.

A-l.

A-3.

C-Z.

Summary of Scheffe contrasts for histo-
chemical Sin"l percent ratings for
statistically significant duration of treat-
ment in plantaris muscle . . . . . . .

Summary of Scheffe contrasts for histo-
chemical Sin"l percent ratings for
statistically significant duration of treat-
ment in soleus muscle . . . . . . . .

Standard eight—week, short-duration, high-
speed endurance training program for post-
pubertal and adult male rats in controlled
running wheels . . . . . . . . . .

Standard eight—week, medium-duration,
moderate-speed endurance training program
for postpubertal and adult male rats in
controlled running wheels. . . . . .

Standard eight-week, long-duration, low-
Speed endurance training program for post-
pubertal and adult male rats in controlled
running wheels . . . . . . . . . .

Standard eight-week, endurance, swimming
training program for postpubertal and adult
male rats . . . . . . . . . . . .

Treatment environment and body weight
values for short, medium and long groups.

Treatment environment and body weight
values for swimming group. . . . . . .

Histochemical ratings for medial gastroc—
nemius muscle presented by animal number,
treatment, and duration . . . . . .

Histochemical ratings for plantaris muscle
presented by animal number, treatment, and
duration . . . . . . . . . . .

Histochemical ratings for soleus muscle

presented by animal number, treatment,
and duration . . . . . . . . .

vii

Page

66

67

107

108

109

110

111

112

117

119

121

 

Table

D-l.

D-Z.

D-3.

... .3: 1.....1 9' a. .Ffllotl. r was:

Table

D-1.

Two—way analysis 0 variance tables
histochemical Sin“ percent ratings
medial gastrocnemius muscle . . .

Two-way analysis of variance tables
histochemical Sin"1 percent ratings
plantaris muscle. . . . . . .

Two-way analysis 0 variance tables

histochemical Sin" percent ratings
soleus muscle. . . . . . . .

viii

Page

for
in

O O O 123
for
in

. . . 126
for
in

. . . 129

Plate

II.

LIST OF PLATES AND FIGURES

Page

Figures 2 through 6 are sections taken from
control animals . . . . . . . . . . 48

Figures 7 through 12 are sections taken
from control animals . . . . . . . . 50

Figure

13.

E-3 0

Frequency of fiber ratings (weighted score)
presented by area, treatment and duration of
treatment for SDH, ATP, PPL and PAS . . . 70

Mean daily per cent shock free time (PSF)
and per cent expected revolutions (PER) for
CRW short I I O O O I O O O O O O 113

Mean daily per cent shock free time (PSF)
and per cent expected revolutions (PER) for ,
CRW medium. I O O O I O O O O O O 114

Mean daily per cent shock free time (PSF)
and per cent expected revolutions (PER) for
CRW long . . . . . . . . . . . . 115

Mean daily total revolutions run (TRR) for
voluntary and CRW short, medium, and long . 116

Mean per cent histochemical fiber ratings
for medial gastrocnemius by treatment and
duration of treatment . . . . . . . . 132

Mean per cent histochemical fiber ratings
for plantaris by treatment and duration of
treatment 0 O O O I O O O I O O O l 3 3

Mean per cent histochemical fiber ratings

for soleus by treatment and duration of
treatment . . . . . . . . . . . . 134

ix

VITA

JAMES F. TAYLOR

Final examination: October 26, 1971, 4:00 p.m.
Dissertation: Histochemical profiles of rat triceps surae
and plantaris after seven exercise regimens.
Major Subject: Anatomy
Biographical items:
Born: January 12, 1942. Manistique, Michigan.
Undergraduate studies:
B.A., Biology; Northern Michigan University,
Marquette, Michigan, 1964.
M.A., Biology; Northern Michigan University,
Marquette, Michigan 1966.
Professional experience:
Graduate assistant, Department of Biology, Northern
Michigan University, 1964—66.
Instructor in Biology, Department of Biology,
Northern Michigan University, 1966-68.
Graduate assistant, Department of Anatomy, College
of Human Medicine, Michigan State University,

1968-71.

Instructor in Anatomy, Department of Anatomy,
College of Human Medicine, Michigan State

University, 1971.

xi

certa

this

CON ,
caw
23c
EST .

LON ,

Mm ,

MG

\.
.{BT

PER .

 

LIST OF ABBREVIATIONS

To prevent confusion, and for the sake of clarity,

certain words and/or phrases are abbreviated throughout

this thesis.

ATP

CDS

CON
CRW
ESC
EST

LON

MG
NBT
PAS

PER

.Intermyofibrillar adenosine
triphosphatase

.Cumulative duration of shock
(seconds) received by both the
experimental, and the control
animals during all work periods
of all bouts of a given training
period.

.Sedentary control

.Controlled Running Wheel

.Electric stimulus control

.Expected swim time (minutes)

.Long-duration, low-intensity
running exercise (long CRW program)

.Medium-duration, moderate-
intensity running exercise (medium
CRW program)

.Medial Gastrocnemius muscle

.Nitro Blue Tetrazolium

.Periodic Acid-Schiff

.Percent expected revolutions;
PER = 100 TRR/TER

xii

PET .

PLT .

PPL .

PSF .

SOL .

SDH .

SHT .

STC.

SWM.

TER .

TRR.

PET

PLT
PPL

PSF

SOL
SDH

SHT

STC
SWM

TER

TRR

TWT

VOL

.Percent expected swim time;
PET = 100 STC/EST

.Plantaris muscle
.Phosphorylase

.Percent shock free time;
PSF = 100 - (100 CDS/TWT)

.Soleus muscle
.Succinate dehydrogenase

.Short-duration, high—intensity running
exercise (short CRW program)

.Swim time completed
.Swimming exercise

.Total expected revolutions that the
animal would run, during all work
periods of all bouts of a given train-
ing period, if he would run at the
prescribed speed.

.Total number of revolutions run by the
experimental animal, during all work
periods of all bouts of a given train-
ing period.

.Total work time (sec) during all work
periods of all bouts of a given train—
ing period.

.Voluntary running exercise

xiii

INTRODUCTION

General Comments

 

Classic investigations in the nineteenth century
(Denny-Brown, 1929a) suggested possible correlations be-
tween certain cytological aspects of “red" and "white"
muscle and functional activity. Since then numerous
physiological and histological studies have documented
differences between "red" and "white" muscle (Denny—Brown,
1929a; Jinnai, 1960; Beatty g£_al., 1963, 1966, 1967;
Blanchaer, 1964; Gauthier and Padykula, 1966; Olson and
Swett, 1966; Adams gt_al., 1967; Arangio and Hagstrom,
1969; Briskey 23 31., 1970; Sandow, 1970; Schiaffino
§E_al., 1970). Recent histochemical investigations
clearly show marked heterogeneity in glycolytic and oxi-
dative properties between red and white muscle (Padykula,
1952; Glock and McLean, 1953; Takeuchi and Kuriaki, 1955;
Wachstein and Meisel, 1955; Ogata, 1958; Nachmias and
Padykula, 1958; Dubowitz and Pearse, 1961; Beckett, 1962,
Ogata and Mori, 1964). Histochemical identification of
enzymatically contrasting muscles, with even more unique
fiber types, indicates the existence of differences in
energy metabolism and energy supplying systems important

to contractile function.

It is presently hypothesized that control of the
metabolic and contractile properties of skeletal muscle
is related to (a) the frequency of impulses in the motor
nerve; (b) or the motoneurone release of "trephic" sub—
stances independent of electrical activity (Gutmann gt 31.,
1956; Guth, 1968; Fex, 1969; Bass gt_al., 1970; Fex and
Sonesson, 1970; Gutmann, 1970; Robert and Oester, 1970).
Employing these basic tenents, investigations have shown
that skeletal muscle prOperties are mutable in atrOphy
(Bajusz, 1964; Fischbach and Robbins, 1969, 1970; Brooks,
1970; Kauffman and Albuquerque, 1970; Klinkerfuss and
Haugh, 1970), denervation (Needham, 1926; Nachmias and
Padykula, 1958; Huls and Leonard, 1961; Gutmann, 1962;
Pelligrini and Franzini, 1963; Hogan et_al., 1965; Engel
22 21., 1966; Prewitt and Salafsky, 1967; Sreter, 1970),
cross-innervation (Buller gt 31., 1960a, 1960b; Buller and
Lewis, 1965a; Smith, 1965; Romanul and VanDerMeulen, 1966;
Dubowitz, 1967a, 1967b, 1967c; Eccles, 1967; Hnik gt_al.,
1967; Prewitt and Salafsky, 1967; Yellin, 1967; Gerebtzoff,
1968; Guth gt 31., 1968; Karpati and Engel, 1968b; Ogata
23 31., 1968; Buller and Mommaerts, 1969; Close, 1969;
Robbins 23 31., 1969; Guth gt_al., 1970) and immobilization
(Bach, 1948; Wells, 1969) as identified by anatomical,
histochemical, biochemical, and physiological measures.

Recent evidence suggests that exercise has similar

effects upon skeletal muscle properties (Gordon, 1967;

Gordc
1970,
Oscai
Short
1970;

Golln

 

inter
an en'
City (
histo.
T0 co:
metabc
knowl:
ident:

i5 ne<

0f 58‘
Chara:
c“Emit

of HO]

Gordon §E_al., 1967a, 1967b; Holloszy, 1967; Barnard §E_al.,
1970, 1971; Edgerton gt 31., 1969, 1970; Holloszy and
Oscai, 1969; Kowalski gt 31,, 1969; Lamb gt_al., 1969;
Short §t_a1,, 1969; Ruhling, 1970; Spurway and Young,

1970; Campbell st 31., 1971; Faulkner gt 31., 1971;
Gollnick, 1971). This body of knowledge is difficult to
interpret because exercise too often has been viewed as

an entity, not as an aerobic-anaerobic continuum. Specifi-
city of exercise as a reality was intimated in a related
histochemical study on heart metabolism (Ruhling, 1970).

To corroborate the emerging histochemical evidence of
metabolic plasticity of skeletal muscle with exercise, a
knowledge of the regional and temporal responses to
identical and diverse physiological stimuli ultimately

is necessary.

Statement of the Problem

 

This study was undertaken to determine the effects
of seven different exercise regimens on the histochemical
characteristics of selected muscle fiber areas of gastro—
<3nemius and soleus (triceps surae), and plantaris muscles

(of normal, adult, male, albino rats.

Rationale

 

The extent to which the principle of specificity
applies to a continuum of exercise is a fundamental
Phfiiological question which must be answered. Histo-

cileamical approaches to investigate this question are

cons
a ra
dist
skel

repr

 

conspicuously absent. This study was designed to permit
a rational statement, in histochemical terms, regarding
distinctive patterns of metabolic alterations in rat
skeletal muscle fibers that are associated with defined,
reproducible exercise regimens.

It was postulated that cellular enzyme and substrate
levels are determined by the frequency, intensity and
duration of contraction, in accord with the metabolic
requirements of the animal. It was believed also that
these cellular responses are not only specific to the
exercise requirements of the total muscle but are identi—
fiable by specific muscle area, and even at the individual
muscle fiber level, as identified in histochemical tissue

sections.

Significance of the Problem

 

Histochemical investigations indicate that basic
differences exist within, as well as between, classic
muscle fiber types. Incorporation of methods of subject—
ing animals to different controlled regimens of reproduci-
ble exercise, which compare favorably with human exercise
programs requiring aerobic, anaerobic, and aerobic—
anaerobic adaptations, should yield: (a) differential
histochemical fiber type alterations produced by specific
regimens of physical activity, (b) new evidence on general
patterns of adaptation to specific exercise regimens and

the effects of different durations of the various exercise

PIOG
scri
and

cell

adapv

 

programs, (c) a body of knowledge for intelligent pre—
scription of exercise programs at the muscle fiber level,
and (d) a stimulus for investigations to elucidate the
cellular mechanisms involved in exercise induced

adaptations.

phys
of vi
surgl
eterJ
histg

variq

Comte:

Sheet

 

REVIEW OF PERTINENT LITERATURE

This review focuses on the fundamental anatomical,
physiological, biochemical and histochemical differences
of vertebrate skeletal muscle fibers; the effect of
surgically induced conditions on these muscle fiber param-
eters; and a consideration of related biochemical and
histochemical studies on skeletal muscle response to
various exercise programs.

Red, Intermediate, and White Skeletal
Muscle Fibers

 

 

Application of diverse biological techniques to
"white" and "red" muscle established new dimensions for

the identification of at least three fiber types.

Anatomical Differences

 

The main differences in fiber types are found in the
content, form, and distribution of the constituent cellu—
1ar organelles and associated tissues. In the red fiber
large spherical mitochondria, with numerous fenestrated
sheet cristae and dense matrices, form an aggregate oxi—
dative machinery layer between the sarcolemma and the

contractile substance (Van Breemen, 1960; Padykula and

GautI

Ogata

 

inter
filam

I
I bag

I
chain

in th
Space
crist
in tin
exist

centre

of mit
of Sme
mYOfik
red fj
1966).

t0 mit

Gauthier, 1963; Gauthier and Padykula, 1966; Murata and
Ogata, 1969; Ogata and Murata, 1969a, 1969b). In the
interior of the fibers on either side of the Z line smaller
filamentous mitochondria are aligned in the region of the
I band. Prominent small mitochondria form longitudinal
chains in the intermyofibrillar space.

In the white fiber, few mitochondria are dispersed
in the subsarcolemmal space or the intermyofibrillar
space, while small to medium mitochondria, with few
cristae and less dense matrices, are occasionally located
in the region of the I band. No striking differences
exist between size and structure of the peripheral and
central mitochondria (Padykula and Gauthier, 1963).

The intermediate fiber occasionally has aggregates
of mitochondria in the subsarcolemmal space. Accumulations
of smaller mitochondria, with fewer cristae, as inter—
myofibrillar chains, are not as conspicuous as found in
red fibers (Gauthier and Padykula, 1966; Shafiq gt 21.,
1966). Lipid dr0plet concentration is directly related
to mitochondrial density (Gauthier and Padykula, 1966).

The sarcoplasmic reticulum of individual red fibers
has received conflicting description. Schiaffino 33 31.
(1970) described a well developed sarc0p1asmic reticulum
in the red fibers of the rat soleus muscle. Padykula and
Gauthier (1970) and Nishiyama (1965a, 1965b), however,

stated that red fibers in the diaphragm and intercostal

musc
ass
defi
The

poor

of s

 

port.

cont

band
termi

trans

 

and s
°Ccas
criSt<
ture.
lunctl

ing at

 

Gauthi

1925),
1970),
more 3
Padqu

and 1e

muscles have poorly developed sarc0p1asmic reticulum. The
assumption that mitochondrial rich fibers have poorly
defined sarc0p1asmic reticulum is not true in all muscles.
The view that both mitochondrial rich and mitochondrial
poor fibers are fast, and have comparably rich deve10pment
of sarc0p1asmic reticulum is consistent with the pro-
portion of motor units with relatively fast speeds of
contraction for rat soleus (Close, 1967a, 1967b).

In the red and white fibers triads occur at the A-l
band junctions, longitudinal tubules extend from the
terminal cisternae toward the H band level to form a
transverse network (Schiaffino et 31., 1970).

The intermediate fiber of extensor digitorum longus
and soleus muscles and the red fiber of the diaphragm
occasionally have T tubules with only one junctional
cristerna (dyad), instead of the familiar triad struc-
ture. Sparse longitudinal tubules extend from the
junctional cisternae into the A band, with limited branch—
ing at the H band level (Ogata, 1964; Padykula and
Gauthier, 1970; Schiaffino gt_al., 1970).

The red fiber usually has more myonuclei (Needham,
1926), wider Z line (Gauthier, 1969; Schiaffino 25 31.,
1970), longer sarcomere length (Schiaffino §£_§13, 1970),
more sarc0plasm (Denny-Brown, 1929a; Gauthier and
Padykula, 1966), and smaller diameter (Denny—Brown, 1929a),

and less connective tissue (Beatty gt 31., 1966) than

whit
to m

than

middi
junc
mic a
crete
cal,
White
of ju
media
with
(Ogaty
and Ge

 

and v

ate tC
fibers

1967;

white fibers. Triglyceride droplets are directly related
to mitochondrial density, and are more abundant in red
than white fibers (Gauthier and Padykula, 1966).

While each 22 plaque endplate is situated in the
middle of the fiber, differences exist in the neruomuscular
junction. The red fiber endplate has the least sarcoplas-
mic and axoplasmic surface, at each contact relatively dis—
crete and separate axonal terminals are small and ellipti-
cal, junctional folds are shallow, flat and sparse. The
white fiber has long flat axonal terminals and the profile
of junctional folds increases in complexity. The inter—
mediate fiber in the diaphragm has large axonal terminals
with the most widely spaced and deepest junctional folds
(Ogata, 1964; Ogata and Murata, 1969a, 1969b; Padykula
and Gauthier, 1970). A clear cut difference in the form
and volume of the junctional invagination is evident.

The capillary to fiber ratio is directly proportion-
ate to oxidative metabolism in red, intermediate and white
fibers (Nishiyama, 1965b; Romanul, 1965; Carrow gt_a1.,

1967; Romanul and Pollock, 1969; Mai gt 31., 1970).

Physiological Differences

 

Physiological studies performed on whole muscle
preparations conform well with morphological investigations
in establishing slow and fast twitch muscles.

At birth all muscles in the kitten are uniformly

slow twitch, and speed of shortening of sarcomeres

10

increases two to three fold within three to four weeks
(Denny-Brown, 1929a, 1929b; Buller gt_al., 1960a; Close,
1967a, 1967b; Close and Hoh, 1967). Slow muscles in
contrast to fast muscles, show little change in speed of
shortening with maturation. Close (1967a, 1967b) examined
the dynamic prOperties of muscles in different species
and found that neither the fast nor the slow muscles had
the same intrinsic speeds. In this connection at least
three types of motor units have been identified in skele—
tal muscles (Henneman and Olson, 1965; Wuerker gt_al.,
1965; Close, 1967b; Edstram and Kugelberg, 1968, 1969).
In a histochemical study on the effect of contraction
induced by low frequency stimulation, Edstrdm and Kugel—
berg (1969) and Kugelberg and Edstrdm (1968) showed that
repetitive stimulation of ventral root, single nerve fiber
or entire nerve trunk, produced preferential changes in
phOSphorylase and glycogen in muscle fibers. Mapping of
the motor unit showed highly intermingled phosphorylase
and glycogen negative fibers, with a more pronounced in-
fluence in white fibers. Wuerker gt 31. (1965), found
differences in contraction speeds of individual motor
units. This information tends to support the concept of a
homogeneous character of the motor unit, as a natural
result of the uniform neural control exerted by its
motoneurone.

In gastrocnemius and plantaris muscles, the type A

motor unit has large, low resistance motoneurones

inne

tens

 

sole
smal

twit

1968
on tl
fibei
units
type
0f 3c

 

Were

milli

and c
V610c
13.5-
milli
Per 8
and 0

gold,

11

innervating fast twitch muscle units with relatively large
tension output (Somjen 32 31., 1964; Burke, 1968). In the
soleus muscle the type B motor unit is characterized by
smaller, higher resistance motoneurones innervating slow
twitch muscle units with small tension output (Burke,
1968). The type B unit characteristics are known from work
on the cat soleus muscle, which has only intermediate
fibers (Henneman and Olson, 1965). A group of intermediate
units remains unidentified, of which some may belong to
type C. Close (1967b) showed in rat soleus muscle, that

of 30 units identified on basis of contraction time, 3

were intermediate (17.5 milliseconds) and 27 slow (38
milliseconds).

Generally fast muscles develOp high initial tension
and contract rapidly (18-129 milliseconds) with conduction
velocity of 95 meters per second and an axon diameter
l3.6-8.3 microns. Slow muscles contract slowly (58-193
milliseconds) with conduction velocities of (51—81 meters
per secOnd) and axon diameter 6.3-2.7 microns (Henneman
and Olson, 1965; Wuerker et_al., 1965; Eberstein and Goodw
gold, 1968).

McComas and Thomas (1968) observed for human gastroc-
nemius muscle a contraction speed of 117.6 milliseconds,
which appears to be considerably slower than that of slow
muscles in other species, such as cat and rabbit.

Rapidly contracting phasic fibers are usually located

near the subcutaneous surface and slow contracting tonic

fibe

 

195;
of a
'whi
and

trac
ceps
fibel
the <
excel
servfi
fast

that

 

muscl
teris
fuhct
of in

aSCer

12

fibers near the deep axial surface (Gordon and Phillips,
1953). Physiological data tend to indicate that "redness"
of a muscle reflects a slow rate of contraction, while
"whiteness" reflects a fast contraction rate. Buchthal
and Schmalbruch (1970) found that histograms of con-
traction times agreed with histochemical findings in tri-
ceps surae muscles. In the tibialis anterior muscle, red
fibers constituted half the fiber population, and half of
the contraction times were longer than 60 msec. However,
exceptions exist. For example, Hall-Craggs (1968) ob-
served that the thyroarytenoid muscle was an extremely
fast twitch muscle, with histochemical characteristics,
that predicted slow twitch, whereas, the cricothyroid
muscle was a slow twitch muscle, with histochemical charac—
teristics found in fast limb muscles. Illustrating
functional activity can only be equated to the activity
of individual fibers after the fiber population has been
ascertained.

Blood flow to individual red and white muscle has
received little attention. Investigators have found that
flow is three times greater in red than white limb muscles.
A direct relationship exists between functional blood flow,
both for substrate supply and removal, capillary per fiber,
myoglobin concentration and the duration of the contraction

(Reis and Wooten, 1970).

en

pr
pl
wh

10
11181

ill-Illa. F ‘
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13

Biochemical Differences

The classic postulate regarding the metabolic differ-
ences between red and white muscle states that white
(tetanic) muscle, capable of rapid but brief contractions,
primarily utilizes the glycolytic system of the sarco—
plasm and associated membranes for energy production,
whereas red (tonic) muscle, which can contract for pro—
longed periods, relies on glycolytic and other oxidative
mechanisms (Beatty gt_31., 1963). In regard to energy-
supplying metabolism, possibly the metabolic type of a
muscle does not cause a distinct type of function, but
rather the function of a muscle implies the development
of a distinct type of energy (Moody and Cassens, 1968).

White muscle has high levels of activity of the
enzymes of glycolysis and glycogenolysis; aldolase, phos-
phorylase, pyruvate kinase and lactate dehydrogenase. Red
muscle has high levels of glycogen synthetase, myoglobin,
hexokinase, acetoacetate, Beta-hydroxy acyl Co-A dehydro-
genase, isocitrate dehydrogenase, succinate dehydrogenase,
glucose-6-phosphate dehydrogenase, cytochrome oxidase, and
glutamic oxaloacetic transaminase (Blanchaer, 1964; Dawson
and Romanul, 1964; Stubbs and Blanchaer, 1965; Beatty
gt 51., 1966; Bocek gt_gl., 1966; Prewitt and Salafsky,
1967; Sigel and Pette, 1969; Jeffress and Peter, 1970).
Lawrie (1952, 1953) found in various species that high

myoglobin content was associated with high capacity for

aer
cyt
ish
spe
to

con
sta

bloc

1965

Misc

 

leve
1970
narr
1970

Phos

leve

14

aerobic energy-rich phosphate formation, particularly
cytochrome oxidase. White muscle is relatively impover—
ished in myoglobin and cytochrome oxidase. Myoglobin
speeds diffusivity of oxygen into red muscle, and serves
to meet sustained demand for oxygen. During sustained
contraction red and intermediate fibers achieve a steady
state in which work output, blood flow and utilization of
blood oxygen are constant (Wittenberg, 1970).

While most investigators (Stubbs and Blanchaer,
1965; Pande and Blanchaer, 1971) claim that only white
muscle has high endogenous muscle glycogen, high glycogen
levels have been reported for red muscle (Gillespie gt_al.,
1970). The concentration of glycogen varies only within a
narrow range under normal conditions (DiMauro gt a1,,
1970). The polysaccharide influences the proportion of
phosphorylase a and b and glycogen synthetase and thus,
its own catabolic and anabolic enzymes. High tissue
levels of glycogen increase the prOportion of phosphorylase
a and exert a double feed back mechanism controlling
glycogen synthesis and degradation. Prenatally, the path—
way of glucose to glycogen, favors glycogen formation in
red and white muscle and neonatally is more active in red
muscle (Bocek et 31., 1966, 1969). Gutmann gt_al. (1969)
stated that glycogen breakdown was determined by the
number of frequency of arriving impulses, and that meta-

bolic recovery processes were dependent upon the functional

15

state of nerve centers. Glycogen concentration only rises
above the initial level after high frequency stimulation.
Bass et_al, (1955) and Stubbs and Blanchaer (1965) found
that stimulation produced a significant conversion of
phosphorylase b to a in only white muscle. White muscle
consumes more glycogen and forms lactate from pyruvate to
a greater extent than red muscle (Domonkos, 1961; Domonkos
and Latzkovits, 1961a, 1961b). The oxygen consumption of
red muscle is three times that of white muscle (Domonkos
and Latzkovits, 1961a). Decreasing hydrogen ion concen-
tration increases glycolysis, but does not affect respira-
tory capacity in red and white muscle (Domonkos and
Latzkovits, 1961a).

Failure of red muscle to utilize glycolysis does not
impair its efficiency. Reserve fat depots and circulating
free fatty acids can be readily used by red muscle
(Romanul, 1964; Reis and Wooten, 1970). Pande and
Blanchaer (1971) found high mitochondrial respiratory
rates with pyruvate in both red and white muscle. Com—
pared to pyruvate, respiratory rate with acetylcarnitine
was only slightly slower in red, but nearly half as great
in white muscle mitochondria. White muscle oxidizes
carbohydrate rather than fat, and in vigorous exercise
glycogenolysis and carbohydrate metabolism predominates

over B-oxidation and fat metabolism.

16

The contractile myosin from fast muscles has higher
enzymatic activity than slow muscle (Margreth gt_al., 1970;
Perry, 1970). Guth and Samaha (1969) reported that acto-
mysin, isolated from cat fast muscle had threefold greater
adenosine triphosphatase (ATPase) activity, and suggested
that neural regulation was based upon specific type, rather
than the activity, of an enzyme. The capacity of the sarco—
plasmic reticulum to remove calcium from the cytOplasm, and
form ATP from creatine phosphate is greater in fast (white)
muscle. The converse applies to slow muscle, as ATP is
formed by the oxidative activity of mitochondria.

Perry (1970) noted that myosin from skeletal muscle
of new born rabbits was similar to that of adult red
muscle, and showed that the capacity of the cell to develop
a greater tension resulted from a rise in the number of
myofibrils, and hence myosin molecules. Forced activity
in new born rabbits caused an early peak of ATPase activity
of the sarcoplasmic reticulum. Thus, specialization appears
in part to be an adaptation to activity pattern character-

istic of the particular muscle.

Histochemical Differences

 

Maturation of the species is a decisive factor in
differentiation of fiber types. Man and guinea pig have
full differentiation of fiber types at birth. In the
mouse, rat, chicken, rabbit and pig differentiation occurs

postnatally (Dubowitz and Pearse, 1960, 1961; Dubowitz,

19

am

 

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hie
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subC
Cal
ten.
mu3<
PAS

pho.

17

1965, 1967a, 1967b, 1967c; Cooper gt_al., 1970; Ashmore
and Doerr, 1971a). In the mammalian embryo all muscle
fibers are equal in size and uniformly non-differentiated
histochemically (Denny-Brown, 1929a; Bowden and Goyer,
1960). Investigators prOpose that the various histo—
chemical types of muscle fibers develop as: separate
populations (Wirsen and Larsson, 1964; Nystrom, 1968),
two p0pu1ations with a possible common origin for red and
intermediate fibers (Germino et_al., 1965; Fenichel, 1966),
intermediate fibers as precursors of white fibers (Vince—
1ette and Jasmin, 1969), or fibers from a common pool
(Dubowitz, 1965; Cooper gt 31., 1970).

Basic biochemical differences exist between red and
white muscles. The advent of histochemical techniques
produced a valuable tool for identification of muscle fiber
types at the tissue level. In adult muscle, histochemical
localization of enzyme systems and other chemical constitu-
ents revealed the existence of two, three or more fiber
type groups. Dubowitz and Pearse (1960) suggested the
subdivision of muscle into two fiber types on the recipro-
cal relationship between phosphorylase and oxidative con-
tent. Stein and Padykula (1962) defined A, B, and C
muscle fiber types from individual fiber profiles, using
PAS, SDH, non-specific esterase and two adenosine tri—
phosphatase techniques. The previous investigators'

muscle fiber profile was based mainly on the amount and

18

distribution pattern of diformazan particles of succinate
dehydrogenase. Romanul (1964) described a spectrum of
eight muscle fiber types, which could be broadly divided
into three groups by correlating the relative intensities
of individual histochemical procedures. Guth and Yellin
(1971) also reported that, in general, three categories
are evident. However, careful analysis revealed diverse
combinations of enzyme activities and more than three
histochemical fiber types in mammalian skeletal muscle.
Other investigators favor the basic classification of only
two fiber types (Engel, 1962, 1965; Karpati and Engel,
1968a, 1968b; Brooke and Engel, 1969; Brooke and Kaiser,
1970). From the preceding, various nomenclatural schemes
have emerged. These are summarized in Table 1.

White muscle fibers are generally characterized by
high glycogen, low lipid, low oxidative enzymes, low
myoglobin, high adenosine triphosphate, high M-type
lactate dehydrogenase, and high phosphorylase. Red muscle
fibers are generally characterized by low glycogen, high
lipid, high oxidative enzymes, low phosphorylase, H—type
lactate dehydrogenase, and high myoglobin. Intermediate
fibers have moderate glycogen, moderate lipid, high oxi-
dative enzymes, and high myoglobin (Briskey g£_al., 1970).

The nomenclature should be based on the muscle
fiber properties being examined and result in a clear cut

differentiation of fiber types (Brooke and Kaiser, 1970).

19

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The relative, or semi-quantitative, histochemical re-
activity can only be indicative of the amount of accumu—
lated end-product. However, direct relative comparison
can be made between fibers in the same muscle (Engel,
1965; Nystrom, 1966, 1968).

Skeletal Muscle Fiber Response to

Experimentally Induced
Conditions

 

 

The requirements placed upon striated muscle in
specific anatomical locales, and the characteristics of
the fiber types involved have been shown to change under

surgically induced conditions.

Denervation

 

Engel gt 31. (1966) found that white fibers are
preferentially affected by denervation, and fiber metabolic
differences are no longer maintained. Nachmias and Pady-
kula (1958) found that succinate dehydrogenase decreased
in red fibers in both soleus and biceps femoris muscles,
and white fibers were essentially unaffected. Guth and
Watson (1967) electrOphoretically determined that after
denervation the protein pattern of the plantaris muscle
resembled that of the soleus muscle, while the denervated
soleus muscle did not change; but when reinnervated by
the nerve supplying the plantaris muscle the soleus muscle

pattern resembled the plantaris muscle. Sreter (1970)

stated that denervation affects both white and red muscles,

21

although the changes in the latter are smaller and confined
to adenosine triphosphatase activity; and indicated that
calcium uptake did not change significantly in red, as
contrasted to profound change in white fibers. Bajusz
(1964) found that red muscle fiber sarcoplasmic granu—
larity was preserved longer. Smith (1965) showed that
glycolytic activity was reduced, and B—oxidation enzymes
were not reduced as much as mitochondrial enzymes. The
basic metabolism resembled cardiac muscle in utilizing

ketones and fatty acids after denervation.

Cross-innervation

 

The cross-innervation studies involve both changes
in specific innervation and physiological activity of the
neuromuscular apparatus. Close (1964, 1965, 1969) re-
ported that changes in intrinsic speeds of shortening are
brought about by a direct effect on the contractile material
in fast muscle. Following cross—innervation of fast and
slow muscle, investigators (Buller gt gt., 1960a, 1960b;
Engel, 1965; Dubowitz, 1967a, 1967b, 1967c; Prewitt and
Salafsky, 1967; Guth gt_gt., 1968, Karpati and Engel,
1968b; Ogata gt_gt., 1968; Fex, 1969; Robbins gt gt.,
1969; Mann and Salafsky, 1970) reported an interconverti-
bility of energy metabolism, contraction times, and
capillary to fiber ratios. After cross—union there was
no appreciable change in conduction velocity, as control

and experimentally cross-united slow and fast nerve fibers

22

had the same conduction velocity on both sides. Muscle
fibers tend to exert little appreciable influence on the
time characteristics of motoneruones under these con—
ditions. Several postulates are possible: (1) special
differentiating action of innervation by slow motoneruones,
with long after hyperpolarization, is due to the slow
frequency of discharge (lO-20/second); (2) muscles become
fast when activated at higher frequencies; (3) or specific
chemical transmitter produced from large and small moto-
neurones influence differentiation. High frequency dis-
charge to slow motor units would merely serve to fatigue
the muscle with no effective return in a higher con—
traction tension.

Buller gt_gt. (1960a, 1960b) found that differenti—
ation of fast muscles was unaffected by spinal cord
transection and dorsal rhizotomy, while differentiation
of slow muscle was greatly depressed and contraction times
resembled normal fast muscle. Guth gt_gt. (1970) reported
that after cross-innervation the qualitative type of
actomyosin ATPase synthesized formerly by fast muscle was
that type formerly characteristic of slow muscle, and
visa versa. Actomyosin adenosine triphosphatase seems
to be specific pH dependent and labile, and suggests
genomic control regulated by neural input.

Directional transformation of muscle fiber types

seems to be specific, as fast (white) muscle transforms

23

to slow (red and intermediate), slow muscle (intermediate)
changes reversibly with slow (red), but slow muscle
(intermediate and red) rarely converts to fast (white)
(Buller and Lewis, 1965a, 1965b, Dubowitz, 1967a; Hnik
gt_gt., 1967; Guth, 1968; Guth gt gt., 1968). Differenti—
ation rather than dedifferentiation indicates interaction
between neural factors and other physiological influences.
Mommaerts gt gt. (1969) agreed that after cross-union
transformation of red to white muscle appeared negligible.

Cross-innervation histochemical muscle fiber type
studies showed a reversal of enzyme profiles, and density
of capillary network present in the fibers of their normal
counterparts (Romanul and Van Der Meulen, 1966, 1967;
Robbins 22.2l'v 1969; Romanul and Pollock, 1969). A
general tendency exists for cross-innervated muscle to
assume the variety of muscle fiber size and histochemical
fiber type relationship observed under normal fast and
slow neuromuscular conditions. One striking feature in
reinnervated and cross-innervation muscle was the absence
of the typical mosaic pattern and presence of "type group—
ing" pattern, possibly brought about by abundant collateral
branching of nerve fibers (Yellin, 1967; Karpati and
Engel, 1968b).

Prewitt and Salafsky (1967), after cross—innervation
of nerves to soleus and flexor digitorum longus muscles,

found an increase in pyruvate kinase and aldolase, and

de<
gex
dig

inc

foli
and

196E

 

 

bee
in t
1970
not

Nels

CO‘W

24

decreases in malate dehydrogenase and isocitrate dehydro—
genase, in the soleus muscle. Conversely, in the flexor
digitorum longus muscle, Edstr6m and Kugelberg (1968)
induced muscle contractions by shock stimulation (10/
second) and found decreased phosphorylase and glycogen
content in white and intermediate muscle fibers. Red
muscle fibers showed increased phosphorylase and glycogen

content .

Tenotomy

 

Bach (1948) reversed positions of red and white
muscle tendons and found conversion of red to pale muscle.
Guth and Yellin (1971) using histochemical tissue tech-
niques found profound changes in actomyosin ATPase, and
succinate dehydrogenase in soleus and plantaris muscles,
following excision of several muscles of the posterior
compartment. Vrbova (1962, 1963, 1966) and others (McMinn
and Vrbova, 1962; Salmons and Vrbova, 1969, Shafiq gt_gt.,
1969) concluded that, with disuse, the soleus muscle
became a fast muscle, and no appreciable change occurred
in the plantaris muscle. Fischback and Robbins (1969,
1970) found that rapid atrOphy alone in slow muscle was
not sufficient to cause a change in contractile properties.
Nelson (1969) could not confirm the findings of Vrbova and

co-workers.

25

Skeletal Muscle Fiber Response to Exercise
Biochemical and Histochemical Studies

 

 

The literature in this area is difficult to interpret
due to vastly different intensities and durations of exer-
cise (for verification see Table 2). Most investigations
have controlled duration (Holloszy, 1967; Edgerton gt gt.,

1969), but few have controlled intensity (Ruhling, 1970).

Biochemical Studies

 

Studies to selectively correlate the effects of
exercise on the myofibrillar and sarcoplasmic protein
content and aerobic and anaerobic capacities are incon-
clusive (Jeffress and Peter, 1970). Prolonged running
increases only the sarcoplasmic proteins (Kendrick—Jones
and Perry, 1965; Gordon gt_gt., 1966, 1967a, 1967b),
whereas forceful exercise increases the number of myo-
fibrillar proteins (Helander, 1961; Kendrick-Jones and
Perry, 1967).

Short gt_gt. (1969) trained rats for eight weeks by
submaximal running (13.7 m/min) on a motor—driven drum,
four hours daily with 5 minutes rest between each 30-
minute period, six days per week. Examination of adductor
magnus tt_!tttg_from these animals showed increased
creatine and decreased lactate levels in the red portion,
but proportionately less than that observed in the white
area. Red muscle from trained animals had greater

glycogen concentration. The decreased lactate

I it i)”. In! I E I

 

 

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26

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27

accumulation might reflect a reduced rate of glycolysis,
increased oxidation of alpha—glycerophosphate, increased
pyruvate oxidation, utilization of lactate in glycogen
resynthesis, or removal of lactate by increased blood flow
to the muscles of conditioned animals. Rawlinson and
Gould (1959) swam three different age group rats daily
for eight weeks for one and two 30—minute periods, and
concluded from biceps femoris homogenates that the total
activity of creatine phosphokinase, adenosine triphos-
phatase, lactate dehydrogenase, malate dehydrogenase, and
phosphorylase enzymes was not affected by either swimming
program in any of the groups.

Lawrie (1952) exercised rats at 2.0 ft/sec and found
an increase in myoglobin with increased duration of treat-
ment. Over a short period, severe exercise elicits no
such response, although glycogen reserves are diminished.
Hearn and Wainio (1956) found no changes in succinate
dehydrogenase levels in the gastrocnemius muscle after
swimming rats 30 minutes daily for up to eight weeks. The
latter authors suggested that existing aerobic systems
are capable of meeting stress induced by moderate exer-
cise. Yakovlev gt_gt. (1963) reported that the enzymes
of aerobic metabolism were the first to increase during
training and the last to decrease on cessation of train—

ing.

I

28

Kendrick—Jones and Perry (1965, 1967) and Lamb
gt gt. (1969) suggested that chronic exercise increased
the capacity of skeletal muscle to work anaerobically.

The previous authors noted that treadmill exercise caused
an increase in hexokinase and glycogen content, and in-
creased the capacity to phosphorylate glucose both during
and after exercise periods. Using intense activity,
Kendrick-Jones and Perry (1965, 1967) found significant
increases in creatine phosphokinase and other sarc0p1asmic
proteins. These authors suggested that the ability of

the myofibril to split adenosine triphosphate was not as
rate limiting as was the supply of adenosine triphosphate
to the enzymatic center.

Gollnick and King (1969) swam rats and found in—
creased mitochondria in the gastrocnemius muscle. Running
to exhaustion prior to sacrifice produced marked swelling
(uncoupled oxidative phosphorylation) in mitochondria,
whereas, swimming to exhaustion produced no such change.
It seems that chronic exposure to exercise increases the
oxidative capacity in some forms of activity because of
the addition of more metabolic apparatus. Holloszy (1967)
and Holloszy and Oscai (1969) found an increased capacity
of the mitochondrial fraction from the gastrocnemius
muscle to oxidize pyruvate after a strenuous program of
treadmill running. Mild exercise did not effect the level

of succinate dehydrogenase. Increase in both mitochondrial

29

enzymatic activity and electron transport capacity was
associated with a concomitant rise in capacity to produce
adenosine triphosphate. Holloszy (1967) observed a change
in the mitochondrial content, and suggested that the
composition of the cristae, rather than a simple uniform
increase in size or number of mitochondria accounted for
the increased aerobic capacity. Wilkerson and Evonuk
(1971) examined calcium-activated adenosine triphosphatase
of the rat gastrocnemius muscle after acute and chronic
programs of swimming. The specific activity of gastroc—
nemius muscle myosin ATPase was only significant when
contrasting control and exhaustive programs at six and

ten weeks. No difference was found for mild exercise at
ten weeks. Previous failure to find significance, as
Holloszy (1967) contended, might reflect the relative
midlness of the program, since rats can easily perform

30 minutes of swimming. One might also speculate that
adequate programs for anaerobic adaptation have not been

utilized.

Histochemical Studies

 

Studies regarding histochemical changes in muscle as
a result of training have steadily emerged within the last
several years. In general, these studies support meta—
bolic alteration of fiber type with various exercise

programs.

30

Investigating the effect of two different duration
running programs on hypertrOphy and transformation of fiber
type in the tibialis anterior muscle, Man—i gt_gt. (1967)
reported that white and red fibers hypertrophied (21% and
11%, respectively) in only the 50-day program. No evi-
dence of change in distribution of various Sudan Black B
fiber types with either training program was noted.

Investigators (Kowalski gt gt,, 1969; Spurway and
Young, 1970) using low-intensity, repetitive running and
weight lifting programs supported an aerobic metabolic
adaptation at the interregional fiber level. Kowalski
gt_gt. (1968) found, using subjective histochemical
measures, an increase in phosphorylase in weight lifting
and an even greater increase in running rats. Both exer-
cises resulted in equal increments for succinate dehydro—
genase and cytochrome oxidase. The greatest relative rise
of succinate dehydrogenase occurred in muscle fibers re-
garded as predominantly anaerobic (white) only with
running.

In a similar study using young mice, Spurway and
Young (1970) indicated that running increased the pro-
portion of fibers with high succinate dehydrogenase, while
weight-lifting decreased this proportion and raised the
percentage of moderate fibers.

The pioneering investigations (Barnard gt_gt., 1970,

1971; Edgerton gt gt., 1969, 1970; Edgerton and Simpson,

31

1969, 1971) have produced the most extensive attempts to
elucidate the influence of exercise on fiber type a1ter~
ation. In an early investigation, Edgerton gt_gt. (1969)
swam lOO-day-old rats for 52 days (one and two 30-minute
exercise bouts each day) and found the proportion of
intermediate and red fibers in the soleus muscle unaltered
in oxidative enzymes, and myosin adenosine triphosphatase
patterns. Moderately and heavily exercised rats showed
an increase in the preportion of fibers having high in—
tensity malate dehydrogenase, succinate dehydrogenase,
and nicotinamide adenine dinucleotide diaphorase in the
plantaris muscle (white and mixed fiber areas). Faulkner
gt gt. (1971) determined the number of red and white
fibers with succinate dehydrogenase from the plantaris
muscle of 6-week and 45-week-old guinea pigs, subjected
to low-speed, high-repetition treadmill running. Total
fiber counts from the plantaris muscle revealed 14—week
and 45-week animals had 20% fewer fibers than 6—week.
Training for 8 weeks enhanced the oxidative capacity by
increasing the number of red fibers, and prevented
attrition of muscle fibers.

In a later comprehensive histochemical, biochemical
and physiological study, Edgerton gt gt. (1969, 1970)
examined the effects of prolonged progressive, low—
resistance, intermittent, repetitive exercise on

gastrocnemius, plantaris and soleus muscles. The

32

5-day per week complex training program for adult male
guinea pigs was progressively increased each week through
9 weeks of training (velocities ranged from 20.8 to 49.3
m/min). Guinea pigs trained to 21 weeks followed the
ninth week program. After 9 weeks of training the mito—
chondria isolated from gastrocnemius and plantaris muscles
showed no significant adaptation, while the 18—week group
showed significantly increased mitochondrial protein con-
centration. The percentage of histochemically demon—
strated red muscle fibers was significantly increased.

No significant differences in myosin ATPase, or con—
tractile properties were observed.

After acute exercise, electrical stimulation of the
plantaris muscle, caused selective depletion of glycogen
and phosphorylase content in aerobic (red) fibers (Edger-
ton gt_gt., 1970). The prOportions of phosphorylase nega—
tive fibers increased with the duration of the exercise.
Kugelberg and Edstrdm (1968) induced muscular contraction
with low frequency stimulation and found that changes in
phosphorylase and glycogen content were most pronounced
in white muscle fibers, less in intermediate, and least
in red muscle fibers. After one and two hours of stimu-
1ation, glycogen negative fibers were identified, but
phosphorylase negative fibers were absent. Edgerton
gt gt. (1970) investigated the question further using

guinea pigs trained for 9 and 18 weeks. Muscular

33

contraction induced by electrical stimulation caused total
phosphorylase activity to be selectively depleted in white
fibers; less in trained than non-trained animals. These
data suggest different effects for normally exercised and
electrically stimulated muscles. The mitochondrial and
glycolytic adaptations might have had a sparing effect on
the metabolic characteristics.

Campbell gt_gt. (1971) obtained biOpsies from the
longissimus muscle of Chester white pigs trained on a
treadmill at 2.2 ft/sec for two weeks, and reported an
increase in the number of alkali-stabile ATPase fibers.
Edgerton gt_gt. (1969) and Rawlinson and Gould (1959)
using histochemical and biochemical techniques, have
reported no changes in myosin adenosine triphosphatase
fiber type or activity.

Recently Dorn gt gt. (1970) using histochemical
techniques reported an increase in glycolytic enzymes and
a decrease in oxidative enzymes for fibers of the rat

soleus and rectus femoris muscles after forced exercise.

MATERIALS AND METHODS

Experimental Animals

 

One hundred and seventy-six normal, 72-day-old, male,
albino rats (Sprague-Dawley Strain)1 were brought into the
laboratory in four shipments. Each animal was randomly
assigned to one of seven treatment groups and then allowed
12 days to become acclimated to laboratory conditions be—

fore treatments began.

Treatment Groups

 

The seven treatment groups used in this study were

as follows:

Control (CON)

 

The animals assigned to the control group received
no special treatment and were housed in standard, indi-
vidual, sedentary cages (24 cm. x 18 cm. x 18 cm.) during

both the adjustment and treatment periods.

Voluntary (VOL)

 

The animals in the voluntary exercise group received

no special treatment during either the adjustment period

 

1Obtained from Hormone Assay Laboratory, Chicago,
Illinois.

34

35

or the treatment period but were housed in standard, indi-
vidual voluntary—activity cages. These dages were identi—
cal to individual sedentary cages except that the animals
were allowed access to freely revolving activity wheels
(13 cm. wide and 35 cm. in diameter). Individual daily
records of total revolutions run (TRR) were recorded on

the attached revolution counter.

Short (SHT)

 

The animals assigned to the short group were housed
in individual, voluntary-activity cages during the adjust-
ment period and in individual sedentary cages during the
treatment period. These animals were subjected to a
short-duration, high-speed endurance program of interval
running. The program was progressive in nature. That is,
the intensity of the program was gradually increased until
on the thirty-seventh day of training, and thereafter,
the animals were expected to complete eight bouts of exer-
cise with 2.5 minutes of inactivity between bouts. Each
bout consisted of six repetitions of 10 seconds of work
alternated with 40 seconds of rest. During the work
intervals, these animals ran at the relatively fast speed

of 5.5 ft/sec. (for compete program, see Appendix Awl).

Medium (MED)
The animals in the medium group were housed under
the same conditions as the SHT animals. However, the

medium (MED) group was subjected to a medium-duration,

36

moderate—speed endurance program of progressive interval
running. By the thirty-seventh day of training, these
animals were expected to complete five 8-minute bouts of
exercise with 5 minutes of inactivity between bouts. Each
bout consisted of eight repetitions of 30 seconds of work
alternated with 30 seconds of rest. During the work inter-
vals, these animals ran at the moderate speed of 4.0

ft/sec. (for complete program, see Appendix A-2).

Long (LON)

 

The animals in the long group were housed under the
same conditions as the SHT and MED groups. However, the
long (LON) group was subjected to a long—duration, low—
Speed endurance program of progressive interval running.
By the thirty-seventh day of training, these animals were
expected to complete four 12.5—minute bouts of exercise
with 2.5 minutes of inactivity between bouts. Each bout
consisted of one repetition of 12.5 minutes of work with
no rest periods. During the work intervals, these animals
ran at the slow speed of 2.0 ft/sec. (for complete pro—

gram, see Appendix A—3).

Electric Stimulus Control (ESC)

These animals were housed in individual voluntary~
activity cages during the adjustment period and in indi-
vidual sedentary cages during the treatment period. Each
ESC animal was permanently paired with a short (SHT)

animal. Only SHT animals were paired with an

37

electrijstimulus—control group (ESC). The SHT program
was selected because these animals received slightly more
shock than the animals on either of the other two forced-
running programs. During the SHT animal's treatment
period, the ESC animals were placed into an attached
adjacent stimulus control cage (21.5 cm. long, 14 cm.
wide, and 10.5 cm. high) with a grid floor comparable to
that of the controlled running wheel (CRW). Each ESC
animal was exposed to the same total light stimulus and

electrical shock as its SHT counterpart.

Swimmingg(SWM)

 

These animals were housed in individual voluntary-
activity cages during the adjustment period and in indi-
vidual sedentary cages during the treatment period.
Animals were swum (28 to 32°C) in individual cylindrical
tanks which measured 28 cm. in diameter and 76 cm. in
depth. On the last four days of the eighth week of this
program, each animal was expected to swim one 60—minute
bout with an attached tail weight equal to 3% of the

animal's weight (for complete program, see Appendix A—4).

Duration Groups

 

To provide chronological perspective of treatment
effects, animals were sacrificed at zero, four, eight,
and twelve weeks after the initiation of treatments. The

respective training requirements (see Appendix A-l through

38

A—4), for the four—week (20 training days), and the eight—
week (40 training days) duration groups (SHT and ESC, MED,
LON, and SWM) were progressively increased. The SHT and
ESC, MED, LON, and SWM twelve-week groups followed their
respective thirty-seventh day schedules during each of the

last 23 training days (see Appendix A-l through A-4).

Treatment Procedures

Treatments began after a lZ—day adjustment period
when all animals were 85 days of age. Animals designated
as zero week control (CON) were sacrificed at the end of
the adjustment period. The SHT and ESC, MED, LON, and
SWM experimental treatments were conducted once a day,
between 12:30 p.m. and 5:30 p.m., Monday through Friday,
in the Human Energy Research Laboratory, Michigan State
University, East Lansing, Michigan. Animal body weights
for SHT, MED, LON, and ESC groups were recorded before
and after each treatment period. Only pretreatment dry
weights were taken for SWM animals.

For each animal in the VOL group, total revolutions
run during the previous 24 hours were recorded, Tuesday
through Friday between 10:00 a.m. and 11:00 a.m.

The SHT, MED, and LON groups and one of the control
treatments (ESC) received treatment in the CRW described
as “ . . . a unique animal—powered wheel which is capable
of inducing small laboratory animals to participate in

highly Specific programs of controlled, reproducible

39

exercise" (Wells and Heusner, 1971). During the first
40-minute learning period in the CRW, the animals ran in
response to shock. The low-intensity, controlled shock
current was applied to the animal through the grid running
surface of the wheel. By the end of the third learning
period, most animals were conditioned to run in response
to a light stimulus which preceded a shock stimulus.

Initially, animals were placed in individually
braked running wheels. For each running period a light
above the wheel signaled the start of a work interval and
remained on for a predetermined time, the acceleration
period. Loss of the light stimulus and application of
the shock stimulus occurred for animals not obtaining
prescribed wheel speeds during the acceleration period.
Animals running slower than the specified speed had the
light and shock sequence repeated. The light was turned
off and no shock current stimulus was applied for animals
obtaining or exceeding prescribed wheel speed. During
the work periods, the wheel was free to turn, while dur-
ing the rest periods, the wheel was automatically braked
to prevent spontaneous activity. A typical running pro—
gram consisted of alternate work and rest periods.

Total revolutions run (TRR) and cumulative duration
of shock (CDS) were recorded from a controlled running
wheel (CRW) attached result unit after each treatment
period for SHT, MED, and LON groups while ESC used SHT

values. These values, with total expected revolutions

40

(TER) and total work time (TWT) (see Appendix A), were
used to calculate percent expected revolutions (PER) and
percent shock free time (PSF). For the SWM group, swim
time completed (STC) was recorded after each treatment
period and used with expected swim time (EST) (see
Appendix A-4) to calculate percent expected swim time

(PET).

Animal Care

 

Since rats are normally more active at night than
during daylight hours, the light sequence in the animal
quarters was automatically timed to reverse the rat's
active period by having the lights off between 1:00 p.m.
and 1:00 a.m. Thus animals were trained during their
active phase and at convenient times.

Standard procedures designed to maintain a rela—
tively constant environment for the animals, such as
daily handling, temperature and humidity control, and
regular cage cleaning, were observed. Throughout the
experiment, all animals had access to water and a com—

mercial animal diet1 at libitum.

Sacrifice Procedures
Fourteen biweekly sacrifices of seven animals of the

same treatment duration were conducted from November 11,

 

.o..- -

lWayne Laboratory—810x, Allied Mills, Inc., Chicago
Illinois.

41

1970 to May 24, 1971. The two initial sacrifices
involved only zero week animals. For all other sacri-
fices, animals were selected after their last treatment
on Friday for sacrifice on the following Monday. Only
animals subjectively determined in good general health
were selected. Performance criteria of 75 percent ex-
pected revolutions (PER) and 75 percent shock-free time
(PSF) were set for the controlled running programs.
Only those SHT, MED, and LON animals whose performance
values were above these criteria were selected for
sacrifice. Proximity to a mean percent expected swim
time (PET) value of 100 was used as a sacrifice selection
criteria to the original animals resulted in a final
sample of 96 animals. The final cell frequencies are
indicated by treatment and duration of treatment in
Table 3. These sacrifices involved one CON animal
plus pairs of animals from one of the following experi-
mental trios; VOL-MED—LON or SWM—SHT-ESC. This sacri-
fice schedule was judged most compatible with treatment
schedules.

Animals were weighed and then sacrificed under
anesthesia by intraperitoneal injection, 4 mg/100 g body

weight of Halatal1 (pentobarbital sodium 64.8 mg).

 

1From Jensen-Salsbery Laboratories, Division of
Richardson-Merrell, Inc., Kansas City, Missouri.

42

TABLE 3.--Final cell frequencies by treatment and duration
of treatment.

 

Duration of Treatment

 

 

Treatment
O-week 4—week 8—week 12-week

Control 2 4 4 4
Voluntary 2 4 4 4
Electric Stimulus

Control 2 4 4 4
Short 2 4 4 4
Medium 2 4 4 3
Long 2 4 3 4

Swimming 2 4 4 4

 

43

Laparotomy, gentle rotation of the abdominal viscera, and
partial removal of parietal peritoneum were performed to
permit withdrawal of 1-2 ml. of blood from the caudal vena
cava. After syringe exchange, 4 m1. of Pelikan ink1 were
injected into the vascular system for subsequent capillary
per muscle fiber calculations. After 3 minutes of 12.2122.
ink circulation, the heart was removed and preserved for
future study.

The right hindlimb was skinned and the superficial
posterior crural muscles were exposed by reflecting the
overlying tissues. The right triceps surae (gastrocnemius
and soleus) and plantaris muscles were removed as a unit.
Similar procedures were followed for the left hindlimb
except the gastrocnemius, soleus, and plantaris muscles
were separated. The left tibialis anterior muscle, the
nerve to the left soleus muscle and the lumbar segments
of the spinal cord were also removed. Only the right
triceps surae and plantaris muscles were used in the
present study, the remaining tissues mentioned above were
preserved for analysis by other members of the research
team.

Upon removal, the right triceps surae and plantaris
unit was rolled in talcum powder. The unit was held with

forceps and lowered, for approximately 60 seconds into

 

1Obtained from John Henschel and Co., Inc.,
Farmington, Long Island, New York.

44

pre-cooled 2-methylbutane (iSOpentane). The iSOpentane
had been previously cooled to a viscous fluid (-l40 to
-185°C) by liquid nitrogen. The frozen muscles were
stored in aluminum 35 mm. film containers in a cryostat
at -20°C until sectioning and histochemical procedures
were initiated. Within 24 hours sandwich blocks (10 mm.
thick) were ablated from the mid-portions of the units
with a pre-cooled stainless steel knife. The sandwich
blocks were frozen onto cryostat chucks using 5% gum
tragacanth. Fresh—frozen, distal-proximal serial cross
sections, were cut at 10 microns using a rotary microtome—
cryostat.1 Sections were mounted on cover glasses and

air-dried for at least one hour.

Histochemical Procedures

Glycogen localization was studied as described by
McManus (1946) using the periodic acid-Schiff reaction
(PAS). Phosphorylase (PPL) was examined by Takeuchi's
(1958) method of inclusion product of polysaccharide and
iodine (clathrate). Succinate dehydrogenase (SDH) locali—
zation was determined using NBT2(2,2'—di-p-nitrophenyl-
5,5'-diphenyl—(3,3'-dimethoxy-4,4'—bipheny1ene)-
ditetrazolium chloride) as the electron acceptor (Barka

and Anderson, 1963). Intermyofibrillar adenosine

 

1International-Harris Microtome--Cryostat, Model CTI
International (IEC) Equipment Co., Needham Heights, Mass.

2Sigma Chemical Company, St. Louis, Missouri.

45

triphosphatase (ATP) localization was investigated employ-
ing the technique described by Wachstein and Meisel (1957)
in calcium—formol fixed fresh frozen sections. Control
sections were included periodically to verify specific
localization patterns.

Incubation times for SDH, ATP and PPL were 45 min,
60 min, and 180 min respectively. Mounting media for the
previous sections was glycerin-jelly while PAS and hema—
toxylin and eosin (H & E) sections were mounted in

Histoclad.

Histological Procedure

 

Harris' alum Hematoxylin andonsin (Gridley, 1960)
was applied to fresh frozen sections for morphological

characteristics.

Tissue Analysis

 

Since muscle fiber pOpulations were known to be
different intermuscularly and intramuscularly, specific
areas of the medial gastrocnemius, plantaris, and soleus
muscles were selected for study (see Figure l). The types
of muscle fibers observed in control animals in the areas
selected were as follows: the medial gastrocnemius muscle
(area I) had high incidence of white fibers; the plantaris
muscle (area II) consisted principally of red and inter—
mediate fibers with occasional white fibers; and the

soleus muscle (area III) had predominantly intermediate

46

fibers with red fibers interspersed. Histochemical charac—
teristics are depicted for these areas in Figures 2 through

12.

Histochemical

 

Microscopic evaluation of succinate dehydrogenase
(SDH), intermyofibrillar adenosine triphosphatase (ATP)
phosphorylase (PPL), and glycogen (PAS) phenotypes was
determined from a group of 50 adjacent muscle fibers in
the three distinctive areas for each animal. To insure
data collection from identical fibers, each group of 50
fibers was traced from the SDH section using a micro-
projector1 at (x208) magnification. Fibers in serial
sections for each histochemical procedure were identified
on the original tracing and rated according to the follow-
ing schema for SDH, ATP and PAS: l = dark intensity, 2 =
intermediate intensity and 3 = light intensity. For PPL
the schema was 1 = blue—violet color, 2 = violet—red to
brown-purple color, 3 = yellow to reddish brown.

Control animals were used to establish individual
sacrifice standards on the basis of reaction intensity
and color for the four histochemical procedures. Except
for the control animals, analyses were performed without
knowledge of the treatment groups. In every case, data
collection was completed before the ensuing biweekly

sacrifice.

 

1Prado Universal, Ernst Leitz GMBH Wetzlar, Germany.

gag dvdlfliljill!
4 . . . .. id.
5.

L:—;’—'—.—'A_L.L ; 'W“

47

PLATE I

Figures 2 through 6 are sections taken from
control animals.

FIGURE 1

A schematic view of the posterior superficial
crural musculature. Histochemical profiles were
classified from intramuscular areas identified
as I, II, and III. (6X).

FIGURES 2, 4, 6

Soleus. PPL, SDH, and ATP respectively. Serial
sections illustrate phenotypic properties of

red (a) and intermediate (b) fibers. Capillaries
(Figures 2 and 6, arrows) and characteristic
subsarcolemmal accumulation of diformazan parti-
cles (Figure 4, arrow) are prominent. (400x).

FIGURES 3, 5

Soleus. SDH and ATP, respectively. End-product
localization identifying typical intermyofibrillar
pattern for red (a) fiber. (500X).

    

GAE’I‘I‘ "L‘I‘I‘LMI U.)

  
          

-_ ‘ h

’f‘.\ 1 0' at!
.01. y

 

49

PLATE II

Figures 7 through 12 are sections taken from
control animals.

FIGURE 7

Medial gastrocnemius. SDH. The large fiber (c)
with a paucity of subsarcolemmal and intermyo-
fibrillar deposition of diformazan particles, is
characteristic of white fibers. The intermediate
fiber (b) has slight subsarcolemmal and moderate
intermyofibrillar end-product deposition. (500X).

FIGURES 8, 10, 12

Plantaris. SDH, ATP, and PPL, respectively. Serial
sections illustrating phenotypic properties of red
(a) and intermediate (b) fibers. Note the marked
heterogeneity of fibers in this muscle compared to
soleus (Figures 2, 4, 6). (400X).

FIGURES 9, 11

Plantaris. SDH and PAS, respectively. Serial
sections illustrating phenotypic properties of
a red (a) fiber. (400K).

50

 

51

Histochemical Interpretation

 

General synopses of the localization and reaction
intensity of end-product in comparable red, intermediate
and white skeletal muscle fibers are indicated in Table 4.
The significance of these histochemical results can only
be interpreted as relative metabolic profile character-
istics brought forth by the particular procedural con-
ditions.

Various techniques (Padykula and Herman, 1955, 1965;
Engel, 1962; Padykula and Gauthier, 1963; Gauthier and
Padykula, 1965; Gauthier, 1967; Karpati and Engel, 1968a;
Severson gt gt., 1968; Brooke and Engel, 1969; Farrell
and Fedde, 1969; Guth and Samaha, 1969, 1970; Tice, 1969;
Eversole and Standish, 1970; Meijer, 1970; Samaha gt_gt.,
1970) have revealed different adenosine triphosphatase
systems and enzymes. In this investigation the Wachstein-
Meisel lead-precipitation technique localized predominantly
intermyofibrillar ATP. The intermyofibrillar region has
many membrane systems (mitochondria, sarc0plasmic
reticulum, and tubular systems) which hydrolyse adenosine
triphosphate or similar engery—rich compounds (creatine
phosphate) to link endergonic processes to others that
are exergonic.

The measure for succinate dehydrogenase (SDH) was
used as an indicator of an oxidation-reduction step in the

tricarboxylic acid cycle. This enzyme catalyzes the

flagellai. I... .-

52

 

 

 

 

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logos cacoHuom
Ac3oun
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onwahuonmmonm
lmcugmaa Amcesneme lflvxume muaeaonoouna . o Lasso
ommumsmmonmauu
enamococm
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ommcomouchcoc
muscAUUSm
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cofluoom mmouo

 

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.Acowuoom mmouov muonau oaomsfi
.oou ca poscoumlcco mo unannoucw cofiuomou can coaumuwHMOOAnl.v wands

53

removal of two hydrogens from succinate to fumarate.
These electrons are carried to the electron transport
system by flavin adenine dinucleotide. Histochemically,
NBT served as the electron acceptor and localization
occurred primarily on mitochondria membranes (Padykula,
1952; Wachstein and Meisel, 1955; Scarpelli and Pearse,
1958; Beckett and Bourne, 1960; Novikoff gt gt., 1961;
Germino gt gt., 1965; Brooke and Engel, 1966; Nystrom,
1966). This aerobic measure was used as an indicator of
resistance to fatigue.

Glycogen (PAS) localization and reciprocal phos-
phorylase (PPL) represented relative anabolic and cata—
bolic processes of a control system. Phosphorylases
it 2332 catalyze reactions leading to formation of
glucose-l-phosphate from glycogen in the presence of
phosphates. The tg'ttttg_reversibility of the reactions
has been used for the histochemical demonstration of
phosphorylase activity (Takeuchi and Kuriaki, 1955;
Takeuchi, 1958; Takeuchi and Sasaki, 1970a, 1970b). This
concentration was measured by the amount of newly formed
polyglucose.

These procedures taken together yield a metabolic
inventory, but did not necessarily include the rate
limiting steps in the various metabolic pathways. It
was unlikely that the histochemical procedures examined

were altered in similar fashion.

54

Statistical Procedures

 

Data were analyzed by treatment groups and durations
by (CDC 3600) computer. Calculations were performed for
means, standard deviations, and simple correlation coef—
ficients for training performance, environmental con-
ditions, and pre- and post-treatment body weights. Since
diameters of wheels attached to the voluntary-activity
cages were less than CRW diameters, daily TRR values of
VOL animals were multiplied by a calibration factor,
0.9163, to equate TRR values for VOL, SHT, MED, and LON
groups.

Mean percent values were calculated for each histo-
chemical rating and the arc sine transformation (angular
transformation) was applied to insure that histochemical
data met the variance homogeneity assumption of analysis
of variance (ANOVA) (Sokal and Rohlf, 1969). This pro—
cedure was repeated for each combination of area and
histochemical technique.

The mean percent values were then analyzed by a
two-way, fixed effects ANOVA model. Complex Scheffé
contrasts were employed to determine the specific signifi—
cant categories within each of the independent variables,
treatment and duration of treatment. For those cate—
gories which were significant, standard Scheffé con-
trasts were used to identify the particular cell means

responsible for the observed significance. The

55

probability of committing a Type I error (a) was set at

.05 for the two-way ANOVA. The probability of committing
a Type II error (8) was set at .25. Significance levels
for the Scheffé tests was held at p = .20 (Scheffé, 1959;

Guenther, 1964).

 

Tit; ..fi.;en-... .11 -

RESULTS

Training Results

Treatments

 

On the basis of the programmed increase of total
expected revolutions (TER) with duration of treatment
(see Appendix A-l through A-3), animals of the long (LON)
group had the largest mean daily total revolutions run
(TRR) increase, medium (MED) next, and short (SHT) the
least. Figure B-4 (see Appendix B), shows that, on the
basis of mean daily TRR, the SHT, MED, and LON animals
met their respective program requirements. Mean daily
TRR of voluntary (VOL) animals did not display a con-
sistent position relative to the programmed running
groups for the first five weeks of training although a
definite trend toward a position between the SHT and MED
groups was evident from six to twelve weeks. This obser-
vation was limited in that the speed and duration of
wheel revolutions run by VOL animals cannot be equated
to those of SHT, MED, and LON animals.

Figures B-l, B—2, and B—3 (see Appendix B) show

that SHT, MED, and LON animals generally maintained

56

57

percent expected revolution (PER) values above 80, thus
exceeding the criterion PER level, 75, of acceptable
execution of program requirements. This level of per—
formance compared favorably with other groups of animals
subjected to similar training programs (Ruhling, 1970).
Figures 8—1, B-2, and B—3 also show that the animals
generally responded to light, rather than electrical
shock, stimuli. Comparisons across treatment duration
of percent shock free time (PSF) values for SHT, MED and
LON groups showed that SHT animals and their electric
stimulus control (ESC) received the most electrical shock.

Almost without exception, percent expected swim
time (PET) values for swimming (SWM) animals were 100
(Table B-2, see Appendix B). Therefore, PET was not
plotted across duration of SWM treatment.

Treatment Environment and
Eddy Weight Values

 

Table B-1 (see Appendix B) shows that SHT, MED, and
LON animals were exercised under conditions of relatively
constant air temperature and barometric pressure and low
humidity. These values did not affect PER and PSF as
reflected in the low correlations among these parameters.
However, animals with relatively high pre-treatment body
weights tended to display low PER and PSF. Animals show-
ing relatively large weight losses tended to display high

PER and PSF. The high correlation coefficient between PER

58

and PSF (Table B-1) confirmed the nearly parallel plot
of these values in Figures B-l, B-2, and B-3 (see
Appendix B).

The SWM animals (Table B-2, see Appendix B) were
exercised under environmental conditions, including water
temperature, comparable to those of SHT, MED, and LON
animals. None of these conditions or pre-treatment body

weight values were highly correlated with PET.

Histochemical Results

 

Individual mean percent values of histochemical
'ratings are tabulated in Appendix C by animal number,
treatment and duration of treatment. The cell mean
percent values of histochemical ratings are depicted by
treatment and duration of treatment in Figures E-l through
E-3 (see Appendix E) for the muscles examined.

Overall Analysis of Variance for Sin"1 Percent

Histochemical Fiber Ratings.--The overall two-way analysis

 

of variance by area, histochemical procedure and rating
are located in Tables D—l through D—3 (see Appendix D).
Complex and Standard Scheffé Analysis for Sin"1

Percent Histochemical Ratings.-—Complex and standard

 

Scheffé contrasts for specific dependent category variable

59

differences within the two independent variables, treat-
ment and duration of treatment1 are located in Tables 5, 6,
7, 8, and 9, 10, ll, 12, respectively. The prominence of
duration, as well as treatment effects, suggested that

the seven different chronic physical activities had
specific effects upon the metabolic characteristics, but
the effects were highly time dependent.

Medial Gastrocnemius.——The presence of significant

 

treatment-duration effects of exercise were evident at
four, eight, and twelve weeks. Similar SDH and ATP
changes occurred for VOL at four weeks, and for LON at
eight weeks with ATP and at twelve weeks with SDH. In—
creases in PAS were found in CON, SHT, MED, and VOL. This
effect occurred for CON and SHT at four weeks, for MED

at eight weeks, and VOL at twelve weeks. The SHT and

SWM groups showed a training effect with PAS measure at
four weeks. Increases in the percentage of intermediate
phosphorylase fibers was seen for ESC at four weeks, and
for MED at eight weeks; while the ESC treatment reverted
to control percentages of phosphorylase fibers at eight
and twelve weeks. Similar ATP changes appeared at twelve

weeks for MED and SWM.

 

1The animals assigned to zero week had received no
experimental treatment. Differences between dependent
categories were not significant. Dependent category values
were pooled, disregarding treatment assignment, to increase
the power of the Scheffé contrasts within duration of
treatment. These animals were designated as zero week
CON (N = 14).

60

TABLE 5.--Summary of Scheffé contrasts for area/histo—
chemical Sin'l percent ratings within duration.

 

Duration (wk)

 

 

 

Area Rating Histochgmical
roce ure 4 8 12
Medial 1 SDH S S N
Gastroc— 2 SDH N N N
nemius 3 SDH N N N
1 ATP N S N
2 ATP N N N
3 ATP N N N
1 PPL S N N
2 PPL S N N
3 PPL N N N
1 PAS S N S
2 PAS S N N
3 PAS N N S
Plantaris l SDH N N N
2 SDH N N N
3 SDH N N N
1 ATP N N N
2 ATP N N N
3 ATP N N N
1 PPL N N N
2 PPL N N N
3 PPL N N N
l PAS N N N
2 PAS N N N
3 PAS N N N
Soleus 1 SDH N N N
2 SDH N N N
3 SDH N N N
1 ATP N N N
2 ATP N N N
3 ATP N N N
1 PPL N N N
2 PPL N N N
3 PPL N N N
l PAS N N N
2 PAS N N N
3 PAS N N N
N = Not significant; 8 = Significant at .20 level.

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61

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63

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64

TABLE 9.--Summary of Scheffé contrasts for area/histochemical Sin"1 percent ratings
within duration of treatment.

 

— - A -—-_- m “—3-

Treatment Group

 

 

CON VOL SHT MED LON ESC SWM
Medial
Gastrocnemius l SDH N s N N N N N
2 SDH N N N N S N N
3 SDH N S N N S N N
1 ATP N S N N S N N
2 ATP N N N N N N S
3 ATP N N N S S S N
1 PPL N N N N N S N
2 PPL N N N S N S N
3 PPL N N N N N S N
1 PAS S S N S N N N
2 PAS S N N N N N N
3 PAS N S N N N N N
Plantaris l SDH N N N N N N N
2 SDH N N N N N N N
3 SDH N N N N N N N
1 ATP N N S N N N N
2 ATP N N S N N N N
3 ATP N S N N N S S
1 PPL N N N N N N N
2 PPL N N N N N N N
3 PPL N N N N N N N
1 PAS N N N N N N S
2 PAS S N N N S N N
3 PAS N N N N N N S
Soleus 1 SDH N N N N S N N
2 SDH N N N N S N N
3 _SDH N N N N N N N
1 ATP N N N N N S N
2 ATP N S N N N S N
3 ATP N N N N N N N
l PPL N N N N N N N
2 PPL N N N N N N N
3 PPL N N N N N N N
1 PAS N N N N S S N
2 PAS N N N N N N N
3 PAS N S N N N S S

 

N I Not significant; S = Significant at .20 level.

65

TABLE lO.--Summary of Scheffé contrasts for histochemical Sin-l
statistically significant duration of treatment in medial gastrocnemius

muscle.

Histochemical

Scheffé Contrast Procedure SDH

 

ATP

percent ratings for

PPL PAS

 

Rating 1 2 3

 

CON-OWK
CON-OWN
CON-OWK
CON-4WK
CON-4WK
CON-BWK

CON-4WK
CON-BWK
CON-IZWR
CON-BWK
CON-IZWK
CON’IZWK

 

VOL-OWN
VOL-OWN
VOL-OWN
VOL-4WK
VOL-4WK
VOL-BWK

VOL-4WK 5+ 5-
VOL-SWK N
VOL-IZWK N
VOL-awn S-
VOL-lZWK s-
VOL-IZWK N

203222

201222

zzzmzz
U)
I

 

MED-OWN
MED-OWN
MED-OWK
MED-4WK
MED-4WK
MED-BWK

MED-4WK .
MED-BWK
MED-IZWK
MED-BWK
MED-IZWK
MED-IZWK

2220122

2222012
2222022

 

LON-OWK
LON-OWN
LON-OWK
LON-4WK
LON-CNN
LON-BWX

LON-4WK N N
LON-8WK N N
LON-IZWK 8+ 5-
LON-BWK N N
LON-12WK 5+ 5-
LON-lZWK N N

22012012

2222012

 

ESC-OWN
ESC-OWN
ESC-OWN
ESC-4WK
ESC-4WK
ESC*BWK

ESC-4WK
ESC-8WK
ESC-IZWK
ESC-BWK
ESC-12W!
ESC-IZWK

0)
+
0)
I
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SWM-OWK
SWM-OWN
SWM-OWN
SWM-4WK
SWM-4WK
SWM-8W!

SWM-4WK
SWM-BWK
SWM-IZWK
SWM-BWK
SWM-IZWK
SWM-IZWK

2220322

 

N:

Not significant

Contrasting significant Scheffé contrasts right to left:
percentage of fibers for a given histochemical procedure/rating at .20; 5+ a increase
in percentage of fibers for a given histochemical procedure/rating at .20.

S- a decrease in

66

TABLE ll.--Summary of Scheffé contrasts for histochemical Sin.l percent ratings for
statistically significant duration of treatment in plantaris muscle.

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. Histochemical
Scheffe Contrast Procedure SDH ATP PPL PAS
Rating 1 2 3 l 2 3 l 2 3
CON-OWN CON-4WK N
CON-OWK CON-BWK N
CON-OWN CON-IZWK S-
CON-4WK CON-BWK N
CON-4WK CON-12WK S-
CON-SWK CON-12WK S-
VOL-OWK VOL-4WK S+
VOL-OWK VOL-BWK N
VOL-OWK VOL-lZWK N
VOL-4WK VOL-BWK N
VOL-4WK VOL-IZWK S-
VOL-SWK VOL-IZWK N
SHT-OWN SHT-4WK N N
SHT-OWN SHT-BWK N N
SHT-OWK SHT-lZWK N N
SHT-4WK SHT—8WK N N
SHT-4WK SHT-IZWK 5- 8+
SHT-OWN SHT-IZWK N N
LON-OWN LON-4WK S+
LON-OWK LON-8WK N
LON-OWK LON-IZWK N
LON-4WK LON-BWK N
LON-4WK LON-IZWK N
LON-OWN LON-12WK N
ESC-OWN ESC-4WK N
ESC-OWK ESC-8WK N
ESC-OWN ESC-12WK 5+
ESC-4WK ESC-BWK N
ESC-4WK ESC-lZWK N
ESC-8WK ESC-lZWK N
SWM-OWN SMW-4WX N S S-
SWM-OWK SWM-BWK N N N
SWM-OWK SWM-lZWK 5+ N N
SWM-4WK SWM-BWK N N N
SWM-4WK SWM-IZWK N N N
SWM-BWK SWM~12WK N N N
N = Not significant.
Contrasting significant Scheffé contrasts right to left: 5- = decrease in
percentage of fibers for a given histochemical procedure/rating at .20; S- = increase

in percentage of fibers for a given histochemical procedure/rating at .20.

67

TABLE l2.--Summary of Scheffé contrasts for histochemical Sin-l percent ratings for
statistically significant duration of treatment in soleus muscle.

 

 

 

 

 

 

Histochemical

Scheffé Contrast Procedure SDH ATP PPL PAS

Rating 1 2 1 2 1 2 1 2 3
VOL-OWK VOL-4WK N S-
VOL-OWK VOL-BWK S N
VOL-OWK VOL-IZWK N N
VOL-4WK VOL-SWK N 5+
VOL-4WK VOL-12WK N N
VOL-BWK VOL-IZWK N N
LON-OWK LON-«WK N N N
LON-OWK LON-BWK N N N
LON-OWK LON-IZWK S+ 5- S
LON-4WR LON-BWK N N N
LON-4WK LON-IZWK S+ S- N
LON-BWK LON-IZWK N N N
ESC-OWK ESC-4WK S- S N N
ESC-OWK ESC-BWK N N S S-
ESC-OWK ESC-IZWK N N N N
ESC-4WK ESC-BWK N N N N
ESC-4WK ESC-IZWK S+ N N N
ESC-BWK ESC-IZWK N N N N
SWM-OWK SWM-4WK N
SWM-OWK SWM-BWK S-
SWM-OWK SWM-IZWK N
SWM-4WK SWM-BWK N
SWM-4WK SWM-IZWK N
SWM-BWK SWM-IZWK N

 

 

N a Not significant.

Contrasting significant Scheffé contrasts right to left: 3- - decrease in
percentage of fibers for a given histochemical procedure/rating .20; 5+ = increase
in percentage of fibers for a given histochemical procedure/rating .20.

68

Plantaris.--None of the Scheffé contrasts within the

 

duration groups for category variables were significant.
Between durations similar PAS changes were found at four
weeks for LON and SWM. ESC and SWM groups produced sig-
nificant increase in the percentage of low ATP fibers at
twelve weeks, while VOL produced the same result at four
weeks. The CON showed a change in the percentage of

intermediate PAS fibers at twelve weeks. The SHT group

 

activity produced a significant increase in the percentage
of intermediate ATP fibers from four to twelve weeks.
Soleus.--None of the Scheffé contrasts within the
duration groups for category variables were significant.
Between durations the LON endurance group required twelve
weeks to produce a directly related change in SDH and PAS.
Similar PAS changes occurred for VOL at four and eight
weeks. VOL activity produced a significant increase in
the percentage of intermediate ATP fibers at eight weeks.
Similar changes were produced in the ESC group at four
weeks but such changes were reversed at twelve weeks.

General Patterns of Metabolic Response.--The promi-

 

nence of duration, as well as treatment, effects sug-
gested further insight into category variables for rela—
tive analysis of the regional, temporal and specific
response to identical and diverse physiologic stimuli.
Frequency weighted scores were calculated (frequency of

fibers times the intensity rating of the enzyme or

69

substrate). These cell weighted values are presented by

area, treatment and duration in Figure 13.

70

 

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DISCUSSION

The attainment of combinations of seven distinct
levels and four durations of chronic physical activity
permitted meaningful interpretation of the histochemical
and exercise related data. The control (CON) animals
were confined to individual sedentary cages, and there—
fore represented a low level activity group. The con-
trolled running and swimming programs were designed to
produce divergent activity levels (see Appendix A—l
through A—4). The performance values (Figures B-l through
B-3, and Tables 8-1 and B-2, see Appendix B) established
that the short (SHT), medium (MED), long (LON), and
swimming (SWM) groups represented four different physio—
logical types and intensities of chronic physical activity.
Figure 8-4 (see Appendix B) indicated a separation of
activity levels, on the basis of mean daily total revolu-
tions run (TRR), for voluntary (VOL), short (SHT), medium
(MED), and long (LON) groups.

The decision to pair electric stimulus control (ESC)
and SHT animals was correct in that the SHT animals

received the largest amount of electrical shock (see

71

 

72

Figures 8-1, B—2, and 8-3, Appendix B). The ESC animals
served as a shock control group, and the CON animals
served as an environmental living control. However, the
amount of muscular activity induced in the ESC animals
was not known due to the static physical response of the
ESC animals to the noxious stimuli.

The histochemical evaluations were made on fiber
types1 selected from distinct intramuscular areas of
medial gastrocnemius, plantaris, and soleus muscles. It
was known that muscle fibers vary intermuscularly and
intramuscularly. The histochemical procedures utilized
elicited markedly distinct patterns and intensities for
the individual muscle cells in these areas.

This study represented an initial step in estab—
lishing histochemical profiles for individual fibers.
Further, this study provided information on the alter-
ation of fiber characteristics and distribution in re-
Sponse to various types and durations of physical
activity.

It was hypothesized that the histochemical profiles
of enzymes and substrates would indicate adaptive re-

sponses by area to the different levels of chronic activity.

 

1The term histochemical fiber type implies a static
situation. This writer, however, acknowledges a classifi—
cation on a more dynamic basis for individual cullular
metabolic activities. In general, distinct intensity
relationships occur between the various histochemical
measures.

 

73

The results obtained were somewhat unexpected in
relation to those of previous investigations (Edgerton
§E_al,, 1969; Kowalski §t_al., 1969; Dorn et 31., 1970;
Spurway and Young, 1970; Campbell gt al., 1971). In the
present study, regional and temporal patterns of change
frequently were different for the various exercise regi-
mens. The prominence of duration, as well as treatment,
effects suggested that the seven different chronic physi-
cal activities had specific effects upon the alteration
of fiber characteristics and fiber type distribution, but
the effects were highly time dependent. Since the program
requirements were progressively increased, it was observed
that weekly changes in programs brought about concomitant
changes in fiber type profiles. Recent evidence suggested
that muscle cells continually undergo alteration in
adaptation to functional demands (Baker and Laskin, 1969;
Guth and Yellin, 1971). It seems reasonable that cardio-
vascular and respiratory factors interact with auto~
regulatory, hormonal, and neurotrophic processes to
influence the regulation of muscle cell metabolism. The
present results supported the concept of alteration of

fiber types as an effect of physical activity.

Patterns of Adaptation

 

Various patterns of adaptation are suggested from
the weighted frequency values for individual areas by

treatment and the duration of treatment as shown in

 

74

Figure 13. It was apparent that the three areas examined
reflected ranges of both local aerobic and anaerobic
related responses. A number of adaptive processes evi—
dently occurred in exercising muscle. However, these
processes did not take place in a continuous linear
fashion, but seemed to proceed concomitantly with meta-
bolic need.

Soleus.--The patterns of adaptation in Figure 13
showed significant temporal changes for periodic acid—
Schiff's reaction (PAS) in soleus fibers for all the
experimental groups. In addition, VOL and SHT groups
showed cyclic increases and decreases in glycogen levels.
Seemingly, an increase in the frequency of stimulation
caused an increase in glycogen content. It was interest—
ing that the long-duration, low—intensity LON and SWM
group and medium duration, moderate intensity MED groups
showed progressive increases in glycogen content. The
increased glycogen content in soleus might be the result
of increased glycogen synthetase activity (synthesis) or
decreased utilization (degradation) of existing glycogen
stores. Both the present results and other findings
(Bass EE.§$°' 1955; Kugelberg and Edstrfim, 1968; Gutmann,
1970) suggested that highly repetitive low frequency
discharge to slow motor units served to increase glycogen
content. Extension of these programs over a longer treat-

ment period should partition the adaptive responses.

 

75

Plantaris.-~The muscle fiber pOpulation examined

 

in the plantaris showed increased glycogen content for
VOL, SHT, MED, LON, and SWM groups at different durations
of treatment. The SWM and ESC groups showed marked in-
creases in glycogen content between zero and four weeks,
whereas, the SHT and MED groups showed marked increases
in glycogen content between zero and eight weeks. The
periodicity of glycogen values illustrated that specificity
of training was a reality. The plantaris thus reflected
that adaptive mechanisms proceed in phases according to
the particular overload stress. For most measures, the
heterogeneous population sampled seemed resistant to
change.

Medial Gastrocnemius.--It was evident that white

 

(anaerobic) fibers acquired red (aerobic) fiber charac—
teristics, as a response to physical activity. The
running groups, MED and LON, had the greatest proportional
increases in intermyofibrillar adenosine triphosphatase
(ATP) and succinate dehydrogenase (SDH) fibers (see
Figure 13). Previously, Carrow et a1. (1967), found an
increase in capillary per fiber ratio and Ashmore and
Doerr (1971b) supported an increase in blood flow in
white muscle with repetitive exercise. Ashmore and
Doerr (1971b), as well as other authors (Edgerton gt_al.,
1969; Kowalski e_t_ a” 1969; Barnard 935 _a_]._., 1970)

hypothesized white to red fiber transformation after

 

76

repetitive exercise. Mai §t_al. (1970) failed to show
increased capillary per fiber ratio for white fibers and
indicated that an increase in capillarity in the white
zone was due to an increase in capillarity of red fibers.
Reference to these facts, indicated that the transfor—
mation of white to red fiber characteristics was probably
facilitated by circulatory mechanisms.

The histochemical changes resulting from this study,
where several intensities and durations of exercise were
considered separately and in combination, supported the
concept of "specificity of exercise." Other authors
(Edgerton st 31., 1969; Kowalski gt_al., 1969; Dorn 33 21.,
1970; Spurway and Young, 1970; Barnard et_al., 1970)
worked with durations from nine to eighteen weeks, and
intensities of 2.7 ft/sec. or less, and reported increases
in oxidative fiber populations but no changes in fiber
proportions of adenosine triphosphatase or phosphorylase.
In contrast, this study showed changes occurred for phos-
phorylase, glycogen, oxidative enzymes, and intermyo—
fibrillar adenosine triphosphatase at durations of four,
eight, and twelve weeks when the velocities were essenti-
ally doubled (4.0 ft/sec. and 5.5 ft/sec.).

It should be noted that biochemical homogenate
studies (Holloszy, 1967; Barnard gt 31., 1970) have
failed to show changes in adenosine triphosphatase after

exercise periods as long as eighteen weeks. The present

 

77

histochemical fiber analysis did reveal changes in
adenosine triphosphatase fiber populations. This finding
could be attributable to the intensity of the programs
used in this study; however, diverse regional histo-
chemical responses to the same stimuli point out that
whole muscles should not be studied as biochemical
entities.

From the results of this study, it can be specu—
lated that the intracellular components are capable of
selective adaptation. If this is the case, the basic
unit underlying specificity of adaptation must be either

the muscle fiber or the highly intermingled motor units.

Interregional Comparisons

 

Several common statements can be made regarding the
areas examined. The fibers selected for study in the
soleus, plantaris and medial gastrocnemius muscles repre-
sented distinct populations. These pOpulations revealed
diverse responses to the same exercise stimuli (see
Figure 13). The metabolic profiles of the muscle areas
under consideration confirmed previous findings (Buller
and Lewis, 1965a; Hnik gt 31., 1967; Guth, 1968; Ashmore
and Doerr, 1971b) that muscle metabolic characteristics
can change. The previous authors established that after
a variety of surgically induced conditions, which modi—
fied the neural input to the muscle, alterations occurred

in energy metabolism. In this study, high work loads

 

78

produced similar results in intact animals. Continual
use of leg muscles in endurance type activity, resulted
in white fibers acquiring red-fiber characteristics.

The presence of significant (P < .05) training,
duration, and interaction effects of exercise on meta—
bolic profiles strongly supports the fundamental principle
of muscle fiber mutability and the hypothesis of specifi—
city of alteration. It should be noted that the observed
differences were specific not only to the types, inten—
sities and durations of exercise employed, but also to
the fiber populations studied. For example, in Figure 13,
the greatest relative rise in ATP occurred in the medium
(MED), and long (LON) running groups. The observed ATP
changes in these two groups took place only in the medial
gastrocnemius muscle and between four and twelve weeks for
the MED group and between zero and eight weeks for the
LON group.

The changes observed reflect the fact that muscle—
fiber metabolic characteristics can be altered under normal
physiological conditions. These alterations in normal
intact animals are in line with the suggestion by Guth
and Yellin (1971) that plasticity is the rule rather than
the exception.

Medial Gastrocnemius.—-Endogeneous glycogen changes

 

paralleled previous histochemical and biochemical findings

(Beatty 23 31., 1966; Edgerton 33 31., 1970). Voluntary

 

79

(VOL), SWM, and LON groups tended to concentrate glycogen,
whereas the SHT and MED groups had less concentration,
indicating active glycogenolysis and glycogenesis. The
phosphorylase values were fairly steady over the twelve-
week treatment period for the VOL, SWM, and LON groups.

It was reaspnable that more glycogen was broken down with
higher frequency activity than lower frequency activity.
Sources of carbon other than glycogen are probably utilized
as white fibers assume characteristics of red fibers.

Fats and free fatty acids are easily obtained and broken
down by oxidative mechanisms. The increased indices
reflecting energy-supplying systems (SDH and ATP) for VOL
at four weeks indicated such a transformation. Further,
the dramatic increase in ATP for the LON group at eight
weeks probably was necessary to supply energy for sus-
tained contraction. The lack of significant changes in
the SHT group would seem to suggest that the recovery
period was of sufficient length for resynthesis of enzymes
and substrates.

Plantaris.--The fact that the fibers tended to

 

become "redder" with the SDH index in response to endur-
ance activity was not totally unexpected. Since Edgerton
2E 31. (1969) and Kowalski et 31. (1969) reported in—
creases in the proportions of red fibers as identified

by malate dehydrogenase and succinate dehydrogenase.

 

 

80

The only significant histochemical differences found
were in endogeneous glycogen and intermyofibrillar ATP.
This normally red and intermediate fiber population
assumed the characteristics of a more uniform population
of intermediate fibers, again suggesting that red and
white fiber characteristics can approach each other. For
the most part, this heterogeneous area had sufficient
enzymatic capacities to maintain equilibrium under varied
exercise stresses.

Soleus.--In the soleus muscle, phosphorylase and
succinate dehydrogenase fiber profiles were relatively
unchanged by the exercise programs. The red fibers of
the soleus muscle had both anaerobic and aerobic capaci—
ties. This finding suggested that these fibers, although
in the minority, were endowed with enzymes to support both
aerobic and anaerobic metabolism and were able to provide
the necessary requirements for both short and sustained
activity. The results confirmed the earlier histochemical
findings of Edgerton g£_al. (1969).

The duration effects were limited to PAS, ATP, and
SDH. All experimental treatments produced an increase in
PAS values at four and twelve weeks in contrast to those
found at zero weeks. Increased glycogen content was more
evident in red muscle. This increase was probably due to
higher levels of glycogen synthetase (Beatty gt 31.,

1963; Gillespie 3g 31., 1970).

 

81

The controlled running groups showed increased SDH
intensity at twelve weeks, but only the LON result was
statistically significant. The LON group probably re-
quired higher resistance to fatigue, and an appropriate
alteration occurred. Overall, it appeared that the soleus
contained glycolytic and oxidative enzymes in a sufficient
number of fibers to cope with the exercise stresses

afforded by these programs. Reed (1971) in an accompany-

 

ing study on succinate dehydrogenase and motor endplate
cholinesterase, found significant duration effects for
the controlled running groups in the soleus SDH measure.
This effect was seen at twelve weeks for the LON group,
and agreed with the present findings. Extension of the
treatment period might reveal even more prominent
treatment-duration differences.

It was obvious that in examining metabolic adaptation
to physical stress at the tissue level multiple factors
must be considered. These factors include: age of animals,
confinement of animals, types of treatment, duration of
the treatment, muscle or region of muscle examined, and
histochemical methods. Furthermore, studies incorporating
only one type or duration of treatment to test the hy-
pothesis that muscle adapts selectively to physical
activity may be either positive or negative in their

findings. A variety of treatments and durations of

 

g}. . V Juana-431.41.!
. w.

82

treatment, as incorporated in this study, provided chrono-

logical evidence to distinguish the various patterns of

response.

Limitations of this Study and Suggestions
for Future Studies

 

Limitations of the Study

 

1.

The relative histochemical reactivity can only be
indicative of the amount of accumulated end-
product, elucidating phenotypic patterns, rather
than necessarily reflecting the amount or location

of the reaction product.

The interpretation of results is restricted to
normal, adult, male, albino rats of the Sprague-

Dawley strain.

The subjective classification for the specific
muscle areas examined are restricted to analysis

of that particular area.

Data were collected and analyzed from only three

subjectively defined muscle areas.

The application of histochemical procedures neces-
sarily limited the collection of physiological

and biochemical data.

 

 

.. My

 

83

Suggestions for Future

 

Studies

1.

A similar study should be completed which encom-
passes a greater variety of low and high intensity
physical activity. The training period should be
extended to determine the duration of treatment F
effect. A six—month program might yield inter-

esting findings.

 

Any further histochemical study should be corre—
lated with apprOpriate physiological analysis of

the motor unit and quantitative enzyme assays.

Study of additional enzymes is necessary for a
more complete inventory to extend the present
findings. The rate limiting step enzymes (lactate
dehydrogenase, glyceraldehyde-3-phosphate dehydro-

genase) should be investigated.

The cellular modifications should be examined by

transmission electron microscopy.

Analysis of histochemical profile of the motor
unit should be delineated in an entire muscle;

rather than area analysis.

The possibility of change in numbers of fibers

per unit area should be investigated.

SUMMARY AND CONCLUSIONS

Summary

This study was undertaken to determine the effects

 

of seven different exercise regimens on the histochemical

 

characteristics and distribution of selected muscle fibers
of the triceps surae and plantaris muscles of albino rats.

One hundred and seventy-six 72-day-old, normal, male,
albino rats (Sprague—Dawley Strain) were randomly assigned
to one of seven treatments. Treatments began after a 12-
day adjustment period when all animals were 85 days of
age. The treatment groups were sedentary control (CON);
voluntary running (VOL); short—duration, high-speed
endurance running (SHT); medium-duration, moderate-speed
endurance running (MED); long—duration, low-speed endur—
ance running (LON); electric stimulus control (ESC); and
long-duration, low-intensity swimming (SWM). Treatments
were administered once a day, Monday through Friday. All
animals had access to food and water ad libitum.

Only animals subjectively determined to be in good
general health were selected for sacrifice. Performance

criteria of 75 percent expected revolutions (PER) and

84

85

75 percent shock-free time (PSF) were set for controlled
running programs. Only those short (SHT), medium (MED),
and long (LON) animals whose performance values were above
these criteria were selected for sacrifice. Proximity to
a mean percent expected swim time (PET) value of 100 was
used as a sacrifice selection criterion for the swimming
(SWM) program. Seven animals, limited to the same dur—
ation, were weighted and sacrificed on Mondays after zero,
four, eight, and twelve weeks of training. The final
sample consisted of 94 animals.

Animals were sacrificed under anesthesia by intra-
peritoneal injection of pentobarbital sodium. Pelikan
ink was injected into the vascular system for capillary
per muscle fiber calculations. The triceps surae and
plantaris muscles were remoVed as a unit, rolled in talcum
powder and frozen in an iSOpentane-liquid nitrogen system.
Fresh-frozen, distal-proximal serial cross sections, were
cut at 10 microns using a rotary microtome-cryostat. Four
histochemical procedures were utilized for identification
of relative fiber type intensities of glycogen (periodic
acid-Schiff), glycolytic enzyme (phosphorylase), oxidative
enzyme (succinate dehydrogenase), and energy producing
systems (adenosine triphosphatase). Fifty muscle fibers,
from each specific intramuscular area of medial gastroc—
nemius, plantaris, and soleus muscles, were graded accord—

ing to intensity and distribution patterns of the various

 

86

histochemical procedures. The percentages of intensity
ratings were recorded for individual animals.

The prominence of duration, as well as treatment,
effects suggested that the seven different chronic physi-
cal activities had specific effects upon the alteration
of fiber characteristics, but the effects were highly
time dependent.

The results indicated there were diverse regional
responses and patterns of change over time to the same
exercise stimulus. Four, eight, and twelve weeks of
various activity programs produced metabolic alterations
in proportions of fiber types in the medial gastrocnemius,
plantaris, and soleus muscles.

In the soleus muscle, similar periodic acid-Schiff
(PAS) changes occurred for the voluntary group at four
and eight weeks. Voluntary (VOL) group activity produced
a significant increase in the percentage of intermediate
ATP fibers at eight weeks, while the electric stimulus
control (ESC) treatment produced a similar significant
increase at four weeks, which was reversed at twelve
weeks (p < .20).

In the plantaris muscle similar increases in PAS
were found at four weeks for long (LON) and swimming (SWM)
groups (p < .20). The electric simulus control (ESC)
and SWM programs produced significant increases in the

percentage of low intensity ATP fibers at twelve weeks,

 

87

while the VOL treatment produced the same result at four
weeks. The short (SHT) group showed a specific pattern
of increasing intermediate ATP fibers from four to twelve
weeks (p < .20).

The general adaptive patterns for the medial gastroc-
nemius muscle showed that anaerobic fibers were able to
acquire aerobic fiber characteristics and supported the
hypothesis of specificity of alteration. The greatest
relative rise in ATP occurred in the medium (MED) and long
(LON) running groups. This change was observed between
four and twelve weeks for the MED group and between zero

and eight weeks for the LON group.

Conclusions

 

1. Histochemical profiles, for medial gastrocnemius,
plantaris, and soleus muscle areas can be influ—
enced differentially by specific types and dur-

ations of physical activity.

2. Alterations in histochemical profiles can be

enzyme or substrate specific.

3. Selective adaptation occurs at the individual
muscle fiber level, or by motor units, but not

homogeneously in an entire muscle complex.

 

 

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APPENDICES

 

 

 

APPENDIX A

TRAINING PROGRAMS

107

IABLE A-I.-- Standard eIght-week, short-duratlon, hlqh—speed endurance tralnlnq
program for postpubertal and adult male rats In controlled runnlnq wheels.

 

 

Intel Iolnl

 

 

ACC- Repe- Tlm IIM Exp. lulal
aler- dork tl- Bet- Run 01 Revo- Work
Day Day ation llme Rest tlons No. ween Speed Prog. Iu- lime
at at Ilme (mln: llme par of Bouts Shock (tt/ (min: tlons (sec)
Hk Uh. Tr. (sec) sec) (sec) Bout Bouts (min) (ma) soc) sec) TER TH?
0 4st -2 3.0 40:00 lo I I 5.0 0.0 I.5 40:00 --- ---
S-F -l 3.0 40:00 I0 I I 5.0 0.0 I.5 40:00 --- ---
I Is" I 3.0 00:l0 IO 40 3 5.0 I.2 I.5 49:30 450 I200
2-T 2 3.0 00:l0 IO 40 3 5.0 I.2 I.5 49:30 450 1200 ”’5
3-W 3 3.0 00:I0 I0 40 3 5.0 I.2 I.5 49:30 450 I200 .
4-7 4 2.5 00:l0 I0 40 3 5.0 I.2 2.0 49:30 600 I200 ’: _':-
5-F 5 2.0 00 I0 IO 40 3 5.0 I.2 2.0 49:30 600 l200 2 4
2 I-M 6 I.5 00 l0 IO 28 4 5.0 I.2 2.5 5|:40 700 Il20 ;
2-1 7 I.5 00 I0 IS 27 4 5.0 I.2 3.0 59:00 8I0 l080 “
3th 8 l.3 00:l0 I5 27 4 5.0 I.2 3.0 59:00 BIO l080 I
4-T 9 I.5 002|0 I5 27 4 5.0 I.2 3.0 59:00 8l0 l080
5-F l0 I.5 00:l0 I5 27 4 5.0 I.2 3.0 59 00 8I0 l080
3 I-M II I.5 00:I0 I5 27 4 5.0 I.2 3.0 59 00 8I0 l080
2-7 I2 I.5 00 IO 20 23 4 5.0 I.2 3.5 59 40 805 920 .:
3-9 I3 I.5 00:I0 20 23 4 5.0 I.2 3.5 59:40 805 920
4-1 l4 I.5 00:I0 20 23 4 5.0 I.2 3.5 59:40 805 920
53F l5 I.5 00 I0 20 23 4 5.0 I.2 3.5 59:40 805 920
4 l-M l6 I.5 00:l0 20 23 4 5.0 I.2 3.5 59:40 805 920
2-T l7 I.5 00 I0 25 20 4 5.0 I.0 4.0 60:00 800 800
3-8 l8 I.5 00:l0 25 20 4 5.0 I.O 4.0 60:00 800 800
4-1 l9 I.5 OO:I0 25 20 4 5.0 l.0 4.0 60:00 800 800
5-F 20 I.5 00:I0 25 20 4 5.0 l.0 4.0 60:00 800 800
5 I-N 2| I.5 00:I0 25 20 4 5.0 l.0 4.0 60:00 800 800
2=T 22 I.5 00 IO 30 I6 4 5.0 I.0 4.5 55:40 720 640
SIN 23 I.5 00:I0 30 I6 4 5.0 I.0 4.5 55:40 720 640
4-T 24 I.5 00 IO 30 I6 4 5.0 I.0 4.5 55:40 720 640
5=F 25 I.5 00 I0 30 I6 4 5.0 I.0 4.5 55:40 720 640
6 I-M 26 I.5 00 I0 30 I6 4 5.0 l.0 4.5 55:40 720 640
2-T 27 2.0 00:I0 35 I0 5 5.0 l.0 5.0 54:35 625 500
s-w 28 2.0 00:l0 , 35 lo 5 5.0 l.0 5.0 54:35 625 500
4-T 29 2.0 00 IO 35 lo 5 5.0 l.0 5.0 54:34 625 500
5aF 30 2.0 00:I0 35 IO 5 5.0 l.0 5.0 54:35 625 500
7 I-M 3I 2.0 00:I0 35 IO 5 5.0 l.0 5.0 54:35 625 500
2-T 32 2.0 00:I0 35 7 8 2.5 l.0 5.0 54:50 700 560
3" 33 2.0 00:l0 35 7 8 2.5 l.0 5.0 54:50 700 560
421 34 2.0 00 lo 35 7 8 2.5 I.0 5.0 54:50 700 560
5-F 35 2.0 00:I0 35 7 8 2.5 I.0 5.0 54:50 700 560
8 II" 36 2.0 00:!0 35 7 8 2.5 I.0 5.0 54 50 700 560
2:1 37 2.0 00:I0 40 6 8 2.5 l.O 5.5 52.l0 660 480
3-8 38 2.0 00 I0 40 6 8 2.5 l.0 5.5 52 I0 660 480
4-T 39 2.0 00:I0 40 6 8 2.5 I.O 5.5 52:l0 660 480
S-F 40 2.0 00:l0 40 6 8 2.5 I.0 5.5 52:l0 660 480

 

Thls standard program was deslgned uslng male rats of the Sprague-Dawley straln.
All anlnals were between 70 and I70 days-ot-age at the beglnnlng ot the program.
The duration and Intenslty ot the program were establlshed so that 75 per cent of
all such anlaals should have P5? and PER scores of 75 or hlgher durlng the llnal
two weeks. Alteratlons In the work tlne, rest tlna, repetltlons per bout, number
of bouts, or tlae between bouts can be used to affect changes In these values.
Other stralns or ages of anlaels could be expected to respond dltterently to the
progral.

All enlaals should be exposed to a mlnlnum ot one week at voluntary runnlng
In a wheel prlor to the start of the program. Fallura to provlde thls adjustaent
perlod wlll laposa a double learnlng sltuatlon on the anlnals and wlll serlously
Impalr the etlectlvaness of the traInlng program.

Standard short-duratlon, hlgh-speed endurance nalntenance prograe tor postpubertal
and adult male rats In controlled runnlng wheels.

 

Total lotal
ACC' Rape- Tlme Tlme Exp. Total
aler- Hort tl- Bet- Run of Ravo- work
atlon Tlae Rest tions No. ween Speed Prog. lu- Tlme
III. (nln: Tine oer ol Bouts Shock (tt/ (min: tlons (sec)

(sec) sec) (sec) Bout Bouts Imlnl (ma) sec) sec) 78R TNT

I.5 00:30 30 6 3 5.0 l.0 4.0 26:30 540 540

108

TABLE 8-2.-- Standard elght-week, aedlum-duratlon, moderate-speed endurance tralnlng
program tor postpubertal and adult male rats In controlled running wheels.

 

Total Total

 

 

Acc- Repe- Tlee Tlme Exp. Total
eler- Hark tl- Bet- Run at Ravo- Hork
Day Day atlon Tlme Rest tions No. ween Speed Prog. lu- Time
of at Tlme (mln: Tlae per ot Bouts Shock Itt/ (mln: tlons (sec)
1k. 8k. Tr. (sec) sec) (sec) Bout Bouts (mln) (ma) sec) sec) 138 THE
0 4-T -2 3.0 40:00 I0 I I 5.0 0.0 I.5 40:00 --- ---
5-F -I 3.0 40:00 I0 I I 5.0 0.0 I.5 40:00 --- ---
I II“ I 3.0 00:I0 I0 40 3 5.0 I.2 I.5 49:30 450 I200
ZIT 2 3.0 00:I0 I0 40 3 5.0 I.2 I.5 49:30 450 I200
3-w 3 3.0 00:I0 I0 40 3 5.0 I.2 I.5 49:30 450 I200
4-T 4 2.5 00:I5 l3 l9 4 5.0 I.2 2.0 52 00 570 II40
5-F 5 2.5 00:I5 I5 I9 4 5.0 I.2 2.0 52:00 570 II40
2 I-M 6 2.0 00:I5 I5 l9 4 5.0 I.2 2.0 52:00 570 II40
2-T 7 2.0 00:I5 I5 l9 4 5.0 I.2 2.5 52:00 7l2 II40
3-8 8 I.5 00:I5 I5 I9 4 5.0 I.2 2.5 52:00 7I2 II40
4'T 9 I.5 00:I5 I5 I9 4 5.0 I.2 2.5 52:00 7I2 II40
S-F I0 I.5 00:I5 I5 l9 4 5.0 I.2 2.5 52:00 7l2 II40
3 I.“ II I.5 00:I5 I5 I9 4 5.0 I.2 2.5 52:00 7|2 II40
2-T I2 I.5 00:I5 I5 I8 4 5.0 I.2 3.0 50:00 8I0 I080
3-I I3 I.5 00:I5 l5 I8 4 5.0 I.2 3.0 50:00 8I0 I080
4-T I4 I.5 00:I3 I5 I8 4 5.0 I.2 3.0 50:00 8l0 I080
5sF I5 I.5 00:I5 l5 I8 4 5.0 I.2 3.0 50:00 8l0 I080
4 I-M l6 I.5 00:I5 I5 l8 4 5.0 I.2 3.0 50:00 8I0 l080
ZIT I7 I.5 00:I5 I5 I8 4 5.0 I.0 3.5 50:00 945 I080
3th I0 I.5 00:I5 I5 I8 4 5.0 I.0 3.5 50:00 945 I080
4=T I9 I.5 00:I5 I5 I8 4 5.0 I.0 3.5 50:00 945 I080
5-F 20 I.5 00:I5 I5 I8 4 5.0 l.0 3.5 50:00 945 I080
5 I-H 2| I.5 00:I5 l5 I8 4 5.0 I.0 3.5 50.00 945 l080
2-T 22 I.5 00:I5 l5 I4 5 5.0 I.0 4.0 53:45 l050 l050
3-I 23 I.5 00:I5 I5 l4 5 5.0 I.0 4.0 53:45 I050 l050
4-T 24 I.5 00:I5 I5 I4 5 5.0 I.0 4.0 53:45 I050 I050
5-F 25 I.5 00:I5 I5 l4 5 5.0 l.0 4.0 53:45 I050 I050
6 III 26 I.5 00:I5 I5 l4 5 5.0 l.0 4.0 53:45 l050 I050
2-T 27 I.5 00:20 20 II 5 5.0 I.0 4.0 55:00 IIOO IIOO
3*! 28 I.5 00:20 20 II 5 5.0 I.0 4.0 55:00 II00 IIOO
4-T 29 I.5 00:20 20 II 5 5.0 l.0 4.0 55:00 IIOO IIOO
5-F 30 I.5 00:20 20 ll 5 5.0 I.0 4.0 55:00 IIOO IIOO
7 l-M 3| I.5 00:20 20 II 5 5.0 I.0 4.0 55:00 IIOO IIOO
Z-T 32 I.5 00:25 25 9 5 5.0 I.0 4.0 55:25 II25 II25
3-i 33 I.5 00:25 25 9 5 5.0 I.0 4.0 55:25 Il25 II25
4-7 34 I.5 00:25 25 9 5 5.0 I.0 4.0 55:25 II25 ll25
5" 35 I.5 00:25 25 9 5 5.0 I.0 4.0 55:25 II25 II25
8 I-M 36 I.5 00:25 25 9 5 5.0 l.0 4.0 55:25 Il25 ll25
2-T 37 I.5 00:30 30 8 5 5.0 I.0 4.0 57:30 I200 I200
3d! 38 I.5 00:30 30 8 5 5.0 I.0 4.0 57:30 I200 l200
4-T 39 I.5 00:30 30 8 5 5.0 I.0 4.0 57:30 l200 I200
5-F 40 I.5 00:30 30 8 5 5.0 l.0 4.0 57:30 l200 I200

 

Thls standard program was designed uslng male rats of the Sprague-Dawley Straln.
All anlmals were between 70 and I70 days-ot-age at the beglnnlng ot the program.
The duratlon and lntenslty ot the program were established so that 75 per cent of
all such anlaals should have PSF and PER scores ot 75 or hlgher durlng the Ilnal two
weeks. Alteratlons In the rest time, repetltlons per bout, number of bauts, or tlme
between bouts can be used to affect changes In these values. Other stralns or ages
at anlmals could be expected to reSpond ditterentIy to the program.

All animals should be exposed to a minlmum at one week of voluntary running In
a wheel prIor to the start of the training program. Failure to provIde thls adjust-
ment perIod wlll Impose a double learnlng sltuatlon on the anlmnls and will serlously
Impalr the ettectlveness of the training program.

Standard medium-duration, moderate-speed
endurance malntenance pr ram Ior
postpubertal and adult male rats In controlled running wheelz?

 

Total Total

Acc- Repe- Time Tlme Exp. Total
aler- Horn tI- Bet— Run of Revo- Work
atlon Tlme Rest tlons No. ween Speed Proq. Iu- Time
Time (mln: Time per ol neuts Shock Itt/ Imln: tlons (sec)

(sec) sec) (sec) Bout Boots (mln) (mu) sec) sec) TER TNT

2.0 00:I0 40 4 6 2.5 I.0 5.5 28:30 330 240

TABLE A-3.-- Standard elght~week, long-duration, law-speed endurance training

109

program for postpubertal and adult male rats In controlled runninq wheels.

 

 

Total Total

Acc- Rope- Time Time Exp. Total

aIer- Work ti- Bet- Run of Revu- Work

Day Day ation Time Rest tions No. ween Speed Prog. Iu- Time
of of Time (min: Time per of Bouts Shock (ft/ (min: tions (sec)

Hk. Wk. Tr. (sec) sec) (sec) Bout Bouts (min) (ma) sec) sec) TER TV?
0 4=T -2 3.0 40:00 I0 I I 5.0 0.0 I.5 40:00 °-- ---
5-F -I 3.0 40:00 I0 I I 5.0 0.0 I.5 40:00 --- ---

I It" I 3.0 00:I0 I0 40 3 5.0 I.2 I.5 49:30 450 I200
2=T 2 3.0 00:I0 IO 40 3 5.0 I.2 I.5 49 30 450 I200

3=“ 3 3.0 00:I0 I0 40 3 5.0 I.2 I.5 49:30 450 I200

4-T 4 2.5 00:20 l0 X) 2 5.0 I.2 I.5 34:40 450 I200

5=F 5 2.5 00:30 I5 20 2 5.0 I.2 I.5 34:30 450 I200

2 I!" 6 2.0 00:40 20 I5 2 5.0 I.2 2.0 34:20 600 I200
2=T 7 2.0 00:50 25 I2 2 5.0 I.2 2.0 34 I0 600 I200

33“ 8 I.5 0I:00 30 IO 2 5.0 I.2 2.0 34 00 600 IAN)

48T 9 I.5 02:30 60 4 2 5.0 I.2 2.0 3It00 non I200

5tF I0 I.0 02:30 60 4 2 5.0 I.2 2.0 3| 00 600 I200

3 I8" II I.0 02:30 60 4 2 5.0 I.2 2.0 3I:00 600 I200
2-T I2 I.0 05:00 0 I 5 2.5 I.2 2.0 35 00 750 I500

3=U I3 I.0 05:00 0 I 5 2.5 I.2 2.0 35 00 750 I500

4=T I4 I.0 05:00 0 I 5 2.5 I.2 2.0 35:00 750 I500

5=F I5 I.0 05:00 0 I 5 2.5 I.2 2.0 35:00 750 I500

4 I=M I6 I.0 05:00 0 l 5 2.5 I.2 2.0 35 00 750 I500
2=T I7 I.0 07:30 0 I 4 2.5 I.0 2.0 37:30 900 I800

3-It I8 I.0 07:30 0 I 4 2.5 I.0 2.0 37:30 900 I800
4=T I9 I.0 07:30 0 I 4 2.5 I.0 2.0 37:30 900 I800
SIP 20 I.0 07:30 0 I 4 2.5 I.0 2.0 37:30 900 i800

5 II" 2I I.0 07:30 0 I 4 2.5 I.0 2.0 37:30 900 I800
2=T 22 I.0 07:30 0 I 5 2.5 I.0 2.0 47: 30 Il25 2250

38! 23 I.0 07:30 0 I 5 2.5 I.0 2.0 47:30 II25 2250

4ST 24 I.0 07:30 0 l 5 2.5 I.0 2.0 47:30 II25 2250

5:F 25 I.0 07:30 0 I 5 2.5 I.0 2.0 47:30 II25 2250

6 It" 26 I.0 07:30 0 I 5 2.5 I.0 2.0 47:30 II25 2250
ZIT 27 I.0 I0 00 0 I 4 2.5 I.0 2.0 47:30 I200 2400

3-H 28 I.0 I0:00 0 I 4 2.5 I.0 2.0 47:30 I200 2400

4=T 29 I.0 I0:00 0 I 4 2.5 I.0 2.0 47 30 I200 2400
5=F 30 I.0 I0:00 0 l 4 2.5 I.0 2.0 47:30 I200 2400

7 ISM 3i I.0 IO 00 0 I 4 2.5 I.0 7.0 47:30 I200 2400
2=T 32 I.0 I0:00 0 l 5 2.5 I.0 2.0 60:00 I500 3000

380 33 I.0 I0:00 0 I 5 2.5 I.0 2.0 60:00 I500 3000

4=T 34 I.0 I0:00 0 l 5 2.5 I.0 2.0 60.00 I500 3000

58F 35 I.0 I0:00 0 l 5 2.5 I .0 2.0 60:00 I500 3000

8 IIM 36 I.0 I0200 0 I 5 2.5 I.0 2.0 60:00 I500 3000
2=T 37 I.0 I2:30 0 I 4 2.5 I.0 2.0 57:30 I500 YXM)

3th 38 I.0 I2230 0 I 4 2.5 I.0 2.0 57:30 I500 3000

4=T 39 I.0 I2:30 0 I 4 2.5 I.0 2.0 57:30 I500 3000

5=F 40 I.0 I2 30 0 I 4 2.5 I.0 2.0 57:30 I500 3000

 

This standard program was designed using male rats of the Sprague-Dawley

strain.

the program.

All animals were between 70 and I70 daySooi-age at the beginning of
The duration and intensity of the program were established so

that 75 per cent of all Such animals should have PSF and PER scores of 75 or

higher during the final two weeks.

Alterations in the work time. number of

bouts, or time between bouts can be used to aflect changes in these values.
Other strains or ages of animals reuld be expected to respond dillerently to
the program.

All animals should be exposed to a minimum at one week of voluntary runninq

in a wheel prior to the start at the program.

Failure to prOVIde this adjustment

period will impose a double learning situation on the animals and will seriiwuly
impair the effectiveness of the training programs.

Standard long-duration, low-speed endurance
and adult male rats In controlled runni

maintenance program for postpubertal

nq wheels.

 

 

Total Total
Acc- Rope- Time Time Exp. Total
oier— Work ti- in»- Run ol Revo- work
ation Time Rest tions Nn. ween 5peed Prog. Iu- Tinm
Time (min: Time par of Bouts Shock (lt/ (min: tions (sec)
(soc) sec) (Sn!) Hunt Bouts (min) (ma) Gut) sec) TER flUT
I.0 I7:K) 0 I 2 7.5 I 0 2.0 21 Y) 750 INX)

 

 

 

 

110

TABLE A—4.-- Standard eight-week, endurance, swimming training program for
postpubertal and adult male rats.

 

 

Expected

Per Swim

Day Day Cent Time

of of Tail (min)

Wk. Hk. Tr. Height EST
I I=M I 0 3D
2=T 2 0 40
3th 3 C' 50
4=T 4 C 60
5=F 5 C 60
2 IIH 6 2 40
2=T 7 2 40
38H 8 2 40
4=T 9 2 45
5=F I0 2 5O
3 I=M II 3 30
2=T I2 3 30
38W I3 3 30
4=T I4 3 35
5=F I5 3 35
4 |=M I6 3 35
2=T I7 3 40
3=w l8 3 40
4=T l9 3 40
5.; 20 3 40
5 IIM 2| 3 40
2=T 22 3 45
3=H 23 3 45
4=T 24 3 45
5-F 25 3 45
6 IIM 26 3 45
2=T 27 3 50
33W 28 3 50
4=T 29 3 50
5=' 30 3 50
7 Ian 3| 3 50
2=T 32 3 55
32k 33 3 55
4-T 34 3 55
5=F 35 3 55
8 I-M 36 3 55
2=T 37 3 60
3=H 38 3 60
4=T 39 3 60
53F 40 3 60

-I-I_II—". d‘ I'VI

 

 

'C = clothes pin only.

This standard program was designed using male rats of the Sprague-Dawley
strain. All animals were between 70 and 90 days-of-age at the beginning of
The duration and intensity of the program were established so
that 75 per cent of all such animals should have PET scores of 75 or higher

the program.

during the final two weeks.

Alterations in the per cent tail weight or
expected swim time can be used to affect changes in these values. Other strains
or ages of animals could be expected to respond differently to the program.

All animals should be exposed to a minimum of one week of voluntary running
in a wheel prior to the start of the program.
pericd will impose a severe, sudden exercise stress upon the animals and will
seriously impair the effectiveness of the training prOgram.

Failure to provide this adjustment

Standard endurance swimming maintenance program for postpubertal and adult

male rats.

 

 

Expected
Per Cent Swim Time
Tail Height (min) EST
2 40

 

APPENDIX B

TRAINING PERFORMANCE AND ENVIRONMENTAL

CONDITIONS

lll

Feibfil ”1'

.mHmEHco Ham now mmoo mcwcwouu Houoem

 

 

 

 

mom.o mmw.o mow.oi wwo.o moo.o moo.o mo.m w.mm meow mmm
mam.o nme.oi moo.o moo.o owo.o om.mw m.woa meow mmm
mmw.oi mwo.o noo.oi moo.o ow.o w.w mmow mmoq
.ooz wmom m
mmo.oi Hmw.o wwo.o ma.aa 0.0mm mmow goo
.umz woom
.uomuaimum
wmw.oi nma.oi ama.oi am.m m.mmw meow Tomaso
.mmmum .uom
mmw.o em.o o.ww meow wuwowesm m
mm.w a.mw meow Aooo
.mea mad
mum mmoa .uos .uoz woom .mmoum wuwoae .oama
aoom w .uoouaioum .wmm ism w :4 .>ma
.coum com: oz manoeuo>
mcowuoamuuoo mHmem
.mmsoum

mood one anome .uuonm wow mosao> unmams

>000 0cm ucmscoww>cm ucmfiummwaii.wim mqmda

112

.mHmecm Ham now mmmo mcwcfimuu Hmuoam

 

 

ewa.o mao.o mmo.o- moo.o meo.ou mn.w m.mm use 9mm
ewo.o meo.o mmw.o mma.o wm.me o.owm she Amo
.umz zoom
.ummualmum
mmm.ou mow.os moo.o ma.m e.mm~ she Ammeeo
.mmmum .me
omH.o mmw.o we.w m.Hw wee aufiowesm m
maa.ou me.a m.ww she Ao.o
.mea HH¢
me.o m.Hm use Aooo
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.umS moon .mmmum muflvfle .mEmB .msma
.ummusnmum .umm lam a Mad umumz .>ma
.cmum cmmz mz manmflum>

 

mcofiumamuuou mHmEHm

 

.msoum mcaEEa3m How mmzam> unmam3 avon new ucoecoufi>cm usmfiummuall.wum mamma

113

50% 36 .2 E8. mszgoam 8886 E8 an. e6 Em“: 2.: as“. .695 28 an 2.8 coozsséum 833m

__« N. no "T 0..“ 1||1cT| n6 MoTI_ o.e H*l_ nu M¢Tl_ on Mil... n. 33:: 1_w>
cm 0. n

 

 

 

_ __ _ o. _ m _ o _ s _ o _ .x; 2.45
no 0.0. 9. cc on on ow 0w 9 >3 2.45
F- - P P P b P p P p n p P L Lr _ - PL P P P h p p b PL P p p p p p _ p p — h P P — p h p p h - h — P — — P P — p b r
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git-7k .. '3 .171r1111 ml .. u H]
.4 .1. . 1
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0—‘0 83d

114

 

2282 2&0 5. Ema: 223.031 8.83m 28 3n. e8 Ewe 2:: out x85 38 5a 2.8 c8211.w1m 33E

j o.e "W on IvTI on lle_n.~ leoW «T10. 33:: 15>
w. w _
on n

 

 

 

 

 

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n on me oe an on ow ow n. o. 0 >3 2.5:
t _ p h b h h _ h _ _ _ _ _ p p p _ _ _ _ _ _ p _ _ b _ _ _ _ _ _ _ p _ _ _ h _ . _ _ p h _ _ _ _ _ h _ _ b _ _ _ _ _
h H
00 1. . . . .1 0v
8_ 1 . . . . 1 on d
. S
A
8. 1 . . 1 8 H
03 1 1 oh
oo. 1 1 8
om. 1 , 1 om
8w 1 g 8.

115

  

, Huler-Ikdn 1 I1. 133 yr ALI—Hf»;

023 2,8 .2 Ema: 293.9% 8.89m E8 Ea uco Emn: me; 3; 62m E8 5a :60 c8: 11.m1m 9.33m

 

 

 

 

 

r ed 11..» n. 33:: .d>
_4 w. _ _ _ _ o. _ m _ m _ s _ w _ n _ e _ m _ w _ _ .xs .235
00 on On me O? on 0» mm ON 0 _ o _ m >40 2.3:
P P r P PL P P P F P P P P P 1? P P P P P P P P P P P P P P P P P P P P P P P P L P P P P P P L P P P P P P P P P P P P
P. 1P
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3 o o O o o o o O o o n O C c o S
no 0 o o o o o o o o o I:
H 8. 1 . . . . . . 1 om H
o! 1 1 o»
8. 1 1 om
I L
om. 1 1 om
r 1
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116

 

0204 v5 53.0%.. .PmOIm imo v8 >m<PZDJO> .2 Ext cam 20:233.... .28. 360 comfiunéum 0.»:va

w. I .o. m o w. m n v n w . .23 2.4m.—
ow on On me. ov mm On ow ON 0. o. n >40 .23...»
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b1 ’ 000.
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204 9116
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2.0.3 I

APPENDIX C

HISTOCHEMICAL FIBER TYPE RAW DATA

 

117

TABLE C—1.--Histochemica1 ratings for medial gastrocnemius muscle presented
by animal number, treatment, and duration.

 

Phenotypic Rating/Histochemical Procedure

 

 

 

 

Animal Treat- Dur.
Number ment (Wk.) 1 2 3

SDH ATP PPL PAS SDH ATP PPL PAS SDH ATP PPL PAS
006 1 0 02 00 47 05 10 06 03 45 38 44 00 00
009 1 0 02 00 47 05 10 06 03 45 38 44 00 00
002 1 4 00 00 45 07 10 07 05 40 40 43 00 03
053 1 4 00 00 49 49 02 02 00 00 48 48 00 00
100 1 4 00 00 49 49 02 02 00 00 48 48 00 00
140 1 4 00 00 49 32 04 02 00 18 46 48 00 00
005 1 8 04 01 49 24 00 05 01 22 46 44 00 04
052 1 8 00 00 49 03 01 04 01 47 49 46 00 00
099 1 8 01 01 45 37 03 03 05 13 46 46 00 00
139 1 8 05 05 45 34 00 12 05 12 45 33 00 04
003 1 12 03 01 45 12 05 16 05 35 42 33 00 02
051 1 12 03 01 45 13 05 16 05 35 42 33 00 02
098 1 12 02 00 48 39 04 02 02 11 44 48 00 00
138 1 12 00 00 49 24 11 06 00 26 39 44 00 00
007 2 0 01 00 41 07 05 05 09 31 45 45 00 12
057 2 01 00 41 07 05 05 09 21 45 45 00 12
069 2 4 10 07 40 29 25 18 09 14 15 25 01 07
070 2 4 10 07 40 29 25 18 09 14 15 25 01 07
147 2 4 00 00 49 12 03 03 00 38 47 47 00 00
149 2 4 10 07 40 29 25 18 09 14 15 25 01 07
064 2 8 00 09 41 15 13 07 09 35 38 34 00 00
068 2 8 00 06 42 07 09 05 08 43 41 39 00 00
148 2 8 00 00 41 24 07 16 09 26 43 34 00 00
150 2 8 01 01 39 30 18 18 10 19 31 31 01 01
062 2 12 00 00 45 43 04 04 05 07 46 46 00 00
072 2 12 00 00 45 43 04 04 05 07 46 46 00 00
145 2 12 00 00 49 28 11 09 00 22 39 41 00 00
152 2 12 00 00 49 28 22 24 00 22 38 26 00 00
055 3 0 08 00 38 24 09 13 12 26 24 22 29 00
058 3 0 02 03 49 15 13 17 01 32 35 30 00 03
014 3 4 00 00 43 11 04 08 07 39 46 42 00 00
015 3 4 00 00 49 13 05 08 00 37 45 42 00 00
107 3 4 00 00 41 05 49 31 09 45 00 19 00 00
108 3 4 00 00 34 05 20 18 16 45 30 30 00 00
020 3 8 00 00 44 06 06 08 06 44 44 42 00 00
022 3 8 00 00 47 15 27 17 03 35 23 34 00 00
106 3 8 15 05 25 27 08 23 22 19 27 22 03 04
110 3 8 07 07 25 21 16 22 22 27 27 21 03 02
018 3 12 15 05 25 27 08 23 22 19 27 22 03 03
021 3 12 00 00 29 12 15 01 21 38 35 49 00 00
101 3 12 00 00 49 28 06 01 01 24 44 49 00 00
104 3 12 00 00 49 26 06 01 00 24 44 49 00 00
059 4 0 01 00 49 03 06 06 00 47 43 44 00 00
011 4 0 00 00 49 27 13 06 00 15 37 44 00 08
074 4 4 00 00 40 14 09 09 10 36 41 41 00 00
077 4 4 00 00 40 12 14 14 10 38 36 36 00 00
154 4 4 00 00 49 19 03 01 01 31 47 49 00 00
159 4 4 00 00 49 19 03 01 00 31 47 49 00 00

118

TABLE C-1.-~Continued.

.P'L—LJ;-- -2 J

lg- = ' ‘

- .

Phenotypic Rating/Histochemical Procedure

 

 

 

 

Animal Treat- Dur.
Number ment (Wk.) 1 2 3
SDH ATP PPL PAS SDH ATP PPL PAS SDH ATP PPL PAS
075 4 8 00 00 44 23 15 22 06 25 35 28 00 02
080 4 8 01 01 44 23 14 21 05 25 35 28 01 02
162 4 8 00 00 45 30 06 22 05 20 44 28 00 00
164 4 8 01 01 29 27 19 23 21 23 30 26 00 00
073 4 12 00 27 33 30 15 22 17 20 35 01 00 00
076 4 12 03 07 38 12 06 40 12 38 41 03 00 00
153 4 12 00 00 49 00 15 16 00 25 35 34 00 25
010 5 0 02 00 47 05 10 06 03 45 38 44 00 00
056 5 0 00 00 41 07 05 05 09 31 45 45 00 12
088 5 4 00 00 31 18 21 19 19 32 29 31 00 00
089 5 4 00 16 20 16 19 29 25 12 31 05 05 11
170 5 4 03 00 29 24 08 12 21 22 39 38 00 04
172 5 4 00 00 47 25 06 07 03 25 44 43 00 00
091 5 8 06 08 43 20 12 08 07 24 32 34 00 06
092 5 8 00 26 41 03 20 22 09 42 30 02 00 05
166 5 8 00 11 31 31 25 33 10 15 25 06 09 04
085 5 12 01 10 35 26 49 00 15 24 00 40 00 00
095 5 12 04 30 37 23 46 20 13 27 00 00 00 00
171 5 12 00 00 38 26 12 14 12 21 38 36 00 03
176 5 12 00 00 45 22 12 17 05 23 38 33 00 05
012 6 0 00 00 49 16 18 Ol 00 34 32 45 49 00
060 6 0 00 00 49 25 05 01 00 25 45 49 00 00
026 6 4 00 09 09 14 11 00 40 23 39 41 01 03
027 6 4 00 09 09 14 11 00 40 23 39 41 01 03
119 6 4 00 00 09 29 05 49 40 21 45 00 01 00
120 6 4 00 00 46 29 04 08 04 21 46 42 00 00
032 6 8 05 01 41 30 08 17 08 19 37 32 01 01
034 6 8 05 01 41 30 08 17 08 19 37 32 01 01
118 6 8 05 01 41 30 08 17 08 19 37 32 01 01
122 6 8 05 01 41 30 08 17 08 19 37 32 01 01
030 6 12 04 03 39 19 11 32 11 31 35 15 00 00
033 6 12 04 03 39 19 11 32 ll 31 35 16 00 00
113 6 12 00 00 49 00 00 00 00 30 49 49 01 20
116 6 12 00 03 47 13 14 32 00 34 36 15 03 03
008 7 0 00 00 49 20 11 00 00 30 39 49 00 00
054 7 05 07 39 25 10 12 05 21 30 31 06 04
041 7 4 00 00 35 12 17 17 14 37 33 33 01 01
046 7 4 00 00 35 12 17 17 14 37 33 33 01 01
135 7 4 00 08 41 26 11 24 09 24 39 18 00 00
136 7 4 00 00 47 17 02 02 03 33 48 48 00 00
045 7 8 00 00 46 29 13 12 14 21 37 38 00 00
047 7 8 00 00 46 29 13 12 04 21 37 38 00 00
131 7 8 00 00 30 27 09 14 20 21 41 36 00 02
132 7 8 00 00 42 23 01 03 07 27 49 47 01 00
037 7 12 00 00 43 49 06 07 07 01 44 43 00 00
038 7 12 06 08 36 45 O9 40 14 05 35 02 00 00
125 7 12 00 00 45 00 22 22 02 44 28 28 03 06
126 7 12 13 04 40 20 12 25 10 30 25 21 00 00
LEGEND: l = Dark staining intensity; 2 = Medium staining intensity; 3 = Light
staining intensity; SDH = Succinate dehydrogenase; ATP = Intermyo-
fibrillar adenosine triphosphatase; PPL = Phosphorylase; PAS = Periodic

Acid-Schiff.

'Ill'llillull

 

119

TABLE C-2.-—Histochemica1 ratings for plantaris muscle presented by animal
number, treatment, and duration.

Phenotypic Rating/Histochemical Procedure

 

Animal Treat- Our. 1

Number ment (Wk.) 2 3

 

SDH ATP PPL PAS SDH ATP PPL PAS SDH ATP PPL PAS

 

006 1 0 24 17 19 10 21 21 21 31 05 12 10 09
009 1 0 07 16 28 11 35 34 16 27 08 00 06 12
002 1 4 25 13 15 17 23 35 24 31 02 02 11 12
053 1 4 19 19 32 11 28 31 12 34 03 00 06 05
100 1 4 26 28 22 19 16 17 21 30 08 05 07 01
140 1 4 25 12 32 16 24 35 11 30 01 03 07 04
005 1 8 24 18 24 03 22 24 21 42 04 08 05 05
042 1 8 23 20 24 07 21 25 23 31 06 05 03 12
099 l 8 24 16 35 22 23 32 19 23 03 02 06 05
139 1 8 24 16 35 22 23 32 09 23 03 02 06 05
003 1 12 19 12 28 24 31 37 15 15 00 01 07 11
051 1 12 19 12 28 24 31 37 15 15 00 01 07 11
098 1 12 24 15 28 25 26 35 09 07 00 00 13 18
138 1 12 14 17 34 24 28 25 13 16 08 08 03 10
007 2 0 12 07 27 13 36 41 13 33 08 02 10 14
057 2 24 23 23 16 26 27 25 24 00 00 02 10
069 2 4 21 16 27 12 27 24 14 36 02 10 09 02
070 2 4 30 18 34 00 18 19 11 30 02 13 05 20
147 2 4 27 15 33 28 23 31 12 20 00 04 05 02
149 2 4 28 23 22 17 22 22 24 28 00 05 04 05
064 2 8 26 17 29 00 23 29 15 22 01 04 06 28
068 2 8 23 21 24 22 21 22 22 22 06 07 04 06
148 2 8 29 26 21 16 19 22 22 33 02 02 07 01
150 2 8 18 20 28 26 32 28 16 18 00 02 06 06
062 2 12 22 22 22 24 28 28 22 21 00 00 06 05
072 2 12 28 26 20 16 22 24 23 29 00 00 07 05
145 2 12 24 24 21 19 22 21 18 29 04 05 11 02
152 2 12 20 11 21 19 26 34 18 29 04 05 11 02
058 3 0 22 15 28 23 23 35 15 20 05 00 07 07
055 3 0 24 22 29 00 26 28 21 30 00 00 00 20
014 3 4 24 21 16 00 26 29 27 43 00 00 07 07
015 3 4 19 18 28 10 30 32 18 25 01 00 04 15
107 3 4 23 21 24 20 27 29 18 26 00 00 08 04
108 3 4 25 22 26 19 22 25 18 18 03 03 06 13
020 3 8 09 16 19 11 41 34 27 37 00 00 04 02
022 3 8 29 19 29 24 21 31 17 26 00 00 04 00
106 3 8 25 14 28 26 24 35 15 20 01 01 07 04
110 3 8 18 06 35 19 29 41 11 23 03 03 04 08
018 3 12 13 10 31 21 37 40 14 19 00 00 05 10
021 3 12 16 07 25 16 33 42 22 34 01 01 03 00
101 3 12 17 07 37 27 31 42 07 16 02 01 06 07
104 3 12 17 07 37 27 31 42 07 16 02 01 16 07
011 4 0 19 17 26 21 17 13 19 23 14 20 05 06
059 4 0 24 21 32 20 26 29 18 27 00 00 00 03
074 4 4 24 22 15 12 26 28 21 38 00 00 14 00
077 4 4 20 16 17 12 29 27 30 22 01 07 03 16
154 4 4 31 17 29 17 19 33 15 11 00 00 06 22
159 4 4 27 12 29 17 22 36 15 11 01 02 06 22
075 4 8 41 20 16 16 09 24 28 19 00 06 06 15
080 4 8 27 18 26 28 23 31 19 11 00 01 05 11
162 4 8 22 13 28 23 28 32 17 19 00 15 05 08

 

1|! 4|. l|l1l||l! .‘Ilull

120

TABLE C-2.-—Continued.

 

Phenotypic Rating/Histochemical Procedure

 

Animal Treat- Our. 1 2 3
Number ment (Wk.)

 

SDH ATP PPL PAS SDH ATP PPL PAS SDH ATP PPL PAS

 

 

164 4 8 37 15 99 18 16 32 99 12 07 03 99 20
073 4 12 34 22 24 99 16 28 22 99 00 00 04 99
076 4 12 37 23 14 17 10 26 25 33 03 01 11 00
153 4 12 14 13 31 28 34 35 11 14 02 02 08 08
010 5 0 27 11 24 07 23 39 18 34 00 00 08 09
056 5 0 16 13 28 24 34 37 11 19 00 00 11 07
088 5 4 23 24 12 13 27 26 27 00 00 00 11 37
089 5 4 25 24 17 17 25 26 21 28 00 00 12 15
170 5 4 29 13 26 14 17 36 19 29 04 01 05 07
172 5 4 27 43 26 35 20 11 18 06 03 36 06 09
091 5 8 17 16 14 22 18 23 30 16 15 11 06 12
092 5 8 35 23 23 06 14 26 18 41 01 01 09 03
166 5 8 22 14 32 26 28 36 15 14 00 00 03 10
085 5 12 04 30 37 23 46 20 13 27 00 00 00 00
095 5 12 37 20 18 20 12 30 26 30 00 00 06 00
171 5 12 09 14 30 21 41 32 10 22 00 04 10 07
176 5 12 15 14 21 26 31 32 21 13 04 04 08 11
012 6 0 19 19 22 09 25 31 18 32 16 00 10 09
060 6 0 20 16 27 07 30 34 15 34 00 00 08 09
026 6 4 16 14 16 20 34 36 22 21 00 00 12 09
027 6 4 16 14 16 20 34 36 22 21 00 00 12 09
119 6 4 25 12 30 23 25 35 10 24 00 03 10 03
120 6 4 29 16 30 23 21 32 14 24 00 02 06 03
032 6 8 25 14 29 13 23 34 11 25 02 02 10 12
034 6 8 15 13 33 35 10 30 15 24 25 07 02 01
118 6 8 25 14 29 13 23 34 11 25 02 02 10 12
122 6 8 17 14 31 34 31 31 13 14 02 05 06 02
030 6 12 20 06 25 27 30 29 14 21 00 15 11 02
033 6 12 20 06 25 27 30 29 14 21 00 15 11 02
113 6 12 21 06 22 23 25 29 24 21 04 15 04 06
116 6 12 10 18 29 24 38 32 11 04 02 00 10 22
008 7 0 33 28 19 00 17 22 21 20 00 00 10 30
054 7 22 18 29 19 28 32 11 23 00 00 10 08
041 7 4 14 15 22 22 36 35 17 27 00 00 11 01
046 7 4 29 27 23 28 21 23 17 22 00 00 10 00
135 7 4 20 19 23 19 30 29 20 25 00 02 07 06
136 7 4 25 18 30 19 25 29 14 30 00 03 06 01
045 7 8 18 13 24 22 32 24 22 27 00 13 04 01
047 7 8 39 23 28 18 11 25 18 30 00 02 04 02
131 7 8 22 28 24 31 26 19 17 19 02 03 09 00
132 7 8 27 14 23 19 19 33 16 24 04 03 11 07
037 7 12 17 16 20 19 23 28 22 24 10 06 08 07
038 7 12 20 14 24 14 24 31 16 32 06 05 10 04
125 7 12 23 14 24 19 25 33 18 22 02 03 08 09
126 7 12 28 15 20 18 22 26 14 26 00 09 16 06

 

LEGEND: 1 = Dark staining intensity; 2 = Medium staining intensity; 3 =
Light staining intensity; SDH = Succinate dehydrogenase; ATP =
Intermyofibrillar adenosine triphosphatase; PPL = Phosphorylase;
PAS 8 Periodic acid-Schiff.

121

TABLE C-3.--Histochemical ratings for soleus muscle presented by animal

 

 

 

 

 

 

number, treatment, and duration.
Phenotypic Rating/Histochemical Procedure
Animal Treat- Dur.
Number ment (Wk.)

SDH ATP PPL PAS SDH ATP PPL PAS SDH ATP PPL PAS
006 1 0 09 07 00 00 41 43 12 20 00 00 38 30
009 1 0 16 12 00 00 34 38 17 19 00 00 33 31
002 1 4 13 37 00 00 37 42 14 26 00 00 36 24
053 l 4 13 09 00 00 37 41 10 07 00 00 40 43
100 l 4 12 07 00 00 38 43 07 07 00 00 43 43
140 1 4 12 11 00 00 38 39 ll 28 00 00 39 22
005 1 8 13 09 00 00 37 41 08 26 00 00 42 24
052 1 8 16 14 00 00 34 36 15 26 00 00 35 24
099 l 8 07 06 02 00 43 44 06 17 00 00 42 33
139 1 8 17 14 00 00 33 36 l6 17 00 00 37 33
003 l 12 14 00 00 03 36 40 08 28 00 00 42 19
051 l 12 14 10 00 03 36 40 08 28 00 00 42 19
098 l 12 14 09 00 00 36 41 41 10 00 00 09 40
138 1 12 10 02 00 00 40 48 04 29 00 00 46 21
007 2 0 07 08 00 00 43 42 08 10 00 00 42 40
057 2 0 21 20 00 00 29 30 22 37 00 00 28 13
069 2 4 24 16 00 01 26 34 15 44 00 00 35 05
070 2 4 09 09 00 09 41 41 17 36 00 00 33 05
147 2 4 16 12 00 00 34 38 12 26 00 00 38 24
149 2 4 22 17 00 00 28 33 16 32 00 00 34 18
064 2 8 17 03 00 00 33 47 06 04 00 00 44 46
068 2 8 15 13 00 00 35 37 14 13 00 00 36 17
148 2 8 10 03 00 00 40 47 48 28 00 00 02 22
150 2 8 10 02 00 00 40 48 05 24 00 00 45 26
062 2 12 12 13 00 00 38 37 12 36 00 00 38 14
072 2 12 15 12 00 00 35 38 09 46 00 00 41 04
145 2 12 06 02 00 00 44 48 06 22 00 00 44 28
152 2 12 ll 07 00 00 39 43 09 47 00 00 41 03
058 3 0 17 06 00 00 33 44 17 24 00 00 33 26
055 3 0 15 10 00 00 35 40 20 09 00 00 30 41
014 3 4 08 10 00 00 42 40 10 09 00 00 40 41
015 3 4 19 16 00 00 31 34 16 38 00 00 34 12
107 3 4 15 09 00 07 35 41 06 23 00 00 44 20
108 3 4 24 12 00 07 26 38 15 23 00 00 35 20
020 3 8 08 03 00 00 42 47 00 03 00 00 49 47
022 3 8 15 07 00 01 35 43 10 29 00 00 40 20
106 3 8 16 08 00 02 34 42 37 43 00 00 13 05
110 3 8 26 05 00 00 24 45 11 32 00 00 39 18
018 3 12 12 07 00 00 38 43 08 30 00 00 42 20
021 3 12 10 04 00 04 40 46 07 46 00 00 43 00
101 3 12 14 03 01 02 36 47 07 24 00 01 43 24
104 3 12 14 03 99 02 36 47 99 24 00 00 99 24
011 4 0 11 10 00 00 39 40 11 33 00 00 39 17
059 4 0 09 11 00 00 41 39 10 32 00 00 40 18
074 4 4 07 05 00 05 43 45 13 42 00 00 37 03
077 4 4 10 09 00 00 40 41 08 21 00 00 42 29
154 4 4 27 11 01 02 23 39 11 27 00 00 39 21
159 4 4 17 08 00 02 33 42 11 27 00 00 39 21
075 4 8 14 06 00 00 36 44 11 32 00 00 39 18
080 4 8 34 05 00 00 16 45 08 33 00 00 42 17
162 4 8 02 02 00 00 48 48 02 38 00 00 48 12

122

TABLE C—3.-—Continued.

_""‘"‘ ‘-“ —' L—t ’3 ‘a;-L—£~‘.-fi;l—; 2.2.; .1 42':- 2..." P‘ - L... J_. :P_. _' P. .‘—.‘. 2- _"’P“’_.:‘.P- ‘P‘_'—..-.' P. PP._ :1":— __..=_..

Phenotypic Rating/Histochemical Procedure

PULP :_" 1 ,.-_ —.._

 

Animal Treat- Dur. 1

Number ment (Wk.) 2 3

 

SDH ATP PPL PAS SDH ATP PPL PAS SDH ATP PPL PAS

 

 

 

11

164 4 8 01 08 01 00 49 42 02 28 00 00 48 22

073 4 12 34 03 00 15 16 47 30 33 00 00 20 02

076 4 12 18 03 00 05 32 47 02 43 00 00 48 02

153 4 12 22 08 00 00 28 42 00 37 00 00 49 13

010 5 0 09 07 00 00 41 43 12 20 00 00 38 30

056 5 0 14 14 00 00 36 36 14 14 00 00 36 36

088 5 4 16 14 00 00 34 36 15 20 00 00 35 30

089 5 4 13 10 00 00 37 40 10 06 00 00 40 44

170 5 4 11 O9 00 01 39 41 11 39 00 00 39 10

172 5 4 11 03 00 02 39 47 07 37 00 00 43 11

091 5 8 14 12 00 02 36 38 11 34 00 00 39 14 g
092 5 8 10 08 00 00 40 42 20 42 00 00 30 08 3
166 5 8 21 16 00 00 29 34 20 31 00 00 30 19 J
085 5 12 14 06 00 24 36 14 10 26 00 00 40 00 '"
095 5 12 43 18 00 17 07 31 23 33 00 01 27 00 U
171 5 12 19 00 00 00 31 49 10 45 00 00 40 05

176 5 12 29 12 00 00 21 38 16 49 00 00 34 01

012 6 0 13 13 00 00 37 37 17 26 00 00 33 24

060 6 0 16 13 00 00 34 37 17 39 00 00 33 11

026 6 4 11 08 06 00 39 42 09 46 00 00 35 04

027 6 4 12 05 00 00 38 45 12 28 00 00 38 22

119 6 4 27 02 00 01 23 48 06 28 00 00 44 22

120 6 4 09 09 00 01 41 41 14 28 00 00 36 22

032 6 8 30 03 02 15 30 47 15 35 00 00 33 00

034 6 8 20 14 00 00 30 36 14 17 00 00 36 33

118 6 8 03 05 00 00 47 45 05 44 00 00 45 06

122 6 8 13 04 00 05 37 46 08 43 00 00 42 02

030 6 12 14 07 01 01 36 43 12 40 00 01 38 10

033 6 12 14 07 00 00 36 43 12 40 00 00 38 10

113 6 12 20 16 00 00 30 34 06 20 00 00 44 30

116 6 12 20 12 00 00 3O 38 15 34 00 00 35 16

008 7 0 16 06 00 00 34 44 13 04 00 00 37 46

054 7 0 ll 11 00 00 39 39 14 10 00 00 36 40

041 7 4 16 15 00 00 34 15 16 41 00 00 34 09

046 7 4 11 13 05 00 39 37 11 04 00 00 34 46

135 7 4 16 08 00 01 34 42 12 27 00 00 38 22

136 7 4 12 07 00 05 38 43 08 32 00 00 42 13

045 7 8 26 00 01 05 24 49 17 44 00 00 33 01

047 7 8 13 06 00 00 37 44 17 09 00 00 33 41

131 7 8 17 11 00 06 33 39 14 44 00 00 36 00

132 7 8 13 08 00 02 37 42 09 28 00 00 41 20

037 7 12 10 03 00 00 40 47 05 49 00 00 45 00

038 7 12 13 02 00 17 37 39 06 33 00 09 44 00

125 7 12 10 04 00 00 40 45 09 30 00 00 41 20

126 7 12 16 07 00 00 34 43 12 26 00 00 38 24

 

LEGEND: 1 = Dark staining intensity; 2 = Medium staining intensity; 3 =
Light staining intensity; SDH = Succinate dehydrogenase; ATP =
Intermyofibrillar adenosine triphosphatase; PPL = Phosphorylase;
PAS = Periodic acid-Schiff.

APPENDIX D

STATISTICAL TABLES FOR MEDIAL GASTROCNEMIUS,

PLANTARIS, AND SOLEUS MUSCLES

 

. III [I'll]! Ill" j.| 1| 11 i

123

TABLE D-1.--Two-way analysis of variance tables for histo-
percent ratings in medial

chemical Sin"1

gastrocnemius muscle.

 

 

 

 

 

 

 

 

 

 

Source SS df MS F
SDH-—Rating 1
A (Training) 278.50 6 46.42 0.663
B (Duration) 375.60 3 125.20 1.789
AB (Training-
Duration) 2666.78 18 148.15 2.117
Error 4618.13 66
Total 7939.01 93
SDH—-Rating 2
A (Training) 2532.17 6 422.03 2.713
B (Duration) 188.68 3 62.89 0.404
AB (Training-
Duration) 3361.93 18 186.77 1.200
Error 10267.14 66_ 155.56
Total 16349.92 3
SDH-~Rating 3
A (Training) 2667.07 6 444.51 2.288
B (Duration) 308.80 3 102.93 0.530
AB (Training—
Duration) 4513.04 18 250.72 1.290
Error 12824.75 66 194.31
Total 2 3.66 93
ATP-~Rating 1
A (Training) 1081.71 6 180.28 1.736
B (Duration) 825.91 3 275.30 2.650
AB (Training—
Duration) 2653.00 18 147.39 1.419
Error 6856.08 66 103.88
Total 11416.70 93

 

124

TABLE D-l.-—Continued.

 

 

 

 

 

 

 

 

 

 

 

 

Source SS df MS F
ATP——Rating 2
A (Training) 1269.91 6 211.65 0.973
B (Duration) 2035.83 3 678.61 3.119
AB (Training-
Duration) 4336.55 18 240.92 1.107
Error 14361.30 66 217.60
Total 22003.59 92
ATP-~Rating 3
A (Training) 3012.10 6 502.02 1.760
B (Duration) 3056.55 3 1018.85 3.572
AB (Training-
Duration) 5935.16 18 329.73 1.156
Error 18827.70 66’ 285.27
Total 25831.51 93
PPL--Rating 1
A (Training) 2129.71 6 354.95 3.039
B (Duration) 1216.22 3 405.41 3.471
AB (Training-
Duration) 3849.16 18 213.84 1.831
Error 7707.98 66‘ 116.79
Total 14903.07 93
PPL—-Rating 2
A (Training) 2303.00 6 383.83 2.797
B (Duration) 1647.30 3 549.10 4.001
AB (Training—
Duration) 5550.69 18 308.37 2.247
Error 9057.11 66 137.23

Total

18558.10 93

 

125

TABLE D-l.-—Continued.

 

 

 

 

 

 

 

 

 

 

 

Source SS df MS F
PPL-~Rating 3
A (Training) 2073.86 6 345.64 4.401
B (Duration) 204.52 3 68.17 0.868
AB (Training-
Duration) 2242.67 18 124.59 1.587
Error 5183.02 66 78.53
Total 9704.07 9
PAS-~Rating 1
A (Training) 1248.36 6 208.06 0.983
B (Duration) 1355.36 3 451.79 2.134
AB (Training—
Duration) 7207.63 18 400.42 1.891
Error 13973.05 66 211.71
Total 23784.40 93
PAS-~Rating 2
A (Training) 1061.75 6 176.96 0.968
B (Duration) 1082.29 3 360.76 1.974
AB (Training-
Duration) 5686.19 18 315.90 1.728
Error 12064.01 66 182.79
Total 19894.24 93
PAS-—Rating 3
A (Training) 892.24 6 148.71 1.714
B (Duration) 150.87 3 50.30 0.580
AB (Training-
Duration) 2846.52 18 158.14 1.823
Error 5726.43 66’ 86.76
Total 9616.06 93

 

 

LEGEND: l = Dark staining intensity; 2 = Medium staining
intensity; 3 = Light staining intensity; SDH =
Succinate dehydrogenase; ATP = Intermyofibrillar
adenosine triphosphatase; PPL = Phosphorylase;
PAS = Periodic acid-Schiff; N = Not significant;
S = Significant at .05 level.

126

TABLE D—2.--Two-way analysis of variance tables for histo—
chemical Sin"l percent ratings in plantaris

 

 

 

 

 

 

 

 

 

 

muscle.
SDH--Rating 1
A (Training) 494.77 6 82.46 1.258 N
B (Duration) 487.40 3 162.47 2.478 N
AB (Training-
Duration) 651.54 18 36.20 0.552 N
Error 4327.76 66 65.57
Total 5961.47 93
SDH—-Rating 2
A (Training) 462.27 6 77.04 1.120 N
B (Duration) 568.34 3 189.45 2.755 N
AB (Training-
Duration) 919.42 18 51.08 0.743 N
Error 4538.62 66 68.77
Total 6488.65 93
SDH—-Rating 3
A (Training) 797.91 6 132.99 1.291 N
B (Duration) 458.49 3 152.83 1.484 N
AB (Training-
Duration) 1789.46 18 99.41 0.965 N
Error 6799.02 66 103.02
Total 9844.88 93
ATP——Rating l
A (Training) 467.27 6 77.88 2.189 N
B (Duration) 236.19 3 78.73 2.213 N
AB (Training—
Duration) 931.73 18 51.76 1.455 N
Error 2348.05 66 35.58

Total 3983.24 93

127

TABLE D-2.--Continued.

 

Source SS df MS F

 

ATP--Rating 2

 

 

 

 

 

 

 

A (Training) 719.53 6 119.92 3.278
B (Duration) 103.66 3 34.55 0.945
AB (Training-
Duration) 1276.12 18 70.90 1.938
Error 2414.42 66 36.58
Total 4513.73 93
ATP--Rating 3
A (Training) 1107.25 6 184.54 2.257
B (Duration) 761.83 3 253.94 3.106
AB (Training-
Duration) 2113.78 18 117.43 1.436
Error 5395.76 66 81.75
Total 9378.62 93
PPL-~Rating 1
A (Training) 220.49 6 36.75 0.816
B (Duration) 123.90 3 41.30 0.917
AB (Training-
Duration) 713.80 18 39.66 0.881
Error 2971.24 66 45.02
Total 4029.43 3
PPL-~Rating 2
A (Training) 205.60 6 34.27 0.789
B (Duration) 58.59 3 29.53 0.450
AB (Training-
Duration) 437.32 18 24.30 0.559
Error 2867.33 66 43.44
Total 3568.84 93

 

influx. in Jug. dfiufilJi

128

TABLE D—2.——Continued.

 

 

 

 

 

 

 

 

 

 

Source SS df MS F P
PPL--Rating 3
A (Training) 597.69 6 99.62 2.965 S
B (Duration) 210.61 3 70.20 2.090 N
AB (Training-
Duration) 628.23 18 34.90 1.039 N
Error 2217.44 66 33.60
Total 3653.97 93
PAS—~Rating 1
A (Training) 565.25 6 94.21 0.747 N
B (Duration) 1665.81 3 555.27 4.403 S
AB (Training-
Duration) 1728.90 18 96.05 0.762 N
Error 8323.35 66 126.11
Total 12283.31 9
PAS-~Rating 2
A (Training) 521.79 6 86.97 0.858 N
B (Duration) 350.76 3 116.92 1.154 N
AB (Training-
Duration) 2333.08 18 129.62 1.279 N
Error 6686.36 66 101.31
Total 9891.99 3
PAS--Rating 3
A (Training) 157.11 6 26.19 0.208 N
B (Duration) 737.77 3 245.92 1.950 N
AB (Training-
Duration) 3350.16 18 186.12 1.476 N
Error 8322.63 ‘66 126.10
Total 12567.67 93
LEGEND: 1 = Dark staining intensity; 2 = Medium staining

intensity; 3 = Light staining intensity; SDH =
Succinate dehydrogenase; ATP

adenosine triphosphatase; PPL
PAS = Periodic acid-Schiff; N
S = Significant at .05 level.

Intermyofibrillar
= Phosphorylase;

Not significant;

129

TABLE D—3.--Two-way analysis of variance tables for histo-
chemical Sin-l percent ratings in soleus

 

 

 

 

 

 

 

 

 

 

muscle.
Source SS df MS F P
SDH—-Rating l
A (Training) 153.92 6 25.65 0.340 N
B (Duration) 128.76 3 42.92 0.568 N
AB (Training-
Duration) 1244.60 18 69.14 0.916 N
Error 4983.19 66 75.50
Total 6510.47 3
SDH--Rating 2
A (Training) 153.92 6 25.65 0.340 N
B (Duration) 128.76 3 42.92 0.568 N
AB (Training-
Duration) 1244.60 18 69.14 0.916 N
Error 4983.19 66 75.50
Total 6510.47 3
SDH-~Rating 3
A (Training) 4.74 6 0.79 1.183 N
B (Duration) 2.63 3 0.88 1.311 N
AB (Training-
Duration) 14.80 18 0.82 1.231 N
Error 44.07 66 0.67
Total 66.24 93
ATP--Rating 1
A (Training) 268.70 6 44.78 0.647 N
B (Duration) 910.17 3 303.39 4.385 S
AB (Training-
Duration) 1237.38 18 68.74 0.994 N
Error 4566.37 66 69.19
Total 6982.62 93

TABLE D-3.--Continued.

 

 

 

 

 

 

 

 

 

Source SS df MS F
ATP--Rating 2
A (Training) 196.25 6 32.71 0.743
B (Duration) 441.42 3 147.14 3.343
AB (Training-
Duration) 910.28 18 50.57 1.149
Error 2905.11 66 44.02
Total 4453.06 93
ATP--Rating 3
A (Training) 24.02 6 4.00 0.429
B (Duration) 60.91 3 20.30 2.176
AB (Training-
Duration) 82.91 18 4.61 0.494
Error 615.88 66. 9.33
Total 783.72 3
PPL——Rating
A (Training) 61.04 6 10.17 0.668
B (Duration) 32.79 3 10.93 0.718
AB (Training-
Duration) 107.57 18 5.98 0.393
Error 1004.99 66 15.23
Total 1206.59 93
PPL--Rating
A (Training). 1102.99 6 183.83 1.652
B (Duration) 685.77 3 228.59 2.054
AB (Training-
Duration) 1289.19 18 71.62 0.644
Error 7345.38 66_ 111.29
Total 10423.33 93

 

131

TABLE D—3.--Continued.

 

 

 

 

 

 

 

 

 

 

Source SS df MS F P
PPL--Rating 3
A (Training) 929.89 6 154.98 1.488 N
B (Duration) 605.99 3 201.99 1.939 N
AB (Training-
Duration) 1106.64 18 61.48 0.590 N
Error 6876.72 66 104.19
Total 9519.24 93
PAS-~Rating 1
A (Training) 370.06 6 61.68 0.699 N
B (Duration) 614.27 3 204.76 2.318 N
AB (Training-
Duration) 1567.42 18 87.08 0.986 N
Error 5827.86 66 88.30
Total 5379161 93'
PAS--Rating 2
A (Training) 2610.10 6 435.02 2.058 N
B (Duration) 2883.87 3 961.29 4.548 S
AB (Training-
Duration) 3222.52 18 179.03 0.847 N
Error 13949.12 66 211.35
Total 22665.61 93
PAS--Rating 3
A (Training) 3182.20 6 530.37 1.894 N
B (Duration) 5443.59 3 1814.53 6.480 S
AB (Training-
Duration) 6189.84 18 343.88 1.228 N
Error 18482.22 66 280.03
Total 33297.85 9

 

LEGEND: l = Dark staining intensity; 2 = Medium staining

intensity; 3 = Light staining intensity; SDH

Succinate dehydrogenase; ATP = Intermyofibrillar

adenosine triphosphatase; PPL = Phosphorylase;
= Not significant;

PAS = Periodic acid-Schiff; N
S = Significant at .05 level.

APPENDIX E

HISTOCHEMICAL FIBER TYPE MEAN DATA

 

 

132

 

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