This is to certify that the
thesis entitled
The Effects of Training and Detraining
on Fiber Splitting
in the Soleus Muscle of the Albino Rat

presented by
Charles W. Beach

has been accepted towards fulfillment
of the requirements for

Ph.D. degree in Physical Education

Major professor

 

My 5: I???

I)ate

 

0-7 639

 

 

L
._.--~

BEACH, C.W. 1;

The effects of:
training and de-?
‘training of f
fiber splitting 1
ib rgw’mflowa

THE EFFECTS OF TRAINING AND DETRAINING
ON FIBER SPLITTING IN THE SOLEUS

MUSCLE OF THE ALBINO RAT

By

Charles W. Beach

A DISSERTATION

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

DOCTOR OF PHILOSOPHY
Department of Health, Physical Education, and Recreation

1978

 

 

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60.3097

ABSTRACT

THE EFFECTS OF TRAINING AND DETRAINING
ON FIBER SPLITTING IN THE SOLEUS
MUSCLE OF THE ALBINO RAT

BY

Charles W. Beach

The purpose of this study was to determine the effects of
detraining on the morphology of previously exercised soleus muscle in
the adult male albino rat. In particular, the study was undertaken
to determine the effects of detraining, or removal of training, on
muscle fiber splitting. The experimental animals were subjected to
either an endurance-running routine or a sprint-running routine. The
training regimens used the Short and Long Controlled-Running Wheel
programs previously reported from this laboratory. These programs
attempted to stimulate either aerobic or anaerobic metabolic processes
in the animals.

Training began when the animals were 84 days of age and con-
tinued for sixteen weeks. This was followed by detraining, in the form
of sedentary living, over the next sixteen weeks. Beginning with zero-
week, sacrifices were held at eight week intervals. In a block with
the plantaris and gastrocnemius muscles, the soleus muscle was surgi-
cally removed from the right hind leg and quick frozen. Serial cross-

sections were then cut and stained. For each animal the microsc0pe

Charles W. Beach

slide of the soleus was projected and each muscle fiber was categorized
as a mature fiber, a split fiber, or a fiber in the process of
splitting. Except for damaged or missing areas, all fibers were
counted.

The results showed that there was an aging effect in the
proportion of splitting or split fibers. Also, there were more
splitting or split fibers in the 8-Week-Exercised animals. After
eight weeks there was a relatively consistent decrease in the preportion
of splitting or split fibers whether the animals continued to exercise
or not. At the end of sixteen weeks of detraining, very few split

fibers were evident.

To Cheryl and my sister, Patricia

ii

ACKNOWLEDGMENTS

I wish to express my sincere appreciation and thanks to Dr.
William W. Heusner for invaluable advice, for guidance, and for keeping
faith while acting as my graduate advisor and director of my doctoral
committee.

Also, I wish to thank Dr. Wayne D. Van Huss for his support
as my advisor during my master's program and for giving me special
guidance on my doctoral dissertation.

In addition, I would like to express special appreciation and
thanks to Dr. Robert Echt for his understanding, encouragement, and
assistance during my research and for serving on my doctoral committee.

I acknowledge and thank Dr. Kwok-Wai Ho, Dr. Roland R. Roy,
and Dr. Gale E. Mikles for their assistance prior to and while serving
on my doctoral committee.

I also thank Mr. David Anderson for assistance in drawing the

figures.

iii

TABLE OF CONTENTS

Page
LIST OF TABLES . . . . . . . . . . . . . . . . . . . . . . . . . . vi
LIST OF FIGURES . . . . . . . . . . . . . . . . . . . . . . . . . vii
Chapter
I. THE PROBLEM . . . . . . . . . . . . . . . . . . . . . . . . 1

Need for the Study

Statement of the Problem
Research Hypotheses .
Research Plan .

Rationale . . .
Significance of the Problem .
Limitations of the Study

#waNNN

0‘

II. REVIEW OF RELATED LITERATURE

Methods of Applying Functional Overload to Skeletal

Muscle . . . . . 7
Effects of Applying Functional Overload to Skeletal

Muscle . . . . . . . . . . . . . . . . . . . 10

III. METHODS AND MATERIALS . . . . . . . . . . . . . . . . . . . 19

Experimental Animals . . . . . . . . . . . . . . . . . . 19
Research Design . . . . . . . . . . . . . . . . . . . . . 20
Training Procedures . . . . . . . . . . . . . . . . . . . 26
Training Programs . . . . . . . . . . . . . . . . . . . . 27
Animal Care . . . . . . . . . . . . . . . . . . . . . . 28
Sacrifice Procedures . . . . . . . . . . . . . . . . . . 28
Fiber Counts . . . . . . . . . . . . . . . . . . . . 30
Statistical Procedures . . . . . . . . . . . . . . . . . 32

IV. RESULTS AND DISCUSSION . . . . . . . . . . . . . . . . . . 33

Training Results . . . . . . . . . . . . . . . . . . . . 33
PRW Results . . . . . . . . . . . . . . . . . . . . . . . 35
SPL Results . . . . . . . . . . . . . . . . . . . . . . . 41
MAT Results . . . . . . . . . . . . . . . . . . . . . . . 41
Discussion . . . . . . . . . . . . . . . . . . . . . . . 51

iv

Chapter Page

V. SUMMARY, CONCLUSIONS, AND RECOMMENDATIONS . . . . . . . . . 54

Summary . . . . . . . . . . . . . . . . . . . . . . . . . 54
Conclusions . . . . . . . . . . . . . . . . . . . . . . . 55
Recommendations . . . . . . . . . . . . . . . . . . . . . 55

REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . 56
APPENDICES
Appendix

A. Training Programs . . . . . . . . . . . . . . . . . . . . 65

B. Analysis of Variance Tables and Planned Comparisons
T-Values and P-Values . . . . . . . . . . . . . . . . . 67

C. Basic Data . . . . . . . . . . . . . . . . . . . . . . . 70

Table

LIST OF TABLES

Original experimental design with cell frequencies .

Concomitant experimental design with cell frequencies

Final experimental design with cell frequencies

A

priori and a posteriori contrasts of the means for the
Proportion of Fibers in the Process of Splitting .

priori contrasts as the difference between means .

priori and a posteriori contrasts of the means for the
Proportion of Split Fibers .

priori and a posteriori contrasts of the means for the
Proportion of Mature Fibers

priori and a posteriori contrasts of the means for the

Proportion of Fibers in the Process of Splitting and
the Proportion of Split Fibers . . .

vi

Page
21
22

24

37

4O

42

45

48

LIST OF FIGURES

Figure
1. Flow chart of criteria for categorizing muscle fibers .

2. Mean daily percent shock-free time (PSF) and percent
expected meters (PEM) for the CRW Short Program .

3. Mean daily percent shock-free time (PSF) and percent
expected meters (PEM) for the CRW Long Program

4. Means and standard errors of the mean for the Proportion
of Fibers in the Process of Splitting by training x
duration with significant (p < .10) DMRT contrasts
shown above the figures and a priori contrasts shown
below the figures .

5. Means and standard errors of the mean for the Proportion of
Fibers in the Process of Splitting by duration x training
with significant (p < .10) DMRT contrasts shown above the
figures and a priori contrasts shown below the
figures .

6. Means and standard errors of the mean for the Proportion of
Split Fibers by training x duration with significant
(p < .10) DMRT contrasts shown above the figures and a
priori contrasts shown below the figures

7. Means and standard errors of the mean for the Proportion of
Split Fibers by duration x training with significant
(p < .10) DMRT contrasts shown above the figures and a
priori contrasts shown below the figures . . . .

8. Means and standard errors of the mean for the Proportion of
Mature Fibers by training x duration with significant
(p < .10) DMRT contrasts shown above the figures and a
priori contrasts shown below the figures

9. Means and standard errors of the mean for the Proportion of
Mature Fibers by duration x training with significant
(p < .10) DMRT contrasts shown above the figures and a
priori contrasts shown below the figures

vii

Page

31

34

36

38

39

43

44

46

47

Figure Page

10. Means and standard errors of the mean for the Proportion
of Fibers in the Process of Splitting and the Proportion
of Split Fibers by training x duration with significant
(p < .10) DMRT contrasts shown above the figures and a
priori contrasts shown below the figures. (This is the
reciprocal of Preportion of Mature Fibers.) . . . . . . . 49

11. Means and standard errors of the mean for the Proportion
of Fibers in the Process of Splitting and the Proportion
of Split Fibers by duration x training with significant
(p < .10) DMRT contrasts shown above the figures and a
priori contrasts shown below the figures. (This is
the reciprocal of Proportion of Mature Fibers.) . . . . . 50

viii

CHAPTER I

THE PROBLEM

Numerous investigators have demonstrated that skeletal muscle
can be modified in response to functional overload. However, these
observations have been the basis for diverse interpretations. In
the case of exercise-related alterations, much of the evidence presented
has been confounding and difficult to interpret because the training
regimens used have not been well defined (22, 23, 37, 38, 88). Other
evidence based on the effects of various surgical procedures has been
equally difficult to interpret (7, 25, 39, 40, 44, 62).

Whether adaptations of functionally overloaded muscle are con-
sidered to be beneficial or deleterious depends upon both the parameter
observed and the interpreter of the data. Conclusions based upon con-
tractile properties of muscle that has undergone hypertrophy are con-
tradictory (l3, 3, 29, 40, 60, 65, 89, 94). The acute and chronic
effects of different types of functional overload on fiber types are
the subject of much current debate (22, 32, 37, 40, 43). The inter-
pretation of changes in the number of muscle fibers also varies. For
example, generation of new fibers, regeneration of damaged fibers,
degeneration of normal fibers, and diseased (dystrophic) fibers all
have been associated with longitudinal muscle-fiber splitting (25, 29,
35, 44, 45, 53, 55, 57, 62, 82, 95, 97).

1

Recent evidence indicates that exercise consists of a continuum
of activity levels each eliciting specific responses within the
organism. The effects of a long-distance, endurance running program,
for example, is quite different from the effects of a short-distance,
high intensity running program. Thus, well defined and closely con-
trolled exercise regimens must be utilized to determine these specific

effects of exercise (103).

Need for the Study

 

Although some nonconclusive information is available concerning
the effects of various training programs on muscle-fiber splitting, a
search of the literature showed that very little is known about the
changes that may occur with detraining. There is an obvious need for
further study of the effects of specific regimens of training and sub-

sequent detraining on the phenomenon of fiber splitting.

Statement of the Problem

 

The purpose of this study was to determine the effects of
detraining on the morphology of previously exercised soleus muscle in
the adult male albino rat. In particular, the study was undertaken to
determine the effects of detraining, or removal of training, on muscle

fiber splitting.

Research Hypotheses

 

It was postulated that if muscle-fiber splitting is a result of
specific functional overload, then the affected muscles would have

program-dependent differential increases in the total number of fibers

at the conclusion of training. No hypothesis was possible regarding

the retention of these fibers during detraining.

Research Plan

 

Ninety-eight normal adult male rats were used as subjects for
this study. The experimental animals were subjected to either an
endurance-running routine (LON) or a sprint-running routine (SHT).
These two training regimens used Controlled-Running Wheel routines
previously reported from this laboratory (106, see Appendix A), and
represented attempts to stimulate either aerobic or anaerobic metabolic
processes in the animals.

The training was administered five days a week for sixteen
weeks. Detraining, in the form of sedentary living, was administered
over the next sixteen weeks. Beginning with zero-week, sacrifices were
held at eight week intervals. In a block with the plantaris and
gastrocnemius muscles, the soleus muscle was surgically removed from
the right hind leg and quick frozen. Serial cross-sections were then
cut and stained. For each animal, the microscope slide of the soleus
was projected and each muscle fiber was categorized as a mature fiber,

a split fiber, or a fiber in the process of splitting.

Rationale

Earlier investigations have lacked an apparatus which could produce
a wide range of exercise intensities in small laboratory animals.
Therefore, while the duration of exercise has been controllable,
the intensity was not. Several years of work at Michigan State
University has resulted in the development of the Controlled-
Running Wheel for Small Animals (CRW) which can be used to simulate
specific human training programs in the rat (51, p. 3).

Thus, both the need and the opportunity now exist to obtain data on

specific training effects on skeletal muscle fibers.

As part of its running action, the rat uses plantar flexion of
the ankle. The soleus muscle is involved with plantar flexion and,
therefore, is assumed to be very active during training in the
Controlled-Running Wheel.

Exercise-induced alterations in muscle fiber number may be
dependent upon the duration of the training program. Thus, zero-week,
eight-week, and sixteen-week periods of training were incorporated
into the research design. Likewise, detraining effects may be dependent
upon duration; therefore, eight-week and sixteen-week periods of de-

training also were incorporated into the research design.

Significance of the Problem

 

Determination of the training-detraining changes in the number
of skeletal muscle fibers should contribute to an overall understanding
of the effects of exercise. The examination of trained and detrained
muscle with respect to the numbers of muscle fibers should provide added
insight into the adaptations that occur during exercise and may supply
information concerning the mechanisms by which exercise can affect

skeletal muscle.

Limitations of the Study

 

1. The results of the study cannot be applied directly to humans
or generally to other types of animals.

2. The exercise programs were designed to stimulate distinct
aerobic and anaerobic metabolic processes. However, at this
time it is impossible to rule out the possibility of overlapping

metabolic requirements.

No control over the shock stimulus to run was included in the
study.

The sample size may have limited the power of the statistical
analyses.

The cross-sections were taken from only one level of the

muscle.

CHAPTER II

REVIEW OF RELATED LITERATURE

During embryonic life single-nucleated, spindle-shaped cells
called myoblasts undergo repeated mitoses. Eventually they stop
dividing and fuse end to end forming elongated structures called
myotubes. The nuclei continue to divide as the striated myotubes
mature into muscle fibers (13, 47, 70, 71).

Postnatal growth in length is largely dependent upon the
addition of new sarcomeres (13, 47). The increase in sarcomere length
accounts for only about 25 percent of lengthening in muscle fibers.
The remaining increment is due to the formation of new sarcomeres near
the site of insertion of the muscle fibers into tendons, the muscle-
tendon junctions (47). Near the muscle-tendon junction the sarcolemma
forms deep clefts in the muscle fiber providing some guidance for the
diameter of the new sarcomeres. The sarcomeres are synthesized and
added to the ends of pre-existing myofibrils (47).

Most authors attribute postnatal growth in width to volumetric
hypertrophy (12, 13, 19, 20, 33, 47, 69, 70).

During the first three months of life, the skeletal muscles of the
rat exhibit harmonious enlargement of perimysium, endomysium and

muscle fibers. Within individual muscle fibers, the sarcoplasm
enlarges and the nuclei increase in number (20, p. 243).

Additionally there is an increase in the number of myofibrils. New
myofilaments, the contractile elements of the muscle fiber, are added
to the sides of the myofibrils. When the myofibrils become so large
that they are no longer functional they begin to split. This process
eventually adds to the total number of myofibrils (29, 32, 47, 84).

There is another method by which skeletal muscle may undergo
postnatal growth in width. Numerical hypertrophy, an increase in
the number of muscle fibers that make up a muscle, may occur. However,
most authors agree that the number of muscle fibers is fixed shortly
after birth and remains constant throughout life (12, 19, 20, 33, 47,
69, 85).

Much research has been done on skeletal muscle hypertrOphy,
volumetric and numerical. These investigations have shown that skeletal
muscle adaptations are affected by workload. The following phenomena
have been found in muscle as a result of heavy functional overload;

(a) increase in weight, (b) increase in muscle force, (c) increase in
fiber diameter, (d) muscle fiber splitting, (e) centrally positioned
nuclei, (f) increased number of nuclei, (g) increased connective
tissue, (h) formation of new capillaries, and (i) formation of longi-
tudinal clefts in individual muscle fibers.

Methods of Applying Functional Overload
to Skeletal Muscle

 

 

Under normal circumstances it is difficult to coerce experi-
mental animals into participating in activities that result in
functionally overloading specific muscles. Therefore, the following
methods have been designed and used in research to produce functional

overload on skeletal muscle.

Surgery

Functional overload can be produced on skeletal muscle by
surgically incapacitating the synergist(s) of the muscle(s) to be
investigated. For example, the plantaris, soleus, and gastrocnemius
are involved with plantar flexion of the ankle in the rat. If the
gastrocnemius and plantaris are surgically incapacitated, then the

remaining soleus is "overloaded” during subsequent plantar flexion.

Tenotomy, Tenectomy, and Tendotomy.--Tenotomy, tenectomy, and

 

tendotomy are synonomous, all referring to the resection, cutting,
or surgical interruption of the tendon of a muscle. This method has
been used on rats (7, 40, 41, 44, 45, 46, 48, 56, 57, 60, 62, 63, 64,

65, 92, 93), mice (84, 86), chickens (41, 42), and cats (58).

Denervation.--Denervation is the deprivation of a nerve supply

 

by incision, excision, or blocking. This surgical method has been used
on rats (3, 48, 62, 63, 79, 81, 93, 104, 107), chickens (42), and mice

(86).

Excision and Extirpation.--Excision and extirpation are synono-

 

mous terms referring to the removal of tissue. This method involves
removing a portion of or an entire muscle and has been used on rats

(39, 4s, 57).

 

Crush Lesion.--A crush lesion is a method of injuring tissue by
crushing it. Crush lesions have been used on portions of muscles in

rats (45, 46).

Exsanguination.--Exsanguination is the deprivation of the blood

 

supply to tissue. Exsanguination of specific muscles has been used on

rats (53).

Exercise

Exercise is used to produce functional overload on skeletal
muscle by inducing animals to be more active than normal. The following
types of exercise have been used to investigate functional overload on
skeletal muscle. In this discussion, references will be provided only

to studies of muscle hypertrophy.

Running.--Treadmill or wheel running forces the animal to use
its normal means of locomotion. When a treadmill is used, both speed
and inclination can be controlled. The treadmill has been used with
rats (3, 52, 63, 73, 74, 79, 81, 104), guinea pigs (20, 21, 68), and
dogs (69). Controlled-Running Wheels for Small Animals (CRW) provide
a means of regulating running speed and permit several training
parameters to be monitored. These wheels have been used only with

rats (11, 26, SO, 51, 54, 87, 88).

Swimming.--Animals may be placed in deep water where they are
forced to swim. The activity of the animals can be increased by
the attachment of weights. Swimming has been used with rats (10, ll,

15, 40).

Weight Training,--For weight training, the animals are taught

 

to pull or push a variable resistance. Weight training has been used

with rats (63), mice (31), hamsters (33), and cats (35, 36).

10

Stretching.--Stretching is similar to weight training, except
that the weights are not "lifted" per se. Instead, they are attached
to a limb of the animal and "carried" during normal activities. This

method has been used on chickens (41, 42, 100).

Combinations

 

Some investigators have used combinations of methods to produce
functional overload in experimental animals. Some of the combinations
were: denervation and treadmill running (3, 104); tenotomy and
swimming (41); tenotomy, denervation, and treadmill running (63); and
denervation, extirpation, and treadmill running (79, 80, 81).

Schiaffino (90) denervated-the gastrocnemius and soleus of a
rat, shortened the tendon of the remaining plantaris, and immobilized
the knee and ankle joints to investigate the effects of mechanical
stretching. Schmalbruch (95) injected hot (60-70°C.) physiological
Ringer's solution into the spaces around the soleus of the rat to
investigate morphological characteristics of degeneration and
regeneration.

Effects of Applyipg Functional Overload
to Skeletal Muscle

 

 

The following phenomena have been found as a result of

functional overload.

Volumetric Hypertrophy

 

Skeletal muscle volumetric hypertrophy refers to increases in
muscle size due to intracellular and/or intercellular development.

This does not include increases in size due to an increase in the

11

number of muscle fibers. Volumetric hypertrophy usually is referred

to simply as, "hypertrophy."

Exercise.--In 1897 Morpurgo (69) found volumetric hypertrOphy
in the sartorius muscles of dogs that had been exercised on a treadmill.
Since that time numerous investigators have found similar results.
Holmes and Rasch (52) trained rats to run on a rotating drum. The
intensity of the program was increased over seven weeks until the
animals were running 28 meters per minute for 30 minutes daily. The
sartorius muscles of the experimental animals had a significantly
greater mean volume than did those of the controls. Maxwell §£_al:
(68) duplicated this result in guinea pig sartorius and soleus muscles
with treadmill running.

In other studies Carrow §t_al: (10) and Edgerton (15) found
volumetric hypertrophy in the plantar flexors of adult male rats that
had been forced to swim with weights attached to their tails. The
animals were required to exercise 30 minutes per day for 35 days.

Goldspink (31) trained female mice and Goldspink and Howells
(33) trained male hamsters to use weighted pulleys to receive food
pellets. These weight-training exercise regimens produced significant
volumetric hypertrophy in the soleus, extensor digitorum longus, and
biceps brachii muscles. Gonyea g£_§l. (35, 36) also used a weight-
training program. Male and female cats were trained to press a bar
for food pellets. After 19 to 46 weeks of training, significant volu-
metric hypertrOphy was found in the limb retractors and wrist and

elbow flexors.

12

However, exercise does not always produce volumetric hyper-
trophy. Edgerton 33-31: (16) trained non-human primates (Galago
senegalensis) by treadmill running. Although there was an improvement
in performance, there was no hypertrophy. Muller (74) trained female
rats on a treadmill for 3, 6, and 12 weeks. The workload was increased
for the first three weeks until the animals could run 36 meters per
minute for two hours on the moving belt that was inclined at 15°. The
mean fiber areas of the rectus femoris, soleus, and gastrocnemius
muscles from the exercised animals were not significantly different
from those of the controls.

Gordon 33.21, (37) trained female rats isometrically by having
them stand on their hind legs with weights strapped to their backs.
DeSpite improved performance, there was no volumetric hypertrophy.
Muller (73) found similar results in female rats that had trained
isometrically by holding themselves on wire grids that were inclined
at 60°. Weights, which were attached to their tails, were increased
each training period until no animal could support itself longer than
5 minutes at a time. Despite this apparently heavy overload, there
was no significant volumetric hypertrophy in the rectus femoris,

soleus, or gastrocnemius muscles.

Surgery.--Significant volumetric hypertrophy has been found in
the rat soleus, plantaris, and/or extensor digitorum longus muscles
by tenotomizing their synergists (7, 41, 44, 45, 46, 48, 56, 60, 62,
64, 65, 92). This same result was found in the soleus of cats (58) and

mice (84, 86) that were subjected to similar surgery. In chickens

13

volumetric hypertrophy was found in the latissimus dorsi posterior
muscle after its synergist was tenotomized (42).

Denervation (101), excision (39, 57), and exsanguination (53)
of synergists has produced significant volumetric hypertrophy in rats.
Hall-Craggs (45) and Hall-Craggs and Lawrence (46) used a crush
lesion on the muscles of the lower legs of rats and found volumetric
hypertrophy in the remaining muscle tissue. After comparing these
effects with the effects of tenotomy (45), it was concluded that the

morphological changes were similar.

Exercise plus Surgery.--Reitsma (79, 80, 81) used excision,

 

denervation, and tenotomy on all synergists of the rat rectus femoris,
plantaris, and soleus muscles. After allowing three weeks for re-
covery from surgery, the animals were exercised by walking on a tread-
mill. Training was conducted 12 hours per day, 5 1/2 days per week, at
a speed of 276 meters per hour for three weeks. The resultant volu-
metric hypertrophy was as much as 28 percent in the rectus femoris,

88 percent in the plantaris, and 111 percent in the soleus (79, 80, 81).
These percentages were calculated by comparing the experimental muscles
with those of the contralateral leg that had not been surgically over-
loaded but were exercised.

Van Linge (104) denervated the triceps surae of the rat. After
allowing twenty days for recovery, the animals were exercised by
walking on a moving treadmill that was inclined at 27°. They
trained four hours per day for 31 days. The volume of the plantaris
increased by 49 percent in just 2 1/2 days and continued to increase

to 90 percent by the Slst day.

14

Stretching.-—Sola e£.a13 (100) investigated the effects of
denervation, denervation and stretching, and stretching without dener-
vation. The surgery was performed on the wing muscles of chickens
and weights were attached to the tips of the wings. The results
showed that the combination of stretching without denervation had the
greatest effect.

Yellin (107) found that the denervated side of the unilaterally
denervated hemidiaphragm from female rats underwent gross hypertrophy.
He attributed this to repetitive, unOpposed stretching resulting from
the rhythmic contractions of the intact, contralateral hemidiaphragm.
However, Feng and Lu (25) previously had shown this hypertrophy to be
associated with contrasting changes of different fiber populations,
some undergoing hypertrophy, other atrophy.

Schiaffino (90) shortened the distal tendon of the plantaris
muscle of the rat and then immobilized the knee and ankle joints. In
a second series of experiments, the shortening of the plantaris muscle
was combined with denervation. The findings appear to show that an
increase in passive tension can produce hypertrophy in skeletal muscle
through a direct effect not mediated by nerves. This finding agrees
with the results obtained in earlier studies by Schiaffino and Hanzli-
kova (93) and Gutmann §£_213 (69).

However, Mackova and Hnik (62, 63) have found that volumetric
hypertrophy induced by tenotomy does not occur when antagonists are
motor denervated. They concluded that the rapid initial weight gain
following tenotomy of synergists is not true working hypertrophy

but is due to passive mechanical factors. This was found to be

15

true even when exercise in the form of "static standing" was used

as an added stress.

Numerical Hypertrophy

 

Skeletal muscle numerical hypertrophy refers to increases in
muscle size due to the addition of muscle fibers. This phenomenon

often is referred to as "hyperplasia."

Exercise.-—Carrow £3.31: (11) found evidence of muscle fiber
splitting in rats that had undergone training in Controlled-Running
Wheels for a period of eight weeks. The training programs that pro-
duced fiber splitting were: a short-duration, high-intensity program
(anaerobic); a long-duration, low-intensity program (aerobic); and a
medium—duration, moderate-intensity program (mixed). Although splitting
was detected in all groups, only the aerobic group had a significant
difference in actual fiber counts when compared to controls. However,

' both the anaerobic and mixed groups had a large percentage of split
fibers.

Edgerton (16) noted evidence of fiber splitting in rats that
had been forced to swim for 30 minutes per day with weights attached
to their tails. The splitting was seen in the soleus, but not in
the plantaris or gastrocnemius muscles.

Gonyea g£_al: (36) trained male and female cats to press a bar
for food pellets. The resistance of the bar was increased gradually,
and after a training period of 19 to 46 weeks the flexor carpi radialis
from the exercised animals showed fiber splitting while none was

observed in control animals.

16

Using a weight training program, Goldspink and Howells (33)
found no significant increase of muscle fiber numbers in the soleus,
extensor digitorum longus, and biceps brachii muscles of hamsters that

had been trained to use weighted pulleys.

Surgery.--Rowe and Goldspink (86) used tenotomy of the gastroc-
nemius muscles of male and female mice to overload the soleus. In
females, but not the males, there was a significant increase in the
number of muscle fibers when compared to controls.

James (57) overloaded the extensor digitorum longus muscle of
rats by excising its synergist, the tibialis anterior. The animals
were sacrificed at ten-day intervals for sixty days post-operatively.
Many muscle fibers were found to possess longitudinally oriented
fissures.

Yellin (107) observed fiber Splits that were similar to fetal
myotubes on the incapacitated side of the unilaterally denervated
hemidiaphragm of female rats. This was attributed to repetitive,
unopposed stretching resulting from the rhythmic contractions of the
intact contralateral side.

After overloading the rat soleus muscle by tenectomizing its
synergists, Hall-Craggs (44) found areas of longitudinal division in
muscle fibers. This phenomenon was found in the same areas with
fiber degeneration and increased numbers of connective tissue nuclei.
In subsequent investigations, Hall-Craggs (45) and Hall-Craggs and
Lawrence (46) used crush lesions of the muscles in the lower legs of
rats and found longitudinal fiber division. However, these new fibers

did not increase the cross-sectional area of the muscle. It was

17

suggested, therefore, that the observed fiber splitting was a
pathological response to trauma.

Schmalbruch (95) produced necrosis in the soleus muscles of
rats by injecting hot (60-700 C.) physiological Ringer's solution into
the space around the muscle. During regeneration the new fibers
appeared to be fragments of 'split' fibers. Therefore, it was concluded
that splitting of muscle fibers indicates regeneration as opposed to

degeneration.

Exercisepplus Surggry.--Reitsma (79, 80, 81) overloaded the

 

rectus femoris, plantaris, and soleus muscles of rats by excising,
denervating, or tenotomizing their synergists. The animals then were
exercised by walking on a treadmill. The investigators found volumetric
hypertrophy, fiber sprouting, fiber splitting, capillaries between
fractionated fibers, longitudinal cleavages, centrally located nuclei,
increased numbers of muscle fiber nuclei, and increased connective
tissue.

Sola EE.§1: (100) used combinations of denervation and/or
stretching by attaching weights to the wings of chickens to produce
muscle fiber nuclear enlargement, muscle fiber nuclear proliferation,
and muscle fiber splitting. Van Linge (104) found increased numbers of
fibers, increased connective tissue, and fiber splitting in the
plantaris muscles of rats. The animals were exercised by walking on a

treadmill after denervation of the triceps surae.

Detraining.--Faulkner pp 21, (23, 24) and Maxwell g£_al: (68)

found that in guinea pigs the number of muscle fibers decreases with

18

age but that treadmill running "delays" this process. Additionally,
during detraining the elevated fiber counts regress to those of

control animals.

CHAPTER III

METHODS AND MATERIALS

Measurements of total-body oxygen debt and oxygen uptake are
used to reflect metabolic responses to physical activity. Repeated
bouts of exhaustive exercise increase oxygen debt tolerance and pre-
sumably develop greater capacity for the generation of metabolic
energy via anaerobic metabolism. Training regimens using this type of
exercise are commonly described as "anaerobic" and are characterized
by maximal workloads with relatively short periods of activity (101).
In contrast, "aerobic" endurance training is dependent upon oxidative
muscle metabolism and tends to increase total-body oxygen uptake
capacity. Moderate or light workloads performed for relatively long
periods of time typify "aerobic" endurance programs (103). This study
was designed to investigate muscle fiber splitting in the soleus muscle
of the male albino rat following various durations of "aerobic" and

"anaerobic" training and subsequent detraining.

Experimental Animals

 

Ninety-eight normal, male, albino rats of the Sprague-Dawley
strain were obtained from Hormone Assay, Inc., Chicago, Illinois.

The animals were assigned to three groups and allowed a 12-day

19

20

period to adjust to laboratory conditions. The training began when the

animals were 84 days old.

Research Design

 

This study was originally organized as 4 two-way (3 x 5)
factorial designs. The first factor, Training Program (TRNPRO), con-
sisted of three levels of physical activity. The three training groups
were: (a) Control (CON)--a nonexercised group that remained relatively
inactive throughout the experiment, (b) Short (SHT)--an exercised group
that was subjected to an "anaerobic" endurance training program composed
of high-intensity workloads maintained for short periods of time, (c)
Long (LON)--an exercised group that was subjected to an "aerobic"
endurance training program composed of low-intensity workloads main-
tained for long periods of time.

The second factor, Duration (DURATN), was represented by five
groups of animals. The five duration groups were: (a) Zero-Week (OWK)-—
a group that was sacrificed after the 12-day adjustment period and thus
received the training regimens for zero weeks, (b) Eight-Week (8WK)--a
group that was sacrificed after receiving the treatments for eight
weeks, (c) Sixteen-Week (l6WK)--a group that was sacrificed after re-
ceiving the treatments for sixteen weeks, (d) Twenty-Four-Week (24WK)--
a group that after receiving treatments for sixteen weeks, remained
relatively inactive in sedentary cages for eight weeks, then was
sacrificed, (e) Thirty-Two-Week (32WK)--a group that after receiving
treatments for sixteen weeks, remained relatively inactive in sedentary

cages for sixteen weeks, then was sacrificed.

21

The design was to have a final replication of n = 4 animals in
each of the fifteen Training-Duration (TRNDUR) cells (see Table 1).
Also, the animals used in this study were subjects for several other
simultaneous investigations. One of these was to examine the acute
and chronic effects of exercise on muscle histochemistry. Therefore,
sacrifices were held fifteen minutes and seventy-two hours after the
last exercise bout for the 8WK and 16WK animals. The research design
for that study also had n = 4 animals in each TRNDUR cell. However,
with the third factor, Sacrifice Time, there were literally n = 8
animals receiving training for eight and sixteen weeks (see Table 2).
Since it was decided that sacrifice time would have no affect on the
muscle morphology, both the animals sacrificed fifteen minutes and
seventy-two hours after the last exercise bout were used for this

study.

Table l.--Original experimental design with cell frequencies.

 

TRNPRO

 

CON SHT LON

 

 

 

 

 

 

owxs 4 4 4

swxs 4 4 4
TREATMENT 16WKS 4 4 4
DURATION

24wxs 4 4 4

szwxs 4 4 4

 

 

 

 

 

 

 

22

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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23

Two extra rats were assigned to each 8WK, l6WK, 24WK, and 32WK
training group. Those animals with the best training performances
were selected for sacrifice. (These criteria will be described later.)

Although eight sacrifices are suggested by Table 2, only seven
were necessary. Two sacrifices of 8WK animals and two sacrifices of
16WK animals were conducted, but only one sacrifice of OWK animals
followed the adjustment period. It was determined that since the OWK
animals received no training, the same animals could be used as both
lS-min and 72-hr subjects. Therefore, to facilitate a balanced sta-
tistical analysis, each OWK animal was assigned randomly to one of the
three treatment groups and then reassigned to a second but different
group. This technique reduced the number of animals needed while
preserving the expected error variance in the OWK cells.

However, a design change was necessitated when tissue samples
were destroyed by freezing artifact and by shredding during sectioning.
Because some of these had to be discarded, the original design would
have been non-orthogonal. Non-orthogonality increases the chance of
significant interaction, when in fact there is none. Therefore, the
research design was changed to 3 one-way (l x 13) analyses of variance.
The one-way design eliminated the problem with non-orthogonality and
through the use of a priori and a posteriori comparisons, internal
validity could be maintained. The final design is shown in Table 3.

The thirteen levels in the analyses of variance were: (a)
Zero-Week (OWK)--a group that was sacrificed after the 12-day
adjustment period and thus received the training for zero weeks, (b)

Eight-Week-Control (8WKCON)--a nonexercised group that was sacrificed

24

Table 3.--Final experimental design with cell frequencies.

 

 

 

 

 

 

 

 

 

 

 

 

 

‘F‘—'—"‘
n
OWK 8
8WKCON 6
8WKSHT 6
8WKLON 5
16WKCON 5
16WKSHT 6
TRNDUR 16WKLON S
24WKCON 3
24WKSHT 4
24WKLON 2
32WKCON 4
32WKSHT 3
32WKLON 4

 

 

 

 

 

25

after eight weeks of relative inactivity, (c) Eight-Week-Short
(8WKSHT)--an exercise group that was sacrificed after eight weeks of
anaerobic training, (d) Eight-Week-Long (8WKLON)--an exercised group
that was sacrificed after eight weeks of aerobic training, (e) Sixteen-
Week-Control (l6WKCON)--a nonexercised group that was sacrificed after
sixteen weeks of relative inactivity, (f) Sixteen-Week-Short (l6WKSHT)--
an exercised group that was sacrificed after sixteen weeks of anaerobic
training, (g) Sixteen-Week-Long (l6WKLON)--an exercised group that was
sacrificed after sixteen weeks of aerobic training, (h) Twenty-four-
Week-Control (24WKCON)--a nonexercised group that was sacrificed after
twenty-four weeks of relative inactivity, (i) Twenty-four-Week-Short
(24WKSHT)--a group that after receiving anaerobic training for sixteen
weeks, remained relatively inactive in sedentary cages for eight weeks,
then was sacrificed, (j) Twenty-four-Week-Long (24WKLON)--a group that
after receiving aerobic training for sixteen weeks, remained relatively
inactive in sedentary cages for eight weeks, then was sacrificed, (k)
Thirty-two-Week-Control (32WKCON)--a nonexercised group that was sacri-
ficed after thirty-two weeks of relative inactivity, (l) Thirty-two-
Week-Short (32WKSHT)--a group that after receiving anaerobic training
for sixteen weeks, remained relatively inactive in sedentary cages for
sixteen weeks, then was sacrificed, (m) Thirty-two-Week-Long (32WKLON)--
a group that after receiving aerobic training for sixteen weeks, re-
mained relatively inactive in sedentary cages for sixteen weeks, then

was sacrificed.

26

Training Procedures

 

The exercise treatments were performed in a battery of indi—
vidual Controlled-Running Wheels (CRW) developed at the Human Energy
Research Laboratory, Michigan State University. The CRW apparatus can
be described as, " . . . a unique animal-powered wheel which is capable
of inducing small laboratory animals to participate in highly specific
programs of controlled, reproducible exercise" (106). The animals
learn to run by avoidance-response operant conditioning. A low-inten-
sity controlled shock current provides motivation for the animals to
run.

A typical training period consisted of alternated work and rest
intervals. The wheel is braked automatically during all rest intervals
to prevent spontaneous activity. A specified number of work and rest
intervals constituted one bout of exercise. A single training period
usually included several such bouts separated by a relatively long rest
time between bouts.

A light above the wheel signaled the start of each work inter-
val. The animal was given a specified period of time (acceleration
period) to reach a preset running speed. If the animal did not reach
the preset speed by the end of the acceleration period, the light
remained on and a controlled shock current was applied through the
running surface. As soon as the animal attained the prescribed speed,
the light was extinguished and the shock was discontinued. If the
animal responded to the light by running at or faster than the preset
speed during the acceleration period, the light was extinguished imme—

diately and shock was avoided. If the animal failed to maintain the pre-

scribed speed while running, the light-shock sequence was repeated.

27

The performance data for each animal were displayed in terms of
the total meters run (TMR) and the cumulative duration of shock (CDS).
The TMR and the total expected meters (TEM) were used to calculate the

percentage of expected meters (PEM):

PEM = 100 (TMR/TEM)

PEM values were the chief criterion used to evaluate and compare
training performance. A secondary criterion is provided by the per-
centage of shock-free time (PSF) which was calculated from the CD8 and

the total work time (TWT):

PSF = 100 - 100(CDS/TWT)

Training Programs

 

In this study, all training programs were administered five
days per week between 12:00 Noon and 9:00 P.M. Body weights of the

exercised animals were recorded before and after each training period.

Short (SHT)

 

This group of animals was subjected to an "anaerobic" training
program composed of high-intensity workloads maintained for short
periods of time. The workload of the program was progressively
increased through the first 37 days until the animals were expected to
complete eight bouts of exercise with 2.5 minutes of inactivity
between. Each bout consisted of six lO-second work intervals alter-
nated with 40-second rest intervals. The required running speed for
the work intervals was a relatively fast 99 meters/minute. (See

Appendix A.)

28

Lopg(LON)

This group of animals was subjected to an "aerobic" training
program composed of low-intensity workloads maintained for long periods
of time. The workload of the program was progressively increased
through the first 37 days until the animals were expected to complete
four bouts of exercise with 2.5 minutes of inactivity between bouts.
Each bout consisted of one 12.5-minute continuous run at a Speed of

only 36 meters/minute. (See Appendix A.)

Animal Care

 

All of the animals were housed in individual sedentary cages
(24 cm x 18 cm x 18 cm) throughout the investigation. Since rats
normally are more active at dusk and during the first few hours of
darkness than they are during daylight hours, the light sequence in the
animal quarters was automatically set to alter the rats' active period
by having the lights off between 1:00 P.M. and 1:00 A.M.

A relatively constant environment was maintained for the
animals by automatic temperature control. All animals were handled
daily. The cages were cleaned regularly. The animals had access to

food (Wayne Laboratory Blox) and water 3d libitum.

Sacrifice Procedures

 

Five sacrifices of twelve animals each were conducted eight
weeks apart. Each of these sacrifices took place on a Monday morning
and included four animals from each of the three training groups:
CON, SHT, and LON. However, there were two additional sacrifices on

the Fridays of the eighth and sixteenth weeks. These sacrifices took

29

place within fifteen minutes of the experimental animals' last exercise
period. Only those animals which were healthy and those of the exercise
groups whose mean PER and PSF values were 75 or higher were selected

for sacrifice.

Final body weights were recorded prior to sacrifice. Each ani-
mal was anesthetized by an intraperitoneal injection (4 mg/100 g body
weight) of a 6.48 percent sodium pentabarbital (Halatal) solution. The
right hindlimb was skinned and the superficial posterior crural muscles
were exposed by reflecting the overlying tissue. The right gastroc-
nemius, soleus, and plantaris muscles were removed as a block.

Upon removal, the right muscle block was rolled in talcum
powder. The block was held with forceps, gently stretched to approxi-
mate its physiological length, and quick frozen in 2 methylbutane
(isopentane). The isopentane had been precooled to a viscous fluid
(-l40 to -l60o C.) by liquid nitrogen. The frozen muscles were stored
in aluminum 3S-mm film containers at -20° C. until sectioning and
histochemical procedures could be initiated. Using precooled stainless
steel knives, muscle blocks approximately 10 to 15 mm thick were cut
from the mid-portions of the frozen muscles. The muscle blocks were
oriented distal end up and frozen on to cryostat chucks using 5 percent
gum tragacanth. Fresh-frozen serial cross-sections, 12 micra thick,
were cut using a rotary microtome cryostat (International-Harris
Microtome). Sections were picked up on cover glasses and fan dried for
one hour. The following series of histochemical stains were then used
for all concomitant studies: Harris' alum hematoxylin and eosin (H G E)

stain for morphology (66); Barker and Anderson's method for succinic

3O

dehydrogenase (SDH) activity (1); myosin adenosine triphosphatase

(pH 9.4) stain (Myosin ATPase) for twitching characteristics (15);
periodic acid-Schiff (PAS) for the presence of intracellular glycogen
(66); and Sudan Black B stain (SUD) for intracellular lipid

localization (66).

Fiber Counts

 

The H 6 E slides were projected using a Prado Microscope
projector with a 10x objective. Each fiber of the soleus was examined,
evaluated, and categorized as: (a) mature, (b) Split, or (c) in the
process of splitting. These subjective evaluations were based on the

following criteria (see Figure 1).

Split
A fiber was categorized as split if, relative to other fibers,

it was smaller and had centrally placed nuclei or was located adjacent

to other similar fibers.

In the Process of Splitting

 

A fiber was categorized as being in the process of splitting
if, relative to other fibers, it was similar in size and had centrally

placed nuclei or sarcolemmal clefts, folds, or splits.

Mature

A fiber was categorized as mature if, relative to other fibers,
it was similar in size, did not have centrally placed nuclei, and had
a normal sarcolemma. If the fiber was smaller than other fibers but

was not adjacent to similar fibers, it was also categorized as mature.

31

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32

Statistical Procedures

 

The proportion of fibers in the process of splitting, the pro-
portion of split fibers, and the proportion of mature fibers were the
dependent variables for the 3 one-way (l x 13) fixed-effects analyses
of variance routine on the Michigan State University Control Data
6500 Computer (CDC 6500). The number of fibers in the process of
splitting (SPLPRO), the number of split fibers (SPLFIB), and the number
of mature fibers (MATFIB) were used to calculate the total number of

fibers (TOTFIB):
SPLPRO + SPLFIB + MATFIB = TOTFIB

The proportion of fibers in the process of splitting (PRO) was calcu-

lated by:
SPLPRO/TOTFIB = PRO

The proportion of split fibers (SPL) was calculated by:
SPLFIB/TOTFIB = SPL

The proportion of mature fibers (MAT) was calculated by:
MATFIB/TOTFIB = MAT

Planned Comparisons were used for a priori tests and were
designed to evaluate differences between pairs of means (99). Duncan
Multiple Range Tests (DMRT) were used for a posteriori tests and were
designed to evaluate differences between pairs of means whenever a
significant F-ratio was obtained (101). The 0.10 level of significance

was established for all statistical analyses.

 

 

 

CHAPTER IV

RESULTS AND DISCUSSION

The results of this study are presented in the following
order: training data from the Controlled-Running Wheel (CRW) programs,
data on the Pr0portion of Fibers in the Process of Splitting (PRO),
data on the Proportion of Split Fibers (SPL), and data on the Proportion
of Mature Fibers (MAT). The F-ratios and P-values for all effects are
summarized in Appendix B and the raw data may be found in Appendix C.
An overall discussion of the findings is presented at the end of this

chapter.

Training Results

 

A criterion of 75 percent of expected meters (PEM) was set as
the minimum acceptable performance level for both CRW training groups.
The training results for the SHT group are shown in Figure 2. The
data indicate that these animals maintained a PEM of approximately 80
during the later stages of training when running velocities of 90 and
99 m/min were required. A search of the literature has revealed no
other programs for the training of laboratory rats which have incor-
porated such high velocities. Percent shock-free time (PSF) values
were approximately 85 to 90 during the more strenuous portions of the

training program. These results show that the animals generally

33

 

 

 

34

 

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35

responded to the conditioned light stimuli rather than to the
unconditioned shock stimuli.

The training data for the Long program are presented in Figure
3. PEM values were approximately 85 for the last fourteen weeks of
training. A running velocity of 35 m/min was required throughout
this period. The PSF values were in the range of 80 to 85 during the
last eleven weeks of training. These results indicate that the animals

responded well to the training programs.

PRO1 Results

 

The final results of the proportion of fibers in the process of
splitting (PRO) are shown in Table 4 and Figures 4 and 5. These results

are summarized here in terms of a priori and a posteriori comparisons.

A Priori Comparisons

 

The 8-Week Short group had a significantly higher mean than the
0-Week, 8-Week Long, and l6-Week Short groups. The 8-Week Control
group mean was significantly higher when compared to the 8-Week Long
group. There were no other significant differences between a priori

contrasts. (See Table 5.)

A Posteriori Comparisons

 

The 8-Week Short group mean was significantly higher than all
other groups except 8-Week Control and 24-Week Long. Also the 8-Week
Control group mean was significantly higher than the means of the

24-Week Short and all 32-Week groups.

 

1Proportion of Fibers in the Process of Splitting.

 

 

 

36

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Table S.--A priori contrasts as the differences between means.

 

Differences Between Means

 

 

A Priori Contrasts PRO SPL MAT
OWK 8WKC¢N -.0031 .0009 .0010
OWK 8WKSHT -.0046@ .0116 .0163@
OWK 8WKLQN .0000 .0146@ .0145

8WKC¢N 8WKSHT -.0015 .0107 .0153@
8WKCQN 8WKL¢N .0032@ .0137 .0134
8WKC¢N 16WKC¢N .0022 .0040 .0032
8WKSHT 8WKL¢N .0047@ .0030 .0019
8WKSHT 16WKSHT .0047@ .0037 .0085
8WKL¢N 16WKL¢N -.0006 .0154@ .0148

16WKC¢N 16WKSHT .0010 .0110 .0100

16WKC¢N 16WKL¢N .0004 .0023 .0019

16WKSHT 16WKL¢N -.0005 .0087 .0081

 

Significant at the .10 level.

 

 

 

 

41

SPL2 Results

 

The final results of the proportion of split fibers (SPL) are
shown in Table 6 and Figures 6 and 7. These results are summarized

here in terms of a priori and a posteriori comparisons.

A Priori Comparisons

 

The 8—Week Long group mean was significantly higher than the
means of the O-Week and 16-Week Long groups. There were no other

differences between a priori contrasts. (See Table 5.)

A Posteriori Comparisons

 

The 8-Week Short, 8-Week Long, and 24-Week Control groups had
significantly higher means than all 32-Week groups. Also the 16-Week
Short mean was significantly higher than the 32-Week Control and

32—Week Long group means.

MAT3 Results

 

The final results of the proportion of mature fibers (MAT) are
shown in Table 7 and Figures 8 and 9. Also the reciprocal of MAT final
results are shown in Table 8 and Figures 10 and 11. The reciprocal
figures were calculated by summing the PR¢ and SPL means and using
the NAT standard errors of the mean.

These results are summarized here in terms of a priori and a
posteriori comparisons. However, for ease of comparison with the PR¢
and SPL results, these summaries are described in terms of the "MAT

reciprocal."

 

2Proportion of Split Fibers.

3Proportion of Mature Fibers.

 

 

 

 

42

 

 

 

 

 

 

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51

A Priori Comparisons

 

The 8-Week Short group mean was significantly higher than the
means of the O-Week and 8-Week Control groups. However, there were
no other significant differences between a priori contrasts. (See

Table 5.)

A Posteriori Comparisons

 

The 8-Week Short, 8-Week Long, and 24-Week Control groups had

significantly higher means than all 32-Week groups. Also, the l6-Week

 

Short mean was significantly higher than the 32-Week Control and

32-Week Long group means.

Discussion

 

In C¢N groups there was a non-significant increase in the
number of fibers in the process of splitting at eight weeks (140 days
of age). The number of fibers in the process of splitting decreased
over the next twenty-four weeks (Figure 4).

A similar, more exaggerated, statistically significant
difference was observed in the SHT group at eight weeks. This group
clearly exhibited greater numbers of fibers in the process of
splitting. Only the 8-Week Control and 24-Week Long groups were not
significantly different. Although the numbers of fibers in the
process of splitting were quite small (<60/1000), there were signifi-
cantly more fibers involved in splitting in the SHT program at eight
weeks. Over the entire thirty-two weeks the pattern of response of the

SHT groups was quite similar to that of the control groups.

 

 

52

However, the pattern of response of the LON groups was quite
different from either the CON or SHT (Figure 4). It appears that during
aerobic training there was suppression of the number of fibers in the
process of splitting. However, these differences cannot be explained
from current evidence.

For the exercised animals the proportion of split fibers was

 

an
greatest at eight weeks (Figure 7). This was followed by a progressive a
reduction in the numbers of split fibers, even though the animals
continued to exercise for another eight weeks. Clearly, there was
a reduction in the numbers of split fibers over time whether the animals '

continued to exercise or not and at the end of sixteen weeks of
detraining very few split fibers were evident.

On the basis of the sixteen-week and thirty-two-week values, a
proportion of approximately .007 would have been expected in the 24-Week
Control group. Therefore, the extremely high value (.0322) would appear
to be due to chance.

Overall, the data for the SHT and LON groups reflect more split
fibers in the exercised animals than in the controls. With the excep-
tion of the 24-Week Control group, every comparison indicates more split
fibers in exercised animals.

It has been postulated that fibers in the process of splitting
are precursors to split fibers and that these in turn become mature
fibers (45, 95, 104). Therefore, one would expect the results observed
in the data on the pr0portion of fibers in the process of splitting
to be reflected in the data on the proportion of split fibers. Except
for the 24-Week Control group discussed earlier, this was true in the

CON and SHT groups. Results of the LON groups, excepting for the

 

 

 

53

32-Week group, showed little consistency between the proportion of
fibers in the process of splitting and those already split. These data
were not readily interpretable. It would appear that either the process
of splitting was delayed by the "aerobic" training or that the process
occured much more rapidly in the LON animals, therefore fewer were
observed.

The prOportion of fibers in the process of splitting added to
the proportion of fibers already split is shown in Figure 11. These
values are the reciprocal of the proportion of mature fibers shown in
Figure 9. There were more fibers splitting or split in the 8—Week
Exercised animals. After eight weeks there was a relatively consistent
decrease in the proportion of splitting and split fibers. Grossly, the
statistical results support this position. The 8-Week Short were sig—
nificantly greater than the O-Week and 8-Week Control and the 8-Week
Short and 8-Week Long proportions were significantly greater than all
32-Week groups. Clearly, there is variability in the data as observed
in the 24-Week Control group, but the trends are evident in the graphs
and there is sufficient statistical evidence to support this position.

No conclusions may be drawn from this study regarding the
total numbers of muscle fibers in the soleus. Improved methods of
fixing and sectioning are necessary to prevent shredding of tissue

sections so that complete fiber counts can be made.

 

CHAPTER V
SUMMARY, CONCLUSIONS, AND RECOMMENDATIONS

Summary

The purpose of this study was to determine the effects of
detraining on the morphology of previously exercised soleus muscle in
the adult male albino rat. In particular, the study was undertaken to
determine the effects of detraining, or removal of training, on muscle
fiber splitting. The experimental animals were subjected to either an
endurance-running routine or a sprint-running routine. The training
regimens used the Short and Long Controlled-Running Wheel programs
previously reported from this laboratory (106). These programs
attempted to stimulate either aerobic or anaerobic metabolic processes
in the animals.

Training began when the animals were 84 days of age and con-
tinued for sixteen weeks. This was followed by detraining, in the form
of sedentary living, over the next sixteen weeks. Beginning with
zero-week, sacrifices were held at eight week intervals. In a block
with the plantaris and gastrocnemius muscles, the soleus muscle was
surgically removed from the right hind leg and quick frozen. Serial
cross-sections were then cut and stained. For each animal the micro-

scope slide of the soleus was projected and each muscle fiber was

54

SS

categorized as a mature fiber, a split fiber, or a fiber in the process
of splitting. Except for damaged or missing areas, all fibers were
counted.

The results showed that there was an aging effect in the pro-
portion of splitting or split fibers. Also, there were more splitting
or split fibers in the 8-Week-Exercised animals. After eight weeks
there was a relatively consistent decrease in the prOportion of
splitting or split fibers whether the animals continued to exercise or
not. At the end of sixteen weeks of detraining, very few split fibers

were evident.

Conclusions

 

1. Greater proportions of splitting and split fibers are evident
in trained animals.

2. The prOportion of split fibers that is evident at eight weeks
of training decreases during the next eight weeks of training
and through the detraining period.

3. There is an aging effect on the proportion of splitting and
split fibers with the peak reached at about 140 days of age and

a progressive decrease thereafter.

Recommendations

 

1. The present study should be repeated with the use of absolute
fiber counts instead of proportional counts.
2. The present study should be extended by reinstating the exer—

cise program after a similar period of detraining.

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10.

11.

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APPENDICES

APPENDIX A

TRAIN ING PROGRAMS

 

 

APPENDIX A
TRAINING PROGRAMS
Standard Eight-Week, Short-Duration, High-Intensity

Endurance Training Program for Postpubertal and
Adult Male Rats in Controlled-Running Wheels

 

 

Total
Acc- Time Time Total
eler- Work Repeti- Bet- of Total Work
Day Day ation Time Rest tions No. ween Run Prog. Exp. Time
of of Time (min: Time per of Bouts Shock Speed (min: Meters (sec)
Wk. Wk. Tr. (sec) sec) (sec) Bout Bouts (min) (ma) (m/min) sec) TEM TWT
O 4-T -2 3.0 40:00 10 1 l 5.0 0.0 27 40:00 --- —-—
5=F -1 3.0 40:00 10 l l 5.0 0.0 27 40:00 --- ---
1 1=M l 3.0 00 10 10 40 3 5.0 1.2 27 49:30 540 1200
2=T 2 3.0 00 10 10 4O 3 5.0 1.2 27 49:30 540 1200
3=W 3 3.0 00 10 10 4O 3 5.0 1.2 27 49:30 540 1200
4=T 4 2.5 00 10 10 40 3 5.0 1.2 36 49:30 720 1200
5:? 5 2.0 00 10 10 40 3 5.0 1.2 36 49:30 720 1200
2 l=M 6 1.5 00 10 10 28 4 5.0 1.2 45 51:40 840 1120
2=T 7 1.5 00 16 15 27 4 5.0 1.2 54 59:00 972 1080
3= 8 1.5 00 10 15 27 4 5.0 1.2 54 59:00 972 1080
4=T 9 1.5 00 10 15 27 4 5.0 1.2 54 59:00 972 1080
5a? 10 1.5 00 10 15 27 4 5.0 1.2 54 59:00 972 1080
3 1=M 11 1.5 00 10 15 27 4 5.0 1.2 54 59:00 972 1080
2=T 12 1.5 00 10 20 23 4 5.0 1.2 63 59:40 966 920
3=w 13 1.5 00 10 20 23 4 5.0 1.2 63 59:40 966 92
4=T 14 1.5 00 10 20 23 4 5.0 1.2 63 59:40 966 920
5s? 15 1.5 00 10 20 23 4 5.0 1.2 63 59:40 966 920
4 1=M 16 1.5 00 10 20 23 4 5.0 1.2 63 59:40 966 92C
2: 17 1.5 00‘ 10 25 20 4 5.0 L. 3 72 60 00 960 soc
3=w 18 1.5 00 10 25 20 4 5.0 1.. 72 60:00 960 8“?
4= 19 1.5 00 10 25 20 4 5.0 1.3 72 0:00 960 80:
5-F 20 1.5 00 10 25 20 4 5.0 1.C 72 60 00 960 800
5 1=M 21 1.5 00 10 25 20 4 5.0 1.C 72 60:00 960 800
2=T 22 1.5 00 10 30 16 4 5.0 1.0 81 55:40 864 642
3=W 23 1.5 00 10 30 16 4 5.0 1.0 81 55:40 864 640
4=T 24 1.5 00 10 3O 16 4 5.0 1.0 81 55:40 864 640
5:? 25 1.5 00 10 30 16 4 5.0 1.0 81 55:40 864 640
6 IBM 26 1.5 00 10 30 16 4 5.0 1.0 81 55:40 864 640
2=T 27 2.0 00 10 35 10 5 5.0 1.0 90 54:35 750 50C
3=W 28 2.0 00 10 35 10 5 5.0 1.0 90 54:35 750 500
4=T 29 2.0 00 10 35 10 5 5.0 1.0 90 54:35 750 500
S-F 30 2.0 00 10 35 10 5 5.0 1.0 90 54:35 750 500
7 l-M 31 2.0 00:10 35 10 5 5.0 1.0 90 54 35 750 500
2-T 32 2.0 00 10 35 7 8 2.5 1.0 90 54:50 840 560
3=W 33 2.0 00:10 35 7 8 2.5 1.0 90 54:50 840 560
4=T 34 2.0 00:10 35 7 8 2.5 1.0 90 54:50 840 560
5:? 35 2.0 00:10 35 7 8 2.5 1.0 90 54:50 840 560
8 l-M 36 2.0 00:10 35 7 8 2.5 1.0 90 54:50 840 560
2-T 37 2.0 00:10 40 6 8 2.5 1.0 99 52.10 792 480
3-w 38 2.0 00:10 40 6 8 2.5 1.0 99 52:10 792 480
4-T 39 2.0 00:10 40 6 8 2.5 1.0 99 52:10 792 480
S-F 40 2.0 00:10 40 6 8 2.5 1.0 99 52:10 792 480

 

This standard program was designed using male rats of the Sprague-Dawley strain. All
animals were between 70 and 170 days-of-age at the beginning of the program. 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 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. Failure to provide this adjustment period will impose a double
learning situation on the animals and will seriously impair the effectiveness of the training
programs.

65

66

Standard Eight-Week, Long-Duration, Low-Intensity
Endurance Training Program for Postpubertal and
Adult Male Rats in Controlled-Running Wheels

 

 

Total
Acc- Time Time Total
eler- Work Repeti- Bet- of Total Work
Day Day ation Time Rest tions No. ween Run Prog. Exp. Time
of of Time (min: Time per of Bouts Shock Speed (min: Meters (sec)
Wk. Wk. Tr. (sec) sec) (sec) Bout Bouts (min) (ma) (m/min) sec) TEN TWT
O 4=T -2 3.0 40:00 10 l l 5.0 0.0 27 40:00 --— ---
5:? —1 3.0 40:00 10 l l 5.0 0.0 27 40:00 --- --—
l 1=M l 3.0 00 10 10 40 3 5.0 1.2 27 49 30 S40 1200
2=T 2 3.0 00 10 10 4o 3 5.0 1.2 27 49 30 540 1200
3=w 3 3.0 00 10 10 40 3 5.0 1.2 27 49 30 540 1200
4= 4 2.5 00 2O 10 30 2 5.0 1.2 27 34 40 540 1200
5s? 5 2.5 00 3O 15 20 2 5.0 1.2 27 34:30 540 1200
2 1=M 6 2.0 00 4O 20 15 2 5.0 1.2 36 34:20 720 1200
2=T 7 2.0 00 50 25 12 2 5.0 1.2 36 34:10 720 1200
3=w 8 1.5 01 00 30 10 2 5.0 1.2 36 34:00 720 1200
4=T 9 1.5 02 30 60 4 2 5.0 1.2 36 31:00 720 1200
=F 10 1.0 02 3O 6O 4 2 5.0 1.2 36 31:00 720 1200
3 1=M 11 1.0 02:30 60 4 2 5.0 1.2 36 31:00 720 1200
2=T 12 1.0 05:00 0 l 5 2.5 1.2 36 35:00 900 1500
3=w 13 1.0 05:00 0 l 5 2.5 1.2 36 35:00 900 1500
4:? 14 1.0 05:00 O l 5 2.5 1.2 36 35:00 900 1500
5=F 15 1.0 05:00 0 1 5 2.5 1.2 36 35:00 900 1500
4 1=M 16 1.0 05:00 O 1 5 2.5 1.2 36 35:00 900 1500
2=T 17 1.0 07.30 0 l 4 2.5 1.0 36 37:30 1080 1800
3=w 16 1.0 07 ’C 0 l 4 2.5 1.0 36 37:30 1080 1800
4=T 19 1.0 07:3“ 0 1 4 2.5 1.0 36 37:30 1080 1800
5=F 20 1.0 07 30 0 l 4 2.5 1.0 36 37 30 1080 1800
5 i=M l 1.0 07:30 O l 4 2.5 1.0 36 37:30 1080 1800
2=T 22 1.0 07:30 0 1 5 2.5 1.0 36 47:30 1350 2250
3=W 23 1.0 07:30 0 1 5 2.5 1.0 36 47:30 1350 2250
4=T 24 1.0 07:30 O l 5 2.5 1.0 36 47:30 1350 225:
5:? 25 1.0 7:30 0 1 5 2.5 1.0 36 47:30 1350 2250
6 l=M 26 1.0 07:30 0 l S 2.5 1.0 36 47:30 1350 2250
2=T 27 1.0 10:00 0 l 4 2.5 1.0 36 47:30 1440 2400
3=W 28 1.0 10:00 O 1 4 2.5 1.0 36 47:30 1440 2400
4:? 29 1.0 10 00 0 1 4 2.5 1.0 36 47:30 1440 2400
5=F 30 1.0 10:00 0 1 4 2.5 1.0 36 47:30 1440 2400
7 1=M 31 1.0 10:00 0 l 4 2.5 1.0 36 47:30 1440 2400
2=T 32 1.0 10:00 0 l 5 2.5 1.0 36 60:00 1800 3000
3=W 33 1.0 10:00 O 1 5 2.5 1.0 36 60:00 1800 3000
4:7 34 1.0 10:00 O 1 5 2.5 1.0 36 60:00 1800 3000
5=F 35 1.0 10:00 0 l S 2.5 1.0 36 60:00 1800 3000
8 1=M 36 1.0 10:00 0 l 5 2.5 1.0 36 60:00 1800 3000
2=T 37 1.0 12:30 0 l 4 2.5 1.0 36 57:30 1800 3000
3=W 38 1.0 12:30 0 1 4 2.5 1.0 36 57:30 1800 3000
4=T 39 1.0 12:30 0 l 4 2.5 1.0 36 57:30 1800 3000
58? 40 1.0 12:30 O l 4 2.5 1.0 36 57:30 1800 3000

 

This standard program was designed using male rats of the Sprague-Dawley strain. All
animals were between 70 and 170 days-of-age at the beginning of the program. The duration and
intensity of the program were established so that 75 per cent of all such animals should have PF?
and PER scores of 75 or higher during the final two weeks. Alterations in the rest time.
repetitions per bout, number of bouts, or time between bouts 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
priq; to the start of the program. Failure to provide this adjustment period will impose a

double learning situation on the animals and will seriously impair the effectiveness of the
training program.

APPENDIX B

ANALYSIS OF VARIANCE TABLES AND PLANNED COMPARISONS

T-VALUES AND P-VALUES

APPENDIX B
ANALYSIS OF VARIANCE TABLES AND PLANNED COMPARISONS

T-VALUES AND P-VALUES FOR THE PROPORTION
OF MATURE FIBERS

ANALYSIS OF VARIANCE TABLE

Degrees Sums
of of Mean
Source Freedom Sguares Sguares F-Ratio F-Prob.
Between Groups 12 .0057 .0005 1.921 .055
Within Groups 48 .0118 .0002 '
Total 60 .0175

PLANNED COMPARISONS

 

Contrast T-Value T-Prob.
OWK - 8WKCON .121 .904
OWK - 8WKSHT 1.926 .060
OWK - 8WKLON 1.616 .113
8WKCON - 8WKSHT 1.688 .098
8WKCON - 8WKLON 1.414 .164
8WKCON - 16WKCON - .340 .735
8WKSHT - 8WKLON - .196 .846
8WKSHT - 16WKSHT - .942 .351
8WKLON - 16WKLON —l.491 .142
16WKCON - 16WKSHT 1.051 .298
16WKCON - 16WKLON .187 .852
16WKSHT - 16WKLON - .856 .396

67

68

ANALYSIS OF VARIANCE TABLES AND PLANNED COMPARISONS

T-VALUES AND P-VALUES FOR THE PROPORTION
OF SPLIT FIBERS

Source
Between Groups

Within Groups

Total
Contrast
OWK - 8WKCON
OWK - 8WKSHT
OWK - 8WKLON

8WKCON - 8WKSHT
8WKCON - 8WKLON
8WKCON - 16WKCON
8WKSHT - 8WKLON
8WKSHT - 16WKSHT
8WKLON - 16WKLON
16WKCON - 16WKSHT
16WKCON - 16WKLON
16WKSHT - 16WKLON

ANALYSIS OF VARIANCE TABLE

Degrees
of

Freedom

12

48

60

Sums
of

Sguares

.004

.009

.014

PLANNED COMPARISONS

7

7

4

Mean
Squares

F-Ratio

 

.0004

.0002

 

T—Value

-1.

-1.
-1

.116

504

.796

299

.588

.459

.349

.445

.704
.273
.256
.006

1.910

T-Prob.

.908
.139
.079
.200
.119
.648
.728
.658
.095
.209
.799
.319

F-Prob.

.057

 

 

 

69

ANALYSIS OF VARIANCE TABLES AND PLANNED COMPARISONS
T-VALUES AND P-VALUES FOR THE PROPORTION
OF FIBERS IN THE PROCESS OF SPLITTING

Source
Between Groups

Within Groups

Total
Contrast
OWK - 8WKCON
OWK - 8WKSHT
OWK — 8WKLON

8WKCON - 8WKSHT
8WKCON - 8WKLON
8WKCON - 16WKCON
8WKSHT - 8WKLON
8WKSHT - 16WKSHT
8WKLON - 16WKLON
16WKCON - 16WKSHT
16WKCON - 16WKLON
16WKSHT - 16WKLON

Sums
of

Sguares

.0002

.0004

.0005

ANALYSIS OF VARIANCE TABLE

Mean
Squares F—Ratio F-Prob.

 

.0000 2.096 .035

.0000

PLANNED COMPARISONS

T-Value

-2.
-3.
.024
.963
.923
.314
.842
.976
.324
.605
.259
.334

NNHH

131
161

T-Prob.

 

.380
.003
.981
.340
.060
.195
.007
.005
.747
.548
.797
.740

APPENDIX C

BASIC DATA

APPENDIX C

 

 

BASIC DATA
Fibers in
Dura- the Process Split Mature Total
Animal Treat- tion of Splitting Fibers Fibers Fibers
Number ment (wk) (SPLFIB) (SPLFIB) (MATFIB) (TOTFIB)

201 Control 00 000 008 2094 2102
202 Control 00 000 017 2743 2762
204 Control 00 002 019 2654 2675
205 Control 00 011 154 2668 2833
207 Control 00 011 113 2659 2783
210 Control 00 003 013 2445 2461
211 Control 00 000 017 2010 2027
212 Control 00 000 029 3067 3096
89 Control 08 009 029 2119 2157
90 Control 08 022 095 2077 2194
91 Control 08 000 021 1705 1726
92 Control 08 000 027 1437 1464
94 Control 08 015 019 1590 1624
95 Control 08 006 017 2220 2243
6 Short 08 000 092 1930 2022
15 Short 08 017 032 1226 1275
19 Short 08 018 070 1585 1673
26 Short 08 006 032 2453 2494
27 Short 08 005 019 1050 1074
34 Short 08 010 069 2405 2484
48 Long 08 000 084 2310 2394
53 Long 08 002 139 2032 2173
57 Long 08 000 027 1142 1169
68 Long 08 005 039 1725 1769
85 Long 08 003 018 1347 1368
97 Control 16 000 046 1742 1788
98 Control 16 000 006 1611 1617
99 Control 16 005 012 1423 1440
100 Control 16 011 046 1414 1471
104 Control 16 000 000 1154 1154

70

 

71
Appendix C Cont'd.

 

 

Fibers in
Dura- the Process Split Mature Total
Animal Treat- tion of Splitting Fibers Fibers Fibers
Number ment (wk) (SPLFIB) (SPLFIB) (MATFIB) (TOTFIB)

2 Short 16 000 017 2208 2225

4 Short 16 001 021 2598 2620
14 Short 16 004 111 1914 2029
17 Short 16 000 043 1294 1337
33 Short 16 008 058 2617 2683
43 Short 16 005 067 2650 2722
47 Long 16 000 004 2230 2234
59 Long 16 002 057 1388 1447
64 Long 16 000 043 2475 2518
66 Long 16 001 019 2328 2348
67 Long 16 018 037 2548 2603
106 Control 24 002 064 1684 1750
107 Control 24 002 061 1737 1800
108 Control 24 002 067 2503 2572
9 Short 24 000 037 2172 2209
16 Short 24 000 007 1293 1300
29 Short 24 004 042 2445 2491
39 Short 24 002 062 2691 2755
80 Long 24 012 079 2324 2415
83 Long 24 001 008 2590 2599
109 Control 32 000 000 1094 1094
110 Control 32 000 000 1085 1085
111 Control 32 000 002 2035 2037
112 Control 32 000 027 2569 2596
13 Short 32 002 020 2890 2912
21 Short 32 000 019 2158 2177
30 Short 32 000 007 2145 2154
49 Long 32 000 010 2582 2592
54 Long 32 000 003 1920 1923
55 Long 32 001 009 1758 1768
71 Long 32 000 019 2620 2639

 

 

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