FIBER SIZE AND CAPILLAEY TO
FIBER RATIO {N THE GASTROCNEMWS
MUSCLE 0F EXERCISED RATS

Thesis $09 the Degree of DH. D.
MICHIGAN STATE UNEVERSITY

Rexford E. Carrow
1965

THESIS

 

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UniVersity

 
 

This is to certify that the
thesis entitled

Fiber Size and Capillary to Fiber Ratio in the

Gastrocnemius Muscle of Exercised Rats

presented bg

Rexford E. Carrow

has been accepted towards fulfillment
of the requirements for

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Datefifipmmber 15 ; 1965

 

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ABSTRACT

FIBER SIZE AND CAPILLARY TO FIBER RATIO IN THE
GASTROCNEMIUS MUSCLE OF EXERCISED RATS

by Rex E. Carrow

Thirty male rats (Sprague-Dawley), 25 days of age were
placed in exercise cages for 7 days. The animals were
assigned to one of three treatment groups: sedentary,
voluntary exercise, and forced exercise. The table of
random numbers was used to assign the animals to their
various groups. For the next thirty—five days the sedentary
group was permitted no exercise other than that allowed by
their small individual cages. The voluntary group remained
in activity cages While the forced group in addition to
being in activity cages swam 30 minutes each day with lead
weights equal to 2% of the body weight attached to their
tails. At the end of the thirty-five days the animals were
sacrificed. The hind limbs were injected with India ink,
the gastrocnemius muscle was removed, embedded in gelatin
and cut on the freezing microtome. The cross—sectional areas
of the red and white muscle fibers from the gastrocnemius
muscles were measured by using a polar planimeter. Ink filled

capillaries were counted in conjunction with fiber measurements.

Rex Carrow

The results of measurements and counts for the seden—
tary, voluntary activity and forced exercise groups were
compared statistically using correlations, analysis of
variance and the Tukey procedures.

The average cross—sectional area of the red fibers per
gram body weight in animals from the sedentary group was
3.82 square microns. In animals which had been forced to
exercise the average red fiber area per gram body weight was
5.72 square microns.

The total per cent differences in the size of the white
fibers (forced-sedentary) and red fibers (forced—sedentary)
were 32.5 and 49.7 per cent respectively. Of the total
differences found, 76.2 per cent was manifest in the animals
permitted to exercise at will (voluntary group). Only a
56.8 per cent increase was produced in the red fibers of
animals from the same group. By comparison, 23.6 per cent
of the total white fiber difference and 43.1 per cent of the
total red fiber difference (forced—sedentary) were found in
the comparisons of the data from the forced exercise and
voluntary exercise groups.

The mean capillary per fiber per gram of body weight
(C/F/G) ratio for animals in the sedentary group was .80.

In animals which had been forced to exercise the C/F/G

Rex Carrow

ratio was 1.0 while in the voluntary group it was .89.

The total per cent differences in C/F/G in red and white
fibers (forced-sedentary) were 25 and 31 per cent. Of the
total differences found 75 per cent was exhibited by the
white fibers of the voluntary group and only 45 per cent was
produced in the red fibers of animals of the same group.
Fifty-five per cent of the total red C/F/G difference and
25 per cent of the total white C/F/G difference (forced-
sedentary) were found when the data from the forced and
voluntary exercise groups were compared.

These results showed that voluntary exercise produced a
greater increase in size of the white than of the red fibers.
The C/F/G ratio in conjunction with these fibers followed the
same pattern. Under the conditions imposed by forced
activity there was a relatively greater increase in the size
of the red than of the white fibers. These differences were
paralleled by commensurate changes in vascular supply.

The circulatory adjustments which accompany changes in
red and white muscle fiber sizes with specific exercise
regimens, guarantee a balance between effective blood flow

and the immediate metabolic needs of the muscle tissue.

FIBER SIZE AND CAPILLARY TO FIBER RATIO IN THE

GASTROCNEMIUS MUSCLE OF EXERCISED RATS

BY

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Rexford E: Carrow

A THESIS

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

DOCTOR OF PHILOSOPHY

Department of Anatomy

1965

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ACKNOWLEDGMENTS

The author is grateful to Dr. Roger E. Brown for the
faith and encouragement he has expressed in me as a future
teacher and research worker. His suggestions on the prepa-
ration of this thesis and the microtechniques involved were
invaluable.

Grateful appreciation is extended to Dr. M. Lois
Calhoun, Professor and Head of the Department of Anatomy.
Her years of personal guidance and confidence in the author
as an individual were primarily responsible for completion
of this program.

Special thanks are also due Dr. Wayne VanHuss, Director
of the Human Energy Research Laboratory for the acquisition
of experimental animals and for his assistance with statis-
tical evaluations and organization.

Many thanks to Dr. Esther M. Smith for her assistance
with the photomicrographic equipment. Her patience in
molding a graduate assistant into a teacher is greatly
appreciated.

Special thanks to Drs. Charles W. Titkemeyer and Robert
F. Langham for serving on the guidance committee and

assuming the responsibility which this entails. The author

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is also indebted to Drs. D. R. Swindler and A. W. Stinson
for their constructive criticism of the manuscript.

Thanks are also extended to Dr. E. A. Roege for her
technical advice, to Mr. Denver Baker, Miss Jean Pajot and
Miss H. A. McCoy for their assistance in preparing the
illustrations. To Mrs. L. J. Wahl and Mrs. L. L. Holmes
for their assistance in preparing the manuscript.

The highest possible tribute is due my wife Pat who,
with understanding, encouragement and sacrifice has con—
tributed so much toward the completion of this work.

Many thanks to sons Rick and Tom who have often accepted
the importance of this study in lieu of a much deserved

camping trip.

iii

A...

VITA
REXFORD E. CARROW

Candidate for the degree of Doctor of Philosophy

Final examination: August 19, 1965. 10:00 a.m.

 

Dissertation: Fiber size and capillary to fiber ratio in

 

the gastrocnemius muscle of exercised rats.
Outline of studies:
Major subject: Anatomy
Minor subject: Pathology
Biographical items:
Born: February 4, 1927. Mt. Pleasant, Michigan.
Undergraduate studies:
B.S., Michigan State University, 1953.
M.S., Michigan State University, 1960.
Professional experience:
Graduate Assistant, Department of Anatomy, College of
Veterinary Medicine, Michigan State University, 1960-
1961.
Instructor, Department of Anatomy, College of Veterinary
Medicine, Michigan State University, 1961-1965.

Member of Society of Sigma Xi.

iv

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TABLE OF CONTENTS

Introduction . . . . . .

Review of Literature . .

Red and White Muscle Fibers

Fiber Size . . . . .

Innervation and Fiber Size

Blood Supply to Muscle

Materials and Methods .

Experimental Animals
Anesthesia . . . . .
Injection Materials
Injection Apparatus
Injection Pressure .

Surgical and Injection
Tissue Procurement and

Measurements . . . .
Statistics . . . . .

Results and Discussion .
General Statement .
Fiber Sizes . . . .
Capillaries Per Fiber
Results . . . . . .

Summary and Conclusions

Literature Cited . . . .

Appendix . . . . . . . .

Methods .
Preparation

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LIST OF TABLES

TABLE

1.

Relative fiber sizes
exercised animals .

Differences in fiber

Relative differences
(relative data) . .

in sedentary and

size (relative data).

in fiber size

Capillaries per fiber and capillaries
per fiber per gram body weight . . . . .

Differences in capillaries per fiber per
gram body weight (relative data) . .

Relative differences in capillaries per fiber
per gram body weight (relative data) . . . .

vi

Page

27

27

27

32

33

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LIST OF PLATES

PLATE
1. Injection apparatus . . . . . . . . . . . .
2. Apparatus for tissue fixation . . . .
3. Left gastrocnemius muscle with mapping
of the cross-section at mid—level . . . . .
4. Cross-sectional View of small, red muscle
fibers and associated capillaries . . . . .
5. Cross-sectional view of several adjacent

fasciculi showing that some have capillaries
filled with ink and others which are devoid
of ink . . . . . . . . . . . . . . . . . . .

vii

Page

57

58

59

61

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LIST OF GRAPHS

GRAPH Page
1. Mean muscle fiber sizes . . . . . . . . . . . . 55
2. Relative muscle fiber sizes per gram

body weight . . . . . . . . . . . . . . . . . . 55
Mean capillaries per fiber . . . . . . . . . . . 56

Relative capillaries per fiber per gram
body weight . . . . . . . . . . . . . . . . . . 56

viii

 

 

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IN T RODUCT I ON

Movements of the body are a result of skeletal muscle
contractions. The energy needed for contraction is ob—
tained from external sources and brought to the individual
muscle cells by the circulatory system. The great range
of activity of the muscular system requires a blood flow
which is capable of adjusting rapidly to provide adequate
nutrients and energy rich materials when the occasion
demands.

Although the muscular and circulatory systems have
been the subjects of many investigations in themselves,
their integrative potential has received comparatively
little attention. Fewer yet are studies which have con-
sidered the relationships that exist between these two sys-
tems when they are subjected to various types of physical
activity.

Furthermore, a review of the literature pertinent to
skeletal muscle and its blood supply in exercised animals
reveals that a great number of techniques as well as a vari—
ety of animals have been used in the investigations. In
maHQr cases the age, sex, or physical condition has been

Omitted from the report. In other instances the limited

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number of animals used invalidated the statistics. As a
consequence of the diversity in the materials and methods
utilized there exists a great deal of confusion as to the
exact meaning and interpretation of the results.

Because of these confusing data, the need for well
controlled experiments on the relationships between skel—
etal muscle and its vascular supply is imperative. Of
equal necessity is the critical examination of materials
and methods in order to obtain accurate and meaningful data.
The present study has been designed to control as many of

the factors in question as possible.

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REVIEW OF LITERATURE

RED AND WHITE MUSCLE FIBERS

Several types of skeletal muscle have been known to
exist since ancient times, probably through the recogni—
tion of light and dark meat. Investigations on this sub-
ject have brought forth numerous terms, i.e., red and white
muscles, light and dark muscles, granular and agranular
muscles, large and small fibers, fast and slow fibers and
tonic and tetnic fibers to describe the differences in
coloration, size and speed of contraction. Reports by
Bell (1911), Needham (1926), Denny-Brown (1929) and Smith
and Giovacchini (1956) included excellent descriptions and

extensive bibliographies on this subject.

FIBER SIZE

The sizes of individual muscle fibers has been the sub-
ject of a great many studies. In human embryos, the fibers
of all skeletal muscles are of approximately the same di-
mensions and appear to grow at a uniform rate until birth
(Halbran, 1894 and Greep, 1954). Soon after birth the
fibers of certain muscles become larger than others. A re-
port by Scott (1957) indicated that in the adult, each

muscle fiber is two or three times as broad in cross-section

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as in the child one year of age and that in the child there
is a great variation between the sizes of the individual
fibers. Similar results have been reported for the rat
(Morpurgo, 1897).

Denny—Brown (1929), found that in the newborn kitten
all muscles were Opaque and the fibers were very small in
diameter. All of the fibers in the gastrocnemius muscle
measured 100 square microns in size. Approximately 50% of
the fibers in the soleus muscle measured 100 square microns
but the remaining fibers measured 220 square microns.
Studies on several species of birds (George and Naik, 1957,
1958, 1959 and Denny-Brown, 1929), and the rat (Nachmias
and Padykula, 1958) and (Stein and Padykula, 1962), also
pointed out the variation in muscle fiber sizes in a single
muscle. In these "mixed" muscles the smaller "red" fibers
were found in the deeper portions, while the large "white"
fibers were located at the periphery.

Denny-Brown's work on the pectoralis muscles of the
pigeon, revealed that the smaller dark fibers averaged 900
square microns, and the cross-sectional areas of the large
clear fibers varied between 3600 and 7200 square microns.
In the same animal, the dark and light fibers of the super-

ficial muscles of the leg were nearly the same size.

Martin _§_g1, (1932) reported fiber sizes of 2300 and
2600 square microns in the semimembranosus and gracilis
muscles of the dog. Valdivia (1958) worked with the guinea
pig and found that red fibers were uniform in size and shape
and averaged 1800 square microns in area. He mentioned that
in mixed muscles the white fibers were more variable in size
and shape and larger than the red fibers. In contrast,
Paff's (1930) report on the guinea pig showed the largest
fibers to be approximately half the size of those measured
by Valdivia. In the rat and cat, he obtained measurements
of 609 and 476 square microns respectively. Additional
measurements on the dark and light muscle fibers in the
gastrocnemius of the rat (Dellasanta, 1964) indicated cross—
sectional determinations of 1353 and 2652 square microns.
Smith and Giovacchini (1956) stated that Arloing and
Lavocat, and Pakual found no differences in muscle fiber
size, while Meyer and Graf observed that red fibers were
larger than white ones. Stoel (1925) counted nearly 3
times as many white as red fibers per square millimeter of
area. Watzka (1939) noted that white fibers shrink more
than red ones during the process of fixation, however, in

the fresh condition, they are nearly the same size.

INNERVATION AND FIBER SIZE

Another important characterization of individual muscle
fibers is their speed of contraction. In 1929, Denny—Brown
showed that all muscles in the two—week-old kitten are slow
in contraction time, but a few weeks later the fibers have
differentiated into the slow acting red.type and the fast
acting white type. Additional works of this nature (Buller
_£_§1, 1960a & b, Hess and Pilar 1963, and Vroba, 1963) in-
dicated that innervation may play an important role in the
differentiation of muscle fibers into fast and slow types.

Bach (1948) reported that in the rabbit the normally
slow acting soleus can be made fast acting by exchanging
its nerve supply with that of the originally fast acting,
white, tibialis posterior muscle. In this reversal the
white tibialis posterior muscle became slow acting and red.
Bajusz (1964) quoted Graf and Kruger who reported that the
type of innervation is one of the factors responsible for
the differentiation of red and white fibers in speed and
duration of contraction. In support of this Bajusz (1964)
demonstrated that nerve impulses are not the same for the
two types of fibers, and that there is relatively greater

dependence of the white than of the red fibers on neuro—

muscular integrity.

In a symposium on "What we need to know about muscle"
(Bennett g£_gl, 1958), Denny-Brown reported that in the
past there was evidence for a uniform speed of contraction
in all fibers of any muscle, but now it seems possible that
there is some variation from fiber to fiber in the larger
muscles. He also mentioned that the sharp distinction of
muscle fibers into red, pale, slow, fast, etc., makes it
natural to seek two different types of muscular function
corresponding to these differences.

In support of this, Bajusz (1963) pointed out that if
red and white fibers differ with respect to speed and dura-
tion of contraction, it would be logical to assume a dif—

ference in innervation.

BLOOD SUPPLY TO MUSCLE

The differences in coloration between muscles and even
muscle fibers brought forth investigations to determine the
significance of the chromatic variation. Smith and Giovac-
chini (1956) quoted Ranvier who proposed that the deeper
color of red muscles was due to some substance within the
muscle fibers which could not be removed by exsanguination.
More recently Millikan (1937) reported the substance to be

myoglobin and that it acted as an oxygen reservoir, While

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Lawrie (1952) mentioned that the red and white fibers prob-
ably had different mechanisms for the utilization of oxygen.
When attention shifted to the oxygen requirements of

muscle, it was necessary to examine the relationship of

the vascular bed with muscle fibers. Spalteholz (1888)
carried out studies which described these relationships in
the rabbit. Krogh (1919) first investigated the problem

in detail. He found that there was 2.0 capillaries per

red muscle fiber and 2.6 per white fiber. In addition, he
pointed out that the number of functional capillaries in
muscles is variable and dependent upon whether the muscle

is resting or active at the time the counts are taken. The
importance of methods in which the capillary to fiber ratios
(C/F) are determined in exemplified by the work of Stoel in
1925 on the rabbit. His figure of 1525 capillaries per
square millimeter of white muscle and 790 capillaries per
square millimeter cross-sectional area in red muscle indi-
cates that there are nearly twice as many capillaries per
square millimeter in white muscle as in red muscle. However,
on a C/F basis these figures revealed 0.7 capillaries per
white fiber and 1.2 capillaries per red fiber. In contrast,
an excellent study (Paff, 1930) on the rat, guinea pig and

cat revealed that the number of capillaries per square

millimeter of cross—section of muscle ranged from 2341 to
3574. Duyff and Bouman (1927) also worked with the rabbit
and reported a C/F ratio of 2.3 for red muscle and 1.3 to
1.5 for white muscle fibers. In Watzka's (1939) work on
the rabbit and the hen, it was reported that red fibers

had a higher concentration of capillaries than did white
fibers. The only other study of this nature on birds was
reported by Bosiger (1950) who found them 3.0 to 6.0 times
as many capillaries per red fiber as for white fibers. A
report on the influence of training on the number of capil-
laries in skeletal muscle by Petren gg_g1, (1936) showed
that the number of open capillaries in the gastrocnemius
muscle of the guinea pig was considerably higher in trained
animals than in controls. Valdivia (1958) worked with
three groups of guinea pigs. Group A consisted of animals
born at sea level in Peru. They were placed in pens and
were permitted to exercise freely throughout their life
span. Group B born in Wisconsin were maintained in small
cages where movement was restricted. Group C guinea pigs
were born in the Peruvian mountains and.kept at an altitude
of nearly 15,000 feet. In examining the C/F ratios in red
luuscles there was no significant difference found between

QINDUps A and B. Group C, the high altitude group, showed

10

an average increase of 1.4 capillaries per fiber over the
other groups. C/F ratios calculated from the dog under
resting conditions by Martin _§_g1, (1932) produced a ratio
of 0.64 while in animals exercised by electrical stimulation,
it was 1.20. By using capillary dilators this ratio in-
creased to nearly 2.5 capillaries per fiber. The authors
made no mention of whether these counts were taken in rela-
tion to red or white fibers. A detailed investigation on
the vascular patterns in red and white muscles of the rab—
bit was carried out by Smith and Giovacchini (1956). They
found C/F ratios of 1.7 and 0.5 in red and white muscle
reSpectively.

The variability in the numbers of capillaries per mus-
cle fiber indicated in the foregoing discussion may be at-
tributed to the different techniques used, and the condi-
tions under which the relationships were obtained. It is
also possible that internal mechanisms as yet unidentified
may regulate the circulation in skeletal muscle (Uvnas,
1960, Hyman, 1963 and Folkow, 1952). In fact it is possible
'that the results obtained by Smith and Giovacchini (1956)

aIKi Martin t g1, (1932) may be attributed to such mechan—

isnus.

After injection with India ink, these authors observed

11

that all muscles of the lower extremities turned black and
appeared to be completely injected. However, upon micro—
scopic examination of tissue sections, it was apparent that
some areas of the muscle were filled while others were de-
void of ink. Recent studies on the circulation to skeletal
muscle (Barlow g; g1. 1961; Hyman and Lenthall, 1962; Hyman
and Paldino, 1962; Hyman ggigl. 1965 and Renkin and Rosell,
1962) suggest that the circulation to skeletal muscle is
influenced by several different mechanisms. The problems
associated with incomplete injections will be discussed

later in this thesis.

MATERIALS AND METHODS

EXPERIMENTAL ANIMALS

Fifty male albino rats (Sprague—Dawley) of the same age
(25 days) were purchased as weanlings. They were numbered
and placed in individual metal cages where they had the
opportunity to become adjusted to the new surroundings.

To avoid isolation stress (Hatch _E_g1, 1963), the animals
were handled daily, but allowed no exercise other than rest
afforded by their confining quarters. All animals were fed
gg_1ibitum from a stock diet and had free access to water.
The temperature in the animal room was maintained between
70 and 72 degrees Fahrenheit. Body weights were recorded
for each animal daily throughout the experiment.

At 31 days of age, each animal was placed in a sponta-
neous exercise cage for seven days. On the basis of the
degree of activity recorded on a counter during the last
four days, the ten most active and ten least active ani-
mals were dropped from the study. The remaining thirty
animals were assigned to one of three treatment groups:
senientary, voluntary exercise and forced exercise. The

tailble of random numbers was used as the basis for assign—

111E? “the animals to the various groups. Under this

12

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procedure the animal possessing the first number chosen from
the table was placed in the sedentary group, the second choice
was placed in the voluntary group and the third selection in
the forced group. This system was carried on until all thirty
animals had been placed in one of the three treatment groups.
They remained in their respective groups for the next 35 days
(63 days from birth). During this time the animals in group I
(sedentary group) were allowed no exercise other than that
available in their individual standard cages (5"x5"x12”); ani-
mals in the voluntary group were housed in individual activity
cages which had a freely rotating drum with counter. Group III
(forced exercise group), in addition to living in activity

type cages, were forced to swim for 30 minutes each day with
lead weights equal to 2% of their body weight attached to their
tails. The water was maintained at body temperature (380 C.)
during the swimming period. At the end of the 35 day activ-
ity period all animals were anesthetized and subsequently in-
jected with India ink. The gastrosnemius muscle was removed

and processed for microscopic examination.

ANESTHESIA

Methoxyflurane* was chosen as the anesthetic agent because

*Pitman-Moore, Co., Indianapolis, Indiana

14

it has excellent muscle relaxant properties and does not

alter heart rate or rhythm except at very deep anesthetic
levels (North g§_g1, 1961; Jones §£_g1, 1962; and Bagwell
and Woods, 1962). In addition, it has a maximum concen-

tration of 3% in air, thus the depth of anesthesia can be
controlled.

The animals were anesthetized in a special chamber
which provided for CO2 absorption and continuous circula-
tion of the anesthetic agent (Heusner, 1965). The desired
level of anesthesia was controlled by maintaining the gas
in the chamber at a concentration of 3%. A Fisher Gas
Partitioner* was used to analyze the gas mixture in the
chamber and a model SR Sargent Recorder** was utilized to
monitor the components resolved in the gas chromatograph
accurately.

After the initial anesthesia, the animals were removed
from the chamber for the necessary surgical and injection
procedures. These usually averaged approximately 25 minutes
per animal and the state of anesthesia was maintained by
using minimum quantities of ether in a nose cone during the

operative period.

* Fisher Scientific Co., Chicago 51, Illinois
**E. H. Sargent and Co., Detroit, Michigan

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INJECTION MATERIALS

The selection of a proper injection material for the
vascular system is of particular importance since several
factors must be controlled to produce accurate results.

The injection mass must contain suspended particles of such
size that they can enter the capillaries without difficulty.
In addition, the material must remain in the vascular bed
during fixation, embedding and sectioning. Rapid fixa-
tion is imperative in order to minimize shrinkage and to
prevent autolysis.

With these points in mind, the Higgin's* ink injection
technique described by Brown (1965) was used. Hartman _§_§1,
(1929) criticized the use of India ink because he observed
the clumping of ink particles at the origina of Open capil—
laries, thus, preventing the fluid from entering the vessels.
However, Brown demonstrated that if care is taken to decant
the ink as it is taken from the bottle, any flocculated ma—
terial will be eliminated, and if the pH of the ink is held
at 9.0, the carbon particles will remain in a colloidal
state and will not aggregate and plug the smaller elements

of the vascular tree.

*Higgins Ink Co., Brooklyn 15, New York

16

Immediately before the injection, commercial formalin
(40%) was added to the India ink to a concentration of 2%
formalin. This aided in a more rapid and even fixation of
the muscle tissue and did not cause precipitation of the
carbon particles. It should be noted that a concentration
of more than 4% formalin does cause the particles to pre-
cipitate.

In order to insure a good injection with India ink, it
is necessary to wash the vascular system free of blood to
prevent flocculation of the ink and plugging of the ves-
sels. Therefore, mammalian Ringer's solution, kept at 380 C.,
was perfused through the rear extremities prior to the in-

jection of the ink.

INJECTION APPARATUS

The apparatus used for perfusion and injection is shown
in Plate I. A sphygmomanometer was used to insure that the
India ink was injected at a constant and even pressure. The
pressure lead of the sphygmomanometer was connected to a
glass T—tube whose branches were in turn connected to pieces
of rubber tubing which led individually to flasks filled with
India ink and Ringer's solution. With this arrangement, any

pressure from the manometer would be equal in both flasks.

17

The filled flasks were immersed in a water bath held at
380 C. so the fluids would be at the normal body tempera—
ture of the animals at all times. A polyethylene tube,
from both the perfusion and injection flasks was joined

to make a common injection cannula. In this way, the same
cannula could be used for both perfusion and injection. A
two way valve (Point C, Plate I) placed at the union of
the two plastic tubes provided a device by which the ink
and perfusate could be turned on or off.

Although the apparatus described is easily constructed
and gives excellent results, there are several problems to
which the investigator must be aware. First, the stoppers
in the fluid filled flasks must be firmly secured so they
will not blow out When pressure is applied at the manometer.
Second, the system should be inspected to see that it is
completely free of bubbles. Finally, it is important that
clamps be placed on the tubing between.the manometer and
flasks (Points A and B, Plate I). This allows for one side
of the system to be closed while pressure is being applied to
the opposite side. If the side containing the perfusate is
left open while the ink is being injected, some of the ink
will be drawn into the bottle containing Ringer's solution.

This will cause the ink to precipitate and plug the vessels

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18

during perfusion of the next animal. A similar problem exists
if the injection side is left open during the perfusing period.
Perfusing fluid drawn into the ink will not only cause the

ink particles to flocculate, but it also dilutes the ink,

thereby, making it difficult to identify in tissue sections.

INJECTION PRESSURE

Several authors (Durant, 1927; Byrom and Wilson, 1938;
and Craig and Shintani, 1964) have noted the normal blood
pressure of the rat to range from 100 to 120 mm of mercury.
This was confirmed recently in a well controlled study by
Dellasanta (1964) whose objective was to determine the num-
ber of capillaries open in skeletal muscle at injection pres-
sures of 40, 120 and 200 mm Hg. The 120 mm level most nearly
approximates the normal blood pressure of the rat, therefore,
all perfusions and injections employed in this study were at

that pressure.

SURGICAL AND INJECTION METHODS

Following anesthesia, a laparotomy was performed by mak-
ing a three to four inch mid-saggital incision in the abdom-
inal wall. The abdominal aorta was exposed caudal to the
renal artery and closed off at this point with a bulldog

.clamp (Plate I). A small opening was made in the wall of

19

the vessel one inch caudal to the clamp, and a polyethylene
tube approximately the same size as the distended diameter
of the aorta was inserted caudad and secured with a liga-
ture. As perfusion with mammalian Ringer's solution at

380 C. (body temperature) started, the inferior vena cava
was cut to allow free flow. When the fluid issuing from
the vena cava appeared was clear the valve (Point C,

Plate I) was turned to shut off the perfusate and permit
the introduction of the ink. Within seconds after the ink
was injected the entire body below the cannulation point
turned black. The animals remained alive during the injec—

tion procedure.

TISSUE PROCUREMENT AND PREPARATION

Immediately following injection, the left leg was re-
moved by disarticulating the limb at the coxofemoral joint.
The skin was carefully removed, exposing the gastrocnemius
muscle.

The Achilles tendon was cut from its attachment on the
fibular tarsal bone and a 7 gram paraffin coated lead weight
was attached to this cut end. Then the muscle, with the
tweight attached, was suspended in 10% formalin for 24 hours.

.It: was held in position at right angles to the upper and

20

lower leg with the femur and tibia supporting the weight by
resting on two metal bars overlying the container of fixa-
tive, Plate II. This served to put a constant tension on the
muscle fibers during fixation, and while it is not likely
that normal tension was duplicated; nonetheless, considerable
distortion was prevented. Also, this controlled tension

gaVe uniformity to all the muscle sections and helped to
validate the statistical analyses of the fiber measurements.

After fixation the muscle was removed from the bone in—
filtrated with 10% gelatin for 24 hours followed by 20% gel—
atin for another 24 hours.

Following infiltration the muscle was removed from the
gelatin, placed on a millimeter ruler to determine the mid-
point. The tissue was cut transversely at this point (Plate
III). From previous work, Dellasanta (1964) it is known
that an area which contains red and white fibers as well as
a mixed fiber area, is located in the middle one-third of this
muscle. The author also found the capillary concentration
to be greatest at this location. To assure uniformity in
both capillary counts and muscle fiber measurements the mid-
,p0int of each muscle was selected for making tissue sections.

The cut surface was placed face down in an embedding mold

‘tvkuich.was filled with 20% gelatin. This was refrigerated at

21

40 C. to harden. The solidified block was transferred to
10% formalin to denature the protein of the gelatin. This
made the blocks firmer and easier to handle. Embedding in
gelatin has proved to be most effective in obtaining sec-
tions because shrinkage caused by paraffin techniques are
avoided (Valdivia, 1958).

The trimmed blocks were placed on the stage of a freez-
ing microtome and sectioned at 16 microns. The sections
were placed on a slide and coverslipped using 20% gelatin

as mounting medium.

MEASUREMENTS

To determine accurate capillary to fiber ratios and to
get repeatable muscle fiber measurements, a micro-projection
method was utilized for recording the various microscope
fields. The unstained sections were viewed on the 5x7 ground
glass of a Bausch & Lomb, Model L, photomicrographic camera.*
By using a 10X eyepiece and an 8 millimeter (20X) objective
in the microscope and adjusting the bellows of the camera to
a height of 55 centimeters a magnification of 296X at the
viewing level was achieved. Frosted acetate sheets were

superimposed over the ground glass and tracings of the various

*Bausch and Lomb Optical Co., Rochester, N.Y.

22

microscopic fields were made Areas which contained pre-
dominantly small red and large white muscle fibers (A and B,
Plate III) were selected from each muscle for tracing.

A total of 50 muscle fibers from each zone were outlined
carefully on the acetate and the ink-filled capillaries
related to the muscle fibers were indicated on the tracings
in their proper locations. Therefore, accurate and func—
tionally realistic capillary to fiber ratios were deter—
mined by counting only those vessels associated with the
muscle fibers used for cross-sectional measurements.

Capillary counts were limited to vessels which measured nine
microns or less. A tendon (Plates III and IV) was used as a
landmark in locating fibers and capillaries in the red area.
After the structures in this area were traced, the section
was moved laterally by microscopic stage so that fibers and
vessels in the white area came into View and could be repro-
duced in a like manner.

The tracings on the acetate sheet provided sharp boundaries
of individual muscle fibers. The cross-sectional areas of the
fibers were measured with a Keuffel and Esser compensating

planimeter. Ten cross-sectional area measurements for each

of the

data w

r—i

spent;

tior

Date

the

Tuk

23

of the fibers in the red and white areas were recorded. The

data was subjected to various statistical analyses.

STATISTICS

The results of measurements and counts for the sedentary,
spontaneous activity and forced exercise groups, were com-
pared statistically using correlations (Edwards, 1964),
analysis of variance (Scheffe, 1959) and the Tukey procedures
(Guenther, 1964) (Appendix A). In the statistical analysis
the average of the ten determinations was used as the repre-
sentative value for that fiber. The fifty fibers measured
for a single area were used as replications in the computa-
tion of correlations and analyses of variance. The Control
Data 3600 Computer was utilized for the calculations. Where
the analysis of variance indicated significance (P = .05),
Tukey's method was used to evaluate the differences between
the means of the three groups.

Analyses were completed comparing the three groups for
the red and white fiber areas. Effects due to differences
in fibers to differences in treatments and to combinations
of muscle fibers and treatments were examined. The raw data
measures, raw area measures per gram body weight and capil-

lary to fiber ratios were analyzed (Appendix A).

 

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RESULTS AND DISCUSSION

GENERAL STATEMENT

It is well known that certain muscles of the body are
made up of one type of fiber i.e., small, dark fibers that
are tonic in nature and serve to maintain body tone by
resisting the forces of gravity. Other muscles contain
only large, pale fibers which come into play in situations
that call for rapid responses of a short term nature. In
addition, some muscles contain a combination of small and
large fibers and are referred to as "mixed" muscles. It
is the opinion of a number of investigators (Denny—Brown,
1929; Vroba, 1963; McPhedran §E_gl, 1965; Wuerker g£_gl,
1965; Henneman and Olson, 1965; Bajusz and Jasmin, 1963;
Buller gt_g1, 1960 a and b) that the two fiber types in
mixed muscles are supplied by separate nerves. Most in-
formation of this type has been gathered as a result of
electromyographic studies or in cases where diseased con-
ditions have caused changes in the individual muscle fibers.

In the information which follows, the effects of selected
exercise regimens on the size and capillarization of both red
and white muscle fibers are considered.

Since muscle fibers are classified using a number of

24

25

criteria, it is pertinent to define the terminology used
in this study. The classical small, slow acting are re-
ferred to as "red” fibers and the large, fast acting, as

"white" fibers.

FIBER SIZES

The mean and relative muscle fiber sizes for both the
control and experimental groups are shown in graphs I and
II. The results of statistical analyses are presented in
tables I-VI. Statistical information and basic data are
presented in appendices A and B.

Correlation analyses (Appendix A) provided important
information with regard to the design of the experiment.
The low correlation values obtained by correlating the
results for the animals' activity to the original selec-
tion indicated that the differences obtained in fiber sizes
could be attributed to the exercise regimens to which the
animals had been subjected and not to variations within the
animals.

The average cross-sectional area of the red fibers per
gram body weight (Table I) in animals of the sedentary group
(SG) was 3.82 square microns. In animals which had been

forced (FG) to exercise the average red fiber/gram body

26

weight was 5.72 square microns. The relative red fiber

size of the naimals forced to exercise was 49.7 per cent
larger than for the sedentary animals (Tables II, III). On
the same basis the cross-sectional area of the white fibers/
gram body weight (Table I) of the forced group showed an
increase of 32.5 per cent over those of the sedentary group
(Tables II, III). In both instances the increases were sig-
nificant (F = 3.00, P = .05; F = 3.00, P = .05).

When the relative red and white fiber sizes from the
voluntary group were compared with those of the sedentary
group, the relative fiber size differences of 24.8 and 28.2
per cent respectively were detected (Table II). These
changes were statistically significant (F = 3.00, P = .05;
F = 3:00, P I .05). When the voluntary and forced groups
relative red and white fiber sizes were compared, no sta-
tistically significant differences were found.

The total per cent differences in the size of the white
fibers (forced and sedentary) and red fibers (forced and
sedentary) was 32.5 and 49.7 per cent, respectively (Table
III). 0f the total differences found 76.3 per cent was
manifested in the animals permitted to exercise at will
(voluntary group). Only a 56.8 per cent increase was pro-

duced in the red fibers of animals from the same group. By

27

Table I

Relative fiber sizes in sedentary and exercised animals

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Mean Mean fiber size/gms.
. . S.D.
Animal group (sq. microns)
group wt.
(gms.) Red White Red White
Sed. 314 3.82 6.24 0.89 0.93
Vol. ex. 295 4.90 7.79 1.33 2.16
For. ex. 262 5.72 8.27 1.75 1.81
Table II
Differences in fiber sizes (relative data)
Grou 3 Red Per cent White Per cent
p fibers difference fibers difference
F-S 1.90 49.7 2.03 32.5
V-S 1.08 28.2 1.55 24.8
F—V 0.82 21.5 0.48 7.7
Table III

Relative differences in fiber size (relative data)

 

 

 

 

 

 

Grou 3 Red Per cent White Per cent
p fibers difference fibers difference
F-S 1.90 49.7 2.03 32.5
V-S 1.08 56.8 1.55 76.3
F-V 0.82 43.1 0.48 23.6

 

 

 

 

 

28

comparison, 23.6 per cent of the total white fiber dif-

ference and 43.1 per cent of the total red fiber differ-
ence (forced—sedentary) were found in the comparisons of
the data from the forced exercise and voluntary exercise
groups.

It was apparent from these figures that voluntary exer-
cise produced a greater increase in size of the white than
in the red fibers. It was further evident that in the ani—
mals that were forced to exercise there was a relatively
greater increase in the size of the red than of the white
fibers.

A reasonable explanation for these changes might be
found in the fact that during voluntary activity the white
fibers were influenced the most and therefore, increased to
a greater extent than did the red fibers. When animals
were subjected to forced activity, the white fibers soon
reached their maximum size, thus the increased load and
constant stress was assumed by the red fibers. Under these
conditions, the red fibers became relatively larger than did
the white fibers. This may be what Morpurgo (1897) saw when
he reported "the fibers that grow the most are those which
were originally thinnest and therefore act as reserve material

for growth. "

29

The greater enlargement of white fibers in relation to
voluntary activity and of red fibers with forced activity
indicated that the two fiber types responded differently
to the imposed exercise programs. While the present study
did not take into consideration the types of innervation
associated with the two types of muscle fibers in question,
it is interesting to speculate on the greater increase in
cross-sectional area of the red fibers when the animal is
under the influence of one activity and of the white fibers
when under the influence of another type of exercise.

Bajusz (1964) reported on the differences in red and
‘white fiber sizes in rats and mice during the course of
<denervation atrophy and found that there was less change
in red fibers than in white fiber sizes. He suggested there-
:fore, that "the white fibers are more dependent on neural
<:ontrol than the red ones." Since the present study has
sahown that white muscle fibers demonstrated the greatest
iJncrease in size when subjected to voluntary activity, it
143 reasonable to expect that if Bajusz had used animals of
tZTIis type, he would have seen a greater decrease in white

'tlléin in red fiber sizes. 0n the other hand, if he had worked
”Tiizh highly trained animals, he might have,seen a greater

d€3<2rease in red than in white fiber sizes.

3O

Denny-Brown (1929) stated that at birth all of the fibers
of the gastrocnemius muscle of the cat were 100 square microns
in cross—sectional area. He cited Banu who said that within
14 days many of these had increased to 220 square microns
while at the same time the smaller fibers maintained their
original sizes. These and other authors (Buller §£_g1, 1960a
and b; Hess and Pilar, 1963 and Vroba, 1963) indicated that
the twitch and tension responses of these fibers increased
with the age of the animal and thereby intimated that the
tension applied as a result of the newly acquired activity
may be responsible for the increase in differentiation in
the size of the fibers. Although electromyographic record-
ings of the individual muscle fiber types were not obtained
in this study, it seems evident that the increased muscular
tension to which the animals were subjected in their exercise
programs was responsible for the differences in muscle fiber

sizes.

CAPILLARIES PER FIBER
GENERAL STATEMENT

Earlier in this paper it was pointed out that several in-
vestigators have delved into the problems associated with the

blood supply to skeletal muscle. It was also emphasized that

31

a variety of animals and techniques had been employed in
these studies. Furthermore, it was noted that in many
cases caution was not taken to incorporate proper controls
into the study. Such conditions have made it difficult
to attach proper significance to the results obtained.

In the results which follow, the effects of selected
exercise regimens on the numbers of capillaries per muscle

fiber are presented.

RESULTS

The mean and relative capillary to fiber data for all
groups of animals are shown in graphs III and IV. Results
of statistical analyses are shown in tables IV—VI and the
statistical information and basic data are given in appen-
dices A and B.

Correlation values (Appendix A) for the capillaries per
fiber (both red and white fibers) per gram body weight were
obtained by correlating the results for the animals per-
experiment activity to the original matching. Since the
animals were originally matched on the basis of the pre-
experimental activity and then randomly placed into groups
the low correlation values indicate that the differences

in capillary to fiber ratios (C/F) could be attributed to

32

 

 

 

 

 

 

 

 

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33

Table V

Differences in capillaries per fiber per gram
body weight (relative data)

 

 

 

 

 

 

 

 

Grou 3 Red Per cent White Per cent
p fibers difference fibers difference
F-S*** .20 25 .12 31
V-S** .09 ll .09 23
F-V* .11 12 .03 6
Table VI

Relative differences in capillaries per fiber per
gram body weight (relative data)

 

 

 

G on s Red Per cent White Per cent
r p fibers difference fibers difference

F-S .20 25 12 31

V-8 .09 45 .09 75

F-V .11 55 03 25

 

 

 

 

 

 

 

*** Forced to sedentary groups.
** Voluntary to sedentary groups.
* Forced to voluntary groups.

34

the exercise programs and not to differences within the
individual animals.

The mean capillary per red and white fiber per gram of
body weight (C/F/G) (Table IV and Graph IV) for animals in
the sedentary group was .80 and .39 respectively. In ani—
mals which had been forced to exercise the mean C/F/G
ratios were 1.0 and .51 while in the voluntary group it
was .89 and .48. The relative C/F/G for the red fibers
in animals of the forced group was 25 per cent greater than
the same ratio in animals of the sedentary group. In com-
parison the C/F/G ratio for the white fibers in animals
forced to exercise was 31 per cent larger than for similar
fibers of the sedentary group (Tables V and VI). In both
instances the differences were statistically significant
(F = 3.35; P = .05; F = 3.35; P = .05) (Appendix A).

When the mean relative C/F/G ratios for the red and
White fibers of the voluntary and forced groups were com-
pared with those of the sedentary group, differences of 9
per cent were found in each case. Comparisons of the mean
C/F/G ratios of the forced and voluntary groups revealed
that the differences were not statistically significant.

The total per cent differences in C/F/G ratios in red

«and.white fibers (forced-sedentary) were 25 and 31 per

35

cent. 0f the total differences found 75 per cent was ex-
hibited by the white fibers of animals in the same group.
In comparison, 55 per cent of the total red C/F/G differ-
ence and 25 per cent of the total white C/F/G difference

(forced-sedentary) is found when comparing the data from

the forced and voluntary exercise groups (Table VI).

These figures indicate that there was an overall in-
crease in the number of capillaries per fiber per gram
body weight in association with both red and white muscle
fibers. However, the greatest relative differences were
produced in conjunction with the white fibers during volun-
tary activity and with the red fibers during forced activity.

Prior to presenting the results of the statistical
analyses for the reader's perspective, a brief review of
the literature on the vascular supply to skeletal muscle
most pertinent to the current problem is presented.

Some authors (Krogh, 1919; Paff, 1930; Smith and Gio-
vacchini, 1956; and Valdivia, 1958) have presented basic
data on capillary to fiber ratios (C/F) in muscles which
are made up of entirely red or white fibers. Results
among these studies vary, but in general, it is recognized
that red muscle which acts slowly but constantly has a

greater number of capillaries per muscle fiber than does

36

white muscle which acts rapidly and for short periods of
time. In addition, it has been found that active muscles
are hyperemic when compared to inactive muscles (Petren
_§_§1, 1936; and Elsner and Carlson, 1962).

Since mixed muscles contain complements of fibers which
are related to different activities it is of interest to
see whether the C/F ratio maintains the same relationship
that it does in muscles which contain only one type of
fiber.

Reports by Krogh 1919, Millikan 1937, Lawrie 1952,
1953, Smith and Giovacchini 1956, Porter and Armstrong
1965 and others indicate the importance of the blood sup—
ply to muscle fibers in relation to the metabolic needs
of the cells. Smith and Giovacchini (1956) found that red
muscle was more vascular than white muscle. They suggested
that since red muscle was also rich in myoglobin (which
acts as an oxygen reservoir) (Millikan, 1937, Lawrie, 1952)
that the combination of these entities provided an arrange-
ment of double assurance. That is "those muscles which
cannot function without a constant supply of oxygen appar-
ently are equipped with a greater capillary bed as well as
oxygen storing myoglobin."

These same authors and Porter and Armstrong (1965) who

37

reported on the sarcoplasmic reticulum in the various types
of striated muscles strongly imply that all muscles do not
have the same mechanisms for satisfying their metabolic
needs.

Indeed, the figures presented in table VI point up the
fact that under conditions of physical activity certain
mechanisms, as yet unidentified, come into play which alter
the blood supply within the muscle thereby providing an
arrangement which is in line with the physiologic needs of
the individual muscle fibers.

Under the influence of forced activity (forced-sedentary)
total differences in C/F/G of 25 and 31 per cent were pro-
duced in the red and white fibers (Table VI). Of the total
differences found, 75 per cent was exhibited by the white
fibers of the voluntary group and only 45 per cent was pro-
duced in the red fibers of the same group. In light of
reports (Denny-Brown, 1929, George and Naik, 1957, 1958,
1959) which indicate that the larger white fibers are more
active during periods of exercise than are the smaller red
fibers, the larger C/F/G in favor of the white fibers during
voluntary activity is reasonable. This would assure that the
increased metabolic needs of the white fibers were met.

Further insight into these differences is revealed by

38

the fact that white fibers contain little myoglobin, few
mitochondria and an extensive sarcoplasmic reticulum (both
of which are associated with the generation and exchange of
energy rich materials). Therefore, they require a greater
blood supply to satisfy their nutritional and energy re-
quirements. In contrast, red fibers are rich in myoglobin,
have many more mitochondria and the sarcoplasmic reticulum
is less well developed. This arrangement provides condi-
tions whereby the smaller C/F/G ratio maintains a constant
environment and assures each tissue its necessary nutrients.

If the differences in C/F/G ratios for the red and
white fibers of the voluntary group (Table VI) are compared
with the differences in mean fiber sizes per gram body
weight for the same group, (Table III), the following re-
sults are revealed.

Of the total difference in the size of the red fibers
56.8 per cent was expressed by the voluntary group. At the
same time a 45 per cent difference of the total C/F/G ratio
was produced in the same animals.

Similarly, the 76.3 per cent difference of the total
mean fiber size per gram body weight in the white fibers
tnas commensurate with a 75 per cent difference from the

total in the C/F/G ratio for the group.

39

It is apparent that the red and White muscle fibers
increased in size in response to voluntary exercise and
furthermore, the increase in fiber size was accompanied
by a comparable increase in blood supply.

Table VI also shows that 55 per cent of the total red
C/F/G difference and 25 per cent of the total white C/F/G
difference (forced-sedentary) was found when the data from
the forced and voluntary groups were compared. These fig-
ures have an inverse relationship to those of the volun-
tary groups.

The figures in table III show that 43.1 per cent of
the total difference in fiber size is related to the red
fibers while table VI shows a total difference of 55 per
cent in C/F/G for these fibers. The white fibers show a
23.6 per cent difference from the total relative difference
in fiber size and a 25 per cent difference in C/F/G. Thus,
under the conditions imposed by forced exercise, changes in
muscle fiber sizes are also paralleled by changes in vas-
cular supply.

The evidence presented here supports the work of Mor-
purgo, 1897; Petren et a1. 1936 and others who have reported
that the number of capillaries which can be opened is greater

in muscles taken from trained than from untrained animals.

40

The differences seen in muscle fiber sizes and capil-
lary to fiber ratios in response to forced exercise can be
explained on the basis of usage. First of all, it is im-
portant to realize that the forced exercise program used
in this experiment was of an endurance nature and not one
which encompassed strength and speed. Therefore, it was
an activity which was constant and of a low level of inten—
sity as far as work was concerned.

Since small, red muscle fibers have been shown to be
more susceptible to discharge and fire more often than
large, white fibers, (Henneman and Olson, 1965; Buller gg
al, 1960a and b; Vroba, 1963) the greater total per cent
difference in the red fiber sizes compared to white fiber
sizes (Tables II and III) would be expected. Of further
significance is the fact that the total per cent difference
in C/F/G in relation to red fibers is greater than it is
with white fibers (Tables V and VI). These results are in
line with those of authors who have reported larger capil-
lary to fiber ratios for red than for white muscle.

An additional point worthy of comment is that in our
preparations, some areas of the muscle appeared to be well
injected while others were devoid of ink (Plate VI).

t g1,

Similar results have been reported by Martin

41

(1932) for the dog, and Smith and Giovacchini (1956) for
the cat. The earlier authors assumed that the fasciculus
reacted as a circulatory unit or that there was alteration
of vascular supply in terms of fasciculi; however, the lat-
ter found no evidence to support this idea.

Based on the results obtained by Dellasanta (1964) on
the effects of various injection pressures on the numbers
of Open capillaries in skeletal muscle, it does not seem
that this factor could be responsible for the condition in
this study.

Studies on nervous control of blood vessels in skeletal
muscle (Folkow, 1952; Uvnas, 1960 and Barlow gt_g1, 1961)
indicate that there are two circulations through muscle and
that they are differently controlled. Others (Hilton, 1959;
Folkow, 1960 and Renkin and Rosell, 1962) point out the re—
lationships of arterioles and pre-capillary sphincters to
blood flow. These authors suggest that the pre-capillary
sphincters monitor normal flow into the "effective circu-
lation" at the samt time shunting more or less blood to the
"by-pass" circulation as the situation demands. These cir-
culatory adjustments in active skeletal muscle would tend to
guarantee a balance between effective blood flow and the im-

mediate needs of the tissues.

42

While the present report is not conclusive in this
respect, it should be noted that the mechanisms involving
the circulation to skeletal muscle are in harmony with
the results of the present investigation which has demon-
strated quantitative changes in circulation with specific
exercise regimens. In light of this information, it is sug—
gested that these mechanisms are responsible for the presence
of ink filled capillaries in some areas of the muscle and

total absence in others.

SUMMARY AND CONCLUS I ONS

Thirty male rats (Sprague-Dawley), 25 days of age were
placed in exercise cages for 7 days. From previous work,
it is known that an area which contains red and white fibers
as well as a mixed fiber area, is located in the middle one—
third of this muscle. The author also found the capillary
concentration to be greatest at this location. To assure
uniformity in both capillary counts and muscle fiber meas-
urements the mid-point of each muscle was selected for
making tissue sections. For the next thirty-five days the
sedentary group was permitted no exercise other than that
allowed by their small individual cages. The voluntary
group remained in activity cages while the forced group in
addition to being in activity cages swam 30 minutes each
day with lead weights equal to 2% of the body weight at-
tached to their tails. At the end of the thirty-five day
forced exercise period, the animals were sacrificed. The
hind limbs were injected with India ink. The gastrocnemius
muscle was fixed, embedded in gelatin and cut on the freez-
ing microtome. The cross-sectional areas Of the red and
white muscle fibers from the gastrocnemius muscles were meas-
ured by using the polar planimeter. Ink filled capillaries

were counted in conjunction with fiber measurements.

43

44

The results of measurements and counts for the seden-
tary, voluntary activity and forced activity groups were
compared statistically using correlations, analysis of
variance and the Tuckey procedures.

The average cross-sectional area of the red fibers
per gram body weight in animals of the sedentary group was
3.82 square microns. In animals which had been forced to
exercise, the average red fiber area per gram body weight
was 5.72 square microns.

The total per cent differences in the size of the red
fibers (forced-sedentary) and white fibers (forced-sedentary)
was 49.7 and 32.5 per cent respectively. Of the total dif—
ferences found, 56.8 per cent was manifest in the red fibers
of animals permitted to exercise at will while a 76.2 per
cent increase was produced in the white fibers of animals
from the same group. By comparison, 23.6 per cent of the
total white fiber difference and 43.1 per cent of the total
red fiber difference (forced—sedentary) were found in the
comparisons of the data from the forced exercise and volun-
tary exercise groups.

These findings support the work of Denny-Brown 1929,
Buller et al. 1960a and b, Hess and Pilar 1963 and Vroba 1963

and have shown that the muscular tension imposed by the

45

various exercise programs was responsible for the differ—
ences in muscle fiber sizes.

In addition, the greater enlargement of white fibers in
relation to voluntary activity and of red fibers with forced
activity indicated that the two types of fibers responded
differently to the imposed exercise programs.

The mean capillary per fiber per gram body weight (C/F/G)
ratio for animals in the sedentary group was .80. In animals
which had been forced to exercise, the C/F/G ratio was 1.0
while in the voluntary group it was .89.

The total per cent differences in C/F/G in red and
white fibers (forced-sedentary) were 25 and 31 per cent.

Of the total differences found only 75 per cent was exhibited
by the white fibers of the voluntary group and only 45 per
cent was produced in the red fibers of animals of the same
group. Fifty-five per cent of the total red C/F/G difference
and 25 per cent of the total white C/F/G difference (forced-
sedentary) were found When the data from the forced and volun-
tary exercise groups were compared.

Comparisons of the differences in C/F/G ratios with the
differences in mean fiber sizes per gram body weight indi-
cated that of the total differences in the size of the red

fibers, 56.8 per cent was expressed in the voluntary group.

46

At the same time, a 45 per cent difference of the total
C/F/G ratio was produced in the same animals.

The 76.3 per cent difference Of the total mean fiber
size per gram body weight in the white fiber was commen-
surate with a 75 per cent difference from the total in the
C/F/G ratio for that group.

Similarly, with forced exercise, the 43.6 per cent dif-
ference in white fiber size was accompanied by a 55 per cent
difference in C/F/G ratio. The red fibers showed a 23.6
per cent difference from the total relative difference and
a 25 per cent difference in C/F/G ratio. Thus, under con-
ditions of voluntary and forced exercise, changes in muscle
fiber sizes were paralleled by prOportionate changes in vas-
cular supply.

The comparable changes in muscle fiber sizes and C/F/G
ratios are in line with the chemical myoglobin) and morpho-
logical (sarCOplasmic reticulum and mitochondria) relation-
ships suggested by Milliken 1937, Lawrie 1952, 1953, and
Porter and Armstrong 1965. Combined, these act to maintain
a constant environment and satisfy the nutritional and
energy requirements of the muscle tissue.

The fact that some areas of the muscle preparations ap-

peared well injected while others were devoid of ink has

47

been noted in this study as well as by other authors. Ade-
quate explanations of this phenomenon have escaped earlier
investigators.

Important contributions to the solution of this problem
may be found in the works Of Folkow 1952, Uvnas 1960 and
Barlow £3.31, 1961, who reported that there are two circu-
latory systems in skeletal muscle and that they are dif-
ferently controlled. In addition, Hilton 1959, Folkow 1960
and Renkin and Rossell 1962, suggested that pre—capillary
sphincters monitored blood flow into the "effective circu-
lation" and the same time shunted greater or lesser amounts
of blood to the "by-pass circulation" as the situation
demanded.

The present study which has demonstrated that quanti—
tative changes take place in the circulation to skeletal
muscle with specific exercise regimens are in agreement with
these reports.

Furthermore, the quantitative changes shown in this
report are in all probability the same as those reported
by Martin gt_§l, 1932 and Smith and Giovacchini 1956 and
it is suggested that the presence of ink filled vessels
in some areas of the muscle and total absence in others are

expressions of mechanisms which affect the blood supply to

48

skeletal muscle as described by Folkow 1952, 1962, Hilton
1959, Uvnas 1960, Barlow §§_g1, 1961 and Renkin and Rosell

1962.

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54

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28:85099.

55

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Plate 1

 

 

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59

   

'4

RED FIBER AREA

 

E WHITE FIBER AREA

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Plate 3

60

PLATE IV

Cross-sectional view of small, red muscle fibers
and associated capillaries. (400 X).

PLATE V

Cross-sectional View of several adjacent fasciculi
showing that some have capillaries filled with ink
and others which are devoid of ink. (620 X).

61

 

APPENDIX A

62

Mean fiber sizes in sedentary and exercised animals

 

 

 

 

Mean Mean fiber size/gms.
. . S.D.
Animal group (sq. microns)
group wt.
(gms.) Red White Red White
Sed. 314 1199 1959 283 283
Vol. ex. 295 1442 2284 386 580
For. ex. 262 1492 2163 447 470

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Analysis of variance, red fibers, raw data
F needed
Source of variance df Mean .fOF . Experi-
square Slgnlfl- mental F
cance
Effects due to
differences in fibers 49 130595 F > 1.35 .89
Effects due to
differences in
treatment 2 12284824 F > 3.00 83.78*
Effects due to
combinations of
muscle fibers and
treatments 98 99905 F > 1.25 .68
Error 1350 146632
Total after mean 1499
* Significant .05
Comparison of means--Tukey Test*
Grou i... i... i i. - i i... — i
p 3 J F J V 3 S
iF 1492 o 51 293*
iv 1441 -51 o 242*
is 1199 -293 —242 o

 

* Significant if > 231

 

 

Analysis of variance, red fibers, relative data

 

 

 

 

 

 

 

 

 

 

F needed
E ' _
Source of variance df Mean .fOF . xperi
square Slgnlfl- mental F
cance
Effects due to
differences in fibers 49 1.66 F > 1.35 .85
Effects due to
differences in
treatments 2 453.55 F > 3.00 233.71*
Effects due to
combinations of
muscle fibers and
treatments 98 1.27 F > 1.25 .65
Error 1350 1.94
Total after mean 1499
* Significant .05
Comparison of means--Tukey Test*
Grou i... 2.. - i i. — 2.. i .. - i..
p 3 J F V J
is 5.73 o .83 1.90*
iv 4.90 -.83 o 1.07*
is 3.83 -1.90 -1.07 o

 

 

 

 

 

 

* Significant if > .84

 

 

 

Analysis of variance, white fibers,
relative data

 

 

 

 

 

 

 

 

 

 

F needed
Source of variance df Mean .for . Experi-
square Slgnlfl- mental F
cance
Effects due to
differences in fibers 49 2.35 F > 1.35 .77
Effects due to
differences in
treatments 2 560.61 F > 3.00 184.14*
Effects to to
combinations of
muscle fibers and
treatments 98 1.87 F > 1.25 .61
Error 1350 3.04
Total after mean 1499
* Significant .05
Comparison of means-—Tukey Test*
Grou i... i... - i i... - i i. - i
p 3 3 F 3 V 3 S
iF 8.27 o .48 2.02*
iv 7.79 -.48 o 1.54*
is 6.25 -2.02 -1.54 o

 

 

 

 

 

* Significant if > 1.06

 

 

Group correlations on fiber sizes

 

 

 

 

Red fibers Red fibers
raw data relative data
Groups r Groups r
F-S*** .17 F-S 05
V-S** -.10 V—S .03
F-V* -.15 F-V -.20

 

 

 

 

 

 

 

 

White fibers

 

White fibers

 

 

 

raw data relative data
Groups r Groups r
F-S 08 F-S -.05
V-S .01 V-S .07
F—V -.04 F-V - 14

 

 

 

 

 

 

*** F-S correlations of fiber sizes of
groups.

** V-S correlations of fiber sizes of
tary groups.

* F-V correlations of fiber sizes of
groups.

 

 

forced and sedentary
voluntary and seden-

forced and voluntary

 

Analysis of variance, red fibers, capillary to fiber
ratios, raw data

 

 

F needed

Source of df Mean .fOF _ Experi-

. square Slgnlfl- mental F
variance

cance

Effects due to
treatments 2 .03908333 F > 3.35 1.61
Error 27 .02426407
Total after mean 29

 

 

 

 

 

 

* Significant .05

Comparison of means--Tukey Test*

 

 

Grou i... i... - i i... — i i... - i
p 3 J F 3 V 3 S
is 2.63 o -.01 10
iv 2.64 .01 o .11
is 2.53 -.10 -.11 o

 

 

 

 

 

 

 

* Significant if > .55

Analysis of variance,

to fiber ratios,

red fibers,

capillary
relative data

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

F needed
Source of Mean for Experi-
. df . . .
variance square Signifi- mental F
cance
Effects due to
treatments 2 .00000975 F > 3.35 20.74*
Error 27 .00000047
Total after mean 29
* Significant .05
Comparison of means--Tukey Test*
Grou i... i.. - i i... - i i.. - i
p 3 J F J V J S
F-S .010064 0 .001055* .002056*
V-S .009009 -.001055 0 .000921*
F-V .008088 -.002056 -.000921 0
* Significant if > .000803

 

 

 

Analysis of variance, white fibers,

capillary

 

 

 

 

 

 

 

 

 

to fiber ratios, raw data
F needed
Source of Mean for Experi-
. df . . .
variance square Slgnlfl- mental F
cance
Effects due to 2 .09430333 F > 3.35 2.38
treatments
Error 27 .03961074
Total after mean 29
* Significant .05
Comparison of means--Tukey Test*
i... ”...—i '. -i i. —i
Group 3 X F X 3 V J S
F-S 1.35 0 -.08 .12
V-S 1.42 .08 O .20
F-V 1.22 .12 -.20 0

 

 

 

 

 

 

* Significant if > .70

 

 

Analysis of variance, white fibers,
fiber ratios,

capillary to

relative data

 

 

 

 

 

 

 

 

 

 

 

F needed
Source of Mean for Experi-
. df . . .
variance square Slgnlfl- mental F
cance
Effects due to
treatments 2 .00000434 F > 3.35 7.03*
Error 27 .00000062
Total after mean 29
* Significant .05
Comparison of means--Tukey Test*
Grou i... i... - i i... - - i... - i
p j 3 F J XV 3 S
F-S .005196 0 .000368 .001280*
V-S .004828 -.000368 0 .000912*
F-V .003916 -.001280 -.000912 0

 

 

 

 

 

 

* Significant if > .000852

 

Group correlations on capillary to fiber ratios

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Red fibers Red fibers
raw data relative data
Groups r Groups r
F-S*** .16 F-S .07
V-S*** .30 V-S -.13
F-V* .55 F-V —.03
White fibers White fibers
raw data relative data
Groups r Groups r
F-S -.27 F-S -.30
V-S .17 V-S ~ .34
F-V .20 F-V .18

 

 

 

 

 

 

 

*** F-S
and
** V-S

and

and

correlations of capillary to fiber ratios of forced
sedentary groups.

correlations of capillary to fiber ratios of voluntary
sedentary groups.

correlations of capillary to fiber ratios of forced
voluntary groups.

APPENDIX B

BASIC DATA

74

 

 

 

 

 

 

 

 

- . Weight Mean Red White Red
Animal at . . . mean
study sacri- daily fibers fibers fiber
number fice revo- cap./ cap./ size
( lutions fiber fiber 2

gms.) (u )

F 4 275 817 2.52 1.24 1205
F 6 244 634 2.38 1.38 1715
F21 247 1401 2.62 1.60 1685
F22 247 685 2.42 1.52 1534
F26 286 964 2.78 1.34 1349
F34 267 1086 2.64 1.12 1400
F37 274 729 2.86 1.68 1298
F39 261 862 2.66 1.04 1673
F42 271 870 2.82 1.28 1925
F47 246 741 2.62 1.34 1063
Mean 262 879 2.63 1.35 1492
V 4 275 5314 2.84 1.19 1785
V 6 293 3635 2.52 1.46 1170
V21 277 517 2.60 1.22 1228
V22 303 1221 2.48 1.18 1308
V26 329 497 2.64 1.36 1731
V34 288 1701 2.74 1.40 1708
V37 322 1562 2.92 1.80 1097
V39 320 2566 2.51 1.34 1693
V42 274 560 2.76 1.28 1275
V47 271 1032 2.46 1.96 1421
Mean 295 1860 2.65 1.42 1442
S 4 303 -- 2.52 1.22 1110
S 6 298 -— 2.56 1.10 1104
$21 308 -- 2.48 1.14 1227
822 332 -- 2.52 1.16 1017
$26 295 -- 2.46 1.28 1182
S34 329 -- 2.44 1.18 1032
S37 327 -- 2.90 1.34 1204
S39 334 —- 2.56 1.42 1178
$42 337 -- 2.34 1.24 1612
S47 279 -- 2.54 1.20 1251
Mean 314 -- 2.53 1.22 1199

 

 

 

 

 

 

 

 

 

 

 

 

 

3.8034

 

 

 

 

Red White White Red White
fiber mean fiber fibers fibers
Size (u ) fiber size (uz) cap./fiber cap./fiber
per gm. size per gm. per gm. per gm.
body wt. (uz) body wt. body wt. body wt.
4.3818 2084 7.5781 .0091 .0045
7.0286 2327 9.5368 .0097 .0056
6.8218 2141 8.6680 .0106 .0064
6.2105 2119 8.5789 .0097 .0061
4.7167 2170 7.5874 .0097 .0046
5.2434 2282 8.5468 .0098 .0041
4.7372 2522 9.2043 .0104 .0061
6.4099 2404 9.2107 .0101 .0039
7.1033 1994 7.3579 .0104 .0047
4.3211 1599 6.5000 .0106 .0054
5.6974 2163 8.2768 .0100 .0051
6.4909 2625 9.5454 .0103 .0043
3.9931 1861 6.3515 .0086 .0049
4.6498 1997 7.2093 .0093 .0044
4.3168 1960 6.4686 .0081 .0038
5.2613 1989 6.0455 .0080 .0041
5.9305 2792 9.6944 .0095 .0048
3.4068 2472 7.6770 .0090 .0055
5.2906 2363 7.3843 .0078 .0041
4.6532 1851 6.7554 .0100 .0046
5.2435 2882 10.6346 .0090 .0072
4.9236 2284 7.7766 .0089 .0048
3.6633 1850 6.1056 .0083 .0040
3.7046 1729 5.8020 .0085 .0036
3.9837 2087 6.7759 .0080 .0037
3.0632 1972 5.9397 .0075 .0034
4.0067 2033 6.8915 .0073 .0043
3.1367 1980 6.0182 .0074 .0035
3.6819 1945 5.9480 .0088 .0040
3.5269 2047 6.1287 .0076 .0042
4.7833 2010 5.9643 .0069 .0036
4.4838 1870 6.7025 .0091 .0043

1959 6.2276 .0080 .0039