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EFFECTS OF EXERCISE AND A VEGETARIAN DIET 0N
CARCASS COMPOSITION, ORGAN WEIGHT 37 AND
SERUM CHOLESTEROL AND TRIGLYCERIDES

Thesis for the Degree 0f Ph.‘ D.
MICHIGAN STATE UNIVERSITY
DANIEI. ALLEN KLEIN!
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thesis entitled

EFFECTS OF EXERCISE AND A VEGETARIAN DIET
ON CARCASS COMPOSITION, ORGAN WEIGHTS, AND
SERUM CHOLESTEROL AND TRIGLYCERIDES

presented by

Daniel Allen Klein

has been accepted towards fulfillment
of the requirements for

___Eh.D.._degree in JealthT—Physical
Education and Recreation

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ABSTRACT
EFFECTS OF EXERCISE AND A VEGETARIAN DIET
ON CARCASS COMPOSITION, ORGAN WEIGHTS, AND
SERUM CHOLESTEROL AND TRIGLYCERIDES
By

Daniel Allen Klein

The purpose of this study was to compare the effects of a strict
vegetarian diet and a control diet containing animal protein (5.23%)
under various conditions of physical activity. The parameters studied
included quantitative responses to exercise, total carcass composition,
organ weights, serum cholesterol, and serum triglycerides.

Fifty, twenty-three-day-old male albino rats (Sprague-Dawley strain)
were randomly assigned to either a control dietary group or to an
experimental vegetarian dietary group. Within each dietary group, the
animals were randomly assigned to one of three exercise programs. Seven
animals from each dietary group were placed into a sedentary subgroup.
Seven animals from each dietary group were placed into a voluntary-exercise
program. The remaining eleven animals, from each dietary group, were
placed into a forced-exercise program. Six animals were desired in each
diet-training subgroup for final analysis. One extra animal was placed
in each sedentary and voluntary-exercise subgroun as a contingency against
injury or illness. Five extra animals were placed in each forced-exercise
subgroup because during a typical forced-exercise training program some

of the animals either become injured or fail to respond to the program

Daniel Allen Klein
and must be dropped from the experiment.

At ninety days of age, blood samples were taken and the animals
were sacrificed. The samples were analyzed for serum cholesterol and
triglycerides. Wet weights were taken of the liver, kidneys, adrenals,
spleen, heart, and testes. The carcasses then were frozen until
determinations of body composition were made.

Higher levels of exercise produced animals with higher per cent
carcass weight due to water, protein, and ash with a lower per cent
carcass weight due to fat.

When the forced-exercise group was compared to the sedentary
group, the active animals were found to have significantly lower relative
liver weights and significantly higher relative adrenals, heart, and
testes weights. Serum cholesterol and triglyceride values were signif-
icantly lower in the same animals.

As compared to the control diet, the vegetarian diet produced
animals that performed at a lower level on the forced-exercise program;
but there was no difference between the two diet groups with respect to
voluntary-activity. The vegetarian animals were fatter with lower amounts
of ash. The animals that were on the vegetarian diet had larger kidneys,
hearts, and smaller adrenals.

Due to the small sample size, the statistical power was less than
0.30 in all analyses where significant differences were not observed.
Therefore, failure to demonstrate significance should not be interpreted,
at this time, as justification for acceptance of the hypothesis of no

treatment effects.

EFFECTS OF EXERCISE AND A VEGETARIAN DIET
ON CARCASS COMPOSITION, ORGAN WEIGHTS, AND

SERUM CHOLESTEROL AND TRIGLYCERIDES

By

Daniel Allen Klein

A THESIS

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

1971

DEDICATION

This thesis is dedicated to my wife, Sharon, for her understanding
inspiration, patience, hard work, and many sacrifices and to my mother
whose encouragement has made it possible to attain this goal in my

education.

ii

ACKNOWLEDGMENTS

Acknowledgment is extended to Dr. w. W. Heusner of the Michigan
State University Human Energy Research Laboratory for providing a
stimulating atmosphere in which to work, setting forth high standards
as a teacher, and for his help in the many facets of conducting research.

Acknowledgment is extended to Dr. w. D. van Huss for sharing his
knowledge and exoeriences freely and for his help in preparing this
thesis.

Acknowledgments are extended to Drs. R. Carrow and C. Whitehair
for their time, suggestions and willingness to help in various ways.

Acknowledgments are also extended to many individuals who helped
in one way or another in the preparation of this thesis: Messrs.

D. Anderson, R. Wells, Mrs. J. LaFay, Miss C. Habenicht and K. Hawks.

iii

TABLE OF

DEDICATION. . . . . . . . . . .
ACKNOWLEDGMENTS . . . . . . . . .
LIST OF FIGURES . . . . . . . . .
LIST OF TABLES. . . . . . . . . .
LIST OF APPENDICES. . . . . . . .
Chapter

I. INTRODUCTION . . . . . .

Purpose of the Study.
'Need for the Study. .

Limitations of the Study.

Definition of Terms .
II. RELATED LITERATURE . . .

Food Shortages. . . .
Deficiency in Diets .
Cholesterol and Diet.
Diet and Disease. . .
Vegetable Protein Sour
Nitrogen Balance. . .
Exercise and Cholester

III. EXPERIMENTAL METHOD. . .
IV. PRESENTATION OF DATA . .

Exercise Analyses . .
Serum Analyses. . . .

Organ Weight Analyses
Discussion. . . . . .

Body Composition Analyses .

V. SUMMARY, CONCLUSIONS, AND
SWY O O O O O O 0
Conclusions . . . . .
Recommendations . . .

iv

CONTENTS

0 O O O O O O O O O O O O O O O 0 iii

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. . . . . . . . . . . . . . . . 21

. . . . . . . . . . . . . . 21
. . . . . . . . . . . . . . . . 28
. . . . . . . . . . . . . . . . 33

RECOMMENDATIONS. . . . . . . . . 36

37

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BIBLIOGRAPHY. O O O O O O O O O O O O O O O O O O O O O O O O O O O 39

APPENDHO O O O O O O O 0 O O O O O O O O O O O O O O O O O O O O C h?

Figure

1.
2.
3.
A.

LIST OF FIGURES

Per cent of EXpected Revolutions (PER)
Per cent Shock Free Time (PSF) . . . .
Total Revolutions Run (TRR). . . . . .
Cumulative Duration of Shock (CDS) . .

Mean Voluntary Revolutions Run (VOL) .

Page
19
l9
l9
19
20

Table

1.

3.
h.
S.

7.

8.

9.
10.
ll.
12.
13.
1h.
15.
16.
17.
18.
19.
20.
21.
22.

Serum Analysis:

Serum Analysis:

LIST OF TABLES

Triglycerides (milligrams per

BodyWelght(ng)...............

Carcass Weight (grams) . . . . . . . . . .

Carcass
Carcass
Carcass
Carcass
Carcass
Carcass
Carcass
Carcass
Carcass

Carcass

Composition by Diet Groups . . .

Composition Data by Exercise Level

hhter (per cent carcass weight).
Water(gram&3)..........
Fat (per cent carcass weight). .

Fat (grams). . . . . . . . . . .

Protein (per cent carcass weight).

”Otein (gram). 0 e o o o o o 0
Ash (per cent carcass weight). .

Ash (grams). . . . . . . . . . .

Organ WEight Data by Diet Groups . . . .

Organ weight Data by Exercise Level. . .

Adrenal Weight (per cent of body weight)

Adrenal Weight (grams) . . . . . . . . .

Heart WEight (per cent of body weight) .

Heart Weight (grams) . . . . . . . . . .

Kidney Weight (per cent of body weight).

Kidney'Weight‘(grams). . . . . . . . . .

vii

Cholesterol (milligrams per cent) .

cent)

Page
22
22
22
22
2h
2h
25
25
25
25
26
26
26
26
29
29
30
3O
30
30
31
31

 

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I I

 

23.
2h.
25.
26.
27.
28.
29.

Liver Weight (per cent body weight) . .
Liver Weight (grams). . . . . . . . . .
Spleen Weight (per cent of body weight)
Spleen Weight (grams) . . . . . . . . .
Testes height (per cent of body weight)
Testes Mbight (grams) . . . . . . . . .

Comparisons of Two Diets. . . . . . . .

viii

31
31
32
32
32
32
3b

I5|[[[lflul‘ ll Ill 1.. fl ' I III A .IIII‘II i I I III: III!

 

Appendix

A.

A.

A.

A.

B.

B.

C.

LIST OF APPENDICES

Table A1 Data on Body and Organ Weights

Control Animals (Wayne Lab Blox). . . . . . . . .

Table A2 Data on Carcass Composition, Serum Cholesterol,

and Serum Triglycerides

Control Animals (Whyne Lab Bldx). . . . . . . . .

Table A3 Data on Body and Organ Weights

Experimental Animals (Vegetarian Diet). . . . . .

Table Ah Data on Carcass Composition, Serum Cholesterol,

and Serum Triglycerides
Experimental Animals (Vegetarian Diet). . . .

Table Bl Raw Data - Activity Record
Forced Exercise Animals . . . . . . . . . . .

Table BZ Raw Data - Activity Record
V01unto3ry Anims O O O O O O O O O O O O O 0

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

Page

1:7

118

D9

50

51

51

S2

CHAPTER I
INTRODUCTION

In 1912, Slenaker reported on the effects of an all vegetable
diet on laboratory rats (81). McCollum, Wu and others used similar
diets with laboratory animals but had little success (68, 107). In
general, they found that animals on vegetarian diets were less active,
contracted more illness, and did not grow as well as the controls.

The fact that peOple in China, Japan, and India, and other countries
used a vegetarian diet, and were able to stay healthy and active,
continued to puzzle these investigators. As an understanding of
nutrition broadened and specific nutritional requirements for various
species were found, diets were developed that were adequate in terms
of both growth and health characteristics.

Exercise also affects the growth and development of animals (13,
49, 93). As the intensity of exercise increases, differences in body
weight (13, 49, 93) and carcass composition (49. 93) are increased.
With the increased knowledge about nutrition and exercise, it is
important that questions about all vegetable diets be studied with

respect to various exercise programs.

Purpose of the Stggy_
The purpose of this study was to determine the effects of a
strict vegetarian diet, with different levels of exercise, on carcass
composition, organ weights, serum cholesterol, and serum triglycerides.

1

2
Need fer the Stugy

Previous investigators have looked at the effects of vegetarian
diets only from the viewpoint of overall growth or weights gains and
voluntary activity (68, 81, 107). Because these variables are easy to
measure and tend to show an overall affect on laboratory animals, early
investigators ceased to look farther. Changes that take place in
carcass composition, organ weights, and serum values would give a more
complete picture as to the affects of diet on the whole animal.

Exercise levels were included so diet affect on voluntary as
well as forced—exercise programs could be measured. Different exercise
levels are necessary to observe diet-exercise interaction effects.

Many times the land area is needed to produce equal amounts of
usable protein from animals as from vegetables (36, 64, 105, 106). If
vegetarian diets can be developed which are as good as omnivorous diets,
more efficient use of our land area can then solve part of the world
fOod problem.

Some vegetable diets have been utilized in reducing serum
cholesterol levels (1, 2,,9, 34, 38) which in turn are associated with
a lower incidence of atherosclerosis and heart disease in man (12, 17,
21, 47, 50, 51, 58, 84, 85, 91). The results are unclear as to the
changes in serum cholesterol levels in man while on an exercise
program (27, 53, 66, 71). Although no attempt was made to measure
atherosclerosis or heart degeneration, diet-exercise effect in altering

serum cholesterol levels might have important implications.

Limitations of the Study
Because of the difficulty of working with an adequate number of

humans, male albino rats were used. Male albino rats can easily be

3
kept on various controlled exercise programs which were of interest in
this study. Changes that might have taken place in the serum cholesterol
and triglycerides with respect to exercise levels and diet groups, could
be easily measured. The rat and man appear to utilize cholesterol in a
similar manner (43, 77). Fbr these above reasons, it was decided to use
the male albino rat for this study. Cbnclusions drawn from animal
studies cannot be directly applied to humans. It will be necessary to
repeat the study controlling the dietary intakes of an experimental
group of human subjects. It should be noted, however, that in many ways
the internal chemistry of man and the rat are similar (73).

The rat requires arginine and histidine in the diet that adult
humans do not need (3). Both diets used in this study contained small
amounts of both these essential amino acids fer the rat.

The control animals were fed Wayne Lab Blox. In this dry block
form, the animals had to eat the mixed block composition if they ate
anything. The experimental animals were fed feed in various ferms and
containers. Some of the food the experimental animals were fed was dry,
some had a high moisture content, and some was in liquid fbrm. It is
not known if the difference in feeding procedures affected the results
of the study.

The Wayne Lab Blox that were fed the control animals contained
approximately 5.26 per cent animal protein. If finances and space had
been available, the use of a feed which contained higher levels of an
all-animal protein should have been used. If a high-animal protein diet
could have been compared directly to a strict vegetable protein diet, the
results might have been more definitive.

The Wayne Lab BloxIdiet contained 4.15 per cent fat. While the

l,
vegetable diet fat content was not controlled, it contained approximately
twice the fat level of the Wayne Lab Blox. It is not known to what
extent the results of the study were affected by this difference, but it

was to be expected that the vegetarian animals would be somewhat fatter.

Definition of Terms

Clarification of several terms used in this report will facilitate
a better understanding by the reader.
Control Animals

Male albino Sprague-Dawley rats fed the standard laboratory diet
of Wayne Lab Blox.
Strictyggetarian Animals

Male albino Sprague-Dawley rats fed the experimental diet which
contained no food of animal origin.
Sedentary Animals

These animals were housed in individual cages approximately ten
inches long, eight inches wide, and seven inches high. ‘Their exercise
was restricted to movement within their cages.
Vbluntary_Exercise Animals

These animals were housed in individual cages approximately ten
inches long, eight inches wide, and seven inches high. Each cage had
an exercise wheel attached so the rats could run at will.
Fbrced Exercise Animals

These animals were housed in individual cages approximately ten
inches long, eight inches wide, and seven inches high. Except for
ferced exercise training periods, the animals were restricted to move-

ment within their cages.

Fbrce Exercise Program
I The medium C-RW exercise program deve10ped at Michigan State

University Human Energy Research Laboratory was used (see Appendix C).
The total training program requires about eight weeks.
Egggtarian Diet

A vegetarian diet consists of food intake primarily of vegetable
origin. Some dairy products as well as eggs are often used to supplement
the diet. When these products are used the diet is often referred to as
a lacto-ovo-vegetarian diet.
Eggetarian or Strict vegetarian Diet

A strict vegetarian diet consists of foods only of vegetable
origin. No animal products or by-products are used in a strict vegetarian
diet.
Egg

This term stands for per cent expected revolutions. This is one
of the measurements taken from the forced-exercise program.
E

This term stands for the mean of the per cent expected revolutions.
‘§§§

This term stands for per cent shock free time. This term is one
of the measurements taken from the forced-exercise program.
E

This term stands for the mean of the per cent shock free time.

This term stands for total revolutions run. This is one of the

measurements taken from the forced-exercise program.

This term stands for the mean of the total revolutions run.

This term stands for cumulative duration of shock. It is one of

the measures taken from the forced-exercise program.

This term stands for the mean of the cumulative duration of shock.
‘XQL

This term stands for the number of voluntary revolutions run in the
voluntary exercise program
:51;

This term stands for the mean of the voluntary revolutions run in
the voluntary exercise program.
N. a. c.‘

This term stands for National Research Council. This council sets

the levels of recommended daily dietary allowances.

CHAPTER II
RELATED LITERATURE

The literature revealed only two types of studies where rats were
maintained on strict vegetarian diets. Several studies used rats to
test the biological value of specific proteins. They were not, however,
concerned with the overall value of a complete diet. Other experiments
studied the effects of cholesterol in the diet and blood. Again, they
were not concerned with the overall quality of a diet, only cholesterol
control.

Therefore, most of the related literature has to do with

observations made on people who were on or given a vegetarian diet.
Fbod Shortages.

One of our most pressing problems today is that of adequately
feeding the peOple of the world. A major reason fer the acute fbod
shortages is that many times the land area is needed to produce an
equal amount of uasble protein in animal form as from vegetables (36,
64, 105, 106). A considerable portion of this problem may be eliminated

through the use of vegetable proteins.

Deficiency in Diets
vegetarian diets can be adequately used by humans but, the most
serious drawback appears to be the lack of vitamin 312 (7, 24, 40, L6,

78, 79, 80, 82, 102, 103, 106). Vitamin 312 anemia is clinically the
7

8
most common deficiency seen in vegetarians. Whenever it is found, the
diet is unbalanced and lacking in several areas (86). Exogenous doses
of even 1 microgram per day of 312 is sufficient to remove the signs of
the anemia (24, 46, 80, 102). vegetarians apparently are more efficient
in the use of the 312 that is in the diet than are non-vegetarians.
With dietary intake well below recommended levels, most vegetarians show
no signs of B12 anemia. Some reports show that alterations in the
intestinal flora help compensate fer the low 812 levels in the diet (79.
103). There is little question that a vegetarian diet supplemented with
small amounts of eggs and milk is more than adequate from a nutritional
standpoint. Several workers have found that a strict vegetarian diet
can safely be used if there is variation in intake and sufficient
calorie content (23, 24, 36, 37, 46, 74, 75, 103, 105).

Advanced cases of malnutrition have been improved by the use of
strict vegetarian diets. Dean (22) and others have shown that recovery
from advanced cases of kwashiorkor was complete when such diets were
used. In fact, recovery is as fast or faster on the vegetarian diet as
when the standard nutritional diet of cows' milk as the main source of
protein is used. The protein source can be either soybeans or other
high vegetable proteins common to the area (4. 10, 22).

Sanchez, Scharffenberg, and Register (77) make the point that
vegetable diets are too low in methionine and or lysine for the rat.
Although similar in metabolism, not enough is known about the human
need for these amino acids. No ill effects are seen in human subjects,
after a prolonged use of a vegetarian diet, that would indicate the need

to increase the amounts of lysine and or methionine.

 

 

9
Cholesterol and Diet

The lowering of serum cholesterol has been closely studied in
regard to a vegetarian diet. The main control of serum cholesterol
levels seems to be the proportion of fat intake that is in the form of
unsaturated fatty acids (1, 2, 5, 9, 34. 38, 41, 55, 60, 61, 62, 63, 78,
90, 95, 97). Most of the unsaturated fatty acids are of vegetable origin.
A vegetarian diet apparently keeps the serum cholesterol levels down
because of the high amounts of unsaturated fats naturally occurring in
the diet. At one time it was thought that the amount of cholesterol in
the diet per se controlled the serum levels, however, this view is no
longer accepted. Some studies have shown that altering the cholesterol
intake in the diet was the controlling factor in maintaining serum
cholesterol levels (14, 29, 56). If cholesterol intake is kept within
the normal range, serum cholesterol levels are independant of cholesterol
intake (48, 57, 59). Generally, serum cholesterol levels vary directly
with the percentage of fat intake that is in the form of saturated fatty
acids. The apparent control of serum cholesterol by the relative amount
of saturated fatty acid in the diet is a real advantage to the vegetarian.
vegetarian diets are low in saturated and high in unsaturated fatty acids.
Studies measuring cholesterol levels in vegetarians as compared to
controls, have found the vegetarian serum cholesterol levels lower (61,

62, 63, 66, 78, 90, 95, 97, 98).

Diet and Disease
Carlson (12), Conner (l7), Hodges (47), Kennel 313;. (50), and
others have shown an association between serum cholesterol levels and
cardiovascular diseases. As serum levels rise, so does the risk of

coronary heart disease and atherosclerosis. Dayton 23.21, (21) in a

 

 

10

well controlled study on 848 volunteers came to the same conclusion. He
divided the volunteers into two matched groups. Control andiexperimental
diets were the same except that the animal fats were replaced by
unsaturated vegetable fats in the experimental diet. The experimental
diet induced an immediate drop in cholesterol levels which continued
throughout the study. Eight years after the start of the study, some
of the results were quite conclusive. When death due to coronary and
cerebral infarction were combined with non fatal coronary and cerebral
infarctions, the experimental dietary group had significantly fewer total
attacks. The experimental diet of vegetable fats replacing animal fats
seems to offer some protection to the user. The experiment was designed
and carried out double blind so that the bias of the examiners was
eliminated. Kannel at 31, (51) has similar data that show, where serum
cholesterol levels are high, there is three times the incidence of
coronary heart disease in men.

Coronary heart disease and atherosclerosis vary across countries.
As a nation ingests more saturated fatty acids the incidence of these
diseases rises (52, 58, 65, 85). Coronary heart disease and atherosclerosis
also vary within a nation with respect to socio-economic level. As the
family income rises, so does the general use of saturated animal fats (90,
91). Although not all the increase in the above diseases can be attributed
to dietary intake, certainly some portion can. The use of fewer calories,
less saturated fat and more unsaturated fat in the diet will promote good
health and reduce the risk of atherosclerosis and coronary heart disease

(84. so).

11
Eggetable Protein Source
Soybeans are a common cource of proteins in vegetarian diets
(18, 32, 35, 39, 64, 104). Other sources have been used (26, 35, 41, 77),
but soybeans are widely grown and easy to handle commercially.
Isolated soybean protein as a substitute for meat protein can apparently

meet all the (N. R. c.) allowances (18, 64).

Nitrogen Balance

Nitrogen balance indicates that humans use plant proteins in
essentially the same way as animal proteins (18, 26, 74, 77). Hegsted
gt_gl, (44) report that it is unlikely that any protein deficiency
will develOp in peOple who are on a vegetarian diet if there are adequate
calories and the intake is somewhat varied. This is substantiated in
studies by Donath gt_a;, (25), Ellis and Mumerd (28), West and Ellis (96),
and others, where they show that there are no clinical differences
between vegetarians and matched controls in the population (23, 37, 67,
70, 75). Hematological and biochemical tests on the blood and serum

are all within normal range.

Exercise and Cholesterol
Physical activity can alter serum cholesterol indirectly. If body
weight is maintained, little or no effect is seen in serum cholesterol
(27, 53, 71). Montoya gt_al. (71) report that a drop may be seen in
individuals with very high initial serum levels who are on an exercise
program. Serum cholesterol levels become lower in exercising individuals

probably because they are lowering body fat and total body weight.

 

CHAPTER III
EXPERIMENTAL METHOD

Fifty male Sprague-Dawley albino rats were purchased from Spartan
Breeders. The animals were twenty-three days of age when brought to the
Michigan State University Human Energy Research Laboratory.

The animals were randomly assigned to either a control dietary
group or to an experimental vegetarian dietary group. Within each dietary
group, the animals were randomly assigned to one of three exercise programs.
Seven animals from each dietary group were placed into a sedentary sub-
group. Seven animals from each dietary group were placed into a voluntary-
exercise program. The remaining eleven animals, from each dietary group,
were placed into a forced-exercise program. Six animals were desired in
each diet-training subgroup for final analysis. One extra animal was
placed in each sednetary and voluntary-exercise subgroup as a contingency
against injury or illness. Five extra animals were placed in each forced-
exercise subgroup because during a typical forced-exercise training program
some animals either become injured or fail to respond to the program and
must be drOpped from the experiment.

Fer final analysis, one animal was randomly selected from each
sedentary subgroup to be discarded from the experiment. One animal from
each voluntary subgroup was chosen to be drapped from the experiment on
the basis of being the most extreme in voluntary activity. The six most

12

13

homogenous animals with respect to activity were chosen for final analysis
from each voluntary subgroup (see Appendix B). There was one animal in
each voluntary subgroup that was extremely high in voluntary activity;
both of these extreme animals were discarded. The six animals, in each
forced-exercise subgroup, were chosen on the basis of performance in the
training program. The six animals, in each forced-exercise subgroup, that
performed the best (see Appendix B) in the C-RW program were chosen for
final analysis.

Sedentary animals were in sedentary cages from the day they were
brought into the laboratory until they were sacrificed.

The voluntary animals were in cages with access to the running'wheels
from the day they were received until they were sacrificed.

Fbrced exercise animals were placed in cages, with access to voluntary
running wheels for approximately ten days. These animals were allowed
the use of exercise wheels so they would learn how to run in a wheel before
going into the training program. At thirty-five days of age, these
animals were started on the C-RW program. When the animals started in
the forced-exercise program, they were changed into sedentary cages. The
exercise they received was limited to the running they did in the interval
training program.

The control animals were fed Wayne Lab Blox ag.libitum. This diet
contained: 24.52 per cent crude protein, 4.0 per cent crude fat, and
4.5 per cent crude fiber. In the control diet 21 per cent of the crude
protein was in the form of animal protein, thus 5.26 per cent of the total
control diet was animal protein. Actual contents of the Wayne Lab Blox

are as follows in decreasing quantity: animal liver meal, fish meal,

l4

dried whey, corn and wheat flakes, ground yellow corn, ground oat groats,
soybean meal, wheat germ meal, wheat middlings, cane molasses, dehydrated
alfalfa meal, soybean oil, brewers dried yeast, vitamin A palmitate,
irradiated dried yeast D-activated animal sterol, vitamin E supplement,
menadione sodium bisulfite, riboflavin supplement, niacin, calcium
panthothenate, choline chloride, thiamine, ground limestone, dicalcium
phosphate, salt manganous oxide, cOpner sulfate, iron sulfate, potassium
iodate, cobalt carbonate, and zinc oxide.

The daily strict vegetarian diet fer each each experimental animal
consisted of:

High Protein Soy Milk

Loma Linda vegeburger two ounces Worthington Soyamel two fluid ounces

Grains Other
one part wheat carrot small slice
one part barley green bean one-third
one part oats lettuce four square inches
one part rice green ripe olive one—half
one part rye apple one-twelfth
one-half part millet pecan meal one-quarter teaspoon

one-fourth part peas
The dry grains were ground and mixed because it was found that the

animals would selectively eat certain grains, which would negate the
necessary protein complementation (11). The grain mixture was placed in
a single container in each cage so the mixture would remain dry. The
grain was placed in the container in sufficient quantity so as to be
continuously available to each animal. The soy milk was placed in a
second small container so it would not be mixed with the other food. The
high protein vegeburger and other foods in the diet were placed in a third
container.

Both the control and exnerimental animals had free access to water.

Ill"!
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15

The experimental group's water intake was subjectively observed to
be much lower than the control group's. This is probably due to the fact
that all the food that the experimental animals had, except the grain,
contained a considerable amount of moisture.

Temperature control was maintained in the animal room at 72° F 1 2°.
In the forced-exercise room, the temperature was maintained at 69° F 1 2°
with no attempt to control humidity.

Lighting in the animal room was maintained on a cycle of twelve
hours of light and twelve hours of darkness.

Body weight for each animal was recorded at the same hour on Sunday
of each week during the experimental period.

The voluntary activity was recorded at the same hour each day as the
number of revolutions the animals had run in the previous twenty-four
hour period. A

The training program for the two forced-exercise subgroups was
the medium endurance control running wheel program. This program was
develOped at the Michigan State University Human Energy Research Laboratory
(see Appendix C).

When the animals were approximately ninety days old, blood samples
were taken and the animals sacrificed. The method for analyzing serum
cholesterol followed the procedure deveIOped by Haung, Wefler, and
Raftery (42). The method for analyzing serum triglycerides followed the
procedure develOped by Van Handel and Zilversmit (92). The animals were
prepared for carcass analysis at sacrifice following the procedure deve10ped
by Mickelsen and Anderson (69), and selected body organs were removed

from the animals and weighed immediately.

16

Diet and training were used in the analysis as independent variables.
The following were used as dependent variables: relative carcass water,
fat, protein, and ash: relative adrenal, heart, kidney, liver, spleen,
and testes; cholesterol (mg %) and triglycerides (mg'fi); final body
weight, PER- last four weeks, PEI-l- last five days, P‘s-F- last four weeks,
'PSE last five days, VOL last four weeks, VOL last seven days. 'The vol-
untary and forced-training data were divided into values above and below
their respective medians and analyzed for differences between the diets
on performance level. Since training causes large differences in body
weight, comparisons of absolute body composition and organ weights were
analyzed to see if growth might be retarded as suggested by previous
workers (92). All relative and absolute values were analyzed for diff-
erences between the two diet groups as well as differences between the
various exercise levels.

Standard one-and two way fixed effects analysis of variance
techniques were employed, where appropriate, to analyze the data. The
Tukey method for multiple comparisons between means was used wherever
necessary. The Sign Test was used to analyze the voluntary and forced-
exercise data. The alph level of'pfihOS was chosen because of the small

number of animals that were used.

CHAPTER IV
PRESENTATION OF DATA

The purpose of this investigation was to compare the effects of a
strict vegetarian diet and a control diet containing animal protein (5.23%)
in young albino rats. Two diet groups were used, both groups receiving
selected exercise programs. The parameters studied included: quantitative
responses to forced exercise, total carcass composition, organ weights,
serum cholesterol and serum triglycerides. The experimental design was
developed to study the parameters as they relate to physical activity as
well as diet.

All of the parameters, except the training data, were analyzed
statistically by two-way analysis of variance to determine the effects
of diet, physical activity, and the interactions between the two. With
two exceptions, there were no significant interaction effects in any of
the analyses. This indicated that except for the effects on serum
cholesterol and relative carcass ash values, the effects of training were
independent of the effects of diet in the variables studied. Since no
other significant interaction effects were observed, the exercise and

diet effects are presented separately in this chapter.

Exercise Analyses
Responses to Forced Exercise
The effects of diet upon the training responses are shown in
Figures 1, 2, 3, 4. These results show that the diet groups signifi-

17

 

 

I I -.l_‘ .

18
cantly differed in their reaponses to training. The per cent of
expected revolutions run (PER), per cent shock free time (PSF), and total
revolutions run (TRR), were all significantly less (Sign Test P(0.0001)
fer the experimental animals on the all-vegetable diet than for the
control-diet animals. The cummulative duration of shock (CDS) was
significantly greater for the experimental-diet group (P40.0001).
These results clearly indicate that the animals receiving the control
diet (5.23% animal protein) responded more favorably to the training
program on all measures: TRR, PER, PSF, and CD8.

In each of Figures 1, 2, 3, and 4 there is a break in the program
at the eighth training day. Some of the animals were not responding well
to the Medium Endurance C-RW program at this time, so the program intensity
was reduced and the number of animals being trained simultaneously was
lowered from six to two or three. After three weeks of this modified
program, the animals had progressed to the level of the nineteenth
training day, and the normal training progression was resumed.

Response to VoluntarygExercise

The mean voluntary revolutions run by the two dietary subgroups
are shown in Figure 5. The diet groups did not differ in their voluntary
activity patterns. The animals in the voluntary exercise groups did not‘
participate in the forced exercise program.

Discussion within Exercise

It is puzzling as to why there should be such a marked difference
in the forced-exercise results and no difference in the voluntary-activity
results. Certainly the performance of the experimental animals in forced
exercise was significantly poorer. This is an important finding, but it

cannot be explained from the present design.

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21
Serum Analyses

Two-way analysis of variance tables are listed for all of the rest
of the data analyzed.
Diet Effects on Serum Cholesterol and Triglygerides

The results of the analyses of serum cholesterol and serum tri-
glyceride levels by diet and exercise groups are shown in Tables 1 and 2.

The mean values for serum cholesterol and triglycerides were lower
for the experimental-diet group, but the difference was not statistically
significant (Tables 1 and 2). However, the results are of interest since
the control diet contained only 4 per cent animal fat, whereas the
experimental diet contained about twice that quantity of vegetable fat.
Exercise Effects on Serum Cholesterol and Triglycerides

With higher intensities of exercise, cholesterol and triglyceride
levels are successively lower (P<0.009 and P(0.0005 respectively). There
also was a decrease in total body fat with increasing exercise intensity,
possibly the decreases on serum cholesterol and triglycerides were due
to lower body fat. The effects of exercise in lowering serum cholesterol
and triglycerides are in line with previous work (72).
Interaction Effect on Serum Cholesterol

There was a significant interaction between diet and training with
respect to serum cholesterol (P(0.04). This was due to the relatively
low value found in the sedentary animals which were on the experimental

diete

Body Composition Analyses
Tables 3 and 4 present the body and carcass weight data analyzed by

diet and exercise.

22

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23
Diet and Exercise Effects on Body Weight and Carcass Height

There was no significant difference between the two diet groups
with respect to body weight or carcass weight.

Exercise had a significant effect on both body weight and carcass
weight. Mean body and carcass weights were progressively lower with
greater exercise intensities.

Diet Effect on Carcass Composition

The summary results of the analyses of both relative and absolute
carcass water, fat, protein and ash are shown in Table 5. The tdewey
analyses for individual variables are in Tables 7 through 14.

There were significant mean differences between the two diet groups
with respect to both fat and ash. The experimental diet used in this
study resulted in a significantly fatter animal. The reason the
experimental animals were fatter was probably due to the higher fat
content in their diet.

The animals on the experimental diet had a significantly lower
level of ash than the control group. The difference observed in the ash
content can not be eXplained from the current data. The difference may
be due to greater skeletal development in the controls or merely to a
greater quantity of other residue. The difference in the ash content
between the diet groups is not a result of greater activity in the control
forced-exercise group. The difference between the two diet groups is
consistent and similar in magnitude at all three levels of exercise
Table 13 and 14).

Exercise Effects on Carcass Composition
The summary results of the analyses of both relative and absolute

carcass water, fat, protein and ash are shown in Table 6. The twosway

24

 

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analyses for individual variables are in Table 7 through 14.

The activity levels used in this study resulted in typical responses
by exercise groups. Relative fat values were lower and relative water,
protein, and ash values were higher with increased levels of exercise
intensity. Overall changes and all but one of the paired comparisons
were significant, with the results appearing to be on a continium. If
the sample size had been larger, the results might have been more clearly
delineated.

The absolute values for carcass composition in Table 6 raise the
question of whether stunted growth occurs with higher intensities of
exercise. The absolute values for water, fat, and protein all show
significantly lower values with increased exercise intensity.

The smaller forced-exercise animals had essentially the same mean
ash level as the significantly larger voluntary animals. Ash levels
appeared to be affected by level of activity rather than body size.

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in exercise intensity. This could indicate that growth was impaired or
merely a decreased need for usable protein, because of the lower levels
of water and fat, in the lighter forced-exercise animals. Comparing
the sedentary and forced-exercise groups, the difference in weight due
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difference in total carcass weight. Especially in view of the absolute
ash values, it is possible that the lower level of protein in the smaller
body size was related to the lower levels of fat and water, rather than

to the stunted growth.

28

Discussion on Carcass Composition

There was no significant difference observed between the two diet
groups with respect to relative or absolute carcass water and protein.
The significantly higher level of carcass fat in the experimental animals
was probably due to the higher percentage of fat in their diet. The
experimental animals were significantly lower in carcass ash values. The
reason for this difference is unclear at this time. Since the difference
in ash values can be seen at all three levels of exercise, between the
two diet groups, the results can not be attributed to the greater exercise
of the forced-exercise control group. Probably, there was something
inherently lacking in the experimental diet that resulted in this lower
value for the experimental animals at all levels of exercise.

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with greater exercise intensities. Relative fat values were significantly
lower with greater exercise intensities. These results are all in line

with previous work (49, 93).

Organ Weight Analyses

The summary results of the relative and absolute organ weight
analyses are shown in Tables 15 and 16. The two-way analyses for
individual variables are in Tables 17 through 28.
Diet Effect on Qrgan Weight

Relative and absolute heart and kidney weights were greater while
the adrenal weights were smaller fer the animals on the experimental
diet. These results are seen at each level of exercise intensity used
in this study.

The significant difference in mean heart weight indicates that some-

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33
thing inherent in the experimental diet must bring about an increase in
heart size even at the lower intensities of exercise. Whether this is
a beneficial or a detrimental effect should be investigated further.
This difference in heart weights may be due to greater fat deposited
within the hearts of the experimental group.

The difference in kidney size is possibly due to the vegetable
proteins placing a greater filtering load upon the kidneys. Histological
work to identify structural differences responsible for the increased
weight is needed before proceeding to any hypothetical explanation.

The adrenal weight differences are most interesting because they
were evident at each of the levels of exercise intensity used. This
fact might indicate the experimental vegetarian diet enabled the animals
to make a more desirable stress response. The adrenal weights were
lower in the experimental group inspite of the fact that the animals
were fatter, which might have resulted in heavier organ weights.
Exercise Effect on Organ Weights

Relative heart, adrenal, and testes weights were all greater and
liver weights were lower with higher exercise intensities. These
responses were similar to the results of previous work (49. 93).

Absolute weights for the heart, liver, spleen and kidneys were
all significantly lower with increased exercise intensities. These
differences probably reflect the smaller body weights found at the
higher exercise levels. The absolute organ-weight results are also in

line with previous work (49, 93).

Discussion
The organ-weight and body-composition effects of exercise found in

this study were completely in line with previous work in the same area

 

34

(L9, 93). Relative protein, water and ash value were higher and
relative fat values lower with greater exercise intensities. Relative
adrenal, heart and testes values were higher and relative liver values
lower with greater exercise intensities. Serum cholesterol and tri-
glyceride values were lower with increased exercise levels. Since these
data are so closely in accord with previous work, they tend to support
the validity of the diet data.

Fbllowing is a brief summary of the comparisons between the two

diet groups which were deemed to be important:

 

Table 29 --- Comparisons of Two Diets

variable Control Vegetarian Interpretation
Weight 0 0 No difference

Fat - + Control animals leaner
Ash + - Control animals more ash
Kidney - + Control-smaller kidneys
Adrenal + - Control-larger adrenal
Heart - + Control-smaller heart
Cholesterol + - Control higher (not sig.)
Forced-Exercise + - Controls ran more

Shock - + Controls got less shock
voluntary Activity 0 0 No difference

In the body composition results, it is likely that the carcasses of
the experimental animals contained more fat because of the higher level
of fat in the diet. The differences observed in the ash content can
not be eXplained from the current data.

The serum cholesterol and triglyceride levels were slightly lower
for the experimental animals (not significant), in spite of the fact that
the eXperimental animals were fatter. It has been shown that relative
fatness of the body is positively related to serum cholesterol levels
(72). This contradiction with previous work indicates that constituents

I

of the control diet (saturated fats) have a greater influence on serum

35
cholesterol levels than does body fat.

The adrenal weights were significantly lower and the heart weights
significantly higher in the experimental animals. These differences
were found at every exercise level, in spite of the fact that the
experimental animals experienced more shock and ran less on the forced-
exercise program. There was no difference between the two diet groups
in activity on the voluntary-exercise program or, of course, in the
sedentary program. Yet, the differences in adrenal and heart size
were found at both of these activity levels. The differences in the
adrenal and heart weight were most interesting and clearly deserve
further investigation. It may be the experimental diet served to block
the stress reaction in some way as evidenced by the smaller adrenals and
the lower cholesterol levels. However, such hypothetical explanations
are premature until analysis of the adrenal gland for ascorbic acid and
cholesterol concentration can be carried out.

Any hypothesis of a direct diet effect on heart hypertrOphy is also
premature. The amount of fat in the carcasses of the experimental
animals was significantly greater than in the control animals and might

have resulted in fatty deposits within the heart.

CHAPTER V

SUMMARY, CONCLUSIONS, AND RECOMMENDATIONS

Summary

The purpose of this study was to compare the effects of a strict
vegetarian diet and a control diet containing animal protein (5.23%)
under various conditions of physical activity.

Fifty, twenty-three-day-old male albino rats (Sprague-Dawley strain)
were randomly assigned to either a control dietary group or to an
experimental dietary group. Within each dietary group, the animals were
randomly assigned to one of three exercise programs. Seven animals from
each dietary group were placed into a sedentary subgroup. Seven animals
from each dietary group were placed into a voluntary-exercise subgroup.
The remaining eleven animals, from each dietary group, were placed into
a forced-exercise subgroup. Six animals were desired in each diet-
training subgroup for final analysis. One extra animal was placed in
each sedentary and voluntary-exercise subgroup as a contingency against
injury or illness. Five extra animals were placed in each forced-
exercise subgroup because during a typical forced-exercise training
program some of the animals either become injured or fail to respond to
the program and must be dropped from the experiment.

At ninety days of age, blood samples were taken fer serum cholesterol

and triglyceride analyses, and the animals were sacrificed. Wet weights

36

37
were taken on the following organs: adrenals, heart, kidneys, liver,
spleen and testes. The carcasses then were frozen until determinations

of body composition were made.

Conclusions

The results of this study, presented with respect to exercise, are
completely in line with previous work (49, 93). Higher levels of
exercise produced animals with higher per cent carcass weight due to
water, protein, and ash with a lower per cent carcass weight due to fat.
When the forced-exercise group was compared to the sedentary group, the
active animals were found to have lower relative liver weights and
significantly higher relative adrenals, heart, and testes weights. There
also were significantly lower serum cholesterol and triglyceride values
in the forced-exercise group.

When the data were analyzed by diet groups, some unusual results
were seen. The mean values for serum cholesterol and triglycerides were
lower for the experimental (vegetarian) dietary group, though the difference
between the means was not statistically significant. However, the results
are of interest since the control diet contained only 4 per cent animal
fat, whereas the experimental diet contained twice that quantity of vege-
table fat. The experimental dietary group also was significantly fatter
which, from previous work, should have resulted in higher serum values (72).

Mean adrenal weight was lower for the experimental dietary group at
each level of exercise used. This fact might indicate the experimental
vegetable diet enabled the animals to make a more desirable stress response.
The adrenal weights were lower in spite of the fact that the animals were

fatter, which might have resulted in heavier organ weights.
I

 

38

Mean kidney and heart weights were significantly higher in the
experimental animals. These differences were found at each intensity of
exercise. This difference might have been related to the change in the
adrenal weights or it may have been due to fatty deposits in the kidney
and heart.

Due to the small sample size, the statistical power was less than
0.30 in all analyses where significant differences were not observed.
Therefore, failure to demonstrate significance should not be interpreted,
at this time, as justification for acceptance of the hypothesis of no

treatment effects.

Recommendations

 

A similar study is needed to confirm the results of this study.

The use of various levels of proteins and fats (animal and vegetable)
should be included to determine their effects on serum cholesterol and
triglyceride.

Ascorbic acid determinations on the adrenal gland should be done to
help indicate its activity. Various kinds of stress could also be used
to help stimulate larger changes in adrenal activity.

Histological work on the kidney should be done to try and identify
any structural changes that might be responsible for the increased weight
in the experimental dietary group.

Histological work on the heart should be done to try and identify
any structural changes or fatty deposits that would account for the

increased weight in the experimental dietary group.

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'46

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Adventist. Cancer. 12:1016, 1959.

APPENDIX

47

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APPENDIX B

TABLE Bl

RAW DATA — ACTIVITY RECORD

FORCED EXERCISE ANIMALS

PERCENT
SHOCK
FREE TIME
MEAN LAST
FOUR WEEKS

083
078
073
082
083
067
067

081
055
057
083
058
053

VOLUNTARY
REVOLUTIONS

MEAN

LAST WEEK

1954
3949
0828
1684
1371
1015

1321
2046
1374
3917

PERCENT PERCENT
EXPECTED EXPECTED
REVOLUTIONS REVOLUTIONS
MEAN LAST MEAN LAST
FOUR WEEKS FULL WEEK
CONTROL 098 101
ANIMALS O92 087
096 086
101 088
089 087
O75 O75
O75 O75
EXPERIMENTAL 100 097
ANIMALS O67 O64
065 066
107 103
O67 068
062 059
TABLE B2
RAW DATA - ACTIVITY RECORD
VOLUNTARY ANIMALS
VOLUNTARY
REVOLUTIONS
MEAN LAST
FOUR WEEKS
CONTROL 1677
ANIMALS 2980
0746
1414
1477
0888
EXPERIMENTAL 1263
ANIMALS 1366
1452
3288
0387

1519

0701
1117

PERCENT
SHOCK
FREE TIME
MEAN LAST
FULL WEEK

088
077
072
080
086
067
067

090
050
058
086
060
045

52

APPENDIX C

STANDARD EIGHT-WEEK, MEDIUM-DURATION, MODERATE-INTENSITY
ENDURANCE TRAINING PROGRAM FOR POSTPUBERTAL AND
ADULT MALE RATS IN CONTROLLED-RUNNING WHEELS

 

Tofal TofaI
Acc- Repe- Time Time Exp. Total
aler- Work fi- Bef- Run of Revo- Work
Day Day afion Time Res? fions No. ween Speed Prog. Iu- Time
of of Time (min: Time par of Bouts Shock (ffl (min: fions (sec)

Wk. Tr. (sec) sec) (sec) Bout Bouts (min) (ma) sec) sec) TER TNT

 

I=M I I.0 00:I0 5 40 3 5.0 I.2 2.0 39:45 600 i200
2=T 2 I.0 00:I0 5 40 3 5.0 I.2 2.0 39:45 600 I200
3-W 3 I.0 00:I0 5 40 3 5.0 I.2 2.0 39.45 600 I200
4-T 4 I.0 00:I0 i0 28 4 5.0 I.2 2.5 5|:40 700 II20
58F 5 I.0 00:I0 IO 28 4 5.0 I.2 2.5 5I:40 700 II20
I'M 6 I.0 00:I0 I0 28 4 5.0 I.2 2.5 5|:40 700 II20
2-T 7 I.0 00:I0 I0 27 4 5.0 I.2 3.0 50:20 8I0 I080
33W 8 I.0 00:I0 IO 27 4 5.0 I.2 3.0 50:20 8I0 I080
4-T 9 I.0 00:I0 I0 27 4 5.0 I.2 3.0 50:20 8I0 I080
5=F I0 I.0 00:I0 I0 27 4 5.0 I.2 3.0 50:20 8I0 I080
I=M II I.0 00:I0 I0 27 4 5.0 I.2 3.0 50:20 8I0 l080
2-T I2 I.5 00:I0 I0 26 4 5.0 I.0 3.5 49:00 9I0 I040
38W I3 I.5 00:I0 I0 26 4 5.0 I.0 3.5 49:00 9I0 I040
4=T I4 I.5 00:I0 I0 26 4 5.0 I.0 3.5 49:00 9I0 I040
5=F I5 I.5 00:I0 I0 26 4 5.0 I.0 3.5 49:00 9I0 I040
I=M I6 I.5 00:I0 I0 26 4 5.0 I.0 3.5 49:00 9I0 I040
2=T I7 I.5 00:I5 I5 I9 4 5.0 I.0 3.5 52:00 997 II40
3=W I8 I.5 00:I5 I5 I9 4 5.0 I.0 3.5 52:00 997 II40
4=T l9 I.5 00:I5 I5 I9 4 5.0 I.0 3.5 52:00 997 II40
5=F 20 I.5 00:I5 I5 I9 4 5.0 I.0 3.5 52:00 997 II40
I=M 2| I.5 00:I5 I5 I9 4 5.0 I.0 3.5 52.00 997 II40
2-T 22 I.5 00:I5 I5 I4 5 5.0 I.0 4.0 53:45 l050 I050
=W 23 I.5 00:I5 l5 I4 5 5.0 I.0 4.0 53:45 I050 I050
4=T 24 I.5 00:I5 I5 I4 5 5.0 I.0 4.0 53.45 I050 I050
5=F 25 I.5 00:I5 I5 I4 5 5.0 I.0 4.0 53:45 I050 I050
I=M 26 I.5 00:I5 l5 I4 5 5.0 I.0 4.0 53:45 I050 I050
2=T 27 I.5 00:20 20 II 5 5.0 0.8 4.0 55:00 IIOO IIOO
3=W 28 I.5 00:20 20 II 5 5.0 0.8 4.0 55:00 IIOO II00
4=T 29 I.5 00:20 20 II 5 5.0 0.8 4.0 55:00 IIOO II00
5=F 30 I.5 00:20 20 ll 5 5.0 0.8 4.0 55:00 IIOO IIOO
I=M 3| I.5 00:20 20 II 5 5.0 0.8 4.0 55:00 Il00 IIOO
2=T 32 I.5 00:25 25 9 5 5.0 0.8 4.0 55:25 II25 II25
3=W 33 I.5 00:25 25 9 5 5.0 0.8 4.0 55:25 II25 II25
4=T 34 I.5 00.25 25 9 5 5.0 0.8 4.0 55:25 II25 II25
5=F 35 I.5 00'25 25 9 5 5.0 0.8 4.0 55:25 II25 II25
ISM 36 I.5 00:25 25 9 5 5.0 0.8 4.0 55:25 II25 II25
2=T 37 I.5 00:30 30 8 5 5.0 0.8 4.0 57:30 I200 I200
3‘" 38 I.5 00:30 30 8 5 5.0 0.8 4.0 57:30 I200 I200
4-T 39 I.5 00:30 30 8 5 5.0 0.8 4.0 57:30 I200 I200
5-F 40 I.5 00:30 30 8 5 5.0 0.8 4.0 57:30 I200 I200

 

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