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Thesis Ear Hie Hem-ea of PE. D.
Michigan Shale Unlvorsily

Perry Brooke JOEMOR

1960

 

This is to certify that the

 

thesis entitled
THE RELATIVE EFFECTS OF EXERCISE AND
CALORIC RESTRICTION IN CONTROLLING
BLOOD CHOLESTEROL IN RATS
presented by

Perry Brooke Johnson ‘

has been accepted towards fulfillment
of the requirements for

211.12. degree mm Education

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LIBRARY

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University _

 
    

 

 

 

 

 

 

 

 

 

4‘

AN D

.4

THE RELATIVE EFEECTS OF EXERCIS‘

CALORIC RESTRICTION IN CCNPRCLLIN

BLOOD CHOLES‘EROL IN RATS

by
PERRY BROOKE JOHNSON

AN ABSTRACT

Submitted to the School for Advanced Graduate Studies of
Michigan State University of Agriculture and
Applied Science in partial fulfillment of

the requirements for the degree of

DOCTOR or PHILoscPHY

§

Department of Health, Physical Education and Recreation

1960 ' -

Approved:

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Perry Brooke Johnson
ABSTRACT

With the felationship between excess serum
cholesterol and atherosclerotic disease well established,1’2
there is a need for investigating all possibilities for con-
trolling serum cholesterol concentrations. There is
evidence that serum cholesterol concentrations rise or fall
paralleling weight gain or 1033.3’4’5 Recent work indicates
that exercise decreases cholesterol level. Is exercise 23;
§g_effective in controlling serum cholesterol, or is it
effective only if weight is lost? Or is weight control by
diet as effective as exercise? The purpose of the present
investigation was to answer these questions.

In an effort to evaluate the relative effects of
exercise and caloric restriction in controlling total blood
cholesterol level, blood samples were taken from eighty-eight
mature male albino rats for serum cholesterol assay. The
animals were divided into four groups equated on the basis of
body weight. One group was sacrificed immediately and the
other three maintained for a fifteen-week experimental
period. One group was sedentary and fed ad libitum, one was
exercised regularly and fed 22 libitum while the third group
was sedentary, but maintained at a mean body weight comparable
to that of the exercise group by careful diet restriction.

In addition, the experiment was designed to test
whether differences in body weight and body fat could account

:finr any differences in blood cholesterol.

 

 

 

 

Perry Brooke Johnson

Control and final total blood cholesterol
concentration and final specific gravity, to be used as an
estimate of body fat, were determined. The under-water
weighing method was used to determine specific gravity.

Control and final body weights also were recorded.

SUMMARY OF MEAN VALUES

 

 

 

 

Grogps

Variable Sedentary Diet Exercise
E1541 Weight 475.6 g 422.2 g 421.2 g
Spec. Gravity 1.0323 1.0256 1.0363
Final Chol.

in mg% 96.41 95.32 75.10
Chol. Change +30.59 +£5.64 +13.85
Wt. Change +148.2 g +95.5 g +92.0 g

 

 

 

 

The data were(analyzed statistically, employing
analysis of variance, analysis of covariance, students "t",
and product-moment correlation coefficients.

It was concluded that caloric restriction was 223.33
effective in controlling total blood cholesterol as was
regular exercise. The mean cholesterol difference between
the diet group and exercise group was significant even after
removing the effects of the control cholesterol level.

The significant positive correlation between control
and final cholesterol concentrations strengthens the theory
that: there is some predetermined, probably inherent factor

Whicli controls cholesterol concentration. However, this

2 s

 

 

    
    

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Perry Brooke Johnson

relationship did not hold true within the exercise group.
It is possible, then, that exercise‘is a factor which can
alter this seemingly predetermined cholesterol concentration.

Although the sedentary group was significantly
heavier than the diet and exercise groups, body weight was
not significantly correlated with final cholesterol concen-
tration. Therefore it cannot be concluded that high blood
cholesterol and high body weight are associated.

Although the nonexercise animals were significantly
less dense than the exercise animals, specific gravity was
not significantly correlated with cholesterol concentration.
Hence, it cannot be concluded that high blood cholesterols
are associated with high percentage of body fat.

There was no significant relationship between
cholesterol change and weight change, which is not surpris-
ing in view of the lack of relationship between final weight
and cholesterol concentration.

The results seem to support the hypothesis that over
and above its effect on weight and obesity, there is some
effect of exercise which helps control blood cholesterol
concentration. .

REFERENCES
‘l. J. Green and j. M. van der Heide, Atherosclerosis and

Coroner °i‘hrombosis, Trans. by J. Winsser, Rotterdam,
‘Tw T_T§156, 12-14. .

 

2- J. W. Gofman, et al., (Technical Group), E. 0. Andrus,
et al., (Comm. on LipOprotiens, etc.), "Evaluation of
Serum Lipoprotein and Cholesterol Measurements as Pre-
dictors of Clinical Complications of Atherosclerosis,"
(lirculation, 14 (1956), 730.

 

3

 

 

 

 

5.

Perry Proohe Johnson

C. V. Mann, et al., "Exercise in the Disposition of
Dietary Calories," New England Journal of hedicine, 253
(1955) , 355. ____..__..

J. T. Anderson, F. Grande and A. Keys, "Serum Cholesterol
Concentration of Men in Semi-Starvation and in Refeed-
ing," Federation_Proceedings, 14 (1955), 426.

J. T. Anderson, A. Lawler and A. Keys, "Weight Gain from
Simple Overeating. II. Serum Lipids and Blood Volume,"

Journal of Clinical Investigation, 36 (1957), 87.

 

 

 

To the memory of my father...

PERRY BROOKE JOHNSON, JR.

 

ACKNOWLEDGEMENTS

The writer wishes to express his sincere appreciation
to the members of his committee, Dr. Henry J. Montoya and
Dr. Wayne D. Van Hues of the Physical Education Department,
Dr. K. J. Arnold of the Department of Statistics, and Dr.

W. Doyne Collings of the Department of Physiology and Pharma-
cology. Without their interest and help, this thesis would
not have been possible.

Grateful appreciation is also expressed to Dr. Evelyn
Jones, for her time and patience in supervising the actual
cholesterol assay, and Wynn Updyke, for his contribution to
the long and tedious hours required during the experimental
period.

111

 

in-

2:2..- 7

 

 

 

 

TABLE OF CONTENTS

LIST OF TABLES . . . . . . . . . . . . .
LIST OF ILLUSTRATIONS . . . . . . . . .
CHAPTER
I. INTRODUCTION . . . . . . . . . . .
The Problem . . . . . . . . . . .
Limitations . . . . . . . . . . .

Exercise and Blood Cholesterol Concentration .

Specific Gravity as an Estimate of Body Fat .

III. PROCEDURE . . . . . . . . . . . .
General Experimental Design . . .
Methodology . . . . . . . . . . .

IV. RESULTS AND DISCUSSION . . . . . .
Results . . . . . . . . . . . . .

Cholesterol . . . . . . . . . .

Body Weight . . . . . . . . . .

Fat . . . . . . . . . . . . . .
Discussion of Results . . . . . .

Cholesterol . . . . . . . . . .

Body Weight . . . . . . . . . .

Fat . . . . . . . . . . . . . .

V. SUMMARY AND CONCLUSIONS . . . . .
Summary . . . . . . . . . . . . .
Conclusions . . . . . . . . . . .
Recommendations for further study

BIBLImRAPHYeoooeeeeeoeooo

.13
.14
.21
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.37
.38
.39
.41

APPENDIX A
APPENDIX B
APPENDIX 0
APPENDIX D
APPENDIX E
APPENDIX E

Sample of Diet Group Ration Calculation .
Composition of Diet .
Total Cholesterol Determination .

RaWDatao e e e 0
Weight Gain Chart .

Correlation Plots .

 

Page
47

48
49
. 51
55
56

 

 

 

 

 

 

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) ._ ..

LIST OE TABLES

TABLE
I. Summary of Mean Values . . . . . . . . . . . . . .

II. Correlation Coefficients, Control Cholesterol vs.
Final Cholesterol . . . . . . . . . . . . . . . .

III. Analysis of Covariance, YéFinal Cholesterol and
x=00ntr01 ChOlGStGI‘Ol‘COVariate) o o o e o e e 0

IV. Analysis of Variance, Final Body Weight . . . . .

V. Correlation Coefficients, Final Body weight vs.
FiflElChOlGStGTOleoeeeeeeoeeeccoo

VI. Correlation Coefficients, Cholesterol.Change vs.
W'eightChangeoeeeeeeeoeeeoeeooe

VII. Analysis of Variance, Carcass Specific Gravity . .

VIII. Correlation Coefficients, Carcass Specific Gravity
VS. Final ChOlesteI'Olo o o e e e e e e e o e o 0

vi

 

Page

. 23

.24

. 26

. 3O

 

LIST OF ILLUSTRATIONS

Page
FIGURE

1 Blood Sampling Technique . . . . . . . . . . . . . . 15

2 Comparison of Mean Values . . . . . . . . . . . . . .29

711

 

 

 

CHAPTER I
INTRODUCTION

In recent years there has been a growing interest in
the relationship between fat metabolism and circulatory dis-
orders. Considerable evidence tends to support the hypoth-
esis that some abnormality in lipid metabolism is closely
linked to atherosclerosis. Cholesterol is one of the lipids
which has been extensively investigated in this connection.

Although there is little direct evidence that a high

blood serum concentration of cholesterol actually causes

 

atherosclerosis or related circulatory disorders, there are
indications that high serum cholesterol concentrations are
related to the atherosclerotic diseases. Green and van der
Heide,1 in their comprehensive review of the total problem.
cite the following factors which point to the possible im-
portance of cholesterol: (1) a high percentage of choles-
terol and its esters in foci of atherosclerosis, (2) experi-
mental atherosclerosis produced in animals by high choles-
terol feedings, and (3) geographical differences--in

countries where atherosclerosis is rare, average blood

1J. Groen and R. M. van der Heide, Atherosclerosis
and Coronory Thrombosis, Health Organization. T. N. 0.,
Publisher WYT, Rotterdam, 1956, trans. Johan flinsser,
12-14-

 

 

2

cholesterol level is lower. In summarizing the relationship
between cholesterolemia and ischemic heart diseases, the
authors sayzz

. . . we think it very acceptable, if not

proven, that an increased blood cholesterol

level promotes the development of athero-

sclerosis and arteriosclerotic heart diseases.

The indications are especially clear when

groups are compared with a very low and a

very high blood-cholesterol level.

Most of the evidence in favor of the importance of
cholesterol is indirect. However, a recent investigation by
Gofman and co-workers indicates a more direct relationship
between high cholesterol concentration and early onset of
atherosclerotic changes.3 They found high serum cholesterol
effective in predicting these degenerative changes.

It is possible, then, that an excess of serum
cholesterol contributes to the degenerative changes seen in
atherosclerosis. On the other hand, there may be no causa—
tive effect, the two changes merely going hand-in—hand. In
either case, with the relationship well established there is
a need for investigating all possibilities for controlling

and lowering serum cholesterol concentrations.

 

2Ibid., 26.

53. w. Gofman, et al., (Technical Group), 3. o.
Andrus, et al., (Comm. on LipOprotiens, etc.), "Evaluation
of Serum LipOprotein and Cholesterol Measurements as
Predictors of Clinical Complications of AtherOSclerosis,"
Circulation, 14 (1956), 730.

 

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a“ I? *‘ ,. .. a?) a“ ~1- --..~.,-. . .

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5
In an effort to determine what variables affect the
serum.cholesterol concentration, much work has been done in
the general area of diet and nutrition. There have been
fewer studies concerning the possible ro1e of physical exer-
cise, but recent investigations seem to indicate that exer-
cise may contribute considerably to the maintenance of normal

4'5'6v7 8 believes

serum cholesterol concentrations. Mann
that "exercise is likely to have a biochemical relation to

lipid metabolism."

I. The Problem

 

There is evidence which indicates that serum

cholesterol concentrations rise along with an increase in

 

4A. L. Myasnikov, "Influence of Some Factors on
Deve10pment of Experimental Cholesterol Atherosclerosis,"
Circulation, 17 (1958), 110.

 

5H. Y. o. Wong, "Hypocholesterolization Effect of
Exercise on Cholesterol-Fed Cockerels," Federation Proceed-
ings, 16 (1957), 158.

 

6H. Montoya, et al., Unpublished data, Michigan
State University, Department of Health, Physical Education
& Recreation, 1959.

7I. B. Page, "Atherosclerosis-~A Commentary,"
Federation Proceedings, 18 (1959), 50.

8G. V. Mann, "Diet, Exercise and Coronary Disease,"
Illinois Vedhcal Journal, 116 (1959), 20.

 

 

 

 

“V“? 1.. ~- .Tf'al'af‘FE-‘LQ

 

 

 

body weight and decrease parallel to weight loss.9'10'll

Recent work indicates that exercise is effective in decreas-
ing serum cholesterol concentration, but only indirectly

through decreasing body weight.12

This evidence brings out
an important question: is exercise pg; as effective in
controlling serum cholesterol, or is it effective only if
weight is lost or maintained? Or, is it possible that

weight control by caloric restriction alone is as effective

as exercise?
Statement of the Problem

it was the purpose of this study to investigate the
relative effects of physical exercise and weight control on
blood serum cholesterol concentrations in mature rats. The
experiment was designed to test for differences in body
weight, total body fat, and blood serum cholesterol concen-

tration among three groups of rats (exercise, sedentary and

 

9G. V. Mann, et al., "Exercise in the Disposition of
Dietary Calories," New England Journal pf Medicine, 255
(1955), 555.

10J. T. Anderson, 3. Grande and A. Keys, "Serum
Cholesterol Concentration of Men in Semi—Starvation and in
Refeeding," Federation Proceedings, 14 (1955), 426.

 

11J. T. Anderson, A. Lawler and A. Keys, "Weight Gain
from Simple overeating. II. Serum Lipids and Blood Volume,"
Journal gj_C1inical Investigation, 56 (1957), 87.

12H. J. Montoye, et al., "The Effects of Exercise on
Blood Cholesterol in MiddIe-Aged Men," American Journal gf_
Clinical Nutrition, 7 (1959), 144.

 

 

‘ _- .
"
B—d‘hJ -

 

 

 

 

 

 

 

5
diet). In addition, an initial control group, sacrificed
at the beginning of the experiment, was available for

comparison with these three groups.
II. Limitations

Due to the ease in controlling such important

factors as amount and intensity of exercise and diet re-

 

striction, rats were chosen for this investigation. It must

be remembered that animal observations cannot be transferred

to human interpretation. However, until evidence to the ~
contrary is presented, phenomena observed in these mammals

are extremely valuable in basic investigations related to

this problem.

 

 

 

CHAPTER II

REVIEJ CE RELATED LITERATURE

I. Exercise and Blood Cholesterol Concentration

Mann,1 reporting the results of a study of three

subjects, states that young men consuming high fat diets
were able to double their caloric intake without increasing
the level of their serum lipids or cholesterol so long as
the surplus of energy was expended as heat and muscular
energy. On the other hand, restricting exercise and allow-
ing fat deposition doubled serum cholesterol concentration.
The serum concentrations were returned to normal by food
restriction and weight reduction.

Montoye and co-workers2 investigated the effects of
supervised exercise on total blood serum cholesterol in 51
sedentary middle-aged men. No effect was observed in mean
serum cholesterol of subjects with "normal" initial serum
levels, but three "high" level subjects showed large de-
creases compared to two controls (no exercise) with high
initial levels, although one control also decreased.
Changes in total serum cholesterol generally accompanied

changes in body weight, regardless of whether the weight

 

1G. V. Mann, et al., "Exercise in the Disposition of
Dietary Calories," New England Journal 2f Medicine, 255
(1955), 555. ——----

 

2H. J. Montoye, et al., "The Effects of Exercise on
Blood Cholesterol in Middle-Aged Men," American Journal of
Clinical Nutrition, 7 (1959),-144.

6

 

 

 

 

 

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7

change was brought about by exercise. They concluded that
exercise was effective in decreasing total serum cholesterol,
but that the effect appeared to be indirect throught weight
reduction.

Comparing ten sedentary subjects with seven active
subjects, all middle-aged, Chailley-Pert and co-workers5
found the active group had considerably lower blood serum
cholesterol concentrations. In addition, three sedentary
middle-aged workers (2 females and one male) with reasonably
high blood cholesterol concentrations were placed on a
conditioning program of daily 5 Km. walks, plus cycling.
Blood cholesterol was appreciably lowered in all three
subjects following the exercise program. In one case the
program lasted six months, in the others, four and two
months.

Mann, Nicol and Stare4

reported differences in
cholesterol concentrations among Nigerian subjects in three
separate areas of the country. Since total caloric and

total fat content of the diet was similiar and since there
was a difference in muscular exercise, they believed that the

physical work patterns may have had an effect in controlling

serum cholesterol concentrations.

 

3Chailley-Bert, a. Labignette and Fabre-Chevalier,
"Contribution a 1'etude des variations du cholesterol
sanguin au cours des activite's physiques," Presse Med., 65
(1955), 415.

4C. v. Mann, B. n. Nicol and r. J. Stare, "The Beta-
Lipoprotein and Cholesterol Concentrations in Sera of
Nigerions," British Medical Journal, 2 (1955), 1008.

 

 

   

 

 

 

 

 

 

 

 

8

In investigating the Bantu, Keys and co-wcrkers5

accounted for large variations in serum cholesterol ngt by
physical activity differences, but by lower fat diets con-
sumed by the heavy manual labor group. They concluded that
the habitual diet, and eSpecially its fat content, has much
more influence on serum cholesterol concentration than
physical activity 223 as.

The effects of daily treadmill walking in nine
university students were studied by Taylor, Anderson and
Keys.6 The exercise consisted of two hours at 3.5 miles per
hour, 10 per cent grade and equalled 1280 calories work.

The diet was increased by 900 calories and the proportion of
fat held constant. There was no change in mean body weight
and no significant change in blood serum cholesterol concen-
trations. The authors conclude that this supports their
hypothesis that serum cholesterol is related to the propor-
tion of total calories derived from fat, not to exercise.
The same authors in a similiar study of nine university
students on a 1500 calorie daily treadmill walking program,
again reported no serum cholesterol changes and no weight

changes.7 They concluded that in calorie balance, serum

 

5A. Keys, et al., "Physical Activity and the Diet in
Pepulations Differing in Serum Cholesterol," Journal 2:
Clinical Investigation, 55 (1956), 1179.

6H. J. Taylor, J. T. Anderson and A. Keys, "Physical
Activity, Serum Cholesterol and Other Lipids in Man," Pro-
ceedings gf_the Societ for Experimental Biology and
Medicine, 95 (1957), 583.

7H. J. Taylor, J. T. Anderson and A. Keys, "Effect
on Serum Lipids of 1500 Calories Daily Talking," Federation
Proceedings, 16 (1957), 128.

 

 

 

 

 

 

 

  

         

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9
cholesterol concentration is independent of caloric intaxe,
absolute fat intake and physical activity level when the xind
of fat and percentage fat calories are constant.

Anderson, Lawler and Keys8 studied twenty physically
healthy schiZOphrenic men, increasing their calorie intakes
without changing physical activity. Average total serum
cholesterol concentrations rose 20 per cent during the first
five weeks, then stayed relatively the same for fifteen
weeks even though weight gain continued. They concluded
that the rise in serum cholesterol tended to be prOportional
to rate of weight gain.

9 used rabbits to determine the

Brown and co-workers
effects of exercise in preventing coronary atherosclerosis,
admitting that there was probably little application to
humans due to the differences in cholesterol metabolism. In
rabbits on diets of 0.1 per cent and 0.5 per cent cholesterol,
there was a lowering of total serum cholesterol after just
four weeks of exercise. At the end of the comparatively
short experiments (6, 8 and 12 weeks) exercise had no effect
on the development of atheromata in the aorta and coronary
arteries. Exercise was also ineffective in speeding dis-

appearance from the vessels once the atheromata were de-

veloped. It should be pointed out, however, that these

 

8Anderson, Lawler and Keys, op. cit., p. e1.

90. E. Brown, et al., "Observations on Blood Vessels
and Exercise," Journal of Gerontologyz 11 (1956), 296.

 

10
rabbits were exercised only twenty minutes daily, which
seems to be far less than a rabbit would normally exercise.
Rabbits were also used in an experiment by

Liyasnikov.lo

These rabbits, however, were exercised to
exhaustion daily for six months. There was a marked decrease
in the serum cholesterol concentration of the twenty-five
exercise rabbits and also some reduction in the develOpment
of atherosclerotic changes.

11 reported a significant reduction in serum

“long
cholesterol concentrations of cockerels fed cholesterol and
exercised daily for seven weeks as compared to a cholesterol
fed, no exercise group. The author also noted a significant
reduction in formation of atheromatous plaques in the
abdominal aortas of the exercised birds.

Results of a recent study by Montoya and co-worxers12
indicate that exercise controls serum cholesterol concen-
tration. Seven rats swum an hour daily for twelve weeks had
a mean serum cholesterol level of 74.72 mg. per cent as

compared to their seven paired littermates' mean level of

95.08 mg. per cent. The difference was significant at the

 

10A. L. Myasnikov, "Influence of Some Factors on "
DeveIOpment of Experimental Cholesterol Atherosclerosis,

Circulation, 17 (1958). 110.

11 V " 1 sterolization effect of
H. Y. 0. long, Hypocho 6
Exercise on Cholesterol-Ted Cockerels," Federatign_Proceed-

ings, 16 (1957), 158.
12 1' Un ublished data, Michigan
H. J. nontoye, et al., p n
State University, Departmen of Health, Physical education
and necreation, 1959.

 

 

 

,_"‘_\’ .. ~

fifth..." ‘

 

 

11

5 per cent level of confidence. The correlation coefficient
between carcass Specific gravity and serum cholesterol con-
centration of 55 rats was -0.42, significant at the 5 per
cent level. This indicates that serum cholesterol concen-
tration is higher in fatter animals, as a low Specific
gravity is indicative of a high percentage of body fat.

Thomas and Garn,13 testing 159 male medical students,
found no significant relationship between serum cholesterol
and body weight. They also correlated serum cholesterol
with body weight/chest breadth and with lower thoracic fat.

Again there were no significant relationships.
II. Specific Gravityas an Estimate of Body Eat

The low density of fat as compared to other body
components logically indicates that there should be an in-
verse relationship between specific gravity and fat content
of the carcass. Liuzzo and co-workers14 reported a -0.99
correlation between carcass specific gravity and direct
chemical analysis of total body fat in rats. Da Costa and

Clayton15 also reported a significant correlation, -0.65, in

 

13C. B. Thomas and S. M. Garn, "Degree of Obesity
and Serum Cholesterol Level," Science, 151 (1960), 42.

14A. Liuzzo, E. P. Reineke and A. M. Pearson, "
"Determination of Specific Gravity by Air Displacement,
Journal gf_Animal Science, 17 (1958), 579.

15E. Da Costa and R. Clayton, "Studies of Dietary
Restriction and Rehabilitation. II. Interrelationships Among
Fat, Water Content and Specific Gravity of the Total Carcass
of the Albino hat," Journal 2: Nutrition, 41 (1950), 605.

 

    

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1:: fig (1 ‘3; f‘ . "' k 'w‘
= 1' .C. .._ 7 K.
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12
the albino rat. They concluded that the regression lines
obtained make indirect determination of fat possible when
specific gravity has been determined. Their work also
indicates that regardless of chronic pathological conditions
affecting the body fat, this relationship still holds true.
Similar studies with cattle16 and guinea pigs17 have also

shown this inverse relationship.

 

16H. F. Kraybill, H. L. Bitter and 0. G. Hankins,
"Body Composition of Cattle. II. Determination of Fat and
Water Content from Measurement of Body Specific Gravity,"
Journal 3: Applied Physiology, 4 (1952), 579.

17E. N. Rathbun and N. Pace, "Studies on Body Compo-
sition. II. The Determination of Total Body Fat by Means of
the Body Specific Gravity," Journal 2: Biological Chemistgy,
158 (1945), 667.

  
  

 

 

 

 

 

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.. Av"'

 

 

 

CHAPTER Iii

PRCCiDURE

General Experimental Design

 

In order to test for the relative effects of
exercise and weight control in controlling serum cholesterol,
eighty-eight mature male albino rats (Carworth) were di- 1

vided into four groups as follows:

Group A An initial control group, sacrificed f
at the beginning of the experiment. I
Group S = A control group, sedentary (con—

fined to small individual cages)

fed 3d libitum, maintained as such

for fifteen weeks.

a
I

Group _ The experimental group, confined
to the small cages, fed E9 libitum,
and subjected to fifteen weeks of
vigorous exercise.
Group D = A second experimental group,
sedentary (confined to cages), no
exercise, and feeding restricted
carefully so that body weight re-
mained comparable to exercise
animals during the fifteen-week
period.
Body weights for the two experimental groups (B and
D) and control group (S) were recorded bi-weekly and 18 t 5

l3

 

14

hours prior to sacrifice. Blood samples were taken at the
beginning of the fifteen-week experimental period and im-
mediately prior to sacrifice to be analyzed for total serum
cholesterol. Carcass specific gravities, determined by
under-water weighing, were used as an estimate of total body

fat.

Methodology

Under light ether anesthesia, a control blood sample

of 4 to 5 ml was withdrawn frOm the orbital sinus1

of each
of the 88 rats (refer to figure 1, page 15). At the time of
sampling, the animals' body weights ranged from 264.0 to
579.2 grams, with a mean weight of 526.8 grams. The samples
were allowed sufficient time to clot, after which they were
centrifuged for fifteen minutes at medium speed. The serum
samples were transferred to vials which were held in frozen
storage for subsequent cholesterol analysis.

After a recovery period of four days, the animals
were divided into four groups equated on the basis of the
body weights recorded at the time the control blood samples
were taken. One group of twenty (referred to as Group A)
was sacrificed immediately. The other three groups, two
consisting of twenty-three rats each and the third, twenty-
two (extras in case of deaths) were maintained throughout

the fifteen-week experimental period..

13. H. Stone, "Method for Obtaining Venous Blood
from the Orbital Sinus of the fiat or House," Science, 119
(1954), 100. —"

 

Blood Sampling Techni

I

f I .' FIGURE 1 /

 

 

 

 

 

 

 

 

16

Bach animal in Group A was weighed. In order to
reduce the amount of food and waste material in the gastro-
intestinal tract, food was withheld for 18 :ls hours prior
to the time of sacrifice. This was deemed essential to
standardized and accurate carcass specific gravity deter-
minations. Nollowing this fast, the animals were killed by
injecting 1 m1 of 5 per cent pentobarbital sodium. While
this injection was taking effect, the electric clippers were
used to remove as much of the animal's hair as possible.
This, again was for the purpose of obtaining more accurate
‘carcass specific gravity measures, as the hair tends to en-
trap air and thereby increase the bouyancy of the carcass.

The heart, thymus, liver, kidney, adrenals, testes
and spleen were removed to furnish the data for a separate
study of the effects of exercise on organ weights. The
carcass was weighed in air and then washed carefully in
detergent ("Blue Cheer") to remove any skin oil which might
affect under-water weight. The carcass was then weighted
with a sinker of known weight and weighed under water. The
temperature of the water was recorded to make corrections in
the calculation of Specific gravity. The carcass Specific

gravity was calculated as follows:

 

Wt. of dry carcass (in air) Temperature
fit. of dry carcass - fit. of Correction
carcass under water sector

The remaining three groups were kept in individual
wire mesh cages, 18 x 16.5 x 24 cm, where the room tempera-

ture was maintained at 21 - 25°C. There was no humidity

 

17

control. Cages were rotated weekly, from top to bottom of
the cage rack.

The twenty-three sedentary animals (Group S, the
control group) were confined to the cages for the entire
fifteen week period. They were fed ad libitum. All groups
were fed a stock diet2 which supplied all of the known
essential nutrients for the rat. (For composition of diet,
see Appendix B.)

The twenty-three experimental exercise animals
(Group B) were also confined to the cages and fed ad libitum,
but they were subjected to fifteen weeks of exercise. The
exercise progressed from an easy five minute swim with no
weights attached, to sixty minutes swimming twice daily,
with 5.5 per cent of the animal's body weight attached to
its tail. The weight was attached as close to the rat's
body as possible by means of a strip of adhesive tape. ihe

entire exercise program was as follows:

First Eour Weeks - Progressed from 5' daily
, to 60' daily

Fifth Week - Swam 60' daily

Sixth & Seventh Veeks - Added 2% of body weight,

progressed from 10' daily
to 60' daily

Eighth Week - 2%. 60' twice daily

2Wayne Laboratory Diets, "Allied Mills, Inc.,
Laboratory Diets Division, Chicago, Illinois.

 

18

Ninth Week - Change to 5 1/2%, 60'
twice daily

- Tried 5%, 2 animals
drowned

Final Six weeks - Changed back to 5 1/2%,
continued 60' twice daily

The animals swam in a separate room in approximately

50 cm of water maintained at 55-5700. They were swum simul-

 

taneously, half in each of two tanks, 60 x 60 cm. Room
temperature in the swimming room was maintained at 51-5200.

during swimming. After swimming, the animals were dried and

 

returned to their respective cages.

‘ in”. .- nun-«mp
,

A second experimental group, Group D, consisting of
twenty-two animals, was maintained in the restrictive cages
for the entire fifteen-week period, just as the control
group, with one exception. By means of food restriction an
attempt was made to maintain the mean weight for this group
as low as that of the exercise group. This was done by
pairing each rat in Group D with a Group E rat of like
weight at the beginning of the experimental period. Weights
were recorded every two weeks, the pairs compared and the
food allowance for each Group D animal for the next two
weeks based on whether it was heavier or lighter than its
mate. Individual differences and unpredictable weight gain
patterns made this a difficult task. Although it was im-
possible to maintain each pair at the sam weight throughout,
the Group D mean weight was controlled quite nicely so that

it was not significantly different from the exercise group.

Fh.v~.

Vm- 2‘ ”-J'QI-F I; a m

 

 

 

   

fps-e.

, i ’ /’
‘;Q 11"" I
A, J . ‘ ‘
- V i“ ~

HIP-

 

19
A Sample of the records kept might best illustrate the
rather tedious method of weight control utilized. (See
Appendix A.)

In the final four weeks, weekly comparisons were
made in order to bring the mean weights of the two groups as
close together as possible. The minimum ration for the
Group D rats was 5.8 cm of Lab-Blox (6 grams). A check of

3 for rate and the

the minimum daily essential nutrients
content of the diet indicated that even at this ration, the
daily essentials were present in sufficient quantity.‘

One rat in Group S became sick and died, reducing
the number in this group to twenty-two. Three exercise rats
drowned during the-course of the experiment, one in the
early weeks before weights were added and two later in the
experiment when 5 per cent was added in all attempt to in-
crease the intensity of the exercise. (See page 18,
exercise program.)

At the end of the fifteen-week experimental period
body weights were recorded and food withheld for 18 i 5
(hours. Following this overnight fast, the animals were
lightly anesthetized, using ether, and 4-5 ml blood samples
again withdrawn from the orbital sinus. The blood samples

were prepared exactly as the control Smnples, and the serum

frozen and stored for later cholesterol analysis. The

 

3J. Q. Griffith and T. J. Farris, The Rat in Labora-
tory Investigation, J. B. Lipoincott 00., Philadelphia,
1942, 97-98.

 

 

w... '- ~.—-.u— -._—— M... xd‘mgfi.’- ’

4"! *1";

 

 

 

 

20

animals were immediately sacrificed and specific gravities
determined following the identical procedure described for
Group A (See page 16).

Total serum cholesterol concentrations were
determined, using the method described by Schoenheimer and

5 and Foldes and Wilson.6

Sperry,4 as modified by Sperry
(See Appendix C for detailed outline of method.) The serum
samples were coded by another person to eliminate any
possibility of bias on the part of the author in carrying
out the cholesterol assay. The assay was carried out in
duplicate, except in a few cases where sufficient serum was
not available. In cases where the second determination did

not agree with the first, a third determination was run in

order to clear up the discrepancy.

4R. Schoenheimer and W. M. Sperry, "A Micro-Deter-
mination for the Determination of Free and Combined Choles-
terol," Journal 2; Biological Chemistry, 106 (1954), 745.

5W. M. Sperry, "The Determination of Cholesterol,"
Journal pf Biological Chemistry, 118 (1957) 577.

6F. F. Foldes and B. C. Wilson, "Determination of
Cholesterol-Adaptation of Schoenheimer and Sperry's Method
to Photoelectric Instruments," Analytical Chemistgy, 22
(1950), 1210.

 

‘ I / 1’, "I ‘ .
1! , -' f7 k [A p “I x.
i !E*ngh'vp , A, _~ / A‘
~ ' r ‘ .. Q Hi. _ ’i ,
.I .‘ '7 _'-I xiii. _ I . ‘ » r.

0

Win

   
 

     

CHAPTER IV
RESULTS AND DISCUSSION
Results

The data were analyzed statistically. Analysis of
variance and analysis of covariance1 were both utilized with
a partitioning of the between means sum of squares for com- I
parisons.2 Students "t" test was used to supplement analysis f“
of variance wherever necessary. The product moment

3

correlation coefficient was employed where applicable. 3

Group means are listed in Table I, page 22, and

presented graphically in Figure 2 on page 29.
Cholesterol

The "t" test indicates that there was no Significant
difference between the final mean cholesterol concentrations
of Group S (96.41 mg%) and Group D (95.52 mg-fé). When the
diet group-~Group D--was compared to Group E (75.10 mg%0 by
analysis of covariance, the difference in mean cholesterols

was Significant (F2 = 10.40).

 

1W. J. Dixon and F. J. Massey, Jr., Introduction to
Statistical Analysis, McGraw-Hill Book Co., Inc., New York?
1957, 2nd Edition, 211-215.

2C. H. Goulden, Methods 2: Statistical Analysis,
John Wiley and Sons, Inc., New York, 1952, 2nd 1 on,
87-88 and 526-527.

3
A. L. Edwards, Statistical Analysis Rinehart and
00., Inc., New York, 195 , - . '

 

 

21

 

TABLE I

SUMMARY OF MEAN VALUES

‘_‘

 

 

 

 

 

 

 

 

Grou 3
Variable A 4306} Diet E5.
) ...... L-fiewl

rinel Wt.(gms) 331.0 475.6 422.2 421.2
‘Spec. Gravity 1.0565 1.0323 1.0256 1.0363
Cont. Chol. 66.55 65.82 69.95 59.25

(mefi)
Final Chol. . . . 96.41 95.52 75.10

mg .
01101. Change 0 o e +30059 +250“ +13085
Wt. Charge 0 o o )+148.2 +9505 +9200

 

‘Significance
of differences

‘.

I

78.
VS.
78.
.786
V3.
V8.
75-

E(ns

D_E*l
D(ns)
S(ns)
s-D*
S vs.
E(ns)
vs. Dgns)
vs. D *

{TC/1UP ENCHU’U '.

 

 

* Sig. at .05 level -

(ns) not significant

** Sig. at .01 level

22

 

 
   
    
 

A

/ I, q - .
I , , . // ’
if: r t ‘3 . ”17, ‘ *’ ‘ r ~ ~‘
-~ e ' - -' -.
- - ' I ‘H. .'.- . v
. “‘ . ‘ 1; .*.r7 if , ‘ " ' I. ‘ .1. ‘2‘- o I) 1"“, . n‘
‘ ‘- -:--.=-a-'_ . _ ‘

g

25

Although the control mean cholesterol concentrations
were not significantly different (P = l.96(ns)), the signifi-
cant positive relationship between control and final choles-
terol levels (+0.41) indicates that the final concentration
was in part determined by the control. Analysis of covari-
ance (see page 24) indicates that the difference between the
Group E and Group D cholesterol concentrations is still
significant after removing the effects of the Control level.
The reduced, but significant "F" ratio (F2 = 5.88) indicates
that there was some significant effect, over and above the
control level effect which contributed to the difference in
final cholesterol concentrations.

When all three Groups (S, D & E) were pooled together,
there was a significant positiye correlation'between control

cholesterol and final cholesterol concentration. Within the

TABLE II

CORRELATION COEFFICIENTS
CONTROL CHOLESTEROL VS. FINAL CHOLESTEROL

 

S,D,E (Pooled) . . . . . . . . . .+ 0.4l**
sedentary O 0 O O O O 0 O O 0 0+ 0040
Diet . . . . . . . . . . . . . .+ 0.41
Exercise . . . . . . . . . . . .+ 0.09

 

**Significant at .01 level

nonexercise groups, the Group S correlation was +0.40 and
the Group D, +0.41. Within Group E, however, the correla-
tion was only +0.09. (None of the group correlations were

significant, due to the smaller N.)

 

TABLE III

ANALYSIS OF COVARIANCE
Y2 FINAL CHOLESTEROL .X= CONTROL CHOLESTEROL(COVARIATE

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Source SSx Sny SSy df MSy "F1"
Between Groups II
S vs. D vs. E 1554.14 2655.54 7158.10 2 5569.00 7.17**
Partitioned A
Comparisons
S vs. D & E . . . . . 1966.56 1 1966.56 5.95(ns)
D vs. E . . . . . 5171.54 1 5171.54 10.40**
Error 12885.66 6875.46 50555.90 61 497.51 . . 0
Total -‘ 14219.80 9529.00 37474.00 55 '.':'IWA. . . '-
Source SSx Sny SSy SSy. df MS "Pg"
8 vs. pa 133.70 161.93 1966.56 1851.05 1 1831.03 4.12*
Error 12885.66 6875.46 50555.90 26667.45 60 444.46 . . .
Total 15019.56 7057.59 52502.46 28498.46 61 . . . . . .
3::s. E 1200.44 2491.61 5171.54 2611.00 1 2611.00 5.88*
Error 12885.66 6875.46 50555.90 26667.45 60 444.46 . . .
Total 14086.10 9567.17 55507.44 29278.45 61 . . . . . .
SSy. = adjusted sum of Squares
"F1" = F ratio unadjusted for control cholesterol
"F2" = F ratio adjusted for control cholesterol

* Sig. at .05 level

Note: "t" for S vs. D = o.15(ns)

24

** Sig. at .01 level

 

   

 

25

Body Weight

The final mean body weights were treated by analysis
of variance. The final mean weight of all three groups

(S,D & E) was significantly greater than that of Group A.

TABLE IV

ANALYSIS OF VARIANCE
FINAL BODY WEIGHT

 

 

Source 88 at ms ‘ "F"

 

Between Groups

A vs. s vs. D vs. E 223460 3 74487 42.47***

 

 

 

Partitioned
Comparisons
A vs. s, D. E 181515 1 181515 103.49*** “
D vs. E 13 1 13 .01(ns)
s vs. D a E 41932 1 41932 23.91***
Error 140286 80 1754 . . . . .
Total 363746 83 . . . . . . . .

 

 

 

 

 

***Sig. at .005 level

The effort to keep the Group D mean weight in line with the
Group E mean was successful [See APPENDIX E for growth chart).
The Group D mean weight of 422.2 grams was not significantly
different‘from the Group E mean of 421.2 grams (F=0.01).

Group S (475.6 grams) was significantly heavier when

compared to E and D.

 

26

Final body weights were correlated with final
cholesterol concentrations. The overall correlation (S. D
& E pooled) was not significant, nor were the correlations

within each group.

TABLE V

CORRELATION COEFFICIENTS
FINAL BODY WEIGHT VS. FINAL CHOLESTEROL

 

 

S,D,E (pooled) .
Sedentary. . .
Diet . . . . .
Exercise . . .

O O O O
O I O O
I O C O
I O I I
O O O O
I O O C
O I O I
O O I O

 

When cholesterol change was correlated with weight
change, neither the pooled nor the individual group

correlations were significant.

TABLE VI

CORRELATION COEFFICIENTS
CHOLESTEROL CHANGE VS. WEIGHT CHANGE

 

 

S,D.E (pooled) . . . . . . . . . + O.
Sedentary . . . . . . . . . . + 0.18
Diet 0 O I O I I O I O O O I I + 0012
Exercise 0 o o o o o o u o c o - 0035

 

Fat, as Estimated by Carcass Specific Gravity

The mean carcass specific gravities are listed in

Table I. These Specific gravities are of the same general

 

27

magnitude as those reported by Montoya and co-workers4
(exercise rats = 1.0429 and sedentary rate = 1.0556). They
are lower than those obtained by Montoye and co-workers5 in

a later study (exercise rats = 1.0761 and sedentary rats =
1.0640). This apparent inconsistency can be explained by the
fact that in both the present study and the first-mentioned
study, the animals at sacrifice were approximately ten weeks
older than in the later study which reported higher values.
There is evidence that in this period of time, a considerable
increase in percentage body fat takes place.6’7

The analysis of variance for specific gravities

indicates that the nonexercise animals (Group S= 1.0525 and
Group D: 1.0256) were not significantly different in body
density. They were significantly less dense (fatter) than

Group E, which had a mean Specific gravity of 1.0565. When

analyzed by the "t" test, Group E was not significantly

 

4H. J. Montoya, et al., "Effects of Exercise on
Endurance and Organ Growth in Rats," Accepted for publi-
cation by Research Quarterly.

5H. J. Hontoye, et al., Unpublished data, Michigan
State University, Department of Health, Physical Education
and Recreation, 1959.

 

6M. C. Conrad and A. T. Miller, "Age Changes in Body
Size, Body Composition and Basal Metabolism," American
Journal 2; Physiology, 186 (1956), 209.

7M. W. Marshall, at al., "Effect of Dietary Fats and
Carbohydrates on DigestibiIIty of Nitrogen and Energy Supply,
and on Growth, Body Composition and Serum Cholesterol of
Rats," Journal 2f_Nutrition, 69 (1959), 571.

 

 

 

denser than Group S. All three groups (S, D & E)were

 

 

significantly fatter (less dense) than Group A (1.0565), the
initial control group sacrificed at the beginning of the
experiment. This is in agreement with previous work which

indicates that the percentage of body fat increases with

 

 

 

 

 

 

 

 

 

 

 

 

 

age.
TABLE VII
ANALYSIS or VARIANCE
CARCASS SPECIFIC GRAVITY
Source SS df MS "F"
IBetween Groups‘ I:
A vs. s vs. D vs. E 10943.26 3 5647.75 26.86**
Partitioned III-A
Comparisons
s vs. D 484.54 1 484.54 5.57(ns)
3 vs. s a D 732.20 1 1 732.20 5.39*
A vs. E, s, D 9727.26 1 9727.26 71.62**
Error 10865.75 80 155.82 . . . .
LITotel ‘ 21809.75 83 . . . . . . .1:
*Sig. at .025 level **Sig. at .005 level

Note: "t" for E vs. S = l.15(ns)

 

81bid.

9Marshall, at al., 22, cit.

 

29

 

 

 

 

 

 

 

 

 

 

 

 

SOO -

300-

100.1

 

 

A S D

Initial weight I

 

   

 

 

 

 

 

 

 

 

 

 

100

60.

20.

 

 

 

 

 

 

 

 

 

1.062 ‘

 

1.60 ‘

1.025 -

 

 

 

 

A s ' D
Carcass Specific Gravity

COMPARISCN 0F MEAN VALUES
FIGURE 2

Weight in grams

Cholesterol in m

Specific Gravity

:1. “(‘52—

u ‘

 

5O

Carcass specific gravity was correlated with final
cholesterol to test the possibility that cholesterol con-
centration is associated with percentage body fat. None of

the correlations were significant.

TABLE VIII

CORRELATION COEFEICIENTS
CARCASS SPECIFIC GRAVITY VS. FINAL CHOLESTEROL

 

 

S,D,E (Pooled) . . . . . . . . 0.00
Sedentary . . . . . . . . . + 0.10
Diet . . . . . . . . . . . . + 0.26
Exercise . . . . . . . . . . + 0.07

 

Discussion of Results
Cholesterol

The basic question involved in the present
investigation, i.e., whether diet would be as effective as
regular exercise in controlling—serum cholesterbl concentra-
tion, can be answered with reasonable certainty. The diet
group, in spIte of having a final mean weight equal to the
exercise group, had a significantly higher cholesterol con-
centration (95.25 mg% as compared to 75.10 mg%). This
difference was still significant even after removing the
contributing effect of the control cholesterol levels.

It is also important to take note of the fact that
the diet group, in spite of being significantly lighter than

the sedentary group, did not have a significantly lower mean

 

51

cholesterol concentration. This tends to support the
evidence which indicates that cholesterol levels increase
with age.10'11’12'15

It is not surprising that the exercise group's mean
cholesterol concentration (75.10 mg%) was significantly
lower than the sedentary group (96.41 mg%). This is in
agreement with the work done by Montoye and c0-workers,14.
where the reported means for exercise and sedentary groups
were 74.72 mg% and 93.08 mg%.

It is interesting to note that, after 15 weeks of
three very different types of living among the three groups,
there was still an overall significant positive relationship
between the control cholesterol concentration and the final

concentration. This lends support to the theory that

cholesterol concentration is somehow predetermined and is

 

10A. Keys, et al., "The Trend of Serum Cholesterol
Levels With Age," Lancet, 265 (1952), 209.

 

11L. A. Lewis, et al., Serum Lipid Levels in Normal
Persons," Circulation, 16 (I957), 256, 244.

12W. M. Sperry and M. Webb, "The Effect of Increas-
ing Age on Serum Cholesterol Concentration," Journal of
Biological Chemistry, 187 (1950), 110.

 

13Marshall, at al., _p, cit.

14H. J. Montoya, et al., Unpublished data, Michigan
State University, Department of Health, Physical Education
and Recreation, 1959.

 

 

‘1' “- 41“ .—

 

 

 

 

 

52
15,16

prObably even inherited. The very low, insignificant
relationship within the exercise group may be an indication
that regular exercise is effective in altering this pattern

of seemingly predetermined blood cholesterol concentration.
Body Weight

Although the exercise and diet groups were equal in
mean weight, and were significantly lighter than the seden-
tary group, there was no significant relationship between
body weight and cholesterol concentration to explain the
obtained differences in mean cholesterol concentration.

This is in agreement with the evidence reported by Thomas and

17 and Lewis and co-workers.18

Garn
The lack of a significant relationship between
cholesterol change and weight change is seemingly contra-

dictory to the work done by Montoya and co-worhers19 and

 

159. M. Sperry, "The Concentration of Total Cholesterol
in the blood Serum," Journal of Biological Chemistry, 117
(1957), 595.

16J. Groen, et al., "The Influence of Nutrition,
Individuality and Some Other Factors, including Various
Forms of Stress, on the Serum Cholesterol; An Experiment of
Nine-L'onth' 8 Duration in 60 Normal Human Volunteers,"
Voeding, 15 (1952), 556.

170. B. Thomas and S. M. Garn, "Degree of Obesity and
Serum Cholesterol Level," Science, 151 (1960), 42.

 

18Lewis, et al., pp, cit.

19H. J. Iontoye et al., "The Effects of Exercise on
Blood Cholesterol in MiddIe -Aged Men, " American Journal of
Clinical Nutrition, " 7 (1959), 144.

 

 

 

 

 

55

others.20'2l'22

However, it should be remembered that the
conditions of the present investigation and the conditions
of the above mentioned studies were not actually comparable.
The studies referred to involved mature, full-grown humans,
capable of increasing 23 decreasing weight by means of
caloric inbalance. The present animal investigation is
complicated by one very important difference. Whereas these
animals were also mature, the only way weight loss could
have taken place would have been through some form of semi-
starvation. This would have further complicated the issue.
So, where the human studies are involved in weight reduc-
tion and weight gain as related to cholesterol reduction or
increase, the present animal study could only be involved in
controlling weight gain and controlling cholesterol increase.
It should also be pointed out that the weight control in—
volved here was gradual, taking place over a period of 15
weeks, and not a case of rapid weight loss as is often the
case in the human studies. We would not expect, therefore,

to obtain similar results from two such different situations.

20G. V. Mann, et al., "Exercise in the Disposition of
Dietary Calories," New Englang Journal 2: Medicine, 255
(1955), 555.

,21J. T. Anderson, F. Grande and A. Keys, "Serum
Cholesterol Concentration of hen in Semi-Starvation and in
Refeeding," Federation Proceedingg, 14 (1955), 426.

 

 

 

22J. T. Anderson, A. Lawler and A. Keys, "Weight Gain
from Simple Overeating. II. Serum Lipids and Blood Volume,"
Journal 2: Clinical Investigatigg, 56 (1957), 87.

 

 

 

Due to the insignificant relationship it is

concluded that control of weight gain did not account for
differences in cholesterol level. The fact that the exer-
cise group was significantly lighter than the sedentary
group cannot be said to account for the fact that their mean

cholesterol concentration also was significantly lower.
Eat, as Estimated by Carcass Specific Gravity

One would expect to find exercise animals leaner
(more dense) than nonexercise animals. Such was the case in
the present study when the exercise group was compared to
the sedentary groups (S and D). The diet and sedentary
groups, in spite of a significant difference in weight, were
223 significantly different in body density. This is an
obvious contradiction to the logical assumption that seden-
tary animals fed 3Q libitum should be fatter because they
presumably eat more and gain more weight. However, there is
no assurance that differences in weight must be fat, for
several reasons. First, it is impossible to know the amount
eaten by the sedentary animals, as they were fed 3g libitum.
It is not entirely impossible that they ate no more than the
diet animals. It is generally recognized that rats, unlixe
man, are more obedient to their homeostatic "built-in hunger
control." And, for this reason, being inactive does not in

itself cause obesity.23 Second, although every precaution

 

23A. C. Guyton, Textbook of Medical Physiology. 7° 3-

Saunders 00., Philadelphflia, 19'5"'8,""837.

 

 

 

 

 

 

55

was taken to insure that the diet rats' minimum daily rations
contained all the ingredients necessary for normal growth,
the constant caloric restriction may have had some effect on
skeletal and, or, muscle development. Third, hunger being a
form of stress, the poorly understood hormonal phenomena
involved in stress may have had some effect here. Increased
fat deposition in the diet group may have taken place in
response to stress. Theoretically, this might occur through
an increased pituitary adrenocortotrophic secretion and its
resulting effect of increased glucocorticoid secretion by the
adrenal cortex.24

Even though the exercise group was more dense
(1.0565) than the sedentary group (1.0525), the lack of a
statistically significant difference here was unexpected.
Of course, the acceptance of the null hypothesis makes this
result inconclusive. It cannot be said with reasonable
certainty that there was a difference, but neither can it be
concluded for certain that pg difference exists. If there
truly was no difference, the one plausible explanation is
that the vigorous forced exercise was a stress and may have
retarded skeletal and, or muscle development in the exercise
rats to some extent. This, then, would mean that the big
difference in body weight was not all due to excess fat.

In spite of the significant specific gravity

difference between the exercise group and the nonexercise

 

24Ibid., p. 902-903.

 

 

 

 

. ‘ _ / h /” . “
‘ I . ~ ‘ P74 L ‘ A) ‘ “I
£4 m ,l m u I“, . -
at); 4'.- ' .7: -':}“.;
0 fi . ’~ ‘ ‘ - . Z) l '3' I . ‘
.. 1-1- __ 0. <0 -‘ . . _. ‘ 94's....- . I _fl 5 ‘
A“ in» h . _ _._ . ‘- _. “- __ s. . :-__... t" :a— -' a I. Le: i. "I A}! . “.1, i ‘

   

56

animals, no significant correlation between specific gravity

and cholesterol concentration was found. This agrees with
the work of Thomas and Garn,:a5 Gofman and J0nes,26

and Keys,27

whose indirect fat measures in humans were not
significantly correlated with blood cholesterol level. One
case of disagreement, however, is with the results of Montoya
and co-workers.28 They reported a significant correlation
of -0.42. This is a difficult contradiction to explain, un-
less the previously discussed diet, caloric restriction and
stress factors somehow affected the results.

At any rate, it cannot be concluded that differences
in body fat account for the obtained significant differences

in mean cholesterol levels.

 

25Thomas and Garn, gp.cit.

25a. w. Gofman and H. B. Jones, "Obesity, Fat
Metabolism and Cardiovascular Disease," Circulation, 5 (1952),
515.

 

27A. Keys, at al., "Studies on Serum Cholesterol and

Other Characteristics of Clinically Healthy Men in Naples,"
Archives gf Internal Medicine, 95 (1954), 552.

28H. J. Montoya, et al., Unpublished data, Michigan
State University, Departmanf of health, Physical Education
and necreation, 1959.

 

 

CHAPTER V

SUMMARY AND CONCLUSIONS

Summary

In an effort to evaluate the relative effects of
regular exercise and diet in controlling total blood choles-
terol level, blood samples were taken from eighty-eight
mature Carworth male albino rats for serum cholesterol assay.
- The animals were then divided into four groups equated on the
basis of body weights. One group, an initial control group
(Group A) was sacrificed immediately. The other three were
maintained for a fifteen-week experimental period. One
group (Group S) was sedentary and fed 2g libitum. One ex-
perimental group (Group E) was exercised regularly and fed .
2g libitum. A second experimental group (Group D) was seden-
tary, but maintained at a mean body weight comparable to
that of Group E by careful diet restriction.

In addition to determining whether weight control by
caloric restriction is as effective as exercise in control-
ling blood cholesterol, the experiment was designed to test
whether differences in body weight and body fat could account
for any differences in blood cholesterol.

Control and final total blood cholesterol
concentrations and final carcass Specific gravities, using
the under-water weighing method, were determined. Control

and final body weights also were recorded.

57

 

f

 

 

(
(J

 

58

The data were analyzed statistically, employing
analysis of variance, analysis of covariance, students "t",
and product-moment correlation coefficients.

After considering the results and their statistical
inferences, the conclusions in the following section were

taken from the more general discussion of results.

Conclusions

 

l. Caloric restriction was not as effective in
controlling total blood cholesterol as was regular exercise.
rhe mean cholesterol difference between the diet group and
exercise group was significant even after removing the
effects of the control cholesterol concentrations.

2. The significant positive correlation between
control and final cholesterol concentrations strengthens the
theory that there is some predetermined, probably inherent
factor which controls cholesterol concentration. However,
this relationship did not hold true within the exercise group.
It is possible, then, that exercise is a factor which can
alter this seemingly predetermined cholesterol concentration.

3. Although the sedentary group was significantly
heavier than the diet and exercise groups, body weight was
not significantly correlated with final cholesterol concen-
tration. Therefore it cannot be concluded that high blood
cholesterol and high body weight are associated.

4. Although the nonexercise animals were

significantly less dense than the exercise animals, specific

 

.E:.W— 3.: '. _g'.i _.J..:-l_- a?
I
)- l

 

 

 

 

 

 

I

 

39

gravity was not significantly correlated with cholesterol
concentration. Hence, it cannot be concluded that high blood
cholesterols are associated with high percentage of body fat.

5. There was no significant relationship between
cholesterol change and weight change, which is not surpris-
ing in view of the lack of relationship between weight and
cholesterol concentration.

6. The results seem to support the hypothesis that
over and above its effect on weight and obesity, there is
some effect of exercise which helps control blood choles-

1

terol concentration. This lends credence to Mann's con-

tention that "exercise is likely to have a biochemical

relation to lipid metabolism."

Recommendations for Further Stugy

 

The evidence indicates that exercise has some direct
effect on blood cholesterol. The next step, if a repeat of
the present study confirms these results, is to investigate
the exact means by which this effect is exerted. In any
subsequent work, it is recommended that a careful check of
food intake be maintained to add to the control of the study.
It would also be extremely valuable to make periodic checks
to assure normal and equal growth (skeletal and muscular) in
each group.

10. v. Mann, et al., "Exercise in the Disposition of
Dietary Calories," New England Journal of Medicine, 253
(1955), 549. ""‘"""

 

 

 

“aL ‘1 _

fill}
~ . .
‘, .
- rdr unr—

 

 

 

 

 

The next step might be to use animals which have

reached a weight gain "plateau." Groups could then be
equated on the basis of control cholesterol concentrations
and one group sacrificed to give an estimate of control
specific gravity. The experiment could then be concerned
with weight reduction and cholesterol decrease and more
closely parallel work with humans.

There is a need for investigating in more detail the
specific gravity results. These results, if confirmed, mean
revising our present beliefs concerning diet, exercise and
body fat. There are several interesting possible explana-
tions, as mentioned in the discussion. However, the first
logical step is to confirm or refute these results. As part
of the overall project, the carcasses were placed in frozen
storage for this purpose. If the direct carcass fat assess-
ments, when completed, confirm the results of the present
study, we can proceed in further investigating this new
concept.

fhe need for investigating the effects of strenuous
exercise on growth has also been pointed up. It is impor-
tant to know the point beyond which exercise becomes too
strenuous to permit normal skeletal and muscular growth.
This is important in ways other than its relationship to
blood cholesterol, and could be very conveniently studied

with rats.

 

 

 

 

 

 

B IBL I (ERAPHY

 

 

“a

“a

 

BIBLIOGRAPHY

Books
Dixon, V. J., and Hassey, F. J., Introduction to Statistical
Analysis. Sew York: McGraw-Hill Book 00., Inc., 1957:

Edwards, A. L., Statistical Analysis. New York: Rinehart
and CO. , Inc- , 19560

 

Goulden, C. H., Methods of Statistical Analysis. New York:
John Wiley and Sons, Inc., 1952.

Griffith, J. Q. and Farris, E. J. (editors). The Rat in
Laboratory Investigation. Philadelphia: J.
Lippincott 00., 1942.

Groen, J., and van der Heide, R. M. Atherosclerosis and

Coroner Thrombosis. Translated by Johan Winsser.
fiotterdam: WYT, I§5 6.

Guyton, A. C. Textbook of Medical Physiology. Philadelphia:
W. B. Saunders Co., 1958.

Pamphlet

Wayne Laboratory Diets. Allied Mills, Inc., Laboratory Diets
Division, Chicago, Illinois.

Articles

Anderson, J. T., Grands, T. and Keys. A. "Serum Cholesterol
Concentration of Men in Semi-Starvation and in Refeed-
ing," Federation Proceedings, 14 (1955), 426.

 

Anderson, J. T., Lawler, A. and Keys, A. "Weight Gain from
Simple Overeating. II. Serum Lipids and Blood Volume."
Journal of Clinical Investigation, 36 (1957), 81-88.

Brown, C. 3., et al., "Observations on Blood Vessels and
Exercise." Journal of Gerontology, 11 (1956), 292-297.

 

Chailley-Bert, Labignette, P.. and Fabre-Chevalier. "Contri-
bution a 1' etude des variations du cholesterol sanguin
au cours des activites physiques," Presse Med.. 63
(1955). 415. M

42

 

 

 

/ ’ ' '
_ / ~~
I - \
.- ‘-\.~

 

43

Conrad, N. G., and Miller, A. T., "Age Changes in Body Size,
Body Composition and Basal Metabolism," American Journal
of Physiology, 186 (1956), 207-210.

 

 

DaCosta, 3., and Clayton, R., "Studies of Dietary Restriction
and Rehabilitation. II. Interrelationships Among Fat,
Water Content and Specific Gravity of the Total Carcass
of the Albino Rat," Journal of Nutrition, 41 (1950),
597-605.

 

Foldes, F. T., and Wilson, B. 0., "Determination of Choles-
terol-Adaptation of Schoenheimer and Sperry's Method to
Photoelectric Instruments," Analytical Chemistgy, 22
(1950). 1210.

Gofman, J. W. et al., (Technical Group), Andrus, E. G.,
et al., (Committee on LipOproteins, etc.), "Evaluation
of Serum LipOprotein and Cholesterol Measurements as
Predictors of Clinical Complications of Atherosclerosis,"
Circulation, 14 (1956), 691-741.

Gofman, J. W., and Jones, E. B., "Obesity, Fat Metabolism
and Cardiovascular Disease," Circulation, 5 (1952),
514-517.

 

Groen, J., et al., "The Influence of Nutrition, Individuality
and Some Other Factors, Including Various Forms of
Stress, on the Serum Cholesterol; An Experiment of Nine-
Month's Duration in 60 Normal Human Volunteers,"
Voedigg, 13 (1952), 556-

Keys, A., et al., "Physical Activity and the Diet in Pepu-
lations Differing in Serum Cholesterol," Journal of
Clinical Investigation, 35 (1956), 1173-1181.

 

Keys, A., et al., "The Trend of Serum Cholesterol Levels
With ng," Lancet, 263 (1952), 209-210.

Keys, A., et al., "Studies on Serum Cholesterol and Other
Characteristics of Clinically Healthy Men in Naples,"
Archives of Internal Medicine, 93 (1954), 328-336.

Kraybill, H. F., Bitter, H. L., and Hankins, O. G., "Body
Composition of Cattle. II. Determination of Fat and
Water Content from Measurement of Body Specific
Gravity," Journal of Applied Physiology, 4 (1952),
575-583.

Lewis, L. A., et al., "Serum Lipid Levels in Normal Persons,"
Circulation, I6 (1957), 227-245.

 

 

44

Liuzzo, J. A., Reineke, 3. P., and Pearson, A. M., "Determi-
nation of Specific Gravity by Air Displacement," Journal
of Animal Science, 17 (1958), 513-520.

Mann, G. V., Nicol, B. M., and Stare, F. J., "The Beta-Lipo-
protein and Cholesterol Concentrations in Sera of
Nigerians," British Medical Journal, 2 (1955), 1008-
1010.

Mann, G. V., "Diet, Exercise and Coronary Disease," Illinois
Medical Journal, 116 (1959), 20-21.

 

Fann, G. V., et al., "Exercise in the Disposition of Dietary
Calories," New England Journal of Medicine, 253 (1955).
349-355.

Marshall, M. w., et al., "Effect of Dietary Fats and Carbo-
hydrates on Digestibility of Nitrogen and Energy Supply,
and on Growth, Body Composition and Serum.Cholesterol
of Rats," Journal of Nutrition, 69 (1959), 371-382.

Montoye, H. J., et al., "The Effects of Exercise on Blood
Cholesterol In Middle-Aged Men," American Journal of
Clinical Nutrition, 7 (1959), 139-145.

Myasnikov, A. L., "Influence of Some Factors on Development
of Experimental Cholesterol Atherosclerosis," Arcula-
tion, 17 (1958), 99-113.

Page, I. H., "Atherosclerosis--A Commentary," Federal Pro-
ceedings, 18 (1959), 47-51.

 

Rathbun, E. N., and Pace, N., "Studies on Body Composition.
1. The Determination of Total Body Fat by Means of the
Body Specific Gravity," Journal of Biological Chemistry,
158 (1945), 667-676.

Schoenheimer, H., and Sperry, W. M., "A Micro-Method for the
Determination of Free and Combined Cholesterol," Journal
of Biological Chemistgy, 106 (1934), 745-760.

 

Sperry, W. N., "The Concentration of Total Cholesterol in the
Blood Serum," Journal of Biological Chemistgy, 117
(1937), 591-395.

Sperry, W. M., and Webb, M., "The Effect of Increasing Age
on Serum Cholesterol Concentration," Journal of Bio-
logical Chemistryg 187 (1950), 107-110.

 

Sperry, w. M., "The Determination of Cholesterol,” Journal of
Biological Chemistqy, 118 (1937), 377-389.

 

 

45

Stone, S. H., "Method for Obtaining Venous Blood from the
Orbital Sinus of the Rat or Mouse," Science, 119 (1954),

100.

Taylor, H. J., Anderson, J. T., and Keys, A., "Effect on
Serum Lipids of 1300 Calories Daily Walking," Feder-
ation Proceedingg, 16 (1957), 128.

Taylor, H. J., Anderson, J. T., and keys, A., "Physical
Activity, Serum Cholesterol and Other Lipids in Man,"
Society for Experimental Biology_and Medicine Proceed-

5

iggs, (19577, 383.

Thomas, C. B., and Garn, S. M., "Degree of Obesity and Serum
Cholesterol Level," Science, 131 (1960), 42.

Wong, H. Y. C., "Hypocholesterolizing Effect of Exercise on
Cholesterol-Fed Cockerels," Federation Proceedings,
16 (1957), 138.

Unpublished Material

Montoya, H. J., et al., "Effects of Exercise on Endurance
and Organ Growth in Rats," Accepted for Publication by
Research Quarteriy.

Montoya, H. J., et al., Unpublished Data from Michigan State
University, Department of Health, Physical Education
and Recreation, Human Energy Research Laboratory.

 

APPEND IX

 

_...L‘a——_-—w

APPENDIX A

SAMPLE OF DIET GROUP RATION CALCULATION

_i_ Difference
14 +12
64 -22
15 +10
80 - 6
53 +12
89 -33
13 + 6
99 +15
4 -44
49 + 7
41 +12

5 + 4
26 0
75 + 4
72 - 4

1 +12
76 + 4
47 -15
68 - 8
86 +14
46 + 6

9 +10

1

1

Last R

2
AL
4
2.5
3.5
AL

9“
mohon-PUIU‘HPNUI

H
O

ation

2

Loss
Gain

c——————-—..

- 5
+ 7

-13

2Last ration: daily ration last week.
inches of Lab-Blox, one inch: aporoximately four grams

of food.

gr

2
AL
2.5
3.5
2.5
AL
2
3.5
AL
4

3
3.5
AL

 

New Ration

Difference: weight difference from Group E paired mate.

Figures are in

5Loss or gain: weight lost or gained since last weighing.

The new ration was based on the last daily ration,
weight lost or gained on that ration, and the weight
difference from the animal's paired mate.

AL: ad libitum

47

the

 

 

COMPOSITION OF DIET

Protein (min)
Fat (min)
Fiber (min)
Ash

Calcium
Phosphorous
Sodium
Potassium
Chlorine
Magnesium
Iron

Copper
Maganese

Zinc

Cobalt

Iodine
Thiamine
Riboflavin
Niacin
Calcium Pantothsnate
Choline
Vitamins E
Carotene
Vitamins A
Vitamins D

APPENDIX B

-. six-‘\‘~.A-\.~_-‘.n
o§o*l.c~l.olo<.c-o\o§o<‘.o§

PPM
PPM
PPM
PPM
PPM
PPM

USP units per 1b.
USP units per 1b.

1

 

24.00
4.00
4.50
8.60
1.30
1.00
0.30
1.00
0.60
0.20

340.00
. 11.00
170.00

30.00
0.60

10.00
6.50
6.50

60.00

18.00

1850.00

35.00
2.00
5600
2000

lWayne Lab-Blox (mice and rats) Allied Mills, Inc.

48

 

 

APPENDIX C

TO”AL CHOLESTEROL DETERMINATION

Reagents

Absolute alchol - Acetone Soln. (1:1)

50% Potassium Hydroxide (5 grams - 10cc water)
Phenolphthalein

0.5% digitonin soln. (500 mg - 100 cc 50$ alchol)

Heat to dissolve

Absolute anhydrous ether

Acetone - absolute anhydrous ether (1:1)
Glacial acetic acid

Con. sulfuric acid

Acetic anhydride

Extraction

1.
2.
3.
4.

5.

Place 3 cc absolute alchol-acetone (1:1) into a 5m1.

vol. flask.

Add 0.4 cc serum to form Ppt.

Bring to boil on steam bath.

0001. Make to volume with abs. alchol-acetone
mixture.

Filter through fat-free filter paper.

Hydrolysis and Precipitation

1.

4.
5.
6.

Washing

1.

2.
3.
4.

Pipette a 1 ml. sample of extract to determine total
cholesterol. (Triplicatss) Place in 5 cc centrifuge
tube.

Add a dr0p of 50p potassium hydroxide and stir.
Hold on water bath (57 to 40°C.) for 45 minutes.
Cool. Add 1 drop of phenolphthalein and titrate
with 105 acetic acid in alcohol. (1 drop excess
acid). Stir.

Add 2 cc of 0.5% alcholic digitonin to each tube.
Stir.

Flocculats, 60°C (one hour). Stir. Remove rods
carefully.

Cover and let stand overnight.

the Precipitatg

Remove stirring rod and place on rack so that the

same rod will return to the same tubs each time
stirring is required.

Centrifuge 15 minutes at a centrifuge setting of 35.
Carefully pour off liquid portion.

Wash with acetone-ether (1:1) (absolute ether)
solution, stirring and then centrifuge for 1C minutes,
and remove liquid by pouring.

49

 

5. Hepeat washing 2 times with absolute anhydrous ether.
5. Dry in sand bath at 110-11500. till no trace of
ether is detected (one hour).

Development of Color

 

l. Pipette 1 cc. glacial acetic acid into centrifuge
tube to dissolve the precipitate. Stir with the
same stirring rod used throughout.

2. 0001 to room temperature.

3. Add 2 ml. freshly prepared cold (use ice bath) acetic
anhydride and sulfuric acid (20:1) solution. Stir.
Read 27 or 30 min. Later in Beckman SpectrOphotometsr
at 620 mu.

Standard Curve

Prepare a series of standards 0.01 to 0.15 mg. choles-
terol per m1. of glacial acetic acid and treat in the
same manner beginning with the deve10pment of color

DTOCGSS.

Plot Optical density us. mg. chol/cc acetic acid.

Calculation

From the reading on the standard curve multiplied by a
factor of 1250 you will get the mg. of cholesterol per
100 cc of blood.

5 ml. (total vol. of extract) X 100 = 1250 (factor)
.4 m1. (Vol. serum) 1

1250 X reading on curve = mg. cholesterol per 100 cc.
of blood.

 

“- ',‘q_upu" '_='

   

APPENDIX D

N = 22
Rat Final1 Carcass Carcass
_y__ wt. Nt.(Dry Wt.(UW)
2 330 . 279.6 14.331
3 343 293.8 12.100
8 318 269.2 12.251
18 302 254.3 13.891
21 285 237.1 11.452
31 300 253.3 16.729
33 337 290.5 17.096
42 296 251.1 14.280
43 300 247.5 15.948
48 360 301.5 20.480
51 325 273.6 15.981
57 324 272.4 18.499
60 346 295.7 19.354
65 323 277.7 15.378
70 309 263.1 16.310
71 342 295.4 19.370
79 382 322.8 19.090
82 355 303.8 8.895
90 373 312.8 11.386
95 370 306.7 14.510
1

RAW DATA- GROUP A

 

Final ‘

 

"7's ight in grams

2 Carcass weight under water

Carcass
sp.cr. 3 0501.4
51 72
41 79
45 4e
55 46
4s 83
69 50
61 72
58 76
67 66
71 7o
60 87
71 66
68 59
57 75
64 51
7o 56
61 59
28 46
56 78
:49 94

3 1.0 is omitted; 1.6., 1.044 is Specific gravity for

rat #6

4 Serum cholesterol in mg%

51

 

 

Hat Init1 Final1 Carcass l Carcassz Carcass3 Control
j:_ Wt. Wt. Wt.(Dry) Vt.(UW) Sp.Gr. Chol.
6 353 598 519.5 22.15 44 58
7 304 467 405.3 11.88 29 90

10 342 520 448.0 19.32 44 68

11 316 425 363.2 16.22 46 55

12 331 447 396.9 14.43 37 65

22 333 494 440.0 10.43 23 112

23 370 554 480.7 13.31 28 49

28 290 446 385.2 14.70 39 70

30 342 482 413.4 21.46 53 42

32 297 460 397.0 15.37 39 54

36 323 462 396.9 15.58 40 80

44 337 539 469.3 10.74 22 75

59 306 434 392.5 6.62 16 77
56 346 494 434.5 11.80 27 65
58 295 451 394.8 11.32 28 56
61 331 466 409.2 15.72 39 62
62 269 375 325.7; 6.92 21 58
63 312 451 393.5 13.88 36 82
66 365 595 524.6 13.95 26 79

73 327 460 417.2 18.02 44 53
74 554 467 409.6- -2.87 -08* 54

87 379 375 325.0 11.90 37 44

1

ZCarcass weight under water

31.0 is omitted; i.e., 1.044 is Specific gravity for $6

Weight in grams

RAJ DATA - GROUP S

= 22

 

*Rat #74 specific gravity = 0.992

4Serum cholesterol in mg%

 

Final
0501.4

Hat

Init

 

 

# Wt.1 Wt.
14 309 366
64 512 400
15 552 455
80 364 462
55 527 438
89 362 465
13 338 421
99 340 425

4 555 452
49 295 591
41 544 422

5 290 425
26 506 407
75 318 398
72 549 495

1 527 459
76 376 462
47 346 442
68 551 455
86 505 571
46 281 553

9 500 406

1.

leight in grams

2Carcass weight under water

53

RAW DATA - GROUP D

Final Carcass

315.0
348.6
380.1
400.5
384.2
411.5
367.0
371.9
372.5
343.1
364.4
363.6
365.8
346.0
428.9
379.0
406.4
388.9
382.8
324.7
298.6
345.8

.(t.(Dry)1 Wt.(UW)2

 

4

 

 

= 22
Carcass Carcass3 Control4 Final
Sp.Gr. Chol. Chol.
6.19 19 64 83
3.07 08 80 103
16.31 44 65 105
9.35 23 43 102
6.58 16 77 58
12.33 30 79 91
8.41 23 57 79
12.64 34 55 56
15.29 42 72 129
8.77 25 72 109
9.09 25 93 143
7.88 21 68 93
9.47 26 53 107
8.75 25 84 81
15.17 36 83 108
12.43 33 70 110
5.32 12 68 83
12.96 34 63 95
10.59 28 122 119
-2.57 -08* 49 86
11.55 39 56 86
9.96 29 66 71

51.0 is omitted; i.e., 1.019 is specific gravity for #14

*Rat #86 specific gravity = 0.992

4

Serum Cholesterol is mg%

fiat Initl
Wt.
16 307
17 304
19 327
24 333
25 340
27 341
29 329
34 337
37 365
38 298
39 333
54 294
67 346
77 338
78 379
84 354
81 333
83 311
85 295
40 320

RAW DATA - GROUP E

 

Finall Carcass
Wt. Wt.(Drz)
364 309.8
429 363.4
402 353.6
438 369.8
427 373.5
484 423.1
430 372.8
415 356.4
476 414.0
377 323.3
416 358.5
392 337.9
491 432.3
428 374.9
452 396.2
458 401.7
436 373.6
365 313.8
353 298.2
390 339.4

1Weight in grams

1

2Carcass weight under water

20

Carcass
Wt.(UW)

12.72
17.90
14.46
14.58
15.12

9.99

5.47
12.07
18.02
13.50

9.62
11.37
17.37
13.96
10.33
12.05
14.98
13.22

9.26
12.77

 

 

 

Carcass Control Final
2 Sp.Gr.3 Chol.4 Chol.4

42 53 85
51 64 51
42 66 46
40 66 64
41 63 49
23 65 59
14 45 44
34 55 5O
45 62 65
43 56 69
28 66 73
34 57 99
41 67 83
38 44 78
26 47 88
30 47 75
41 63 91
43 67 90
31 62 95
38 70 108

31.0 is omitted; i.e., 1.042 is specific gravity for #16

4

Serum cholesterol in mg%

 

om

APPEND IX E

 

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