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ABSTRACT

FOOD INTAKE, BODY WEIGHT GAIN, AND BODY COMPOSITION
OF
THE YOUNG OBESE (ob/ob) MOUSE

BY
PI-YAO LIN

Estimates of food intake and body weight gain were obtained in obese
(ob/ob) mice from 7 to 56 days of age. Milk intake was daily estimated
from 7 to 21 days of age by removing the pups from their mothers' cage for
6 hours, then returning them and measuring their increase in body weight
during the next hour for nursing. There was no difference between obese
and lean mice in milk intake. From 14 to 21 days of age, obese mice gained.
more body weight than lean mice. At 21 days of age, obese mice contained
about twice as much body fat as lean mice did, whereas the body protein con-
tent was not different. Mice were weaned at 21 days of age, and individual-
ly fed a commercial diet or a high-fat diet. During the first several days
after weaning, obese males failed to consume as much food as did the lean
mice. Obese mice consumed equal quantities of food, but gained more body
weight than lean mice from 35-42 days of age for males, or from 28-35 days
of age for females when they were fed the commercial diet. After that, obese
mice consumed more food and gained more weight. Obese mice fed the high-.

fat diet started to eat more food and gain more weight from 28 days of age.

Pi-Yao Lin

At 56 days of age, the carcass of obese mice contained 4-5 times as much
fat as did lean mice, but contained significantly less protein than lean
mice. For the 5 week post-weaning period, obese mice converted about 3-4
times as much dietary energy to body energy as did lean mice, whereas
obese mice consumed only 20-40% more energy. At the same time, obese mice
converted only about 70% as much dietary protein to body protein as did
lean mice. The high-fat diet enhanced the energy efficiency in mice. The
present studies suggest that alterations in energy metabolism, as well as
protein metabolism may play a primary role in the development of obesity

in these mice. Hyperphagia may be of secondary importance.

Key indexing workd: food intake, body weight gain, body composition,
obesity, ob/ob mice.

FOOD INTAKE, BODY WEIGHT GAIN, AND BODY COMPOSITION
OF
THE YOUNG OBESE (ob/ob) MOUSE

BY

PI-YAO LIN

A THESIS

Submitted to

Michigan State University
in partial fulfillment of the requirement
for the degree of

MASTER OF SCIENCE

Department of Food Science and Human Nutrition

1977

ACKNOWLEDGMENTS

The author wishes to express his sincere appreciation to his academic
advisor, Dr. Dale R. Romsos, for guidance and advice throughout this study:
and for aid in preparing this manuscript. He is also indebeted to Dr. Wanda
L. Chenoweth, and Dr. Werner G. Bergen for their suggestions during the
preparation of this manuscript, and for their serving as members of the
graduate guidance committee. Appreciation is also extended to Dr. John L.
Gill for help with statistical analysis.

The author feels deeply grateful to the Tunghai University in Taiwan,
Republic of China for an educational leave, and to the Oberlin Shansi
Memorial Association in Oberlin, Ohio for financial assistance granted to
him during his studies at this University.

My parents, Mr. and Mrs. Ching-Chuang Lin, deserve Special acknowled-
ments for their unending patience, help and understanding during my pro-
gram.

To my wife, Sheng-Hsiang, and my children, Jen-Tzu and Kuang-Hsing,

I give my Special thanks as their love, encouragement and patience made

everything worthwhile.

ii

TABLE OF CONTENT

INTRODUCTION. 0 O O O O O I O O O O O O O O O O O O O O 0 O
I. . Obesity. . . . . . . . . . . . . . . . . . . . .

II. The Genetically Obese Mouse . . . . . . . . . .

III. Effects of Food Intake and Dietary Nutrients on The

Deve10pment of Obesity . . . . . . . . . . .

IV. Objectives . . . . . . . . . . . . . . . . . . .

MATERIMS AND METHODS O O O O O O C . O O O O C C C O C .

RESIJIJTS O O O O O O O O O O O O O O O I O O O O O 0 O O O 0

Milk Intake, Growth Rate, and Body Weight Loss of Mice“

Before Weaning . . . . . . . . . . . . . . .
Body Composition of Mice at 21 Days of Age . . . . .
Food Intake, and Growth of Mice After Weaning . . . .

Fat, Energy, and Protein Retention in Mice from 21

to 56 Days of Age . . . . . . . . . . . . .
DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . .
SUMMARY . . . . . . . . . . . . . . . . . . . . . . . . . .
REFERENCES . . . . . . . . . . . . . . . . . . . . . . . .

APPENDIX. 0 O O O O O O O O O O O O O I O O O O O O I O O 0

iii

LIST OF TABLES

Page

1. ApprOXimate diet CWPOSition 000.00.00.0000.00.0000... 11

2. Milk intake, body weight gain and body weight loss
during fasting inmice 00.00000000000000000000.0000000 1-5

3. Body weight, fat, protein, and energy content of
mice at 21 days Of age 00000000000000.0000000000000000 1-8

4. Effect of diet on body weight gain and food intake

0f mice 000.00.00.0000.000000.000000000000000000000000 20

5. Effect of diet on weekly body weight and food
intake of mice 0000000.0000000.00.00.0.000.000.0000.00 2]-

6. Effect of diet on energy, fat and protein retention
in mice from 21 to 56 days of age..................... 25

iv

LIST OF FIGURES

Figure Page
1. Lactation curve of 15 dams ........................... l3
2. Growth curves of mice before weaning ................. l6
3.

Growth curves of mice after weaning .................. 23

Appendix

I.

II.

III.

IV.

Analysis of
A. For milk
B. For body

C. For body

Analysis of
energy

Analysis of
A. For body

LIST OF APPENDICES

Variance

Variance

intake of mice .

weight loss of mice during a fast

Variance for body weight, fat, protein and
content of mice at 21 days of age.

weight gain of mice from 7 to 20 days of age

weight at 21 days of age in mice for feeding
experiments

B. For body weight gain of mice from 21 to 28 days of age.

C. For food intake of mice from 21 to 28 days of age. .

Analysis of Variance

A.

B.

A.

P'JUOW

For

For

For

For

weekly body weight gain of mice from 28 to 56 days
of age .

weekly food intake of mice from 28 to 56 days of

age .

total food intake of mice from 21 to 56 days of age
total protein intake of mice from 21 to 56 days of

age .

0

, Analysis of Variance

For energy retention of mice from 21 to 56 days of age.

For
For
For

For

energy efficiency of mice from 21 to 56 days of age
fat retention of mice from 21 to 56 days of age.
protein retention of mice from 21 to 56 days of age

protein efficiency of mice from 21 to 56 days of

age .

vi

Page

40
4O
41

42

43

45

46

48

50

INTRODUCTION

I. OBESITY

Obesity, which can be defined as an excessive accumulation of adipose
tissue in the body(l,2), is a common and serious nutritional disease
eSpecially in affluent countries. The prevalence of obesity in children and
adolescents depends upon various socioeconomic conditions. It is estimated
‘that about 2.7% of all school children are obese in England, about 2% in
Sweden, about 8% in Canada, and about 11% in the United States( reviewed
in Ref. 3). The importance of childhood obesity as a forerunner of obesity
in adult life has been documented. Approximately 80% of all obese children
remain so as in adults(2,4). If adults who exceed 20 to 25% body fat are
classified as obese, then, about 15 to 40% of the adult pOpulation in the
United States would be classified as obese(5). The data from various other
countries are based on somewhat different criteria of obesity and thus are
not strictly comparable with the U.S. data. Roughly, 20-40% of the adult
p0pulation in industrial countries suffer this nutrition related problem.

Obesity is, or at least is suSpected to be a risk factor in many
disorders including diabetes mellitus(6) and cardiovascular diseases such
as vascular lesions of the central nervous system, hypertension and coronary
heart disease(6,7). The prominence of stria blood vessels are common in the
grossly obese. A few obese develOp carbon dioxide insensitivity, hypoventi-,

lation, uneven ventilation and somnolence(8). In obesity, there is an

increase in the severity of complications of established diseases as well
as an enhanced risk during surgery and recovery(9). Psychological disorders
may be initiated of enhanced by obesity, and these disorders may alter
metabolic patterns.

Obesity has been classified according to the following mechanisms,
although some overlapping exists, a) regulatory or metabolic(lO-lZ), b)
hyperplastic or hypertrOphic adipocyte growth(10,l3), c) period of onset
as juvenile, mature, or pregnancy(l3,l4), and d) actual stage of obesity
as static or dynamic(13). Regulatory obesity is a result of a primary
impairment in the regulation of food intake; and metabolic obesity is
caused by an inborn or acquired error in the regulation of metabolism in
tissues other than those directly regulating food intake. With reSpect to
the deve10pment of the adipose tissue, hypertrOphic obesity is described
as a marked cell enlargement; and hyperplastic obesity, as a marked
increase in the cell number. The obese-hyperglycemic mouse is classified
as an example of hypertrOphic-hyperplastic obesity, which is the result of
both hyperplasia and hypertrophy of varying degrees in adipocytes. Johnson
and Hirsch(1972) indicated that animal models having obesity of the hyper-
trophic-hyperplastic type might present the most suitable model for studies
directed at understanding the human condition becauSe of the similarities
of this model to childhood onset or the hyperplastic type of human obesity
(15). Therefore, the obese-hyperglycemic mouse has been utilized extensively

during the past 20 years as an animal model in obesity studies.

II. THE GENETICALLY OBESE MOUSE

The obese mouse(ob/ob) was first identified in the V stock at the.
Jackson Laboratories, Bar Harbor, Maine in the summer of 1949(16). These
obese mice were first distinguishable at about 4 to 6 weeks of age. Obese
mice increase body weight rapidly until they are up to four times the
weight of normal animals. The recessive gene(ob) of the mutant causes
sterility in the homozygote. Infertility is characteristic of the.ob/ob
females(17,l8). The ovaries and uterus in adult mice were atrophic, a
finding not usually present in gold thioglucose(GTG) or hypothalamic
obese(HTO) mice(l9), and thus may reflect the genetic derangement in this
species. The general characteristics of the obese syndrome are described
by the following features: obesity, hyperglycemia, insulin resistance, and
hyperinsulinemia(20). The activity of glycolytic enzymesin the livers of
the obese mice is significantly higher than that in lean controls(21). In
contrast to expectations, the increased insulin levels do not suppress
hepatic gluconeogenic enzymes, except phOSphoenolpyruvate carboxykinase
(PEPCK) in obese mice(22). Activity of the lipogenic enzymes is also
increased in the livers of the obese mice(23-25).

Hellman(26), and Westman(27) studied the development of the obese
syndrome in these mice and found the obesity could not unequivocally
identified before 26 days of age. Recently, increased body fat deposition
in the obese mice by 21 days of age has been suggested by Chlouverakis, et

al.(28) based on the analysis of body composition in a group of 21 female'

offspring from known heterozygotes. Joosten, et al.(29) measured the fat

cell diameters of 12-17 day old obese pups and indicated the obese mouse
had enlarged adipocytes. This finding has been further studied and confirmed
by Kaplan, et al.(l976)1. Total fat content in the hindlimbs of obese mice
was significantly higher at 3-4 weeks of age than their lean counterparts(30).
Besides the differences in body fat, obese mice also show other
different features before weaning. The body temperature of obese mice was
about 0.3° lower from 8 to 16 days of age, and about l.0° lower onwards
than in their lean siblings(31). A lower locomotor activity has been detected
in obese mice at only 7 days of age(32). The fact that young adult obese
mice consumed only one-half as much oxygen as did lean mice(33,34) has been
applied to predict obesity in young obese mice at 3 and 4 weeks of age(35).
Based on studies of thyroid, the obese mouse may be hypothyroid as early as
5 weeks of age(3l). The plasma-insulin concentration of these mice has also
been measured. Hyperinsulinemia is not manifested before the beginning of
the fourth week. Elevated levels of circulating inSulin are present by one
month of age, and reach a peak level by 6 months(36). Hyperglycemia, a
prominent feature in fully develOped obese mice(37), does not appear until
after the onset of obesity and hyperinsulinemia, and reaches its peak by
12 weeks of age(26,38). ”Obese-hyperglycemic" has been commonly used to
Specify this genetically obese mice. However, recent evidence suggests that
metabolic alterations may already be present in these animals at a very.

early age.

 

1Kaplan, M.L., Trout,J.R., and Leveille, G.A. unpublished observations.

 

III. EFFECTS OF FOOD INTAKE AND DIETARY NUTRIENTS
ON
THE DEVELOPMENT OF OBESITY

With reSpect to food intake, obese mice have been reported to
consume significantly more food than lean mice. For example, Mayer, et
al.(39) in 1951 found that obese mice, placed on a "free-choice diet",
selected more fat, and ate about 25% more diet than non-obese mice. Even
though animals were exercised, obese adult mice still consumed more food
and gained more weight than their lean sibs(40). When the diet was diluted
with cellulose, or made bitter, obese mice at 7-15 weeks of age still
consumed more food than lean mice, suggesting that a defect exists in the
regulation of food intake in obese mice(4l). The obese syndrome in these
mice was thought at one time to be similar psychOphysiologically to
hypothalamic obesity in rats. However, genetically obese mice do not display
the behavior patterns associated with the rodents made obese by hypothalamic_
damage(4l-43). Thus, the normal physiological and behavior correlates
involved in the regulation of food intake have been suggested to remain
intact in the genetic obesity.

Obese mice can become obese even in the absence of excessive food
intake(41). When food is restricted, the body weight of the obese mouse
returns to normal or becomes lighter, whereas the body fat concentration
of obese mice still remains higher than that in lean mice(42,43). Similar
results have also been found in some other studies in which food restriction
was utilized to study energy metabolism(44-46). Therefore, hyperphagia is
not necessary factor to cause obesity in these obese mice. The studies of

Bray and his coworkers(47) also indicate that some differences in

hypothalamic endocrine function may not result from hyperphagia, but may
be secondary to an alteration of the homoeostatic level of "adipose tissue
mass"(4l). In all these studies, adult animals have been used; in which
obesity was already present. There is a paucity of data on food intake of
the young obese mouse, in which the obese syndrome has not been complicated.
Studies on the young obese are thus needed to clarify to the role of energy
intake in the deve10pment of obesity in these animal models.

Several environmental factors interplay in the deve10pment of obesity.
Diet is one of those factors, which has been well demonstrated to induce
obesity in rodents(reviewed in Ref. 48). Before weaning, milk is normally
the main diet for pups. Nutrients in milk vary with the food components
which dams consumed(49). When dams were fed a 15% fat diet, the fat con-
centration(wet weight) in the milk produced by those dams is about 25%.
When dams were fed a fat-free diet, their milk containes about 16% fat.
Although it has been suggested that obese pups may have more milk intake
due to their high growth rate(32), the effect of milk intake on the deve-.
10pment of obesity in mice has not been elucidated, possibly because
Simplified techniques for measurement of 24-hour milk intake of pups are
not available. Nowadays, a pr0per technique for the estimate of milk intake
of pups is to seperate pups from dams for a short period, then to return
them and measure the body weight difference due to nursing(50). The milking
procedure did not affect the growth rate of these pups.

After weaning, the diet effect on the deve10pment of obesity depends

on the nutrients contained in the diet. When a normal diet containing

3-4 kcal/g, such as a commercial diet, is diluted with indigestible
material, such as cellulose or kaolin, the mass of food ingested is adjusted
to provide maintenance of total caloric intake(Sl). Whendiets with an
increased caloric density are made by increasing the percentage of fat,
'some Species of animals will show precise adjustment and will maintain
body weight in spite of the increased caloric density of the diet(52).
However, most strains of rats are unable to make a complete adjustment and
will store excessive quantities of energy and become obese. The obesity
induced by high-fat diets in normal animals is called dietary obeSity. In
genetically obese mice, obesity develOped when only carbohydrate was
provided as caloric source. When only 5% fat was added the fat-free diet,
obese mice contained about the same body fat as other obese mice fed a

30% fat diet(53). In another words, obese mice are more efficient in converting
dietary energy to body energy than lean mice, apparently regardless of the
diet components(44,48). Most of this increased body energy contributes to
fat accmmulation. On the contrary, lean body mass of the obese mice is
lower than that of their lean counterparts(42, 54,55). Bergen, et al.(30)
studied muscle growth in obese mice and indicated that a possible defeCt
in muscle may play a role in the etiology of genetic obesity. However, the
possible role of nutrition in the early development of obesity has nOt

been determined.

IV. OBJECTIVES

The present study was designed to follow the food intake and growth
pattern of obese and lean mice from 1 to 8 weeks of age. Before weaning
at 3 weeks of age, milk intake of the pups was estimated daily. from 3 to 8
weeks of age, obese and lean mice were fed a high-carbohydrate commercial
diet or a high-fat semipurified diet. Food intake and body weight changes
were monitored. In order to observe the differences in energy retention,
fat retention, and protein retention between obese and lean mice, body

composition was determined in 3 and 8 week old mice.

MATERIALS AND METHODS

Heterozygote breeding mice(C57BL/6J, ob/+)2 were housed in solid
bottom cages with wood shavings for bedding,'and fed ad libitum a commercial
diet3. Pregnant dams were removed from the breeding cages and placed in
separate similar cages. The average litter contained 6-7 pups. Litters
containing less than 4 pups or more than 9 pups were not utilized in the
iexperiment. Pups were individually identified at 5 days of age. Room lights
in the temperature-controlled(22-24°) were turned on at 0700 hours and off
at 1900 hours each day. All animals had free access to water.

From 7 to 21 days of age, estimates of milk intake and growth rate
were recorded daily. To obtain estimates of milk intake, pups were separated
from their mother at 0900 hours daily and returned to the mother's cage 6
hours later. Pups were allowed access to water, but not to food during this
time. Pups were weighed at 0900 hours, 6 hours later, and again after one
hour nursing. The body weight gain of each pup during one hour of nursing
was utilized as an estimate of the pups' milk intake(50). The sum of all
the pups' milk intake in one litter was utilized as an estimate of milk
production of the dam. Body weights measured daily at 0900 hours were utilized

to determine the growth rate of the pups. Body weight differences between

 

2 .
Jackson Laboratory, Bar Harbor, Maine.

3Wayne’Lab-Blox, Allied Mills, Inc., Chicago, Illinois.

10

0900 hours and 6 hours later were recorded as body weight loss during
fasting. The obese(ob/ob) and 1ean(ob/+ or +/+) mice were identified
retrospectively after it became obvious by visual inspection which mice
were obese.

At 21 days of age, obese were identified by their lower oxygen con-
sumption(35) and by their relatively fat appearances. From 21 to 56 days
of age, male and female obese mice and their lean lettermates were indi-
vidually housed in metal cages with wire-screen bottoms. They were fed
a pelleted commercial diet or a high-fat diet(Table 1). Identification
of the obese mice utilized in this feeding experiment was also confirmed
retrOSpectively by visual inspection.

Male and female obese mice and their lean littermates were killed
at 21 and 56 days of age. Food residue in the stomach was removed from
the carcass. The carcass was then softened in an autoclave at 100° for 15
minutes prior to homogenization. The homogenate was heat in a water bath
at 50°, mixed, and sampled. A cholroform/methanol (2:1) mixture was used
to extract fat from the carcass homogenate. Carcass fat was determined
gravimetrically. The nitrogen content of the carcass was determined byja
Semimicro-Kjeldahl method(58). The protein content was computed by multi-
plying the nitrogen content by 6.25. A bomb calorimeter4 was utilized to
determine the energy content of mouse carcasses. The carcass homogenates
were dried(59) in a vacuum oven at 50° for 24 hours prior to combustion in

A

4Parr Adiabatic Calorimeter with Oxygen Bomb (Parr 1241). Parr Instrument
.Co., Moline, Illinois.

11

the calorimeter.

Statistical analysis of the results was conducted with a 2 or 3-

factor model of crossed classification5(60).

Table 1

Approximate diet composition

 

% of Energy.d

 

c
Gross energy

 

(kcal/g) Fat Protein ‘ Carbohydrate
Commercial dieta 4.19 25 23 52
High-fat dietb 7.16 82 18 0

 

aWayne Lab-Blox. Allied Mills, Inc., Chicago, Illinois.

bWeight percentage: casein 31.2; tallow 47.0; corn oil 7.8; methionine
0.5; mineral mix 6.3 (see ref.56); vitamin mix 0.6 (see ref. 57);
choline 0.3; cellulose 6.3.

cDeterminedrin a bomb calorimeter.

dComputation based on 4 kcal per g protein and carbohydrate and 9 kcal
per g fat. Carbohydrate content of the commercial diet computed by
difference. It was assumed that the cellulose in both diets was not
utilized by the mouse.

 

5Gill, J.L. (1977) Crossed Classification: Factorial Experiments. In:

Design and Analysis of Experiments in the Animal and Medical Sciences..
Iowa State University Press, Ames. ( In Press).

RES ULTS

The milk production of 15 dams is shown in Figure l. The rate of
milk produced for the one hour period increased from 7 to 15 days of
lactation, and then declined rather rapidly, especially from 19 to 21
days of lactation. The maximum milk yield per hour appeared at about
14 days of lactation.

From 7 to 20 days of age, young obese and lean mice consumed an
equal amount of milk during one hour nursing periods. Similar results
were obtained when pups nursed for 90 minutes before weighing. However,
obese mice gained more body weight than their lean sibs(Table 2). As
shown in the normal growth pattern(6l), mice grew faster from 7 to 14 days
of age than from 14 to 20 days of age. The growth curves of the mice
during this period are shown in Figure 2. At 21 days of age, the body
weights of obese mice were heavier (p<0.05) than their lean counterparts.
The young mice lost body weight when they were isolated from their mother's
cage for 6 hours. Obese mice lost less weight than lean mice from 7 to 14
days of age but not from 14 to 20 days of age. All mice lost less body
weight from 7 to 14 days of age than from 14 to 20 days of age.

At 21 days of age, both male and female obese mice contained about
twice the carcass fat and total body energy observed in their lean litter-
mates; the carcass protein content was not influenced by the phenotype(
Table 3). 'On a concentration basis, obese mice contained about 27% fat,

and 16% protein; lean mice contained about 14% fat, and 18% protein.

12

13

Figure 1.

Lactation curve of 15 dams. Each dam nursed 5 to 8 pups. Each point

is mean I SEM of 15 dams.

14

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16

Figure 2.

Growth curves of mice before weaning. Each point is mean I SEM.
There are 15 obese males, 30 lean males, 10 obese females, and 30

lean females.

Body Weight (gm)

17

A. Male 8. Female

 

 

 

 

 

. 4.
—Obese
3 --- Lean '
. 2-
7 9 ll l4 I6 l8 2| 7 9 ll l4 l6 l8 2

Age (Days) Age (Days)

18

Table 3

Body weight, fat, protein and energy content of mice

at 21 days of age.

1.—

 

 

 

Male Female
Body Obese Lean Obese Lean MSEc ANOVAd
Weight, g 9.203 8.18 9.17 7.85 1.59 P
Fat, g 2.58 1.19 2.25 0.94 0.17 P
Protein? g 1.43 1.48 1.47 1.42 0.04 NS
Energy, kcal 26.0 13.4 25.6 12.8 18.6 P

 

IMean for eight mice per group.
Protein = nitrogen cantent x 6.25
cMSE = Mean Square Error.

Analysis of Variance.
effect. NS indicates not significant.

P designates significant(p < 0.05) phenotype

19

During the first several days immediately after weaning, the mice
showed some differences in adaptation to the dietS( Table 4). From 21
to 23 days of age, obese males consumed less energy and gained less body
weight than lean males, while obese females consumed less energy but gained
as much weight as did their lean counterparts (results based on average
of both diets). At the same time, the diet fed did not affect the body
weight gain of mice. However, mice consumed more energy when fed the
commercial diet than fed the high-fat diet. From 23 to 25 days of age, obese
mice consumed less energy and gained less body weight than lean mice when
the results from both diets were averaged. Male and female mice fed the
commercial diet consumed equal energy and gained equal weight, while males
fed the high-fat diet consumed less energy and gained less weight than
females. From 25 to 28 days of age, obese mice fed the commercial diet
still consumed less energy but started to gain more weight than lean mice.
Obese mice fed the high-fat diet consumed more energy and gained more
weight than their lean sibs. After 28 days of age, the effects of the diet and
the phenotype on body weight gain and food intake in mice became more
pronounced (Table 5). The obese mice gained more body weight. From 35-42
days of age, obese mice fed the commercial diet started to consume more
energy than lean mice, whereas obese mice fed the high-fat diet started to
consume more energy than lean mice at an earlier age (25-28 days of age).

Before 21 days of age, the sex of the mice did not influence the
observations. From 21 to 56 days of age, the sex of mice had only a minimum

effect on food intake, but body weight gain of the mice was influenced by

20

Table 4

Effect of diet on body weight gain and food intake of mice

 

 

 

 

 

 

 

 

 

Days
of Male Female
Age Dieta Obese Lean Obese Lean MSEc ANOVAd
Body Weight at Day 21 (g)
' cu 9.2b 8.0 8.4 8.0 1.25 P
HFD 7.3 6.5 8.0 7.2 1.39
Body Weight Gain (g)
21-23 CD 0.1 1.0 0.4 0.8 0.29 P,PxS
HFD -0.2 0.4 1.0 0.7
23-25 CD 0.9 1.4 1.0 1.3 0.20 P,S,DxS
HFD 0.4 0.9 1.7 1.7
25-28 CD 1.8 1.8 1.9 1.5 0.20 P,D
HFD 3.1 2.4 3.0 2.7
Food Intake (kcal)
21-23 CD 10 18 12 19 40.4 P,D,S
HFD < l 5 10 9
23-25 CD 14 22 16 22 48.4 P,S,DxS
HFD 9 14 25 21
25-28 CD 28 33 32 36 67.9 D,S,PxD
HFD 39 32 44 40

 

aCD = Commercial Diet; HFD = High-Fat Diet.
bMean for eight mice per group.

cMSE = Mean Square Error.

dAnalysis of Variance. P,D, and S designate significant (p < 0.05)

phenotype, diet and sex effects, reSpectively.

21

Table 5
Effect of diet on weekly body weight gain and food intake of mice'

 

 

 

 

 

 

 

Days Male Female
Of a c d
Age Diet Obese Lean Obese Lean MSE ANOVA
Weekly Body Weight Gain (g)
28-35 on 4.0b 5.2 4.9 3.6 2.4 P,D,S,PxD,PxS
HFD 10.1 7.4 9.2 5.5
35-42 CD 5.2 3.1 5.6 2.3 1.7 P,D,S,PxD,DxS
' HFD 9.9 4.6 8.4 1.7
42-49 CD 5.0 1.7 4.6 0.4 1.5 P,D,PxD,PxS
HFD 6.2 2.4 7.4 1.1
49-56 CD 4.2 1.1 3.7 0.5 1.0 P,D,S,PxD
HFD 6.8 1.3 5.2 0.9
Weekly Food Intake (kgal)
28-35 CD 82 93 97 94 404 P,D,PxD
HFD 136 104 138 106
35-42 CD 118 110 131 102 376 P,D,PxD
HFD 166 130 164 118
42-49 CD 141 112 145 101 248 P,D,PxD,PxS
HFD 167 123 178 112
49-56 CD 150 108 145 100 227 P,D,PxD
HFD 174 118 179 117
Total Food Intake (kcal)
21-56 CD 541 496 577 474 4574 P,D,PxD
HFD 694 528 737 522
Total Protein Intake (3)
21-56 CD 31.9 29.2 34.2 27.0 10.45 P,D,PxS
HFD 25.8 19.6 27.4 19.4

 

acn = Comercial Diet; HFD = High-Fat Diet.
bMean for eight mice per group.

c
MSE=Mean Square Error

Analysis of Variance. P, D, and S designate significant (p < 0.05)
phenotype, diet, and sex effects, reSpectively.

22

their sex. Male and female obese mice gained body weight at similar rates,
but lean males gained more weight than lean females.

Growth curves of the mice from 21 to 56 days of age are summarized
in Figure 3. Consumption of the high-fat diet increased the body weight
of the obese mice whereas lean mice fed the high-fat diet had body weights
similar to those observed in lean mice fed the commercial diet. Obese mice
fed the high-fat diet weighed more than lean mice by 35 days of age,.whereas
obese mice fed the commercial diet weighed more than lean mice after 42-49
days of age.

Energy, fat, and protein retention of the mice from 21 to 56 days of
age were computed from the results of carcass analysis, and are shown in
Table 6. At 56 days of age, obese males contained 52% fat, and 12% protein;
lean males contained 16% fat, and 20% protein. For the females, obese mice
contained 57% fat, and 10% protein; lean mice contained 21% fat, and 18%
protein. These results are computed from the average data which both diets
were utilized . The three retention values , in terms of energy, fat, and
protein, are resulted by substracting the mean value at day 21 from the
individual value at day 56. Clearly, the obese mice retained more energy
and exhibited a higher energy efficiency than lean mice. Obese mice retained
4-5 times as much energy and were about 3 times as efficient in converting
dietary energy to body energy as their lean counterparts. Consumption of
the high-fat diet enhanced the energy retention and energy efficiency to
a greater extent in obese mice than in lean mice. As eXpected, fat retention

was closely related to energy retention. Both obese and lean females

23

Figrue 3.

Growth curves of mice after weaning. Each point is mean I SEM

of eight mice per group.

BODY WEIGHT (GM)

24

I A. Commercial Die?
42 -

   

on
O

 

obese males

3
l

I I U '

 

 

2| 28 35 42 49 56

I c. High Fat Diet
42»

   
    

38 b
obese males

‘a"
‘0'

I
’ o

x! lean males

I I U I

 

J l l l l

 

2I 28 35 42 49 56

46F

3

U I I I

 

memmo

8. Commercial Diet

     
 

obese females

lean females
,.r-"""’

 

46F
42~

 

2| 28 35 42 49 56

0. High Fol Diet

 
 
 
    
 

obese females

”4"”.

r", lean females

I

 

35 42 49 56

2| 28

AGE (DAYS)

25

Table 6

Effect of diet on energy, fat and protein retention in mice
from 21 to 56 days of age

 

 

 

 

 

 

 

 

 

Male Female
Dieta Obese Lean Obese Lean MSEc ANOVAd
Energy Retention (kcal)
cu 108b 35 118 28 188.4 P,D,PxD,PxS
HFD 210 45 233 42
Energy Efficiengyd(%)g
CD 19.9 7.3 20.4 6.3 2.1 P,D,PxD,PxS
HFD 30.4 8.4 31.7 8.0
Fat Retention (g)
CD 11.9 2.5 13.5 2.6 3.2 P,D,S,PxD,PxS
HFD 20.0 2.9 23.6 3.8
Protein Retention (g)
CD 2.4 3.2 2.0 2.0 0.1 P,D,S,PxS
HFD 3.0 3.4 2.5 2.2
Protein Efficiengy (%)f
CD 7.6 11.0 6.0 7.3 1.4 P,D,S,PxD,PxS,DxS
HFD 11.5 17.5 9.2 11.6

“I;

aCD = Commercial Diet; HFD = High-Fat Diet.
bMean for eight mice per group.
CMSE = Mean Square Error.

dAnalysis of Variance. P, D, and S designate significant (p < 0.05)
phenotype, diet and sex effects, reSpectively.

eEnergy retention (kcal) / Energy intake (kcal).

fProtein retention (g) / Protein intake (3).

26

retained more fat than did their male counterparts.

Protein intake of mice was calculated and is shown in Table 5. Obese
mice had a higher protein intake than lean mice. Mice consumed more protein
when fed the commercial diet than when fed the high-fat diet; the protein
to energy ratio was also higher in the commercial diet. Obese males retained
less protein than did lean males; however, differences were not observed [1
when obese and lean females were compared. Regardless of diet fed, obese

mice were less efficient in converting dietary protein to body protein than

 

lean mice. However, the high-fat diet caused a higher protein retention

 

 

" .-

and protein efficiency than the commercial diet in mice.

DISCUSSION

As early as 7-14 days of age, small differences in body weight gain
were evident in the obese mice. It has been suggested that increased
milk intake may be a congenital factor in the development of obesity (32).
However, no differences in milk intake were observed, suggesting that the
obese mice might have a greater feed efficiency than lean mice. It is also

possible that the one hour estimate of milk intake did not reflect the

 

total daily milk intake. The lactation curve obtained was similar to pre-
vious reports (50, 62), although the daily milk yield was somewhat lower
than reported previously (50,63). Genetic background(64) as well as.the
number of pups suckled per litter (65) would influence the results. Losses
in body weight of the pups due to urination were not considered in the
estimation of milk intake. Milk intake data for the let day were not
presented because solid food was found in the gut of pups at this age. Pups
have been reported to eat solid food when they were 15-16 days old (66)..
However, intake of solid food in pups less than 21 days of age was not quan-
titated in the present study.

Obese pups from 7 to 14 days of age lost less weight than lean mice
during 6 hours of fasting; an observation similar to that found in adult
obese and lean mice (67). Adult obese mice excrete much less creatinine

than lean mice, during the first 36 hours of fasting, suggesting that the

obese mice may utilize less muscle mass than lean mice during the fast(68).

27

28

Possibly obese mice rely to a greater extent on fat metabolism during a

fast than do lean mice. However, the composition of the weight loss in
the young pups during the fast is unknown. When the ratio of the daily
weight gain to weight loss during the fast was calculated from 14 to 20
days of age, the values were 1.65 I 0.12 and 0.84 I 0.07 for the obese and
leanmice, respectively. When values within the 99% confidence intervals A
were then used to predict the phenotype of an additional 7 litters, 6 of
the 7 obese pups were correctly identified whereas 37 of the 39 lean mice
were prOperly identified. Two lean mice were mistakenly identified as
obese and one obese nouse was incorrectly identified as lean. When the
values were calculated for the two week period (7 £0 20 days of age),
similar results were also obtained. It may be possible to refine this
technique into a useful tool for screening and identifying young obese and
lean mice. It would be eSpecially useful when large numbers of animals
are needed; individual oxygen consumption measurements (35) would be im-
practical in this Situation.

At 21 days of age, obese mice contained about 130% more body fat, but'.
were only 14% heavier than lean mice; an observation in agreement with a
.report by Chlouverakis, et a1. (28). Adipocyte cell size is increased in
obese mice as early as 12-17 days of age (29). Similarily, the genetically
obese rat (fa/fa) may have an increased body fat content as early as 14 days

of age6. These observations suggest that significant differences in energy

 

‘6Be11, G.E., and Stern, J.S. (1976) Development of obesity and hyper-

insulinemia in the Zucker obese rat (fa/fa). Federation Proc. 35, 657.

29

metabolism probably occur very early in the life of the obese mouse.

The time required for mice to adapt to a new diet immediately after
weaning varied with the diet consumed and the sex of the mice. Obese males
just maintained their body weight immediately after weaning whereas both
obese and lean females started to gain weight immediately after weaning,
especially when the high-fat diet was fed. Mice were placed in wire bottom-

ed cages in a room maintained at 22-24° when weaned. This may have stressed

 

the obese males, which are known to adapt poorly to cold (69,70), to a

greater extent than the other mice. The critical temperature, which is

 

defined as the temperature below which there is an increase in the metabolic
rate, for young mice is 30-32° (71,72).

In the present study, obese mice did consume more food than the lean
mice during the 5 weeks feeding period. However, even when obese and lean
mice were pair-fed from 24 days of age (41) or when the body weight of the
adult obese mice was reduced to the level of the lean mice (42,44,45) obese
mice still contained more fat than lean mice-. These studies as well as
those of others(43,46) indicate that excess food intake can enhance the
degree of the obesity, but may not be necessary for its development. Hyper-
phagia is suggested to be secondary to a primary metabolic defect (41,73).

The obese mice, in agreement with earlier reports ( reviewed in ref.
48), were much more efficient in retaining dietary energy than the lean
mice when either one of the experimental diets was used. Comparisons between
the commercial diet and the high-fat diet have to be made with reservation
because one was formulated with natural ingredients and the other with semi-

purified ingredients. Nevertheless, obese mice fed the high-fat diet were

more efficient than were obese mice fed the high-carbohydrate commercial

3O

diet. The high-carbohydrate commercial diet was selected because it was
available in pelleted form, which greatly facilitated collection of food
intake data in these young mice. In other studies a high-fat diet has

also been shown to potentiate the development of obesity in rodents (53,
74-76). The obese mouse has a lower oxygen consumption (33,34), 3 lower
body temperature (31), and lower locomotor activity (32,77,78) than lean
mice. The involvement of these factors in the dietary-induced potentiation’

of obesity is not clear.

 

Obese animals also showed differences in their ability to retain
dietary protein. While obese mice consumed more protein than lean mice,
they retained less protein. Lean mice retained 35-45% their body energy
as protein, whileas obese mice retained only 7-11%. These values are
similar to those reported by others (44,46). Direct estimates of muscle mass
also indicate a difference in protein accumulation between obese and lean
mice (30). The role of protein metabolism in the development of obesity

in these mice remains to be elucidated.

SUMMARY

From 7 to 20 days of age, obese mice consumed an equal quantity
of milk but gained more body weight than lean mice. When mice were fasted
for 6 hours every day, obese mice lost less weight than lean mice from 7
to 14 days of age. It is possible that obese utilize less muscle mass,‘
and rely to a greater extent on fat metabolism during a fast than do lean
mice. The ratio of the daily body weight gain to the weight loss during‘
a fast also Showed a significant difference between obese and lean mice.
This may be a useful tool for screening and identifying the phenotype of
mice, especially when large numbers of animals are needed. At 21 days of
age, the fat content of obese mice was double that of lean mice, whereas
the protein content was not changed. These observations suggest that
significant differences in energy metabolism probably occur very early in
the life of the obese mouse.

During the first several days after weaning, obese males failed to
consume as much food as did the lean males. Obese mice consumed equal
quantities of food, but gained more body weight than lean mice from 35-42
days of age for males, or from 28-35 days of age for females when they
were.fed the commercial diet. After that, obese mice consumed more food
and gained more weight. Obese mice fed the high-fat diet started to eat

more food and gained more weight from 28 days of age. The present obser-

 

 

vations imply that excess food intake can enhance the degree of the obesity,

but may not be necessary for its development. Hyperphagia is suggested to

31

32

be secondary to a primary metabolic defect.

l

Obese mice, in agreement with early reports, were much more

efficient in retaining energy than lean mice when either one of the eXperi-

mental diets was used. The high-fat diet has
the deve10pment of obesity in mice. When the
were averaged, obese males contained 52% fat,

contained 16% fat, and 20% protein at 56 days

for the females, obese mice contained 57% fat,

contained 21% fat, and 18% protein.

been shown to potentiate
reSults of carcaSs analysis
and 12% protein; lean males
of age. At the same age and

and 10% protein; lean mice

Obese mice also showed differences in their ability to retain diet-

ary protein. Obese males retained less protein than did lean males;

however, differences were not observed when obese and lean females were

compared. Regardless of diet fed, obese mice were less efficient in

converting dietary protein to body protein than lean mice. These findings

support the suggestion that a possible defect

in protein metabolism may

play a role in the etiology of the genetic obesity.

 

 

10.

ll.

12.

l3.

14.

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APPENDIX

39

Appendix

A.

1. Analysis of Variance.

For milk intake of mice

For

 

 

 

 

 

 

 

 

 

Source df Mean Square F ratio p
1). From 7 to l4gd§ys of agg
Phenotype 1 0.0039 0.0218 0.88
Sex 1 0.3952 2.1834 0.14
Phenotype x Sex 1 0.0250 0.1380 0.71
Error 81 0.1810
2). From 14 to 20 days of age
Phenotype 1 0.2789 0.7230 0.99
Sex 1 0.4470 1.1590 0.28
Phenotype x Sex .1 1.2776 3.3123 0.07
Error 81 0.3857
body weight gain
Source df Mean Square F ratio p
1). From 7 to 14 days of age
Phenotype 1 0.0439 6.7177 0.01
Sex 1 0.00005 0.0074 0.93
Phenotype x Sex 1 0.0025 0.3700 0.54
Error 81 0.0065
2). From 14 to 20 days of egg
Phenotype. 1 0.0702 5.6575 0.02
Sex 1 0.0023 0.1831 0.67
'Phenotype x Sex 1 0.00079 0.0635 0.80
Error ' 81 0.01242

 

40

Appendix 1. Analysis of Variance. (continued).

~ a C. For body weight loss during a fast

 

 

 

 

Source df Mean Square F ratio p
1). From 7 to 14 dgys of age
Phenotype 1 0.2441 15.6028 < 0.0005
Sex 1 0.0014 0.0887 0.77
Phenotype x Sex 1 0.0032 0.2046 0.65.
Error 81 0.0156
2). From 14 to 20 days of age
Phenotype 1 0.9751 3.5682 0.06
Sex 1 0.1720 0.6294 0.43
Phenotype x Sex 1 0.0220 0.0805 0.78
.Error 81 0.2733

 

41

Appendix II. Analysis of Variance for body weight, fat, protein and

energy content of mice at 21 days of age.

 

 

 

 

 

Source df Mean Square F ratio p

1). Body weight.
Phenotype 1 10.8461 6.8240 0.02
Sex 1 0.2538 0.1596 >0.50
Phenotype x Sex 1 0.1846 0.1161 >0.75
Error 28 1.5894

2). Body fat.
Phenotype 1 14.5672 84.6909 <0.0005
Sex 1 0.6694 3.8915 0.06
Phenotype x Sex 1 0.0133 0.0774 0.78
Error 28 0.1720

3). ngy protein.
Phenotype 1 0.00003 0.0008 0.98
Sex 1 0.00195 0.0563 0.81
Phenotype 1 0.02023 0.5841 0.45'
Error 28 0.03464

4). Body energy_content.
Phenotype 1 1,297.0381 69.5544 1<0.001
Sex 1 2.1636 0.1160 0.75
Phenotype x Sex 1 0.0223 0.0011 >0.75
Error 28 18.6478

 

42

Appendix III.

A.

B.

Analysis of Variance.

For body weight at 21 days of age, mice for feeding experiments.

For

Source df Mean Square F ratio p
Phenotype 1 10.1442 6.2256 0.53
Sex 1 0.6480 0.3977 '0.02
Phenotype x Sex 1 0.2525 0.1550 0.70
Error 60 1.6294
body weight gain of mice,

1). From 21 to 23 days of age
Source df Mean Square F ratio p
Diet 1 0.0492 0.2463 0.62
Sex 1 0.5898 2.9540 0.10
Diet x Sex 1 0.4550 2.2785 0.14
Error1 28 0.1997
Phenotype 1 0.5902 6.8717 0.01
Diet x Phenotype 1 0.2817 3.2797 0.08
Sex x Phenotype 1 0.3546 4.1288 0.05
Diet x Sex x

Phenotype 1 0.0515 0.5999 0.44

Error2 28 0.0859

 

 

 

 

 

 

43

Appendix 111. Analysis of Variance. (continued).

‘ ' B. For body weight gain of mice,(continued),

 

 

 

 

Source df Mean Square F ratio p
2). From 23 to 25 dgys of agg

Diet 1 ‘ 0.0019 0.0158 '0.90
Sex 1 1.0842 8.9485 0.006
Diet x Sex 1 1.1718 9.6716 0.004
Error1 28 0.1212
Phenotype 1 0.3813 4.6656 0.039
Diet x Phenotype 1 0.0276 0.3382 0.57
Sex x Phenotype 1 0.1234 1.5096 0.23
Diet x Sex x

Phenotype 1 0.0315 0.3855 0.54
Error2 28 0.0817

3). From 25 to 28 dgys of age

Diet 1 2.0682 19.4992 <0.0005
Sex 1 0.0005 0.0049 0.94
Diet x Sex 1 0.0235 0.2218 0.64
Error1 28 0.1061
Phenotype 1 0.2437 8.9802 0.006
Diet x Phenotype 1 0.0437 1.6118 0.22
Sex x Phenotype 1 0.0009 0.0340 0.86
Diet x Sex x

Phenotype 1 0.0558 2.0548 0.16
Error2 28 0.0271

 

44

Appendix 111. Analysis of Variance. (continued).

C. For food intake of mice.

 

Source df Mean Square F ratio p

 

1). form 21 to 23 days of egg

 

 

Phenotype 1 364.5732 9.0285 0.003
Diet 1 1151.7868 28.5234 <0.0001
Sex 1 295.3878 7.3152 ~0.008
-Phenotype x Diet 1 134.5935 3.3331 0.08
Phenotype x Sex 1 113.8932 1.1782 0.26
Phenotype x Diet x

Sex 1 24.0397 0.5953- >0.25
Error 56 40.3804

2). from 23 to 25 days of age
Phenotype 1 202.9400 4.1967 <0.05
Diet 1 36.1092 0.7467 >0.25 ‘
Sex 1 567.7390 11.7405 0.001
Phenotype x Diet 1 162.3743 3.3578 >0.05
Phenotype x Sex 1 110.1548 2.2779 >0.10
Diet x Sex 1 416.5340 8.6137 <0.005
Phenotype x Diet x ‘

Sex 1 41.8452 0.8653 >0.25

Error 56 48.3573

3). from 25 to 28 days of age
Phenotype 1 3.0055 0.0443 >0.75
Diet 1 604.0487 8.9009 0.004
Sex 1 364.4429 5.3702 0.025
Phenotype x Diet 1 468.9448 6.9101 0.01
Phenotype x Sex 1 5.0784 0.0748 >0.75
Diet x Sex 1 34.0746 0.5021 >0.25
Phenotype x Diet x

Sex ~ 1 12.1805 0.1795 0.75.

Error 56 67.8638

 

45

Appendix IV. Analysis of Variance.

‘ 0

A. For weekly body weight gain. I

 

Source df Mean Square F ratio p

 

1). from 28 to 35 days of age

Diet 1 209.9239 90.0296 <0.0005
Sex 1 11.8078 5.0640 0.03
Diet x Sex 1 ‘ 4.6494 1.9940 0.17
Error1 28 2.3317
Phenotype 1 40.4337 19.1735 <0.0005
Diet x Phenotype 1 41.8447 19.8426 <0.0005
Sex x Phenotype 1 12.2763 5.8214 0.02
Diet x Sex x

Phenotype 1 2.4846 1.1782 0.29
Error2 28 2.1088

2). from 35 to 42 days of age

 

Diet 1 69.3056 42.9081 <0.0005
Sex 1 22.6100 13.9982 0.001
Diet x Sex 1 14.7264 9.1173 0.005
Error1 28 1.6152
Phenotype 1 302.4121 169.5491 <0.0005
Diet x Phenotype 1 42.6736 23.9252 <0.0005
Sex x Phenotype 1 6.6693 3.7392 0.06
Diet x Sex x -
Phenotype 1 0.0210 0.0118 0.91
Error2 28 1.7836

 

46

Appendix IV. Analysis of Variance. (continued).

‘ ' .A. For weekly body weight gain.(continued).

 

Source df 'Mean Square F ratio p

 

3). from 42 to 49 days of age

 

Diet 1 27.4576 17.0462 <0.0005-
Sex 1 2.7390 1.7004 0.20
Diet x Sex 1 2.5921 1.6092 ' 0.22
Error1 28 1.6108-
Phenotype 1 314.8850 201.6399 <0.0005
Diet x Phenotype 1 7.0225 4.4969 0.04
Sex x Phenotype 1 11.5600 7.4026 0.01
Diet x Sex x

Phenotype 1 3.0276 1.9388 0.18
Error2 , 28 1.5616

4). from 49 to 56 days of age

Diet 1 21.5528 19.5784 <0.0005
Sex 1 9.4403 8. 5754 0.007
Diet x Sex 1 0.6520 0.5923 0.45
Error1 28 1.1008

Phenotype 1 260.7418 255.3022 <0.0005
Diet x Phenotype 1 11.7478 11.5027 0.002
Sex x Phenotype 1 1.1289 1.1054 0.30
Diet x Sex x '

Phenotype 1 1.1790 1.7125 0.20

Error2 28 ' 1.0213

 

.47

Appendix IV.

~ 0

B.

Analysis of Variance. (continued).

For weekly food intake of mice.

 

 

 

 

 

Source df Mean Square F ratio p
1). from 28 to 35 days of age
Phenotype 1 3,054.7768 7.5654 0.01
Diet 1 14,218.5377 35.2133 <0.001
Sex 1 393.8216 0.9753 >0.25
Phenotype x Diet 1 5,171.0093 12.8064 <0.001
Phenotype x Sex 1 168.7930 0.4180 >0.50
Diet x Sex 1 160.5047 0.3975 >0.50
Phenotype x Diet x ,
Sex 1 225.3448 0.5581 >0.25
Error 56 403.7836
2). from 35 to 42 days of age
Phenotype l 14,220.5589 37,7742 <0.001
Diet 1 13,843.7132 36.7732 <0.001
Sex 1 81.8611 0.2174 >0.50
Phenotype x Diet 1 2,016.0863 5.3554 0.025
Phenotype x Sex 1 945.9063 2.5126 >0.10
Diet x Sex 1 402.7055 1.0697 >0.25
Phenotype x Diet x
Sex 1 145.7417 0.3871 >0.50
Error - 56 376.4619
3). from 42 to 49 days of age
Phenotype 1 32,733.6349 131.8903 <0.001
Diet 1 7,190.6092 28.9724 <0.001
Sex 1 105.1365 0.4236 0.50
Phenotype x Diet 1 1,217.2640 4.9046 0.03
Phenotype x Sex 1 1,507.7565 6.0750 0.02
Diet x Sexr 1 10.0311 0.0404 >0.75
Phenotype x Sex x
Diet 1 64.1228 0.2584 >0.50
Error 56 248.1884

 

48

Appendix IV. Analysis of Variance. (continued)

‘ ' 28. For weekly food intake of mice. (continued).

 

Source df Mean Square F ratio p

 

4). from 49 to 56 days of age

Phenotype l 41,122.4498 180.9054 <0.0005
Diet 1 7,221.2759 ' 31.7678 <0.001
Sex 1 88.7014 0.3902 >0.50
Phenotype x Diet 1 1,052.2350 . 4.6290 0.03
Phenotype x Sex 1 97.7378 0.4300 0.50
Diet x Sex 1 267.0496 1.1748 0.25
Phenotype x Diet x

Sex 1 9.7340 0.0428 >0.75
Error 56 227.3146

 

C. For total food intake of mice from 21 to 56 days of age.

 

 

Source df Mean Square F ratio p
Phenotype l 265,812.3274 58.1135 <0.001
Diet 1 181,643.2575 39.7120 3' <0.001
Sex 1 6,793.1161 1.4851 0.25
Phenotype x Diet 1 40,241.1151 8.7977 0.004
Phenotype x Sex 1 5,519.2147 1.2066 >0.25
Diet x Sex M l 1,319.7581 0.2885 >0.50
Phenotype x Diet x M

Sex 1 461.8543 0.1009 0.75
Error 56 4,574.0139

 

49

Q

Appendix IV.

D.

For total protein intake of mice from 21 to 56 days of age.

Analysis of Variance. (continued).

 

 

Source df Mean Square F ratio p
Phenotype 1 581.5392 55.6525 <0.001
Diet 1 903.4217 86.4562 <0.001
Sex 1 2.0838 0.1994 >0.50
Phenotype x Diet 1 17.7010 1.6940 >0.25
Phenotype x Sex 1 41.7457 3.9550 0.05
Diet x Sex 1 1.5920 0.1524 >0.50
Phenotype x Diet x

Sex 1 7.0578 0.6754 >0.50
Error 56 10.4495 4

 

50

Appendix V.

A.

Analysis of Variance.

For energy retention of mice from 21 to 56 days of age

For

 

 

 

 

 

Source df Mean Square F ratio p
Phenotype 1 269,740.6864 1,431.8289 <0.0001
Diet 1 58,052.5994 308.1529 <0.001
Sex 1 561.8344 2.9823 0.08
Phenotype x Diet 1 38,164.8620 202.5855 <0.001
Phenotype x Sex 1 1,894.9905 10.0589 <0.01
Diet x Sex 1 267.9560 1.4223 >0.10
Phenotype x Diet x
Sex 1 88.7201 0.4709 >0.25

Error 56 188.3889
energy efficiency of mice.
Source df Mean Square F ratio p
Phenotype 1 5,254.6588 2,588.7894 <0.0001
Diet 1 607.7261 289.2281 <0.001
Sex 1 0.1920 0.0913 >0.75
Phenotype x Diet 1 353.4738 168.2247 <0.001
Phenotype x Sex 1 10.7676 5.7676 0.02
Diet x Sex 1 1.7324 0.8244 >0.25
Phenotype x Diet x

Sex 1 0.0521 0.0247 >0.75
Error 56 2.1012

 

51

Appendix V.

C.

Analysis of Variance. (continued).

For fat retention of mice from 21 to 56 days of age.

For

 

 

 

 

 

 

 

Source df Mean Square F ratio p
Phenotype l 3,318.5135 1,026.0376 <0.0001
Diet 1 390.1550 120.6304 <0.001
Sex 1 35.7279 11.0465 0.002
Phenotype x Diet 1 276.5699 85.5115 <0.001
Phenotype x Sex 1 15.0477 4.7638 0.04
Diet x Sex 1 8.5025 2.6288 0.10
Phenotype x Diet x

Sex 1 1.0714 0.3312 >0.50
Error 56 3.2343
protein retention of mice from 21 to 56 days of age
Source df Mean Square F ratio p
Phenotype 1 0.8101 7.2654 0.008
Diet 1 2.5049 22.4654 <0.001
Sex 1 10.3472 92.8000 <0.0005
Phenotype x Diet 1 0.3466 3.1085 0.07
Phenotype x Sex 1 2.5422 22.8000 <0.001
Diet x Sex 1 0.0002 0.0017 >0.75
Phenotype x Diet x

Sex 1 0.0346 0.3103 >0.50

Error 56 0.1115

 

52

Appendix V.

E.

For protein efficiency of mice from 21 to 56 days of age.

Analysis of Variance. (continued).

 

 

Source df Mean Square F ratio p
Phenotype 1 175.8939 123.4949 <0.001
Diet 1 323.0854 226.8380 <0.001
Sex 1 180.4636 126.7033 <0.001
Phenotype x Diet 1 14.0093 9.8359 <0.005
Phenotype x Sex 1 33.9394 23.8288 <0.001
Diet x Sex 1 8.4511 5.9335 0.02
Phenotype x Diet x

Sex 1 1.9092 1.3404 0.25
Error 56 1.4243

 

53

 

MICHIGAN STATE UNIVERSITY LIBRAR

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