EFFECT OF 2, 2, 9, 9-TETRAMETHYL- l, 10-
DECANEDIOL (CI- 720) ON BLOOD, EGG YOLK, AND
EXCRETA CHOLESTEROL, BLOOD TRIGLYCERIDES, FEED
CONSUMPTION, EGG PRODUCTION, AND EGG
COMPONENT WEIGHTS

Thesis for the Degree of M. S.
MICHIGAN STATE UNIVERSITY
RICHARD DOUGLAS REYNNELLS
1976

 

 

.....

ABSTRACT

EFFECT OF 2,2,9g9-TETRAMETHYL-1,10-DECANEDIOL (CI-720)
ON BLOOD, EGG YOLK, AND EXCRETA CHOLESTEROL,
BLOOD TRIGLYCERIDES, FEED CONSUMPTION,

EGG PRODUCTION, AND
EGG COMPONENT
WEIGHTS

by
Richard Douglas Reynnells

01-720 (2,2,9,9-tetramethyl-1,10-decanediol; supplied
by the Parke-Davis Company) was administered to Single Comb
White Leghorn laying hens to determine if the drug would
produce the antilipemic effect (of decreased free and total
plasma cholesterol, and plasma triglycerides) that have
been reported for the rat, mouse, and rhesus monkey.

All hens were in their first year of lay, caged
individually, and had feed and water administered ad libitum.
Three hens were used in each of the four treatments of
experiment 1, which consisted of feed and capsule adminis-
tration of the drug, plus the respective controls. In the
second experiment, eight hens were used in the control and
in each of the two drug treatments. I I

In this study, administration of 01-720 during
experiment 1 resulted in declines in total (133 to 114 mg/dl
; 5.6 pooled SEM) and free (107 to 79 mg/dl‘i 5.7 pooled
SEM) plasma cholesterol, which were significant as calculated
by the split plot statistical analysis. These differences

Richard Douglas Reynnells

were not significant statistically when calculated as a
percentage, using the hen as her own control. In
experiment 2, the total plasma cholesterol was significantly
decreased by 01-720 (155; 111; 101 mg/dl 2: 5.3 pooled SEM,
for treatments of 0, 2600, and 5200 ppm of the diet,
respectively). Free plasma cholesterol was lowered by the
drug (85; 50; 38 mg/dl 1 5.7 pooled SEM, for the same
respective treatments). During both experiments, drug
treatment caused a significant decrease in the percent free
of total plasma cholesterol, versus the control hens.

Plasma triglycerides were not determined in the first
experiment, but were significantly decreased by the drug in
experiment 2. Treatments of 0, 2600, and 5200 ppm drug 31a
the diet resulted in means of 1524; 613; 228 mg trigly-
cerides/d1 plasma‘: 96 pooled SEM, for respective treat-
ments.

The total yolk cholesterol was increased in the first
experiment, but was not changed in the second.

Hen body weight was not altered in experiment 1, but
was lowered in experiment 2 by the drug. A related para-
meter, percent change in body weight, showed no difference
between treatment means during the first experiment, but
during the second experiment the drug treated hen's body
weight varied significantly more than control hens.

The percent egg production was lowered very signifi-
cantly (to zero in some cases) in both experiments by 01-720.

During the second experiment, the drug treated hens changed

Richard Douglas Reynnells

in the amount of weekly percent egg production very
significantly more than control hens. There was no
difference between the four treatment means for this latter
parameter during the first experiment.

Comparisons of the total egg weight, component weights,
and their percent of total egg weight were not significantly
different among treatments during either experiment,
although the eggs from drug treated hens of experiment 2
tended toward lower egg weight and yolk weight than those
from the control hens.

The statistical analysis showed that the drug lowered
the feed consumption during both experiments toward or below
the maintenance level of 70 g feed consumption/hen/day.

There was no difference between the four treatment
means of excreta cholesterol, although the actual amount of
excreta cholesterol was possibly low.

Before any concrete statements about the degree of
01-720 effectiveness are made, a study using a pair-feeding
technique should be accomplished. The reason for this lack
of complete confidence in the indicated statistical results
of the drug effect, is due to the lowered feed consumption
for most of the drug treated hens. With lowered feed intake
one would expect blood lipids, as well as egg production and
body weight to decline. Probably the drug effect is
confounded with the lowered feed consumption.

This research was supported in part by grant number

1818, and the Parke-Davis Company.

EFFECT OF 2,2,9.9-TETRAMETHYL-1,10-DECANEDIOL (CI-720)
ON BLOOD, EGG YOLK, AND EXCRETA CHOLESTEROL,
BLOOD TRIGLYCERIDES, FEED CONSUMPTION,

EGG PRODUCTION, AND
EGG COMPONENT
WEIGHTS

by

Richard Douglas Reynnells

A THESIS

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

MASTER OF SCIENCE
Department of Poultry Science

1976

to my family and parents

11

ACKNOWLEDGMENTS

I would like to express my very sincere appreciation
to those who have offered the numerous helpful criticisms
and encouragements throughout the past two years.

This research was supported in part by NIH Training
Grant GMO-1818.

iii

LIST OF TABLES

TABLE OF CONTENTS

LIST OF FIGURES O O O O O O O O O O O O O O 0 O 0

LIST OF FLOW CHARTS . . . . . . . . . . . . . . .

INTRODUCTION

REVIEW OF LITERATURE O O O O O O O O O O O O O O

ChOlCSterOIoooooooooooooooo

1.
2.

3.

4.
5.

6.
7.
8.

EffBCt Of 01-720 0 o o o o o o o 0
General . . . . . .

A. Agents used in an attempt to lower

plasma cholesterol . . . . . .
B. Factors which may alter plasma
cholesterol, or atherosclerosis

Specific Drugs and Other Compounds Used
in Plasma and Tissue Cholesterol Level

manipulations o o o o
A. Cholestyramine .

B. Clofibrate . .
C. Triparanol . .
Hormones

Diet Effect on Blood Lipids . . . .
A. General observations . . . . .
B. Egg in the diet . . . . . .

C. Crystalline cholesterol supplementa-

tion to the diet . . .
Sitosterol and Other Plant Sterols
Turnover of Cholesterol . . . . . .
Summaries of Cholesterol or Athero-
sclerosis . . . . . . . . . . . . .

Atherosclerosis . . . . . . . . . . . . . .

OBJECTIVES

1.
2.

Effect of Hormones . . . . . . . .
Plaque 8 O 0 O O O O O O O O O O O 0

iv

MATERIALS AND METHODS . . . . . . . .
Experiment 1 . . . . . . . . . .

1. General Comments . . .
2. Total Plasma Cholesterol
3. Free Plasma Cholesterol
4. Plasma Triglycerides .
5. Egg Parameters . . . .
6. Excreta Cholesterol . .
7. StatiStics o o o o o o

Experimentzoooooooo.o

1. General Comments . .

2. Total and Free Plasma ChOlesterol I

3. Total Yolk Cholesterol
4. Plasma Triglycerides .

O O O O O O O
O O O O O O O
O O O O O" O O
O O O O O O O
O O O O O O O
O O O O O O O
O O O O O O O

5. Egg Weight; Shell, Albumen, and Yolk

WeightB........
6. Statistics 0 o o o o 0

RESULTS AND DISCUSSION . . . . . . .
Total Plasma Cholesterol . . . .

1. Experiment 1 . . . . .
2. Experiment 2 . . . . .
3. Discussion . . . . . .

Free Plasma Cholesterol . . . .

1. Experiment 1 . . . . .
2. Experiment 2 . . . . .
3. Discussion . . . . . .
Free Cholesterol as a Percentage
Cholesterol . . . . . . . . . .

1. Experiment 1 . . . . .
2. Experiment 2 . . . . .
3. Discussion . . . . . .

Plasma Triglycerides . . . . . .
1.' Experiment 1

2. Experiment 2
3. Discussion . . . . . .

of Total Plasma

Page

76
76
79
86
87
87

9O

Total Yolk Cholesterol .

1. Experiment 1 . . . . . . . .
2. Experiment 2 . . . . . . . .
3. Discussion . . . . . . . . .
Body Weight (in grams) . . . . . . . .
1. Experiment“ 0 o o o o o o o
2. Experiment 2 . . . . . . . .
Percent Change in Body Weight . . . .
1. Experiment1........
2. Experiment 2 . . . . . . . .
3. Discussion . . . . . . . . .
Weekly Percent Egg Production . . . .
1. Experiment 1 . . . . . . . .
2. Experiment 2 . . . . . . . .
3. Discusaion o o o o o o o o 0
Change in Weekly Percent Egg Production
1. Experiment 1 . . . . . . . .
2. Experiment 2 . . . . . . . .
3. Discussion . . . . . . . . .

Total Egg Weight:

1.
2.
3.

Experiment 1 .
Experiment 2 .

Discussion

Feed Consumption . . . .

1.
2.
3.

Total Excreta Cholesterol

1.
SUMMARY . .

Experiment 1 .
Experiment 2 .

Discussion

Experiment 1 .

vi

Yolk, Albumen, and Shell Weights,
Plus Their Percent of Total Egg Weight

Page

92
92
97
108

130
130

131
132

133
133
135
136
136
139
144
149
149

151

Page
APPENDICES
A. FOOTNOTES 0 O O O O O O O O O 0 O O ' O O O O O 154
B. RECOVERIES O 0 O O O O O O O O O O O O O O O 155
C. Appendix Table 1. Split plot analysis of
variance table--a comparison between the
computer and hand calculated results . . . . 156
D. SPLIT PLOT STATISTICAL MODEL . . . . . . . . 157

E. 'SPLIT PLOT STATISTICAL ANALYSIS, SUMS OF
SQUARES O O O O O O O O O O O O O O O O O O O 158

F. TUKEY HSD, MODIFIED TUKEY HSD, AND DUNNETT
TEST OF MEANS PLUS fmax-TEST FOR HOMOGENOUS
VARIANCE O O O O O O O O O O O O O O O O O O 159

G. Appendix Table 2. Re-evaluation of free
plasma cholesterol values of experiment 2 . . 161

H. Appendix Table 3. Coefficient of variation
for parameters of experiments one and two . . 162

I. Appendix Table 4. Plasma triglyceride values
(in mg/dl) from experiment one . . . . . . . 163

BIBLIOGRAPHY O O O O O O O O O O O O O O O O O O O O O 1 66

General Bibliography . . . . . . . . . . . . . . . 176

vii

3.

4.

5.

7.

8.

9.

10.

11.

12.

13.
14.

LIST OF TABLES

Compounds reported to alter various cholesterol
levels in different species . . . . . . . . . . .

Factors which may influence plasma cholesterol
or atherosclerosis . . . . . . . . . . . . . . .

Effect of dietary cholesterol supplementation on
various cholesterol levels in the chicken . . . .

Factors which may or may not alter different
species chances of circulatory disease . . . . .

MSU Breeder Ration 72-10 0 o o o o o o o o o o 0

Chicken total plasma cholesterol values taken
from the 1 i te rature O O O O O O O O O O O O O O 0

Experiment 1. Percent free of total plasma
cholesterol . . . . . . . . . . . . . . . . . . .

Experiment 2. Percent free of total plasma
cholesterol . . . . . . . . . . . . . . . . . . .

Relationship between plasma and yolk total
CheleSterOJ. O O O O O O O O O O O O O O O O O 0 0

Total egg yolk cholesterol in mg cholesterol/g

yOlk O O O O O O O O O O O O O O O O O O O O O 0

Total yolk cholesterol values reported in the
literature . . . . . . . . . . . . . . . . . . .

Body weight (+1 g) of hens at the end of each

treatmentweeEooooooooooooooooo

Egg production record in total eggs per week . .

Experiment 1. Change in percent egg production

from the pro-treatment level . . . . . . . . . .

viii

Page

23

30
33

61

80

83

100

103

107

109
120

124

Table Page

15. Experiments 1 and 2. Means for egg weight; and
the egg component weights, plus their percent
of total egg weight . . . . . . . . . . . . . . . 134

16. Experiment 1. Results of statistical analysis
by the Tukey HSD test of means, comparing the
feed intake of the four treatments . . . . . . . 140

17. Experiments 1 and 2. Intake of 01-720, as
calculated from the feed consumption means in
each experiment. Also, the intake of drug based
on the body weight in a treatment in experiment 1 145

Appendix
Table
1. Split plot analysis of variance table--a com-
parison between the computer and hand calculated
results 0 O O O O O O O 0 O O O O O O O O 0 O O O 156

2. Re-evaluation of free plasma cholesterol values
of experiment 2 . . . . . . . . . . . . . . . . . 161

3. Coefficient of variation for parameters of
experiments one and two . . . . . . . . . . . . . 162

4. Plasma triglyceride values (in mg/dl) from
experiment one . . . . . . . . . . . . . . . . . 163

ix

LIST OF FIGURES
Figure Page

1. Experiment 1. Total plasma cholesterol in
mg/dl O O O O O O O O O O O O O 0 O O O O O O 0 O 54

2. Experiment 2. Total_plasma cholesterol in
mg/dl O O O O O O O O O O O O O O O O O O O O O O 5 7

3. Experiment 1. Free plasma cholesterol. Plot
of method of drug administration x time inter-
action; according to the split plot statistical
analySisoooooooooooooooooooo 65

4. Experiment 1. Free plasma cholesterol.
Three-way interaction of method of drug adminis-
tration x presence or absence of drug x time . . 68

5. Experiment 1.- Free plasma cholesterol in mg/dl . 70

6. Experiment 2. Free plasma cholesterol in mg/dl
plasma 0 O C O O O O C O O C C C O C C C O C C O 73

7. Experiment 1. Percent free of total plasma
cholesterol . . . . . . . . . . . . . . . . . . . 77

8. .Experiment 1. Percent free of total plasma
cholesterol. Results of the split plot
statistical analysis . . . . . . . . . . . . . . 81

9. Experiment 2. Percent free of total plasma
cholesterol . . . . . . . . . . . . . . . . . . . 84

10. Experiment 2. Plasma triglycerides in mg/dl . . 88

11. Experiment 1. Egg yolk total cholesterol in
mg/g yOJ-k C O I O O O O O O C O C O O O O O O O O 93

12. Experiment 1. Egg yolk total cholesterol percent
of the individual hen's pre-treatment level . . . 95

13. Experiment 2. Individual hen values of total
cholesterol/g yolk, from an egg laid on or about
the 21st day of drug treatment . . . . . . . . . 98

Figure Page
14. Experiment 2. Body weight in grams (:1) . . . . 112
15. Experiment 1. Effect of time on percent egg

production, according to the split plot

statistical analysis . . . . . . . . . . . . . . 118
16. Experiment 1. Percent egg production average for

the three hens/treatment, at each week of the

experiment 0 O O O O O O O O O O O O O O O O O O 1 22
17. Experiment 2. Percent egg production . . . . . . 126

18. Experiment 1. Feed consumption, average of
three hens per treatment . . . . . . . . . . . . 137

19. Experiment 2. Feed consumption average of the

eight hens/treatment, as calculated at the end
of each week of drug treatment . . . . . . . . . 142

xi

3.
4.
5.

LIST OF FLOW CHARTS

Total and free plasma cholesterol determination
by the Searcy and Bergquist (1960) procedure . .

Sample preparation for plasma triglyceride
analysis on the Technicon Autoanalyzer . . . . .

Egg parameters . . . . . . . . . . . . . . . . .
Excreta processing . . . . . . . . . . . . . . .

Triglyceride analysis by the Mendez gt El. (1975)
procedure 0 O O O O O O O O O O O O O C C C O O O

xii

Page

37

39
42

46

50

INTRODUCTION

The compound 2,2,9,9-tetramethyl-1,10-decanediol
(CI-720), has been found to be an effective antilipemic
agent (decreases plasma triglycerides and cholesterol) in
rats, mice, and rhesus monkeys (Parke-Davis, 1974). Because
of these results, the researchers at Parke-Davis have pro-
posed that CI-720 be used in humans to treat hyperlipemias
II, III, IV, and V of the Fredrickson classification. These
classes are outlined in a bulletin by the World Health
Organization (1973). The basis of this classification is
the use of blood levels of triglycerides and cholesterol as
a method of detecting and defining defects in lipid
metabolism.

Although much information exists which fails to support
the much commercialized saturated fatty acid/cholesterol
theory of atherosclerosis etiology (Pinckney and Pinckney,
1973; Kaunitz, 1975), these data are generally disregarded
(Stare gt_gl., 1974; Palmer, 1975). Hence, the degree of
cholesterol involvement in atherosclerosis is still
unresolved.

Nevertheless, there is a small portion of the human

population that does experience various pathologies of

lipid metabolism. These people may benefit from 01-720 or
other antilipemic compounds.

In the research reported in the present paper, 01-720
was administered to Single Comb White Leghorn laying hens in
order to determine if the antilipemic effects of this drug,
as observed by the Parke-Davis scientists, were also
manifested in the chicken. Other parameters of interest
were total yolk cholesterol, feed consumption, egg produc-

tion, egg component weights, and excreta cholesterol levels.

REVIEW OF LITERATURE

Cholesterol
1. Effect of 01-720

No research on the antilipemic effect of 01-720 has
been completed except that with the rat, mouse, and rhesus
monkey. Based on the findings from these species, Parke-
Davis Company researchers reported that CI-720 was about
ten-fold as active in decreasing plasma triglycerides as was
clofibrate®(1mperial Chemical Industries Inc., Ltd.). At
high levels (150 mg 01-720/kg body weight), 01-720 caused
plasma cholesterol to be lowered in the monkey and rat
(Parke-Davis, 1974).

The mechanism of action of 01-720 as an antilipemic
drug is unknown. This drug has not been found to increase
the fecal cholesterol content. In vivo, CI-720 has been
found to inhibit long-chain fatty acid incorporation into
triglycerides in the liver and plasma. 01-720 does not
prevent the incorporation of acetate into either sterols or

triglycerides.

2. General

A. A ents used in an attem t to lower lasma
cfioIesEeroI

Due to the general presumption of cholesterol as either
a predisposing or direct cause of atherosclerosis, several
drugs (which should include foodstuffs when used as such)
have been used clinically or in experiments with animals in
an attempt to lower plasma cholesterol. Obviously, research
is conducted in these areas for other reasons; for example,
to delineate the causes and cures of various diseases of
lipid metabolism such as xanthamatosis.

Table 1 lists a number of compounds or conditions which
alter the plasma cholesterol levels in various species.

B. Factors which may alterAplasma cholesterol,_or
atherosclerosis

I Table 2 lists some factors which are known to have an
effect on plasma cholesterol or atherosclerosis. In general,
age, stress, and immediate effect of exercise, increase
plasma cholesterol; while weight stability and continued
daily exercise, tend to maintain or lower blood cholesterol,
and at times, lower the severity of atherosclerosis. Some
researchers have found the Caucasian race to have higher
plasma cholesterol than the Negroid (Pinckney and Pinckney,
1973), and familial differences in plasma cholesterol level

are evident in animals and humans.

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Table 2. Factors which may influence plasma cholesterol or
atherosclerosis.

 

Item ‘ Reference

 

Exercise Ratcliffe and Cronin (1958)
Fisher and Leveille (1957)
Weight stability Pinckney and Pinckney (1973)
Palmer (1975)
Stress Kaunitz (1975)
Thornberry (1970)
Increase in age Weiss (1957)
Roberts and Straus (1965)

Being male or female Wood gt al. (1961)
Bartov 33 al. (1970)
Genetics Miller and Denton (1962)

Stare (1974)

Tissue versus blood '
cholesteroI Ievel Jurgens gt 21° (1971)

 

10

3. Specific Drugs and Other Compounds Used in Plasma and
_issue ho estero Level Manipulations

A. Cholestyramine

 

When Tennent gt 3;. (1961) used cholestyramine (MK-135)
to lower plasma cholesterol in the normocholesterolemic dog,
they found an additive effect of this drug with either of
the hepatic cholesterol synthesis inhibitors, MER-29 or
benzemalecene. In the normocholesterolemic cockerel, these
workers found that either MK-135 or benzemalecene decreased
plasma cholesterol; and when used together, these compounds
had an additive effect in lowering plasma cholesterol.

When Jones (1969) used cholestyramine, he found a highly
significant decrease in the plasma cholesterol of the hen,
but no change in the yolk cholesterol. This is in agreement
with the earlier work of McGinnis and Ringer (1963), who in
addition, found no change in body weight and an increase in
the number of light colored yolks when they used this
quaternary ammonium anion exchange resin.

Epley (1971) explained the decrease in plasma choles-
terol of cholestyramine-treated cockerels by way of a
decrease in cholesterol esters.

Epley and Balloun (1970) reported that when diets were
supplemented with cholesterol, MK-135 caused a reduction in
total plasma cholesterol and cholesterol esters of cockerels.
If there were no cholesterol supplementation, there was still
a decrease in the total cholesterol of the cockerel's blood.

The free cholesterol was not changed significantly by this

11

drug. In another trial by Epley and Balloun (1970), when
MK-135 was administered with the control diet, the serum
cholesterol was not significantly lowered. These latter
results are in disagreement with the other research, but are
supported by the findings of Reynolds 33 gl. (1971). Rey-
nolds and co-workers fed four hypocholesterolemic compounds
(triparanol, cholestyramine, 80-12937, or 80-10644),
separately, to diethylstilbestrol-treated (DESB) cockerels.
With the DESB treatment, all four compounds caused a
decrease in plasma cholesterol toward normal. Without DESB,
each drug caused only slight decreases in the plasma
cholesterol, which were dose dependent.

Tennent gt 3;. (1960) made a comparison of MK-135 with
another bile acid binding, polymeric organic base, MK-325.
They found that the effect of each compound was to inhibit
what they considered to be cholesterol-induced
hypercholesterolemia and aortic plaque formation in
cockerels. They also noted a decrease in plasma cholesterol
of normocholesterolemic cockerels and dogs that had been
treated with either of these drugs in another trial. As
evidence of the antiabsorbing action of these drugs on
cholesterol, Tennent 31 gl. (1960) also observed an increase
in fecal and biliary acid sterol levels in both species.
Tennent gt'gl. (1959) also reported the antihypercholes-

terolemic action of this drug in cockerels.

12

B. Clofibrate

When Herman gt al. (1970) fed ethyl-p-chlorophenoxy-
isobutarate (clofibrate) to humans, they found some subjects
to have a lowered triglyceride fraction of their plasma
lipids. The mechanism of action of clofibrate was re-
portedly not known. Because of an excess of lipids present
in their blood samples, especially triglycerides, the blood
of some individuals is excessively turbid. This extreme
blood turbidity was not corrected in all individuals by
using clofibrate. Although some people have been successful
in using a low carbohydrate type of diet to correct this
hyperlipemic condition, this type of treatment does not
work for all individuals.

IUsing rats, Thorp and Waring (1962) found clofibrate to
be the most active of the aryloxyisobutyric acids in de-
creasing total lipid and cholesterol in blood and liver.
They also found opposite effects within or between different
species when this drug was used. Clofibrate enhanced the
estradiol or testosterone reduction of plasma cholesterol in
rats. In the monkey and chicken, these same investigators
discovered that the increase in serum cholesterol due to an
atherogenic diet (which has been defined as one low in
protein and containing cholesterol) was exacerbated by
clofibrate. In rats, clofibrate plus androsterone (which
may function more as a metabolic regulator than as an

androgen) was effective in decreasing plasma and liver

13

lipids, whereas clofibrate or androsterone alone caused no
effect on the plasma or liver lipids.

According to Thorp and Waring (1962), clofibrate caused
no inhibition of the acute hyperlipemia or hypercholes-
terolemia in rats which were intravenously given the surface

active polymer, Triton WR-1339.

0., Triparanol

After feeding 1-(p-(beta-diethylaminoethoxy)-phenyl)-1-
(p-tolyl)-2-(p-chlorophenyl) ethanol (also called triparanol
or MFR-29, patented to the Wm. S. Merrell Co.) to 24-week-
old hens, Burgess et al. (1961) reported that triparanol
induced an 85% decrease in yolk cholesterol, and the re-
placement of this cholesterol with desmosterol. Also
observed after feeding the large quantity of MER-29 was a
decrease in egg weight, but yolk weight was not altered.

The ovaries of hens on the triparanol treatment declined
from a normal weight Of 69 g to 3 g Per ovary.

Wong et 3;. (1963) reported an increase in total serum
sterols, and the desmosterol component of the blood sterols,
in both cockerels and laying pullets given triparanol via
the feed. Desmosterol represented 76% of the total blood
sterols of triparanol-treated cockerels, while none was
detected in the blood of control cockerels. Desmosterol
was also present (67% of sterols) in treated birds aortas,
and not in control cockerels. The laying pullet's response

to triparanol was the same as the cockerels, but was more

14

pronounced. Triparanol stopped egg production in all drug
treated laying pullets. The degree and incidence of
atherosclerosis of the chicken aorta was increased by
triparanol.

Nelson gt al. (1962) fed laying hens triparanol in the
diet. The findings included: no alteration of Ca++ level
in the plasma; a decrease in egg size, production rate, and
yolk size; and an increase in plasma cholesterol by 90%.
Following withdrawal of this drug, the plasma cholesterol
level returned to normal. Perhaps the discrepancy with the
other findings can be explained as a difference in dosage,
as observed in the rat by Blohm gt 3;. (1959), or to a
mechanism of action for the increase in plasma cholesterol
similar to nicarbazin (Weiss, 1960).

Nichols gt al. (1961) fed two-week-old chicks a diet
including triparanol. They initially observed a decrease in
plasma cholesterol, then the cholesterol levels increased
about as often as they decreased, and showed no consistent
pattern. Growth was decreased with increasing amounts of
triparanol, and toxic levels were reached. The egg weight
of layers fed HER-29 decreased to five ounces/dozen. At
higher levels, Nichols and co-workers found egg production
ceased, with no change in serum or yolk cholesterol.

Blohm and MacKenzie (1959) found MER-29 decreased plasma
cholesterol by decreasing the incorporation of acetate-1-140
into cholesterol in the liver and intestine of rats. Blohm
gt 3;. (1959) reported this inhibition in plasma cholesterol

15

was specific for cholesterol, and occurred at a late stage
in the cholesterol synthesis pathway, after formation of a
steroid nucleus had taken place. Blohm 23,31. (1959) found
the plasma cholesterol of rats and monkeys to be decreased,
the rat having several tissues with lowered cholesterol.
Avigan.gtflgl. (1960) administered MER-29 to rats, and
discovered a decrease in serum sterols. There was
replacement of 27-79% of serum and tissue sterols with

24-dehydro-cholesterol.

4. Hormones

The site of carbohydrate and lipid metabolism in the
chicken differs markedly from that in mammals. Work by
Leveille 22.210 (1975) supported the view that the liver is
the primary site of lipid biosynthesis in the chicken, and
that adipose tissue is relatively unimportant here. In
addition, insulin was reported to increase free fatty acids
in the blood of fowl, which is opposite to most species.
Insulin may stimulate glucagon release, with the glucagon
being lipolytic in the chicken. Glucagon may be one of the
most important endocrine secretions controlling avian
metabolism.

According to Grande (1968), birds (geese, roosters,
ducks, and turkeys) given crystalline glucagon intravenously
showed prompt plasma increases in free fatty acids and sugar.
He also concluded that because catecholamines have not I

changed plasma free fatty acids of the domestic fowl, the

16

effect by glucagon probably is not mediated through
catecholamines (epinephrene) in birds. He referenced
several papers to report that glucagon produces a decrease
in plasma free fatty acids in the dog and man.

Goodridge (1968) reported that in pigeon adipose tissue
the lipase was stimulated by both epinephrine and glucagon;
but in the chicken, lipolysis was stimulated by glucagon and
not by epinephrine. He found no change in lipolysis
(free fatty acid and glycerol release to the bloodstream) in
the adipose tissue when insulin was administered to embryos,
7-8-day-old chicks, or to 28-day-old chicks. In these 7-8-
day-old chicks, insulin increased adipose tissue response to
the lipolytic action of glucagon was about ten-fold, but did
not alter the response of 28-day-old chicks. When treated
with glucagon, the chick embryo tissue showed only a slight
increase in lipolysis, but the other age treatments showed
marked acceleration of lipolysis.

Goodridge and Ball (1966) also reported that the
synthesis of fatty acids from glucose or pyruvate by pigeon
adipose tissue ($3,1itgg) proceeded at a low rate, and was
unaffected by insulin. They concluded that the adipose
tissue of pigeons may serve largely as a depositary for fat
synthesized elsewhere, probably in the liver.

Caldwell and Suydam (1960) found that exogenous
estrogen induced more rapid plaque formation in the blood

vessels of cockerels than did dietary cholesterol, and that

17

the degree of hypercholesterolemia did not determine the
extent of atherosclerosis.

Stamler gt 3;. (1954) suggested the increased resis-
tance to cholesterol-induced atherogenesis of mature egg
producing hens (intact or after oviduct ligation) was due to
their endogenous estrogen secretion. Pinckney and Pinckney
(1973) reported that this same Stamler advised male human
patients to treat their high blood cholesterol (and there-
fore supposedly arteriosclerosis) with estrogens. These
authors did not state which the men found more disconcerting,
the possibility of arteriosclerotic plaques, or the changes
that occurred in their voices and chests.

Thorp and Waring (1962) found that subcutaneous
implantation of estradiol or testosterone in the intact or
gonadectomized rat, caused a decrease in blood cholesterol.

Hardy 23 gl. (1962) found a statistical difference
between the serum cholesterol of high- or low-cholesterol
strains of three-week-old chicks, but that the response to
treatment with different hormones was similar. In one
experiment, the treatments were: control (safflower oil
carrier alone, was injected), cortisone, diethylstilbestrol,
or testosterone. There were no line by treatment inter-
actions. In a second experiment, estriol benzoate had a
significant line by treatment interaction in addition to
increasing serum cholesterol. Cortisone and thyroxine also

increased the serum cholesterol in the second experiment.

18

Weiss 31 gl. (1965) used D-thyroxine, which is about
1/5 as active as the L- form, to lower plasma and increase
egg yolk cholesterol. Weiss 23 gl. (1967) also reported
these results, and in addition they found that after a
change of about 30% in the plasma or yolk cholesterol, the
level of cholesterol of these parameters plateaued,
regardless of the amount of thyroxine administered.

In the thyroidectomized chick, both D and L forms of
exogenous thyroxine are capable of stimulating cholesterol
biosynthesis, release, and turnover in a manner comparable
to that in a normal chick (Lepp gt_gl., 1964). These
researchers also found that thyroidectomy of chickens with-
out iodoamino acid treatment would increase these chicks
serum cholesterol; and that iodoamino acid treatment with or
without thyroidectomy of the chicks did not change liver
cholesterol or liver weight versus euthyroid chicks. Lepp
and his co-workers reported the accepted mode of action of
thyroxine, and it's analogs, in cholesterol metabolism is at
the biliary excretion and/or degradation stage.

When Chung gt 3;. (1967) added diethylstilbestrol,
cholesterol, and/or various fats to the feed of cockerels,
they detected changes in the types of fatty acids which
were esterified to plasma cholesterol.

Sturkie (1965) mentioned that the method of
diethylstilbestrol administration was important in its effect
of increasing blood lipids. When injected or implanted,

19

diethylstilbestrol has the best action, but is relatively

ineffective when given orally.

5. Diet Effect on Blood Lipids

A. General observations

Lorenz gt‘gl. (1938) reported that in laying hens
maintained on very low fat diets, enormous concentrations of
lipids may appear in their blood, and that the total fatty
acids and free cholesterol varied with the dietary fat level.
In the immature female bird, dietary fat level did not change
the various plasma lipid levels. The male chicken showed an
increase of cholesterol ester when fed a high fat diet; but
phospholipid, neutral fat or free cholesterol were not
changed by dietary fat level. Lorenz and his associates
showed that the laying hen experienced a decrease in
variability in neutral fat and free cholesterol when given a
high fat diet (by replacing carbohydrate), and reported that
an interaction existed between dietary fat and ovarian
activity.

Leveille gt gl. (1975) reported a decrease in hepatic
lipogenesis of chicks fed a diet high in fat or protein
(at the expense of carbohydrate).

Price 23 gl. (1957) found that 0-14% poultry oil in the
diet of caged or floor raised layers did not change the hen's
plasma cholesterol level.

Dietary supplementation with menhaden oil, when fed to

laying hens, caused a cessation of their egg production

20

(Weiss 33 al., 1967). They also found that safflower oil at
7.5% or 15% of the diet had little effect on egg choles-
terol concentration, but at 30%, there was an increase in

the egg cholesterol.

B. Egg in the diet

Corn oil, corn oil low-melting margarine (which would
be high in unsaturated fatty acids), butter, beef tallow, or
pork lard fed individually to rats at 10% of the basal diet,
all increased serum cholesterol. Males were affected by
these treatments more than females. Wachholz (1972) also
found that there was no difference between the effects of
fat sources or levels of supplemental cholesterol on the
rat's serum cholesterol. Cholesterol was supplied either as
2% of the diet in the crystalline form, or as 10% of the
diet as whole egg.

Pair-fed cockerels given 10% of the diet as dried whole
egg had lower plasma cholesterol-ester and plasma total
cholesterol, and gained more weight than those given
crystalline cholesterol plus soy oil dietary supplementation
(Epley and Balloun, 1970). These workers found no
difference in the severity of atherosclerosis which resulted
from the dietary supplementation of either coconut or soy
oil.

El-Maguid and Quisenberry (1968) fed dried whole egg to
laying hens, the amount in the diet was equivalent to 1, 2,
4, or 10 eggs/man/day. The hens had lowered plasma

21

cholesterol at the high egg level, and only slightly in-
creased plasma cholesterol at the lower levels. Yolk
cholesterol did not change. Two caloric levels for the
diets were used, and with the high egg supplementation, the
hen's performance was improved at each caloric level, versus
the other egg treatments. Performance was measured by
increased egg production, increased feed efficiency, and
decreased mortality. El-Maguid and Quisenberry (1968) did
not report the fecal sterol level, but perhaps the reason
for the lowered blood cholesterol level at the higher dosage
of egg equivalents was that: the excess cholesterol was in
such a large amount that a portion was not absorbed
(Heftman, 1970); and/or the amount of cholesterol that was
absorbed from the high egg equivalent diet was sufficient to
completely shut down the liver cholesterol production at the
beta-hydroxy-beta-methyl glutaryl-Co A step of cholesterol
8yrithesis. The sum of blood cholesterol from exogenous and
erldogenous sources then was not enough to maintain the
Previous level of the hens treated with the high number of
egg equivalents. The cholesterol provided by the lower
leVela of dietary egg equivalents could have been enough to
inhibit a portion of the cholesterol synthesis by the liver,
the overall sum of plasma cholesterol was a biological
rather than a pharmacological type of increase over the
PreVious level. Additional results from the previously
mentioned work of Epley and Balloun (1970) support the
findings of El-Maguid and Quisenberry.

22

0. Crystalline cholesterol supplementation to the diet
Table 3 summarizes results of several researchers who

added crystalline cholesterol to various feeding regimes.

6. Sitosterol and Other Plant Sterols

Weiss gt El- (1967) cited a personal communication with
Dr. T. A. Miettinen to report that campesterol, stig-
mosterol, and beta-sitosterol totaled 1.2% of the sterols in
a batch of commercial eggs.

Clarenburg gt al. (1971) found layers to have lower
intestinal absorption of sitosterol than did non-layers (60%
versus 85% absorption, respectively), and discovered a 35%
decrease in egg yolk cholesterol content as a result of
dietary sitosterol supplementation. This loss of yolk
sterol was replaced by beta-sitosterol. These workers also
reported that plasma sterols were decreased with
sitosterol in the diet.

Bartov 33 gl. (1971) concluded that the response
difference of laying hens cholesterol levels to dietary soy
oil, coconut oil, and safflower oil was not due to the
different oil's sterol content. Dietary coconut or
safflower oil significantly increased the yolk cholesterol,
but the soy oil had no effect. In another series of trials,
only coconut oil increased the laying hen's plasma
cholesterol. These researchers stated that "it appears that
plant sterols do not interfere with the cholesterol meta-

bolism in the laying hen". Their results support the

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24

assumption that the anticholesterolemic effect of plant
sterols is based mainly on the inhibition Of cholesterol
absorption rather than the metabolism of cholesterol.

Diller gt'al. (1960) found that dietary beta-sitosterol
fully returned the cholesterol-induced hyperlipemia of
chickens to normal. Also, at 4% of the diet, beta-
sitosterol prevented or reversed an increase in lipid and
cholesterol concentration in the liver and aorta of these
chickens.

Weiss 33 al. (1965) found that 1% beta-sitosterol in
the diet decreased hens blood cholesterol when given a
cholesterol-containing diet.

Weiss et_§l. (1967) reported that in normal laying
hens, 1% dietary beta-sitosterol had little effect on blood
or egg sterol concentrations. They again reported that 1%
beta-sitosterol in the diet would decrease egg and blood
cholesterol if the hen was given a cholesterol-containing
diet. The egg cholesterol was increased 60% when the hen
was fed cholesterol at 1% of the diet. They found no
detectable beta-sitosterol in the egg of hens fed 1% beta-
sitosterol and 5% lecithin, or safflower oil as 30% of the
diet, but attributed this absence to technical error.

Boorman and Fisher (1966) reported that with no dietary
sterols, all sterols present in the egg were cholesterol.
Also, when hens were fed a diet having a mixed sterol con-
tent, cholesterol was preferentially absorbed from the gut

versus phytosterols; and that more campesterol was absorbed

25

than beta-sitosterol. The extent to which absorption of
plant sterols occurs is in part dependent on the fat con-
tent of the diet, plus phytosterols and cholesterol com-
petitively inhibit the absorption of the other.

Konlande and Fisher (1969) showed evidence for a non-
absorptive antihypercholesterolemic action of phytosterols
in the chicken. Campesterol appeared as the major active
component of soy and wheat sterols in relation to the

antihypercholesterolemic effect of these sterols.

7. Turnover of Cholesterol

In his review, Kaunitz (1975) stated that the
cholesterol turnover in the body of man was 1000-2000mg/day.
The plasma turnover amounts to about 1000 mg/day. The
usual American diet supplies 300-800 mg cholesterol/day,
of which only about 150-300 mg are absorbed. About 2% of
the tissue cholesterol is used in steroid hormone
synthesis in humans.

According to Andrew et al. (1968), the laying fowl has
two main excretory pathways for the elimination of
cholesterol. These pathways are the egg formation mechanism
and the feces. They showed that there was a plasma
"isotopic steady state" in hens that were orally administered
labelled cholesterol. They used the steady state data to
conclude there is little ovarian cholesterol synthesis.

Andrew and co-workers calculated the half-life of

26

cholesterol in the laying hen to be about 36 hours, and
stated the cholesterol half-life in humans and dogs was

8. Summaries of Cholesterol or Atherosclerosis

Kaunitz (1975) has reviewed atherosclerosis etiology,
and has presented alternate theories to those currently
being presented.

An atherosclerosis monograph guest edited by Stare
(1974) and provided by Best Foods, Inc., a division of
CPC (Corn Products Corporation) International, dealt mostly
with lipid metabolism in adult humans, and was obviously
biased toward an unsaturated fatty acid cure-all for
atherosclerosis.

Palmer (1975) also discussed dietary aspects of human
atherosclerosis. She attempted to link intake of
cholesterol and saturated fatty acids with the morbidity and
mortality from coronary disease.

A book by Roberts and Straus (1965) categorized
atherosclerosis according to animal types and humans, giving
differences between them in many aspects of this disease.

Page (1954) summarized investigational and clinical
trends of the research concerning atherosclerosis.

Kritchevsky, as well as Cook, has written a book (both
in 1958) which dealt with cholesterol history and various
aspects of cholesterol metabolism.

Pinckney and Pinckney (1973) have written a book which

is understandable by the lay-man concerning the role of

27

cholesterol and saturated fatty acids in atherosclerosis.
They expose fallacies of the theory that "normal" amounts of
cholesterol or saturated fatty acids are detrimental to ones
health. They also detail some of the politics behind this
cholesterol controversy.

Recently Brown and Goldstein (1976) offered their
explanation for cholesterol metabolism and control.

The World Health Organization (1973) issued a helpful
memorandum which classified types of hyperlipidemias and

hyperlipoproteinemias.

Atherosclerosis

1. Effect of Hormones

Stamler gt 2;. (1954a) reported that in the male
chicken, cortisone caused a small increase in blood pressure
and an intensification of atherosclerosis, but was rela-
tively inactive as a glycocorticoid. They also found that
hydrocortisone caused no increase in atherosclerosis (aortic
or coronary artery) or hypertension of cockerels with
steroid-induced diabetes and hyperadrenalism, which was
associated with an enhancement of hypercholesterolemic
hyperlipemia. ACTH (corticotropin) action was the same as
that of hydrocortisone.

2. Plagues
A writer for the monograph guest edited by Stare (1974)

stated that the first step in the atherosclerotic process is

28

the accumulation of lipid deposits in smooth muscle cells
that have begun to proliferate in the intima.

Fox (1938) reported that the simplest atherosclerotic
change is intimal hyperplasia. Some animals with the worst
cases of atherosclerosis had lived the longest and did not
die from atherosclerosis. Furthermore, he found differences
between birds and mammals in the type of arteriopathy they
exhibited, and that the atherosclerotic type vascular
changes occurred in all animals with increasing age.

Palmer (1975) suggested that plaque lipids are derived
chiefly from serum lipids and accumulate and stimulate an
initial change in smooth muscle proliferation.

Palmer (1975) and Fox (1938) both mentioned that the
primary atheroma sites are those where blood flow is turbu-
lent and damage to epithelium is mechanically more probable.
The theory of Kaunitz (1975) that cholesterol increase in an
area is part of the body's response to injury, would seem
applicable here. Fox (1938) stated that in the bird, the
area just above the heart is the point in the vascular
system stressed most, and perhaps more in the chicken than
in any other animal. ,

' Kaunitz (1975) indicated that cholesterol may be
present in huge amounts in some pathological conditions, as
in scars, fibroids, and granuloma, and that cholesterol is
usually associated with Ca++ in atherosclerotic plaques.

Feizenbaum.gt El- (1961) stated that day-old-male

chicks on a coconut oil plus cholesterol supplemented diet,

29

"showed a clear trend toward greater atherosclerosis" than
those fed corn oil plus cholesterol type diet. The
atherosclerosis observed in the chicks was most prevalent
in the abdominal section of the aorta, versus the thoracic
segment. These researchers also reported that a low
protein diet and/or dietary cholesterol caused an increase
in plasma and aortic cholesterol.

Thorp and Waring (1962) cited a personal communication
with R. S. F. Campbell g§_§l. (1960) to report that in the
monkey, aortic atheroma increased in proportion to the rise
in serum cholesterol; but in the chicken, aortic or
coronary atherosclerosis was not changed by an increase in
serum cholesterol.

Ratcliffe and Cronin (1958) have proposed a hypothesis
of atherosclerosis etiology which states that social
pressure may create a stress, and thereby an imbalance of
adrenal secretions. These secretions would then cause the
plaque formation. They reported that atherosclerosis
presence or absence was independent of age or diet, but
was associated with an increased population density of
various animal species in a zoo. Their hypothesis was based
on conclusions they drew from data of 55 years of autopsy
records of these animals and birds.

In Table 4 are listed factors that may or may not alter
an organism's susceptibility to circulatory pathologies.

30

 

Table 4. Factors which may or may not alter different
species chances of circulatory disease.
Item Reference

 

Ascorbic acid; water
hardness; diet fiber

High calorie diets

Egg consumption

Alcohol (drinking)
011 type in diet

Blood pressure

Cholesterol biosynthesis
in the aorta
Vitamin A

Diet with or without
cholesterol

Palmer (1975)
El-Maguid and Quisenberry (1968)

Epley and Balloun (1970)
Fisher 21 al. (1963)

Kritchevsky (1958)

Epley and Balloun (1970)
Swell 33 a1. (1960)
Chung gt KI. (1965)
Banerjee e: 31;. (1965)
Fisher et a1. (1963)
Kaunitz—(1975)

Krista 23 a1. (1970)
Miller and—Balloun (1968)
Eisley and Pritham (1955)
Azarnoff (1958)

Bayer 212 al. (1972)

Fisher at El- (1961)
CaldwelI-and Suydam (1960)

 

OBJECTIVES

The purpose of this study was to discern the effect of
CI-720 (an antilipemic agent in other species) on the
following parameters of the Single Comb White Leghorn
laying hen:

1. Total and free plasma cholesterol

2. Plasma triglycerides

3. Total yolk cholesterol

4. Body weight

5. Percent egg production

6. Total egg weight; plus component weights and their

per cent of total egg weight

7. Feed consumption

8. Excreta cholesterol

31

MATERIALS AND METHODS

Experiment 1
1. General Comments

Twenty-four Single Comb White Leghorn (SCWL) hens in
their first year of lay were brought from the Poultry
Science Research and Teaching Center to a controlled-
environmental cage room maintained at about 22°C. The hens
were allowed to acclimate in 40 x 20 x 40 cm individual wire
cages before being randomly assigned to treatments. The
hens were housed in these same type cages during the
experiment. Light was provided 17% hours each day (0630 to
2400 hours).

Feed and water were available fig libitum. Care was
taken, by the treatment space allocation, to avoid cross
contamination of feed. Individual daily feed intake was
recorded, usually before 0800 hours.

The diet used was MSU Ration 72-10 (Table 5), which was
blended in a Mix Mill model 911 A2 nutri blender.

For the drug feed treatment, 01-720 was blended upward
at 2600 ppm with a portion of the control diet. A tumble
type mixer was used for the final blending.

During the pre-treatment period, all placebo capsules
were partially filled with corn starch and collectively

32

33

Table 5. MSU Breeder Ration 72-10

 

 

Ingredient Parts/1000
Corn, #2 yellow 576.2
Soybean meal, 48% 200.0
Meat and bone meal 30.0
Alfalfa, 17% 40.0

Tallow, stabl.

Methionine hydroxy analog
Dicalcium phosphate
Limestone

Salt, iodized

Choline chloride, 50%
Vitamin mix

Mineral mix

U'l

m4

wumwtomov:
O

OOOOOOGiO

.1

Vitamin mix (supplies per kg diet): Vitamin A--1O OOO I.U.;
Vitamin D--1,000 I.C.U.; Vitamin E--10 I.U.; Vitamin K-
-4.0 mg; Thiamine--3.0 mg; Riboflavin--10.0 mg;
Pantothenic acid--15.0 mg; Niacin--100.0 mg;
Pyridoxine--6.0 mg; Biotin--150.0 mcg; Folacin--3.0 mg;
Vitamin B12--15.0 mcg; Ethoxyquin--125.0 mg; Dist. dr.
solubles* to 5.0 g.

Mineral mix (supplies per kg diet): Manganese--55 mg:
Magnesium--500 mg; Iron-—80 mg; Copper--11 mg;
Zinc--80 mg; Dist. dr. solubles* to 5.0 g.

*with 4% corn oil.

Specifications: Ca--%. 3.5; P--%, 0.59: Fat--%, 8.3;
Fiber--%, 2.8; Meth.--% of protein, 2.0;
PrOtQ/EO, 5083; PrOtein--%, 1609; 39000 1:03.]. MoEo/
kg diet.

 

34

stored in a clean, dry screw-cap bottle. Drug treatment
capsules were packed with 01-720 at 150 mg CI-720/kg body
weight. During the first week of drug treatment capsules
were filled with drug, based on the weight recorded for
respective birds, for the first day of pre-treatment.
Successive weekly individual body weight determinations were
used to calculate the drug dosage for that hen in respective
treatment weeks. Drug capsules for a treatment week were
stored in containers, individually labelled for each hen.

All capsules used were size 1, made of gelatin, and
produced by the Eli Lilly Company.

The new drug level in the capsules started the day of
body weight determination, at the end of each treatment
week.

The daily capsule was administered to the appropriate
hen by pushing it into the esophagus, aided by water as a
lubricant.

Egg production was recorded at approximately 1600 hours
daily. All eggs were marked with the corresponding date and
cage number and stored at 4°C until processed for total yolk
cholesterol, total egg weight, and yolk, albumen, and
shell weights. Evans 33 9;. (1967) found no consistent
change in lipid distribution of eggs after storage for six

and twelve months, versus fresh eggs.

Twelve hens with egg production over 75% were selected

and randomly distributed to treatments, namely: control

35

feed; drug feed (CI-720 0 2600 ppm); control per 25
capsules containing starch); drug pg; gg (CI-720 in
capsules at 150 mg/kg body weight). Three hens were in
each treatment.

Five ml heparinized blood samples were withdrawn from
the brachial vein of each hen, between 0800-1000 hr. on
15 May (day zero of treatment with drug), and days 4, 7,
14, and 21 of treatment, and on day 7 of the post-treatment
period. Body weight was also determined at these times
(118).

After all blood samples were withdrawn, they were
immediately centrifuged at 2000 rpm (using an International
Centrifuge model SBV size 1) for thirty minutes.

An aliquot of plasma was aspirated to a screw cap tube
and diluted one to ten with acetone:ethanol (1:1), according
to the method of Searcy and Bergquist (1960), for each
sample. The diluent was then stored in a refrigerator at
5°C until processed for total and free cholesterol. Two
dilutions were processed for each hen's plasma sample at
each data collection date.

Plasma samples for triglycerides were processed
following the method of Technicon Instruments (no date
noted), at the same time as the total and free plasma
cholesterol determinations were made.

In order to determine the total cholesterol content,
total excreta was collected on days 15 through 21 of drug

treatment.

36

IClass A Mohr pipettes plus PropipetQ§>(Spectronics
Corp.) were used for most volume transfers, except the
ferrous sulfate-acetic acid reagent and the concentrated
sulfuric acid of the Searcy and Bergquist procedure color
reaction. For these latter volume transfers, a RepipetCE)
(Lab-Industries), or a glass syringe plus needle was used
for the injection of reagent into the test tubes,

respectively.

2. Total Plasma Cholesterol
Flow Chart 1 outlines the Searcy and Bergquist (1960)
procedure used to determine the total and free plasma

cholesterol.

3. Free Plasma Cholesterol

See Flow Chart 1 for this procedure.

4. Plasma Triglycerides

After processing the plasma to a stop point, the samples
were stored in refrigerators at 18°C or 1°C. The modified
version of the Technicon Auto-analyzer method (no date noted)
that was followed is outlined in Flow Chart 2. Due to
analytical difficulties, most sample data were discarded.
Meaningful statistical analysis of the remaining samples
was not possible. The values determined for the remaining

data are included in the appendix for those interested.

37

Flow Chart 1. Total and free plasma cholesterol determina-
tion by the Seardy and Bergquist (1960)
procedure.

 

.3 ml plasma + 2.7 ml acetone:ethanol (1:1)

thoroughly mix

centrifuge 10 minutes at 2000 rpm

aspirate supernatant

discard precipitate

store excess at 5°C

Total Choleéterol
0.4 ml to test tube

add 6.0 no FeSO4-acetic
acid reagent

add 2.0 ml Concentrated
H2304 .

wait at least 10 minutes
for color development

read absorbance of unknowns
against a 0.4 ml acetone:
ethanol (1:1) + color
reagent blank*

determine sample mg/dl
cholesterol from the
regression line of
standard increments
read**

Free Cholesterol

1.0 ml to a cOnical
centrifuge tube

add 1.0 ml digitonin sln.

thoroughly blend on a
vortex type mixer

wait at leastoone hour
for precipitate forma-
tion

centrifuge 10 minutes
at 1700 rpm

aspirate supernatant

resuspend preCipitate in
ca. 2.0 ml acetone:etha-
nol (v/v)

centrifuge 5 minutes at
1700 rpm

aspirate, discard
supernatant

 

Flow Chart 1 (cont'd.).

38

 

Free Cholesterol
(cont'd.)

dissolve precipitate in
6.0 ml FeSO4-acetic acid
reagent

add 2.0 ml concentrated
H2504 .
determine optical
density as in total
plasma cholesterol
determination

 

*All total or free cholesterol determinations of either
experiment were made on a Hitachi Perkin-Elmer Model 139
UV-VIS Spectrophotomer, the wavelength was set at 490 nm

**Standards were prepared by adding known amounts of
crystalline cholesterol to known volumes of acetone:

ethanol (1:1)

39

Flow Chart 2. Sample preparation for plasma triglyceride
analysis on the Technicon Autoanalyzer.

 

add 0.3 m1 plasma to 5.7 ml isopropanol*
in a screw cap tube

cap

0
vortex 30 sebonds
0

add 1-1.2 g zeolite mixture

cap

vortex 30 seconds

let stand 30 minutes

during this time, invert tube and gently
resuspend mixture three times

centrifuge tube at 2000 rpm for 10 minutes

0
O
0

store

 

*redistilled at 82°C to 85°C

4O

5. Egg;Parameters

Eggs were removed from storage and segregated by hen,
for the entire experiment. The procedure used for selecting
eggs to be processed was: the egg laid on the day of blood
sample collection was used when possible. If no egg was
laid on that date, the egg from the following day was used.
Again, if there was no egg from that date, the egg laid
the day preceeding blood withdrawal was used. If none of
these conditions could be met, the egg closest to the blood
sampling date was chosen (randomly if there were two eggs
the same distance from the data collection date).

0n the rare occasion the yolk broke during its
separation from the albumin, a pasteur pipette with the tip
filed off at the base was used in conjunction with a
Propipet®to salvage enough uncontaminated yolk for a total
yolk cholesterol determination.

A single procedure for the determination of total yolk
cholesterol was not used. Instead, the modified Zlatkis
method of Weiss gt 9;. (1964) was utilized through the
filtering and dilution phase, then the extraction and
saponification of cholesterol was as reported by Abell gt 5;.
(1952). The color reaction and total cholesterol determi-
nation of Searcy and Bergquist, as previously discussed, was
followed for the final steps. There was also limited input
by other researchers. For instance, Brown (as cited by
Weiss etugl., 1964), stated that "a wide range of conditions

may be used for saponification in a constant volume without

41

necessarily altering the accuracy of the determination of
blood cholesterol" and suggested "saponification at 65°C
for one hour with the same concentration of alcoholic
potassium hydroxide used by Abell 33.3l. (1952), in order
to be certain that all the cholesteryl esters present were
saponified". Also, Fisher and Leveille (1957) did not wait
between the addition of chloroformtmethanol (at 2:1) to the
yolk, and the subsequent filtration. Weiss gt_gl. (1964),
however, waited several hours. For these experiments, the
yolk/solvent mixture stood about one hour before being
filtered. The recovery for this combination of procedures
was 102%.

The column used in the filtration of denatured protein
from the solvent was a 22 x 450 mm chromatography column,
having a tapered tip and sintered glass filter immediately
above the taper. The tip was fitted with a cork, which was
firmly inserted into a 125 ml microfiltering flask with
tubulation. The apparatus was held in place by a Thomas
support stand and utility clamps. A vacuum was used to
facilitate the filtering process.

Flow Chart 3 shows the method used to determine total

yolk cholesterol, plus egg and component weights.

6. Excreta Cholesterol
The excreta from individual hens was air dried about 16
weeks. Then the feathers and other extraneous material were

separated from the excreta and discarded. The excreta was

42

 

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43

Flow Chart 3 (cont'd.).

 

thoroughly stir, scrape
inner surfaces of beaker
let stand one hour
pour beaker contents into column

rinse beaker and column
to a final filtered
volume of 90-95 ml

rinse flask to ca.
100 ml

bring to 100.0 ml in a
graduated cylinder

thoroughly mix

0
store excess

' tart procedure
a transfer 0.5 ml to s
at 13 C glass stoppered ?¥9g?§ll 53 El"

centrifuge tube

add 5.0 ml freshly prepared
alcoholic KOH

stopper andfshake well
incubate in a hot water béth at 60°-65°C for 1 hour
cool to roomftemperature
add 10.0 $1 hexane*

thoroughly mix

CONTINUED

 

44

Flow Chart 3 (cont'd.).

 

add 5 ml water

shake vigorously for 1 minute

centrifuge at slow Speed for 5 minutes

transfer hexane layer to storage bottle

0
O....0...O...0.0.00.00.00.00...0.0.0....0..
O C
C 0

cap and seal transfer 1.0 ml to
3 test tubes (tripli-
store at 13°C °at°)

a stream of filtered,
compressed air aided
the evaporation of

the aliquot in water
bath--60°0

cool to room
temperature

use the Searcy and
Bergquist (1960)
procedure for the
color reaction as
described in the
total plasma cho-
lesterol determina-
tion (Flow Chart 1)

 

*68.7 ;,1.4°C boiling point

**Recovery was 102%--see Table 2 of Appendix for all
recoveries.

45

then weighed to 1 .01 g. The total amount of excreta was
ground to a fine consistancy in a hand operated meat
grinder. The product of this grinding was transferred to
Whirl Pak® (NASCO) bags and stored at room temperature
until processed for total cholesterol. The excreta cho-
lesterol was extracted in a manner similar to that of
Fisher gt gl. (1961). Flow Chart 4 outlines the steps used

in this process.

7. Statistics

A split plot method of analysis (Gill, personal
communication) was used for all information with full
replication. These data were processed using a computer
program. As a check of the computer results, the plasma
total cholesterol data were manually analyzed by this
method. The appendix contains a table comparing these
results.

The data put on the computer were: 1. total plasma
cholesterol 2. free plasma cholesterol 3. percent free of
total plasma cholesterol 4. change in total plasma choles-
terol using the pre-treatment value as 100% 5. same as 4, but
for free plasma cholesterol 6. body weight 7. percent change
in body weight by week 8. percent egg production 9. change
in percent egg production.

The rest of the data were highly unbalanced and were
analyzed statistically using a one-way ANOVA for each time

period, in an attempt to segregate time trends.

46

Flow Chart 4. Excreta processing.

 

add 10+.01 g processed excreta
to a 300 ml erlenmeyer flask

add 100.0 ml CH013:Me0H (2:1) to flask

seal flask with a cork
plus parafilm wrapping

shake ca. 24 hours on a
mechanical shaker

filter supernatant only

rinse funnel and filter with a
portion of the 100 ml solvent

bottle filtrate and add remainder of Solvent
rinsings to flask
store at 13°C shake to total of 48 hours,

counting from start of the
first shaking period

filter contents of flask

measure total filtrate

thoroughly mix

evaporate a 1.0 ml sample
of filtrate at about 60°C,
using a stream of com-
pressed, filtered air
color reaction and total
cholesterol determination
by the Searcy and Berg-
quist procedure (Flow
Chart 1 )

 

47

When an f-value indicated possible differences in
treatment means at a date, the f-max test (Gill, personal
communication) for homogenous variance was used; if variances
were equal, the Tukey HSD test was used, and if unequal, the
Dunnett test of means was a better one to use. For
unbalanced data within a time period, a modified Tukey HSD
test of means was followed. All statistical tests were from
personal communication with Gill, and are summarized in the
appendix, as is the split plot model and sums of squares

formulas.

Experiment 2
1. General Comments

The preparation and physical environment of the SCWL
laying hens Studied were as in experiment 1. The animals
were in their first year of production, and from two
different strains. Hens known to be in laying condition were
selected and placed on three dietary treatments, according to
the previous 13 days egg production. Hens from each level of
production were placed in each treatment, ensuring that each
group started with hens of similar production. Eight hens
were used per treatment. '

The control diet was the same as in experiment 1. The
low drug treatment was the control diet plus 01-720 added at
2600 ppm. The drug was blended upward with control diet, and
mixed with an amount of control diet sufficient for the

entire experiment in the nutri blender described in part one.

48

The high drug treatment level had 01-720 at twice the
I concentration (5200 ppm) as the low drug treatment feed,
and was prepared in the same manner.

Using values of total plasma cholesterol, egg produc-
tion, and other factors, the end of the third week of drug
treatment was determined to probably be the best time to
sample the blood of hens for this one-time plasma choles-
terol measurement.

Only a weekly mean treatment value for feed consumption
was recorded; at this time, the body weight was also
recorded (3 1 g). The birds were generally fed before
0800 hr. daily, with egg collection at about 1600 hr. each
day. Eggs of the collection week (days 15 through 21 of
drug treatment) were marked with the date and cage number and
stored with the excess eggs from experiment 1. All other
eggs from the drug treated groups were destroyed.

Between 0800-1000 hr. on day 21 of drug treatment, 10 ml
heparinized blood samples were taken either from the brachial
vein or by heart puncture from all hens of this experiment.
The blood was centrifuged directly after all samples were
taken, as in experiment 1, and the plasma frozen in indivi-

dual screw-cap tubes.

2. Total and Free Plasma Cholesterol
Total and free plasma cholesterol were determined as in

experiment 1.

49

Procedural changes from experiment 1 were: 1. all
volume transfers 1 ml and under were made using Micro-
pettors® (Scientific Manf. Industries), in place of
pipettes, whenever possible; 2. a Repipet®was substituted
for the syringe and needle, with beaker reservoir, used for

the concentrated sulfuric acid of the color reaction.

3. Total Yolk Cholesterol

Total yolk cholesterol was determined as in experiment 1.

4. Plasma Triglycerides

Plasma triglycerides were analyzed by the method
reported by Mendez gt_§l. (1975), which is diagrammed in
Flow Chart 5.

Rather than the tri-olein standard Mendez and his co-
workers recommended, corn oil in isopropanol was used. Corn
oil is about 97% compounds which have a glycerol base. The I
glycerol moiety is the part reacted and determined in this
procedure, so the decision was made that this degree of

purity was satisfactory.

5. Egg Weight; Shell, Albumen, and Yolk Weights

These parameters were all determined as in experiment 1.

6. Statistics

Because low egg producing hens were thought to be
affected more by the drug for some traits than were high
producers, the blocking of hens on egg production does not

constitute valid blocking. The reason is that a basic

50

Flow Chart 5. Trigl ceride analysis by the Mendez gt gl.
(1975 procedure.

 

blank* standards* unknowns
add 0.5 ml water add 0.5 ml of each add 0.5 ml un-
' standard to the known plasma

respective tube

add 0.5 ml water

add 2.0 ml heptane

add 5.5 ml add 3.0 ml add 3.5 n1
isopropanol isopropanol isopropanol

add 1.0 m1 dilute H

2504
mix 30 seconds on a vortex
type mixer

let phases separate without
centrifugation

transfer 0.2 ml heptane layer to a
clean dry screw cap tube

add 2.0 ml isopropanol

add 0.6 ml saponification reagent

thoroughly mix

let stand at room temperature
not less than 5 minutes

add 1.5 ml sodium metaperiodate
reagent

CONTINUED

 

51

Flow Chart 5 (cont'd.).

 

thoroughly mix

add 1.5 ml acetylacetone reagent

thoroughly mix

cap each tube

place in’hot water bath (65°-70°C)
for not less than 15 minutes

cool tubes to room temperature

read absorbances at 415 nm,
within 45 minutes**

determine unknown sample values
from the regression line of
standard optical density readings

 

*15 ml screw cap tubes were used for all observations

**used the previously described Spectrophotometer
(Flow Chart 1)

52

premise of blocking has been violated; that a nuisance
variable not interact with treatments. All data were
analyzed as a one-way analysis of variance (anova). If the
f-statistic was significant, the f-max test for homogenous
variance was applied. For equal variances, the Tukey HSD
test of means was employed; if unequal, Dunnett's test of
means was used. Unless noted to the contrary, the Tukey HSD
test was the statistical analysis used.

For all the egg data, there was not balanced replica-
tion, so a modified Tukey HSD test of means was used, as in
the first experiment. In addition, the values for the only
egg from the high drug treated group of hens was in the
midst of those from the low drug level. Therefore, the
values from drug treated hens were combined, and the
statistical analysis done as a comparison of the control
group of hens versus the hens in the combined drug

treated groups.

RESULTS AND DISCUSSION

Total Plasma Cholesterol
1. Experiment 1

According to the split plot statistical analysis of the
data, the presence of CI-72O significantly decreased
(I“<.O46) the hen's total plasma cholesterol, from 133 to
114 mg/dl plasmali 5.6 as the experiment standard error of
the mean.

Figure 1 shows a comparison of the plasma cholesterol
treatment means from hens on sample dates of experiment 1.
From this graph, one can see a possible reason for the
significance of a drug effect. The three hens on the drug
pgg_g§ treatment started out with low total plasma choles-
terol values, and did not change much. This would cause
the final mean for the drug treated hens to be predictably
lower than the final mean for those hens not given the drug.
When these drug treatment means did change over time, their
respective control values generally changed in a similar
manner, so there was no interaction over time between the
treatments.

When the hen was used as her own control (the choles-

terol levels expressed as a percent of their respective

53

54

Figure 1. Experiment 1. Total plasma cholesterol in mg/dl.
Values represent the mean of three hens per
treatment. The standard error for the

experiment was 5.7.

laSt day of pre-treatment

day 4 of drug treatment

day 7 of drug treatment

day 14 of drug treatment

day 21 of drug treatment

'13 t=J U 0 w >-
II

a day 7 of post-treatment

 

nus-oeso-uao.-.ovo

 

 

 

loses-e

 

55

n I a . c e c . . . ..
u e c n a s o a o n
c .

        

 

u a n c u n c u a
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a a a o c n c u n
I .

 

  

I70

Echo—a 529330.050 .22 GE .

 

 

ABCDE F ABCDEF ABCDEF

ABCDEF

Drug Per 05

Drug Feed Control Peros

Treatment

Control Feed

Treatment

Treatment

Treatment

(Cl-720 a1 (Cl-720 at

(CI-720 a1

(cu-720 01 Oppm)

150 mQ/kg bod
weighty

0mg/kg bod
weighty

2600 ppm)

LeHer code on opposite page

56

pre-treatment level) no difference could be detected between
any of the four treatments, for any split plot factor.

The values for total plasma cholesterol of hens in all
treatments were examples of considerable biological

variation.

2. Experiment 2

Statistical analysis of treatment means by the Tukey HSD
test of means indicated that hens of the control group had
higher total plasma cholesterol than hens of either of the
drug treated groups of hens (P<<.01).

In Figure 2 are shown the total plasma cholesterol
level from hens in each treatment. In general, there was
little difference between the total plasma cholesterol level
of hens in either of the drug treatments. Two hens of the
control treatment are shown to have total plasma cholesterol
values close to the means of the drug treated groups.

Total plasma cholesterol values for individual hens
were, in general, higher in the second than in the first
experiment. For example, the mean of all the control hen's
values were 137 and 155 mg/dl plasma for experiments 1 and
2, respectively, for total plasma cholesterol. Possible
explanations are, that mixed strains were used in the second
experiment, and all hens were of the same strain and age in
experiment 1. Also, these values may have been evidence of
seasonal variation, as Weiss (1957) suggested. He

additionally listed reproductive state, diet and analytical

57

Figure 2. Experiment 2. Total plasma cholesterol in mg/dl.
These are the individual hen levels on the 21st

day of drug treatment. The standard error for

the experiment was 5.32

mg total cholesterol/d1 plasma

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58

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.....................................
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---------------------------------
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---------------------------------
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M‘OI‘QOOO
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Drug FeedTreatment

(Cl-720 at 2600 ppm)

Numbers represent the hen number

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O4
16
17
31
37
38
141
142

‘- F 1- 1- F ‘-
Drug Feed Treatment
(cu-720 at 5200 ppm)

59

technique as factors in the variability of chicken total
plasma cholesterol. Another consideration is that there
was only one observation over time in experiment 2, while
the overall mean was calculated from hen levels at six
dates in the first experiment. Also, three hens were used
per treatment in experiment 1, and eight hens per treatment

in experiment 2.

3. Discussion

Johnson 33 El- (1959) suggested that due to the great
variability of laying chicken's plasma cholesterol, conclu—
sions based on the average values obtained from unreplicated
small groups of birds are not dependable. The total plasma
cholesterol coefficient of variation (8 x 100/y) for these
hens was 29.5fi. For a coefficient of this general magnitude,
they reported that they would have required about 70 hens
per treatment to detect a difference of 15% in blood
cholesterol levels as a result of different treatments, with
statistical significance of (P<:.O1), nine times out of ten.
The total plasma cholesterol coefficient of variation for
experiments 1 and 2 was about 23 and 21%, respectively.

Shrewsbury (1967) found the coefficient of variation to
be much greater for plasma cholesterol than for egg total
cholesterol. The data from experiments 1 and 2 supported the
findings of Shewsbury (see Table 3 of the appendix).

In experiment 1, the range of all values of total
plasma cholesterol was 75-209 mg/dl, with a mean of

60

123 mg/dl. In experiment 2, hens had a total plasma
cholesterol range of 71-195 mg/dl, with a mean of 122 mg/dl.
Table 6 gives values for total plasma cholesterol others
have found.

In experiment 2, many of the hens on drug treatment
were "off feed" and out of production. This was most
evident in the group of hens given CI-720 via the feed at
5200 ppm, whose feed intake was about half the maintenance
level of about 70 g feed intake/hen/day for SCWL (Miller
and Denton, 1962), during the week of sample collection.

In experiment 1, the feed consumption average dropped for
hens given either drug treatment, but few were below the
maintenance level. With insufficient food for the body
functions, lipids would be used for energy so the blood
levels of these lipids would be expected to decline.
Acetate then would not be available for cholesterol synthe-
sis, and other lipids, and one would expect those hens with
below maintenance feed intake to have lesser plasma choles-
terol, triglycerides, and other blood lipids. Also, the
inability of the hens to provide adequate nutrients for the
egg would cause the egg production to cease.

Lorenz gt 3;. (1938) found the total plasma cholesterol
changed less than any other lipid during the laying period.

Bartov gt al. (1971) observed that plasma cholesterol
concentration exhibited a pronounced rhythmic change
associated with the egg formation cycle, and that these

fluctuations were not reflected in the yolk cholesterol

61

 

 

Table 6. Chicken total plasma cholesterol values taken
from the literature.
Cholesterol
Range or Mean
Type in mg/dl blood Reference
SCWL hens 54-422, §=211

SCWL hens +5% of diet
as dried egg yolk

Barred Plymouth Rock hen

Barred Plymouth Rock hen+

5% of diet as dried egg
yolk

SCWL floor layers

SCWL floor non-layers
SCWL floor layers 17%
protein; 937-1050
Cal./1b. diet

18% protein; same energy

SCWL non-layers; 17%
protein; same energy

18% protein; same energy
14-day-old SCWL males
4-week-old to 28-day-
old SCWL males

SCWL laying hen

SCWL 2-week-old males

140-220, 5:184
88-238, §=137
138-210, 5:165
248

272

255—340
285-294

368-470
518-645

86:34
ca. 55-95
178-285

141112

Miller and Denton
(1962)

Price _e_t_ §_]_._.
(1957)

Peterson (1951)

Peterson
(1952)

Wagh and Hinners
(1961)

Banergee
(1965

L.
I;

I:
I?

 

Table 6 (cont'd.).

62

 

Type

Cholesterol
Range or Mean
in mg/dl blood

Reference

 

Laying hen--meat pop.
Laying hen--mixed pop.

Laying hen
Range layer

New'Hampshire

New Hampshire cage layer

Laying hen

Laying hen low fat diet
Laying hen high fat diet
Immature nonlaying hen
Male chicken

low fat diet

Male chicken
high fat diet

169159
239165

76-156
116-288, 5:205
110-450, 5:217

88
73-215
79-242
52.312133
102-147

103-187

Washburn and Nix
(1974)

Walker _e_l Q.
(1951) 1

Johnson
(1959)

Dua st §l° (1966)

t

'P

Lorenz gt 9;.
(1938)

 

63

concentration. There appeared a definite increase in plasma
cholesterol when no egg was laid, even though one was pre-
sent in the shell gland. Bartov and co-workers also noted

a marked decline in blood cholesterol after repeated daily
(six) blood withdrawal.

By comparing layers zgr§u§_non-layers, Price £3 5;.
(1957) found that layers have a lower plasma cholesterol
level than non-layers; and for these latter birds in cages,
they reported a 50-250% increase in total plasma cholesterol
over non-layers on litter. In the present study, the values
for the control hen's total plasma cholesterol did not
reflect the inverse relationship Price and associates
reported. The reason could be that none of the hens were
completely out of production; the only hens of these
experiments to have ceased production were hens given the
CI-720. The wide difference between the control hens total
plasma cholesterol values would then be explained by
biological variation.

The CI-720 administration appears to have results
similar to those observed following the administration of
cholestyramine, regarding total plasma cholesterol and
total yolk cholesterol, 1.9., to lower the plasma choles-
terol, and have no effect on yolk cholesterol. The fact
that hens in one drug treatment from the first experiment
did produce eggs with significantly higher yolk cholesterol
than the eggs produced by hens of a control treatment at one

sample date, should be mentioned to qualify this comparison

64

‘with cholestryamine. The treatment means for yolk choles-
terol were nearly identical in experiment 2.

Weiss gj_§l. (1967) have reported results which
indicate that clofibrate may decrease plasma total choles-
terol of hens, but they did not report statistical analyses
for the data because of the intense individual variability

of the subject‘s plasma cholesterol.

Free Plasma Cholesteggl
1. Experiment 1

Statistical analysis using the split plot model with a
computer, showed a highly significant (P< .009) lowering of
the hen's free plasma cholesterol by administration of
01-720, from 107 to 79 (3.5.7 SE14) mg/dl.

The method of drug administration over time was signi-
ficant (P<1.044). The drug given via the feed seemed to be
more variable in effect than when the capsule was used to
administer the drug. Free plasma cholesterol values for all
six data collection dates are reported in Figure 3. The
response to method of drug administration over time is about
the same except at the end of week one of treatment. This
time has no particular significance as far as any treatment
change is concerned, and was probably due to chance. This
one out-lying value was undoubtedly influential in causing
the level of significance to be present.

The only other item of interest for this parameter is

the three-way interaction of method x time x drug (P<1.014).

Figure 3.

65

Experiment 1. Free plasma cholesterol. Plot

of method of drug administration x time
interaction; according to the split plot
statistical analysis. The means plotted
represent the data from six hens, as the control
and drug feed treatments were compared against

respective per os (capsule) treatments.

mg Free cholesterol/dl plasma

66

I20

"O

'00

9O

 

 

70
6O
50
4O

3O

20 Method x Time Interaction

 

Day Day Day Day Day Day
0 4 7 I4 21 7
of , g . of
drug treatment ' ' post—treatment

 

 

-_ :2 Feed Treatments,combined

. _ _ = Capsule Treatments, combined

67

Plotting the difference in response between presence and
absence of the drug over time (Figure 4), one can see that
a difference does exist between feed or capsule treated
groups of hens, and that they also do not react in the same
way with time. .

Figure 5 illustrates the antilipemic effect of CI-720
by using the hen means of free plasma cholesterol at each
treatment date. The antilipemic effect of CI-720 was
apparent for the hens of the drug feed group, which also
showed a rebound to the pro-treatment level when the drug
was removed; this rebound effect was not evident for hens in
the drug per as treatment. Hens of the drug pgr os treat-
ment had levels of free plasma cholesterol during the
treatment periods which were higher than the pre-treatment
reading, as was true for the total plasma cholesterol
levels. This drug per as group of hens had started out with
low total and free plasma cholesterol, and remained that
way. These drug per 08 treated hens were probably pri-
marily responsible for the overall indication of lowered
free plasma cholesterol, as a result of administering CI-720.

When the hen was used as her own control, (all subse-
quent free plasma cholesterol levels taken as the percent
of the respective hen's pre-treatment value) no difference
could be found in comparisons between treatments, using the

split plot statistical analysis.

68

Figure 4. Experiment 1. Free plasma cholesterol.
Three-way interaction of method of drug

administration x presence or absence of

drug x time.

mg tree cholesterol/d1 plasma

+70

+60

+50

+40

+30

69

 

Feed Method of ' Capsule Method of
Drug Administration Drug Administration
Day Day Day Day Day Day Day Day Day Day Day Day
0 4 7 I4 2] 7 0 4 7 I4 21 7
of oi of of
— drug treatment — post-a — drug treatment — post-
treatment treatment

VALUES REPRESENT THE DIFFERENCE IN RESPONSE (No DRUG MINUS
DRUG TREATED HEN VALUES), AS SEGREGATED av METHOD or DRUG

ADMINISTRATION

Figure 5.

70

Experiment 1. Free plasma cholesterol in mg/dl.
Values represent the mean of three hens per

treatment. The standard error for the experiment

was 11.6.

A = day 6 of pre-treatment

B = day 4 of drug treatment

0 a day 7 of drug treatment
D = day 14 of drug treatment
E = day 21 of drug treatment
F = day 7 of post-treatment

mg tree cholesterol/dl plasma

4

      
  
  
 
  

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Treatment
(CI-720 at

150 mg/lxg body
weight)

72

2. Expgriment 2
As in the first experiment, the statistical analysis

indicated that the drug lowered the free plasma cholesterol
from the control hen's level, this time at a 99% confidence
level. The values for treatments of 0, 2600, or 5200 ppm
CI-720 were 83, 50, and 38 mg free cholesterol/d1 plasma,
respectively. The two treated groups of hens were also
tested statistically by the Tukey HSD test of means, and
were found to be similar.

When the single observation of free plasma cholesterol
for each hen was compared with that hen's egg production
(Figure 6), there seemed to be an indication that the
effect of CI-720 was associated with the hen's capability
of producing eggs. This would logically be an effect of a
drug that altered plasma lipid levels, as the yolk is about
32% lipid, and the egg about 11% lipid. In the high drug
level treatment, only hen 104 was in laying condition, and
in the 2600 ppm treatment the hens in cages 106, 107, 108,
and 110 were the only ones in production. These hens had
free plasma cholesterol levels approximately 1.5 to 5
times that of the remainder of the hens given the drug.
These drug treated hens, with the slightly depressed from
control levels of free plasma cholesterol, could have had a
higher tolerance toward the drug. Those hens with low
plasma cholesterol and low egg production, also were not

eating at their normal level. Therefore, the change in diet

73

Figure 6. Experiment 2. Free plasma cholesterol in mg/dl
plasma. Plotted are the individual hen values
for the eight hens per treatment on day 21 of
drug treatment. The standard error for the

experiment was 4.02.

mg tree cholesterol/dl plasma

120

ITO

100

90

8°

7O

60

5°

40 .352

30

2°

'0 .;.;.;.

74

In
0
F

Control Feed Treatment Drug Feed Treatment
(Cl-720 at 0 ppm) (CI-720 at 2600 ppm)

Numbers represent the hen number

 

h
M
F

Drug Feed Treatment
(Cl-720 at 5200 ppm)

75

could explain the decline in egg production and plasma

cholesterol, at least in part.

3. Discussion

Lorenz gt 3;. (1938) found considerable variation in
the laying hen's free plasma cholesterol. The immature non-
laying female chicken's free plasma cholesterol was found
to have a range of 72-106 mg%. The range for males was
68-106 mg%, when on a low fat diet. On a high fat diet,
the male chickens had free blood cholesterol values between
74 and 100 mg%. In laying hens on a low fat diet, Lorenz
gt al. (1938) found the free cholesterol range for plasma was
67 to 206 mg%; and on the high fat regime, the free plasma
cholesterol was 73 to 190 mg%. In the laying bird, the
large increase in total plasma lipid versus non-layers of
comparable age, was confined to the free cholesterol,
neutral fat (triglycerides) and phospholipid. Thus, the
free percent of total plasma cholesterol would increase
with the onset of egg production. These changes associated
with maturity were not uniform, even for birds on the same
diet.

Caldwell and Suydam (1960) found free plasma cholesterol
in cockerels to be about 24 to 31 mg%.

The values in either of the current experiments are in
the range of free plasma cholesterol levels reported by

other researchers for both layers and non-layers.

76

Because of the low free plasma cholesterol values and
low free percent of total plasma cholesterol, the data of
the second experiment appeared to be in error, therefore,
the plasma diluent of each hen was re-evaluated using
freshly prepared reagents. In table 2 of the appendix is
given a comparison of these results; this table also gives
an indication of the high degree of precision of this

procedure.

Free Cholesterol as a Percentage of Total Plasma Cholesterol
1. Experiment 1

This parameter showed responses which were similar to
changes in free plasma cholesterol. The split plot statis-
tical analysis indicated a highly significant decrease in
the percent free of total plasma cholesterol due to CI-720
administration (P<.013), and the three-way interaction had
a significance level of .029. The time effect was also
highly significant (P<.003).

Figure 7 illustrates the variability of the hen's
percent free of total plasma cholesterol values over time.
Because the pattern of each control group was similar in
appearance to it's drug counterpart (but not in magnitude),
the statistical significance of a drug effect may not have
been entirely accurate. Also, the hens given the drug in
the feed started with and maintained low values for this
parameter throughout the experiment; and the drug treated

groups of hens had values on treatment dates greater than

Figure 7.

77

Experiment 1. Percent free of total plasma
cholesterol. There were three hens per

treatment.

= day 6 of pre-treatment

= day 4 of drug treatment

= day 7 of drug treatment

day 14 of drug treatment
= day 21 of drug treatment

'11 {11 U 0 w >
II

= day 7 of post-treatment

78

100

Percent Free at Total Plasma Cholesterol

ABCDEF ABCDEF

Control Feed Drug Feed

Treatment Treatment

(CI 720 at 0 ppm) (CI-720 at
2600 ppm)

letter code on opposite page

ABCDEF

Control Per os
Treatment
(CI-720 at

0 mg/kg body
weight)

 

ABCDEF

DrUg Per os
Treatment
(Cl-720 at

150 mg/kg body
weight)

79

the respective pre-treatment level. Table 7 is the
counterpart of Figure 7.

Figure 8 shows the difference in response (no drug
minus drug) when interacting with feed or capsule administra-
tion, over time. This figure shows that the no drug/drug
treatments differ with time when in the feed or capsule,
and the manner in which they differ over time is not the
same.

Figure 8 shows that the significance of time is pro-
bably due to the markedly low value of this parameter on
day 4 of treatment when compared with the other rather
consistent values.. As far as is known, this day 4 value

was a chance variation.

2. Experiment 2

Because of non-homogenous variance, as indicated by the
f-max test on the three treatment variances, the Dunnett
method of testing means was used. The control hen's values
333535 the low drug level treated hens, and the latter
zgrggg the hens given the high CI-72O dosage were not
significantly different from each other (P7>.05). But the
control hens mean for the percent free of total plasma
cholesterol was greater than the mean of the hens given the
high drug level (P< .01).

Table 8 shows the percent free of total plasma choles-
terol for experiment 2, and also contains the values for

total and free plasma cholesterol for each hen. Figure 9

80

Table 7. Experiment 1. Percent free of total plasma
cholesterol.

 

 

Hen # Day 0 Day 4 Day 7 Day 14 Day 21 Day 7 5
Control Feed Treatment (CI-720 @ 0 ppm)

1 100 62 95 85 81 6O 82

2 86 57 102 72 86 71 79

3 86 71 97 92 80 68 82
mean 91 63 98 83 82 66 81
Drug Feed Treatment (CI-720 @ 2600 ppm)

6 96 6O 75 49 80 74 72

7 57 46 63 51 52 78 58
8 54 67 68 72 80 72 69
mean 69 58 69 57 71 75 66
Control pg£_g§ Treatment (CI-720 @ 0 mg/kg body weight

9 82 82 76 9O 96 87 86
10 71 75 85 89 89 68 79
11 80 68 76 67 87 87 78
mean 78 74 79 82 91 81 81
Drug pg£_gg Treatment (CI-720 @ 150 mg/kg body weight)

14 8O 48 71 74 78 70 70
15 58 54 78 81 70 56 66
16 94 84 67 88 86 76 83
mean 77 62 72 81 78 67 73

 

Figure 8.

81

Experiment 1. Percent free of total plasma
cholesterol. Results of the split plot

statistical analysis.

A = the effect of time on the percent free of
total plasma cholesterol
B = the three-way interaction of method of drug

administration x presence or absence of

drug x time

82

100

>

 

 

 

 

.2 .0
,2 ; 80
“6 3
3 :2 70
u“. u ..
I 2 60
§ .. Time meet
0 3
a a.
Day Day Day Day. Day Day
0 4 7 I4 21 7
at ot
—— drug treatment post-
treatment
+30
+20
‘2
.13 +10
3
o
6 0
c 2 Feed Method of Drug Administration
'2 ‘6
g E
2 _
o 2
:z o
u. h-
0 u.
0 +30
I
2
‘f. +20
C
o
‘3.
,3 +10
0

Capsule Method of Drug Administration

 

 

Day Day Day Day Day Day
0 4 7 l4 2' 7
of ol
—— drug treatment post—
treatment

B: VALUES REPRESENT THE DIFFERENCE IN RESPONSE (No DRUG MINUS DRUG TREATED
HEN VALUES), As SEGREGATED av METHOD OF DRUG ADMINISTRATION

83

Table 8. Experiment 2. Percent free of total plasma
cholesterol.*

 

Control Feed Treatment (CI-720 @ 0 ppm)
Hen Number 439, 102 103 115 121 122 124 125

Total Plasma
Cholesterol 179 190 164 151 161 195 101 100

Free Plasma
Cholesterol 90 97 86 81 91 113 54 48

Percent Free

of Total 50 51 52 54 56 58 53 48
Low Drug Level Treatment (CI-720 @ 2600 ppm)

Hen Number 105 106 10] 108 109 110 136 139

Total Plasma
Cholesterol 97 108 98 158 103 111 109 100

Free Plasma
Cholesterol 29 61 52 95 30 60 33 37

Percent Free

of Total 30 56 53 6O 29 54 30 37
High Drug Level Treatment (CI-720 @ 5200 ppm)

Hen Number 104 116 117 131 ‘137 138 141 142

Total Plasma
Cholesterol 121 97 97 95 119 118 71 93

Free Plasma
Cholesterol 71 36 34 32 35 38 28 30

Percent Free
of Total 59 37 35 34 29 32 39 32

 

*Included are the individual values for the total and free
plasma cholesterol.

Figure 9.

84

Experiment 2. Percent free of total plasma
cholesterol. Values are calculated as the
difference between the total and free plasma
cholesterol levels on day 21 of drug treatment.

Experiment standard error was 1.96.

Percent Free of Total Plasma Cholesterol

85

 

see—P-23 22-22-20 2—-—-cpz
Control Feed Treatment Drug Feed Treatment Drug Feed Treatment
(CI-720 at 0 ppm) (CI-720 at 2600 ppm) (CI-720 at 5200 ppm)

Numbers represent the hen number

86

is a graph of only the percent free of total plasma
cholesterol for the individual hens.

3. Discussion

Lorenz gt 5;. (1938) observed levels of 57-99 percent
free of total plasma cholesterol when they fed a high fat
diet to laying hens; and on a low fat diet, the range was
63% to 105%.

Walker gt_al. (1950) found the laying hen to have a
percent free of total plasma cholesterol range of 66%-94%.
Four-to-twenty-five-week-old cockerels tested by

Caldwell and Suydam (1960) had 24.5% of the total plasma
cholesterol in the free form. This value is similar to the
26.6% for day-old chicks reported by Chung £3 El. (1965)
who also found that laying hens had about 72% of the total
cholesterol in the free form.

Kaishio (1933) has reported that the plasma ratio of
free cholesterol to total cholesterol in non-laying hens
was within the limits of variation of the male birds he
used. His range for esterified cholesterol for hens was
11.8% to 70.2%, with a mean of 45.7%. Therefore, the
percent free of total plasma cholesterol range was about
29 to 88%. '

Human circulating cholesterol is about 2/3 esterified,
and 1/3 in the free form, according to information in the

monograph which was guest edited by Stare (1974). This is

87

comparable to non-laying hens and immature chickens, but is
the inverse of laying hens.

In experiment 2, the reason is not know why the percent
free of total plasma cholesterol values did not exceed 60%
even for the control hens. This is about the lower limit of
the range observed by others for laying hens.

In the first experiment, the hens out of production
only had values of 40%—50% free of total plasma cholesterol.
The second experiment had non-laying hens with a range of
29%-39% free of total plasma cholesterol. Although the
magnitude is different, the trend of hens out of production

is the same in both experiments.

Plasma Triglycerides
1. Experiment 1
Due to technical difficulties, most sample results were
discarded. Because some values fell within the range as
reported by others, the data from this experiment are
included in Table 4 of the Appendix, but final conclusions

are left to the reader.

2. Experiment 2 _

The plasma triglycerides statistical analysis (Tukey
HSD), indicated the mean for the control group of hens was
higher than the mean for either group of hens on drug
treatment (P < .01).

Figure 10 is a plot of the individual hen triglyceride
values obtained for this one-time blood withdrawal. This

Figure 10.

88

Experiment 2. Plasma triglycerides in mg/dl.
Plotted are the individual hen values at day
21 of drug treatment. There were eight hens
per treatment. The standard error for the

experiment was about 96 mg/dl.

A a control feed treatment (CI-720 @ 0 ppm)

B a drug feed treatment (CI-720 @ 2600 ppm),
also referred to as the low drug level
treatment

0 a drug feed treatment (CI-720 @ 5200 ppm),
also referred to as the high drug level

treatment

89

 

x at» at
930.33.01.00 _

9O

figure indicates there is considerable individual variation
‘of plasma triglyceride levels among the control hens. Also
shown is the biological variation in response to the drug.

All hens below 300 mg triglycerides/d1 plasma were at
zero production for at least a week prior to the blood
withdrawal, except hen 105, which laid one egg at the first
of that week, then ceased to produce. This time period also
coincided with the below maintenance level of feed intake
recorded for both drug treated groups of hens.

Conversely, hen 108 laid only two eggs for the days 14
through 21 of CI-720 treatment, but had the highest plasma
triglycerides of any drug treated hen. The next time period
this hen laid at her previous rate of 4 eggs/week. An
explanation of the high level of blood lipids from hen 108
could be that she was not entirely out of production, there-
fore her blood lipids were maintained at a high level, and
may not have been materially affected by the drug. Both
hen 108 and 105 were from the low drug level treated group.

3. We

The effect of the drug probably is confounded with that
of the acute starvation which was experienced by most hens
that were out of egg production. The below maintenance
level of food intake would decrease hepatic fatty acid
synthesis and increase plasma free fatty acids (Leveille
21H§;., 1975). Earlier work by these same researchers

supported the concept that the liver is the major site of

91

avian lipid biosynthesis (Leveille gt‘al., 1968). These
changes would mean that plasma triglycerides should decrease
by fasting. Fasting also inhibits liver cholesterol
synthesis at the beta-hydroxy-beta-methylglutaryl-Co A step.
This fasting effect is apparent when comparing blood lipid
levels with that of feed intake.

Herman gt‘al. (1970) found that dietary carbohydrate
removal caused a decrease in some human serum triglycerides.
Muruiri‘gt'al. (1975) reported that when meal fed
chicks are fed, their plasma triglycerides were increased

from 38:4 mg% toward the level of the agilgb. group of
chicks, which was 117116 mg%.

Walker gt‘al. (1950) reported the total lipid of the
laying hen ranged from 652 to 2308 mg%. 0f the total lipid,
neutral fats (triglycerides) were about 62% (404 to 1431
mg%), phospholipids were about 30%, and total cholesterol
about 7% (46 to 162 mg%).

Lorenz 33 al. (1938) reported hens to have higher
total lipids than the hens Walker and his associates used.
The former researchers found that the lipid levels in the
blood of laying hens were variable, as evidenced by the
range of a single hen, over about a three month period of
time, which was recorded to be from 1030 to 4129 mg% total
lipid. Lorenz 23,9l. (1938) also reported that by using
analysis of covariance, there was no relationship between

duration or intensity of production and the plasma lipid

92

level, provided the hen was in production at the time of

the observation.

Total Yolk Cholesterol

1. Experiment 1

Although Figure 11 shows that the hens given the drug,
on the average, had higher yolk cholesterol than hens used
as their control, only the comparisons of control pgr os
versus the drug feed treatment groups of hens, or the control
pgr os versus the drug pgg_g§ treatment for hens on day 21
of drug treatment were statistically significant (P<.05).
The Tukey HSD test of means was used to analyse the data.

Figure 12 shows the values for total egg yolk choles-
terol which was determined at the end of each treatment
week and post-treatment week as a percent of the respective
hens pro-treatment level of yolk cholesterol. Because of
the intense individual variation of the percent of pre-
treatment values, statistics were not used to evaluate these
results. In Figures 11 and 12 it can be seen that the pro-
bable reason for the significance of the above comparisons
was the below 100% of pre-treatment yolk cholesterol values
of hens in the control per os treatment, and the much
greater than pre-treatment level of several of the drug
treated hens. Also, according to these figures, the drug
may have been more effective in altering the yolk cholesterol
levels when the capsule was used for CI-720 administration,

than via the feed.

93

Figure 11. Experiment 1. Egg yolk total cholesterol in
mg/g yolk. Plotted are the means of three

hens/treatment.

mg total cholesterol/g yell:

94

 

Day Day Day Day Day Day

 

 

0 ,4 7 14 21 7
at at
drug Treatment post-
treatment

— Control Feed Treatment (Cl-720 at Oppm)
"' - ' Drug Feed Treatment (Cl-720 at 2600 ppm)

- -Control Per os Treatment (Cl-720 at 0mg/kg bo y
we ght

----- Drug Per os Treatment (Cl-720 at 150 mg/kg 50 V
weight

Figure 12.

95

Experiment 1. Egg yolk total cholesterol
percent of the individual hen's pro-treatment

levele

day 4 of drug treatment

a day 7 of drug treatment
= day 14 of drug treatment
= day 21 of drug treatment

IIIUOUZII>

a day 7 of post-treatment

(1) Control Feed Treatment (CI-720 @ 0 ppm)
Hen # 1
IIIII-IIII 2 H811 # 2

 

-——- = Hen # 3
(2) Drug Feed Treatment (CI-720 @ 2600 ppm)
Hen # 6
.......... = Hen # 7
-——- = Hen # 8

(3) Control Per 08 Treatment (CI-720 o 0 mg/kg
body weig

III-III... . Hen # 1O
-——- = Hen # 11

(4) Drug Per 03 Treatment (CI-720 0 150 mg/ks
body weight)

 

= Hen # 14

 

.II“IIII| 8 H811 # 15
——- 3 H611 # 16

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97

Other than biological variation, the reason why hens
of the control feed group showed so much change from the
pre-treatment level of yolk cholesterol over time, as well
as the downward trend of yolk cholesterol levels of hens

in the control per as treatment, is not known.

2. Experiment 2

No change was noted in the yolk cholesterol of hens of
any treatment. The final treatment mean of the drug
treated hens had only slightly more cholesterol/g yolk than
did the control hens (Figure 13). In the high drug level
treatment, the only hen to lay an egg had about an average
amount of cholesterol for these drug treated hens. There-
fore, the drug may have a maximum effect on the amount of
cholesterol placed into the yolk, if there is any effect
at all.

3. Discussion

If the drug had caused the hen to excrete cholesterol
through the egg, those hens given the CI-720 would have been
expected to have higher levels of cholesterol in the yolk
than the control hens. This appeared to be the case only in
experiment 1. For hens of any treatment in experiment 1,
the change in the amount of cholesterol deposited in the
yolk during the following week's drug treatment was usually
opposite in direction to the change in total plasma choles-
terol (see Table 9). This inverse relationship cannot be

evaluated for experiment 2, because this was a one-time

Figure 13.

98

Experiment 2. Individual hen values of total
cholesterol/g yolk, from an egg laid on or
about the 21st day of drug treatment. Because
only hen 104 yielded information of this
parameter, and the value was close to the mean
of the hens given the low drug treatment, all
drug treated hens yolk cholesterol values were
combined. The standard error of the experiment

was 0.57.

mg Total Cholesterol/g Yolk

 

99

ONMID
GOO!-
FFF

CONTROL FEED DRUG FEED
TREATMENT TREATMENTS
CI-720 atOppm COMBINED

Cl- 720 at 2600 ppm
except hen 104 with
5200 ppm

Numbers represent the hen number

100

Table 9. Relationship between plasma and yolk total

 

 

cholesterol.

Day* Day . Day Day
Treatment 0-7 7-14 . 7-14 14-21

Plasma Yolk . Plasma Yolk
Control Feed 1 d . d 1
Drug Feed dd ii I dd i
Control per 03 d d : i d
Drug per as d i : i d

 

Legend: i=increase, ii=large increase, dadecrease,

dd=large decrease.
*The days represent the days of treatment with CI-720.

There is a 7-10 day period of time required for the forma-
tion of the yolk, therefore, the change in the blood
cholesterol would not be reflected in that day's egg, but
in the egg laid sometime during the following week.

For example, the change in blood cholesterol from day 0 to
day 7 of drug treatment may show an increase; and because
the blood lipids of the hen during this week are forming
a future yolk, which is not completed until the following
week, then this level of blood cholesterol would not be
reflected in the yolk cholesterol until the following week.
This time period for the yolk would be from day 7 to 14 of

drug treatment.

101

blood withdrawal and egg sample. The experiment 1 control
per on treatment is the only group of hens in this
experiment not to follow this trend of an inverse relation-
ship between blood and yolk cholesterol. Marion gt 2;;
(1960) found a significant inverse relationship between
serum cholesterol and the cholesterol present in the yolk
(-.288 correlation), when dietary fats and fatty acids

were manipulated.

Others have noted that if the blood is overloaded with
cholesterol, as in the addition of cholesterol to the diet,
this cholesterol supplementation will cause an increase in
the yolk cholesterol content. For example, Weiss gt_§l.
(1967) concluded that egg cholesterol concentration did not
parallel the blood levels when there were different types
of fat in the diet except in those cases where hypercholes-
terolemia was produced by feeding diets that contained
cholesterol.

Singh and Naber (1970) fed a hypocholesterolemic drug
(20,25-diazacholesterol, also called 50-12937) and reported
that at .1 to 5 ppm, blood sterols were increased and egg
sterols were decreased, but long term feeding of 5 ppm
caused an increase in egg sterols above the control hen's
sterol level.

Bartov gt 3;. (1971) found that yolks from hens of about
equal rates of production vary significantly in their total
yolk cholesterol concentration, but the yolk cholesterol

from the same hen was quite uniform. They reported that the

102

yolk cholesterol values were independent of egg production,
but that the group of lowbproducing hens contained more
hens yielding high yolk cholesterol values than did the
group of high-producing hens. These researchers indicated
that these "good" layers excreted more cholesterol per
week through the egg than did those hens laying less eggs.

Jones (1969) noted that there was a slight difference
in the yolk cholesterol content between strains of birds,
but variation from egg to egg, even between birds of the
same strain was enough to prevent any statistical
differences. Turk and Barnett (1971) also reported con-
siderable variation among individual hens from the same
flock, regardless of age, feed, management, strain of layer,
or geographic location, and therefore no difference statis-
tically between these. Clarenburg gt al. (1971) also made
the observation of lack of uniformity of hen's yolk
cholesterol.

In the present studies, the hens producing at lower
rates tended to have more cholesterol/g yolk than the
better producers. Fluctuations in production tended to
create opposite changes in the total yolk cholesterol con-
centration. These data are in quasi-agreement with Bartov
gt‘al. regarding the egg production:yolk cholesterol
relationship, but the eggs produced by control hens was less
uniform than that reported by Bartov £1.§l- and other
authors. Table 10 gives the means for each hen in both

experiments.

103

Total egg yolk cholesterol in mg cholesterol/g

 

 

 

Table 10.
yolk.
Hen # day day 4 day 7 day 14 day 21 post- mean
zero trt.
Experiment 1:
Control Feed Treatment (CI-720 @ 0 ppm)

1 14.2* 14.1 15.4 15.9. 14.5 11.8 14.0

2 14.4 11.2 12.4 9.2 14.1 14.7 12.7

3 13.9 14.7 14.3 16.2 15.0 13.9 14.7
mean 14.2 13.3 14.0 13.1 14.5 13.5 13.8
SEM 0.15 1.08 0.88 2.06 0.26 0.86
Drug Feed Treatment (CI-720 @ 2600

6 14.3 14.5 15.7 n/e 18.2 14.8 15.5

7 15.5 n/e n/e n/e n/e 17.4 16.5

8 15.0 13.8 13.4 16.0 14.7 14.8 14.6
mean 14.9 14.2 14.6 16.1 16.5 15.7 15.5
SEM 0.35 0.35 1.15 0.00 1.75 0.87
Control ng_g§ Treatment (CI-720 @ 0 mg/kg body weight)

9 12.7 13.0 13.4 12.5 12.0 12.8 12.7
10 12.4 12.3 12.3 12.1 9.8 9.2 11.4
11 14.8 13.8 15.3 15.4 14.2 12.7 14.0
mean 13.3 13.0 13.7 12.7 12.0 11.6 12.7
SEM 0.75 0.43 0.88 0.38 1.56 1.18

 

*all are means of triplicate readings

n/e . no egg

104

Table 10 (cont'd.).

Hen # day day 4 day 7 day 14 day 21 post- mean
zero trt.

 

Drug Per 03 Treatment (CI-720 @ 150 mg/kg body weight)
14 13.4 13.8 14.3 15.9 14.8 13.8 14.3

15 14.8 14.6 16.3 16.6' 14.7 15.8 15.5
16 12.7 14.1 16.1 16.7 17.3 14.1 15.2
mean 13.6 14.2 15.6 16.4 15.6 14.6 15.0
SEN 0.62 0.23 0.64 0.25 0.85 0.62

Experiment 2:

Hen # 99 102 103 115 121 122 124 125
mean 22.4 16.3 15.3 19.6 16.3 15.7 17.8 17.0
group mean‘; SEM 17.55:0.85

105 106 107 108 110 104
mean 20.9 16.3 16.5 18.9 17.5 18.3
group mean 2 SEM 18.110.70

 

105

Harris and Wilcox (1963b) found a seasonal difference
in yolk cholesterol, which increased from August to Feb-
ruary, then decreased in April and June. They also
calculated a negative correlation between yolk size and
its cholesterol concentration, regardless of the season.

In this work, the first experiment was conducted in
May and June, and the second experiment occurred in October
and November. The information reported by Harris and
Wilcox may explain in part the larger values for yolk
cholesterol observed in the second experiment. In a
companion paper, Harris and Wilcox (1963a) reported that
environmental influences contribute a greater effect on
yolk cholesterol than does genetics. Others who found a
seasonal difference in yolk cholesterol levels were Jones
(1969); and Whiteside and Fluckinger (1965) for total
yolk sac cholesterol.

Edwards gt g1. (1959) found a positive correlation of
.44 between weight of the hen and the egg cholesterol level,
and a value of .38 for the correlation between yolk choles-
terol and the yolk iodine number.

Weiss gt_§;. (1963) reported that the Zlatkis method
"as adopted to the determination of cholesterol in eggs
gave abnormally high values when compared to the method of
Abell." The values of the present experiments tend to
support this statement, when a comparison is made between

these values and those reported in the literature when the

106

Zlatkis method was used; this is especially true in the
first experiment.

The values of total yolk cholesterol obtained in the
present experiments are in the range of values found by
others. The yolk cholesterol of the first experiment was
lower and the range larger than in the second experiment.

In neither experiment were the values of the range marked
by dramatic differences between adjacent values over time.

Comparing experiment 1 egg yolk cholesterol values with
those of experiment 2, there is some difficulty in making a
definite statement of the effect of CI-720 on yolk choles-
terol levels. The effect of CI-720 (assuming it's existence)
is comparable to the drug used by Singh and Naber (to
decrease the blood cholesterol and move it to the yolk) in
only the first of two experiments. There did not appear to
be any drug effect in experiment 2. Egg production of most
hens treated with CI-720 in experiment 2, was stopped by
the drug; this could have been due to a lack of feed intake
or to altered lipid metabolism so the yolk may not have been
formed. The first yolks produced after the egg formation
mechanism was stopped, tended to be higher in yolk choles-
terol (see hen #6 of experiment 1 in Table 9).

Table 11 lists yolk cholesterol concentrations

reported in the literature.

107

Table 11. Total yolk cholesterol values reported in the
literature.

 

Description

mg Cholesterol
per gram yolk

Reference

 

Experiment 1:
Laying hen
Experiment 2:
Good layer

Poor layer

SCWL
BPR

Laying hen

Laying hen
Laying hen
Laying hen,
mixed strain

(Athens-
Canadian)

mixed strain
(American
breeds)
Laying hen

SCWL

11.7-14.8

11e3-14e
mean=12.

1
5
11.3-14.7
mean=13.2

15I6-tOeS--SIDI
17.3:3.2"-SIDI

29e4'3001
11.2-13.7

14-14.5

19-27 family
range

mean=22.8_t2.8 SOD.
17.5-21.5 family

range

mean:19.2:1.2 S.D.

17.8

21.830.22 (1957)
25.2;0.19 (1958)

Bartov gt_al. (1971)

Miller and Denton
(1962)

Combs and Helbacka
(1960)

Weiss 31 El- (1967)

Clarenburg _e_t a}.
(1971)

Washburn and Nix (1974)
Dua gt a}. (1966)

Harris and Wilcox
(1963a)

 

108

Bodngeight (in grams)

1. Experiment 1

The split plot statistical analysis showed no f-value
to be even close to being significant. The hens of the drug
treatments did decrease in body weight, possibly because of
lowered feed consumption. The hens given CI-720 showed a
return toward the pre-treatment level the week after drug
withdrawal. Body weights of individual hens are listed in
Table 12. The split plot model was used for the observed

values only.

2. Experiment 2
Figure 14 shows hen treatment means of body weight at

each data collection date, the individual hen values used to
calculate these means are in Table 12.

In both experiments, the CI-720 seemed to cause a
decrease in body weight, which then either plateaued, or the
hen partially recovered the weight before the experiment
ended. The body weight changes paralleled feed consumption
patterns.

The body weight of hens in the control group was
reasonably uniform, and in most cases these hens gained
weight throughout the experiment.

For the last two weeks of drug treatment, the control
hens were significantly heavier than the hens given the high
dosage of CI-720 (P‘<.02), using the Tukey HSD test of means.

109

Table 12. Body weight (+1 g) of hens at the end of each
treatment week.

 

Day 7
Day 0 Day 0 Day 7 Day 14 Day 21 Post-
Hen # Expt. Trt. Trt. Trt. Trt. trt.

 

Experiment 1:
Control Feed Treatment (CI-720 @ 0 ppm)

1 2544 2570 2524 2502 2243 2236
2 1707 1728 1784 1880 1904 1920
3 2174 2184 2200 2210 2161 2177

Drug Feed Treatment (CI-720 @ 2600 ppm)

6 2069 2046 2087 1926 2005 2057
7 2274 2191 2163 2176 2197 2224
8 1868 1841 1762 1802 1818 1817

Control Per 08 Treatment (CI-720 @ 0 mg/kg body weight)

9 1764 1744 1739 1694 1699 1738
10 1826 1777 1791 1787 1816 1843
11 2114 2158 2243 2198 2267 2267

Drug ng_pg Treatment (CI-720 @ 150 mg/kg body weight)
14 1920 1871 1794 1858 '1847 1866
15 2177 2093 2130 2107 2098 2027
16 2216 2198 2140 2098 2162 2238

110

Table 12 (cont'd).

Day 0 Day 0 Day 7 Day 14 Day 21 Day 28

Hen # Expt. Trt. Trt. Trt. Trt. Trt.

v—v+ +v +7

Experiment 2:

Control Feed Treatment (CI-720 @ 0 ppm)

99
102
103‘
115
121
122
124
125

Drug Feed Treatment (CI-720 @ 2600 ppm)

105
106
107
108
109
110
136
139

fir

2537
1771
1874
2210
2275
2286
1869
2467

2355
1845
2109
2312
2212
1934
1823
1800

2563
1871
1880
2251
2267
2315
1968
2489

2045
1868
2221
2171
2081
1854
1548
1546

2682
1881
1917
2226
2373
2411
2010
2489

2043
1887
2161
2299
2018

' 1885

1527
1725

2714
1868
1884
2257
2358
2399
1965
2526

2054
1822
2170
2266
1926
1838
1566
1805

Table 12 (cont'd.).

111

 

 

 

Day 0 Day 0 Day 7 Day 14 Day 21 Day 28

Hen # Expt. Trt. Trt. Trt. Trt. Trt.
Egpgpgment 2 (cont'd.)z

Drug Feed Treatment (CI-720 @ 5200 ppm)

104 2309 2303 2206 2182
116 2190 1949 1857 1923
117 2009 1836 1676 1723
131 2082 1930 1836 1850
137 1800 1645 1557 1446
138 1934 1535 1597 1627
141 2155 1935 2054 1889
142 2163 ‘ 2108 2007 2071

 

112

Figure 14. Experiment 2. Body weight in grams (:1).

BODY WEIGHT- - IN GRAMS
OHENS/TREATMENT

2300
2200
2100
2000
1900

1800

113

I
:7.“
~§..I.
\ I..

‘4.

‘I —-——'-——-_--—-

I
Iona-0....-
.-..-IIIII.II.I.I..IIIII

 

DAY DAY DAY DAY
7 I4 21 28

OF DRUG TREATMENT

 

 

INITIAL BODY WEIGHT WAS NOT RECORDED

—= CONTROL FEED TREATMENT (CI- 720 at 0 ppm)
. _ _= DRUG FEED TREATMENT (CI-720 at2600 ppm)

------ = DRUG FEED TREATMENT (CI-720 at 5200 ppm)

114

The body weight of hens in the control treatment was not
greater than that of hens of the low drug treatment
(P>.05), for all dates--see Figure 14. The Tukey test
was used here also. The hens on drug treatment started with
body weights that were slightly less than the control hens,
which would cause over confidence in the decrease in body
weight caused by the drug, but the trend seems to be clear
from Figure 14.

During the second and third weeks of drug treatment,
the feed consumption of hens given the high drug level
(5200 ppm) was about half the maintenance level. Obviously,
if feed is not available to the hen's body tissues, the
energy stored as adipose tissue will be used to maintain
life. There w0uld thus be a reduction in the hen's body
weight. .

Percent Change in Body Weight

1. Experiment 1

The absolute value (mathematically) of the percent
change in body weight of individual hens was calculated on
a weekly basis, throughout the experiment. Considerable
variation was evident in all treatments. Nothing in the
computer analysis of this variable, using the split plot
procedure, was close to having a significant f-ratio.

Using the hen as her own control, and calculating the
percent of pre-treatment body weight value at each date,

for each hen, no difference could be detected in the means

115

of any treatment given the hens. The Tukey HSD test of
means was also used for the evaluation of the data. Con-
trol groups were combined and compared against the combined
drug group, due to the split plot model characteristics,
for means in this analysis.

When the change in body weight was evaluated as the
initial minus final body weight, there was obviously no
difference between the treatment group of hen's means
because the ranges overlapped, if the hen #1 values were
included; and there probably was a difference between treat-
ments if values of hen #1 were excluded. Hen # 1 was injured
on day 14 of drug treatment, and so lost body weight even
more than the drug treated hens. Although some hens given
this drug gained weight, they usually lost at the rate of
3-4%. The control hens fluctuated about the pre-treatment
level, and their maximum loss was about 4% of their body
weight during a week. There was a 2.0 vs. a 2.4% change in
body weight for control and drug treated hens, respectively;
SEM = 0.259.

2. Experiment 2

Comparing the overall hen mean of the absolute change
in body weight from the preceeding week, for each treatment
hens on the control treatment were found to be significantly
less changed than those on either level of drug treatment

(P<<.05) for the days 8-14 of drug treatment period. Hens

116

on the drug treatments were not different from each other
in any comparisons.

Because of non-homogenous variance, the Dunnett test
of means was used for the statistical analysis of the data
from the second week of treatment; the Tukey HSD test of
means was used for all other comparisons. In this second
experiment, there was no weight recorded at the start of
the experiment, 80 the first change in body weight is from

the day 8 through 14 time period.

3. Discussion
During experiment 1, the body weight fluctuations
(on a weekly basis) were very similar for hens on all four
treatments. Considering the overall percent change for
the combined control treatments, three hens increased, one
did not change, and two declined in body weight. One of
the drug treated hens of this experiment gained weight, four
lost body weight, and one did not change in body weight.
Hens in experiment 2 showed similar responses in the
weekly change for the 0 ppm and 2600 ppm drug treatments,
but the 5200 ppm drug treated hens usually declined in body
weight. Results of the overall weight change for hens
of this second experiment showed the control hens all
gained weight, the 2600 ppm treated hens all lost body
weight except for one hen that did not change, and one that
gained weight. The 5200 ppm drug treated hens all lost

body weight, some as much as 300 grams.

117

Weekly Percent Egg Production

1. Experiment 1

According to the split plot statistical analysis, the
effect of time and of CI-720 presence was significant
(P(.0005; and P<.019, respectively), for this parameter.

For the time effect, the second week of drug treat-
ment was undoubtedly the point that caused such a high
level of confidence (see Figure 15). During this second
week of drug treatment, two of the three hens in the drug
feed treatment were out of production, and the other hen
laid only one egg. This treatment had hens which were
adversely affected by the drug, but the other drug treat-
ment did not have hens which showed such a dramatic effect
of CI-720. Table 13 shows the number of eggs produced by
each hen during each treatment week, for both experiments.

The mean of the three hens/treatment at each sampling.
date is plotted in Figure 16. The capsule drug treated hens
did not seem to be affected by CI-720 except at day 14 of
treatment (also see Table 14) but on this treatment date,
all groups of hens also experienced a decrease in percent
egg production, with the treated hens being affected more
than the control hens. The hens given the drug feed treat-
ment produced eggs at a rate (zero then rebounded) which
would suggest that there was a definite lowering effect of
01-720 on egg production. The rebound of egg production
after the withdrawal of CI-720 commenced too quickly for the
effect of CI-720 to be totally mediated through the lowered

Figure 15.

118

Experiment 1. Effect of time on percent egg
production, according to the split plot
statistical analysis. All hen values were
combined and segregated according to time for
this parameter. n=12 hens at 6 times; SEM

for the experiment was 10.5.

PERCENT EGG PRODUCTION

100

90

80

70

60

50

40

30

20

TO

TIME EFFECT

A

119

A- DAY 0 OF DRUG TREATMENT

3-
c-
D-
5-

II

II

POST TREATMENT

120

Table 13. Egg production record in total eggs per week.

 

pre- days days days post-
Hen # trt. 0-7* 8-14 15-21 trt.

 

Experiment 1:
Control Feed Treatment (CI-720 0 0 ppm)

1 5 5 5 5 6
2 7 7 4 7 8
3 6 5 2 4 5

Drug Feed Treatment (CI-720 0 2600 ppm)

6 6 5 0 0
7 3 0 0 0 0
8 6 4 1 5 5

Control Per 08 Treatment (CI-720 0 0 mg/kg body weight)

9 7 5 5 6 6
10 6 6 4 6 6
11 6 6 4 6 6

Drug For as Treatment (CI-720 0 150 mg/kg body weight)

14 6 6 2 6 6
15 5 6 3 5 p 4
16 5 4 3 3 4

 

*days of drug administration

121

Table 13 (cont'd.).

Hen # days 0-7* days 8-14 days 15-21 days 22-28

 

Experiment 2:
Control Feed Treatment (CI-720 0 0 ppm)

99 5 5 3 5
102 6 4 6 6
103 4 5 5 5
115 1 2 3 5
121 6 6 7 6
122 7 6 7 7
124 6 6 4 S
125 6 6 6 6
Drug Feed Treatment (CI-720 O 2600 ppm)

105 6 4 1 O
106 7 6 6 7
107 5 4 6 4
108 4 6 2 4
109 5 1 O O
110 3 5 5 6
136 4 5 O 0
139 5 1 O 0
Drug Feed Treatment (CI-720 G 5200 ppm)

104 6 5 6 5
116 4 O O O
117 2 O O O
131 5 O O 0
137 4 1 O O
138 5 4 O 0
141 6 1 O O
142 2 O O O

 

*days of drug administration

122

Figure 16. Experiment 1. Percent egg production average
for the three hens/treatment, at each week

of the experiment. SEM for the experiment

was 10.5.

PERCENT EGG PRODUCIION

100

90

80

7O

60

50

4O

3O

20

10

123

 

ABCDE_ABCDE ABCDE

A- FIRE-TREATMENT WEEK

B- DAY 0 through 7 or DRUG TREATMENT

C- ., 8 n 14 u u ”

D- n 15 ,. 21 u u .,

E - POST-TREATMENT WEEK

1 -CONTROL FEED TREATMENT; Cl-72O atOppn

2-DRUG FEED TREATMENT; cu-no at 2600 ppm

3 -CONTROL PER 05 TREATMENT; cu-720 at 0 mg/kg body weight

4-DRUG PER 05 TREATMENT; cu- 720 at 150 rug/kg body weight

Three hens per treatment

124

Table 14. Experiment 1. Change in percent egg production
from the pre-treatment level.

 

Control Feed Trgggment

 

 

51-725 0 5ppm

Hen # 1’ 2 3 5
change

Time
Period
end
pre-trt.* 71 100 86
end week 3 71 100 57
change 0 O 29 9.6
end
post-trt. 86 114 71
change 14 14 14 14

Control Per os

 

 

5

Treatment
Hen t 9 1O 11
Time
Period
end
pre—trt. 100 86 86

end week 3 86 86 86

change 14 0 0
end

post-trt. 86 86 86
change 14 0 0

"percent egg production

4.7

4.7

Dru Feed Treatment
51-625 @ 2655 ppm

6 7 8 i
change

86 43 86
O 0 71
86 86 15 62

71 O 71
15 43 15 24

DruggPer os
Treatment

14 15 16 i

86 71 71
86 71 43
O O 28 9.3

86 57 57
O 14 14 9.3

125

feed intake or any resorption of egg yolk, or probably even

an alteration in the lipids produced for the yolk.

2. Experiment 2

The percent production mean of the eight hens fed each
of three treatments is shown in Figure 17, apparently there
are significant differences between the treatments.
Tukey's HSD test of treatment means showed no difference
between any of the treatments during the first week. But
in the second week of treatment, egg production of the
control hens was significantly (P‘<.O1) greater than that
of the high drug level treated group of hens. This is the
same period of time after drug administration that the hens
of the first experiment were initially affected. In
experiment 2, when a hen given a drug treatment went out of
production, she did not return. Possibly the post-treatment
occurred at such a time to allow the hen to recover from
low production in experiment 1; but in experiment 2 there
was no post-treatment. Half of the hens on each drug
treatment molted in experiment 2. This could have been due
to a direct effect of the drug, or to the stress CI-720
may have produced, or to lowered feed intake. In general,
the egg production of the poorer layers seemed to be
affected more than the "good" layers egg production for
experiment 2.

For this second week of drug administration, the hens

given the high level of CI-720 had less eggs produced than

126

Figure 17. Experiment 2. Percent egg production. The
standard error of the mean was about 8.7

for the experiment.

PERCENT EGG PRODUCTION

100

90

 

127

.....
.....
.....
.....
.....
.....
.....
.....
.....
.....
.....
.....
.....
.....
.....
.....
.....
.....
.....
.....
.....
.....
.....
.....
.....
.....
.....
.....
.....
.....
.....
.....
.....
.....
-----
.....
.....
.....
.....
.....
-----
.....
.....
.....
.....
.....
.....
.....
.....
.....
.....

.........
uuuuuuuuu
---------
.........
.........
---------
.........

.........
.........
.........
---------
---------
.............
..............
.............
..............

..................
..................
..................
..................
..................
..................
------------------

ABCD ABCD ABCD

A- DAYO through7OF DRUG TREATMENT

B - n 8 u 14 u I! n
c - I: Is I: 2] u II II
D - u 22 n 28 n n n

1 - CONTROL FEED TREATMENT (CI-720 at 0 ppm)
2 'DRUG FEED TREATMENT (CI-720 at 2600 ppm)

3 ' " " " ( N at 5200 ppm)

EIGHT HENS PER TREATMENT

128

the hens treated with the low level of CI-720 (P (.05).
For both of the last two weeks of drug treatment, the
control hens produced more eggs than the hens given CI-720
at 5200 ppm, (P (.01). For the last two weeks of CI-720
administration, the control hens laid more eggs than did
the hens on the low level of drug (P (.05).

The overall mean weekly percent production showed a
highly significant difference between the hens of the high
drug level, and those given the control treatment. The
control hens had a higher rate of production.

In Figure 15, the hen in cage #104 was the only reason
there was a positive percentage to graph for the last two
weeks of the experiment for the 5200 ppm drug treated hens.

The Dunnett test of means was used for the means of
the last week only, the rest of the data was tested by the

Tukey HSD test of means.

3. Discussion

As may be the situation with CI-720, many compounds
that alter blood lipid levels (by various mechanisms), also
stop egg production. Some of these compounds are: nicar-
bazin (Weiss, 1960; Konlande and Fisher, 1969); lithocholic
acid (Edwards, 1962); DEAE Sephadex (Turk and Barnett, 1972);
or 20,25-diazocholesterol (Singh and Naber, 1970).

CI-720 may act by depressing feed consumption, in
possible combination with other mechanisms to cause a

decrease in the egg production, such as the disruption of

129

lipid metabolism. Obviously, for egg production, hens

must have sufficient levels of nutrients available to her
body for self preservation and then the extra demands of
reproduction. Walker 33 El- (1950) state that a laying fowl
may eliminate about 5 g of fat daily; while Lorenz 23 al.
(1938) placed the figure at about 4 g, but for a lighter
bird.

Because 7-10 days are required for a yolk to mature in
the ovarian follicle (Card, 1972), the effect of increased
feed consumption in experiment 1 (during or after drug
administration) on increasing egg production should not
occur until at least this period of time had elapsed. For
example, in experiment 1, hen number 8 (drug feed treatment)
had returned to her previous level of egg production after
a week of essentially zero egg production, all during the
time the drug was being administered and the feed intake was
below her normal level, but above that required for main-
tenance for this breed of chicken. Also hen number 6
returned immediately to her previous level of egg production
and feed intake, after the CI-720 was withdrawn. Hen
number 6 was at zero production the two previous weeks. In
experiment 2, no return of egg production accompanied the
increase in feed intake during the fourth week of drug
treatment (which was still below the 70 g feed intake/hen/-
day required for maintenance), at the end of which the
experiment was terminated. Apparently the CI-720 has some

direct effect on the egg production in some hens.

130

Perhaps the reason the hens given CI-720 via the
capsules were not affected as much as the ones given the
per as drug treatment, may be due to the fact that the drug
given orally was in one massive dose. Therefore, the
absorption time for the CI-720 was much different for hens
given per 08 versus drug feed treatment. In experiment 1,
the drug per 03 treated hens had CI-720 administered at a
rate of about 1.5 times that of the drug feed treated hens
(based on the overall averages of 309 and 202 mg CI-720/hen/-
day). The difference in the amount of CI-720 administered
is due to the fluctuation in feed consumption due to the
presence of the drug; the hen could not control the amount
of CI-720 injested when the drug was in the capsule. The
egg production in the capsule treated group of hens was
not affected as much as the 2600 ppm drug treated group of
hens, but the hens given the latter treatment in either
experiment showed similar responses for this parameter.

Whether the effect of CI-720 is direct, is mediated
through the low feed consumption, alterations in the lipid
metabolism, some other mechanism, or a combination of these,
CI-720 is clearly at least a predisposing cause of a decline

in egg production.

Change in Weekly Percent Egg Production
1. Experiment 1
No difference in the change in percent egg production

(using absolute values, as in the percent change in body

131

weight) was detected by using the split plot statistical
analysis. The absolute value (mathematically) of the
change in weekly percent egg production was calculated for
individual hens.

Hens in drug treatments generally declined in egg pro-
duction from week to week. There was an overall decrease
in egg production for four of these hens, while one hen did
not change, and one increased while on drug treatment. The
control hen's egg production generally did not change, or
was increased, between the different weeks. There was no
overall change in egg production for four of the control
hens, with one decreasing and one increasing.

Probably the natural variation in egg production was
sufficient to hide the fact that hens of the drug feed
treatment were out of production versus their much higher

previous record.

2. Experiment 2

A comparison of the average change in percent egg pro-
duction (calculated as in experiment 1) for control hens for
the first week of drug treatment, versus that of the 2600 ppm
CI-720 treated hens, showed no statistical significance
even though this low drug level treated group mean change was
about twice that of the control treated hens. The control
versus the high drug level treatment comparison showed the
control hens changed less (the control hens had higher egg
production) than the hens of this drug treatment (P1<.01).

132

No other comparison was significant. The Tukey HSD test
of means was used for all comparisons of these means.

All hens administered CI-720 at 5200 ppm had an
overall decline (no production for 7 of 8 hens) in egg
production. Five hens treated with the low level of drug
decreased, two did not change, and one increased in overall
egg production. One control hen decreased, two increased,

and five showed no change in their overall egg production.

3. Discussion

For experiment 2, the changes in the treated groups of
hen's egg production were associated with a 61% decline in
feed consumption after the hens were given CI-720 @ 5200
ppm, and with a decline of about 30% in feed intake for the
hens fed CI-720 @ 2600 ppm.

The normal positive and negative fluctuations in the
egg production of control hens were apparently sufficient to
mask the continued decline in egg production to zero of
several hens. In experiment 1 only hens of the drug feed
treatment were out of production for a week (plus or minus).
In experiment 2, several hens of the low drug treatment, and
all the high drug level treated hens (except at the first
week of treatment) but one had stopped producing eggs by the
third week of treatment.

Judging by the average amount of change from the pre-
treatment period to either the third week of drug treatment,

or to the end of the succeeding post-treatment week (see

133

Table 14) the CI-720 seems to have an effect only in the
hen's egg production of the drug feed treatment. The
other drug treatment change was almost identical to the
control hen's change.

Total E Weight: Yolk,gAlbumen,gand Shell WeightsJL

lus Their Percent of Total Egg Weight

1. Experiment 1

When computing an analysis of variance for the eggs
laid on the day which ended a treatment week, no f-value
was significant for any of the parameters of this section.
See Table 15 for the mean values and ranges for these

parameters for either experiment.

2. Experiment 2

All these parameters were statistically tested and
found to have means which were not significantly different
from each other, according to the f—ratio or by the Tukey
HSD test of means if the f-value was significant. Only the
total egg weight and yolk weight means showed any tendency
toward statistical significance (P<.2 and P 4.1,
respectively).

Only eggs laid (by the eight hens per treatment) on
the day ending the third week of drug treatment were
evaluated for these parameters. Only one egg was available
from the hens given the high drug treatment, and only

five eggs from those given the lower drug treatment.

Table 15o

Experiments 1 and 2.

134

Means for egg weight; and

the egg component weights, plus their percent
of total egg weight.*

 

Experiment 1

Experiment 2

 

Control Drug Control Drug
Eg weight
(g? mean** 55.910.59 55.7:0.59 62.411.60 57.212.40
range 48.1-62.3 49.9-63.0 55.9-69.9 52.5-67.5
Yolk weight
(g) mean 1709:0025 17.519‘22 1902:0043 17e4i0035
Shell weight
(g) mean 5.210.12 5.410.09 5.0:0.19 4.630.30
range 4e0-605 4e9’6e5 405-5e9 307-505
Albumen
weight
(8) mean 3206:0e30 3300:9047 380511037 3502:7083
range 29.0-36.4 28.3-39.0 32.5-43.9 32.0-43.5
Yolk weight
percent of
total egg
mean 32.1:0.20 31.4;0.35 30.810.63 30.630.73
range 30.1-34.4 28e0-3405 2804-3208 2706-3209
Shell weight
percent of
total egg
mean 9.430.15 9.810.15 8.0:0.30 8.010.29
range 709-1103 8e9-11e3 700-904 7.0-8.9
Albumen weight
percent of
total egg
mean 58.630.25 58.8:9.33 61.3;Q.77 61.410.59
range 56.7-61.7 55.1-64.7 56.6-63.0 60.2-64.3

 

*Experiment 1 had three hens per treatment and six dates on

which eggs were collected for evaluation.

Experiment 2 used

eight hens per treatment, and only one egg per hen (where
available) was analyzed.

**;$.E.M.

There were unequal numbers in each.

135

If the yolk and/or egg size were decreased by CI-720,
the mechanism may be as that proposed by Burgess gt 3;.
(1962) for triparanol. Triparanol supposedly interrupted
the estrogen production in the ovary, and inhibited the
primary maturation of ova. The percent of the yolk of
total egg weight was not changed by CI-720 administration.
Probably these marginal significance levels were due to
chance fluctuations in the egg and yolk sizes, as these
values were very similar in the first experiment, and the
experiment 2 albumen did not appear to compensate for the

yolk size difference.

3. Discussion

The values of the egg and components reported for hens
of both experiments are well within the ranges reported in
the literature for control hens.

Romanoff and Romanoff (1949) stated that the actual and
relative weights of the egg's structural elements,
especially the shell, may deviate rather widely; even the
weights for eggs of a "single individual hen".

Romanoff and Romanoff (1949) have cited other authors
to report values that are pertinent to this discussion, they
are as follows: V. S. Asmundson, for shell percent of total
egg weight of 11.0, and values of 61.6 and 27.4 percent of
total egg weight for albumen and yolk, respectively; N.
Olsson found the shell to be 10.9 and 10.6 percent of the

total egg weight, with the albumen percent being 61.1 and

136

59.6 percent, and the yolk 28 and 29.8 percent of total egg
weight. In their table 11, which was composed of data from
various sources, these authors reported that for a 58'g
egg, the albumen percent of total egg weight was 55.8

(32.4 g): yolk was 31.9 percent (18.5 g); and shell was
12.3 percent (7.1 g) of the total egg weight.

The mean shell percent of total egg weight 0. C. Mor-
gan (1932) reported was 9.72, with a range of 6.76 to
12.5 percent.

Asmundson and Baker (1940) reported a range of shell
percent of total egg weight of 5.28 to 9.46.

In 1961 Chung and Stadetman reported that the albumen
represented 56.3 percent, and the yolk 30.4 percent of the
total egg weight in the chicken. By simple arithmetic, the
shell then was about 13.3 percent of the total egg weight.
These authors found that the total egg weight was highly
correlated with the albumen and yolk weights, as would be
expected.

Weiss £3.2l0 (1967) believed that individual thyroid
activity in a hen, with it's seasonal variation, probably
influenced the level of the hen's egg production, egg yolk

size, and yolk cholesterol content.

Feed Consumption
1. Experiment 1
Figure 18 deals with the feed intake of hens used in

experiment 1, using corrected values to eliminate the data

Figure 18.

137

Experiment 1. Feed consumption, average of
three hens per treatment. For the last two
weeks of the experiment only the values of two
hens were used to calculate the mean for the
control feed treated hens for this figure, as
indicated by the numeral 2 on the line in the

graph.

138

 

120
2
no 7 N
10° ‘\ ’ — #0 .—
.90. \ fl — — fl ’ .0... .
9o — *8", I
e\ --..-IIIIIIIIIIUIIIIO ”
eguuei".--- ’ ’
\

g FEED CONSUMPTION/HEN/DAY
(3 HENS/TREATMENT)

 

A B C D E

A: FEED CONSUMPTION MEAN DURING PRE-TREATMENT PERIOD

B: u .. .. .. WEEKIDRUG TREATMENT
C= .. .. .. .. .. 2
= .. .. n .. .. 3
E= .. ~ .. .. POST TREATMENT PERIOD

— = CONTROL FEED TREATMENT (C|~ no at 0 pm.)
- — - = DRUG FEED TREATMENT (Cl-720 at 2600 ppm)
— - = CONTROL PER 05 TREATMENT (CI—720 at DMQ/kg BODY WEIGHT)

------- = DRUG PER 05 TREATMENT (CI: 720 at 150 lug/kg BODY WEIGHT)

139

of hen # 1 for the last two weeks of the experiment. This
hen was injured on day 14 of drug treatment and was off
feed from that date until the post-treatment week. The
feed intake analysis was calculated with the values of the
injured hen considered as valid, and with her observations
taken as the mean of the other two hens for that date.

Hen # 1 had maintained this exact level of feed consumption
for each of the previous weeks. By these manipulations,
the reader has the option of viewing the data as it was
recorded, as well as what probably would have happened.
These alterations resulted in no change in the post—
treatment period statistical results, but importantly
altered the results from week three of drug treatment. See
Table 16 for a comparison of these data.

The three control hens' feed intake was found to be
significantly (P< .05) greater than that of the drug per 08
treated hens, on day 21 of drug treatment. For this same
date, as well as the second week of drug treatment, the
control feed treated hens consumed more feed (P< .01)

than the hens of the drug feed group.

2. Experiment 2

There was pronounced individual variation among the
birds given the drug treatments, especially those hens
offered the high level of drug (5200 ppm), where hen 104
ate at nearly a normal rate, and some hens consumed

essentially zero feed for several days, as indicated by the

140

Table 16. Experiment 1. Results of statistical analysis
by the Tukey HSD test of means, comparing the
feed intake of the four treatments.*

 

End of Treatment Week

 

 

using

Treatment post- expt.

Comparison 1 2 3 trt. trt. F

Control feed gg. 1 2 3 3

drug feed NS sig. sig. NS sig.

.01 .01 NS .05

NS

Control feed xg.

control per 03 NS NS NS NS NS
NS NS

Control feed 1g.

drug per 08 NS NS sig. NS NS
.05
NS

Drug feed Kg. -

control per 08 NS NS NS NS NS
NS NS

Drug feed XE-

drug per 08 NS NS NS NS NS
NS NS

Control per os zg.

drug per 08 NS - NS NS NS NS
NS NS

 

*The data tested was the mean of feed intake for the three
hens of a treatment. Because hen # 1 was injured on day 14
of drug treatment, and was thereafter off feed, the data
was calculated with her feed consumption as valid as well as
corrected to what it had been previously.

1NS=not significant P>.05
2

.Bstatistical analysis including the hen in cage # 1 as a
valid observation is below the results computed from data
using the corrected values (the mean of hens 2 and 3).

sig.=significant P<.05

141

amount of feed left in the container each day. Even if
hen 104 ate only at the maintenance level (70 g/hen/day),
that left the other seven hens with much less than the
approximately 36 g/hen/day calculated as the mean for that
treatment in each of the second and third weeks of drug
administration.

By the fourth week of drug treatment, those hens
eating feed with CI-720 at 5200 ppm (see Figure 19) appeared
to bounce back from the near starvation level of feed intake
of the preceeding two weeks to almost a maintenance level.
This rebound effect was evident for hens given the low drug
treatment, in both experiments (compare Figures 18 and 19).
After a decline in feed intake of 30 g/hen/day, those hens
given the 2600 ppm treatment started an upward trend to
near their pre-treatment level by the time the experiment
had ended.

Analysis of the final treatment means by Tukey's HSD
test of means indicated the control group of hens had higher
feed intake than those of either of the drug treatments
(P‘<.05). There was no difference statistically between the
drug treatments. Statistical analysis for means of the
hen's feed intake at the end of each treatment week was not
performed. Instead, Figure 19 serves to better illustrate
the dramatic change in feed consumption using the mean for
feed intake at the end of each treatment week. Any time
the feed intake values drop below the individual hen's

Figure 19.

142

Experiment 2. Feed consumption average Of the
eight hens/treatment, as calculated at the end

of each week of drug treatment.

Feed Consumption in groms/hen/day

143

I20

 

110

100 ,

~O
O
/

o
o"

80 \ ...O e'....

70 \ 9.. --.---I.-IIIIII /
/

60 \ x

50 \ ’

40 \ II

h—————_-’

 

week week week week
I 2 3 ~4

—— : Control teed treatment (Cl-7200t0ppm)

III...- 2 Drug " ” ( II II 2600 ppm)

-—- _'—_ H II II ( " " 5200 ppm)

144

requirements which allow her to be of economic value,

that is significant, whether or not it is statistically.

3. Discussion

Table 17 lists the amount Of CI-720 taken in by indi-
vidual drug treated hens of experiment 1 as a mean for the
week, plus the mean of the eight hens per drug treatment in
the second experiment. Hens given the CI-720 via the
gelatin capsule injested the drug in larger quantity (107 mg
as the difference between treatment means) than those hens.
receiving CI-720 in the feed. The effect of the CI-720
bolus was not as great as when this drug was given in the
feed, in lowering the feed consumption.

The rationale behind these drug levels was that at
2.6 g CI-720/kg diet, an average hen would consume about
260 mg CI-720/hen/day (depending on energy content of the
diet). An average S.C.W.L. laying hen might weigh 1.8 kg,
therefore at 150 mg CI-720/kg body weight, the amount of
drug consumed based on these two different calculations,
would be about equal (for experiment 1). The assumption was
made that feed treated hens would ingest about 260 mg
CI-720/hen/day; and the hens treated according to their body
weight would be administered about 270 mg CI-720/hen/day.

By doubling the dosage of the first experiment for the
feed method of drug administration, the second experiment

in part, attempted to find an upper limit of CI-720

145

Table 17. Experiments 1 and 2. Intake of CI-720, as
calculated from the feed consumption means
in each experiment. Also, the intake of drug
based on the body weight in a treatment in
experiment 1.

 

Experiment 13:

Drug Feed Treatment (CI-720 @ 2600ppm)

 

 

 

Hen # 6 7 8
b CI-72oC CI-720 01-720
Feed Con- Feed Con- Feed Con-
Intake sumed Intake sumed Intake sumed
Day 1.7d 998 257 63 164 80 208
Day 8-14 68 177 68 177 80 208
Day 15-21 85 221 71 185 87 226
Mean 84 218 67 175 82 214
Treatment
Mean 202

Drug Per 03 Treatment (CI-720 @ 150 mg/kg B.W.f)

 

 

 

Hen # 14 15 16
CI-720 CI-720 CI-720
cap- cap- cap-

Weighg f sule sule sule

date B.W. (mg) B.W. (mg) B.W. (mg)

9 May 1.920 288 2.177 327 2.216 332

15 May 1.871 281 2.093 314 2.198 330

22 May 1.794 269 2.130 320 2.140 321

Mean 1.862 279 2.133 320 2.185 328

Treatment

Mean 309

 

146

Table 17 (cont'd.).

 

Experiment 2a:

Drug Feed Treatment (CI-720 @ 2600ppm)

 

 

. Mean feed b Mean CI-720 c
intakelhenlday_ intake/hen/day
Day 1-7d ‘ 98 255
Day 8-14 67 174
Day 15-21 73 190
Day 22-28 88 229
Mean 82 212

Drug Feed Treatment (CI-720 @ 5200ppm)

Day 1-7d 93 484
Day 8-14 37 192
Day 15-21 36 ' 187
Day 22-28 71 369
Mean 59 308

 

a = 3 and 8 hens/treatment in experiments 1 and 2,
respectively.

in g feed consumed/hen/day.

(feed intake) x (2600 g CI-720 per kg diet) =

g CI-720 consumed x 1000 = mg CI-720 consumed.

days of drug treatment.

all values are rounded off.

hen body weight in kg+1 g.

the date weight used To calculate 01-720 packed into
the capsule for 1-3 treatment weeks.

00’
II

Ohhtwtl

147

administration. This doubled dose of CI-720 (5200 ppm)
was enough to inhibit the feed intake of all but one of
the eight hens of that treatment.

The response of the 2600 ppm CI-720 treated hens in
each experiment showed almost identical patterns of feed
consumption (see Figures 18 and 19), even to the average
amount of feed eaten over the length of the experiment.
Hens in experiment 1 that were given this particular treat-
ment averaged about 81 g feed consumed/hen/day, and the
hens in experiment 2 for this treatment averaged 82 g
feed consumed/hen/day.

In experiment 2, comparing the amount of drug consumed
by the 2600 ppm treated hens with the amount consumed by
those hens given the high level of drug for the second and
third weeks of drug treatment, the values were about the
same. Yet the feed intake was about completely inhibited
for some hens of the high drug level treatment, and not in
the lower drug level treatment. The level of feed con-
sumption of hens of the 2600 ppm drug treatment was about
twice that of hens of the 5200 ppm drug treatment. If the
effect of CI-720 was to influence the appetite center in
the brain, then the large amount of CI-720 ingested during
the first week by the hens given the higher level of drug
could have been enough to nearly stop the desire to eat
(except hen 104). Then at the lower levels of drug which
occurred later because of no appetite, the effect of CI-720

may have dissipated enough to allow some food consumption.

148

Alternatively, the hens may have come to a point of "eat
or die", and the drug was not strong enough to force the
latter choice.

Apparently the effect of CI-720 on feed consumption
is not only on palatability, as those hens given the drug
in the capsule were similarly effected as those given
CI-720 in the feed, but to a lesser magnitude. Possibly an
action of CI-720 was the result of an inhibition of the
appetite center. Because the capsule treated hens injested
about 120 mg CI-720 more than the corresponding drug feed
treated hens, on a regular basis, and yet maintained a feed
intake that was greater than those hens eating the con-
taminated feed, a safe assumption could be that the effect
of CI-720 is at least partially mediated through a
palatability and/or instinctive rejection of foreign
substances.

Several hens molted in the drug treated groups, in both
experiments but more so in the second. This could have been
due to an action Of the drug, but more likely to the acute
starvation or below maintenance levels of feed intake of the
hens. In experiment 1, hen # 7 started to molt by the end
of the first week of drug treatment; during this time, she
averaged about 63 g of feed intake per day. By day 17 of
drug treatment, hen # 6 started to molt, the preceeding week
her feed intake averaged about 68 g. Both hens were from

experiment 1.

149

In experiment 2, records of individual feed intake were
not kept, but for the 2600 ppm treatment, four hens showed
evidence of molting (cessation Of egg production, and
dropped feathers) the week after their feed consumption had
declined to 66 g feed consumed/hen/day, and several showed
signs of illness (watery droppings, ruffled feathers,
crouched position, eyes closed). Four hens on the 5200 ppm
treatment dropped feathers the third week of treatment;
this group of hens had taken in only about 36 g feed/hen/day

average during the second and third weeks of treatment.

Total Excreta Cholesterol

1. Experiment 1

Excreta samples were collected during the days 15
through 21 of drug treatment; in experiment 1, only. The
collection pan served as the storage container for air
drying.

When the excreta cholesterol was expressed as
mg cholesterol/g dry excreta, there was no difference between
the different treatment means, as judged by the insignificant
f-value of the analysis of variance. The mean for the three
hens/treatment were: 1) control feed treatment (CI-720 at
0 ppm)--- 4.1; 2) drug feed treatment (CI-720 at 2600 ppm)--
-3.4; 3) control per os treatment (CI-720 at 0 mg/kg body
weight)---3.5; 4) drug per os treatment (CI-720 at 150 mg/kg
body weight)---3.2 mg cholesterol/g dry excreta. For an

approximation of these values when based on wet excreta

150

weight, simply divide by four. The standard error for the
experiment was 0.144.

March gt El. (1964) expressed their results as g sterol
per kg diet. They reported values of 4.89 and 5.86 as
control hen's means. When the values of the present
experiment are expressed as March and co-workers did, these
values are much lower, more on the order of 1+ g cholesterol
excreted/kg diet.

This may indicate a technical error in this experiment,
or degredation of the cholesterol as Clarenburg gt,al.
(1971) have suggested.

Konlande and Fisher (1969) reported that control chicks
had values of 6.2 mg cholesterol/g excreta, but did not
state whether these values were based on wet or dry excreta
weights.

Because of the extended period of time (about 18 weeks)
between time of sample collection and processing, the
cholesterol content could have been reduced due to bacterial
degredation (Clarenburg gt §;., 1971). Also worms were
very abundant in the excreta collection trays, and they
probably consumed part of the excreta, thus making-total

excreta erroneous.

SUMMARY

During two experiments, laying hens were administered
an antilipemic compound (CI-720) for the purpose of
determining its effect on a number of blood, repro-
ductive, and physical parameters. The results were

as follows:

A. Total plasma cholesterol was lowered by CI-720
from 133 to 114 mg/dl plasma in experiment 1,
and from 155 to 111 and 105 mg/dl plasma in
experiment 2. The first experiment values could
have been an artifact of the random distribution
of the hens, as could the free plasma cholesterol
values from experiment 1.

B. CI-720 caused a decline in free plasma cholesterol
from 107 to 79 mg/dl plasma in the first experi-
ment, and from 85 to 50 and 38 mg/dl plasma
during the second.

C. The percent free of total plasma cholesterol was
similar in response as the free plasma cholesterol.
A larger percentage was found to be in the free
form in the control hens than in drug treated

hens, during both experiments.

151

H.

152

Plasma triglycerides were not determined in the
first experiment, but were found to be signifi-
cantly depressed by the drug in the second
(1524;613;228 mg/dl plasma, for treatment of 0,
2600, and 5200 ppm of the diet, respectively).
The total yolk cholesterol was increased in the
first experiment, but no difference was found
between treatment means during the second.

Hen body weight was not changed by the drug
treatment in experiment 1, but was lowered in
experiment 2.

The percent change in body weight was not changed
in experiment 1 by CI-720, but the drug treated
hens showed more variation during experiment 2.
Weekly percent egg production declined to zero
for some drug treated hens, but not all, during
both experiments.

There was no difference between treatments in
the amount of change in weekly percent egg pro-
duction during experiment 1. In the second
experiment, the drug treated hens were shown to
have changed more in weekly percent egg produc-
tion, than those given control treatments.
Total egg weight; yolk, albumen, and shell
weights, plus their percent of total egg weight
was not altered by drug administration during

either experiment. The drug treated hens of

153

experiment 2 showed a trend toward lowered egg
weight and yolk weight compared to that of
control hens.
K. Feed consumption was lowered by the drug, during
both experiments.
The conclusion drawn from these experiments was that
CI-720 probably had an antilipemic effect in the
chicken, but that this effect was confounded with
the lowered feed intake. Also, CI-720 did not lower
the egg cholesterol. Therefore, this drug probably
has little direct application for the poultry
industry.

APPENDICES

2.

3.

4.

154

APPENDIX A

FOOTNOTES

Three classes of hypocholesterolemic compounds:

A.

B.

2 (p-(2-diethylaminoethoxy) phenyl) benzimidazole
monohydrochloride
1'-(2-(1-naphthylamino)ethyl)-1,4'-bipiperidine
dihydrochloride
4-(2-chlorophenyl)-alpha-(p-methoxyphenoxymethyl)-

1-piperazine ethanol

80-12937 is 20,25-diazocholestenol dihydrochloride

50-10644 is 2-(1-pyrrolidinyl) ethyl triphenyl

methane-4 carboxylate hydrochloride

beta-diethylaminoethyl diphenyl propylacetate

hydrochloride

155

 

 

APPENDIX B
RECOVERIES
Parameter Percent Recovered
Total Plasma Cholesterol 86.7
Free Plasma Cholesterol 86.6
Plasma Triglycerides 91.4
Total Yolk Cholesterol 102.9

An example of the method used for these determinations
was to split a sample of plasma, and to one aliquot 100 mg
crystalline cholesterol (or triglyceride aliquot) was added.
The samples were processed as outlined in Flow Chart 1 (for
total plasma cholesterol, as an example). The difference
between the calculated amount of cholesterol of the
"spiked" and the original sample was 86.7 mg cholesterol.
This represents 86.7 percent of the added cholesterol

recovered.

156

APPENDIX 0

Appendix Table 1. Split plot analysis of variance table--
a comparison between the computer and

hand calculated results.*

 

 

 

 

 

 

Mean Square f-Statistic
Source of Variation d.f.
com. man. com. man.
AmongSubjggtg
method of
administration (a) 1 3727 3727 3.24 3.24
drug/no drug 66) 1 6421 6422 5.6 5.9
x interaction 1 251 249 .21 .22
error a (hens) 8 1150 1149
total 11
Within Subjggtg
time (v) 5 972 972 1.1 1.5
«xx Y interaction 5 751 751 1.2 1.2
flix Y interaction 5 418 418 .66 .66
4x fl x7 " 5 1037 1038 1.6 1.6
error b 40 630 630
subtotal 60
total 71

 

*Total plasma cholesterol was the data used.

157

APPENDIX D

SPLIT PLOT STATISTICAL MODEL

Split Plot Model:

Yijkl

Where:

/%j
003%:
(“V315

(4 Y)”
OGYAM

(o‘fl .0131

1'5 KB)

fl + at; +flj + D(«'J)K I (“flLJ f Y! + (‘3 7);} + (YD)("J')KI

+- («fly)/J,e + {(15411)

population mean

method of drug administration
drug; no drug treatments

Error a---hens nested in treatment
method x drug interaction

time effect

method x time interaction

drug; no drug x time interaction

method of drug administration x drug; no
drug x time interaction

random experimental error---confounded
with this is the hen x time interaction

158

APPENDIX E

SPLIT PLOT STATISTICAL ANALYSIS, SUMS OF SQUARES

53~=(§"'°"/“)W 1 "’ "2 f“;
j a 1,2 (1
(\01- '-
.. 1 I , y.... /N k 8 1,2,3 (11)
55/3: (Elfin/nht) - Y'” /N 1 a 1,2,...,6 (t)

53.9. = (if my“)- (£5, y.-...%Ihf) _ (3‘25. y-J.-‘/m M) 1* y-mz/N

”1.5:!

HIJS =1 [eldel

33°:(fi‘): fax-1%?) “(.21-2:: Van/hf)

SS). 3 (g; 7....17ka) "' y...."/N

#11:;

“cw “(55 W/‘Jkéi Yr-oo‘/°’*‘~f)‘(}§‘, 7'"; Yea/1% air/N

'- z 1 ‘ l ‘2.
5%? = (f; .J.’ez/hfl‘\) "(JZ;;Y.J., /mht)-é-‘ Y...1/MJ‘\)+ Yoooo/N

wt!”

53 74551“: ivy—27")“(ff yt-of/JK) ‘(55 70-7/1“)-

dfi c=IJ=I 1:: AIM «=1 .1“

.... (i Z‘ Y.J.xt/MK)+(;Z: ”....wxfl) + (5;: Y°--,¢‘/MJA) +

IF! 1:!

1" (2.:- YOJHT/mk‘t) ‘— Y.-..1/N

d"

53 = ssY - 55‘ — 55/, - ss“fl - 550- $5,, “554);-

159

APPENDIX F

TUKEY HSD, MODIFIED TUKEY HSD, AND DUNNETT TEST OF MEANS
PLUS fmax-TEST FOR HOMOGENOUS VARIANCE

Tukey HSD Test of Means:

._ /

test statistic: f;-— yg__
V/1SE/F

where: t = the number of means being compared

 

n - t = the degrees of freedom for error
critical value: Igwd,t, n-t

from table A.8---Gill

Modified TukengSD Test of Means:
' __ __ /
test statistic: .be-a Y"

«V795E/7}

where: r8 = the smaller of the r1 being compared

 

 

critical value: same as above

Dunnett Test of Means:

 

test statistic: y" _ Y" a
MOTSE(E%'+I%’)
where: m = the number of experimental groups
1): the degrees of freedom for error
critical value: tn, 0, , m, V

from table A.9.1---Gill

160

f -Test for Homogenous Variance:

 

max
test statistic - T——32 max
S min

where: t = the number of groups
v a the degrees of freedom for error =r7-1
critical value: fmax’cx , t,))
from table 6.1---Gill, or Table T from Rohlf and Sokal

161

APPENDIX G

Appendix Table 2. Re-evaluation of free plasma cholesterol
values of experiment 2.*

 

 

 

 

_ Initial
Hen # Rep. 1 Rep. 2 y Determi-
nation
Control Treatment(CI-720 @ 0 ppm) in mgldl
99 93 92 93 90
102 96 95 96 97
103 88 82 85 86
115 83 81 82 81
121 88 94 91‘ 91
122 133 128 131 113
124 55 55 55 54
125 so 46 49 48
Low Drug_LevelpTreatment(CI:120 @ 2600 ppm) in mgldl
105 34 32 33 29
106 60 63 62 61
107 52 51 - 52 52
108 -- 95 95 95
109 31 31 31 30
11D 60 '61 51 5°
136 36 36 36 33
139 40 37 39 37
Hi h Dru Level Treatment CI-720 @ 200 m in m dl
104 74 78 76 71
116 40 40 4O 36
117 36 38 37 34
131 36 32 34 32
137 37 33 35 35
13s 44 41 43 38
141 30 27 29 28
142 33 31 32 30

 

*Data is in mg/free cholesterol/d1 plasma. There were eight
hens in each of the three treatments. Values labelled Rep.
1 and 2 were calculated from different curves, on the same
day. The values labelled initial determination are the
means obtained as a final value at a previous time. All
determinations were from the same plasma sample.

162

APPENDIX H

Appendix Table 3. Coefficient of variation* for parameters

of experiments one and two.

 

 

Parameter Experiment 1 Experiment 2
Total plasma cholesterol 23.7% 21.4%
Free plasma cholesterol 31.1 34.6
Percent free of total

plasma cholesterol 17.8 21.5
Plasma triglycerides ----** 59.8
Body weight (in grams) 10.9 12.3
Weekly egg production 44.3 49.9
Total yolk cholesterol 10.0 11.9
Total egg weight -6.1 8.8
Shell weight 11.0 13.1
Albumen weight 6.8 11.3
Yolk weight 7.5 5.9

 

*calculated as: (standard deviation) x (100)
if

**data discarded

163

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