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REPRODUCTIVE PERFORMANCE OF MICE FED WITH GREAT-LAKES CARP
AND FISH FARM RAISED CARP

BY

Yu-Min Kuo

A THESIS

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

MASTER OF SCIENCE

Department of Animal Science

1994

 

ABSTRACT

REPRODUCTIVE PERFORMANCE OF MICE FED WITH GREAT-LAKES CARP
AND FISH FARM RAISED CARP

BY

Yu-Min Kuo

Three successive studies were performed to determine the
reproductive toxicity of Great Lakes fish on mice. Mice were
randomly divided into three main treatment groups: lab Chow
(C), farm raised carp (A), and Great Lakes carp (G). The
treatment diets were provided immediately after the dams (F-O)
gave birth through lactation. Both F-l and F-z were fed the
treatment diets from weaning to termination. Reproductive
performance of both generations were examined in vivo and in
vitro. Increased liver weight and decreased hepatic vitamin
A and E were observed in both fish-eating groups. The highest
liver weight/body weight ratio was found in group G. Group A
consistently had the lowest body weight in F-l and F-2 males.
There were no differences in fecundity, lactation index,
litter size, thymus and testis weights, and caudal epididymal
sperm concentration, motility, and in vitro fertilization
ability in both F-l and F-2 males. No difference in body,
liver, thymus weight, and.in vitro fertilization in F-l and F-
2 females was observed. Based on the parameters observed in
this study, Great Lakes carp did not alter the reproductive

performance in young mature mice for two generations.

 

LIST OF TABLES . . .
LIST OF FIGURES . .
INTRODUCTION . . . .

LITERATURE REVIEW .

TABLE OF

CONTENTS

I. Polyhalogeneted Aromatic Hydrocarbons

(PAns) . .

A. Polychlorineted Biphenyle (pcne) .

Introduction

Physical and Chemical Properties
Sources of Exposure .
Contamination Level .

Absorption . . . . . . . .
Distribution . . . . . .
Metabolism and Elimination
Toxicity . . . . . . . . .

a. Clinical Cases . . .

b. Effects on Enzyme Activity

c. Carcinogenicity . . .

d.
e.

Summary of PCBs .

Immune Alterations .

Reproductive Toxicity

O . o O . O O . O O . Q . 0 . O

B. Polychlorineted Dibenzo-p-dioxins
(PCDDS).

II.

Chlorinated Pesticides
A. nexachlorobenzene (EOE)

B. Aldrin And Dieldrin

III. Mercury .

Introduction

Physical and Chemical Properties
Sources of Exposure .

C. Dichlorodiphenyltrichloroethene (DDT).

Absorption and Distribution .
Metabolism and Elimination
Toxicity

iii

vii

25

29
29
32
35

39
39
39

41
41
42

 

PEASE

PHASE

PHASE

Statement of Problem . .
Objective . . . . . . .

I STUDY . . . . .
MATERIALS AND METHODS .
Animals . . . . . .
Treatment Diets . .
Mouse Liver Analysis
Medium and Chemicals
Experimental Design
Sperm Collection . .

In Vitro Fertilization
Statistical Analysis .

Results . . . .
Nutrients Content of
F-O Dams . . . .

F-l Males . . . . .
F-l Females . . . .
Discussion . . . . . . .
II STUDY . . . . . . . .
Materials And Methods .
Animals . . . . . .
Treatment Diets . .
Experimental Design
Results . . . . . . . .
Discussion . . . . . . .

III STUDY . . . . .
Materials And Methods .
Animals . . . . . .
Treatment Diets . .
Medium and Chemicals
Experimental Design

In Vitro Fertilization

Statistical Analysis
Results . . . . . . . .
Nutrients Content of
F-O Dams . . .

F-l Males . . . . .
F-l Females . . . .
F-2 Males . . . . .
F-Z Females . . . .
Discussion . . . . . . .

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APPENDICES .

APPENDIX

1.

APPENDIX 2.

APPENDIX 3.

APPENDIX

APPENDIX

APPENDIX

APPENDIX

APPENDIX

APPENDIX

APPENDIX

APPENDIX

APPENDIX

APPENDIX

APPENDIX

APPENDIX

APPENDIX

4.

5.

10.

11.

12.

13.

14.

15.

16.

O O O O O O O O O O C O O O O O O O O O

ORGANOCHLORINATED PESTICIDES AND PCBS
IN FISH OIL O O I O C O O O O O O O O O

NUTRIENT REQUIREMENTS OF'MICE . . . . .
DEFINITION OF SPERM MOTION PARAMETERS .

BODY AND LIVER WEIGHTS OF F-O
LACTATING MICE . . . . . . . . . . . .

LIVER VITAMIN A AND E CONCENTRATIONS
OF LACTATING F-O FEMALES . . . . . . .

BODY WEIGHTS OF F-l MALE MICE FROM
BIRTH TO 23 WEEKS OF AGE . . . . . . .

LIVER AND TESTIS WEIGHTS OF F-l
MALES O O O O O O O C O O O C O O O O O

LIVER VITAMIN A AND E CONCENTRATIONS
IN F-l “LES O O O O O O O O O O O O O

CAUDAL EPIDIDYMAL SPERM CONCENTRATIONS
OF F-1 MALES AT 6, 7, 8, 15, 23, 32,
AND 34 WEEKS OF AGE . . . . . . . . .

EPIDIDYMAL SPERM MOTION QUALITY OF
F-l MALES . C C O O O C O O O b O O C .

REPRODUCTIVE PERFORMANCE OF F-l
MALES . O O O C C O O C O C C O C O O O

REPRODUCTIVE PERFORMANCE OF F-1
FMES O O I O O O O O O O O O O I O O

LIVER VITAMIN A AND E CONCENTRATIONS
OF F-1 MALES AFTER HIGH DIETARY
VITAMIN E SUPPLEMENTATION . . . . . .

BODY AND LIVER WEIGHTS OF F-l
MALES AFTER HIGH DIETARY VITAMIN
E SUPPLEMENTATION . . .... . . . . . .

BODY AND LIVER WEIGHTS OF F-O
LACTATING FEMALES . . . . . . . . . . .

BODY WEIGHTS OF F-1 MALE MICE FROM
BIRTH TO 20 WEEKS OF AGE . . . . . . .

116

116

117

118

120

121

122

123

125

126

127

128

129

130

131

132

133

' .

APPENDIX 17. LIVER, THYMUS, AND TESTIS WEIGHTS
OF F-1 MALES. . . . . . . . . . . . . . 134

APPENDIX 18. LIVER VITAMIN A AND E CONCENTRATIONS
IN F-l MES . O O O O O O C O O O O O 13 5

APPENDIX 19. CAUDAL EPIDIDYMAL SPERM
CONCENTRATIONS OF F-1 MALES AT 8
AND 11 WEEKS OF AGE . . . . . .... . . 136

APPENDIX 20. EPIDIDYMAL SPERM MOTION QUALITY OF
F-l “LES O O O O O O O O O O O O O O 137

APPENDIX 21. REPRODUCTIVE PERFORMANCE OF F-l
“LES O O C O C O C O O O O O O O O O I 138

APPENDIX 22. BODY, LIVER, AND THYMUS WEIGHTS OF
F-l FWES C O C O O O C O O O O O O 13 9

APPENDIX 23. IN VITRO FERTILIZING ABILITY OF F-l
FWES . O C C O C O C O O I C O O O O 14 0

APPENDIX 24. BODY WEIGHTS OF F-2 MALE MICE FROM
BIRTHTOBWEEKSOFAGE. . . . . . . . 141

APPENDIX 25. LIVER, THYMUS, AND TESTIS WEIGHTS
OF F-Z MES O O O O O O O O O O O O O 142

APPENDIX 26. LIVER VITAMIN A AND E CONCENTRATIONS
IN F-Z MES. . O O C C O O O O O O O O 144

APPENDIX 27. CAUDAL EPIDIDYMAL SPERM CONCENTRATIONS
OF F-2 MALES AT 6, 7, AND 8 WEEKS
OF AGE 0 O O O O O C O C O O O C O O O 14 5

APPENDIX 28. EPIDIDYMAL SPERM MOTION QUALITY OF
F-z MALES O O O I O C O O O O O O O O 146

APPENDIX 29. REPRODUCTIVE PERFORMANCE OF F-2 MALES . 147

APPENDIX 30. BODY, LIVER, AND THYMUS WEIGHTS OF
F-Z FWES e e e e e e e e e e e e e e 148

APPENDIX 31. LIVER VITAMIN A AND E CONCENTRATIONS
IN F-2 FEMALES . . . . . . . . . . . . 149

APPENDIX 32. IN VITRO FERTILIZING ABILITY OF F-2
FWES O O O C O O C O O C O O O O O O 150

LIST 0! Rnrnmczs I O C O C O O O O O O O O O O O I O C 1 5 1

vi

TABLE

TABLE

TABLE

TABLE

TABLE

TABLE

TABLE

TABLE

TABLE

TABLE

TABLE

TABLE

TABLE

TABLE

8.

9.

10.

11.

12.

13.

14.

LIST OF TABLES

MEAN CONCENTRATIONS OF PCBS IN FISH AND
HERRING GULL EGGS FROM GREAT LAKES . . . . . .

NUTRIENT ANALYSIS OF TREATMENT DIETS . . . . .

MINERAL ANALYSIS OF THREE TREATMENT DIETS AND
MINERAL REQUIREMENT FOR MICE . . . . . . . . .

ORGANOCHLORINATED PESTICIDES AND PCBS IN
RAW CARP AND MOUSE DIETS . . . . . . . . . . .

ORGANOCHLORINATED PESTICIDES AND PCB RESIDUES
IN F-l MALE LIVER O O O O O O O O O O O O O O

NUTRIENT CONTENTS OF THE TREATMENT DIETS . . .

ORGANOCHLORINATED PESTICIDES AND PCBS IN
FISH OIL O O O O O O O O O O O O O O O O O O
NUTRIENT REQUIREMENTS OF MICE . . . . .

OPERATING SETTING OF THE CELLSOFT VIDEO-
MICROGRAPHIC INSTRUMENT USED FOR COMPUTER-
ASSISTED SEMEN ANALYSIS . . . . . . . . . . .
BODY AND LIVER WEIGHTS OF F-O LACTATING MICE .

LIVER VITAMIN A AND E CONCENTRATIONS
OF LACTATING F-O FEMALES . . . . . . . . . .

BODY WEIGHTS OF F-l MALE MICE FROM BIRTH
TO 23 WEEKS OF AGE . . . . . . . . . . . . .

LIVER AND TESTIS WEIGHTS OF F-1 MALES . . . .

LIVER VITAMIN A AND E CONCENTRATIONS IN
F-l MALES O O O O O O O O O O O O O O O O 0

vii

55

56

57

66

90

116

117

119

120

121

122

123

125

TABLE

TABLE

TABLE

TABLE

TABLE

TABLE

TABLE

TABLE

TABLE

TABLE

TABLE

TABLE

TABLE

TABLE

TABLE

TABLE

TABLE

TABLE

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

25.

26.

27.

29.

30.

32.

CAUDAL EPIDIDYMAL SPERM CONCENTRATIONS
OF F-l MALES AT 6, 7, 8, 15, 23, 32, AND

3 4 WEEKS OF AGE 0 C O O O O O O O O O O O O
EPIDIDYMAL SPERM MOTION QUALITY OF F-l MALES
REPRODUCTIVE PERFORMANCE OF F-l MALES . . .
REPRODUCTIVE PERFORMANCE OF F41 FEMALES . .
LIVER VITAMIN A AND E CONCENTRATIONS OF F-l
MALES AFTER HIGH DIETARY VITAMIN E
SUPPLmENTATION . O O O O O O O O O O C O O

BODY AND LIVER WEIGHTS OF F-1 MALES AFTER
HIGH DIETARY VITAMIN E SUPPLEMENTATION . . .

BODY AND LIVER WEIGHTS OF F-O LACTATING
FWES C O O O C O O O O C O O C O O O O O

BODY WEIGHTS OF F-l MALE MICE FROM BIRTH
To 2 0 WEEKS OF AGE 0 O O O O O O O O O O O O

LIVER, THYMUS, AND TESTIS WEIGHTS OF F-l
MES C O C O O O O O O O O O O O O O I O O

LIVER VITAMIN A AND E CONCENTRATIONS IN
F-l WES O O O O O O O O O O O O O O O 0

CAUDAL EPIDIDYMAL SPERM CONCENTRATIONS
OF F-l MALES AT 8 AND 11 WEEKS OF AGE . . .

EPIDIDYMAL SPERM MOTION QUALITY OF F-1
MALES O O O O O C C C O O O O O O O O O O O

REPRODUCTIVE PERFORMANCE OF F-l MALES . . .

BODY, LIVER, AND THYMUS WEIGHTS OF F-1
FMES C O O O O O O O O O O O O O O O O O

IN VITRO FERTILIZING ABILITY OF F-l FEMALES

BODY WEIGHTS OF F-2 MALE MICE FROM BIRTH
TO 8 WEEKS OF AGE . . . . . . . . . . . .

LIVER, THYMUS, AND TESTIS WEIGHTS OF F-2
“LES O O O O O O O O C O O O O C O O O O O

LIVER VITAMIN A AND E CONCENTRATIONS IN
F-z MALES O O C O O C O O O O O O O O O I

126

127

128

129

130

131

132

133

134

135

136

137

138

139

140

141

142

144

 

‘le

TABLE

TABLE

TABLE

TABLE

TABLE

TABLE

33.

34.

35.

36.

37.

38.

CAUDAL EPIDIDYMAL SPERM CONCENTRATIONS
OF F-2 MALES AT 6, 7, AND 8 WEEKS OF AGE .

EPIDIDYMAL SPERM MOTION QUALITY OF F-2
“LES C O C C O O O C O O I O O O O O O O

REPRODUCTIVE PERFORMANCE OF F-2 MALES . . .

BODY, LIVER, AND THYMUS WEIGHTS OF F-2
FMES O O O O O O O O O O O O O O O O O O

LIVER VITAMIN A AND E CONCENTRATIONS IN
F-z FMES O O O O O O O O O O O O O O O O

IN VITRO FERTILIZING ABILITY OF F-2 FEMALES

ix

145

146

147

148

149

150

PI

PI:

FIC

m

FIGURE

FIGURE

FIGURE

FIGURE

FIGURE

FIGURE

FIGURE

FIGURE

FIGURE

FIGURE

FIGURE

FIGURE

FIGURE

1.

2.

8.

9.

10. LIVER VITAMIN A AND E CONCENTRATIONS OF

11.

12.

13.

LIST OF FIGURES’

BODY AND LIVER.WEIGHTS OF F-O LACTATING MICE

LIVER VITAMIN A AND E CONCENTRATIONS OF

LACTATING F-O FEMALES

BODY WEIGHTS OF F-l MALE MICE IN PHASE I
FROM BIRTH TO 23 WEEKS OF AGE

LIVER WEIGHTS OF F-l MALES AT 7,
15 WEEKS OF AGE

8, AND

LIVER WEIGHT OVER BODY WEIGHT RATIOS
OF F-l MALES AT 7, 8, AND 15 WEEKS OF AGE

LIVER VITAMIN A AND E CONCENTRATIONS IN

F-1 MALES .

CAUDAL EPIDIDYMAL SPERM CONCENTRATIONS
OF F-1 MALES AT 5, 6, 7, 8,

AND 34 WEEKS OF AGE

15' 23,32,

REPRODUCTIVE PERFORMANCE OF 52-DAY-OLD

F-1 MALES .

REPRODUCTIVE PERFORMANCE OF F-1 FEMALES

F-1 MALES AFTER HIGH DIETARY VITAMIN E
SUPPLEMENTATION

BODY AND LIVER WEIGHTS OF F-O LACTATING

FEMALES . .

BODY WEIGHTS OF F-l MALES IN PHASE III FROM

BIRTH TO 20 WEEKS OF AGE .

O

LIVER WEIGHTS OF F-1 MALES AT 8 AND 11

WEEKS OF AGE

59

6O

61

62

63

65

67

68

7O

81

91

92

94

FIGURE

FIGURE

FIGURE

FIGURE

FIGURE

FIGURE

FIGURE

FIGURE

FIGURE

FIGURE

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

LIVER VITAMIN A AND E CONCENTRATIONS

IN 8-WEEK-OLD F-1 MALES

CAUDAL EPIDIDYMAL SPERM CONCENTRATIONS
OF F-1 MALES AT 8 AND 11 WEEKS OF AGE .

REPRODUCTIVE PERFORMANCE OF 8-WEEK-OLD
F-l MALES . . . . . . . . '

BODY AND LIVER WEIGHTS OF 8-WEEK-OLD
F-l FMLES . O O O O O O

LIVER WEIGHTS OF F-2 MALES AT 5, 6, 7,

AND 8 WEEKS OF AGE . . . .

O

LIVER VITAMIN A AND E CONCENTRATIONS

ON 8-WEEK-OLD F-2 MALES

CAUDAL EPIDIDYMAL SPERM CONCENTRATIONS
OF F-2 MALES AT 5, 6, 7, AND 8 WEEKS

OF AGE 0 O I O O I O O O O

BODY WEIGHTS OF 7-DAY-OLD F-3 PUPS .

BODY AND LIVERflWEIGHTS OF F-2 FEMALES

LIVER VITAMIN A AND E CONCENTRATIONS

IN 8-WEEK-OLD F-2 FEMALES

xi

95

96

98

99

100

102

103

105

106

107

INTRODUCTION

The. Great Lakes represent one of the world's most
significant inland freshwater systems and one of sizeable
economic and ecological importance to both the United States
and Canada. Unfortunately, increased commercial production
and widespread use of synthetic compounds since the 19405 have
contaminated this ecosystem, The widespread contamination of
the Great Lakes basin has led to concern about the overall
effects on the health of human and animal populations
associated with this fresh water environment (Government of
Canada, 1991).

Eleven persistent toxic chemicals have been identified as
critical Great Lakes contaminants by the International Joint
Commission (1987) . They are polychlorinated biphenyls (PCBS) ,
chlorinated pesticides (including DDT and its metabolites),
dieldrin, toxaphene, tetrachlorodibenzo-p-dioxin (TCDD),
tetrachlorodibenzo-furan (TCDF), mirex, hexachlorobenzene
(HCB), mercury, alkylated lead, and benzo[a]pyrene (B[a]P).
The physical and chemical properties (e.g., low volatility,
chemical stability, lipid solubility, slow' rate of
biotransformation and degradation) of these synthetic
chemicals and heavy metals allow them to gain entry into

1

2
organisms. In biological systems some persistent metabolites
bioaccumulate. The biomagnification of chemicals such as
PCBS, dioxins, and organochlorine pesticides from the lowest
trophic levels (phytoplankton) to top predators (fish-eating
animals and humans) has been observed in the Great Lakes food
web.

Several recent studies have shown that humans and fish-
eating'animals that consume significant.amounts.of Great Lakes
fish accumulate many contaminants found in the Great Lakes.
These contaminants, such as PCBS and TCDD, are known to be
detrimental to the reproductive system (Poland and Knutson,
1982). Some fish predators of Great Lakes fish like mink,
bald eagles, herring gulls, lake trout, and lake turtles have
experienced adverse effects from the Great Lakes contaminants
on reproduction and consequent declines in populations
(Government of Canada, 1991).

Reproduction is a complex, stepwise, cyclic process
involving gametogenesis, gamete interaction, implantation,
embryonic development, and sexual maturation of the offspring.
Reproductive toxins may act directly, by virtue of either
structural similarity to endogenous compounds such as hormone
or chemical reactivity with biological component like
alkylating agent and chelator (Welch et a1., 1971; Allen et
a1., 1979; Anderson et a1., 1980). Other reproductive toxins
may act via metabolic processing within the organism before

exerting toxic effects. The metabolite formed may then exert

 

me:
ha:
Gre

QXa

3
its toxic effect. through the 'mechanisms of reproductive
toxicity described above. Other indirect-acting reproductive
toxins may produce alterations in physiological control
mechanisms of the organism, such as enzyme induction or
inhibition.

Reproductive toxins may exert adverse effects through
more than one mechanism. 1For example, PCBS may act indirectly
by induction of microsomal monooxygenases or UDP-glucuronyl
transferase (Poland and Knutson, 1982). PCBS may also act
directly by virtue of steroid hormone agonist properties
(Bitman and Cecil, 1970).

The study is designed with intention to answer the
question, "does consumption of chlorinated hydrocarbon and
mercury contaminated Great Lakes fish pose a reproductive
hazard to mammals?" Using mice as the model, the effects of
Great Lakes carp-containing diets on reproduction were

examined.

LITERATURE REVIEW

1. Polyhalogenated Aromatic Rydrocarbons (PAEs)

Polychlorinated biphenyls (PCBs), dibenzo-p-dioxins
(PCDDs), dibenzofurans (PCDFs), azo(xy)benzenes, and
naphthalenes collectively termed PAHs, have received increased
attention in the scientific literature and popular press over
the past 25 years as toxic environmental pollutants. This
group of chemicals is usually considered together, because (1)
their chemical structures are similar, i.e. they are
approximate isostereomers; (2) they produce similar
characteristics of toxic responses, although they vary greatly
in potency; and (3) they are believed to act through a common

mechanism (WHO, 1989c).

A. Polychlorinated Biphenyls (Pens)
W

The synthesis of PCBS was first described in 1881 by
Schmidt and Schultz (Peakall and Lincer, 1970). The
industrial potential applications of ZPCBs ‘were not, well
realized until about 1930. PCB mixtures quickly gained wide
acceptance in industrial products where nonflammability and
heat-resistant properties were desired. Specific industrial
applications of PCBS includes hydraulics, lubricants,
transformers, capacitors, plasticizer applications, petroleum

additives, and constituents of heat transfer systems. The

of
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hm
suc
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5

increased use of PCBs generated to a series of commercially
available raw materials marketed under various trade names:
Aroclor (Monsanto, U.S.A.), Clophen (Bayer, West Germany),
Phenoclor (Caffaro, Italy), Kanechlor (Kanegafuchi, Japan),
Pyralene (Prodelec, France), and Sovol (U.S.S.R.) to name a
few. Domestic sales of the Aroclors in the U. S. increased
from 32 million lbs. in 1957 to almost 80 million lbs. in 1970
(Nisbet and Satofin, 1972).

Unfortunately, the characteristics that make this class
of compounds desirable for industrial use at the same time
make them extremely resistant to spontaneous degradation in
the environment. As a result, residues have been detected in
human and animal tissue as well as in environmental specimens
such as soil and river and lake sediments from diverse
locations around the world. Concerns over PCB contamination
of the environment and the associate toxic effects first
became apparent when PCBS in fish and wildlife were reported
by Jensen in 1966. This discovery prompted monitoring efforts
in the United States. It soon became obvious that
uncontrolled and widespread use of PCBS had led to their
incorporation into the global environment as persistent and
ubiquitous contaminants (Holmes et a1., 1967; Koeman et a1.,
1969; Risebrough et a1., 1969; Risebrough and Waid, 1982).

e c o e 'e
The most popular blend of industrial grade PCBS in North

America was Aroclor 1242 (Waid, 1990). Aroclors are

6
designated by four-digit numbers; the first two digits (12)
following the trade name specify that the compound is a
chlorinated biphenyl mixture; the last two digits (42) give
the approximate percent of chlorine by weight in the compound.
Aroclor 1016 which contained about 41 percent chlorine was an
exception to this nomenclature system.

The PCBs, systematically called 1,1'-biphenyl, chloro-
derivatives, consist of a group of 209 congeners ranging from
monochloro- to decachloro-biphenyl. PCBs are colorless
crystals when isolated in pure form by recrystallization. The
solubility of PCBS in water is extremely low, especially for
higher chlorinated congeners. However, all PCBS are soluble
in organic solvents, oils, and fats.

Chlorinated biphenyls are quite stable to chemical
alteration. From an environmental point of view, air
oxidation, aqueous hydrolysis, and photochemical reactions in
sunlight are potential reactions to be investigated. Existing
studies showed that PCBS are fairly resistant to oxidation and
hydrolysis under conditions of light, high temperatures, and
enzyme degradation (Trotter, 1975).

W

PCBS were included into the environment by many routes.
Sources of PCBS included industrial leaks and accidents, and
contamination from food-packaging materials containing PCBS.

PCBS became the most abundant of the chlorinated aromatic

pollutants in the ecosystem (Risebrough et a1., 1968). Biota

7
potentially acquired PCBs from three sectors of the
environment: atmosphere, water, and food (Biros et a1., 1970;
Risebrough and Brodine, 1970).

Duke et a1. (1970) found the source of mollusk and fish
PCB (Aroclor 1254) contamination at a Florida industrial plant
that leaked PCBs into the river. In 1970, Holden also
reported PCB-contaminated shellfish and marine life along
Scotland's Atlantic coast. He found that the contamination
occurred when sludge from nearby sewage treatment plants was
dumped in a deep-water estuary. Several recent surveys
indicated that PCB residues occur in human populations from
both industrialized and developing countries (Jensen, 1987).

eve

Due to the physical and chemical properties of PCBs, once
they enter organism, bioaccumulation occurs in the food.chain.
The biomagnification of PCBs from the lowest trophic level to
the top predators has been observed in the Great Lakes food
web (Government of Canada, 1991) . Since aquatic animals
excrete organochlorine chemicals very slowly or not at all,
these contaminants build up to higher concentrations at each
step in the food web. By 1972, significant concentrations of
PCBs had been discovered in animal feeds and many foods
including milk, poultry, dairy products, eggs, and freshwater
fish (Gold, 1983). The most contaminated food was freshwater
fish” Concentrations of PCBs‘were‘determined in samples taken

at different times and in different Great Lakes regions in

kthLhme
HP.

10

f0:

31:

8
fish and herring gull eggs, the results are summarized in
Table 1” In lake trout, total PCB concentrations are the
highest in Lake Michigan and lowest in Lake Superior.
Biomagnification of PCB concentrations also can be observed,
from low to high, in spottail shiners, lake trout, and herring

gulls eggs in the Great Lakes.

 

 

 

Table 1. Mean concentrationfl'of PCBs in fish and herring gull
eggs from Great Lakes.
Lake
.Eanflss QTQEEQ InshLMUI axis BEEN: Emanier
Spottail 0.028b NA? , 0.15* < 0.015 < 0.01b
Shiners (1979-1983) (1982- (1979- (1983)
1983) 1981)
Lake Trout‘ 2.6 4.5 NA 0.6 0.2
(1988)“ (1984) (1987) (1986)
Herring 15 9.7 19 13 7
Gull eggs' (1989) (1989) (1989) (1989) (1989)

 

 

 

'All values are in parts per million (ppm) on a wet-weight basis.
hGovernment of Canada, (1991).

°Data are not available.

‘Environmant Canada and Department of Fisheries and Oceans, (1982).
'The numbers in parentheses refer to when the sample collected.
'Source: Canadian Wildlife Service, (1989).

Several researchers examined correlations between PCB
body burdens in women who eat fish and different geographic
locations surrounding the Great Lakes. Monitoring programs
for PCBs in humans have mainly been restricted to samples of
adipose tissue, breast milk and blood. Until now, human milk
was investigated most frequently (Jensen, 1983; Jensen and

Slorach, 1989) . The differences in sampling and analysis

9

protocols made it extremely difficult to compare data among
studies. Chemical residues measured in these studies may not
truly represent body burdens in women and consequently, the
degree of exposure to infants. ‘ In addition, PCB
concentrations in milk samples taken from women in the same
region consuming similar amounts of fish varied enormously.
For example, the mean PCB concentration in milk samples from
women in Toronto consuming similar amounts of fish was 25 ppb
with a standard deviation of 23 ppb (Frank et 81., 1988).
This variation may be due to the different sampling time
relative to the stage of lactation, the number of previous
breast-fed infants, place of residence and life history of
exposure to PCBs.
Miss

The uptake of PCBs could occur by three mechanisms (Shaw
and Connell, 1990):
1. absorption of PCBs in the atmosphere/water through lungs in

mammals or birds and gills of fish.
2. absorption of PCBs in the atmosphere through the epidermis.
3. consumption of food containing PCBs and.passage through the
gastrointestinal tract.

The atmospheric route through the lungs and epidermis is of
little significance due to the very low concentrations of PCBs
in the atmosphere. A primary route of uptake by fish was
absorption by the gills, since the gills represent 8 active

membrane surface for water exchange (Addison, 1976; Nadeau and

10
Davis, 1976; Phillips, 1980). Once absorbed by the gills,
PCBs are then partitioned into the blood and then to other
organs. ‘

The major route of PCBs acquisition by animals is through
contaminated foods Gastrointestinal absorption of commercial
PCB mixtures and individual congeners has been investigated
extensively in laboratory animal studies (Albro and Fishbein,
1972; Allen etial., 1974a,b; Matthews and Anderson, 1975; Gage
and Holm, 1976; Tanabe et 81., 1981). A commercial PCB
mixture containing Kanechlor-300, 400, 500 and 600 in corn oil
was administrated orally to immature male Wistar rats for 5
days (Tanabe et 81., 1981). More than 84 percent of the total
dose was absorbed from the gastrointestinal tract with less
than 16 percent of the dose excreted in the feces.

The major structural determinant that governs absorption
efficiencies is the degree of chlorination. Quantitative
studies of PCB absorption indicate that.most, if not all, PCBs
containing six or fewer chlorine atoms are efficiently
absorbed by intestine in rats (Albro and Fishbein, 1972; Van
Miller et a1., 1975). Dermal studies with PCB congeners or
mixtures demonstrate that these compounds are readily absorbed
and elicit toxic and biologic effects at dermal and distal
sites (Nishizumi, 1978; Puhvel et 81., 1982; Wester et a1.,

1983).

 

 

 

ea
EC
1a
50
ti.
19'

in

ind

11
E' ! I1 !°

For more than two decades it has been known that PCBs
accumulate in human tissue. In the body, PCBs are associated
with lipids; the highest concentrations are found in fat-rich
tissues such as adipose tissue and milk (Jensen, 1989) .
However, steady state concentrations in blood, muscle, adipose
tissue, and human milk are on the similar calculation basis
(Brown and Lawton, 1984; Birnbaum, 1985). The initial PCB
distribution in serum is dependent on blood-flow rates, blood
volumes, PCB-serum absorption affinities, tissue/blood
partition ratios, perfusion rates and tissue volumes (Matthews
and Dedrick, 1984).

PCB transport from the application site to the distal
sites occurs via several processesa 'The absorption of PCBs is
consistent with passive absorption into the lipophilic cell
membranes followed by transport to all tissues via the blood
(Maliwal and Guthrie, 1982). Liver and muscle are the primary
early depots because the liver is highly perfused and has
moderate affinity for these Compounds. Muscle makes up the
largest tissue volume. However, since PCBs are highly lipid-
soluble compounds, they have a higher affinity for lipid-rich
tissues other than the liver and muscle (Hansen and Welborn,
1977) . Therefore, these compounds are eventually concentrated
in adipose tissue and the skin (Matthews and Tuey, 1980).

It was also apparent that tissue persistence of

individual PCB congeners are dependant on their structure and

ex
av.
9a]
197
197
a1.
gen.
Wit)

and

12
the metabolism rate. In addition to the degree of
chlorination of individual PCBs, orientation and chlorine
substitution patterns are also important structural features
that determine tissue persistence (Safe, 1989).

Generally, the tissue mixtures from humans and animals
containing PCBs from environmental exposure have GC patterns
resembling those of PCB mixtures with more than 50 percent
chlorination (Matthews et 81., 1978). This is in marked
contrast to the major manufactured products containing 42
percent or less chlorine. While one cannot rule out the
possibility that differential uses favor introduction of more
highly chlorinated materials in the environment, the
observation has led to the general belief that less-
chlorinated components are more readily metabolized than
highly chlorinated congeners.

Environmental observations have been confirmed in many
experimental feeding studies using a number of mammalian and
avian species. The absence or'diminished concentration of the
early eluting peaks were reported in rats (Curley et 81.,
1971), rabbits (Grant et 81., 1971), cows (Fries et 81.,
1973a), quail (Bailey and Bunyan, 1972), and hens (Fries et
81., 1973b) . The peaks absent in tissues and products
generally are the early eluting peaks that correspond to PCBs
with lower degrees of chlorination. In a rat study, Matthews

and Anderson (1975) injected intravenously “C-labeled PCBs

13
(4-chloro-, 4,4'-dichloro-, 2,3,5,2',5'-pentachloro- and
2,4,5,2' ,4' ,5'-hexachlorobiphenyl) at a dose of 0.6 mg PCBs/kg
of body weight to determine the total radioactivity in major
organs and tissues from 15 minutes to 42 days later. An
increase in PCB retention with increasing ring chlorination
was reported in this study. This observation was consistent
with the belief that the metabolism rate of PCBs decreases
with increasing chlorination. Studies of single PCB homologs
at various degrees of chlorination have shown that those with
five or fewer chlorine atoms are more readily metabolized and
excreted than those with higher chlorination (Hutzinger et
81., 1972; Berlin at 81., 1975).

The effects of chlorine substitution patterns in sites of
oxidation have not been studied systematically. Examination
of the results in the literature, however, suggest the
following: (1) hydroxylation is favored at the para position
in the least-chlorinated phenyl ring unless this site is
sterically hindered; (2) in the lower chlorinated biphenyls,
the para position of both biphenyl rings and carbon atoms,
which are para to the chloro substitute are all readily
hydroxylated; (3) the availability of two vicinal
unsubstituted carbon atoms, particularly C-5 and C-4 in the
biphenyl nucleus, also facilitates oxidative metabolism of the
PCB substrate, but is not a necessary requirement for
metabolism; (4) as the degree of chlorination increases on

both phenyl rings, the rate of metabolism decreases; and (5)

14
the metabolism of specific PCB isomers by different species
may result in considerable variations in metabolite
distribution (Safe, 1989).

The metabolism of individual PCBs by induced and non-
induced rodent and human liver microsomes (Ghiasuddin et 81.,
1976; Shimada, 1976; Kennedy et 81., 1980; Kaminsky et 81.,
1981) and rat hepatocytes (Vickers et.81., 1986) was reported.
In the studies by Kaminsky et 81. (1981), it was evident that
the metabolism of several dichlorobiphenyls was dependent on
the substrate structure and the type of cytochrome P-450
isozymes. For example, the major phenobarbital-induced
cytochrome P-450 isozyme preferentially'metabolizes di-ortho-
chloro-substituted biphenyls, whereas the B-naphthoflavone-
induced cytochrome P-450 isozymes primarily metabolize
dichlorobiphenyls, which do not contain ortho substitutes.
Both cytochrome P-450 isozymes catalyze the oxidation of mono-
ortho-chloro-substituted dichlorobiphenyls.

Due to their' high lipid solubility, PCBs are ‘most
concentrated in substances with a high lipid content. They
have been detected.in oils on human and animal hairs (Matthews
at 81., 1976). However, the major carriers for PCB
elimination are substances with the greatest volume and/or
lipid content. Lactation is the most important elimination
route for such chemicals (Brilliant.et 81., 1978; Yakushiji et
81., 1979; Nau et 81., 1986). Through the transfer of

residues accumulated over a lifetime from mother to Offspring,

15
PCBs may be retained in animal populations for several
generations. Vodicnik and Lech (1982) found lactating mice
transferred most of the body burden to their nursing young
during a period of 20 days, whereas the body burden of the
non-lactating animals remained essentially constant.

Eggs, particularly egg yolks, represent another carrier
of newly synthesized lipids that may serve as a route for PCB
elimination. PCBs have been detected in the eggs of fish
(Johnson and Morris, 1974) and wild birds (Koivusaari et 81.,
1972). Trout exposed to PCBs experimentally had a more rapid
elimination rate in both sexes during spawning; this appeared
to be primarily due to the voiding of PCB-containing eggs and
sperm (Harding and Addison, 1990).

1198121322
8. Clinical Cases:

The first incident in which PCBs were recognized as the
causative agent in intoxicating the general public happened in
Japan in 1968 (Higuchi, 1976). A PCB mixture used in a rice
oil plant's cooling system leaked into rice oil subsequently
consumed by over 1,600 persons. Another accidental exposure
of humans to PCBs occurred in Taiwan in 1979 (Chen et 81.,
1980). The PCB concentrations in toxic rice oils causing PCB
poisoning were about 830 to 1,030 ppm in Japan (Nagayama et
81., 1975) and 53 to 99 ppm in Taiwan (Chen et 81., 1981).
The blood-PCB concentrations of PCB-intoxicated patients in

Japan were about 5.9 ppb (Koda and Masuda, 1975), much lower

 

16

than those of Chinese patients. The mean blood PCB value of
Chinese patients was 49 ppb (Chen et 81., 1980). This large
difference is presumably due to the difference in time lags
between PCB intoxication and blood-PCB analysis. For Japanese
patients, the blood PCB analysis was done about 5 years after
toxic rice oil ingestion; for Chinese patients the blood PCB
measurement was made 9 months to 1 year after intoxication.
The patients reported in these two cases developed a spectrum
of adverse symptoms, such as chloracne, gum and nailbed
discoloration, joint swelling, waxy secretions of the glands
in the eyelids, lethargy and joint pain (Urabe et 81., 1979;
Chen et 81., 1981).

An ongoing study involving the offspring of women who
consumed Lake Michigan fish demonstrates the occurrence of
several effects“ The children were born to women who consumed
at least 11.8 kg of Lake Michigan fish, equivalent to 2 to 4
fish meals monthly, over at least 6 years. Maternal serum PCB
concentrations averaged 5.5 i 3.7 ng/mL; umbilical cord serum
PCB levels averaged 2.5 i 1.9 ng/mL (Fein et 81., 1984). In
this study, 242 women showed significantly decreased in infant
birth weight (160-190 g lighter than controls), gestational
length (average 4.9 days less than controls) and head
circumference (average 0.6 cm smaller than controls) (Fein et
81., 1984). Consumption oftcontaminated fish also contributed
to motor immaturity, poorer lability of states, a greater

amount of startle and more abnormally weak reflexes. The most

DC
in
nu
p0:

in;

ind
31.
hicl
demc
SinC
(Lit.

19746

17

highly exposed infants were frequently described as
"worrisome" (Jacobson. et 81., 1984). The infants were
retested at 7 months of age and a significant decrease in
visual recognition of novel visual stimuli was associated with
increased levels of PCB cord serum (0.2 to 7.9 ng/ml). This
decrement did not correlate with fish consumption during
pregnancy or lactation (Jacobson et 81. , 1985) . Further
studies of these children have demonstrated that PCBs are
still detectable in a significant proportion (4.18 i 3.29
ng/ml) of the serum samples from the children at 4 years of
ages. Mothers' milk was the primary source of exposures
(Jacobson et 81. , 1989) . However, prenatal exposure predicted
poor short-term memory on both verbal and quantitative tests
in a dose-dependent manner (Jacobson et 81., 1990). Although
much larger quantities of contaminants are- transferred
postnatally via lactation, prenatal exposure seems more
important in terms of developmental effects.

b. Effects on Enzyme Activity:

One of the major biochemical effects of PCBs is the
induction of microsomal enzymes in the liver. Risebrough et
81. (1968) suggested that PCBs have the capability to induce
microsomal enzyme activities. Street at 81. (1969)
demonstrated the induction of liver enzymes in rats by PCBs.
Since then, many articles have been published on this subject
(Litterst and Van Loon, 1972; Chen et 81., 1973; Allen et 81.,

1974a; Turner and Green, 1974). The enzyme systems studied

18
have included mainly hydroxylases, N- and O-demethylases and
nitroreductases and, to a lesser extent nonspecific
carboxylesterase, bromosulfophthalein-glutathione conjugating
enzyme, p-nitrophenol UDP-glucuronyl transferase and EPN-
detoxification systems.

Two types of enzyme inducers are reported in the
literature. One group, to which phenobarbital belongs,
increased cytochrome P-450 content, and increased benzopyrene
hydroxylase and ethylmorphine demethylase activities in the
liver (Alvares et 81., 1970; Bresnick, 1978; Cinti, 1978).
The second group stimulated the formation of cytochrome P-448
and an increase in hydroxylation but not demethylation
(Conney, 1967; Lu et 81., 1980). Alvaers et 81. (1973)
reported that rats treated with PCBs produced an increase in
cytochrome P-448, hydroxylase, and demethylase. Therefore,
PCBs display induction behavior typical of both groups.

c. Carcinogenicity:

Most chemicals are not carcinogenic unless they undergo
metabolic activation to become highly reactive electrophilic
intermediates with the capacity to bind covalently to cellular
constituents such as RNA, DNA, and proteins (Heidelberger,
1975). The formation of an arene oxide intermediate during
the metabolism of PCBs was proven previously (Safe et 81.,
1975; SundstrOm and Hutzinger, 1976) . Arene oxides are
electrophilic in nature. They may be toxic by forming adducts

with macromolecules in cells and interfere with cellular

19

functions. They may also detoxify other toxicants by
facilitating the formation of glutathione conjugates and other
phase II conjugates. The in vivo binding of 2,2',4,4',5,5'-
and 2,2 ' , 3 , 3 ' ,5, 5'-hexachlorobiphenyl to hepatic proteins, RNA
and DNA in mice has been demonstrated (Morales and Matthews,
1979). The greatest binding was observed in RNA, followed by
protein and DNA, respectively; In vitro studies using
mammalian cells in culture showed DNA damage mediated by some
PCB congeners and their metabolites (Seymour et 81., 1976;
Shimada and Sato, 1978). The DNA was isolated and examined
for strand breaks by centrifugation techniques. All of the
compounds tested (2,5,2'5'-tetrachloro biphenyl, 4'-chloro-4-
biphenylol, 4'-chloro-3-methoxy-4-biphenylol, 4'-chloro-4—
methoxy-4-biphenylol, 4'-chloro-3,4-biphenyldiol) induced
single-strand breaks in L-929 cell DNA.

d. Immune Alterations:

Polyhalogenated aromatic hydrocarbons (PCBs, PBBs, PCDDs,
PCDFs, etc.) have immunotoxic properties (Vos and Luster,
1989). The first suggestion that PCBs might affect the immune
system stemmed from an observation on weight and histological
changes of lymphoid organs, including the thymus, spleen and
lymph nodes (Vos et 81., 1980). More direct evidence that
PCBs may alter immune responses was obtained using functional
(antibody titration) tests (Vos and de Roij, 1972). Exposure
of guinea pigs to Clophen A60 for 4-8 weeks reduced serum

antibody titers to tetanus toxoid. Loose et 81. (1977)

511.
VIII
(A1
Ppn

COR

20

reported that exposure of mice to Aroclor 1242 for 6 weeks
suppressed both primary and secondary antibody responses to
sheep red blood cells and abolished memory cell functions. In
addition, they reported that PCB exposure lowered circulating
immunoglobulin levels. Acute PCB exposure also reduced
antibody synthesis. In general, the immune alterations in
exposed animals resulting from PCB poisoning included
depression of serum immunoglobulin levels and delayed-type
hypersensitivity reactions,‘ as well as enhanced in vitro
proliferation responses of T-lymphocytes.

e. Reproductive Toxicity:

PCB effects on reproduction were first shown by Gilbert
(1969). Later, a severe decrease in the reproduction and
survival rates of mink were observed in commercial ranches
where a diet containing PCB-contaminated fish was used
(Aulerich et 81., 1971). Then, Ringer et 81. (1972) fed 10
ppm Aroclor 1254 to mink, the same concentrations as those
contained in Lake Michigan Coho salmon, and confirmed the
hypothesis that PCBs were causative agents of reproductive
failure in mink. When groups of 12 female and 4 male mink
were fed diets containing 0, 1, 5, or 15 ppm Aroclor 1254 for
4 months and were mated, a dose-related impaired reproduction
(reduced number of females whelped and reduced kit/female
ratio) occurred at 5 ppm, with total inhibition of
reproduction at 15 ppm (Aulerich and Ringer, 1977).

Adverse effects of PCBs on the estrous cycle, fertility,

T8

to

ob:
0f

the
co,
“Ur:
i131
day
SDe;

in

21
growth and mortality were reported in rats (Brezner et 81.,
1984). Female Wistar rats were orally administered 30 mg
Aroclor 1254/kg body weight for one month. The estrous cycle
was prolonged in 67 percent of the rats. Sexual receptivity
and litter size decreased. Vaginal bleeding was frequent
during gestation and parturition was delayed. Offspring,
whether exposed prenatally and/or postnatally, demonstrated
lower body weight gain than controls and a higher mortality
rate.
In contrast to rats, Swiss-Webster mice fed 10, 100 or
250 ppm Aroclor 1254 alone or in combination with lead for 12
weeks prior to breeding and during gestation and lactation
showed no adverse effects (Talcott and Koller, 1983). No
effects were observed on reproduction or the pups' immune
response to antigen challenges.

' A single dose of Aroclor 1254 (125-1000 ppm) administered
to male mice had no effect on sperm head abnormalities
observed 5 weeks later (Tophan, 1980). Studying the effects
of PCBs on sperm production, Sanders et 81. (1977) reported
that Aroclor 1254 in the diet of adult albino mice at a
concentration of 200 ppm for 15 days significantly reduced the
number of spermatozoa in testes, but did not affect gonadal
weights. Nor did they find any effects at 50 ppm after a 15-
day treatment period. A similar effect on reduction of
spermatozoa from 400 ppm Aroclor 1254 in the diet was observed

.in adult, white-footed male mice (Sanders and Kirkpatrick,

6T
Af
Vi
th
tr:
ef]
tre

in;

0f 1
aet.
air,
Gel]

(Pul

22
1975). No decreases in the 100 and 200 ppm treatment groups
was observed.

In a study of the effects of PCBs on fertility, Orberg
(1977) administered daily oral doses of 50 pg of either
trichlorobiphenyl or hexachlorobiphenyl to mice from days 5 to
19 of gestation or from birth through day 12 of lactation.
Male pups were subsequently mated with nontreated females.
The numbers of pups sired were not affected" Kihlstrom«et 81.
(1975) injected postpartum NMRI strain mice with 50 mg Clophen
A-60/kg body weight. Treatment began on the day of delivery
and then weekly for 3 successive weeks during lactation.
After reaching sexual maturity, the male offspring were mated
with nontreated females. The frequency of implantation and
the resorption rate were not different from controls. When
treated females were mated to control males, there was no
effect on implantation, either. Interestingly, when both
treated males and females were mated, the number of
implantations decreased significantly.

Despite numerous studies concerning the adverse effects
of PCBs on mammalian reproduction, mechanisms and/or sites of
action remained unknown. Reproductive failure may be caused
directly via a toxic PCB action on reproductive organs and
cells or indirectly via a lesion in the neuro-gonadal axis
(Fuller and Hobson, 1986). Demonstrations of PCB accumulation
in gonads in conjunction with reports of altered steroid

hormone regulations in various mammalian models suggest

23
effects of PCBs on endocrine function (Fuller and Hobson,
1986).

An intriguing effect of PCBs on steroidogenesis was
reported by Fuller et 81. (1980). Luteinized ovaries were
induced by .PMSG-hCG priming of immature albino rats.
Following formation of corpora lutea, 20 mg Clophen A-30/kg
body weight/day were administered intraperitoneally for 2
consecutive days. The ovaries were removed, sliced, and
incubated for 2 hours in the presence of luteinizing hormone
(LH). LH has the ability to increase ovarian progesterone
synthesis (Hadley, 1988). Addition of LH to vials containing
PCB-exposed luteal tissue increased progesterone synthesis
almost 100% above basal levels; whereas, there was only a 31%
increase progesterone from luteal tissue in the presence of LH
alone. This suggested a synergistic relationship between PCB
and LH.

Low chlorinated PCBs possess weak estrogenic activity.
Bitman and Cecil (1970) found that both DDT and PCBs (Aroclors
1221, 1232, 1242, and 1248) had estrogenic activity which was
reflected by an increase in glycogen content of the uterus of
immature rats. Ecobichon and MacKenzie (1974) tested the
effects of several Aroclors and isometrically' pure
chlorobiphenyls (mono-, di-, tetra-, and hexa-CB) on uterine
'weight, water content, and glycogen concentration in immature
rats. In all Aroclor-treated rats, except Aroclor 1016, the

literine weight was increased. 0f the pure chlorobiphenyls,

24

only those chlorinated in the 2 position affected the uterus.
Among those compounds tested, decreasing estrogenic activity
was noted with increasing chlorination, except 2 , 4 , 6 , 2 ' , 4 ' , 6 ' -
hexachlorobiphenyl, which increased uterine weight.

Recently, it was demonstrated that PCBs will bind to
estrogen receptor. Korach et 81. (1987) tested a series of
PCBs for their binding activities to soluble uterine estrogen
receptor protein and found that conformational restricted
hydroxy PCBs were shown to be particularly effective binding
ligands for estrogen receptors. It was reported that
conformational restriction may improve the estrogen receptor-
binding activity of the biphenyl molecule. The degree of
enhancement appears to be near maximum with one ortho-chlorine
and with slight improvement with two ortho-chlorines (Korach
et 81., 1987).

'Therefore, weak estrogenic effects of PCBs could affect
endocrine function by competitive affinity for hormone
receptors. Altered receptor availability and/or increased
hormone metabolism could change trophic hormone thresholds,
thus could disrupt feedback patterns between the endocrine
organs and hypothalamus. This may directly or indirectly
modify some phase of the reproductive process.
W

Although PCB production in the United States was banned
in 1977, use of PCBs in other countries has continued. PCBs

are persistent in the environment and in organisms. 'There are

25
considerable differences in toxicity among congeners and, for
a given congener, there are significant species differences in
sensitivity. Although metabolism and excretion of PCBs are
not efficient, they are considered important modes of PCB
detoxification. PCB accumulation in target organs to a great
extent is closely related to chemical solubility and
lipophilicity. Solubility tends to decrease with increased
halogenation of PCBs, the rate of metabolism and therefore
elimination. Thus, highly halogenated PCBs tend to be more

persistent and have greater potential for bioaccumulation.

B. Polychlorinated Dibenzofip-dioxins (PCDDs)

PCDDs, the most potent compounds of PAHs, serve as the
prototype of halogenated aromatic hydrocarbons. There are 75
possible chlorine-substituted dibenzo-p-dioxin isomers. These
compounds are not synthesized for commercial purposes, but are
formed as trace contaminants in the synthesis of several
commercial products, most importantly, chlorophenols.

There is a commonality Of toxic responses produced by
halogenated aromatic hydrocarbons. This is difficult to
appreciate in the literature because toxic effects observed
after administration of a given compound vary with the dose,
duration of exposure and, most importantly, with the species
of animals used" Species vary greatly in their sensitivity to
halogenated aromatic hydrocarbons. The oral LD,0 (in ug/kg

Jbody ‘weight) of TCDD ‘varies over a 5000-fold range in

26

different species: guinea pig, 1; rat (male), 22, (female),
45; mouse, 114; rabbit, 115; hamster, 5000 (Schwetz et 81.,
1973; Vos et 81., 1974; Henck et 81., 1981). The large
species variation of TCDD sensitivity is not accounted for by
an appreciable difference in the rate of TCDD metabolism. The
LDws in the guinea pig and hamster differed by more than 3
orders of magnitude, but the whole body half-life in them
differed only 3 fold (Olson et 81., 1980).

After an acute lethal dose of TCDD or other dioxin
congeners, tested animals (chicken, cow, guinea pig, hamster,
rabbit, rat) have a latent period of a week or more prior to
death in which. weight loss or reduced weight gain was
accompanied by a depletion of adipose tissue. By death, the
loss in body weight may be as great as 50% (McConnell, 1980).
Reduction in body weight and adipose tissue were partially
attributed to lack of food intake (Harris et 81, 1973).

In all species studied, TCDD and other congeners produced
a loss of lymphoid tissue, especially in the thymus, and also
in the spleen and lymph nodes (Poland and Knutson, 1982). The
thymus may be reduced to 0 percent of the normal size, with a
loss of cortical lymphocytes. In young animals (rats, mice,
and guinea pigs), TCDD-produced thymic loss was accompanied by
suppression of the immune response (Faith and.Moore, 1977; V05
et 81., 1980). The lack of effects of TCDD on primary
lymphocytes and thymic cell lines in vitro suggests that

halogenated aromatic hydrocarbons do not act directly on T

27
cells (Vos et 81., 1980), but may reduce thymocytes by acting
indirectly. Lethally exposed animals, however, generally do
not die from infections, nor does a germ-free environment
protect them from death (Greig et 81., 1973).

TCDD and related compounds produce hepatomegaly in all
species, even at doses well below the lethal dose (McConnell,
1980). The enlarged liver is due to hyperplasia and
hypertrophy of parenchymal cells, due especially to
proliferation of the smooth endoplasmic reticulum (Fowler et
81., 1973). This morphologic change is accompanied by
increased microsomal monooxygenase activity (Poland and
Knutson, 1982). In the rat liver, TCDD increased DNA
synthesis and the content of DNA (Dickins et 81., 1981).

Chronic exposure to halogenated aromatic hydrocarbons
impairs reproduction (Poland and Knutson, 1982) . Compared
with controls, female rats fed.diets containing TCDD had fewer
vaginal plugs when caged with males, longer intervals between
mating and parturition, and smaller litter sizes at birth
(Murray et 81., 1979). Decreased uterine size, decreased
number of corpora lutea, and aberrant ovarian stroma cells
observed in TCDD- and PCB-exposed rats suggest that estrous
cycles may be altered (Kociba et 81., 1978). Decreases in
plasma progesterone concentrations were observed in both TCDD-
treated monkeys (Barsotti et 81., 1979) and PCB-treated rats
(Jonsson et 81., 1976).

Enzyme induction is the most studied and best understood

28

response produced by PAHS, and provides the basis for
subsequent formulation of their mechanism of toxicity. The
microsomal monooxygenase system metabolized most exogenous
lipophilic chemicals to more polar and readily excretable
products (Nebert.et 81., 1981). This enzyme complex, embedded
in the endoplasmic reticulum, consisted of a flavoprotein
NADPH-cytochrome P-450 reductase, and a group of hemoproteins,
collectively termed cytochromes P-450 (Nebert et 81., 1981).

Much of the research reported to date has focused on the
ability of TCDD and PAHS to bind to a specific cytosolic
receptor, aromatic hydrocarbon receptor (AhR), that activates
a particular enzyme locus. The Ah receptor was detectable in
many tissues and organs. The best understood activity of the
receptor concerns its role in the induction of cytochrome
P4501A1 gene -- termed xenobiotic responsive elements -- and
the stimulated transcription of this gene (Hoffman et 81.,
1991). The receptor also mediates induction of cytochrome
P450IA and several other enzymes that metabolize xenobiotics.
Many pathological effects of the polychlorinated aromatic
compounds depend on the action of the Ah receptor, but the
pathogenetic mechanism is still unknown (Poland and Knutson,
1982). These pathological effects include hyperplasia and
necrosis in the liver, proliferation in the epithelial lining
in the urinary tract, loss of lymphoid tissue, and hyperplasia
and hyperkeratosis in interfollicular epidermis.

TCDD produced a dose-related induction of hepatic aryl

29
hydrocarbon hydroxylase (AHH) activity in chicken embryos
(Poland and Glover, 1973). The ED5o was 0.3 X 10‘9 mol/kg body
weight. The most important observation from these studies was
that for chlorinated dibenzo-p-dioxin congeners, there was a
high correlation between their potency to induce AHH activity
and their toxic potency (Poland and Glover, 1973; Kende et

81., 1974).

II. Chlorinated Pesticides

A. nexachlorobenzene (HCB)

Hexachlorobenzene (HCB) is a white crystal with a
molecular weight of 284.80 and.a melting point of 231°C (Merck
Index, 1989). Chlorinated hydrocarbons were used widely as a
fungicide in the world during the late 19405 and 19505. It is
also an impurity in several widely used pesticides such as
tetrachlorobenzene and pentachlorobenzene (Morris and Cabral,
1986). In most countries, HCB has not been produced as a
fungicide since the mid-19705 to early 19805. Today, the
major source of HCB appears to be industrial wastes
originating from the manufacture of many chlorinated
compounds.

HCB is highly resistant to environmental degradation.
.Like many other chlorinated compounds, it accumulates in food
chains (Ault et 81., 1985). Using chemical and environmental

:modelling, the behavior and fate of HCB in the environment

30

have been demonstrated. HCB has been identified in the air,
water and soil, translocating from one medium to another
(Eisenreich et 81., 1981; Rippen et 81., 1984). For example,
HCB may move from soils or other point sources via water run-
off and concentrate in reservoirs; there it may be further
taken ‘up and concentrated. by various biota and aquatic
species. HCB may therefore enter the food chain via both
aquatic and terrestrial plants and animals (Ault et 81.,
1985).

During 1979-1981, HCB was detected in nearly 100 percent
of the human population Sampled in U.S.A., prompting efforts
to identify its sources (Mack and Mohadjer, 1985). Using
annual market-basket analysis, HCB residues were found
regularly in cooking oil, fish, poultry, dairy products, and
root vegetables (Corneliussen, 1972).

»Between 1955 and 1959, the consumption 'of seed-wheat
treated with HCB led to severe illness in 4,000 persons in
Turkey. The estimated intake of HCB was 50 to 2,000 mg/day
for a long time during this period (Schmid, 1960). The
illness was characterized by symptoms of porphyria (Cam,
1960). Hepatomegaly was also observed in most hospitalized
patients; some 30 percent of the cases showed enlarged thyroid
glands (Cam and Nigogosyan, 1963) . Young children were
particularly at risk, nursing infants developing a lesion
known as "pink sore" that was associated with a 95 percent

mortality rate. HCB ingestion was related to transplacental

31
and milk acquisition from women who had consumed contaminated
grain.

In animal models, several adverse effects were observed.
A dose-dependent increase in hepatic and thyroid tumors was
observed in hamsters during a chronic (70-week) study
(Lambrecht et 81., 1982). With continued administration for
3 to 12 weeks, the activities of ethoryresorufin O-deethylase
and glutathione-S-transferase were induced by HCB (Wada et
81., 1968; Sweeney et 81., 1971; Debets et 81., 1981), with an
increase in liver size and weight (Debets et 81., 1981).
Cutaneous lesions may occur, as well as the onset of porphyria
(Smith, et al, 1979; Debets et 81., 1981). Gross and
microscopic observations indicated marked hepatosplenomegaly,
and congestion in most abdominal and thoracic organs as early
as 4 weeks after beginning the exposure to HCB (den Tonkelaar
and van Esch, 1974). Enlargement of the thymus, spleen, as
well as lymphoid centres and nodes were also observed.

HCB has been reported to be teratogenic in some animal
species. Exposure during gestation resulted in enlarged
kidneys in pups on days 1, 15 and 20 after birth. In a two—
generation study in rats with concentrations of 0.3-40 ppm HCB
in the diet, chronic nephrosis was observed, particularly in
the males (Arnold et 81, 1986). Parathyroid adenomas were
observed in males receiving the highest dose, while neoplastic
nodules in the liver (Smith et 81., 1986) and adrenal

pheochromocytomas were observed in females receiving the

32

highest dose (Kuiper-Goodman and Grant, 1975).

B. Aldrin and Dieldrin

Aldrin and dieldrin are organOchlorine pesticides
manufactured since 1950 and were used throughout the world
until the early 19705. Both compounds were used as
insecticides totcontrol soil pests and to preserve seeds (WHO,
1989a). While readily metabolized to dieldrin in plants and
animals, aldrin is rarely found in food (Worthing and Walker,
1983). Dieldrin, however, is extremely persistent in the
environment and is commonly found in dairy products, meat
products, fish, oils, and root vegetables (Johnson and Manske,
1977; Wessels, 1978; Zabik et 81., 1979; Frank et 81., 1985).
Dieldrin/aldrin has a low propensity for movement away from
treated soils (Herzel, 1972; El Beit et 81., 1981). Dieldrin
is mainly and rapidly adsorbed on soils with a high organic
matter content (Powell et 81., 1979).

Since the early 19705, both aldrin and dieldrin have been
restricted or banned in a number of countries because of
persistence in the environment (IARC, 1974). Neither aldrin
nor dieldrin is currently produced in the United States
(Meister, 1987).

Concentrations of dieldrin in the adipose tissue of
humans were declined from the mid-19705 to the early 19805 in
the Netherlands, the United Kingdom, and U.S.A. (GIFAP, 1984) .

Data on dieldrin in human milk were below 10 ug/kg milk (limit

33

of detection) in the U.S.A. during the period 1971-1979
(National Food Administration, 1982). As a result of
transplacental and lactational transfer, dieldrin was detected
in the blood and adipose tissue of fetuses and newborn infants
(Eckenhausen et 81., 1981; WHO, 1989a). The concentrations
were one-tenth to one-half of that in maternal tissues.

Oral LO” of dieldéin in mice and’rats ranges from 40 to
70 mg/kg body weight (Cholakes et al. , 1981) . Like most other
chemical substances, dieldrin has multiple targets of
toxicity. The liver is a major target organ of dieldrin
toxicity in rats and mice. An increased liver/body weight
ratio and hypertrophy of the centrilobular hepatocytes were
observed (Benitz et 81., 1977). The other target is the
central nervous system. In humans and other vertebrates,
intoxication following acute or long-term exposure to dieldrin
is characterized by headache, nausea and vomiting, followed by
involuntary muscle movements and epileptiform convulsions.
Death may result from cerebral anoxaemia (Jager, 1970; Joy,
1976; Hayes, 1982). t

In the liver of dieldrin-treated immature male rats,
there was increased activity of microsomal N-demethylase,
.dimethylaminoantipyrin, aldrin epoxidase and cytochrome P-450
contents (Campbell et 81., 1983) . In rhesus monkeys, the
activity of liver microsomal monooxygenase and cytochrome P-
450 was increased by dieldrin at daily feeding doses of 1.75

or 5 mg/kg diet for approximately 6 years (Wright et 81.,

a]

fc

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dii
Kai
inc
cor.
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at

inc:

34
1978). In humans, no evidence of enzyme induction was
observed in a group of 10 workers in a manufacturing plant
with a mean exposure equivalent to a daily oral intake of 17
ug/kg body weight (maximum 24 ug/kg body weight) (Hunter and
Robinson, 1967; Hunter et 81., 1969).

A number of long-term carcinogenicity studies of aldrin
or dieldrin in different strains of mice were conducted. In
all studies, benign and/or malignant liver cell tumors were
found, Females are less sensitive than males (NCI, 1978). No
other types of tumors were induceda ‘Using cultured mouse FM3A
cells, Morita and Umeda (1984) reported that aldrin and
dieldrin were weak mutagens. However, in a study at the
National Institute of Public Health of the Netherlands, no
increased incidence of tumors was found in rats fed diets
containing 75 ppm dieldrin for 2 years (Van Genderen, 1979).
When Syrian golden hamsters were fed diets containing dieldrin
at 20, 60, or 80 ppm for 10 weeks, there was no significant
increase in tumor incidence (Cabral et 81., 1979). Therefore,
from long-term feeding experiments, it seems that aldrin and
dieldrin may be carcinogenic in mice, but not in rats or
hamsters. There was inadequate evidence of carcinogenicity in
humans, and only limited evidence for carcinogenicity in
experimental animals.

In a study by Keplinger et 81. (1970), Swiss mice were
fed a diet containing 3 ppm-dieldrin for 6 generations and

there were no effects on fecundity, gestation period, or

35

litter sizes” IHowever, there was an increase in the mortality
rate in pre-weaning pups. Increased pre-weaning mortality in
the F-1 litter was also observed in the study of Eisenlord et
81. (1967). They did not observe any effect on fertility or
litter size in a three-generation feeding study in Long-Evans
rats fed 0.1, 1.0, or 2.0 ppm dieldrin in the diet. The no-
observed-adverse-effect-levels for reproductive toxicity of
dieldrin are 2 ppm in the rat diet and 3 ppm in the mouse
diet, which are equivalent to a daily intake of 0.1 and 0.4
mg/kg of body weight, respectively.

No evidence of teratogenicity of dieldrin was found in
the mouse, rat, or rabbit, after ingestion up to 6 mg/kg of
body weight (Dix and Wilson, 1971; Dix et 81., 1978; Coulston

et 81., 1980).

C. Dichlorodiphenyltrichloroethane (DDT)
Dichlorodiphenyltrichloroethane (DDT) was one of the most
widely used pesticides to control insects on agricultural
crops that carry diseases (WHO, 1979). DDT is a white,
tasteless, nearly odorless crystal. DDT is stable under most
environmental conditions and is resistant to complete
breakdown by the enzymes present in soil microorganisms and
higher organisms (WHO, 1979; 1989b). High lipid solubility
and low water solubility of DDT and its metabolites enable
these compounds to be readily taken up by organisms and lead

to retention in the fatty tissues of fish, birds, and mammals

36
(Edmundson et 81., 1972). Rates of accumulation into
organisms vary with the species, duration and concentration of
exposure, and environmental conditions. In general, organisms
at higher trophic levels tend to contain higher concentrations
of DDT-type compounds than those at lower trophic levels
(Johnson et 81., 1971; WHO, 1989b).

DDT was introduced into North America in 1946 and was
banned in the United States in 1972. The levels of DDT and
its principal metabolite, DDE, in the Great Lakes basin have
decreased significantly since 1978 (Government of Canada,
1991). Since the 19705, the level of DDT in lake trout has
declined followed by a leveling off in the Great Lakes. In
Lake Michigan trout, DDT concentration was decreased from 7
ppm in 1979 to 2 ppm in 1984. From recent data,
concentrations of DDT/DDE in the lake trout are below'2 ppm in
the Great Lakes and do not pose a hazard to human health
(Government of Canada, 1991).

DDT is highly toxic to fish (Mayer, 1987). Although the
body burden of DDT/DDE in lake trout can be more than 7 ppm,
the acute LD_.,o reported in rainbow trout exposed to a single
oral dose of DDT was 0.007 ppm (Pimentel, 1971). Adipose
tissue is believed to be the site of DDT/DDE storage. Smaller
fish are more susceptible than larger ones of the same
species. Cellular respiration may be the main target of DDT
toxicity'iniaquatic animals since there are reports of adverse

effects on ATPase. It was demonstrated that the inhibitory

37
activities of DDT on ATPase eventually result in impairment of
fluid absorption of intestine (Koch, 1969; Cutkomp et 81.,
1971; Desaiah et 81., 1975).

A single oral dose of 237 ppm DDT caused death in mice
(Bathe et 81., 1976). The primary effects associated with DDT
and DDE exposure in experimental animals, such as rats, mice,
guinea pigs, and rabbits, were liver and neurological effects
(EPA, 1989). Liver effects ranged from increased P-450
enzymes to necrosis and tumors, and neurological effects,
including tremors and convulsions.

Reproductive effects were observed in experimental
animals following exposure to DDT. Decreased testicular
weight and sperm concentration were reported in male rats
after oral exposure to 500 mg DDT/kg body weight/day on days
4 and 5 of age, then 200 mg DDT/kg body weight/day from days
4 to 23 (Krause et 81., 1975). Female mice exposed to 1.3 to
6.5 ppm. DDT for' 5 generations had increased abortions,
stillbirths, and pup mortality. Most of the females in the
6.5 ppm group died before parturition (Shabad et 81., 1973).
Increased uterine weight and glycogen content were reported by
Clement and Okey (1972) in immature rats that ingested 50 mg
DDT/kg body weight for 7 days.

DDT and its metabolites can lower the reproduction of
birds by causing eggshell thinning, leading to egg breakage,
and embryo death (Lincer, 1975; Heinz, 1976). Different

species of birds vary greatly in sensitivity to these

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38

chemicals. Predatory birds are extremely sensitive and often
show marked shell thinning, whereas gallinaceous birds are
relatively insensitive (WHO, 1989b). As for the mechanism of
the eggshell thinning phenomenon, no‘ generally accepted
explanation exists. There are reports that DDT inhibits
carbonic anhydrase which is important in forming eggshell
(Peakall, 1970). 10thers believed 'that DDT's estrogenic
activity' causes Ihormone. imbalance, thus changing' calcium
partition (Matsumura, 1985).

DDT and its metabolites have been found in human blood,
placental tissue, and umbilical cord blood. Saxena et 81.
(1983) reported increased DDT levels in maternal blood ranging
from 7.4 to 393.8 ppb and levels in placental tissue ranging
from 19.8 to 16.2 ppb in mothers who gave birth to premature
infants or who spontaneously aborted fetuses. O'Leary et a1.
(1970) reported a difference in DDE blood levels between full-
term and premature infants. Women who delivered early had
infants with mean DDE blood levels of 19 to 22.1 ppb, while
women who delivered at full-term had infants with mean DDE
blood levels of 4.9 to 6.1 ppb. IHigh DDE level in breast milk
was found to be associated with hyporeflexia in newborn
infants (O'Leary et 81., 1972). Further investigation is
needed to substantiate this finding and to«determine if DDE is

the causative agent.

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39
III. Mercury
Intrednstian

Sediments in areas downstream from municipal, and
industrial complexes showed higher concentrations of mercury,
zinc, arsenic, lead, copper, chromium and cadmium than
sediments in locations unaffected by human activities (Mckee
and Wolf, 1963). Fish from locations showing' elevated
concentrations of heavy metals in the sediments had higher
concentrations of Hg, Zn, Cr, and Cu than fish from control
locations (Mckee and Wolf, 1963). In the flesh of Michigan
fish, only mercury approached concentrations presently
considered by the 0.8. Food and Drug Administration to be
harmful to human health (Michigan Water Resources Commission,
1972).

E] i J i :1 . 1 E !'

Mercury is a metal which is liquid at room temperature
and pressure. Mercury can exist in a wide variety of physical
and chemical state. There are three forms of mercury:
elemental, inorganic, and organic compounds (Goldwater, 1972) .
Metallic or elemental mercury in the atmosphere is the major
pathway for global transport of mercury. Metallic mercury may
be oxidized to inorganic divalent mercury, particularly in the
presence of organic material, such as in the aquatic
environment. Divalent inorganic mercury may, in turn, be
methylated to dimethyl-mercury by anaerobic bacteria. If

taken up by fish, dimethylmercury may eventually cycle through

40

higher levels of fish-eating animals or humans (Smith and
Carson, 1981).
§22£2§§_2£_§522§2£§

Metals are redistributed naturally in the environment by
both geologic and biologic cycles (Beijer and JernelOv, 1986) .
For the geologic cycles, rainwater dissolves rocks and ores
and physically transports material to streams and rivers,
adding and deleting from adjacent soil, and eventually to the
ocean to be precipitated as sediment or taken up in rainwater
to be relocated elsewhere on earth (Li, 1981). The biologic
cycles include bioconcentration by plants and animals and
incorporation into food cycles (Friberg et 81, 1986). One of
the most significant effects of mercury pollution is that
aquatic organisms can absorb and accumulate mercury in their
tissues, leading to increasing concentrations in the food
chain. A

Methyl mercury is the most important form of mercury in
terms of toxicity effects from environmental exposures (Goyer,
1991). Except in fish, mechry concentrations in food are
very low, generally in the range of 5 to 20 ug/kg (NAS, 1977).
Mercury concentrations in large carnivorous fish like pike and
swordfish have been found to exceed 1,000 pg/kg (EPA, 1984).
Therefore, consumption of fish by humans is the most important
source of general population exposure to mercury. The mercury
content in the fillet of lake trout in Lake Ontario was about

170 ug/kg in 1982 (Government of Canada, 1991). Between 70

and
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41
and 90 percent of the total mercury detected in fish muscle
tissue is in the form of methyl mercury (WHO, 1976).
El !' i I' ! .1 !'

The physical and chemical properties of mercury allow
them to gain entry into organisms, resist being metabolized or
excreted, and to bioaccumulate (Gleason et 81., 1969).
Elemental mercury volatilizes to mercury vapor at ambient air
temperatures, and. most. human exposure is by inhalation.
Mercury vapor readily diffuses across the alveolar membrane
and is lipid soluble. Therefore, it has an affinity for red
blood cells and the central nervous system (Roels et 81.,
1985). For inorganic mercury and methylated mercury,
gastrointestinal absorption is still the major route. Cells
that are involved in the transport of mercury, such as
gastrointestinal, liver, and renal tubular cells, have more
chances to contact mercury than other cells (Thayer, 1984).
Hence, liver and kidney are the primary organs for mercury
accumulation (WHO, 1989c).

All forms of mercury can cross the placenta to the fetus
in experimental animals. In rats, fetal uptake of elemental
mercury has been shown to be 10 to 40 times higher than uptake
after exposure to inorganic salts probably because of lipid
solubility (Lauwerys, 1983).

MW
Within cells, mercury may bind to a variety of enzymes

including those in microsomes and mitochondria and will

42

produce nonspecific cell injury or cell death (Luckey et 81.,
1975). Mercury has a particular affinity for ligands
containing sulfhydryl groups. In liver cells, methyl mercury
forms soluble complexes with cysteine and glutathione, which
are secreted in bile and reabsorbed from the gastrointestinal
tract (Mottet et 61.; 1985). Organomercurial diuretics are
thought to be absorbed in the proximal tubule-binding specific
receptor' sites. that inhibit. sodium 'transport (Skerfving,
1974).

Biologic half-times are available for a limited number of
mercury compounds. The biologic half-time for methyl mercury
is about 70 days; the half-time for retaining salts of
inorganic mercury is about.40 days (WHO, 1976). There are few
studies on biologic half-time for elemental mercury or mercury
vapor, but it also appears to be linear with a range of values
from 35 to 90 days (WHO, 1976). 4
W

The major human health effects of mercury are neurotoxic
effects in adults (Bakir et’ 81., 1973), and toxicity to
fetuses of mothers exposed to methyl mercury during pregnancy
(Cox et 81., 1989). The fetal brain is much more susceptible
to methyl mercury than that of adults (Clarkson, 1987). This
difference may be due to the fact that this toxicant
depolarizes microtubules, thus interfering with cell division
and migration, both of which are essential in the proper

development.of fetal brain (Paton and Allison, 1972; Clarkson,

43
1987). Clinical manifestations of neurotoxic effects in
adults include parathesia, ataxia, vision and hearing loss,
tremor, and finally, coma and death (Goyer, 1991).

Mercury has been shown to alter the reproductive function
in rodents. A single intraperitoneal dose of 2 ppm mercuric
chloride given to female mice 0.5 to 4.5 days before mating
decreased the number of implants, living embryos, and
increased embryo death (Suter, 1975). Lee and Dixon (1975)
Observed decreased fertility in male mice receiving a single
intraperitoneal dose of 1 ppm mercuric chloride. The
decreased fertility was observed between days 8 and 49 post-
treatment and was associated with altered spermatogonia.
Khera and Tabacova ( 1973) reported that daily oral gavage
doses of 1, 2.5, and 5 mg methylmercuric chloride/kg body
weight for 7 consecutive days administered to male rats caused
a dose-related reduction in mean litter size. Reduced number
of viable embryos was attributed to pre-implantation losses
resulting from abnormal spermatogenesis due to mercury

treatment of the parent male rat.

44

Statement of Problem

There have been many reported observations of adverse
effects on reproductive performance of animals exposed to
contaminants of the Great Lakes. Several epidemiological
studies of the predators of the Great Lakes fish, including
humans, have also showed less than optimum reproduction or
declines in population in the past 20 years. Based on the
physical and chemical properties and the toxicities of PCBs,
TCDDs, HCB, aldrin, dieldrin, DDT, and mercury, the Great
Lakes contaminants are a threat to this ecosystem.

Whereas much has been learned.about.the.effects of single
chemical on reproduction through high doses administered to
animals or cultured cells, little is known of the effects of
collective contaminants from Great Lakes on reproduction and
other physiological functions. Long term, multi-generation,
exposure to low concentration of environmental contaminants is
an additional knowledge gap concerns most of the researchers
todayu Therefore, this study is designed to examine the long-
term, low-dose exposure of the laboratory animals to Great

Lakes carp.

45

Objective

This study was designed to study the effects of Great
Lakes contaminants on reproductive perfOrmance. The specific
aims were to determine: (1) the sperm quality of male mice and
the oocyte fertilizing quality of female mice fed with fish
diet prepared from Great Lakes carp; ( 2) the sexual maturation
of the male offspring from the fish-fed mice; and (3) the
effects of the treatments on hepatic vitamin A and E
concentrations and the development of some other reproductive

function related organs.

46

PHASE I STUDY

MATERIALS AND METHODS

Animals

Forty-eight 12-week-old CBA/CAJ female mice, purchased
from Jackson Labs, Bar Harbor, ME, were bred with 16 CBA/CAJ
male mice. The study was conducted beginning at October 16,
1991. The mice were housed in an environmentally controlled
facility with a 12 hours light/12 hours.dark cycle.and at 21°C
in the Food Science & Nutrition Building at Michigan State
University (MSU). At birth, litters were assigned randomly

into three treatment groups.

Treatment Diets
‘Each treatment group received one of the following diets

formulated on a dry-matter basis:

5?

100% Lab Mouse Chow #5015 (control)
A: 47% farm-raised carp + 40% Lab Mouse Chow #5015
+ 13% fish oil

G: 60% Great Lakes carp + 40% Lab Mouse Chow #5015

Lab Mouse Chow #5015 was purchased from Purina Mills
Inc., St. Louis, MO. Farm-raised carp (Cypinus carpio) were
purchased from the Billy Bland Fishery Inc., Taylor, Ark.

Great Lakes carp were collected in August 1 and October 31,

47
1990 at the mouth of the Saginaw River with the assistance of
the Fisheries Division of the Michigan Department of Natural
Resources“ Fish.oil was kindly donated.by Zapata Haynie Cop.,
Reedville, VA. The analysis of organoChlorinated pesticides
and PCBs in the fish oil is shown in Appendix 1.

Carp from both sources were ground, loaded into sausage
casings and cooked at 170°F in the smoke house of the MSU’Meat
Lab. for 30 minutes to inactivate thiaminase (Gnaedinger and
Krzeczkowski, 1966). All treatment diets were adjusted to
contain 40 to 45% water. Major nutrients were balanced based
on the Nutrient Requirements of Laboratory Animals (Appendix
2; NRC, 1978) and analyzed by Litchfield Analytical Services,
Litchfield, MI. Biotin, folacin, niacin, riboflavin, thiamin,
vitamin B6, vitamin 8,2, vitamin E, vitamin K“ a-cellulose, and
fish oil were used to balance the diet ration. Total vitamin
A and E concentrations in the diets were analyzed by the
Animal Health Diagnostic Lab., Nutrition Section, MSU. Total
vitamin A concentration was calculated from the concentrations
of retinol and retinyl palmitate. Additional mineral analyses
were performed by the Animal Health Diagnostic Lab.,
Toxicology Section, MSU. Analysis of organochorinated
pesticides and PCBs in the ground carp was performed before
cooking and in the complete diets by the Aquatic Toxicology

Lab. , MSU.

48
Mouse Liver Analysis
Total vitamin A and E concentrations in mouse liver were
analyzed by the Animal Health Diagnostic Lab., Nutrition
Section, MSU. Analysis of organochorinated pesticides and
PCBs in mouse liver was performed by the Aquatic Toxicology

Lab., MSU.

Medium and Chemicals

Brinster's Medium for Ova Culture (BMOC-3) , used for
sperm capacitation and in vitro fertilization, was purchased
from the Gibco Life Technologies, Gaithersburg, MD. BMOC-3
contains 189 mg/L CaClz, 356 mg/L KCl, 162 mg/L KI-I,P0,, 294
mg/L MgS04-7H20, 5546 mg/L NaCl, 2106 mg/L NaHCO3, 5000 mg/L
bovine serum albumin, 1000 mg/L D-glucose, 2253 mg/L D,L-
sodium lactate, 56 mg/L sodium pyruvate, 3.3 mg/L streptomycin
sulfate, and 3.3 mg/L penicillin potassium (Brinster, 1971).
BMOC-3 was equilibrated in 5% C02 in air at 37°C overnight
before use.

Folacin, niacin, riboflaVin, thiamin, vitamin B6, vitamin
B12 , and a-cellulose were purchased from ICN Biomedicals, Inc. ,
Costa Mesa, CA. Biotin and vitamin K, were purchased from
Hoffman-LaRoche Chemical Division, Nutley, NJ; ‘Vitamin E was
obtained from Rhone-Poulenc Co., Atlanta, GA.

Pregnant.mare serumlgonadotropin (PMSG), human chorionic
gonadotropin (hCG), bisBenzimide Hoechst No. 33258, and all

other chemicals used in this study, except where indicated,

49

were purchased from Sigma Chem. Co., St. Louis, MO.

Experimental Design

Diets were provided ad libitum to dams (F-O) immediately
after parturition and through lactation. Thus pups (F-l) were
treated by the milk. Water was provided ad libitum. Pups (F-
1) were culled to the size of 6 or 7 per litter. Body weights
of pups were measured at days 1, 4, 7, 14 and 21. At 21 days
of age, F-1 pups were weaned, sexed, and.weighed, then housed
in groups of 3 or 4 mice per cage. F-O females were
sacrificed by cervical dislocation. Liver weights, liver
vitamin A and E concentrations of F-O females were measured at
weaning. The F-l received their respective treatment diets
continuously to termination.

The body weights of the F-1 males were recorded at 3, 4,
5, 6, 7, 8, 10, 14, 15, 21 and 23 weeks of age. Three livers
from the F-1 male mice from each treatment were weighed at 7,
8 and 15 weeks of age. Liver vitamin A and E concentrations
were analyzed at 19 and 32 weeks of age. At 34 weeks of age,
five livers from each treatment were analyzed for
organochorinated pesticides and PCBs.

Epididymal sperm concentration and motion were measured
at 5, 6, 7, 8, 15, 23, 32 and 34 weeks of age. At 8 weeks of
age, fertilizing ability of sperm from 3 F-1 males from each
treatment were tested in vitro with oocytes from non-treated

B6D2-F1 females.

50

At 7.5 weeks of age, 7 F-1 males were paired individually
with previously non-treated mature CBA/CAJ females for 5 days.
These females received the same diets as F-1 males during the
pairing and were later housed individually. The bred females
were continuously provided with treatment diets throughout
gestation and lactation. At birth and weaning, litter size
and viability of the offspring (F-2) were measured. Body
weights of the F-2 pups were recorded at days 1, 4, 7, and 14
during lactation.

F-1 females, at.5 and 10 weeks of age, were superovulated
(see Method for in vitro fertilization on p.51). The oocytes
were inseminated in vitro with epididymal sperm from
previously non-treated B6D2-F1 males. Ovulation and
fertilization rates were recorded.

At 10 weeks of age, 7 F-l females from each treatment
group were bred with non-treated and proven fertile B6D2-F1
males for 5 days and sacrificed by cervical dislocation 10
days after the end of breeding period. The number of fetus

was recorded.

Sperm Collection

Caudal epididymal sperm were collected for analysis and
in vitro fertilization. Each pair of the epididymides was
placed in the inner well of a Falcon Organ Tissue Culture Dish
(60 x 15 mm style with a center well, #3037, Becton Dickinson

Labware, Lincoln Park, NJ) containing 1.0 ml BMOC-3. The

51

outer well contained 3 ml BMOC-3 without bovine serum albumin
(BSA). Epididymides were poked 30 times each with a 25 G
needle to release the sperm. The dish, with the epididymides
in the center well, was then incubated at 37°C, with 5% C0213:
air and 100% humidity for one half-hour before recovered sperm
concentration and motion analysis or use for in vitro
insemination.

For recovered sperm concentration and motion analysis, 20
ul of the sperm suspension was placed on a CellSoft 20 u
chamber and examined under a CellSoft Video-Micrographic
Computer-Assisted Semen Analyzer (CRYO Resources Inc., New
York, NY). A minimum of 100 sperm cells were analyzed for
each sample. Percentage of recovered sperm concentration,
motile cells, velocity, linearity, amplitude of lateral head
displacement and beat/cross frequency were assessed. See

Appendix 3 for set up and definition of the parameters.

In Vitro Fertilisation

Oocytes were collected from female mice superovulated by
intraperitoneal injection of 10 IU PMSG followed 48 to 50
hours later by 10 IU hCG. Twelve to 14 hours after hCG
injection, oviducts were removed and washed in 3 ml BMOC-3
without BSA in the outer well of a Falcon Organ Tissue Culture
dish and then placed in the inner well with 1 ml BMOC-3. The
ampulla portion of the oviducts were teased open to release

the cumulus mass of oocytes. The cumulus masses were then

52
transferred to a second petri dish and placed in the center
well containing 1 ml BMOC-3. Fifty ul of sperm suspension (1-
3 x 107 cells/ml) that was incubated for one half-hour was
then placed in the oocyte-containing well and incubated for 24
hours before examination of fertilization. The final sperm
concentration at insemination was 0.5-1.5 x 10‘ cells/ml.

To determine fertilization, 100 pl of 370 NM bisBenzimide
Hoechst No. 33258, a chromosome stain (Coming, 1975) , was
added to the petri dish and incubated for an additional 30
minutes. The Hoechst stain allows observation of the
chromosome within nucleus. The oocytes were then washed by
transferring them to 1 ml fresh BMOC-3 and transferred again
onto a microscope slide. Oocytes/embryos were classified on
a Nikon Optiphot microscope equipped with a 100 W mercury
bulb, 365/10 nm excitation filter, 400 nm dichroic mirror, and
400 nm barrier filter. Two-cell embryos with a nucleus in
each cell were classified as fertilized. Zygotes with one
cell and two pronuclei were also classified as fertilized.
Oocytes containing only one cell and one nucleus were
classified as non-fertilized. Fragmented oocytes or those
that lost their cytoplasm were classified as degenerated and

non-fertilized.

Statistical Analysis
Data, except the superovulation rate, were analyzed using

the Statistical Analysis System (SAS Institute, Inc., Cary,

53
NC). The effects of treatments were determined by one-way
analysis of variance (one-way ANOVA) . Significant differences
among treatment groups were determined by least significant
differences (LSD) for the comparisons of multiple means. For
superovulation rate, Chi square (X2) test was applied to
determine if differences were significant. All statements

regarding significance are based on p value < 0.05.

liu‘

pe:
V11
ab:

00]

die

mic

lOt
cor

and

and
are
Lak
Ark
C011
C011

1.7

54

RESULTS

Nutrient Contents of Treatment Diets

Diets were analyzed for nutrient, and organochorinated
pesticides and PCBs. All diets contained about 45% water.
‘Vitamin (Vit) E concentrations in both.A and G fish diets were
about 83% of that in diet C (Table 2). Vitamin A
concentration in diet A was about the same as diet C; in diet
G, however, it was only about two-thirds the concentration of
diet C.

Mineral analysis of diets and mineral requirement for
mice are shown in Table 3. Diets C and G contained similar
amounts of major mineral with.the exception of higher iron and
lower sodium in diet G. Compared to diets C and G, diet A
contained higher concentrations of calcium, iron, phosphorus,
and aluminum. 3

The organochlorinated pesticides and PCBs in Great Lakes
and Arkansas carp before cooking and in the treatment diets
are shown in Table 4. The PCB concentrations in the Great
Lakes carp before cooking was 7.12 ppm, whereas the amount in
Arkansas carp was non-detectable (< 1 ppb) . The diet G
contained 2.54 ppm PCBs. Dieldrin, p,p'-DDE and o,p'-DDD
concentrations were 7.14, 50.44, and 97.07 ppb in diet G and

1.79, 2.21, and 3.01 ppb in diet A, respectively.

55

 

 

 

Nutrients” Diet C Diet A Diet 8
Fat, 8 11.15 22.45 25.25
Crude protein, 4 18.75 ~30.56 31.88
Crude fiber, 8 2.00 2.50 2.40
Calcium, % 0.85 2.21 1.20
Phosphorus, % 0.59 1.25 0.86
Potassium, t 0.78 0.58 0.67
Magnesium, % 0.16 0.13 0.12
Sodium, % 0.47 0.38 0.32
Selenium, ppm 0.15 0.56 0.98
Iron, ppm 181.00 449.00 132.00
Manganese, ppm 130.00 128.00 59.00
Copper, ppm 17.00 12.00 11.00
Zinc, ppm 128.00 84.00 183.00
Ash, 4 5.60 9.90 5.90

Vitamin A (TU/kg) 45331 42709 30636

Vitamin E (IU/kg) 52.06 42.85 44.03

   

' The nutrient analysis was determined by Litchfield Analytical Services,
Litchfield, MI. Selenium and Vitamin A and E analyses were
determined by Dr. Stowe's lab., the Animal Health Diagnostic Lab.,
Nutrition Section, MSU.

‘ Data expressed on dry-matter basis.

56

Table 3. Mineral analysis of three treatment diets and
mineral requirement for mice%

 

Minerals” Diet C Diet A‘ Diet 6‘ Requirement‘
B 7.00 3.16 3.36 ‘ NS‘
Ba 4.72 66.0 4.25 NS
Ca 8010 21700 8040 4000
Cu 17.0 10.8 10.0 4.5
CO 0.520 0.487 <0.200 NS
PO 68.3 . 291 290 25
M9 1610 1220 1020 500
Mn 129 147 53.2 45
MO 1.91 0.678 0.791 NS
P 5310 12400 7160 4000
Zn 127 82.8 170 30
A1 155 769 114 NS
Sb (2.00 <2.00 <2.00 NS
AS <1.00 <1.00 <1.00 NS
Cr 1.15 1.45 1.00 2
Cd <0.200 <0.200 <0.200 NS
Hg «.00 «.00 <4.00 NS
Pb (1.00 <1.00 (1.00 NS
SO (8.00 <8.00 <8.00 NS
T1 <5.00 (5.00 <5.00 NS
Na 5190 4170 3760 NS

4470 5310 2000

K 5650

     

' The mineral analysis was determined by Dr. Braselton's lab.,
the Animal Health Diagnostic Lab., Toxicology Section, MSU.

‘ Data expressed as ppm on dry-matter basis.

° Diets A and G contained no visible bone.

‘ Mineral requirement for mice is based on Nutrient Requirements
of Laboratory Animals (Appendix 2; NRC, 1978).

' Data are not available.

 

 

 

Table 4.

Organochlorinated pesticides and PCBs in raw carp and
mouse diets.‘”

  
 

 

 

 

 

 

Arkansas_sars_ 9 t 3 9 06

Before Diet A Before Diet G

cooking cooking
PCBs (total) <IDL <IDL 7120 2542
Endosulfan I <IDL <MDL <MDL <IDL
Dieldrin <MDL 1.79 <MDL 7.14
Endrin <IDL <IDL <IDL <IDL
Endosulfan II <IDL <MDL <IDL <MDL
Methoxychlor <IDL <MDL <IDL <IDL
Heptachlor 1.58 <IDL 1.54 <IDL
Heptachlor epoxide <MDL <MDL <MDL <MDL
Lindane 1.81 <MDL <MDL 2.62
Oxychlordane <IDL <MDL <MDL <MDL
Gama-chlordane 1.11 <MDL 3.01 1.31
Alpha-chlordane <IDL <MDL 2.73 <IDL
t-nonachlor <MDL <MDL 10.65 18.46
p,p'-DDT <IDL <MDL <MDL 2.75
p,p'-DDE 38.6 2.21 377.13 50.44
o,p'-DDE <IDL 1.32 <MDL 7.19
p,p'-DDD 1.24 <MDL 6.13 6.25
o,p'-DDD 1.28 3.01 45.95 97.07

 

' The organochlorinated pesticides and PCBs analyses were determined by
Dr. Giesy's lab., the Aquatic Toxicology Lab., MSU.

‘ Data expressed as ppb on wet-weight basis. Pesticides that were not
detected by gas chromatography are labeled <IDL (instrument detective
limit). Pesticide concentrations in the sample that are < 1 ppb are
marked <MDL (method detective limit). Minimum detection level is 1 ppb
and the quantification limit is 5 ppb. '

58

P-O Dams

F-O dams were sacrificed when the offspring were weaned
at 3 weeks postpartum. There was no difference in body
weights (BW) of F-O dams among all three groups (Figure 1;
Appendix 4). Livers from the group G females were heavier
than those in group A, which in turn were heavier than those
in group C (Figure 1). In both fish treatment groups G and A,
liver vitamin A concentrations were about 50% lower, and liver
vitamin E concentrations were about 80% lower than those in
group C (Figure 2; Appendix 5) . Because there was only 1
mouse per treatment group for this measurement, no statistical
analysis is shown here. There was no difference in 21-day
body weights of F-l among all treatment groups (Figure 3;

Appendix 6).

F-l Males

After weaning, BW of F-l males was not significantly
different in groups G and C (Figure 3). From 14 weeks of age,
males in group A, however, I had significantly lower body
weights than those in groups G and C (Figure 3). Liver
weights (LW) were not different at 7 and 8 weeks of age among
all treatment groups. At 15 weeks, LW of F-1 males in
treatment A were less than males in treatment C and G (Figure
4; Appendix 7). The LW/BW ratios, however, were greater in
groups A and G than in group C at week 7 and 8, and similar

mong all treatment groups at 15 weeks of age (Figure 5;

 

 

 

      

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les were rovi e wi
immediately after par urition an

-0 fema
through lactation

nd E concentra ' s

' er vitamin A a
0 females.
ent diets

treatm

 

61

 

CDC V7A V'A’

 

 

 

 

 

 

 

 

50 | I I l
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E

40 e —
«5 /§
S—a
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v

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+2 i/i
.EP *
Q) 20 r
3
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"U 10 -
O
m

0 l l l

O 5 1O 15 20 25

Age (Weeks)

Figure 3. Body weights of F-1 male mice in Phase I from birth
to 23 weeks of age. There was no significant
difference in body weights among treatment groups
prior to 10 weeks of age. At weeks 14, 21, and 23,
body weights of Group C were significantly larger
than those of Group A and G.

62

 

 

 

\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\n
m.//////////////////////////////////////

       

%/////////////////////////////////////w

 

 

 

o 000
ms“...

0'3, 0 35.5 .0 «season

a: Significantly “Home fro- m“: C and G, p<0.05

 

 

Figure 4. Liver weights of F-1 males at 7, 8, and 15 weeks of

age.

63

 

 

 

 

          

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W ////////////////////////////////W

/Z\

/////////////////////////////////w

.\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\h
.%////////////////////////////////w

 

 

mmumumo

o..—u> O .7qu no 2.3.3.—

.: Significantly different fro- group C, p<us

 

 

Figure 5. Liver weight over body weight ratios of F-l males at

7, 8, and 15 weeks of age.

64
Appendix 7). There was no significant difference in testis
weights of F-1 males among all treatment groups (Appendix 7).

Similar to the F-0 dams, liver vitamin A and E
concentrations of F-l males were significantly lower in group
G and group A than in group C (Figure 6; Appendix 8). The
concentrations of organochlorinated pesticides and PCBs in the
liver of F-l males are shown in Table 5. Livers from group G
had the highest concentrations of PCBs (4.2 ppm), dieldrin
(9.29 ppb), oxychlordane (18.3 ppb), p,p'-DDE (49.59 ppb),
chlordane (17.83 ppb) and. HCB (13.44 ppb) among the 3
treatment groups.

For the epididymal sperm analysis, no sperm cells were
observed at 5 weeks of age in samples from any treatment
groups (Figure 7; Appendix 9). A steady increase in sperm
concentration was observed from 6 to 8 weeks of age. The
concentrations reached a plateau at 8 weeks} of age. No
apparent difference in sperm concentrations or motion
parameters were observed among the treatment groups at the
ages examined (Appendix 10). ’In vitro fertilizing ability of
the sperm from F-1 males at 7.5 weeks of age did not differ
among all treatment groups (Appendix 11).

In the breeding study, fecundity and litter size were not
significantly different among all treatment groups (Figure 8) .
Viability of the pups (F-2) at 7 days of age in groups G and
A, was lower than that in group C (Figure 8); the viabilities

in groups G, A, and C were 45%, 12%, and 94%, respectively.

65

 

 

 

 

 

Ic IA Io

 

A ////////E

         
 

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m ////////////////....

\x\\\\\\\
m /////,

 

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m /////////t

 

120

M
1.

AAA...»

31> U enema 1 «look».—

Vi/t/Ai
a: 32 week- ot age

\A

u.

e
t
c.
o
w
w
0,
1.
.V......

 

 

Figure 6. Liver vitamin A and E concentrations in F-l males.

66

Table 5. Organochlorinated pesticides and PCB
residues in Fé1ma1e liverff.

    
   

 

 

Group C Group A Group
G'

PCBs <IDL 150 4200
Endosulfan I <IDL <IDL <IDL
Endosulfan II <IDL 0.32 0.91
Aldrin <IDL <IDL <IDL
Dieldrin 1.04 2.06 9.29
Endrin <IDL <IDL <IDL
Methoxychlor ’<IDL 0.45 <IDL
Heptachlor epoxide 1.31 0.87 <IDL
Oxychlordane <IDL 1.52 18.30
Y-chlordane <IDL <IDL 0.61
A-chlordane 0.34 1.72 17.83
t-nonachlor <IDL <IDL 0.25
Hexachlorobenzene 0.22 0.59 13.44
Heptachlor <IDL <IDL <IDL
p,p'-DDT <IDL <IDL <IDL
o,p'-DDD 0.21 <IDL <IDL
p,p'-DDD <IDL <IDL 0.51
o,p'-DDE <IDL 0.30 _ 1.89
p,p'-DDE 0.47 2.85 49.59

' The organochlorinated pesticides and PCBs analyses were determined
by Dr. Giesy's lab., the Aquatic Toxicology Lab., MSU.

‘ Data expressed as ppb on wet-weight basis. The water content in three
diets was similar and was 458. Pesticides that were not detected by
the gas chromatography are labeled <IDL (instrument detective limit).
Pesticide concentration < 0.2 ppb and PCB concentration < 0.03 ppm in
samples are below the quantification limit.

° F-l male mice were 239 days old (32 weeks).

67

 

 

°°noentraflon (mllllonlml)
a on 3

8
.0 IA fie

 

 

  

  

 

/

  

 

3‘ “hmks

/

I :t:"

a
a»
7‘\
a:
d
a
:0
N

 

 

Figure 7.

Caudal epididymal sperm concentrations of F-1 males
at 5, 6, 7, 8, 15, 23, 32, and 34 weeks of age.
Sperm suspension was collected as follows: both of
the epididymides were placed in a Falcon Organ
Tissue Culture Dish (60 x 15 mm, Becton Dickinson
Labware) and poked with a 25 G needle to release
the sperm into 1.0 ml BMOC-3 medium. After a 30—
min incubation at 37°C, 5% CO2 in air and 100%
humidity, 20 pl of the sperm suspensions were
placed on a CellSoft 20u-chamber and examined under
a CellSoft Video-micrographic Computer-Assisted
Semen Analyzer (CRYO Resources Inc., New York, NY).
A minimum of 100 sperm cells were analyzed for each
sample.

68

 

 

 

     

m m ./ m

. \\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\

///////////////////////////////m

 

 

 

o o o
8 m m 2

os1> D 95.5 as «soon».—

n: Significantly different from gronp C, p<0.|5

 

 

Figure 8. Reproductive performance of 52-day-old F-1 males.

69

F-i Females

To test oocyte fertilizing ability, F-1 females at 5 and
10 weeks of age were superovulated and the oocytes were
collected and inseminated in vitro with epididymal sperm from
non-treated males. Significantly fewer females in groups A (4
of 6) and G (0 of 7) than in group C (7 of 8 females)
responded to the superovulation treatment (Figure 9; Appendix
12). Fertilizing ability of oocytes collected from groups A
did not differ C showed no difference in vitro (Appendix 12).

To study the reproductive performance, 10-week-old F-1
females were paired with proven fertile males for 5 days. Ten
days after the breeding period, females were sacrificed to
determine the number of fetuses. Groups A and G had about a
50% pregnancy rate; whereas, all 7 females in group C were
pregnant (Figure 9). The number of fetuses per dam was not

different among treatment groups (Figure 9).

 

7O

 

 

   

W\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\,m
m////////////////////////////////.

Litter/

.\\\\\\\\\\\\\\\\\\\\\

 

 

 

w.

u

G m
a 2m
m /m
c m
I . . _ . _ w
m m m m u n as

31> O .7qu no “eunuch

 

Fecundity refers to number of females

carrying fetus 10 days after breeding period/

ovulated/ number of females superovulated with
number of females bred.

Superovulation rate refers to number of females
PMSG/hCG.

a: Significantly different from group C, p<0.05
II: Significantly different he. granps C and A, p<0.05

 

Figure 9. Reproductive performance of F—1 females.

71

DISCUSSION

The fish-containing diets were designed to contain the
highest possible carp content and still maintain palatable
texture for mice. Based on the results of a preliminary
palatability study, 75% carp on a wet-weight basis was used to
formulate the treatment diets. The carp content in the final
diets was 60% on a dry-matter basis.

The PCB concentration was 7.12 ppm in the Great Lakes
carp before cooking and 2.54 ppm in diet G containing 75%
cooked Great Lakes carp (Table:4). Since diet.G contained 75%
cooked Great Lakes carp on a wet-weight basis, the PCB
concentration in the 100% cooked Great Lakes carp was
calculated to be about 3.39 ppm. The decrease in PCB
concentration from 7.12 ppm to 3.39 ppm apparently was due to
the cooking process.

Enlarged livers were observed in both fish treatment
groups (Figures 1 and 5). In F50 lactating mice, although
liver weights from group A were less than those from group G,
both were significantly heavier than those from group C
(Figure 1). This change in liver weight, however, was not
detected in F-1 males (Figure 4). Nevertheless, when data of
F-1 males were expressed as LW/BW’ratio, groups A and G showed
significantly higher ratios than group C at 7 and 8 weeks of
age, although this higher LW/BW ratio was less apparent in F-l

males at 15 weeks of age (Figure 5).

72

Increased liver weights were also observed in other
studies where animals were exposed to PCBs, TCDD or other
related compounds (Risebrough et a1., 1968; Fowler et a1.,
1973; McConnell, 1980). This may relate to an increase in
total liver lipid and triglyceride contents (AzaIs-Braesco et
a1., 1990) or a proliferation of the smooth endoplasmic
reticulum (Fowler et a1., 1973; Buchmann et a1., 1991) . Since
no detectable PCBs and less than 7 ppb DDT metabolites were
observed in diet A (Table 4), the statement above does not
explain the enlarged livers in group A mice observed in both
F-0 dams and F-1 males. Therefore, ingestion of 60% fish diet
on dry matter basis will cause liver enlargement in mice.

Decreased liver vitamin (Vit) A concentration in PBBs-
and PCBs-exposed animals was reported previously (Spear et
a1., 1988; Mercier et a1., 1990). PCBs were shown to alter
Vit A metabolism and distribution, leading to'a reduction in
Vit A storage in the liver. When the liver Vit A storage was
decreased, the storage form of Vit A, retinyl esters, which
normally represent 95% of the hepatic Vit A contents, was
diminished (Mercier et a1., 1990). Such transformation of
retinyl esters to retinol (mobile form) should involve
enhanced hydrolysis of retinyl esters into free retinol
(Mentlein and Heymann, 1987). In 1984, retinyl palmitate
hydrolase was the only enzyme shown to be active in the
reaction in vivo (Goodman and Blaner, 1984). More recently,

another enzyme, namely retinyl ester hydrolase, was also found

73
to possess the characteristics of retinyl esters hydrolysis in
male Wistar rat liver homogenate and microsomes (Mercier et
a1., 1990). Both enzymes, belonging to the cytochrome P-450
system, could be affected by PCBs, DDT or TCDD, and may have
had the potential to change hepatic Vit A storage (Mercier et
a1., 1990).

Because of the higher concentrations of PCB or DDT
metabolites in diet G than in diet A, it is reasonable to
hypothesize that mice from group G would have lower liver Vit
A concentrations. However, F-0 dams and F-1 males in group A,
which consumed a diet containing no detectable PCBs and only
trace amounts of other organochlorinated pesticide
metabolites, had lower liver Vit A than those of group G
(Figures 2 and 6) . Therefore, effects of liver Vit A
discussed above does not explain the lower Vit ~A
concentrations in group A livers than those in groups C and G.
One possible cause might be the high fish-oil content of the
fish diets. Fish oil, highly unsaturated fatty acid, if
undergoing active oxidative lrancidity, could cause Vit E
destruction (Scott et a1., 1982). Under continuously
oxidative stress, once the majority of Vit E was depleted, Vit-
A will soon be attacked by newly generated radicals (see Phase
II-Discussion).

The concentrations of organochlorinated pesticides and
PCB residues in F-1 male livers were directly related to those

in their respective diets (Table 5). In both fish eating

in
mo
be
or
lac
the
by
emb
the
fan
the

diff

74
groups, the liver residues of PCBs, dieldrin, oxychlordane, A-
chlordane, and p,p'-DDE were higher than those of diet C.

In this study, caudal epididymal sperm concentrations and
sperm motion were not significantly different among the three
groups at all ages measured. These findings were similar to
the work of Sager et a1. (1991), who observed no differences
in caudal sperm reserves, sperm production, epididymal sperm
morphology, and epididymal sperm motility in male Sprague-
Dawley rats when exposed to Aroclor 1254 at.doses of 8, 16, 32
or 64 ug/g of body weight on days 1, 3, 5, 7, and 9 during
lactation. However, they did report abnormal sperm function
that was tested by breeding with non-treated females followed
by embryo recovery. Increases in the percentage of abnormal
embryos from.pronuclear to blastocyst stages were observed in
the 32 and 64 pg/g groups. Based on the in vitro
fertilization (IVF) assay; I did not observe any difference in
the percentage of fertilized oocytes, nor did I observe any
difference in litter size in the breeding study.

The survival rate of Fl-2 pups from both carp-eating
groups was significantly lower than that of the lab chow
controls. Nutrient differences between lab mouse chow and
both fish diets should be responsible for this observation.
Survival rates for F-2 and body weights of F-1 were the lowest
in those mice receiving diet A. The reason for these
observations is not clear. Since mineral concentrations, such

as calcium, phosphorus, iron, manganese and zinc were the

V

4!
le
ir
su
PC
ef
ac
fur
rec
dis
org
The.

die1

Pr0v
the l

diet

75
major differences between diets A and G, high minerals in diet
A should account for the effects observed in the animals
received diet A.

No F-1 females in group G respOnded to the PMSG/hCG-
inducing superovulation (Figure 9) . Other studies have
demonstrated that PCBs could interfere with ovulation in two
ways. First, PCBs accumulation would disturb the normal
reproductive development and ovulation by stimulating the P-
450 enzyme system, which metabolizes the steroid hormones
leading to a change of the concentration of these substances
in the body during the critical neonatal period when the young
suckle PCB-contaminated milk (Lucier et a1., 1977). Second,
PCBs and DDT may interfere with ovulation by their estrogenic
effects (Fuller and Hobson, 1986) . The weak estrogenic
activity of PCBs and other pesticides could affect endocrine
function by their competitive affinities for estrogen
receptors within reproductive organs and, furthermore, by
disrupting the feedback control between the reproductive
organs (or other endocrine organs) and hypothalamus.
Therefore, I hypothesize that PCBs and other pesticides in
diet G were responsible for the failure of ovulation in group
G.

The F-1 females were bred with previously non-treated and
proven fertile males. The results, however, did not support
the previous hypothesis that PCBs and other pesticides in the

diet G were responsible for the failure of ovulation in group

76
G. In the study of reproductive performance of F-1 females,
over 50% of the group G females were pregnant. One possible
reason for failure to collect any oocyte from group G mice may
be that. Great. Lakes contaminants changed. the timing' of
ovulation after PMSG/hCG injection. Thus, no oocytes could be
collected from the oviduct at the expected time.

Pregnant rate of F-1 females in group G were much lower
than those of the control group (Figure 9). Similar findings
were also reported in other PCB studies. Kihlstrom et a1.
(1975) administered a PCB mixture to pregnant mice on the day
of delivery and repeated this 3 times weekly. The
reproductive capacity' of F-1. males was 'measured by the
frequency of implantation in F-1 males mated females that were
non-treated previously. The reproductive capacity was 94% in
the control F-l males, and was about 20% lower than that in
control males in the PCB-treated males. Orberg and Kihlstrom
(1973) reported prolonged estrus and decreased reproductive
capacity after administration of PCBs (Chlophen-A-60) to mice.
On the contrary, R6nnbéick (1991) found no indication of
altered litter size in neonatally exposed females mice (15 ppm
tetrachlorobiphenyls/day) . The treated females showed no
indication of developmental toxicity: no gross malformations,
no obvious changes in size, and no increased mortality were
observed during the lactationjperiod, In this study, not only
those females in group G had lower fecundity, but also those

in group A had similar results. It is apparently that there

77
were critical factors other than the Great Lakes contaminants
causing these adverse effects. .Again, ingestion 60% fish.diet
may be the reason for low fecundity observed in both fish
eating groups.

Recently, a study of delayed first ovulation by a diet
containing high percentage of fish oil was published (Zhang et
a1., 1992). This impaired gonadal development, retarded or
prevented the first ovulation in female rats by 21% fish oil
in the diet was not due to nutritional imbalance. It was
believed that eicosapentaenoic acid, a fatty acid in the fish
oil, could compete for cyclooxygenase and reduce the synthesis
of dienoic prostanoids including prostaglandin E2 which plays
an important role in the estrogen-stimulated release of
hypothalamic GnRH. In this study, the percentage of fish oil
in diets A and G were 22.45% and 25.25%, respectively.
Therefore, fish oil could be a possible factor causing the low

reproductive performance in groups A and G.

7S

PHASE II STUDY

In the Phase I Study, the results of Vit A and E
concentrations in. mouse livers 'showed that Vit (A
concentrations in groups A and G were about 50% or lower than
that in group C; whereas Vit E concentrations in groups A and
G were less than 20% of that in group C (Figures 2 and 6).
Because decreases in Vit A and E concentrations in the liver
were observed in both groups A and G, factors other than the
Great Lakes contaminants were thought to be causing the
altered metabolism and/or distribution of the vitamins.

The hypothesis was that oxidative stress generated from
the fish oil exhausted Vit E which is an antioxidative agent.
Following the depletion of Vit E,‘Vit.A would also be depleted
by the oxidative stress. This may occur both during feed
storage and in vivo. 3

The objectives of this Phase II study were to determine:
1) whether increased dietary Vit E concentration will elevate
the low hepatic Vit E concentration, and 2) whether increased
dietary Vit.E concentration will in.turn raise the hepatic‘Vit

A concentration in both groups A and G .

79

HATSRILLS AND METHODS

Animals
Nine 81-day-old F-l male mice (3 from each group) from

the first phase were used in the second phase of study.

Treatment Diets

Two new diets, A+ and G+, which contained Vit E
concentrations 5 times greater than those of the original
diets (A and G) were made for this study. One hundred and
five ug/kg Vitamin E, which was 4 times the concentration of
previous diets, was added to diets A and G to make the final
Vit E concentrations 5 times greater than the original diets.
Group C'was under the same treatment diet (diet C) as in Phase

I study.

Experimental Design

There were 3 treatment groups, C, A+, and G+, in this
study. Three mice each from the previous groups A and G were
fed the high Vit E diets (diets A+ and 6+); whereas, 3 mice
from group C were fed diet C continuously. Animals were
sacrificed at 10, 30, and 156 days after consuming the high
Vit E diets. Liver vitamin A and E concentrations were

analyzed as in Phase I.

RESULTS

Liver Vit E concentrations in both groups A+ and G+ were
higher than those of group C after being supplied with high
Vit E diets for 10 days and remained higher at day 30 and day
156 (Figure 10; Appendix 13).

The liver Vit A concentrations in both groups A+ and G+
reached about 60% of those in the control after the mice were
fed diets with high Vit E for 10 days (Figure 10). After 30
and 156 days of the high Vit E supplement, liver Vit A
concentrations in groups A+ and G+ were higher than those of
group C. When the data were expressed as a percentage of
group C, the liver Vit A concentrations in group A+ were 54%
higher after 30 days and 82% higher after 156 days of the Vit
E supplement. Concentrations of liver Vit A in group G+ were
about the same as those in group C after 30 days and 47%
higher after 156 days following the treatment.

Although no significant difference in LW/BW ratio among
treatment groups, same as F-1 males in Phase I study, there
was still a trend that.mice in group G had larger LW (Appendix

14).

 

81

 

 

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Figure 10. Liver vitamin A and E concentrations of F-1 males

82
DISCUSSION

The NRC (1978) requirement for vitamin (Vit) E for mice
is 20 IU/kg in the diet. However, Vit E requirements are
exceedingly difficult to determine because of their
interrelationships with other dietary factors, such as
polyunsaturated fatty acid (PUFA), other antioxidants, sulfur
amino acids and selenium (Se) (Church and Pond, 1985).

Vitamin E is very unstable; its oxidation is increased by
the presence of PUFA, oxidizing agents, and trace minerals
(eg. Fe), and decreased with increasing levels of fat-soluble
antioxidants, sulfur amino acids, or Se (Church and Pond,
1985) . Requirements of both Vit E and Se are greatly
dependent on each other's dietary concentrations. Low Se
content in the diet was believed to cause Vit E deficiency
(Trapp et a1. , 1970) . In the present study, Se concentrations
were the highest in diet G and lowest in diet C (0.15, 0.56,
and 0.98 ppm in diets C, A, and G, respectively) (Table 2).
Therefore, the possibility of lower liver Vit E concentrations
caused by low Se concentrations in the diet can be ruled out.

The high concentrations of PUFA found in unsaturated oils
such as cod-liver oil, corn oil, soybean oil, sunflower-seed
oil and linseed oil can increase Vit E requirements. This is
especially true if these oils are allowed to undergo oxidative

rancidity in the diet or are in the process of peroxidation

83

after consumption by the animal (Scott et a1., 1982). If a
diet becomes completely rancid before ingestion, the only
damage done is the destruction of the Vit E present in the oil
and in the feed containing the rancidifying oil. But if a
diet undergo active oxidative rancidity after consumption, it
will cause destruction of body stores of Vit E (Scott et a1.,
1982).

Dietary supplementation of Vit E not only raised the
hepatic Vit E concentrations in the mice, but also elevated
the hepatic Vit A concentrations (Figure 10). Since dietary
Vit E can protect or spare body supplies of oxidizable
materials, such as Vit A and C, by interruption of fat
peroxidation, it is reasonable to deduce that the lower liver
Vit A observed in the previous study was mostly caused by the
oxidative rancidity. After higher Vit E was supplied, it
protected Vit A (and perhaps many other nutrients) from the

PUFA-generated radicals.

S4

Phase III Study

After reviewing the nutrient contents in the 3 treatment
diets of Phase I (Table 2), it was thought that the higher
calcium, phosphorus, iron, manganese and lower zinc
concentrations in diet A than those in diet G may have caused
the lower body weights of F-1 and decreased viability in F-2
pups of group A. In trying to make diets A and G nutrients
more alike, two diets, A' and G', were designed to eliminate
the differences described above.

Another factor that should be addressed here is that, in
the Phase I study, CBA/CAJ female mice did not ideally respond
to PMSG/hCG stimulation at the expected time. The average
number of oocytes collected by superovulation treatment was
about 6 per mouse in non-treated CBA/CAJ females; whereas, in
our experience, collecting more than 30 oocytes in B6D2-F1
females‘was common (B6D2-F1 is named.after its maternal strain
C57BL/§_J and paternal strain QBA/ZJ) . Although there were 87%
of the females in group C responsed to superovulation during
Phase I (Appendix 11), more than 50% ovulated only 3 to 4
oocytes. At this point, it is not certain whether the 0%
ovulation rate in the group G females during Phase I (Figure
9) showed truly biological significance or not. Therefore, it
was decided to change the mouse strain from CBA/CAJ to BGDz-Fl

during this experiment phase.

MATERIALS AND METHODS

Animals

Thirty C57BL/6J female mice (Jackson Labs, Bar Harbor,
ME) were bred with 15 DBA/2J male mice. One male was randomly
caged together with 2 females. The mice were kept in the same
facilities and under the conditions as described for Phase I.
When the females were found pregnant, they were randomly

divided into 5 treatment groups-- C, A, A', G and 6'.

Treatment Diets
Five treatment groups, C, A,.A', G, and.G', were included
in this phase of the study. Each group received one of the
following diets formulated on a dry-matter basis:
C : 100% Lab Mouse Chow #5015 (control)
A : 47% farm-raised carp + 40% Lab Mouse Chow #5015
+ 13% fish oil
A': 44.2% farm-raised carp + 40% Lab Mouse Chow #5015
+ 15.8% fish oil + 100 ppm Zn
G : 60% Great Lakes carp + 40% Lab Mouse Chow #5015
6': 58.65% Great Lakes carp + 40% Lab Mouse Chow #5015

+ 0.95% Ca + 0.40% P + 317 ppm Fe + 70 ppm Mn.

The mineral sources of Zn, P, Fe and Mn were from ZnSQU
CaHPO“ FeSO4, and MnSO“ respectively. Calcium, however, was

supplied from 2 sources: CaCO3 and CaHP04. All 5 mineral

SS
supplements were purchased from ICN. Lab Mouse Chow #5015,
fish oil, farm-raised carp and Great Lakes carp used in this
trial were the same as in Phase I. The diets were prepared in

the same manner as in Phase I.

Medium and Chemicals

BMOC-3 (for sperm capacitation and in vitro
fertilization), PMSG, hCG, and Hoechst #33258 bisBenzimide
stain were described previously in the Medium and Chemicals
section of Materials and Methods of the Phase I study (pp.

43).

Experimental Design

The treatment diets were provided immediately after the
dams (F-O) gave birth and.through lactation. lBody weights and
liver weights of the F-0 females were measured at 21 days
after parturition.

At.21 days of age, F-1.mice (B6DZ-F1) were weaned, sexed,
weighed, and then housed in groups of 3 or 4 mice per cage.
The offspring (F-1) received their respective treatment diets
continuously to termination.

Body weights were recorded every week before 10 weeks of
age, and every 5 weeks after 10 weeks of age. Liver weights,
thymus weights and testis weights were measured at 8 and 11
weeks of age. Liver concentrations of vitamin A and E were

analyzed at 8 weeks of age. Sperm concentration and motion

87
were measured at 8 and 11 weeks of age under the same
procedures as in Phase I. Sperm fertilizing ability of F-1
males, at 8 weeks of age, was tested in vitro with oocytes
from non-treated B6D2-F1 females.

At 8 weeks of age, 8 F-l males were paired each with a
previously non-treated mature B6D2-F1 female mouse for 5 days.
The females, received the same treatment diet as the males
when they were housed together, and when housed individually
through gestation and lactation. Litter size, body weights,
gestation index (survival rate to day 4) and lactation index
(survival rate to day 21) of the offspring (F-2) were
recorded. The dams were sacrificed by cervical dislocation at
21 day postpartum to measure body and liver weights.

F-2 mice, at 21 days of age, were weaned, sexed, weighed,
and then housed in groups of 3 or 4 mice per cage. The F-2
males received the same diet as dam continuously to
termination. Body weights were recorded every week until
termination (8 weeks of age). Liver vitamin A and E
concentrations were measured at 6 weeks of age. Liver
weights, thymus weights, testis weights and sperm
concentration and motion were measured at 5, 6, 7, and 8‘weeks
of age. Sperm fertilizing ability of the F-2 males, at 8
weeks of age, was tested in vitro. At 7 weeks of age, 6 F-2
males were paired individually with a previously non-treated
mature B6D2-F1 female mouse. Litter size and viability index

(survival to day 7) of the offspring (F-3) were recorded.

SS
F-l females were fed their treatment diets the same as
the F-1 males. At 52 to 58 days of age, 15 females from each
treatment groups were superovulated by PMSG/hCG. Ovulation
rates and in vitro fertilization rates were recorded. Body

weights, liver weights and thymus weights were also recorded.

F-2 females*were fed their treatment diets under the same
conditions as the others. Between 31 to 44 days of age, they
were superovulated. Ovulation and in vitro fertilization rate
were recorded. Body weights, liver weights and thymus weights
were recorded. Liver vitamin A and E concentrations were

analyzed at 44 days of age.

In vitro Fertilisation
The procedure of in vitro fertilization was described
previously in the Materials and Methods section of the Phase

I study (pp. 51).

Statistical Analysis
Data were analyzed as previously described in the Phase

I study (pp. 52).

RESULTS

Nutrient Contents of Treatment Diets

The nutrient contents for diets C, A, A', G and G' are
shown in Table 6. The nutrients in diets A' and 6' listed in
Table 6 were based on the calculation from diets A and G, and

their respective additives.

E-O DIII

After 21 days of lactation, there was no difference in
body weights (BW) among the F-0 lactating dams (Figure 11;
Appendix 15). Although the liver weights (LW) of the mice in
groups G, G' and A! were heavier than those in groups A and C,
the differences were not statistically significant (p = 0.10) .
When data were expressed as LW/BW ratios, groups G, G' and A'

were significantly larger than those of groups C and A.

P-l Males

After' weaning, F-1. males *were fed. their' respective
treatment diets continuously to termination. Although the BW
of F-l males in groups A, A' and G' were constantly smaller
than those of group C and G, the differences were not
significant until after 15 weeks (Figure 12; Appendix 16).
The group C mice were the heaviest; while those in groups A
and A' were the lightest. No differences in the BW were

observed between groups G and G' at 15 and 20 weeks of age.

90

 

Table 6. Nutrient contents of the treatment dietsh

 

 

Nutrientsb C A A' G G'
Dry Matter % 55.00 53.96 53.96 54.82 54.82
Fat % 11.15 22.45 25.25 25.25 25.25
Crude Protein A 18.75 30.56 30.56 31.88 31.88
Crude Fiber % 2.00 2.50 2.50 2.40 2.40
Ash t 5.60 9.90 9.90 5.90 9.90
Calcium % 0.85 2.21 2.21 1.25 2.20
Phosphorus % 0.59 1.25 1.25 0.86 1.26
Potassium % 0.78 0.58 0.58 0.67 0.67
Magnesium % 0.16 0.13 0.13 0.12 0.12
Sodium % 0.47 0.38 0.38 0.32 0.32
Iron ppm 181.00 449.00 449.00 132.00 449.00
Manganese ppm 130.00 128.00 128.00 59.00 129.00
Copper ppm 17.00 12.00 12.00 11.00 11.00
Zinc ppm 128.00 84.00 183.00 183.00 183.00

 

‘ The nutrient analysis of diets C, A and G was determined by
Litchfield Analytical Services, Litchfield, MI. The nutrient
concentrations for diets A' and G' are based on calculation
from diets A and G, and their respective additives.

‘ Data expressed on dry-matter basis.

91

 

 

 

 

 

 

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a: Significantly different from grenpe C and A, p<0.05

 

 

Figure 11. Body and liver weights of F-o lactating females.

F-O females were provided with treatment diets

immediately after parturition and through

lactation.

92

 

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Age (Weeks)

Figure 12. Body weights of F-l males in Phase III from birth
to 20 weeks of age. No difference in body
weights among treatment groups was observed before
10 weeks of age. At 15 and 20 weeks of age, mice
in Group C showed significantly larger body
weights than those in other groups; mice in Groups
G and G' were not significantly different in body
weights, and.were both larger than those of Groups
A and A'.

 

93
There were no differences in BW between groups A and A' ,
either.

Liver, thymus and testis weights were measured and
analyzed in F-l males at 8 and 11 weeks. Livers in groups G
and G' were heavier than those of group A' , which were heavier
than groups C and A (Figure 13; Appendix 17). At 11 weeks of
age, however, LW’in.group>G‘was heavier than all others. When
data were expressed as LW/BW ratio, there were no differences
in all 5 groups at 8 and 11 weeks of age. There was no
difference in thymus or testis weights among all 5 groups at
8 weeks of age. .At 11 weeks of age, the thymus of mice in all
4 fish-diet groups weighed less than those in group C. Mice
in group G' had smaller testis than those in groups C, A' and
G at 11 weeks.

Liver vitamin (Vit) E concentrations in F-1 males of
groups G and G' were lower than those in groups C, A and A'
(Figure 14; Appendix 18) . Liver Vit A concentrations in
groups C and G were similar and were the highest among the
treatment groups. Liver Vit A concentrations in groups A and
A' were similar and were lower than those in groups C and G,
but higher than those in group G'.

To study the reproductive performance of F-l males,
epididymal sperm concentration, motion quality, in vitro
fertilizing ability and breeding ability were studied. There
was no difference in sperm concentration (Figure 15; Appendix

19) or the other parameters measured by the Sperm Analyzer at

 

 

94

 

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a: Significantly different freln grenpe C and A, p<0.05
h: Significantly different from grenpe C, A and A', p<0.05

age .

 

Figure 13. Liver weights of F-l males at 8 and 11 weeks of

95

 

 

 

 

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Figure 14. Liver vitamin A and E concentrations in 8-week-old

 

F-1 males.

96

 

 

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Figure 15. Caudal epididymal sperm concentrations of F-l males
at 8 and 11 weeks of age. Each column represents
the mean of 3 samples. Sperm suspension was
collected as following: both epididymides were
placed in a Falcon Organ Tissue Culture Dish (60 x
15 mm, Becton Dickinson Labware) and poked with a
25 G needle to release the sperm into 1.0 ml BMOC-
3 medium. After a 30-min incubation at 37°C, 5%
CO2 in air and 100% humidity, 20 ul of the sperm
suspensions were placed on a CellSoft 20u-chamber
and examined under a CellSoft Video-micrographic
Computer-Assisted Semen Analyzer (CRYO Resources
Inc., New York, NY). A minimum of 100 sperm cells
were analyzed for each sample.

97
both 8 and 11 weeks of age (Appendix 20). Similar to the F-1
males in Phase I, there was no difference in the in vitro
fertilizing ability of the sperm in all treatment groups
(Appendix 21). When mated with non-treated females, only 2
females in group G' were found not pregnant. At weaning,
groups G and A' showed smaller litter size than those in group
C (Figure 16). There were no significant differences in the

gestation index and lactation index.

F-1 Females

There were no differences in body and thymus weights
among the 5 groups (Figure 17; Appendix 22) . Groups G and G' ,
similar to the F-1 males in this phase of study, had larger LW
than those of groups C and A (Figure 17) . There was no
difference in the superovulation rate or in vitro

fertilization rate (Appendix 23).

F-z Males

The BW of F-2 males from birth to 8 weeks of age is shown
in Appendix 24. Although the BW of group A' were consistently
lower than those of the other groups, no significant
difference was observed among groups.

The liver, thymus and testis weights of F-2 males were
measured at 5, 6, 7 and 8 weeks of age and are shown in
Appendix 25. At weeks 5~and 8, groups G and 6' showed larger

liver weights than those in group C (Figure 18) . However,

98

 

 

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Birth Li ter Size Lactation Index Weaning Litter Size

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a: Significantly different from grenp C, p<0.05

 

 

 

Figure 16. Reproductive performance of 8-week-old F-1 males.
Fecundity refers to number of females giving
birth/number of females bred. Lactation index
refers to percent of F-2 pups survived to the age
of 21 days.

 

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Figure 17. Body and liver weights of 8-week-old F-l females.

 

 

100

 

 

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In: Significantly different from groups C and A, p<0.05

a: Significantly different from group C, p<0.05

of age.

 

Figure 18. Liver weights of F-2 males at 5,

101

when the data were expressed as a LW/BW ratio, no differences
were observed at 5 to 8 weeks of age. At week 7, groups A'
and G' showed significantly larger thymus weights than groups
C and G, but there were no differences at weeks 5, 6 and 8.
The testis weights of the mice in groups G and G' were
significantly larger than those of groups C and A at 5 weeks
of age. There were no differences in testis weight at weeks
6 and 7. Although group G showed significantly larger testis
weights than groups A' and G' at 8 weeks of age, no
differences were observed when the data were expressed as
testis/body weight ratio.

The liver Vit A concentrations of the F-2 males, when
expressed as a percentage of group C, were 23.7% in group A,
13.5% in group A', 51.9% in group G ,and 38.8% in group 6'
(Figure 19; Appendix 26) . Liver Vit E concentration was
highest in group A (20.33 ug/g of dry LW), followed by 13.02
ug/g in group C, 7.52 ug/g in group A', 5.27 ug/g in group G,
and was lowest in group G' (2.79 ug/g).

The assessment of the effects of consuming Great Lakes
carp on reproductive performance of the mice was carried on to
the second. generation (F-2). There ‘was no significant
difference in F-2 male sperm concentrations (Figure 20;
Appendix 27) or other motion parameters measured at 5, 6, 7,
and 8 weeks of age (Appendix 28). In vitro fertilizing
ability did not differ (Appendix 29). When bred with non-

treated females, only 2 females in group C were found not

102

 

180

Is IA @A' IG EG'

Percent of Group C Value

 

Vitamin A Vitamin E

 

 

 

Figure 19. Liver vitamin A and E concentrations on 8-week-old
F-2 males.

103

 

 

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Figure 20. Caudal epididymal sperm concentrations of F-2 males
at 5, 6, 7 and 8 weeks of age. Each column
represents the mean of 3 samples. Sperm
suspension was collected as follows: both
epididymides were placed in a Falcon Organ Tissue
Culture Dish (60 x 15 mm, Becton Dickinson
Labware) and poked with a 25 G needle to release
the sperm into 1.0 ml BMOC-3 medium. After a 30-
min incubation at 37°C, 5% CO2 in air and 100%
humidity, 20 pl of the sperm suspensions were
placed on a CellSoft 20u-chamber and examined
under a CellSoft Video-micrographic Computer-
Assisted Semen Analyzer (CRYO Resources Inc., New
York, NY). A minimum of 100 sperm cells were
analyzed for each sample.

104
pregnant (Appendix 29). The fecundity of groups A, A', G and
G' was 100%. There was no difference in the survival rate at
7 days, although one litter in group A' was found dead at
birth. There were no difference in the BW of the pups (F-3)
in groups C, A and G, but lower BW were observed in groups A'

and G' (Figure 21).

r-z Females

The body, liver and. thymus weights of F-2 females
continuously exposed to treatment diets after weaning were
measured at 31 to 44 days of age. Similar to the F-1 and F-2
males, group A' had the lowest BW in 5 groups. No differences
were found in the BW among the other 4 groups (Figure 22;
Appendix 30). The liver weights in group G were higher than
those of groups A and A' (Figure 22). However, when the data
were expressed as a LW/BW ratio, all the fish-eating groups,
.A,.A', G and G', showed larger LW/BW ratios than group C.
There'were noidifferences in the thymus weights among all five
groups. I

Liver Vit A and E concentrations of the F-2 females are
shown in Figure 23 and Appendix 31. Similar to the F-1 males
in this phase (III), the total liver Vit A concentrations in
groups C (883.7 ug/g of dry LW) and G (576.3 ug/g) were higher
than those in groups A (346.3 ug/g) and.G' (329.3 ug/g), which
were higher than those of group A' (164.3 ug/g). Liver Vit E

concentrations in group C (22.56 ug/g) were the highest among

105

 

 

 

 

 

 

 

 

 

 

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o 8 6 ‘

no .I.G.IA.@A.'.I.G.EG!. .

os1> O macaw as 13.3.—

a: Significantly different from groups C, A and G, p <0.05

 

 

Figure 21. Body weights of 7-day-old F-3 pups.

 

 

106

 

 

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///////////////////////////////._

 

140 .CAgA'GfiG'k

 

 

os1> D assume no 33.—o.—

 

F-2 females

2 females.

a: Significantly different from group G, p<0.05

b: Significantly different non: groups C and A, p<0.05
c: Significantly different from group C, p<0.05

were between 31 to 44 days old.

 

Figure 22. Body and liver weights of F

107

 

 

 

 

 

Ic IA @A' IG EG'

 

 

 

 

 

 

 

 

Vitamin E

 

.......................

..................................
.............................

...............................
..............................

..............................

...............................
...............................
................................
eeeeeeeeeeeeeeeeeeeeeeeeeeeeeee
eeeeeeeeeeeeeeeeeeeeeeeeeeeeee
ooooooooooooooooooooooooooooooo

eeeeeeeeeeeeeeeeeeeeeeeeeeeeee
oooooo
eeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee

\\\\\\\\\\\\\\\\\\\\\\\\\\\t

 

////////////////v

 

120

o o o o
8 6 .4 2

os1> 0 9.9.9 as «noose.—

 

 

Figure 23. Liver vitamin A and E concentrations in 8-week-old

F-2 females.

100
the 5 groups. Liver Vit E concentrations in group G was 8.49
ug/g of dry LW, which was about one-third the concentration of
group C, and was higher than those of groups G', A and A'.
There were no differences in the superovulation rates and
the in vitro fertilization rates of the F-2 females (Appendix

32).

109

DISCUSSION

Diets A' and G' were formulated to minimize the
differences in nutrient contents between'the Arkansas carp and
the Great Lakes carp. Because it is difficult to remove
excess compounds from the diets, the diets were adjusted by
simply adding 100 ppm Zn and 2.8% fish oil to diet A and 0.95%
Ca, 0.4% P, 317 ppm Fe, and 70 ppm Mn to diet G (Appendix 13).

The LW in groups C, A and.G of the F-0 dams in this phase
(Figure 11) were similar to the previous findings of F-0 dams
in Phase I (Figure 1). The LW of groups G and G' were the
highest of the 5 treatments and lowest in group C. There was
no difference in the LW of groups G and G', indicating that
the minerals added to diet G had no effect on liver weight.
The same situation was also found in groups A and A'.

In the Phase I and III studies, LW were larger in the
carp-eating groups than in the lab chow control, indicating
that the fish diets used in these two studies could cause
liver enlargement. The causative factors within the fish
diets are still under investigation. On the other hand, the
LW of F-O dams in group G were consistently larger than those
in group A in both Phase I and III, suggesting that some
component of the Great Lakes carp, other than in the Arkansas
farm-raised carp may be responsible for this additive liver
enlargement in the fish-eating mice.

However, in the F-1 males of the Phase I study, there

110

were no differences in the LW between groups A and G (Figure
4). These findings in the LW between groups A and G were also
in agreement with the observations on the F-1 females (Figure
17), F-2 males (Figure 18), and F-2 females (Figure 22) in
Phase III. There are two possible explanation for this
inconsistency between F-0 and other generations. First, the
variation of the BW in F-0 dams among treatment groups was
much less than that in other generations. Although data
expressed as LW/BW ratio reduced the variations caused by the
BW to some extent, it was not perfect enough. When the mice
reached 10 weeks of age, body fat accumulations were found in
groups C, G and G', but not in groups A and A'. The body fat
would decrease the LW/BW ratio by increasing the BW and thus,
diminishing the differences between groups A and G. The
reason why groups A and A' had less body fat storage is
unknown. Second, F-O dams were exposed to treatment diets
right after parturition and through lactation. The different
hormone, glucose, and immunoglobulin concentrations, and liver
metabolism in gestation and lactation could intense the liver
response.

Lower BW in groups A, A' and G' was observed in the F-1
(Figure 12) and F-2 males (Appendix 24) during this phase of
the study. In the F-1 males, the BW of groups A and G' were
similar and lower than those of group G before 10 weeks of
age, indicating that the minerals (Ca, P, Fe and Mn) added to

diet G' played some role in decreasing the BW. However, this

3‘.
Tl
( 2
fL

11)

a1
Hc
we

an

re
811

Ho

31

111

finding was only observed before 10 weeks of age. This could
also be explained by the body fat deposition. Thus, group G'
began showing larger BW levels than group A after 10 weeks of
age. The lowest BW were observed consistently in group A'.
This may suggest that the extra fish oil supplementation
(2.8%) and 100 ppm zinc added in diet.A' may even lower the BW
further.

Decreasing thymus weight in polyhalogenated aromatic
hydrocarbons (PCBs, PBBs, PCDDs, PCDFs, etc.)-treated animals
has been reported by others (Faith and Moore, 1977; Vos et
a1., 1980; Poland and Kuntson, 1982; Vos and Luster, 1989).
However, I did not observe any constant change in the thymus
weight, nor did I observe any differences in testis weight
among the treatments.

Lower liver Vit A and E concentrations in fish-eating
groups, similar to those found in the F-0 dam (Figure 2) and
F-l male mice (Figure 6) in Phase I, were also observed in the
F-1 males (Figure 14), F-2 males (Figure 19) and F-2 females
(Figure 23) during this phase of study. According to the
Phase II study, oxidizing agents such as PUFA may be
responsible for lowering the liver Vit A and E concentrations.
Supplemental nutrients did not interfere with this event.
However, the total liver Vit A and E concentrations in group
C during this phase were much lower than in Phase I. This
alteration may be due to the different mouse strains or other

unknown factors.

112

The results of caudal epididymal sperm concentrations and
motion parameters of F-l and F-2 males in Phase III showed no
differences among the 5 groups at all ages measured. There
was also no difference in the caudal epididymal sperm
fertilization ability in vitro. These findings were in
agreement with the Phase I study results. Therefore, it may
be concluded that eating Great Lakes carp has no effect on
caudal epididymal sperm reserves, production, motility, and in
vitro fertilization ability in mice.

Smaller litter sizes at weaning were observed in groups
A' and G in the F-1 males of this phase. The small litter
size of group A' at weaning was obviously due to the low
survival rate of the F-2 pups (Figure 16). The lower survival
index at day 21 in group A' when compared to that in group A
suggested that extra fish oil (2.8%) and/or 100 ppm zinc may
have some impact on either lactating dams, growing pups or
both. Smaller litter size of group G at weaning was mainly
due to the smaller litter size at birth. (Although there were
no significant differences in litter size at birth, group G
had about 2 pups less than the control groups (7.9 v.s. 9.8).
The consequences of smaller litter size in group G was
enhanced by a slightly decreased pup survival rate (83.5% i
14.5 vs. 89.6 t 14.6), hence a significant difference in
litter size at weaning was shown. The low-birth litter size
of F-1 males in group>G apparently was not caused by the Great

Lakes carp, because a decrease in litter size did not occur in

113
group G'.

In Phase I, the survival rate of the F-2 pups from both
carp-eating groups were lower than that of the lab chow fed
controls. The viability of F-l male pups in group A was only
12%. In this phase of the study, although F-2 pups in groups
A and A' still exhibited low survival rates (74.4 t 16.2 in A
and 64.6 i 28.0 in A' vs. 89.6 i 14.6 in C), neither was
different from the other groups (Appendix 21). The use of a
different mouse strain in Phase III could be one of the
reasons contributing to the difference. The different
survival rate of the group G F-l male pups showed in Phase I
and III could also be explained by the changed mouse strain.

The effects of the fish diets on reproductive performance
of female mice was also studied at this phase. In Phase I, F-
1 females of group G failed to respond to PMSG/hCG-inducing
superovulation at expected time (Figure 9) . As discussed
previously (pp. 84), the low superovulation rate and numbers
of oocyte collected from CBA/CAJ mice may not have been the
proper strain for this study; In this phase, there were no
differences in F-l and F-2 females' superovulation rates and
in vitro fertilization rates. Thus, ingestion of Great Lakes
carp had no effect on reproductive performance in vivo or in

vitro for F-l and F-2 female mice.

114

SUMMARY

Increased liver weight was observed in the F-0 dams of
both carp-eating groups with the effects being more pronounced
in the Saginaw Bay carp-fed group. I propose that
environmental contaminants within the Saginaw Bay carp, in
addition to some indigenous factors in both sources of carp,
were responsible for the higher liver weights in the Saginaw
Bay carp-fed mice. The increased liver weights, however, were
not significant in the F-1 and F-2 generations. The lack of
statistically significance in latter generations was probable
due to increased variations in both liver weights and body
weights. On the other hand, physiological changes such as
hormone, immunoglobulin concentrations, and liver metabolism
during gestation and lactation may have enhanced the liver
response of F50 dams to the fish diet and the Great Lakes
contaminants.

Among all treatment groups, the lowest body weight was
observed in the Arkansas carp-fed groups. Adding minerals
(Ca, P, Fe, and Mn) to the diet composed of 60% Saginaw Bay
carp reduced the body weights of Saginaw Bay carp-fed mice to
the weights observed in Arkansas carp-fed mice. Decreased
body weight in Arkansas carp-fed mice may have been caused by
the high mineral contents in the Arkansas carp diets.

Low hepatic vitamin A.and E was observed consistently in

both fish-eating groups. Oxidizing agents such as fish oil

115

may be responsible for these effects. Although decreasing
thymus‘weight.ingpolyhalogenated.aromatic.hydrocarbon-treated
animals was repeatedly reported by others, I did not observe
any consistent change on thymus weight, nor did I observe any
significant difference in the testis weights among treatments.

From this study, I demonstrated that the diet containing
60% Saginaw’ Bay carp had no adverse effects on caudal
epididymal sperm reserves and motility and in vitro
fertilizing ability of sperm from the treated mice. No
differences in litter size and. offspring ‘viability ‘were
observed in all three generations. No differences in
superovulation rates and in vitro fertilizing ability of F-1
and F-2 females suggest that ingestion of Saginaw'Bay carp had
no any adverse effects on the reproductive performance of

female mice in vitro.

APPENDICES

116

APPENDIX 1.

_
Table 7. Organochlorinated Pesticides and PCBs in Fish

 

 

 

 

Oil.
Pesticide Concentration (ppm)
PCBS <IDL
a-BHC <IDL
B-BHC <IDL
Lindane <IDL
Heptachlor <IDL
Heptachlor Epoxide <IDL
Aldrin <IDL
Technical Chlordane 0.17
p,p'-DDE 0.05
o,p'-DDD 0.02
p,p'-DDD 0.02
o,p'-DDT <IDL
p,p'-DDT <IDL
Methoxychlor <IDL
Dieldrin 0.05
Endrin <IDL
Malathion <IDL
Toxaphene <IDL
Source:

The pesticides and PCBs analysis were performed by Thionville
Laboratories, Inc. , New Orleans, LA. PCBs and pesticides that
were not detected are labeled <IDL. The minimum detection
level for PCBs and toxaphene was 0.5 ppm; the minimum
detection level for the other pesticides was 0.02 ppm.

APPENDIX 2.

 

Table 8. Nutrient Requirements of Micefl
—

 

 

 

Minerals Requirement Vitamins Requirement
Eifzgzmlnfizfllfi 0.4 Vitamin A 500.0
Calcium ( IU/kg)
Magnesium 0.05 Vitamin D 150.0
(IO/kg)
Phosphorus 0.4 Vitamin E 20.0
(IU/kg)
Potassium 0.2 Vitamin K1 3.0
(mg/kg)
nigzg;m1ne;g1§ Biotin 0.2
11091391 (1119/ kg)
Chromium 2.0 Choline 600.0
Copper 4.5 Folacin 0.5
Iodine 0.25 Niacin 10.0
Iron 25.0 Pantothenate 10.0
Manganese 45.0 Riboflavin A 7.0
Zinc 30.0 Thiamin 5.0
Vitamin B6 1.0
Crude protein 18.0%' Crude fat 11.0%
Crude fiber 3.0% Ash 6.5%
Source:

‘ NRC. 1978. Nutrient Requirements of Laboratory Animals.

In

118

APPENDIX 3.

Definition of sperm motion parameters:

Number of sperm recovered from epididymides which were
poked 30 times and cultured for 30 min within 1 ml of medium.

W
The percentage of sperm that travel more than 20
microns/sec.

e 0
Average of the distance traveled by motile sperm in
microns/sec.

1.1mm

A.measure of the straightness of a sperm track on.a scale
of 0 to 10: 10 indicates a perfectly straight line and 0
indicates a circular track.

a e ' e

A measure of the displacement of the sperm head from a
computer-calculated curval mean of its track in microns/sec.
In some species, high A.L.H. appears to be linked with the
process of capacitation.

W

The beat/cross frequency (BCF) is reported as the number
of beats (or crosses) per second. Every time the sperm cell
crosses the computer-calculated curval mean, the computer
counts that crossing as one heat. In some studies BCF appears
to be reversely related to ALH.

119

APPENDIX 3. (cont'd)

 

Table 9. Operating Setting of the CellSoft
Videomicrographic Instrument Used for Computer-
Assisted Semen Analysis'

 

 

 

Parameter” Value
Number of frames per second 30
Number of frames to analyze 15
Maximum velocity (um/s) 300
Minimum track points for calculation of motility 2
Minimum track points for calculation of velocity 3
Minimum track points for calculation of amplitude

of lateral head displacement 7
Minimum velocity for calculation of amplitude of

lateral head displacement (um/s) 20
Minimum linearity for calculation of amplitude of

lateral head displacement 3.5
Source:

‘ CellSoft; CRYO Resources, New York, NY.
” Ginsburg et a1. (1990).

.120

IUEPIIflIIXIII.

—
Table 10. Body and liver weights of F-O lactating

 

mice“.
_
Group C Group A Group G

Body weight

Grams 26.16 i 1.84 26.23 i 2.33 26.56 i 2.13

( % )° (100.0) (100.3) (101.5)
Liver weight

Grams 1.84 :t 0.29 2.269: 0.31 2.53“: 0.24

( % ) (100.0) (123.0) (140.9)
Sample size 14 14 16

 

' F-0 females were provided with treatment diets immediately
after delivery and through lactation.

Data presented as mean 1 standard deviation.

° \ refers to percent of Group C.

Significantly different from Group C, p<0.05.
Significantly different from Group A, p<0.05.

I’

I.

JAPPTHHIEX 5.

121

 

Table 11.

   

Group A

Liver vitamin A and E concentrations of
lactating_Ff0_fem§lesT“.

 

Group C Group G
Retinol 309.0 105.0 91.5
( t )‘I (100.0) (34.0) (29.6)
Retinyl Palmitate 5678.5 2707.5 3104.5
( % ) (100.0) (47.7) (54.7)
Total Vitamin A 5987.5 2812.5 3196.0
( % ) (100.0) (47.0) (53.4)
Vitamin E 46.81 6.92 9.74
( % ) (100.0) (14.8) (20.3)
Sample size 1 1 1

 

' Vitamin A and E analyses were determined by Dr. Stowe's lab.,
the Animal Health Diagnostic Lab., Nutrition Section, MSU.
’ F-O females were provided with treatment diets immediately

after delivery and through lactation.

° Data expressed as pg/g of dry liver weight.
‘ % refers to percent of Group C.

122

APPENDIX 6.

 

Table 12. Body weights of F-1 male mice from birth to 23
°f

Body weight (Grams)

     

 

Age Group C Group A Groqu

Day 1 1.28 i 0.10 1.28 i 0.11 1.27 i 0.10
Day 4 1.98 i 0.35 1.80 i 0.36 1.88 i 0.25
Week 1 3.33 i 0.62 3.18 i 0.49 3.15 i 0.32
Week 2 6.83 i 0.50 5.92 t 0.55 6.46 i 0.55
Week 3 8.73 i 0.78 7.30 i 0.96 8.26 i 0.61
Week 4 13.70 i 1.22 10.88 i 1.26 12.98 i 1.27
Week 5 18.47 i 1.48 15.72 i 1.65 17.38 i 1.14
Week 6 21.00 i 1.41 18.01 i 1.46 19.48 t 1.47
Week 7 22.76 t 1.63 20.02 i 1.20 21.52 t 1.32
Week 8 24.34 t 2.13 21.60 i 1.36 23.22 t 1.09
Week 10 26.03 i 1.84 23.71 i 1.44 25.98 i 1.62
Week 14 31.24 i 0.35 26.20 i 1.70 33.62 i 2.13
Week 15 30.23 i 2.76 27.58 i 1.83 30.51 i 0.78
Week 21 36.54 i 0.77 29.33 i 2.48 32.28 t 1.34
Week 23 39.76 i 1.26 31.43 t 0.27 31.44 i 2.04

' Data presented as mean 1 standard deviation.

JAPPIBHIEX 7.

Table 13. Liver and testis weights of F-l malesh

 

 

Group C Group A Group G

W
Liver weight

Grams 1.42 t 0.22 1.46 t 0.10 1.50 t 0.10

( e )b (100.0) (102.8) (105.6)
Body weight

Grams 23.14 t 0.99 19.65 t 1.60 19.92 t 1.87

( e ) (100.0) (84.9) (86.1)
Liver/body
weight ratio 0.061 0.074‘ 0.075°

( t ) (100.0) (121.3) (123.0)
Testis weight

Grams 0.12 t 0.01 0.11 t 0.01 0.11 t 0.01

( % ) (100.0) (91.7) (91.7)
Sample size 3 3 3
We:
Liver weight

Grams 1.52 i 0.14 1.49 t 0.08 1.63 t 0.08

( t ) (100.0) (98.0) (107.2)
Body weight

Grams 24.89 t 2.00 22.34 i 0.26 24.25 t 0.62

( t ) (100.0) (89.8) (97.4)
Liver/body
weight ratio 0.061 0.067c 0.067c

( t ) (100.0) (109.8) (109.8)
Testis weight

Grams 0.12 t 0.01 0.12 t 0.01 0.12 t 0.01

( a ) (100.0) (100.0) (100.0)
Sample size 3 3 3

124

Table 13 (cont'd)

1§_ussks_2f_ase
Liver weight
Grams 1.81 :t 0.15 1.47‘ 10.16 1.86 t 0.11
( t ) (100.0) (81.2) (102.8)
Body weight
Grams 34-50 1 2-13 26.89‘I t 1.03 31.28 1: 1.86
( A ) (100-0) (77.7) (90.4)
Liver/body
weight ratio 0-052 0.054 0.059
( t ) (100-0) (103.8) (113.5)
Testis weight
( t ) (100-0) (92.3) (100.0)
Sample size 3 3 3

Data presented as mean 1 standard deviation.

8 refers to percent of Group C.

Significantly different from Group C, p<0.05.
Significantly different from Groups C and G, p<0.05.

‘0'.

.125

.APTIHHIEX 8.

Table 14. Liver vitamin A and E concentrations in

F-1 males“.
m

 

 

Group C GroupA Group G
(.12_£§§B§_QI_§Q§
Retinol 147 86 109
( t )c (100.0) (58.5) (74.1)
Retinyl Palmitate 7754 1906 2979
( t ) (100.0) (24.6) (38.4)
Total Vitamin A 7901 1992 3088
( t ) (100.0) (25.2) (39.1)
Vitamin E 77.79 9.26 12.96
( t ) (100.0) (11.9) (16.7)
Sample size 1 1 1
12.xeeks_ef_ess
Retinol 273 182 166
( % ) (100.0) (66.7) (60.8)
Retinyl Palmitate 3781 1432 2004
( % ) (100.0) (37.9) (53.0)
Total Vitamin A 4054 1614 2170
( % ) (100.0) (39.8) (53.5)
Vitamin E 36.74 6.90 13.77
( % ) (100.0) (18.8) (37.5)
Sample size 1 1 1

' Vitamin A and E analyses were determined by Dr. Stowe's lab.,
the Animal Health Diagnostic Lab., Nutrition Section, MSU.

” Data expressed as pg/g of dry liver weight.

° t refers to percent of Group C.

     

     
   

126
JAPPTBUIEX 9.

Table 15. Caudal epididymal sperm concentrations of F-1
males at 6, 7, 8, 15, 23, 32, and 34 weeks of

Sperm concentration (million/ml)

 

 

Age (weeks) Group C Group A Group G
6 0.97 i 0.15 0.63 i 0.15 1.70 i 0.17
7 6.47 i 3.18 3.63 i 0.93 3.47 i 1.50
8 12.44 i 0.95 10.59 i 0.87 13.76 i 5.43
15 11.42 i 3.78 15.93 i 1.35 13.63 i 2.18
23 11.74 i 0.80 15.00 t 8.48 7.06 i 0.46
32 8.61 i 2.17 15.41 i 1.50 15.08 i 0.90
34 -L 10.67 i 2.26 10.26 t 4.28 8.70 i 0.57

 

G'.

 

Data presented as mean t standard deviation.

Sperm suspension was collected as followings: both of the epididymides
were placed in a Falcon Organ Tissue Culture Dish (60 x 15 mm, Becton
Dickinson Labware) and poked with a 25 G needle to release the sperm
into 1.0 ml BMOC-3 medium. After a 30-min incubation at 37°C, 5% CO2 in
air and 100% humidity, 20 pl of the sperm suspensions were placed on a
CellSoft 20p-chamber and examined under a CellSoft Video-micrographic
Computer-Assisted Semen Analyzer (CRYO Resources Inc., New York, NY). A
minimum of 100 sperm cells were analyzed for each sample.

APPENDIX 10 .

—
Table 16. Epididymal sperm motion quality of.F-1 males“h

 

Parameters‘ Group C Group A Group G
§_ueske_ef_ase

t motile sperm 74.07 t 3.84 50.90 1 27.27 78.70 t 5.48
Velocity(pm/s) 172.07 1 24.14 98.60 1 11.32 230.16 1 69.71
Linearity 6.65 t 2.07 4.30 t 1.95 5.22 t 1.11
A.L.H.‘ (pm/s) 4.44 1 3.37 5.77 1 6.36 1.89 1 1.06
8./c. ya: (Hz) 12.81 1 2.21 12.58 1 1.76 16.07 1 8.13
Sample size 3 3 3
Z_seehe_ef_ase

% motile sperm 74.30 t 6.32 60.50 t 6.33 45.47 t 5.82
v816c11y(pm/8) 170.13 1 8.06 160.27 1 16.79 143.56 1 44.05
Linearity 4.55 t 0.25 4.48 t 0.60 4.33 t 1.91
A.L.H. (pm/s) 5.08 t 2.31 3.56 t 0.84 2.71 t 0.39
B./C. r. (Hz) 11.59 1 1.05 13.13 1 2.10 10.65 1 1.34
Sample size 3 3 3
15.2:eke_ef_ase

t motile sperm 81.82 t 4.35 81.68 t 3.94 82.15 t 5.69
velocity(pm/s) 220.88 1 20.16 228.17 1 24.43 226.60 1 20.13
Linearity 4.78 t 0.91 4.45 t 0.32 4.83 t 0.50
A.L.H. (pm/s) 7.72 1 1.69 10.24 1 3.51 9.52 1 0.33
s./c. r. (Hz) 12.74 1 0.81 12.43 1 1.23 9.46 1 2.72
S:Eple size 3 3 3

' For sperm motion quality analysis, 20 pl of the sperm suspension were
placed on a CellSoft 20 p chamber and examined under a CellSoft Video-
micrographic Computer-Assisted Semen Analyzer (CRYO Resources Inc.,

New York, NY).
sample.

A minimum of 100 sperm cells were analyzed for each
Sperm suspension was collected from epididymides and had been

incubated in BMOC-3 medium for one half-hour at 37°C, 5% CO2 in air, 100%

humidity.

‘ Data presented as mean 1 standard deviation.

No statistical significance was observed among the groups, p > 0.05.

0 B0

See Appendix 3 for definition of parameters.
A.L.H. represents amplitude of lateral head displacement.
B./C. F. represents beat/cross frequency.

128

IUEPlflflDIIIIL1.

 

 

No. of oocyte tested 22.5 t 2.1 33.3 t 11.4 27.0 t 7.8
IVF‘ rate ( t ) 86.4 1 7.5 73.6 1 9.65 83.6 1 14.57
Sample size 3 3 3
MW
Fecundity' 7/7 7/7 7/7
Viability‘ 94.3 t 10.2 12.2” 1 32.4 44.6II :1: 43.2
Sample size 7 7 7
—

 

' Data presented as mean 1 standard deviation.

‘ F-l males were 52 days old (7.5 weeks).

° To determine the in vitro fertilizing ability, sperm was used to
inseminate oocytes collected by superovulation from previously non-
treated femalea. The sperm suspension was collected from treated male
epididymides and had been incubated in BMOC-3 medium for one half-hour
at 37°C, 5% co, in air, 100$ humidity. Oocytes were inseminated with 50
pl of sperm suspension (1-3 x 105 and were co-incubated at 37°C, 5% CO2
in air, 100% humidity for 24 hours before examination of fertilization.

‘ IVF refers to in vitro fertilization.

' For breeding, 7 F-l males were each paired with a previously non-treated

mature female. The females began to receive the same treatment diets as

the»males when they were housed together for 5 days. These females were
then housed individually and continuously provided with treatment diets
through gestation and lactation.

Fecundity refers to number of females giving birth/number of females

bred.

' Viability refers to percent of F-2 mice that survived to the age of 7
days. '

“ Significantly different from Group C, p<0.05.

‘

129

IUEPlnflDIIIJLZ.

 

     

 

 

Table 18. Re roductive performance of F-l females.

Parameters Group C Group A Group G

W

o c '

Superovulation rateb 7/8 4/6 0/7

% females ovulated 87 67' O“

No. of oocyte ovulated 9.6 1 7.9 9.8 1 3.3 0

% oocytes fertilizedc 62.9 1 19.1 59.8 1 22.8 ---

Sample size 8 6 7

W111:

Fecundity‘ 7/7 3/6 4/7

t of Group C 100 50.0' 57.1'

Litter size‘ 7.0 1 2.6 6.3 1 1.5 6.0 1 0.82

Sample size 7 6 7
— _

 

 

 

‘ To determine the oocyte in vitro fertilizing ability, oocytes were
inseminated with sperm from previously non-treated males. Oocytes
were collected from PMSG/hCG superovulated treated females. The ages
were 5 and 10 weeks old. The sperm suspension was collected from mature
male epididymides and had been incubated in BMOC-B medium for one half-
hour at 37°C, 5% cxx in air, 1008 humidity. Oocytes were inseminated
with 50pl of sperm suspension (1-3 x 105 and.were co-incubated at 37°C,
5% CO2 in air, 100% humidity for 24 hours before examination of
fertilization.

Superovulation rate refers to number of females ovulated/number of
females superovulated with PMSG/hCG. ‘

‘ Data presented as mean 1 standard deviation.

For breeding, F-l females were paired each with a previously non-treated
mature male. The males have never received any treatment diet even
during the 5-day breeding period.

Fecundity refers to number of females carrying fetus 10 days after
breeding period/number of females bred.

' Significantly different from Group C, p<0.05.

‘ Significantly different from Groups C and A, p<0.05.

b

I.

APPENDIX 13.

 

 

Table 19. Liver vitamin A and E concentrations of F-l
males after high dietary vitamin E
Supplementation“c .

Group C Group A+ Group G+

Retinol 309.0 629.0 419.0

( t )‘ (100.0) (203.6) (135.6)

Retinyl Palmitate 6133.0 3785.0 3382.0

( % ) (100.0) (61.7) (55.1)
Total Vitamin A 6442.0 4414.0 3801.0
( % ) (100.0) (68.5) (59.0)
Vitamin E 19.32 76.19 112.89
( % ) (100.0) (394.4) (584.3)
Retinol 130 544 162
( t ) (100.0) (418.5) (124.6)
Retinyl Palmitate 3797 5522 3760
( % ) (100.0) (145.4) (99.0)
Total Vitamin A 3927 6066 3930
( % ) (100.0) (154.5) (100.1)
Vitamin E 40.79 97.72 129.45
( t ) (100.0) (239.6) (313.4)
1§§_Q§Y§
287 585 464
Retinol (100.0) (203.8) (161.7)
( % ) 2700 4855 3924
Retinyl Palmitate (100.0) (179.8) (145.3)
( t ) 2987 5440 4388
Total Vitamin A (100.0) (182.1) (146.9)
( t ) 30.57 77.30 98.30
Vitamin E (100.0) (252.9) (321.6)
( % )

 

 

Vitamin A and E analyses were determined by Dr. Stowe's lab.,

the Animal Health Diagnostic Lab., Nutrition Section, MSU.

‘ Mice were fed high Vit E diets (A+ or G+) beginning at 81 days of age.
From birth to 81 days of age, mice were fed treatment diets A or
G, respective.

° Data expressed as pg/g of dry liver weight.

8 refers to percent of Group C.

131

APPENDIX 14.

Table 20. Body and liver weights of F-l males after
high dietary vitamin E Supplementationfl.

 

Group C Group A+ Group G+
Body weight
Grams 37.40 27.43 30.62
( t )“ (100.0) (73.34) (81.87)
Liver weight
Grams 1.837 1.429 1.540
( t ) (100.0) (77.79) (83.83)
Liver/body
weight ratio 0.049 0.052 0.050
( t ) (100.0) (106.12) (102.04)
Body weight
Grams 38.32 26.96 33.72
( % ) (100.0) (70.35) (88.00)
Liver weight
Grams 1.854 1.156 1.856
( t ) (100.0) (62.35) (100.11)
Liver/body
weight ratio 0.048 0.043 0.055
( t ) (100.0) (89.58) (114.58)
1§§_QQ¥§
supplement
Body weight 42.61 36.90 37.76
Grams (100.0) (86.60) (88.62)
( % )
Liver weight 2.181 1.723 1.949
Grams (100.0) (79.00) (89.36)
( % )
Liver/body 0.051 0.047 0.052
weight ratio (100.0) (92.16) (101.96)
( % )
1 1 1

Sample size

 

' Mice were fed high Vit E diets (A+ or 6+) beginning at 81 days of age.

From birth to 81 days of age, mice were fed treatment diets A or

G, respective .

” % refers to percent of Group C.

132

JKPPlIflIIIIJLS.

Table 21. Body and liver weights of F-O lactating females“h

Group C Group A Group A' Group G Group G'
Liver weight

Grams 1.64 1.79 1.95 2.03 1.95

1 0.30 1 0.25 1 0.19 1 0.26 1 0.27

( 8 )° (100.0) .(109.1) (118.9) (123.8) (118.9)
Body weight

Grams 24.75 24.06 25.00 25.12 25.08

1 1.97 1 1.30 1 1.38 1 0.60 1 2.26

( e ) (100.0) (97.2) (101.0) (101.5) (101.3)

Liver weight
[Body weight

Ratio 0.066 0.074 0.078‘l 0.081‘ 0.078‘
1 0.01 1 0.01 1 0.00 1 0.01 1 0.00
( t ) (100.0) (112.1) (118.2) (122.7) (118.2)
Sample size 5 6 6 7 7

' F-0 females were provided with treatment diets inanediately after delivery
and through lactation.

‘ Data presented as mean 1 standard deviation.

° 1 refers to percent of Group C.

‘ Significantly different from Groups C and A, p<0.05.

133

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134

APPENDIX 17.

 

Table 23. Liver, thymus and testis weights of F-l malesh
Grog C Group A Group A' Group G Group G '
Liver weight
Grams 1.29 1.28 1.49“ 1.74“ 1.55“
1 0.14 1 0.13 1 0.22 1 0.15 1 0.09
( a )" (100.0) (99.2) (115.5) (134.9) (120.2)
Liver/body
weight ratio 0.053 0.071 0.064 0.055 0.063
( t ) (100.0) (140.0) (120.8) (103.8) (118.9)
Thymus weight
Milligrams 38.1 32.9 36.4 35.4 37.9
1 0.8 1 2.5 1 4.6 1 4.5 1 9.2
( s ) (100.0) (86.4) (95.5) (92.9) (99.5)
Testis weight
Grams 0.19 0.20 0.19 0.21 0.17
1 0.01 1 0.03 1 0.01 1 0.01 1 0.04
( t ) (100.0) (105.3) (100.0) (110.5) (89.5)
Sample size 3 3 3 3 3
W
392
Liver weight
Grams 1.42 1.52 1.46 1.85‘ 1.57
1 0.33 1 0.29 1 0.32 1 0.29 t 0.14
( t ) (100.0) (107.0) (102.8) (130.3) (110.6)
Liver/body
weight ratio 0.051 0.059 0.055 0.066 0.060
( t ) (100.0) (115.7) (107.8) (129.1) (117.6)
Thymus weight
Milligrams 41.0 25.7‘ 28.9’ 29.3‘ 28.5‘
1 7.4 1 5.0 1 6.0 1 9.3 t 5.0
( t ) (100.0) (62.7) (70.5) (71.5) (69.5)
Testis weight '
Grams 0.22 0.20 0.22 0.23 0.17'
1 0.02 1 0.04 1 0.03 1 0.02 1 0.05
( t ) (100.0) (90.9) (100.0) (104.5) (77.3)
Sample size 3 3 3 3 ___ 3

 

 

 

' Data presented as mean 1 standard deviation.

8 refers to percent of Group C.

Significantly different from Groups C and A, p<0.05.
Significantly different from Groups C, A and A', p<0.05.
Significantly different from Group C, p<0.05.
Significantly different from Groups C, A' and G, p<0.05.

I'

name

135

IUEPIUUIEX 11!.

 

Table 24. Liver vitamin A and E concentrations in F-l males““.

Group C Group A GrogA' Group G Group G'

Retinol 247 224.5 252.5 333.5 177
( t )‘I (100.0) (90.9) (102.2) (135.0) (71.7)

Retinyl

Palmitate 941 695.5 500 688 388
( t ) (100.0) (73.9) (53.1) (73.1) (41.2)

Total Vitamin A 1188 920 752.5 1021.5 565
( t ) (100.0) (77.4) (63.3) (86.0) (47.6)

Vitamin E 15.91 14.25 13.18 9.48 9.97
( t ) (100.0) (89.6) (82.8) (59.6) (62.7)

Sample size 2 2 2 2 2

' Vitamin A and E analyses were determined by Dr. Stowe's lab.,
the Animal Health Diagnostic Lab., Nutrition Section, MSU.

‘ F-l males were 8 weeks of age.

° Data expressed as pg/g of dry liver weight.

‘ 8 refers to percent of Group C.

136
APPENDIX 19.

 

Table 25' Caudal Epididymal Sperm concentrations of F-1
_ -_ 1 males at 8 and 11 weeks of agefin

   

 

Sperm concentration (million/ml)

 

 

Age Group C Grofiup A Group A' Group G Group G'

8 weeks 15.32 12.70 17.51 16.16 9.90
i 0.15 i 5.29 i 2.58 i 5.20 i 4.15

Sample

size 5 5 5 5 5

11 weeks 19.49 16.20 25.49 20.34 16.21
16.06 t 8.12 i 4.88 i 5.97 112.53

Sample 8 8 8 8 8

size

 

' Data presented as mean 1 standard deviation.

” Sperm suspension was collected as followings: both of the epididymides
were placed in a Falcon Organ Tissue Culture Dish (60 x 15 mm, Becton
Dickinson Labware) and poked with a 25 G needle to release the sperm
into 1.0 ml BMOC-3 medium. After a 30-min incubation at 37°C, 5% CO2 .in
air and 100% humidity, 20 pl of the sperm suspensions were placed on a
CellSoft 20p-chamber and examined under a CellSoft Video-micrographic

Computer-Assisted Semen Analyzer (CRYO Resources Inc., New York, NY).
A minimum of 100 sperm cells were analyzed for each sample.

137

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APPENDIX 26.

 

144

Table 32. Liver vitaminh and Sconcentrations in F-2 males”“.A

 

 

 

Group C Grouf A Grouf 3' Gran? G Group G'
Retinol 994.3 199.0 134.0 502.7 423.3
( s )‘ (100.0) (20.0) (13.5) (50.6) (42.6)
Retinyl Palmitate 216.7 87.5 8.2 126.3 46.7
( t ) (100.0) (40.4) (3.8) (58.3) (21.6)
Total Vitamin A 1211.0 286.5 163.7 629.0 470.0
( t ) (100.0) (23.7) (13.5) (51.9) (38.8)
Vitamin E 13.02 20.33 7.52 5.27 2.79
( t ) (100.0) (156.1) (57.8) (40.5) (21.4)
Samgle size 1 1 1 l 1

' Vitamin A and 8 analyses were determined by Dr. Stowe's lab.,
the Animal Health Diagnostic Lab., Nutrition Section, MSU.
Data expressed as pg/g of dry liver weight.

° P-2 males were 8 weeks old.

‘ 8 refers to percent of Group c.

f

145

APPENDIX 27.

Table 33. Caudal epididymal sperm concentrations of F-2
males at 6, 7, and_8 weeks of age“. ‘

     

Sperm concentration (million/ml)

 

 

Age Group C Group A Group A ' Group G Group G '
6 weeks 2.35 1.75 4.01 2.73 4.34

i 1.28 i 0.48 i 2.05 i 0.97 t 0.96
7 weeks 7.69 6.74 7.16 9.54 6.36

i 4.79 i 2.92 i 3.65 i 4.64 i 1.70
8 weeks 9.60 6.15 6.03 5.35 6.76

i 0.94 i 1.36 t 3.64 i 1.37 i 4.62
Sample 3 3 3 3 3
size

 

' Data presented as mean 1 standard deviation.

5 Sperm suspension was collected as followings: both of the epididymides
were placed in a Falcon Organ Tissue Culture Dish (60 x 15 mm, Becton
Dickinson Labware) and poked with a 25 G needle to release the sperm
into 1.0 ml Buoc-a medium. .After a 30-min incubation at 37°C, 5% 00,13:
air and 100% humidity, 20 pl of the sperm suspensions were placed on a
CellSoft 20u-chamber and examined under a CellSoft Video-micrographic
Computer-Assisted Semen Analyzer (CRYO Resources Inc., New York, NY).
A minimum of 100 sperm cells were analyzed for each sample.

146

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Table 37. Liver vitamin A and 8 concentrations in F-2
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Group C

 

Group.h GroupiA' Grogp G Group G'

Retinol 784.0 328.3 155.0 540.0 300.3
( t )‘ (100.0) (41.9) (19.8) (68.9) (38.3)
Retinyl Palmitate 99.7 18.0 9.3 36.3 29.0
( t ) (100.0) (18.1) (9.3) (36.4) (29.1)
Total Vitamin A 883.7 346.3 164.3 576.3 329.3
( s ) (100.0) (39.2) (18.6) (65.2) (37.3)
Vitamin E 22.56 3.43 3.99 8.49 5.98
( t ) (100.0) (15.2) (17.7) (37.6) (26.5)

”8ample size 3 3 3 3 3

   

 

Vitamin A and E analyses were determined by Dr. Stowe's lab.,
the Animal Health Diagnostic Lab., Nutrition Section, MSU.
Data expressed as pg/g of dry liver weight.

P-2 males were 8 weeks old.

8 refers to percent of Group C.

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