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This is to certify that the

thesis entitled

STUDY OF THE DEVELOPMENTAL STAGES OF BLUEGILL
(LEPOMIS MACROCHIRUS) EGGS USING SELECTED
HISTOLOGICAL TECHNIQUES

 

presented by

Yarisa Montes—Brunner

has been accepted towards fulfillment
of the requirements for

Master of Science Fisheries and Wildlife

degree in

 

 

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= . r professor

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Date §A7€/9 2’

0-7639 MS U i: an Affirmative Action/Equal Opportunity Institution

 

PLACE ll RETURN BOX to move this checkout from your record.
TO AVOID FINES return on or before duo duo.

DATE DUE DATE DUE DATE DUE

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

MSU I. An Alfirmdivo Action/Equal Opportunity Intuition
cWM‘I

STUDY OF THE DEVELOPMENTAL STAGES OF BLUEGILL
(LEPOMIS MACROCHIRUS) EGGS
USING SELECTED HISTOLOGICAL TECHNIQUES

BY

Yarisa Montes-Brunner

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

MASTER OF SCIENCE

Department of Fisheries and Wildlife

1992

ABSTRACT

STUDY OF THE DEVELOPMENTAL STAGES OF BLUEGILL (LEPOMIS
MACROCHIRUS) EGGS USING SELECTED HISTOLOGICAL TECHNIQUES

BY

Yarisa Montes-Brunner

Bluegill are important gamefish for stocking farm ponds
and lakes; but, their high reproduction rates can cause
stunting. Polyploid induction may be used for population
control. The objectives of this study were: 1) to develop
histological techniques to monitor egg development to
determine the critical time for the production of polyploid
bluegill and 2) to observe differences between the
development of normal eggs and eggs that received polyploid
induction treatments.

Standard histological techniques and laser scanning
confocal microscopy (LSCM) were not effective in monitoring
chromosomal changes in fertilized eggs. However, LSCM could
be used to observe developmental changes in the eggs.
Blastodiscs were not observed to divide in eggs that
received induction treatments during the first 60 minutes
postfertilization. Polyploid induction treatments may have
produced a shock to the cell that required a recuperation
period, cell division was inhibited in polyploid eggs, or

treatments had a lethal effect on the eggs.

This thesis is dedicated to my loving husband Bryan R.
Brunner and to my little boy Bryan José for all the reasons

that only they know.

iii

ACKNOWLEDGEMENTS

I thank the members of my committee, Dr. Donald
Garling, Dr. James Asher, and Dr. Niles Kevern for their
guidance. Special thanks to Dr. Garling for his continued
help and support. Special thanks also to Dr. Asher for the
use of his laboratory and histological equipment and Dr.
Whallon for assistance in using the laser scanning confocal
microscope.

Many fellow graduate students helped care for fish and
provided support. Special thanks go to Andy Westmaas, Paul
Wilbert, and Laurel Ramseyer.

I also wish to thank my parents, Jose and Lydia Montes,
whose moral support helped me through these years.

Finally, I would like to thank those individuals and
organizations who helped fund this research project.
Funding was provided by the citizens of the Chicago area
through their generous donations to the Howard Tanner
Fisheries Research Fund, the North Central Regional
Aquaculture Center, the Office of Urban Affairs, and the

Michigan Agricultural Experiment Station.

iv

TABLE OF CONTENTS

LIST OF TABLES . . . . . . . . . . . . . . . . . . . . . vi
LIST OF FIGURES . . . . . . . . . . . . . . . . . . . . Vii
KEY TO SCIENTIFIC NOMENCLATURE . . . . . . . . . . . . . iX
INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . 1

LITERATURE REVIEW . . . . . . . . . . . . . . . . . . . . 7

Control of Reproduction in Fishes . . . . . . . . 8
Use of Polyploidy for Control of Reproduction in
Fishes . . . . . . . . . . . . . . . . . . . 13

Naturally Occurring Polyploid in Fishes . . . . . . 17
General Description of Teleost Egg and Sperm . . . . 17
Fish Chromosomes: Numbers and Size . . . . . . . . 22
Techniques for Chromosome Observation in Fishes . . 24
Laser Scanning Confocal Microscope . . . . . . . . . 24
MATERIALS AND METHODS . . . . . . . . . . . . . . . . . . 27
RESULTS 0 O O O O O O O O O O O O O O O O O O O O O O O O 3 5
DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . 49
SUMMARY AND CONCLUSIONS . . . . . . . . . . . . . . . . . 57
APPENDIX A . . . . . . . . . . . . . . . . . . . . . . . 60

LIST OF REFERENCES . . . . . . . . . . . . . . . . . . . 67

LIST OF TABLES

Table Page
1. Techniques used to control overpopulation

in aquaculture and natural fish communities . . . . . . . 9
2. Naturally occurring polyploid fishes . . . . . . . . 18

3. Tissue preparation techniques for
chromosome observation in fishes . . . . . . . . . . . . 25

4. Composition for Bouin's and Farmer's fixatives
used to fix fertilized bluegill eggs . . . . . . . . . . 30

5. Dehydration and paraffin infiltration procedures for
bluegill eggs fixed at 50, 40 and 30 min

postfertilization . . . . . . . . . . . . . . . . . . . . 61
6. Paraffin imbedding of bluegill eggs . . . . . . . . . 62
7. Procedure for cutting paraffin blocks with microtome 64

8. Staining of slides prepared from paraffin blocks . . 65

9. Light microscope instructions (Kohler illumination) . 66

vi

LIST OF FIGURES

1. Bluegill egg showing the zona radiata (zr) and outer
membrane (cm), 30 minutes postfertilization . . . . . . . 20

2. Blastodisc (b) near the micropyle (m) of a normal
bluegill egg, 20 minutes postfertilzation . . . . . . . . 40

3. Perivitelline space (ps) between the egg membrane (em)
and yolk (y) of a normal bluegill egg, 20 minutes
postfertilization, enlarged giving way to protoplasmic
accumulation . . . . . . . . . . . . . . . . . . . . . . 42

4. View of the micropyle of a normal bluegill egg, 30

minutes postfertilization . . . . . . . . . . . . . . . . 42
5. Polar body (pb) being extruded from the nucleus (n) of
a bluegill egg, 35 minutes postfertilization, after triploid
induction treatment . . . . . . . . . . . . . . . . . . . 43
6. First mitotic division of normal bluegill egg, 35
minutes postfertilization at 229C, blastodisc is divided
into two blastomeres of equal size . . . . . . . . . . . 43
7. Seams in the cleavage furrow of a normal bluegill egg,
35 minutes postfertilization . . . . . . . . . . . . . . 44

8. Blastodisc with incomplete cleavage furrow (cf) from a

normal bluegill egg, 35 minutes postfertilization . . . . 44
9. Blastodisc showing two nuclei (n) in each blastomere of
a normal bluegill egg, 50 minutes postfertilization . . 45

10. Second mitotic division of a normal bluegill egg, 55
minutes postfertilization, resulting in four blastomeres of
equal size . . . . . . . . . . . . . . . . . . . . . . . 47

11. Bluegill egg that received a pressure shock of 8000
psi, triploid induction treatment, 45 minutes
postfertilization . . . . . . . . . . . . . . . . . . . . 47

12. Bluegill egg that received a cold shock of 5%:,

tetraploid induction treatment, 55 minutes
postfertilization . . . . . . . . . . . . . . . . . . . . 48

vii

13. Bluegill egg that received a pressure shock of 8000
psi, tetraploid induction treatment, 40 minutes
postfertilization . . . . . . . . . . . . . . . . .

viii

48

KEY TO SCIENTIFIC NOMENCLATURE

Common name
Atlantic salmon
Bluegill

Blue tilapia
Brook trout
California roach
Channel catfish
Chinook salmon
Common carp
Fathead minnow
Green sunfish
Largemouth bass
Nile tilapia

Rainbow trout

ix

Scientific name

Salmo salar

 

 

Lepomis macrochirus
Oreochromis aureus
Salvelinus fontinalis
Hesperoleucus symmetricus
l2£§l2£2§ 222§£2£2§
Oncorhynchus tshawytscha
Cyprinus gaggig
Pimephales promelas

Lepomis cyanellus
Micropterus salmoides

Oreochromis niloticus

Oncorhynchus mykiss

INTRODUCTION

The Centrarchid family is indigenous only to North
America and consists of 30 fish species. Several species
have been studied extensively by fishery biologists and
their systematics have been carefully considered (Roberts,
1964). However the family has received only scant attention
from cytologists (Roberts, 1964). The bluegill (Lepomis
macrochirus, Rafinesque) belongs to this family and is the
largest lepomine sunfish found in Michigan (Janssen, 1974).
It has a wide range of distribution and is found in lakes
and streams from western New York throughout the Great Lakes
region and Mississippi Valley to northern Mexico, and along
the Atlantic Coast all the way from New York to Florida
(Morgan, 1951). In Michigan it is found primarily in lakes
and reservoirs and is less abundant in the larger rivers;

Bluegill are the favorite panfish of North American
anglers and are the most frequently pursued gamefish in the
United States (Janssen, 1974). Because it easily adapts to
pond conditions, it is also one of the favorite fishes for
stocking farm ponds (Morgan, 1951). They may grow to 4
inches and reproduce in 1 year, and some may spawn more than

once during the summer (Morgan, 1951).

2

The high fecundity of bluegill is an important factor
in its selection as a forage fish for largemouth bass in
lakes and ponds (Morgan, 1951). In spite of their desirable
traits, their early sexual maturity and high rates of
reproduction can cause overpopulation problems and
negatively influence growth rate (Childers, 1967; Murnyak et
al. 1984). Once sexual maturity is reached, fish must
expend metabolic energy between somatic and reproductive
growth; this results in a reduction in growth rate (Murnyak
et al. 1984).

Frequently bluegills dominate the biomass of the lakes
and ponds they inhabit. Where they predominate numerically,
they are prone to stunting. Murnyak et al. (1984) defined
stunting as an exhibition of an individual growth rate below
the potential for the species. Some of the factors that can
cause stunting are high reproductive rates, low predation
rates and limited supply of food items. In the case of
bluegills in overcrowded situations, the cessation of growth
occurs when 3 to 5 inches in length is attained. Although
the average length for males is 5.76 inches and for females
6.62 inches, adult bluegill have the potential to grow to
lengths over 10 inches and a weight of over a pound (Morgan,
1951). The world record hook-and-line bluegill was 4 lb. 12
oz. caught at Ketona Lake, Alabama on April 9, 1950 by T.S.

Hudson (IGFA, 1990).

3

Overpopulation and subsequent stunting can be a problem
in both aquaculture and natural fish communities (Lagler and
Steinmetz, 1957; Childers, 1967; Wohlfarth and Hulata, 1983;
Murnyak et al. 1984). Stunting affects fisheries by
reducing the number of acceptable sized bluegill.

Strategies that have been developed to overcome this problem
include nongenetic and genetic techniques (Wohlfarth and
Hulata, 1983). Seining, trapping and partial or complete
poisoning of bluegill populations and nests are some of the
nongenetic methods that have been used to reduce bluegill
numbers; but, these techniques are labor intensive and
costly. Chromosomal manipulation by polyploid induction is
one of the most recent genetic techniques used as a means of
population control in fishes (Bidwell et al. 1985; Myers,
1986; Thompson et al. 1987; Don and Avtalion, 1988; Ihssen
et al. 1990).

Temperature (cold and heat), chemicals and hydrostatic
pressure shocks have been used to induce polyploidy in
fishes (Ihssen et al. 1990). In order for these methods to
be successful, an understanding of the pre- and
postfertilization stages of development of the egg is
necessary. Little work has been done in this area with the
bluegill.

Interest in reproductive control by polyploid induction
arises from the fact that triploidy in fish causes

sterility. It has been argued that sterility has a positive

4
impact on food conversion and growth rate because the energy
normally used for gametogenesis in a fertile fish may be
used in triploids to increase the quantity of edible tissue
(Don and Avtalion, 1986).

Triploidy provides functional sterility because the
normal pairing of chromosomes during meiosis I is impeded by
the presence of three homologous chromosomes. This results
in the uneven or aborted separation of chromosome triplets
which inhibits the early development of gametes (Myers,
1986).

Purdom (1983) found that the sterility effect of
triploidy was manifested in different ways depending on the
fish species. Some species of fish may exhibit retarded and
reduced gonad development and the production of nonviable
gametes, other species may exhibit degenerated gonads and
gametes and some may exhibit sexual characteristics and
normal gonads but produce nonviable gametes. In the cases
where triploidy causes gonad development to be reduced, an
increase in somatic growth has been reported (Myers, 1986).
Examples of gonad reduction in size and function are found
in triploid Atlantic salmon, rainbow trout and Coho salmon
(Benfey and Sutterlin, 1982).

Conflicting reports of triploid growth potential are
found throughout the literature. In animals, the nuclear
and cell sizes increase in proportion to the increase in

chromosome number, so it is expected that triploids should

5
show an increased size over normal diploids (Gold, 1979).
Some researchers state that triploids grow faster and attain
greater weights than diploids (Valenti, 1975) while others
argue that differences in growth rate can only be seen after
sexual maturity (Gervai et al. 1980; Don and Avtalion,
1986). However, for some fish species it has been observed
that the increased cell size of triploid individuals is
compensated by a reduction in the number of cells per organ;
therefore, they are not bigger than the normal diploid
individuals (Gold, 1979).

Two methods for inducing triploidy have been suggested:
1) inhibition of the second meiotic division in the
fertilized eggs and 2) crossing a tetraploid with a diploid
organism (Myers, 1986). Both of these techniques have been
used to produce triploid fishes with varying levels of
practicality and success (Ihssen et al. 1990).

The inhibition of the second meiotic division will
result in eggs with two sets of maternal chromosomes.
Fertilization by haploid sperm then produces triploid
individuals. In fishes, the second meiotic division is
completed shortly after fertilization (Ginzburg, 1972).
This division can be prevented by using a treatment that
causes depolymerization of microtubules which form the
spindle apparatus (Purdom, 1983). The spindle apparatus is
responsible for separation of chromosomes during cell

division.

6
The use of tetraploids as a method of producing
triploids in fish has been suggested as a way of avoiding

conventional induction techniques (Chourrout et al. 1986:

Chourrout and Nakayana, 1987). It has been argued that

conventional triploid induction techniques may cause

abnormal embryonic development (Myers, 1985). Tetraploidy
has been achieved by shocking the fertilized eggs before
metaphase of the first mitotic division (Myers, 1986). The
success of tetraploid induction for each species depends on
the timing (before mitotic metaphase) of the treatment,
intensity of the treatment and the duration of exposure

(Myers, 1986).

The objectives of this study were:

1) To develop histological techniques to monitor egg
developmental stages in order to determine the critical
stage for production of triploid and tetraploid
bluegill.

2) To observe and describe differences, if any, between
the development of normal eggs and the development of
eggs that received pressure or cold shock treatment for

the production of triploid and tetraploid bluegill.

LITERATURE REVIEW

Reproduction control is desirable to channel available
energy to promote efficient growth in fishes. Growth is
controlled to some extent by environmental conditions such
as availability of food and space. Limitation of these
factors can result in slow growth and stunting (Murnyak et
al. 1984). There is an inverse relationship between fish
density and growth rate (Smith, 1980). Density affects
growth through food and space shortages. Growth is
density-dependent with greater growth at lower fish density
(Murnyak et al. 1984).

Based on scale analysis, Murnyak et al. (1984) found
that growth rates of non-stunted and stunted bluegill were
similar before stunting. They also found that under
improved environmental conditions non-stunted bluegill grew
significantly better than stunted bluegill. They argued
that stunting could have prolonged detrimental effects on
bluegill by reducing their capacity for growth. Stunted
bluegill grew more slowly and did not realize their full

growth and yield potential.

8

Control of Reproduction in Fishes

 

Research efforts to improve fish growth rate have
concentrated on different methods of reproduction control.
Techniques for controlling reproduction may be classified as
genetic and nongenetic.

Some of the nongenetic techniques to control bluegill
reproduction have been seining, trapping and poisoning of
populations and nests. These methods are labor intensive and
costly. Other methods that have been used with bluegill and
other species, like tilapia, include the use of predators,
monosex culture, reproductive sterilization, cage culture
and high density stocking rates (Table 1).

The use of predators to control bluegill reproduction
has been shown to be inadequate or too effective under
different ecological situations and stocking densities
(Huet, 1970). For example, in very shallow and weedy
waters, the use of largemouth bass as a predator may be
inadequate because the bluegill can make themselves
inaccessible by hiding in the weeds. Conversely, if
predators are stocked with bluegill in water where there is
little or no cover, predators can cause a "negative" effect
on the prey population. The use of predators in this case
would be considered too effective (Huet, 1970).

Monosex culture has been used with tilapia in an effort

to control overpopulation. This type of culture can be

Table 1. Techniques used to control overpopulation in
aquaculture and natural fish communities.

 

Technique

Author

 

Nongenetic

Seining, trapping and
poisoning of populations
and nests

Predators

Monosex culture
Reproductive sterilization
Cage culture

Genetic

Hybrids

Sex-reversal

Chromosomal manipulation by
polyploid induction

Childers, 1967

Murnyak et al. 1984

Huet, 1970

Pandian and Varadaraj, 1987
Nelson et a1. 1976

Rifai, 1980

Childers, 1967
Dawley, 1987

Pandian and Varadaraj, 1987
Thorgaard et al. 1981

Purdom, 1983
Myers, 1986

 

10
achieved by sexing the fingerlings and stocking one sex, or
by eliminating one sex from the population by sex reversal
or hybridization (genetic methods). Sexing tilapia
fingerlings can be accomplished by examination of the
urogenital papillae: two orifices are present in the female
and one in the male. The major disadvantage of this method
has been human error. One female introduced into a pond can
undo all the labor involved in sexing the fish (Pandian and
Varadaraj, 1987). Also, not all juvenile fish exhibit
sexual dimorphism. This is true for bluegill and many other
fishes (Morgan, 1951).

Reproductive sterilization by irradiation has not
yielded practical results (Nelson et al. 1976). Examination
of the gonads from irradiated fish indicated neither obvious
depletion of the germ cells nor evidence of necrobiosis even
when the irradiation levels were greater than the single
lethal dose for the fish (Nelson et a1. 1976). This
indicated that germ cells are very resistant to radiation.

Reproduction can be controlled in culture ponds by cage
culture. However, cage culture appears to be of limited
commercial value due to the additional cost of the materials
needed to build the cages and the amount of cages necessary
to raise enough fish to make a profit (Rifai, 1980).
Controlling reproduction by high density stocking rates has
the disadvantage of producing small fish size at harvest

time.

11

Genetic methods to control reproduction include
interspecific hybridization, the use of sex reversed
individuals as brood stock and chromosomal manipulation
(Table 1). The only genetic method for reproductive control
attempted with sunfish has been the production of hybrids
(Childers 1967; Dawley, 1987). ~

Some interspecific and intergeneric hybrids that result
in nearly all male progeny have been reported for bluegill
(Childers, 1967). Although hybridization techniques to
produce nearly all male fish have been developed during the
last 20 years, their applicability to monosex culture has
remained limited due to: 1) the difficulty in maintaining
pure parental stocks that consistently produce 100% male
offspring, 2) poor spawning success (Childers, 1967) and 3)
incompatibility of breeders resulting in low fertility
(Childers, 1967).

Prior to the discovery that some sunfish hybrids were
fertile, their use was proposed as a way to increase fish
size and control reproduction. Hubbs and Hubbs (1933)
concluded that sunfish hybrids were sterile. In more recent
studies, it has been found that the majority of the sunfish
crosses are fertile (Krumholz, 1950: Lagler and Steinmetz,
1957: Dawley, 1987) and that in some cases the total length
of the hybrids was not significantly different from their

parents (Childers, 1967).

12

The use of sunfish hybrids has sometimes resulted in
the same difficulties as using the parental species because
their reproductive capacity and their ability to survive
allows them to become so abundant that they are unable to
grow to sizes large enough to be of value for fishermen
(Childers, 1967). However, not all hybrids show high
reproductive capabilities. Dawley (1987) reported that some
hybrids show reduced fertility and Childers (1967) reported
that for some hybrids the sex ratio is almost 100% males.
Childers (1967) argued that some F1 hybrids appear to be
highly vulnerable to capture by hook-and-line and that large
populations of hybrids can be almost completely eliminated
in a few days when subjected to moderate fishing pressure.
Childers (1967) also found that certain kinds of F1 hybrids
appeared to be unable to produce sizeable F2 populations in
ponds containing largemouth bass: but, this can vary with
vegetation abundance.

Hormone treatment for producing sex-reversed
individuals has been widely use with tilapia (Pandian and
Varadaraj, 1987). This technique interferes with the
sex-determining mechanism in half of the population. For
example, if a population of only males is desired, an
androgen is administered through diet or water to a normal
population of young fish. The genetic females are induced
to develop as functional males, while genetic males are not

affected. Because gonads are irreversibly differentiated

13
during a specific period in the larval development, the sex
determination must be manipulated during the post-hatching
differentiation period (Nelson et al, 1976). The desired
hormonal action depends on the efficacy and dose of the
steroid, method of administration, and time and duration of
the treatment.

Some researchers have failed to obtain 100% sex
reversed progeny using hormone treatments primarily for two
reasons: 1) failure to synchronize the hormone treatment
duration with that of gonadal differentiation and/or 2)
failure in the feeding regime. Fry in the last peck of
hierarchal order fail to receive the effective minimum
hormone treated diet to ensure complete sex reversal. In
these individuals, the differentiation process results in
the production of intersex or even female fish. Stunted
growth and liver malformation have also been observed in sex
reversed fish subjected to high doses of androgens (Pandian

and Varadaraj, 1987).

use of Polyploidy for Control of Reproduction in Fishes

The use of induced polyploidy as a genetic technique
for reproductive control has attracted considerable
attention in recent years (for review see Ihssen et a1.
1990). The majority of studies have dealt with the
induction of triploidy in a wide variety of fish. Producing

tetraploid fish is fairly recent and remains a desirable

14
goal because of its potential for further production of
triploids (Thorgaard et al. 1981: Purdom, 1983: Myers,
1986). Several techniques have been used to induce
polyploidy in fishes. These treatments have included
temperature (cold and heat), chemicals and hydrostatic
pressure shocks (Ihssen et al. 1990).

Polyploid organisms possess complete chromosome sets in
excess of the two sets normally found in diploids.
Triploids have showed enhanced growth due to their sterility
and altered cell physiology (Valenti, 1975). Two methods
have been used to induce triploidy: 1) inhibition of the
second meiotic division in the fertilized eggs and 2)
crossing a tetraploid and a diploid organism (Myers, 1986).

For the induction of triploidy, temperature shock has
been the most commonly used method due to its simplicity.
This treatment causes the inhibition of the second meiotic
division in the fertilized egg. In salmonids, a heat shock
of 27-32°C applied 10 to 20 min postfertilization produced a
satisfactory survival and high induction efficiency
(Chourrout, 1980). Heat shock has been successful with the
common carp (Gervai et al. 1980) and with Oreochromis sp.
(Valenti, 1975). Wolters et al. (1982) obtained 100%
induction of triploidy in catfish with the use of cold
shock. Valenti (1975) found cold shocks of 119C (75%
triploid) were superior to heat shocks at 38%: (10%

triploid) in Q. aureus. However, Chourrout and Itskovich

15
(1983) obtained 95% triploid in Q. niloticus using a heat
shock of 40 . 5°C.

Hydrostatic pressure has been used to induce triploidy
primarily in the salmonids. Allen and Myers (1985) found
that pressure treatments of 8000 psi for 10 min in Chinook
salmon resulted in more consistent triploid induction than
with a heat shock of 29%: for 15 min. But Benfey and
Sutterlin (1982) found heat shocking Atlantic salmon eggs
was more effective than pressure treatment in inducing
triploidy.

It has been argued that the treatment for triploid
induction (temperature, pressure or chemical shock) may
cause abnormal embryonic development for which the organism
is unable to compensate (Myers, 1985). Crossing tetraploids
with diploids as a method of producing triploids in fish has
been suggested as a way of avoiding the negative side
effects of conventional induction techniques. It has been
reported that the growth of triploid fish produced by
crossing tetraploids with diploids was superior to triploids
produced via inhibition of the second meiotic division in
the fertilized egg (Myers, 1986).

Tetraploidy has been achieved via suppression of the
first postfertilization mitotic division. The use of
temperature shocks, cytochalasin B and hydrostatic pressure
to inhibit first cleavage have been attempted in a variety

of fishes (Ihssen et al. 1990) and amphibians (Fischberg,

16
1959). Fischberg (1959) was able to achieve 93.7%
tetraploidy in newts by applying a 36 to 37°C shock for 10
min during the formation of the cleavage furrow. Thorgaard
et al. (1981) produced tetraploid rainbow trout by shocking
eggs at 36%: for 10 min at 5 to 6 hours postfertilization.
Tetraploidy can also be induced in rainbow trout by applying
a less intense shock, 28%:, for 14 min at the time of first
cleavage (Chourrout, 1982).

Cytochalasin B acts primarily in preventing the
formation of actin fibers in the cleavage furrow but also
affects respiration, glucose uptake and protein synthesis
(Grant, 1978). Refstie et al. (1977) and Allen and Stanley
(1979) produced mosaic salmonids after cytochalasin B
treatments. The mosaics were comprised of haploid, diploid,
triploid and tetraploid cells. The production of mosaics
could have been the result of lack of synchronization
between the stage of development of the egg and the
application of the treatment.

Hydrostatic pressure has proved to be an effective
tetraploid induction method when the shock has been applied
during the metaphase—anaphase period prior the cleavage.
Chourrout (1984) used a hydrostatic pressure shock treatment
of 7000 psi for 4 min to achieved 100% tetraploid induction
in rainbow trout. At one year of age some tetraploid males

reached maturity and were mated with diploid females.

l7
Triploids produced this way were larger than those produced

via meiotic inhibition (Chourrout, 1984).

Naturally Occurring Polyploid in Fishes

The increased interest in the study of fish chromosome
and cytology may be caused by the new awareness of the
benefits derived from genetic manipulation in fish. As
previously mentioned, chromosomal manipulation by polyploid
induction is one of the most recent genetic techniques used
as a means of population control in fishes. Natural
polyploidism is extremely rare among bisexual vertebrates
but rather common in plants (Gold, 1979). Muller (1925)
stated that one reason for this is that animals usually have
two sexes which are differentiated by means of a process
involving the diploid mechanism of segregation and
combination. However, natural polyploidy has been found in
annelids, insects, crustaceans, fish and amphibians. In
fish, some natural polypoids have been reported (Table 2).
The evolutionary significance of this natural occurrence is

not yet fully understood.

General Description of Teleost Egg and Sperm

In a typical teleost egg, 44 to 84% of the fresh weight
is water, there is a great variation in protein and fat
content and the yellowish color is due to soluble

carotenoids (Ginzburg, 1972). The number and size of the

18

Table 2. Naturally occurring polyploid fishes.

 

Common Name N1 Reference

Atlantic salmon Nygren et al. 1968

 

Brook trout Allen and Stanley, 1978

 

California roach Gold and Avise, 1976

 

Fathead minnow Gold, 1986

 

Poeciliids Schultz, 1967

 

UUNUUoh

Rainbow trout Cuellar and Uyeno, 1972

Thorgaard and Gall, 1979

 

 

 

 

 

1N = ploidy level of fish

19
vacuoles increases in subsequent stages of oocyte
development. New rows of vacuoles are added from the
periphery to the nucleus until vacuoles occupy most of the
cytoplasm (Ginzburg, 1972). Yolk granules store nutritive
substances (proteins and lipids) and are concentrated in the
vegetal pole. In some fish species, including bluegill,
yolk granules unite toward the end of oogenesis and form a
homogeneous mass (Morgan, 1951).

The egg membrane has two parts: the zona radiata and
the outer membrane (Figure 1). The zona radiata protects
the egg from external influences and is pierced by thin
.tubules where water and nutritive substances enter. The
outer membrane is sperm impermeable and very sticky. The
micropyle at the animal pole is a funnel shaped pit in the
membrane that passes into a tubule that opens to the
cytoplasm. The diameter of the micropyle corresponds to the
width of the sperm head. The cortical alveoli have the
function of protecting the egg from penetration by multiple
sperm. After fertilization they discharge their contents
into the micropylar region and block sperm penetration
(Ginzburg, 1972). During ovogenesis, reserve substances
(yolk granules and lipids) accumulate in the cell to provide
nutrients for the embryo.

Female meiosis provides an example of deviation from
symmetric division. Females commit their resources to

produce one large egg and the three unused chromosome

20

 

Figure 1. Bluegill egg showing the zona radiata (zr) and
outer membrane (om), 30 minutes postfertilization.

21
complements are discarded in small cells called polar bodies
(O'Farrell et al. 1989). A maturation promoting factor
(MPF) stimulates the oocyte to enter into meiosis (Murray
and Kirschner, 1989). As the nuclear membrane dissolves,
the spindle of the first meiotic division forms near the
karyosphere which is a small dense body with chromosomes
distributed on it. The chromosomes are arranged on the
equatorial plane of the spindle and form the metaphase
plane. with the separation of the first polar body, the
spindle of the second meiotic division appears at metaphase
II: meiosis is halted and ovulation occurs (Ginzburg, 1972).

The fish sperm is composed of three parts: spherical
head, middle piece and tail. Milt is a suspension of sperm
and seminal fluid where the sperm is immobile and needs
water for activation. In fresh water fishes, sperm is
motile in water for only two to three minutes. There are
long and short lived sperm and there is variation in the
period of active movement for sperm of the same batch
(Ginzburg, 1972).

In teleost fish with external fertilization, the sperm
has lost the acrosomal reaction that was once necessary to
penetrate the egg membrane. The egg membrane is impermeable
to sperm but it has a micropylar system in the animal pole.
The sperm penetrates the egg through the micropyle and the
nucleus and internal organelles are absorbed by cytoplasm

(Ginzburg, 1972). After fertilization, cortical granules

22
are discharged into the micropylar region and block sperm
penetration. Fusion of pronuclei occur after gametic
association: the sperm head is transformed into a pronucleus
and fuses with the female pronucleus restoring the diploid
condition (Ginzburg, 1972). Following a meroblastic
cleavage pattern only a portion of the egg divides. The
high concentration of yolk impedes the division of the whole
egg and the furrows do not extend beyond the blastodisc

(Morgan, 1951).

Fish Chromosomes: Numbers and Size

Chromosome numbers in fish cover nearly the whole scope
of vertebrate chromosome number, ranging from n=8 in
Notobranchius rachowi to n=82 in Ichthypucon gggpi (Post,
1973). Peak frequencies of chromosome number are found at
n=20 to 28 (80%) and n=40 to 52 (5-6%). Chromosome number
for Lepomis macrochirus is reported to be 2n=48 (Roberts,
1964). Apart from microchromosomes in birds and some
reptiles, fishes have the smallest chromosomes in
vertebrates (Post, 1973). The average length of fish
chromosomes is between 2-5 um, and many species possess
numerous small chromosomes of 2 pm or less (Gold, 1979).
For Centrarchids, chromosome sizes ranging from 1.2 to 1.8
um have been reported (Roberts, 1964). Considering the vast
number of living fish species, their diversity in morphology

and the antiquity of the group as a whole, many orders are

23
relatively uniform in karyotype (Gold, 1979). However,
Roberts (1967) suggested that fish are likely to have more
intraspecific chromosome polymorphism than other vertebrates
and that karyotypic variation may occur in different
individuals of the same species. He also states that the
relative similarity of karyotypes in the Centrarchid family
with only two chromosome numbers (2n=46 and 48), contrast
sharply with the wide range of karyotypes among the Salmonid
family. However, even in the Centrarchid family, Lepomis
cyanellus is considered to be a typical example of
Robertsonian variation (Roberts, 1964). This type of
rearrangement may bring changes in chromosome number by
fusing two small chromosomes together or dividing a large
one and forming two (Gold, 1979).

Karyotype work in fishes has long been impeded by lack
of adequate techniques to permit examination of the large
number and the small size of their chromosomes (Booke,
1968). Obtaining consistent chromosome spreads of good
quality is one of the limiting factors in the study of
chromosomes in fishes (Gold, 1979). Throughout the
literature, it is obvious that technical difficulties have
resulted in reports of chromosome numbers and morphology

that are now considered incorrect.

24

Technigpes for Chromosome Observation in Fishes

Several techniques to study cell chromosome in fishes
(Table 3) have been used with a wide range of results
(McPhail and Jones, 1966; Kligerman and Bloom, 1977). Some
of these techniques include tissue sectioning (Nogusa,
1960), air drying cells of different tissues (Chen, 1970;
Denton and Howell, 1969: Howell, 1972: Gold, 1974),
culturing leukocytes (Labat et al. 1967; Heckman and
Brubaker, 1970), culturing fibroblasts (Roberts, 1964: Chen,
1970; Chen, 1975), colchicine injection followed by squashes
of different tissues (Wright, 1955: Roberts, 1964: Simon,
1964; McPhail and Jones, 1966) and solid tissue techniques

(Kligerman and Bloom, 1977).

Lpser Scanning Confocal Microscope

Although the use of light microscopy has been very
common in the study of fish chromosomes, the use of laser
scanning confocal microscope offers significant promise for
advances. The laser scanning confocal microscope is a
relatively new instrument that allows the examination of
microscope specimens to an unusual depth. In the
microscope's name "laser" and "scanning" refer to the method
of illumination, "confocal" refers to the method of image
formation (Dr. J. Whallon, personal communication).

The term "confocal" indicates that the microscope is

aligned so that the illuminated spot and the imaged spot

Table 3. Tissue preparation techniques for chromosome

observation in fishes.

 

Technique

Author

 

Tissue sectioning

Air drying cells of different
tissues

Culturing leukocytes

Culturing fibroblasts

Colchicine injection and
squashes of different tissues
(gonads, gill epithelium and
blastodiscs)

Solid tissue techniques

Nogusa, 1960

Denton and Howell, 1969
Chen, 1970

Howell, 1972

Gold, 1974

Labat et al. 1967
Heckman and Brubaker,
1970

Roberts, 1964
Chen, 1970, 1975

Wright, 1955

Roberts, 1964

Simon, 1964

McPhail and Jones, 1966

Kligerman and Bloom, 1977

 

26
coincide precisely. This is not the case in conventional
light microscopes, in which the light from out-of—focus
areas limits the depth to which a specimen can be examined
to just a few microns. The traditional method to try to
solve this problem has been to embed the sample in resin or
paraffin and then to slice it in thin sections for
sequential viewing.

In a confocal instrument, the specimen is scanned with
a finely focused laser beam. A pinhole aperture is placed
directly in front of the detector at the focal point of
light coming from the in-focus part of the specimen. The
.effect of these modification is to block light from
out-of—focus regions. The result of this is an increase in
resolution and contrast.

In the process of optical sectioning, the microscope
collects a series of images from progressively greater
depths in the sample and these images can be stored on the
computer hard disk attached to the instrument. These images
can also be used to form a three-dimensional composite if

this is desirable.

MATERIALS AND METHODS

This study was designed to develop histological
techniques to monitor egg developmental stages in order to
determine the critical stage for production of triploid and
tetraploid bluegill. Secondly, we planned to use these
histological techniques to observe and describe differences,
if any, between the development of normal eggs and the
development of eggs that received pressure or cold shock
treatment for the production of triploid and tetraploid

bluegill.

Experimental animals

Bluegill ready to spawn were collected by hook-and-line
from Lake Lansing (near Haslett, Michigan) and identified
using an appropriate reference (Hubbs and Lagler, 1964).

The fish were transported to the Michigan State University
Aquaculture Center in aerated 163 L coolers. At the Center,
they were maintained in a 1900 L circular tank with a water
temperature of 22°C +/- 2°C. They were spawned soon after

arrival.

27

28

Spawning selection

The method used for spawning was described by Rothbard
and Pruginin (1975). Females and males previously selected
for ripeness were strip-spawned. Ripe fish chosen for
stripping must be ready for natural spawning. Ripe
bluegills can be easily identified by intense pigmentation
(especially males) and a swollen genital area.
Checking for ripeness

Fish were considered ready to spawn when a gentle
squeezing of the abdominal area caused the eggs and the milt
to flow from the genital pore. Mature eggs have yolk that
is slightly granular with a large oil globule. These eggs
are perfectly spherical (0.85-1.36 mm), strongly adhesive
(Banner and Hyatt, 1975) and have an amber color. Ripe milt
has a white color.
Egg fertilization

Dry fertilization was used to prevent premature closing
of the micropyle and to enhance the ability of the sperm to
penetrate and fertilize the egg. Female bluegills were
dried around the genital area and the eggs were expelled
into a dry watch glass. Milt was collected from the male
using a pipet. Milt and eggs were mixed with a glass rod.
When the milt was added to the eggs a stopwatch was started
to time egg development. One minute postfertilization,

water at 22°C was added to activate the eggs.

29
Histological techniques tggmonitor qu deyelopmental stages
Light microscope observations
Experiment I: The use of Bouin's solution as a fixative and
the use of carmine stain for whole eggs and blastodiscs at
different developmental stages of the fertilized egg

Fertilized eggs were transferred to concave microscope
slides using a wide mouth pipet. Samples were fixed by
adding Bouin's solution (Table 4) to the slides at 30, 40 or
50 min postfertilization. Fixed samples were submerged in a
Coplin jar containing Bouin's solution for at least 24 hr.

Whole eggs were rinsed in distilled water to remove
excess Bouin's solution and dipped in a Coplin jar
containing carmine for 5, 10 or 15 min. They were
sequentially rinsed with distilled water to remove excess
stain and dipped in Coplin jars containing 30%, 50%, 70%,
95%, 95%, 100% and 100% EtOH and 100% and 100% xylene for
10, 30 or 60 min for dehydration.

With the use of forceps and under the dissecting
microscope, blastodiscs were dissected from whole eggs. The
blastodisc membrane was removed before staining and the
carmine was applied directly to the blastodiscs for 5, 10 or
15 min. Excess carmine was removed by rinsing the
blastodiscs with distilled water. Dehydration was attempted
as described for whole eggs.

Slides were prepared by air drying the samples for a

few minutes. Three drops of permount were added to the

30

Table 4. Composition for Bouin's and Farmer's fixatives

used to fix fertilized bluegill eggs.

 

 

Reagent Composition ml

Bouin's Solution Picric Acid 75
Formalin 25
Glacial Acetic Acid 5

Farmer's Solution 95% EtOH 75
Glacial Acetic Acid 25

 

 

 

 

 

31
slide and a coverslip was placed over the sample. The
slides were then placed on a heat plate at 48%: for 24 hr.
After this, slides were ready to be examined under the light

microscope.

Experiment II: The use of Bouin's solution as fixative for
eggs at different developmental stages and the use of
conventional histological techniques

Fertilized eggs were transferred to glass vials using a
wide mouth pipet. Clumped eggs were dispersed and the
samples were fixed by adding Bouin's solution to glass
vials. The eggs used in this experiment were fixed with
Bouin's solution 30, 40 or 50 min postfertilization. They
remained in the solution for at least 24 hr. The
conventional histological techniques used in this experiment
are explained in Appendix A, Table 5 to 10. Slide

preparation was as described in Experiment I.

Laser scanning confocal microscope observation

Experiment III: The use of Bouin's and Farmer's solutions

as fixatives and the use of ethidium bromide to stain

fertilized eggs at different developmental stages
Fertilized eggs were transferred to glass vials using

a wide mouth pipet. Clumped eggs were dispersed and excess

water removed. Samples were fixed by adding Farmer's

fixative (Table 4) to glass vials. They remained in the

32
fixative for at least 24 hr. Another set of samples were
fixed by adding Bouin's solution. Samples were fixed at 10
min intervals (from 1 min to 60 min postfertilization).

The fertilized eggs were transferred to concave
microscope slides and rinsed with distilled water to remove
excess Farmer's fixative or Bouin's solution. They were
stained with 0.0025% ethidium bromide for 10 to 20 min and
then rinsed three times (for 10 min each) in distilled
water. Slide preparation was achieved by placing 8 to 10
eggs on a concave microscope slide and adding glycerol or

distilled water.

Experiment IV: The use of Farmer's solution as a fixative
and the use of acetocarmine to stain fertilized eggs at
different developmental stages

The samples used in this experiment were fixed as
described in Experiment III using Farmer's fixative.
Samples were transferred to concave microscope slides and
rinsed with distilled water to remove excess Farmer's
fixative. Fixed samples were stained with acetocarmine for
10 to 20 min and rinsed with distilled water.

The acetocarmine stain was prepared by boiling 100 ml
of 45% acetic acid and adding 1 g of certified carmine dye.
After filtering the dye, more acetic acid was added (to

bring to 100 ml) and the solution was placed in a bottle.

33
Several staining procedures were used including: 1)
staining for 5, 10 or 20 min, 2) staining for 30 s, 3)
staining for 30 s and applying 95% ethanol and 4)staining
for 30 s, applying 95% ethanol and squashing the sample.

Slide preparation was as described in Experiment III.

Experiment V: The use of Farmer's solution as a fixative
and the use of Feulgen technique to stain fertilized eggs at
different developmental stages

The samples used in this experiment were fixed as
described in Experiment III using Farmer's fixative. The
fertilized eggs were transferred to concave microscope
slides and washed with distilled water to remove excess
Farmer's fixative. The eggs were stained using the DNA
specific Feulgen technique for 10 to 20 min and rinsed with
distilled water.

The Feulgen stain was prepared by pouring 200 ml of
boiling distilled water over 1 g of basic fuchsin. After
filtering the stain, 30 ml of 1 N HCl and 3 g of potassium
metabisulfite (Kzszos) were added to the filtrate. The
solution was decolorized for 24 hr in a tightly-stoppered
bottle in the dark. After this, 0.5 g of carbon (Norit) was
added, then filtered through coarse filter paper and stored
in the dark. After the stain application, slides were

prepared as described in Experiment III.

34

Comparison between the development of normal eggs and eggs

that received pressure or cold shock treatment for the
production of triploid and tetraploid bluegill

To achieve the second objective of this study a
sequence of photomicrographs of developing normal eggs was
produced. These eggs were fixed at 10 min intervals (from 1
min to 60 min postfertilization) with Farmer's fixative and
stained for 10 min with 0.0025% ethidium bromide.
Comparisons were made between the normal eggs and the eggs
that received the induction treatment (Westmaas, 1992). The

induction treatments were as follows:

Triploid treatment: Pressure shock of 8000 psi for 5 min
applied to eggs at 1.5 min postfertilization. Eggs were
fixed at 35, 45 and 55 min postfertilization.

Tetraploid treatment I: Cold shock of 5°C for 10 min
applied to eggs at 35, 40 and 45 min postfertilization.
Eggs were fixed at 45, 50 and 55 min postfertilization,
respectively.

Tetraploid treatment II: Pressure shock of 8000 psi for 5
min applied to eggs at 35, 40 and 45 min postfertilization.
Eggs were fixed at 40, 45 and 50 min postfertilization,

respectively.

RESULTS

Development of histological techniques to monitor eqq
developmental stages

The success of polyploid induction techniques depends
on the timing of application of the treatment to the
developing egg, intensity of the treatment and the duration
of exposure for each species. To achieve the first
objective of this study, several histological techniques
were tested to determine their effectiveness in monitoring
the developmental stages of fertilized bluegill eggs. These
techniques included standard light microscopy and the use of
laser scanning confocal microscopy. The procedures for the
preparation of the samples were developed to study
chromosomal changes in the nucleus of the blastodisc in the

fertilized egg.

Light microscope observations

Experiment I: The use of Bouin's solution as a fixative and
the use of carmine stain for whole eggs and blastodiscs at

different developmental stages of the fertilized egg

35

36
Whole eqqs:

Samples fixed with Bouin's solution at 30, 40 and 50
min postfertilization were used. Samples were stained with
carmine for 5, 10 and 15 min. There was very little
infiltration of the stain in the samples stained for 5 min
and too much infiltration in the ones stained for 15 min.
From direct observation under the light microscope of the
samples stained for 10 min, the micropyle and the blastodisc
were easily identified but no other organelles (including
the nucleus) were seen.

Blastodisc:

The same staining procedure was used for blastodiscs
dissected from whole eggs that were fixed with Bouin's
solution. The dehydration procedure could not be completed
with the techniques that were available due to difficulty in
handling. Furthermore, blastodiscs were damaged due to the

effects of surface tension and handling.

Experiment II: The use of Bouin's solution as a fixative
for eggs at different developmental stages and the use of
conventional histological techniques
Eggs fixed at 50 min postfertilization:

The blastodisc, micropyle, yolk pieces and granular
structures (probably oil droplets) were visible only in a
few slides of the serial sections examined under the light

microscope. No nuclei inside the blastodisc, or

37
mitochondria or other organelle in the blastodisc cytoplasm
were identified. The blastodisc appeared as a elevated area
in the animal pole close to the micropyle of the cell.
qus fixed at 40 min postfertilization:

The samples were subjected to an increase in
dehydration and infiltration time and also the use of
terpineol in an attempt to improve paraffin infiltration.
From the study of the slides only pieces of blastodiscs and
yolk could be identified.
qus fixed at 30 min postfertilization:

Although the samples used in this procedure were
subjected to an increase in dehydration and infiltration
time, the results were very similar to the results of the
procedure used for eggs fixed at 50 min postfertilization.
Some complete structures like blastodisc, micropyle, yolk
and granular structures were observed in the slides, but no

other structures could be identified.

Laser scanning confocal microscope
Experiment III: The use of Bouin's and Farmer's solutions
as fixatives and the use of ethidium bromide to stain
fertilized eggs at different developmental stages

Samples used in this experiment were fixed at 10 min
intervals (from 1 to 60 min postfertilization) with Farmer's
and Bouin's solutions. They were stained for 10 and 20 min

with ethidium bromide and for the slide preparation glycerol

38
or distilled water was added directly to the samples. It
was found that Bouin's solution produces a coarse structure
in the cytoplasm and increases the "natural fluorescence" of
the cell to a point where it was not possible to distinguish
any structures inside the cell. Farmer's solution was a
better alternative as a fixative. Staining by adding
0.0025% ethidium bromide for 10 min was found to produce the
best image and distilled water kept the samples hydrated
better than glycerol. No chromosomes were observed in any

of the eggs examined.

Experiment IV: The use of Farmer's solution as a fixative
and the use of acetocarmine to stain fertilized eggs at
different developmental stages

Samples were stained with acetocarmine for 5, 10 and 20
min. From the study of these slides, it was found that 5
min in the acetocarmine caused too much infiltration of the
stain so different staining times were tried. Eggs were
stained with acetocarmine for 30 s, with or without 95%
ethanol and then some eggs were squashed. From the eggs
stained with acetocarmine for 30 s, a fluorescence similar
to the one produced by ethidium bromide was observed: but,
it was less intense. The use of 95% ethanol did not improve
the image. No chromosomes were identified in the squashed

blastodiscs or in any of the eggs observed.

39

Experiment V: The use of Farmer's solution as a fixative
and the use of Feulgen technique to stain fertilized eggs at
different developmental stages

Although Feulgen stain is specific for DNA, the results
obtained were very similar to those observed in Experiments
III and IV. The images produced by the stain were
satisfactory but inferior to those produced by ethidium
bromide. No chromosomes were observed in any of the eggs

examined.

Comparison between thegdevelopment of normal eggs and eggs

that received pressure shock treatment for the induction of

triploid and tetraploid bluegill

The second objective of this study was to observe and
describe differences between the development of normal eggs
and eggs that received pressure or cold treatments for the
production of triploid and tetraploid bluegill. This was
achieved by comparing sequences of photomicrographs of
normal eggs and of eggs that received the shock treatments.
Stage I-Fertilization to 30 min postfertilization:

From the sequence of photomicrographs it is apparent
that immediately after fertilization the blastodisc appears
to constrict at the margin of the animal pole close to the
micropyle, forming a white cap (Figure 2). The
perivitelline space between the egg membrane and the yolk

enlarges and gives way to protoplasmic accumulation in the

4O

 

Figure 2. Blastodisc (b) near the micropyle (m) of a normal
bluegill egg, 20 minutes postfertilzation.

41
region (Figure 3). Small oil droplets were observed
distributed over the surface of the yolk. After the
formation of the blastodisc, no external changes in the
fertilized egg were evident until the time of the first
mitotic division. With the use of the LSCM, it was possible
to measure blastodiscs and micropyles. Blastodiscs diameter
ranged from 0.426 mm to 0.523 mm with an average of 0.476 mm
(n=11). Micropyle (Figure 4) diameter ranged from 0.191 mm
to 0.221 mm with an average of 0.209 mm (n=4). Some polar
bodies were observed being extruded from the eggs that
received the triploidy induction treatment (Figure 5).
Stage II-First mitotic division:

Following the pattern of meroblastic cleavage in
teleost eggs, the first mitotic division occured 35 min
postfertilization at 22°C, and the furrow divides the
blastodisc into two blastomeres of about equal size (Figure
6). Figures 7 and 8 show the seams in the cleavage furrow
and a blastodisc with an incomplete cleavage furrow,
respectively.

Stage III-Second mitotic division:

At 50 min postfertilization, it was observed that
although the cells had not completed the second mitotic
division, four distinct nuclei, two in each blastomere,
could be easily recognized (Figure 9). This may indicate

that anaphase was probably complete but telophase was yet to

42

 

Figure 3. Perivitelline space (ps) between the egg membrane
(em) and yolk (y) of a normal bluegill egg, 20 minutes
postfertilization, enlarged giving way to protoplasmic
accumulation.

 

Figure 4. View of the micropyle of a normal bluegill egg,
30 minutes postfertilization.

43

 

Figure 5. Polar body (pb) being extruded from the nucleus
(n) of a bluegill egg, 35 minutes postfertilization, after
triploid induction treatment.

 

Figure 6. First mitotic division of normal bluegill egg, 35
minutes postfertilization at 22°C, blastodisc is divided
into two blastomeres of equal size.

44

 

Figure 7. Seams in the cleavage furrow of a normal bluegill
egg, 35 minutes postfertilization.

 

 

Figure 8. Blastodisc with incomplete cleavage furrow (cf)
from a normal bluegill egg, 35 minutes postfertilization.

45

 

Figure 9. Blastodisc showing two nuclei (n) in each
blastomere of a normal bluegill egg, 50 minutes
postfertilization.

46
occur. At 55 min postfertilization the second cleavage
furrow appears at a right angle to the first furrow and this
resulted in four blastomeres of equal size (Figure 10).

After this stage, cell division becomes more rapid,
irregular, and the blastomere size started to become more
heterogeneous. Not all the fertilized eggs developed at the
same rate, some reached early blastula stage while others
were in two or four cell stages.

No dividing blastodiscs were observed in the eggs that
received the pressure or cold treatments for the production
of triploid and tetraploid bluegill. Blastodics and eggs
seemed to be normal in size, shape and general appearance
but no cell division had occurred in the blastodiscs of
these eggs (Figs. 11, 12, and 13). At least two mitotic
divisions were expected since the first mitotic division
occurred at 35 min postfertilization and the second at 55
min. The treated eggs were fixed in this same range from 35

to 55 min postfertilization.

47

 

Figure 10. Second mitotic division of a normal bluegill
egg, 55 minutes postfertilization, resulting in four
blastomeres of equal size.

 

Figure 11. Bluegill egg that received a pressure shock of
8000 psi, triploid induction treatment, 45 minutes
postfertilization.

 

Figure 12. Bluegill egg that received a cold shock of 5°C,
tetraploid induction treatment, 55 minutes
postfertilization.

 

Figure 13. Bluegill egg that received a pressure shock of
8000 psi, tetraploid induction treatment, 40 minutes
postfertilization.

DISCUSSION

The bluegill belongs to the Centrarchid family, is the
largest lepomine sunfish found in Michigan, the favorite
panfish of North American anglers and the most frequently
pursued gamefish in the United States (Janssen, 1974). Due
to its early sexual maturity and high rate of reproduction,
bluegill frequently dominate the biomass of the lakes and
ponds they inhabit (Morgan, 1951). Where it predominates
numerically, it is prone to stunting (Murnyak et al. 1984).
Strategies to overcome this problem have included nongenetic
and genetic techniques. Chromosomal manipulation by
polyploid induction is one of the most recent genetic
techniques proposed as a means of population control in
fishes (Bidwell et al. 1985: Myers, 1986; Thompson et al.
1987; Don and Avtalion, 1988).

Several techniques to study cell chromosomes in fishes
have been used with a wide range of results (McPhail and
Jones, 1966: Kligerman and Bloom, 1977). For the purpose of
this study standard histological procedures, light and laser

scanning confocal microscopy, were used.

49

50
Standard histological technigpes and light microscopy

In Experiment I, whole eggs and blastodiscs were fixed
with Bouin's solution at 30, 40 and 50 min
postfertilization. The samples were stained with carmine
for 5, 10 or 15 min. The poor stain infiltration observed in
whole eggs may have indicated a permeability problem of the
egg membrane. The literature suggests steps that could be
taken in order to improve infiltration (Drewry, 1965: Booke,
1968; Gold and Price, 1985). These may include: 1) an
increase in dehydration time, 2) the use of different
dehydrating chemicals and 3) staining of serial sections of
the samples.

The blastodisc dehydration procedure could not be
completed due to difficulty in handling. The small size of
the blastodiscs made them very difficult to manipulate and
the effect of surface tension and handling damaged them.

In Experiment II, Bouin's solution was used as a
fixative and conventional histological techniques were
applied to fertilized bluegill eggs fixed at 30, 40 and 50
min postfertilization. A possible explanation for not
having been able to identify any organelle in the cells may
be that the procedures were too harsh for the eggs. The
excessive hardening of the egg prevented suitable sectioning
by the microtome and only scattered pieces of eggs were

recovered on the slides.

51

Hardening of the blastula tissue prevented effective
spread of chromosomes. Drewry (1964) found that fixatives
containing alcohol caused hardening of the tissue and
prevented suitable examination and squashing. To remedy
this situation Booke (1968) suggested the addition of 1 drop
of 50% acetic acid to soften the tissue 1 min prior to
staining. Also different types of fixatives have been used
including methanol-formaldehyde (9:1) which proved to be
very effective in preserving tissue (Gold and Price, 1985);
however, the use of fixatives containing formaldehyde can
increase the natural fluorescence of the cell (Dr. J. Asher,
personal communication).

The results obtained indicated that standard
histological techniques used in Experiments I and II were
not effective in monitoring the developmental stages of
fertilized bluegill eggs. If standard techniques are to be
used, a change in protocol including the use of different
fixatives and chemicals for dehydration and for improved
infiltration, and an increase in time for each step is
advisable. Disadvantages of these techniques are that they
are tedious, time consuming, and there is no guarantee that
good quality slides will be achieved.

Roberts (1967) found that some of the disadvantages of
using embryonic material to study fish chromosomes were
that: 1) in blastomeres the chromosomes tend to be elongated

making the counting and determination of the centromere

52
difficult, 2) the cytoplasm has high affinity for
chromosomal stain (due to its high mitochondrial DNA
content) and no sharp contrast between the cytoplasm and
chromosomes is obtained, 3) in small eggs it is very
difficult to separate the blastomere from the yolk, 4) the
yolk interferes with staining and obscures mitotic
structures and 5) in late embryos the percentage of dividing
cells is reduced. One or more of these factors may have
interfered with the success of the histological techniques
used to monitor chromosomal changes in the developing

bluegill eggs.

Laser scanning confocal microscopy

The staining procedures applied included the use of
ethidium bromide in Experiment III, acetocarmine in
Experiment IV and Feulgen stain in Experiment V. The
identification of chromosomes in the developing eggs was not
possible with any of the staining procedures.

Although it was not possible to monitor chromosomal
changes in the cell, the laser scanning confocal microscope
(LSCM) was an excellent instrument to observe developmental
changes in fertilized bluegill eggs. With the use of LSCM
it was found that blastodiscs and micropyles measured 0.476
mm (n=11) and 0.209 mm (n=4), respectively. With the LSCM
it was observed that in normal, fertilized bluegill eggs the

first cleavage furrow forms 35 min postfertilization at

53
22°C. Not all eggs from the same batch developed at the
same rate. A number of factors may have affected the rate
of meiosis and mitosis in bluegill eggs. Some of these are:
1) the internal temperature of the parental fish, 2) the
temperature of the eggs at the moment of fertilization and
3) the incubation temperature of the developing eggs. An
increase in any of these temperatures would be expected to
produce a faster rate of development.

Although for the purpose of this study the eggs were
observed only up to 60 min postfertilization (4 cell stage),
Morgan (1951) found that, with an incubation temperature of
22°C, at 1 hr and 40 min postfertilization early blastula
was evident and that cells of the blastoderm were quite
small. In 3 hr, the marginal cells began to enclose the yolk
and by 4 hr the marginal cells had reached the equator of
the egg. Between 9 and 12 hr, the yolk was almost
completely covered by the marginal cells and an outline of
the developing embryo could be seen through the egg
membrane.

The LSCM was also a useful tool to study the eggs that
received the polyploid induction treatments. No dividing
blastodiscs were present in the eggs that were examined.
The precise reason for the inhibited cell division in
treated eggs is unknown, but it can be argued that the
treatment may have produced a shock to the cell system that

required a period of recuperation. The various induction

54
treatments may have interrupted the synthesis of some of the
molecules (e.g., proteins and nucleic acids) or the
replication of organelles necessary for cell division. The
cell may have needed time to "recuperate" before continuing
with its normal development.

It was not possible to determine exactly when the first
mitotic division occured in the bluegill eggs that were
exposed to polyploid induction techniques. The eggs
receiving the tetraploid treatments were fixed at 40, 45, 50
and 55 min postfertilization, immediately after receiving
the pressure or cold shocks. Eggs receiving the triploid
treatments were fixed at 35, 45 and 55 min
postfertilization. Cell division had not yet occurred by 55
min postfertilization.

It was also possible that these eggs were polyploid and
that, specifically in the case of tetraploid eggs, cell
division had been inhibited. However, it is not known
whether or not theses cells were polyploid. To determine
ploidy level, a study of the chromosome complements would be
needed or at least some measure of the DNA content of the
cell would have to have been obtained. For the production
of tetraploid cells, the induction treatment must be applied
prior the first mitotic division of the normal developing
zygote (Bungenberg De Jong, 1957; Purdom, 1983; Myers,

1986). For bluegill eggs incubated at 22°C, the first

55
mitotic division occurred at 35 min postfertilization
(Morgan, 1951).

Another possibility for the observed inhibited cell
division in eggs that received the polyploid induction
treatments could have been that the eggs were dead.
Westmaas (1992) observed that the relative survival of
bluegill eggs to 4 days posthatching after exposure to the
same pressure treatments used to induce triploidy in this
study, ranged from 59.4 to 83.7 percent. Relative survival
was defined as the ratio of the percent survival of shock
treated eggs and control eggs from individual females times
100. Control survival ranged from 76.7 to 87.9 percent
(Westmaas, 1992). Consequently, at least some of the eggs
exposed to triploid induction treatments should have
survived.

Westmaas (1992) also observed that the survival rates
of eggs exposed to tetraploid induction treatments were
extremely low, usually less than 1 percent. Consequently,
few, if any, of the eggs exposed to tetraploid treatments
should have been expected to survive.

However, normal eggs quickly change in appearance after
they are dead. Dead eggs become cloudy and yellowish in
color. These changes were not observed in the eggs that
received the induction treatments during the time they were

examined.

56

The analysis of chromosome complements is the most
direct assay for ploidy determination (Purdom, 1983). In
fish, chromosome analysis can be difficult due to the large
number of chromosomes and their small size (Roberts, 1964:
Booke, 1968: Gold, 1979). The development of successful
methods for observation of fish chromosomes will undoubtedly
be beneficial for systematics and for fish phylogeny
(Ivanov, 1972), as well as for timing of ploidy induction

treatments.

 

SUMMARY AND CONCLUSION

The objectives of this study were:

1) To develop histological techniques to monitor egg
developmental stages to determine the critical time for
production of triploid and tetraploid bluegill and

2) To observe and describe differences between the
development of normal eggs and eggs that received
induction treatments for the production of triploid and
tetraploid bluegill.

Several histological techniques were tested to
determine their effectiveness in monitoring the
developmental stages of fertilized bluegill eggs. These
techniques included standard light microscopy and the use of
laser scanning confocal microscopy. The procedures for the
preparation of the samples were developed to study
chromosomal changes in the nucleus of the blastodisc in the
fertilized egg.

The results obtained from the standard histological
techniques indicated that these techniques were not
effective in monitoring the developmental stages of
fertilized bluegill eggs. The application of conventional

staining techniques to piscine chromosomes was not

57

58
effective because chromosomes could not be differentiated
from the rest of the cytoplasm. Stains that produce intense
differentiation between chromosomal and mitochondrial DNA
need to be used. If standard techniques are to be used, a
change in protocol including the use of different fixatives,
chemicals for dehydration, and techniques to improve
infiltration is advisable.

The second objective of this study was partially
achieved by comparing sequences of photomicrographs of the
development of normal eggs and eggs that received treatment
for the production of triploid and tetraploid bluegill
(Westmaas, 1992). Although it was not possible to monitor
chromosomal changes in the cell and to determine when
metaphase of the first mitotic occurred, the laser scanning
confocal microscope (LSCM) was an excellent instrument to
observe developmental changes in the fertilized bluegill
egg.

With the use of LSCM, it was found that blastodiscs and
micropyles measured 0.476 mm (n=11) and 0.209 mm (n=4),
respectively. The LSCM made it possible to observe polar
bodies that were being extruded from some eggs that received
the triploidy induction treatment. These polar bodies may
have been from the first meiotic division.

With the LSCM, first cleavage furrow was observed at 35
min postfertilization in normal bluegill eggs incubated at

22°C. Consequently, the bluegill eggs that had been shocked

59
to induce tetraploidy in this experiment were treated after
the critical developmental stage to induce tetraploidy.

The LSCM was also a useful tool to study the eggs that
received the induction treatments. It was observed that
pressure and cold shocks inhibited cell division, as no
dividing blastodiscs were present in the eggs that were
examined. The precise reason for this is unknown but it can
be argued that: 1) the pressure or cold shock treatments may
have produced a shock to the cell system that required a
period of recuperation, 2) the eggs were polyploid and cell
division had been inhibited, and 3) the eggs were dead as a
result of the induction treatment.

Observation of fish chromosomes in the fertilized egg
may be possible with the use of available techniques
(histological techniques or the use of LSCM), but
considerable effort will have to be expended in order to
identify appropriate chemicals and/or the proper protocols
to be used. The development of successful methods for
observation of fish chromosomes will undoubtedly be
beneficial and will enable researchers to transfer the
triploid and tetraploid induction techniques to other
gamefish that are prone to stunting like crappie, perch and

bullhead.

APPENDIX

Appendix A
Standard histological techniques

used to monitor developmental stages
in fertilized bluegill eggs

6O

61

 

Table 5. Dehydration and paraffin infiltration procedures
for bluegill eggs fixed at 50, 40 and 30 min
postfertilization.

Stage Procedure for dehydration and paraffin
infiltration

A. Eggs fixed at 50 min postfertilization

1. Transfer fertilized eggs into tissue basket

2. Wash in distilled water to remove excess Bouin's
solution

3. Dehydrate by placing the tissue basket in beakers
containing 50 ml of 30%, 50%, 70%, 95%, 95%, 100%
and 100% EtOH for 1 hr each

4. Continue dehydration in two series of 100% xylene
for 1 hr each

5. Place tissue basket into three series of small
beakers containing 100% melted paraffin for 1 hr
each in the vacuum oven

B Eggs fixed at 40 min postfertilization

1. Repeat steps 1 and 2 as described in A

2. Dehydrate by placing tissue basket in beakers
containing 50 ml of 30%, 50%, 70%, 95%, 95%, 100%
and 100% EtOH for 3 hr each

3. Continue dehydration in two series of 100% xylene
for 3 hr each

4. Place tissue basket into small beakers with

different paraffin:terpineol proportions in the
vacuum oven (1 hr a to c; 1.5 hr d to f)

1/4 paraffin: 3/4 terpineol (12.5:37.5ml)
1/2 paraffin: 1/2 terpineol (25:25ml)

3/4 paraffin: 1/4 terpineol (37.5:12.5m1)
100% paraffin (50ml)

100% paraffin (50ml)

. 100% paraffin (50ml)

HHDQJOU‘DJ

TABLE 5.

C. Eggs
1.
2.
3.

62

Continued.

fixed at 30 min postfertilization

Repeat steps 1 and 2 as described in A

Repeat steps 2 and 3 as described in B

Place tissue basket into three series of small
beakers containing 100% melted paraffin for 3 hr
each in the vacuum oven

63

Table 6. Paraffin imbedding of bluegill eggs.

Step

1.

Description

Transfer tissue basket from the vacuum oven and Open it
on the paraffin stove

Deposit melted paraffin into metal boats

Using heated wide mouth pipet, transfer eggs into metal
boats

Place plastic square over metal boats and fill slowly
with melted paraffin to avoid overflow

Wait at least 24 hr before removing the paraffin blocks
from the metal boats

64

Table 7. Procedure for cutting paraffin blocks with

10.

microtome.

Description

To cut the paraffin block using the microtome, place
paraffin block (excess paraffin should be removed
first) in the microtome and tighten

Clean the blade with xylene (wipe clean one way)

Place the fuzzy side of the blade to the right and
tighten the screws

Move the blade close to the block and lock it using the
screws

Use 2 brushes to remove the paraffin strips and to keep
them away from the blade

Scrape the longer side of the block if necessary to
make straight strips

Using a small brush, separate strips for the slides,
maintaining proper sequence

Apply albumin to microscope slides and when dry place
the paraffin strips on slide

Apply water to the slide using a fine brush (without
forming bubbles)

Place on heat plate at 48°C for at least 24 hr.

65

Table 8. Staining of slides prepared from paraffin blocks.

In this procedure the slides are placed in Coplin jars
containing several reagents for different lengths of time:

Reagent Time
Xylene 4 min
Xylene 4 min
100% EtOH 2 min
95% EtOH 2 min
95% EtOH 2 min
70% EtOH 2 min
Tap water 3 min
Hematoxylin 5 min
Tap water 20 5
Tap water 20 s
Acid alcohol* 1 dip
Tap water 20 s
1% Amn. water** 20 5
Tap water 20 s
Eosin 5 s
D water 1 dip
95% EtOH 1 min
95% EtOH 1 min
100% EtOH 2 min
100% EtOH 2 min
Xylene 3 min
Xylene 3 min

Xylene store for mounting

*Acid alcohol; 75% EtOH and lml HCl
** 1% Amn. water; 99ml water and 1ml ammonia

66

Table 9. Light microscope instructions (K6hler

10.

illumination).

Description

Turn on light

Use low power objetive

Focus in structure (fine focus)

Use next power if desirable and focus again
Close the diaphragm

Focus the condenser until sharp image attained

Center image of the diaphragm with X hair in the center
of image (move with screws, they are perpendicular)

Open diaphragm until image fills field of vision (no
edge of diaphragm seen)

Remove the ocular and move condenser diaphragm, close
1/2 to 2/3 and put ocular back

Procedure needs to be repeated with every change in
power

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