Fish Physiology Ii (242-303)

5
5
HORMONAL
HORMONAL S EX C
SEX ONTROL AND
CONTROL AND ITS
ITS
APPLICATION
APPLICATION TO FISH
FISH C ULTURE
CULTURE
A. HUNTER AND EDWARD M
GEORGE A. M.. DONALDSON
West Vancouver
Vancouver Laboratory,
Laboratory, Fisheries
Fisheries Research
Research Branch
Dept.
Dept. of
of Fisheries
Fisheries and
and Oceans
Oceans
West Vancouver, British Columbia,
Vancouver, British Columbia, Canada
Canada
Introduction.. ....................................................
I. Introduction .................................................. 223
223
II. Determination and Differentiation ..............................
11. Sex Determination ............................ 225
225
A. Genotypic
Genotypic Sex Sex and and Sex Sex Chromosomes
Chromosomes.. . . .........................
....................... 225
B. Models
B. Determination ..................................
Models of Sex Determination ................................ 226
226
C.
C. Models Differentiation ..................................
Models of Sex Differentiation ................................ 229
229
D. Summary ....................................................
D. Summary .................................................. 241
241
III. Hormonal Sex Control
111. Control.. . . .. .. .. .. .. .. ....... .. .. .. .. .. .. .. . . . . . . . .. .. .. .. .. .. ..... .. .. .. .. . . . . . 242
A. Management .................................................
A. Management ............................................... 243
243
B. Treatment ...................................................
B. Treatment ................................................. 250
250
C. Evaluation
C. Evaluation.. . . .. .. .. .. .. .. .. .. .. . . . ...........................
................................... 268
268
IV. Economically Important Species. Species. ...................................
................................. 268
268
A. Cichlids . . . . . . . . . . . .. .. .. .. .. .. .. .. .. . . . . . . . . . . . . . . . . . . . .. .. .. .. .. ....... .. .. .. .. . 269
269
B. Salmonids . . . . . . . . . ..........................................
........................................ 276
C. Cyprinids .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
C. Cyprinids 288
288
Conclusions .....................................................
V. Conclusions ................................................... 290
290
References .. .. .. . . . . . . . . . .. .. .. .. .. ......... .. .. .. .. .. .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References 291
291
I. INTRODUCTION
INTRODUCTION
Studies involving experimental sex manipulation by hormones in fish

have become increasingly popular. This is attributable largely to the tremen
tremen-
dous economic potential that sex-control techniques have for the culture of
economically important species of fish. For the purposes of this discussion,
hormonal sex control refers only to the general control of sexual processes
which
which can
can be achieved
achieved byby manipulating
manipulating gonadal
gonadal sex.
sex. The
The hormones
hormones pri
pri-
marily involved in experimental studies of the manipulation of gonadal sex
have
have been
been the sex
sex steroids.
steroids.
The first
first major
major proliferation
proliferation of
of studies
studies directed
directed toward
toward the
the influence
influence of
of
hormones on the gonads of fish occurred in the late 1930s
1930s and early 1940s.
1940s.
223
223
FISH PHYSIOLOGY. VOL. IXB
PHYSIOLOGY, VOL. Copyrig
Copyrightht ©
0 1983
1983 by Academic Press.
Press, Inc.
Inc.
All rights
rights of reproduction in
in any form reserved.
reserved.
ISBN 0·12-350429-5
0-12-350429-5
224 GEORGE A.
GEORGE A. HUNTER
HUNTER AND
A N D EDWARD M.. DONALDSON
EDWARD M DONALDSON
Although
Although most most ofof these
these studies
studies examined
examined the the effects
effects ofof the
the sex
sex steroids
steroids on
on
secondary sexual characteristics, several suggested the possibility that func func-
tional
tional sex
sex inversion
inversion could
could bebe achieved
achieved in fish. In
in fish. In the
the following
following 22 decades
decades the
the
efficacy
efficacy of hormonal sex control was demonstrated in several gonochorist
species.
species. Within
Within this
this period
period anan extensive
extensive series
series of
of studies
studies were
were conducted on on
the
the medaka,
medaka, Oryzias Zutipes (Yamamoto,
Oryzius latipes (Yamamoto, 1953,1953, 1955,
1955, 1958,
1958, 1959a,b, 1961,
1959a,b, 1961,
1962,
1962, 1964a,b,
1964a,b, 1965,
1965, 1967,
1967, 1968;
1968; Yamamoto et al. al.,, 1968;
1968; Yamamoto
Yamamoto and
Matsuda,
Matsuda, 1963; 1963; Yamamoto
Yamamoto and and Suzuki,
Suzuki, 1955),
1955), which
which formed
formed the basis of
the basis of aa
comprehensive
comprehensive review review of of the
the subject
subject (Yamamoto,
(Yamamoto, 1969).
1969). Synthesizing
Synthesizing the
results of these numerous studies of the effect of gonadal steroids on sex
differentiation
differentiation in in fish
fish with
with the concept
concept of of aa dual
dual inductor
inductor system
system ofof sex
sex
differentiation
differentiation proposed
proposed for for the
the amphibians
amphibians by by Witschi
Witschi (1929),
(1929), Yamamoto
Yamamoto
concluded that the sex steroids, androgens and estrogens, were in fact the
respective male male and
and female
female sex
sex inducers
inducers responsible
responsible for for gonadogenesis
gonadogenesis in in
fish.
fish. Further, he laid down specific criteria for successful successful treatment
application.
The establishment of an effective protocol for sex manipulation in fish
marked a divergence between those studies concerned with the fundamental
aspects of hormonal sex control and those directed toward the optimization
of treatment, usually in economically important species. The former studies,
which were primarily concerned concerned with the elucidation of the role of sex
steroids with respect to sex determination and sex differentiation, were also
reviewed by Vanyakina (1969) (1969) and later by Harrington
Harrington (1974).
(1974). Of additional
value are several publications dealing with the role of sex steroids in induced
or natural sex inversion in hermaphroditic
hermaphroditic species (Chan,(Chan, 1970,
1970, 1977;
1977; Chan
et al. 1975; Reinboth, 1970,
al.,, 1975; 1970, 1972).
1972).
Schreck (1974)
(1974) provided the first review of the literature in the light of of
the newly established economic objectives. Several recent articles have
elaborated the specific
specific genetic and hormonal techniques applied in general
(Yamazaki,
(Yamazaki, 1983) 1983) or specifically
specifically to the economically important cichlids (Shel (Shel-
ton
ton et al.al.,, 1978;
1978; Guerrero,
Guerrero, 1979)
1979) and
and salmonids
salmonids (Donaldson
(Donaldson and and Hunter,
Hunter,
1982a).
1982a).
A discussion of hormonal sex control inherently involves consideration of
the
the assumptions on on which
which the
the action
action of
of the
the hormone
hormone is is based,
based, ultimately
ultimately the
mechanisms of sex determination and sex differentiation.
differentiation. Although pursued
intensively during the 1900s, 19OOs, a unifying model of sex determination and sex
differentiation has proven illusive. This is, perhaps, not surprising in light of of
the bewildering diversity of sexual expression found within the vertebrates,
particularly the largest group the Pisces. Therefore, the discussion first con con-
centrates
centrates on on the
the theoretical
theoretical context
context within
within which
which hormonal
hormonal sex sex control
control stud
stud-
ies are conducted. Second,Second, the various components of hormonal sex control
studies are examined with particular reference to those factors which influ-
5.
5. HORMONAL
HORMONAL SEX
SEX CONTROL
CONTROL AND
AND ITS
ITS APPLICATION
APPLICATION TO
TO FISH
FISH CULTURE
CULTURE 225
ence treatment success.

success. Finally, studies that have involved several eco
eco-
nomically important species
species are discussed in detail.
It is notable that the majority of studies on hormonal sex control have
involved gonochorist species. Therefore, these species are given emphasis.
Hermaphroditic species are considered where applicable;
applicable; however, these
species are discussed in greater detail in Chapter 4, this volume.
II.
11. SEX DETERMINATION AND
DIFFERENTIATION
DIFFERENTIATION
Within the Pisces, a wide range of reproductive strategies

strategies are encoun
encoun-
tered.
tered. The great majority of fish are either differentiated or undifferentiated
undifferentiated
gonochorists. However, numerous species exhibit synchronous, pro pro-
togynous, or protandrous hermaphroditism. Further, a number of species
are capable of gynogenetic reproduction
reproduction.. The diversity of physiological re
re-
productive systems and ethological sexes
sexes has been the topic of numerous
reviews including Atz (1964),
(1964), Yamamoto (1969),
(1969), Chan (1970,
(1970, 1977),
1977), and
Reinboth (1970).
(1970). Although it is
is generally accepted that in fish, as in other
vertebrates, sex has a genetic basis, the specific mechanisms by which genet
genet-
ic sex is determined and expressed are unclear.
A. Genotypic
Genotypic Sex
Sex and
and Sex
Sex Chromosomes
Chromosomes
Among the vertebrates the evolutionary tendency has been to aggregate

all those genes responsible for sexual development onto a single pair of
heteromorphic sex chromosomes, thereby providing a vehicle for the inheriinheri-
tance of sex as a Mendelian trait. The two two basic sex chromosomal systems
which
which have
have thus
thus evolved
evolved are
are either
either male
male heterogametic-female
heterogametic-female homogame
homogame-
tic (XY:XX)
(XY:XX) as in the case of the mammals or male homogametic-female
homogametic-female
heterogametic (ZZ:WZ)
(ZZ:WZ) as in the birds (Ohno,
(Ohno, 1967).
1967).
Approximately 10001000 species of fish have been examined cytologically.
cytologically.
The results of these investigations indicate that very few of these species
contain
contain easily
easily discernable
discernable heteromorphic
heteromorphic sex sex chromosomes
chromosomes (Voronstov,
(Voronstov,
1973; Yamazaki,
1973; Yamazaki, 1983).
1983). However, recent advances in cyotogical
cyotogical techniques
have permitted the identification of heterosomes in species such as the
rainbow trout, Salmo gairdneri, which are barely discernable from auto
S a l m gairdneri, auto-
somes and are presumably in the early stages of heteromorphic evolution
(Thorgaard, 1977). Both male and female digamety systems
(Thorgaard, 1977). systems have been dem
dem-
onstrated as well as several variations of these basic models including species
with multiple sex chromosomes.
chromosomes. Overall, the XY:XX
XY:XX system appears to be
most prevalent in fish (Yamazaki,
(Yamazaki, 1983).
1983).
226 GEORGE
GEORGE A.
A. HUNTER
HUNTER AND
A N D EDWARD
EDWARD M.
M. DONALDSON
DONALDSON
Although
Although in in the majority
majority ofof species
species heterosomes
heterosomes are are not
not distinguishable
distinguishable
by
by cytological
cytological techniques,
techniques, the presence
presence ofof heterosomal
heterosomal systems
systems have
have been
been
demonstrated
demonstrated by by genetic
genetic techniques.
techniques. Aida
Aida (1921,
(1921, 1936)
1936) first
first demonstrated
demonstrated
sex linked color genes and the male heterogametic-female
heterogametic-female homogametic sex sex
chromosomal system in the medaka. Vanyakina Vanyakina (1969)
(1969)and Yamamoto
Yamamoto (1969)
(1969)
have
have reviewed
reviewed the the use
use of
of similar
similar techniques
techniques to to describe
describe the heterosomal
heterosomal
systems
systems inin several
several tropical
tropical fish,
fish, notably
notably in
in the
the family
family Poeciliidae.
Poeciliidae. Yamamoto
Yamamoto
(1969)
(1969)also
also provides a brief,
brief, but interesting,
interesting, review of the early work involvinvolv-
ing inter- and intraspecific matings which has provided much of our current
knowledge
knowledge of of these systems. Of
these systems. Of particular
particular interest
interest is is the
the demonstration
demonstration of of
both XX:XY
XX:XY and ZZ:WZ ZZ:WZ (YY:WY) systems in the M exican and British Hon
Mexican Hon-
duran races of platyfish Xiphophorus (Platypoecilus) maculatus (Gordon,
(Platypoecilus) maculatus
1947;
1947; Kallman,
Kallman, 1965).
1965). Similar
Similar systems
systems have
have been
been reported
reported forfor Oreochromis
Oreochromis
mossambicus
mossambicus (Tilapia rnossambica) (Hickling,
(Tilapia mossambica) (Hickling, 1960).
1960). Bull
Bull and
and Charnov
Charnov
(1977)
(1977) have explored the possible transition from male to female hetero hetero-
gamety or vice versa through a intermediate polygenic system of sex deter deter-
mination. More recently, analysis of the sex of progeny following gynogen gynogen-
esis has proven useful for the determination of of female homogamety in
several species including grass carp, Ctenopharyngodon
Ctenopharyngodon idella (Stanley, (Stanley,
1976),
1976), common carp, Cyprinus carpio (Nagy (Nagy et al.al.,, 1978, 1981), and coho
1978, 1981),
Oncorhynchus kisutch (Refstie et al.
salmon, Oncorhynchus al.,, 1982).
1982). A detailed examination
of this technique
technique is presented in Chapter 8, this volume. Further, the analy analy-
sis of the progeny produced by the matings of hormonally sex-inverted and
sis
untreated individuals, first described in the medaka by Yamamoto Yamamoto (1953),
(1953),
has been used effectively for the identification of heterosomal systems
(Table
(Table I).
I).
The control
control over
over sex
sex determination
determination exerted
exerted by by mechanisms
mechanisms associated
associated
with the sex chromosomes is relatively strict within the higher vertebrates.
This also appears to be the case in a number of fish species examined.
However,
However, in in several
several species
species that
that have an
an apparent
apparent heterosomal system, sex sex
determination does not appear to be strictly bound to the sex chromosomes.
B. Models of
of Sex Determination
Several models of sex determination have been proposed since the dis
dis-
covery early in the century of
of the sex chromosomes. The models proposed
for the vertebrates, developed primarily from studies on the amniotes have
been based either on chromosomal or genic inheritance.
l. CHROMOSOMAL
1. CHROMOSOMAL INHERITANCE
INHERITANCE
Sex determination based on chromosomal inheritance was proposed
proposed by
Mittwoch (1971)
(1971) for the higher
higher vertebrates. The model stated that the pres-
5.
5. HORMONAL
HORMONAL SEX
SEX CONTROL AND ITS
CONTROL AND ITS APPLICATION
APPLICATION TO
TO FISH
FISH CULTURE
CULTURE 227
Table
Table I
Demonstration
Demonstration of
of Heterosomal Systems by tbe
the Mating of Untreated
Untreated and Sex-Inverted
Sex-Inverted Individuals
Species
Species Chromosomal
Chromosomal mechanism
mechanism Reference
Reference
Oryzias
0y z i a s latipes XX:XY
xx:XY Yamamoto
Yamamoto (1953)
(1953)
Oreochromis mossambicus
mossambicus XX:XY
xx:XY Clemens and
Clemens and Inslee
Inslee (1968)
(1968)
Carassius
Carassius auratus XX:XY
xx:XY Yamamoto
Yamamoto and and Kajishima
Kajishima (1969)
(1969)
Oreochromis niloticus
niloticus XX:XY
xx:XY Jalabert al. ((1974)
Jalabert et al. 1974)
H emihaplochromis multicolor
Hemihaplochromis XX:XY
xx:XY Hackmann
Hackmann and and Reinboth
Reinboth (1974)
(1974)
Oreochromis aureus
aureus wz:zz
WZ:ZZ Guerrero (1975),
(1975), LiuLiu (1977)
(1977)
Poecilia reticulata XX:XY
xx:XY Takahashi
Takahashi (19715
(197%) a)
Salmo gairdneri XX:XY
xx:XY Okada al. (1979),
Okada et al. (1979),
Johnstone
Johnstone et al.al. (1979a)
(1979a)
Oncorhynchus kisutch
Oncorhynchus kisutch XX:XY
xx:XY Hunter al. (1982a)
Hunter et al. (1982a)
Oncorhynchus
Oncorhynchus tsawytscha XX:XY
xx:XY Hunter al. (1983)
Hunter et al. (1983)
ence or absence of the whole Y or W chromosome determines

determines the respective
dominant male or female sex. sex. The resulting differential chromosome vol vol-
umes along with Y- Y- or W-linked RNA synthesis were presumed to induce
gonadogenesis
gonadogenesis mediated by a higher mitotic rate in the heterogametic sex. sex.
In mammals and birds heterogamety had been previously previously correlated with
early sex differentiation (Hamilton,
(Hamilton, 1965).
1965).
This model probably does not apply to the teleosts. First, as previously
mentioned, the majority of fish species do not have heteromorphic chromo chromo-
somes. Second, early differentiation of germ cells apparently does not corre
somes. Second, corre-
late with the heterogametic sex. (1965) reported early
sex. Eckstein and Spira (1965)
differentiation in the female cichlid, Oreochromis aureus (Tilapia
(Tilapia aurea),
aurea),
which has been demonstrated
demonstrated to be heterogametic (Guerrero, 1975). 1975). How
How-
ever,
ever, differentiation occurs first in the homogametic female Oryzias
O y z i a s latipes
(Satoh and Egami, 1972;1972; Onitake, 1972;
1972; Quirk and Hamilton, 1973),
1973), the
cichlid, Oreochromis mossambicus (Nakamura and Takahashi, 1973)
cichlid, 1973),, and
'
the goldfish, Carassius auratus (Nakamura, 1978). Early differentiation of
(Nakamura, '1978).
the female has also
also been reported in the cichlid, Hemihaplochromis multi multi-
color (Muller,
(Miiller, 1969),
1969), Ti tapia zillii (Yoshikawa
Tilapia (Yoshikawa and Oguri, 1978),
1978), the trout,
Salmo trutta (Ashby, 1957), Ctenopharyngodon idella, (Shelton and Jensen,
(Ashby, 1957),
1979), and the salmonids, Oncorhynchus masou,
1979), masou, Oncorhynchus keta, and
leucomaenis (Nakamura, 1978),
Salvelinus leucomuenis
Salvelinus 1978), and the threespined stickleback,
Gasterosteus aculeatus (Shimizu
(Shimizu and Takahashi, 1980). Early differentiation
Takahashi, 1980).
of the male in a differentiated gonochorist teleost has not been reported.reported.
Although a correlation between hetero- or homogamety is not evident, evident, de de-
veloping germ cells of many species examined do demonstrate a differential
(1972) observed that following the oral administration
mitotic rate. Onitake (1972)
228 GEORGE A. HUNTER
GEORGE A. HUNTER AND
A N D EDWARD M. DONALDSON
EDWARD M. DONALDSON
of estrone to genetically male medaka the germ cells began an an atypical rapid
proliferation which preceded sex differentiation. Onitake suggested that the
rapid mitotic increase was necessary to to the process of differentiation. In an
intensive examination of factors affecting sex inversion in the cyprinodont,
mumoratus, Harrington
Rivulus marmoratus, Harrington (1975)
(1975) reported
reported that
that rearing
rearing temperatures
temperatures
of
of 19°C
19°C or
or 26°C
26°C resulted
resulted in
in higher
higher proportions
proportions of
of primary
primary males
males and
and her
her-
maphrodites, respectively,
respectively, and these results were correlated with mitotic
activity in the developing gonad. The role of differential mitotic growth in
the
the process
process of
of sex
sex differentiation
differentiation remains
remains to
to be determined.
determined.
2. GENIC INHERITANCE
2. GENIC INHERITANCE AND POLYGENIC
AND POLYGENIC SEX
SEX
DETERMINATION
DETERMINATION
The concept
concept ofof genic
genic balance
balance established
established byby Bridges
Bridges (1925,
(1925, 1936)
1936) was
was
initially modified by Winge (1934)(1934) to apply to sex determination in Poecilia
(Lebistes).Winge proposed that the X and Y chromosomes contained superi
(Lebistes). superi-
or
or male
male and
and female
female sex-determining
sex-determining genes.
genes. Minor
Minor male
male and
and female
female sex
sex-
determining factors were held in the autosomes.autosomes. Normally the autosomal
genes are maintained in balance allowing sex to be determined by the het- het
erosomal mechanism. However, in exceptional individuals, autosomal com com-
binations or recombinations may occur that result in an excess excess of autosomal
factors
factors of one sex capable of overriding the heterosomal mechanism. The
outcome is an individual with a phenotypic sex differing from its heterosomal
sex. Similar polygenic sex-determining systems have been hypothesized for
sex.
several
several xiphophorin
xiphophorin fishes
fishes (Kosswig
(Kosswig and
and Oktay,
Oktay, 1955;
1955; Kosswig,
Kosswig, 1964;
1964; Anders
and
and Anders, 1963;1963; Dzwillo
Dzwillo and
and Zander,
Zander, 1967), lutipes (Yamamoto,
1967), Oryzias latipes (Yamamoto,
1963, 1969), and Betta
1963, 1969), Bettu splendens (Lowe and Larkin, 1975). 1975). Exhaustive re re-
views
views of of the
the early
early research
research onon the development
development of of the
the polygenic
polygenic concept
have been provided by Kosswig (1964) (1964) and Yamamoto (1969).(1969). Yamamoto
(1969)
(1969)summarized
summarized the results obtained
obtained byby asserting that
that "sex
“sex determination
determination
in gonochorists is polyfactorial
polyfactorial with or without epistatic sex genes in sex
chromosomes.
chromosomes.”" When the total strength of the male factors exceeds that of of
the female factors, the zygote will be male and vice versa. Therefore, de de-
pending on the control exerted by epistatic genes on the sex chromosomes
the
the stability
stability of
of sexual
sexual determination
determination will vary
vary from
from species to to species.
species.
Within the tilapia, interspecific crosses of several types result in non non-
Mendelian sex ratios (Pruginin
(Pruginin et al. 1975). Avtalion and Hammerman
al.,, 1975).
(1978)
(1978)examined an extensive series of hybrid crosses between homozygous
(Tilapiu) hornorum males and Oreochromis mossambicus
Oreochromis (Tilapia)
females and between homozygous Oreochromis macrochir mucrochir males and
Oreochromis niloticu8 (Tilapia niliotica) females conducted by Chen (1969)
niloticus (Tilapia (1969)
and
and Jalabert et al.al. (1971),
(1971), respectively. Assuming that the Y and Z chromo-
HORMONAL SEX
5. HORMONAL SEX CONTROL
CONTROL AND
AND ITS
ITS APPLICATION
APPLICATION TO
TO FISH
FISH CULTURE
CULTURE 229
somes were identical and that an autosomal influence exists, Avtalion and
Hammerman proposed that the simplest sex-determining mechanism in
Hammerman
tilapia would consist of three gonosomes (X, (X, W, Y) in pairs of of two (XX, XY,
XW, WY, WW, and YY) YY) similar to the system proposed proposed for Xiphophorus
Xiphophorus
macuiatus by Gordon (1947)
maculatus (1947) but with the addition of of a pair ofof autosomes.
(AA,
(AA, Aa, and aa). The resulting 18 possible possible combinations of of autosomes and
gonosomes could be used to predict predict the results obtained by Chen, but not of of
Jalabert et al. (1971). Hammerman and Avtalion (1979)
ai. (1971). (1979) suggested that the
results of of Jalabert et al. (1971) could be explained by assigning different
ai. (1971)
strengths to each of of the chromosomes involved. involved. Analysis of of the sex-deter-
sex-deter
mining mechanism has been hampered because of of the fact that in tilapia, sex
chromosomes cannot be identified by karyotypic karyotypic analysis and no sex-linked
color markers are present. Avtalion et al. ai. (1975)
(1975) and Hardin (1976) (1976) have
identified a male-specific electrophoretic marker in adult male Oreochromis
(Sarotherodon)
(Sarotherodon)aureus. aureus. However, it has only been found in sexually mature
individuals and may, therefore, be hormonally induced. The usefulness of
individuals
these
these markers
markers is is dependent
dependent on on whether
whether theythey are
are sex
sex linked
linked asas opposed
opposed to to sex
sex
limited. For further review, the reader is referred to Wolhfarth and Hulata
(1981)
(1981) and Avtalion (1982). (1982).
The
The concept
concept of of aa polygenic
polygenic mechanism
mechanism in in which
which autosomal
autosomal genesgenes may may
play
play aa decisive
decisive role
role inin the
the process
process of of sex
sex determination
determination has has dominated
dominated the the
research
research on on fish.
fish. However,
However, no no single
single polygenic
polygenic system
system has
has been
been capable
capable of of
being
being reconciled
reconciled withwith allall empirical
empirical datadata (Harrington,
(Harrington, 1974).
1974). Despite
Despite excep
excep-
tions,
tions, the
the concept
concept of of aa polygenic
polygenic system
system remains
remains suited
suited to
to the
the majority
majority of of
data
data from
from studies
studies of of fish.
fish. An
An interesting
interesting example
example is is the
the recent
recent report
report by by
Streisinger
Streisinger et al. (1981).
et al. (1981).In In this
this study,
study, clones
clones ofof homozygous
homozygous diploid
diploid female
female
zebrafish,
zebrafish, Brachydanio rerio, rerio, were
were produced
produced by by hydrostatic
hydrostatic pressure
pressure or or
temperature
temperature shocksshocks administered
administered to to ova,
ova, activated
activated byby ultraviolet
ultraviolet (UV)-treat
(UV)-treat-
ed
ed sperm.
sperm. SomeSome of of the
the clones
clones were
were predominately
predominately male male and
and produced
produced high high
proportions
proportions of of males
males in in subsequent
subsequent generations.
generations. Such Such results
results are
are difficult
difficult toto
reconcile
reconcile with
with any
any ofof the
the current
current models
models of of sex
sex determination
determination otherother thanthan aa
polygenic
polygenic system.
system. FromFrom the the analysis
analysis of of single
single gene
gene mutations
mutations it it is
is clear
clear that
that
even
even inin the
the mammals,
mammals, genetic genetic sex
sex cannot
cannot be be explained
explained by by the
the constitution
constitution of of
the
the sex
sex chromosomes
chromosomes alone. alone. Autosomal
Autosomal genes genes which
which may
may play
play an
an important
important
role
role in
in gonadal
gonadal differentiation
differentiation have have been
been reported
reported in in pigs
pigs Gohnston
(Johnston et et ai.
al.,,
1958),
1958), goats
goats (Hamerton
(Hamerton et al.,, 1969),
et ai. 1969), and
and mice
mice (Cattanach
(Cattanach et al.,, 1971).
et al. 1971).
C.
C. Models
Models of
of Sex
Sex Differentiation
Differentiation
Several
Several models
models of
of sex
sex differentiation
differentiation have
have been
been proposed
proposed which
which are
are com
com-
patible
patible with
with single
single or
or multiple
multiple gene
gene action.
action.
230
GEORGE A. AND
HUNTER A N D EDWARD M.. DONALDSON
EDWARD M DONALDSON
1.
1. It-Y
H-Y ANTIGEN
ANTIGEN
Sex differentiation
differentiation based on the action of an individual gene or genes has
recently been
been given considerable
considerable support
support in in the aves
aves and
and mammalia
mammalia with with the
the
discovery ofof the male specific histocompatibility-Y
male specific histocompatibility-Y chromosome
chromosome (H-Y) (H -Y) anti-
anti
gen.
gen. The antigen,
antigen, first
first discovered
discovered inin mice by by Eichwald and and Silmser
Silmser (1955)
(1955)isis
ubiquitously
ubiquitously associated
associated with with the heterogametic
heterogametic sex sex in
in mammalian
mammalian and and some
some
nonmammalian
nonmammalian vertebrates.
vertebrates. TheseThese observations
observations havehave ledled toto the
the hypothesis
hypothesis
that
that the
the antigen
antigen plays
plays aa major
major role
role in
in gonadal
gonadal sexsex differentiation.
differentiation. The The current
current
hypothesis is that a gene or genes on the Y or W chromosome code for the H- H
Y antigen
antigen and
and the presence
presence of of the antigen
antigen on on the
the surface
surface of of somatic
somatic cells
cells of
of
the indifferent gonad results in the development of the heterogametic gonad
(Wachtel et al. 1975; Zenzes et al.
aZ.,, 1975; 1978). However, the mechanics of the
al.,, 1978).
action or regulation of H-Y antigen with respect to gonadal differentiation
have yet to be discovered.
discovered. Ohno et al. al. (1978)
(1978) have demonstrated that the
membrane H-Y antigen receptor is present only on gonadal cells in both
sexes. Ohno
sexes. Ohno andand co-workers
co-workers usedused H-Y antibody
antibody to to strip
strip H-Y
H-Y antigen
antigen from
from
mice
mice gonadal cells of known genetic constitution. Culture of the cells re re-
vealed that removal of of the H-Y antigen resulted in the development of of
spherical aggregates that resembled ovarian follicles. follicles. Un stripped cells
Unstripped
formed cylindrical tubular structures morphologicallymorphologically similar to semi semi-
niferous tubules. Ohno and co-workers concluded that there was a causal
relationship between the presence of the H-Y antigen on the surface of
gonadal cells and the development of the testes testes.. They further suggested that
the H-Y antigen could act as a short-range hormone-inducing specific specific gene
expression.
al. (1979)
Miiller et al. (1979) inverted the sex of of chicken embryo testes (ZZ) with
estrogen. He found that the normally H-Y (W) (W) antigen-negative testes were
positive after sex inversion, which indicated that the gene for the antigen
was expressed in the absence of of the W chromosome and, therefore, must be
present in both sexes, supporting the report by Ohno et al. al. (1978).
(1978).Assuming
the the presence of the H-Y (W) (W) antigen was responsible for the formation of
the ovary, M iiller et al.
Muller al. (1979)
(1979) suggested that the hormone-induced sex
inversion was an indirect effect mediated by H-Y (W) (W) antigen. They also
indicated that there was a correlation between morphogenetic changes and
H-Y (W)
(W)titer in the gonad.
gonad. Therefore, Miiller
Muller and co-workers suggested that
the induction of the H H-Y-Y (W)
(W) antigen by estrogen did not operate as a strict
on-off
on-off switch mechanism, again similar to the conclusion arrived at by Ohno
al. (1978).
et al. (1978). Similarily,
Similarily, induced H-Y antigen-positive ovarian tissue in the
ovaries of sex-reversed (ZZ) (ZZ) individuals has been demonstrated in Zenopus
laeuis (Wachtel et al.
laevis al.,, 1980)
1980) and Pleurodeles waltlii (Zaborski
(Zaborski and Andrieux,
1980).
1980). In a series of experiments reviewed by Zaborski (1982) (1982) involving
5.
5. HORMONAL
HORMONAL SEX
SEX CONTROL AND ITS
APPLICATION TO
TO FISH
FISH CULTURE
CULTURE 231
231
amphibians,
amphibians, H H-Y-Y expression was repressed in Pleurodeles waltlii ovaries ovaries
(ZW)
(ZW) or Rana ridibunda testes (XY) (XY) by the administration
administration of dihydrotesto
dihydrotesto-
sterone or estradiol,
estradiol, respectively.
respectively. Therefore;
Therefore; although
although in the lower verte verte-
brates the sex hormones
hormones appear to be inducers of H H-Y-Y antigen,
antigen, an inhibitory
control of its expression
expression is also suggested.
suggested. Because
Because both androgens
androgens and
estrogens
estrogens are produced in each sex, sex, the nature of hormonal controlcontrol of the
antigen system is presumed to be quantitative.
antigen system However, recent research
quantitative. However,
with rainbow trout gonadal
gonadal homogenates
homogenates suggests
suggests that during the period of
sex differentiation
differentiation the gonads
gonads may not be capable of estrogen production
(van
(van den Hurk et al. al.,, 1982).
1982).
Sex-specific
Sex-specific antigens have been detected in nonmammalian vertebrates
including
including birds (Bacon,
(Bacon, 1970;
1970; Wachtel
Wachtel et al.al.,, 1975;
1975; Muller et al. al.,, 1980),
1980),
amphibians pipiens, Xenopus laevis (Wachtel
amphibians such as Rana pipiens, (Wachtelet al. 1975), and
al.,, 1975),
reptiles such as Emys orbicularis (Zabroski
(Zabroski et al. 1979). Recently,
al.,, 1979). Recently, Shalev
Shalev
and Huebner (1980),
(1980), citing unpublished work, have reported the presence
of the antigen in invertebrates.
The presence of the antigen system has also been demonstrated in sever sever-
al species
species of fish.
fish. Muller and Wolf Wolf (1979)
(1979) tested absorption of mammalian
mammalian
anti-H-Y
anti-H-Y antiserum in the teleosts teleosts Salvelinus alpinus,
alpinus, Salmo
Salmo gairdneri,
Rutilus rutilus,
rutilus, Carassius auratus, Barbus tetrazona, Poecilia reticulata
(Lebistes reticulatus), and Xiphophorus helleri.
(Lebistes reticulatus), helleri. The gonads
gonads of the more
primitive orders Ostariphysi, represented by Rutilus, Carassius,
Carassius, and Bar
Bar-
bus, and Isospondyli,
Zsospondyli, represented by Salvelinus and Salmo,Salmo, absorbed anti
anti-
H-Y antiserum; however,
however, a clear sexsex difference
difference was not observed.
observed. In the
more advanced poeciliids Poecilia and Xiphophorus, the anti-H-Y
advanced poeciliids anti-H-Y antiserum
was absorbed almost exclusively
exclusively by the gonadal
gonadal tissues of the male but not
the female.
female. AsAs previously mentioned, Poecilia, Salmo, and Carassius all
Poecilia, Salmo,
have male heterogametic-female homogametic systems. systems. The sex-determin
sex-determin-
ing mechanism in Xiphophorus
Xiphophorw does not appear to be strictly heterosomal
(Kosswig,
(Kosswig, 1964).
1964). The presence of the antigen systemsystem in Poecilia reticulata
has been recently confirmed by Shalev Shalev and Heubner (1980).
(1980). Further sup
sup-
port for
for a sex-specific
sex-specific antigen expression in advanced species
species comes
comes from
research by Pechan et al. al. (1979).
(1979). In this study, anti-H-Y antiserum absorp
absorp-
tion was found exclusively
exclusively in male cells of Xiphophorus maculatus,
maculatus, Haplo
Haplo-
chromis burtoni, Oryzias latipes, and several
several tilapia hybrids.
hybrids. The H-Y anti
anti-
gen was detected most readily in Xiphophorus maculatus males (YY). Male
heterogamety has not yet been determined for the cichlid Haplochromis
Haplochromis
burtoni.
burtoni.
The presence of of the sex-specific
sex-specific antigen system in fi sh is certainly
fish certainly in
in-
teresting from an evolutionary
evolutionary perspective. Further examination
examination of the anti
anti-
gen system
system in the lower vertebrates may provideprovide an answer to the basic
question
question of when in the course
course of evolution
evolution the antigen system
system assumed a
232
GEORGE A. AND EDWARD
HUNTER AND M.. DONALDSON
EDWARD M DONALDSON
primary
primary role
role in
in the
the process
process ofof sex
sex differentiation.
differentiation. The
The demonstration
demonstration H-YH-Y
antigen
antigen expression
expression inin the
the ovaries
ovaries of
of homozygous
homozygous chickschicks and
and amphibians
amphibians sex
sex
inverted
inverted with
with estradiol
estradiol suggests
suggests aa major
major rolerole of
of the
the antigen
antigen in
in the
the process
process of
of
hormonal
hormonal sex
sex inversion
inversion inin these
these groups.
groups. Examination
Examination of of the
the role
role of
of the
the
antigen
antigen system
system inin the natural or
the natural or hormonally
hormonally induced sex inversion
induced sex inversion of
of both
both
hermaphroditic
hermaphroditic andand gonochorist
gonochorist fish
fish is
is aa promising
promising area
area of
of study.
study.
2. THE
2. THECORTICOMEDULLARY
CORTICOMEDULLARYINDUCTOR
INDUCTORMODELS
MODELS
The corticomedullary inductor model was proposed by Witschi (1929) (1929)to
explain sex differentiation in amphibians. Witschi observed that the primor
primor-
dium of the amphibian gonad, like those of of most vertebrates, is comprised of of
both
both an
an outer
outer cortex
cortex and
and inner medulla
medulla ultimately
ultimately derived
derived from
from the
the germinal
germinal
epithelium. During differentiation, either the cortex or the medulla devel devel-
ops at the expense of the other resulting in the development of an ovary or
testis, respectively. Witschi theorized
theorized that the genetic male and female
factors
factors embodied
embodied byby the balance
balance theory
theory of of sex
sex determination
determination were
were phe
phe-
notypically manifested in the dualistic character of the primordial gonad.
The action of these genetic factors results in the production of an embryonic
cortexin or medullarin, which in turn initiated ovarian and testicular differ
differ-
entiation, respectively. The existence of of these hypothetical inductors re re-
mains to be demonstrated. As a result, consideration of the suitability of of
Wistchi's
Wistchi’s model has remained a conceptual debate.
In later publications, Witschi (1965,
(1965, 1967)
1967) modifi ed his theory to include
modified
an
an antagonist action of the inductors. The theory of antagonism has been
illustrated in the amphibians by ablation of the dominant component of the
gonad which results in differentiation of the remaining component (Haffen,(Haffen,
1977).
1977). Witschi (1967)
(1967) has suggested that the interaction ofof the inductors is
similar to an immune reaction. Therefore, each has the capability of inhibit
inhibit-
ing or destroying the other.
other. Reinboth (1982)
(1982) has recognized the similarity
between Witschi's
Witschi’s (1967)
(1967)model and the model of mammalian sex differentia
differentia-
tion involving the H-Y antigen system and a presumptive ovarian factor. The
existence of the ovarian factor and the nature of its interaction with the H-Y
antigen system remain a subject of study and debate (Wachtel and Koo,
1981).
1981).
With regard to fish, the debate surrounding the dual-inductor concept
has centered on reconciliation of the model with the proposed unitary origin
of the teleost gonad and the common occurrence within the teleosts of
various forms of hermaphroditism.
various
The discrete topographical division of of the primoridal amphibian gonad
provided Witschi with strong support for his dual-inducer concept. This dual
embryonic nature appeared ideally suited to providing the separate chemical
HORMONAL SEX
CONTROL AND
AND ITS
ITS APPLICATION
APPLICATION TO
TO FISH
FISH CULTURE
CULTURE 233
environments necessary
environments necessary for for the
the divergent
divergent development
development of of either
either male
male or
or
female gonia. Within the fish, a dualistic structure structure of of the primordial gonad
has
has been reported in the elasmobranchs (Chieffi, (Chieffi, 1959).
1959). However, unlike
this group and the rest of of the vertebrates the gonads of of cyclostomes and
teleosts have reported to develop from a unitary
have been reported unitary primordium homolo-homolo
gous to the cortex of of other vertebrates
vertebrates (D’Ancona,
(D'Ancona, 1941,1941, 1949).
1949). D’Ancona’s
D'Ancona's
observations have
observations have received
received general support (Hoar,
general support (Hoar, 1969).
1969). However,
However, the the
reconciliation of of a dual-inductor
dual-inductor system with with a single primordial source has
been difficult.
been difficult. First,
First, neither D'Ancona nor
neither D’Ancona later investigators
nor later have been
investigators have been able
able
to explain how
to explain the development
how the development of aa single primordium could
single primordium could provide
provide aa basis
basis
for
for two distinct cell lines lines producing
producing two antagonistic
antagonistic inductors. Second,Second,
discrete
discrete male
male and
and female
female territories
territories are
are found
found as early as
as early as the
the first
first indication
indication
of
of sex
sex differentiation
differentiation in in many hermaphroditic species.
many hermaphroditic species. Further,
Further, although the
although the
classification
classification of many hermaphroditic
of many hermaphroditic species remains in
species remains in doubt, it is
doubt, it is clear
clear
that
that they
they are not restricted
are not restricted to to aa few isolated advanced
few isolated advanced families
families as
as suggested
suggested
by
by D'Ancona (1949). Smith
D’Ancona (1949). Smith (1975)
(1975)hashas provided
provided an an excellent
excellent review
review ofof their
their
distribution.
distribution. Because
Because of of these
these difficulties
difficulties and
and aa recognition
recognition of of the problems
problems
associated
associated with
with the
the examination
examination of of the
the peritoneal
peritoneal embryonic
embryonic differentiation
differentiation
of
of interrenal,
interrenal, nephric, and and gonadal
gonadal elements as as described
described by by Hardisty
Hardisty
(1965), Harrington
(1965), Harrington (1974,
(1974, 1975)
1975) has suggested that
has suggested that aa reevaluation
reevaluation of of the
single
single primordium
primordium hypothesis
hypothesis may may be in in order.
order.
An additional problem presentedpresented by the hermaphrodites is the method
by which
which the
the proposed
proposed antagonistic
antagonistic inductors
inductors maymay be compartmentalized
compartmentalized in in
aa gonad
gonad containing
containing bothboth testicular
testicular and
and ovarian
ovarian areas.
areas. The gonadal
gonadal organiza
organiza-
tion
tion of
of some
some species
species offers
offers aa certain
certain degree
degree of of spatial
spatial isolation
isolation in
in the case
case of
of
protogynous
protogynous or or protandrous
protandrous serranid
serranid or or sparid
sparid species
species (Atz,
(Atz, 1964)
1964) and
and the
the
synchronous
synchronous hermaphrodite
hermaphrodite Rivulus marmoratus (Harrington,
Riuulus mannoratus (Harrington, 1971).
1971). How
How-
ever,
ever, in
in the grouper Epinephelus, there
the grouper there appears
appears to to be
be nono discrete
discrete segrega
segrega-
tion
tion of
of male
male and
and female
female territories.
territories.
3.
3. SEX
SEX STEROIDS
STEROIDS
Although
Although the two two presumptive
presumptive inducers
inducers have
have never
never been
been identified,
identified,
Yamamoto
Yamamoto (1969)
(1969)concluded
concluded thatthat based
based on
on his
his work
work with Oryzias latipes the
with Oryzias the
two
two inductors,
inductors, which
which hehe termed
termed the
the gynotermone
gynotermone andand androtermone,
androtermone, were were
in
in fact
fact estrogens
estrogens andand androgens,
androgens, respectively.
respectively. Yamamoto's
Yamamoto’s synthesis
synthesis of
of the
the
hormone
hormone andand inductor
inductor models
models has
has dominated
dominated the
the work
work on
on sex
sex control
control in
in fish.
fish.
However,
However, thethe origins
origins ofof the
the hormonal
hormonal model
model are
are much
much older.
older.
In
In the
the six
six decades
decades since
since Lillie
Lillie (1917)
(1917)postulated
postulated sexsex steroid
steroid involvement
involvement
in
in his
his explanation
explanation of of the
the free-martin
free-martin effect
effect in
in cattle,
cattle, exhaustive
exhaustive studies
studies have
have
been
been conducted
conducted in in an
an attempt
attempt toto determine
determine thethe specific
specific role
role of
of hormones
hormones in in
the
the process
process ofof sex
differentiation. Numerous
Numerous experiments
experiments involving
involving classic
classic
HUNTER AND EDWARD M
M.. DONALDSON
DONALDSON
castration
castration and
and replacement
replacement have
have confirmed
confirmed that
that the
the sex
sex hormones
hormones mediate
mediate
the development of of secondary
secondary sexual
sexual characteristics
characteristics inin mammals
mammals andand birds
birds
Gost,
(Jost, 1965;
1965; Goldstein and Wilson, 1975).
1975). In both mammals (Price, 1970)1970)and
birds (Haffen, 1975), biological,
(Haffen, 1975), biological, biochemical, and histochemical tests indi indi-
cate hormonal activity in the indifferent gonad of the dominant sex. sex. Howev
Howev-
er, evidence for a role of the sex steroids in sex differentiation resulting from
steroid administration has been inconclusive. In the marsupials, Burns
(1950)
(1950) working with opposum, Didel phys virginiana,
Didelphys virginiuna, was able to demon
demon-
strate the formation of ovotestes under the influence of of estradiol administra
administra-
tion. However, in eutherian mammals, numerous experiments involving the
tion.
uioo or in vitro
in vivo uitro administration of exogenous sex steroids have been ineffec
ineffec-
tive (Burns,
(Burns, 1961;
1961; McCarrey and Abbott, 1979).1979).
Feminization as a result of estrogen administration to the male embryo
has been achieved in the chick (Narbaitz
(Narbaitz and De Robertis, 1970)1970) and quail
(Haffen, 1969); however, the inversions are often transitory. The administra
(Haffen, 1969); administra-
tion of a variety of androgens have produced either simple masculinizing
effects
effects or masculinizing and feminizing effects.
effects. Testosterone and its esters
act similarily to the natural hormones producedproduced by the embryonic male
gonad masculinizing the genital ducts but not influencing the female gonads. gonads.
Some androgens including androstanedione, androstenedione, androstene androstene-
diol, and trans-hydroandrosterone
truns-hydroandrosterone masculinize female genital ducts but fem fem-
inize male genital ducts and gonads (Haffen and Wolff, 1977). 1977). Numerous
studies have demonstrated that (1) (1)feminized male gonads can secrete a
hormone similar to the sex reversing hormone, (2) (2) embryonic gonadal secre
secre-
tions from the medulla which have the same effect as steroid hormones, and
(3)
(3) indifferent avian gonads synthesize and secrete steroids, steroids, all of which
Haffen and Wolff
Wolf€claimed support the steroid-inductor model. Somatic sex
inversion has been achieved in cultured embryonic left testes administered
exogenous androgens or estrogens (Carlon and Erickson, 1978). 1978). Carlon and
Erickson suggested that it is the absence of steroidogenesis during the indif indif-
ferent period which is necessary for testicular development.
Similarily,
Similarily, androgen administration to several species of reptiles has re re-
sulted in variable results (Haffen
(Haffen and Wolff, 1977). Estrogen administration
Wolff, 1977).
to the green lizard,
lizard, Lacerta
Lacertu viridis resulted in partial or complete inhibition
of testicular development in some individuals to produce an ovotestis and
complete inhibition in others to produce an ovary (Raynaud,(Raynaud, 1967).
1967).
Within amphibians a relatively large number of studies involving hor hor-
monally induced sex inversion have demonstrated that the administration of
exogenous estrogens and androgens results in functional feminization in
urodeles and masculinization of ranid anurans, respectively. However, para para-
doxical
doxical actions of steroid treatment have been reported in several species
(Burns,
(Burns, 1961;
1961; Haffen and Wolff,
Wolff, 1977).
1977).
HORMONAL SEX
CONTROL AND
AND ITS
ITS APPLICATION
APPLICATION TO
TO FISH
FISH CULTURE
CULTURE 235
amphibia, sex steroids

In teleost fish as in the amphibia, steroids are capable of of influencing
the course of of sex differentiation.
differentiation. Yamazaki (1983) (1983) reported
reported at least 15 15 species
in which functional sex inversion has been been achieved.
achieved. However, these species
are primarily gonochoristic teleosts within a small number number of of families.
families .
Chieffi. (1967)
Chieffi (1967) reported the effects of of androgens and estrogens on the elas- elas
mobranchs, primarily Scyliorhinus canicula.
mobranchs, canicula. Both estrogens and androgens
influence the differentiation
influence differentiation of the genital ducts; however, only estrogen
influences the gonad in this species producing prodUcing ovotestes.
ovotestes.
The small number of of hermaphroditic species which have been treated
with steroids to manipulate natural sex inversion have responded inconsis- inconsis
tently. Reinboth (1962,(1962, 1975)
1975) used a single 2-mg injection of of testosterone to
mimick sex inversion in several species of of protogynous hermaphrodite
(Labridae). He concluded that, in general, androgens induced pre-
wrasses (Labridae). pre
inversion in protogynous species, but this evidence alone did not
cocious sex inversion
support the steroid-inductor model (Reinboth, (Reinboth, 1970).
1970). Chen et al. (1977) (1977)
reported thatthat two of Epinephelus tauvina, also a pro
of three groupers, Epinephelus pro-
togynous
togynous hermaphrodite, fed 80 mg methyltestosterone over 30 days initi- initi
ated sex inversion.
inversion. Subsequently,
Subsequently, all 25 fish given a dosage of of 11 mg meth
meth-
yltestosterone/kg diet 3 3 times per week over a 2-month 2-month period underwent
sex
sex inversion.
inversion.
The
The rice field eel, Monopterus
Monopttwus albus, albus, has perhaps been the most inten inten-
sively studied protogynous hemaphrodite (Chan, (Chan, 1977;
1977; Chan et al. al.,, 1977).
1977).
Biochemical
Biochemical and histochemical techniques have demonstrated 3j3-hy 3P-hy-
droxysteroid dehydrogenase an
droxysteroid dehydrogenase and d 17j3-hydroxysteroid
17s-hydroxysteroiddehydrogenase
dehydrogenase activity
and
and aa large increase in
large increase in the
the production
production of of androgens
androgens during
during natural
natural sexsex inver
inver-
sion (Chan
(Chan et al.al.,, 1975;
1975; Tang et al.al.,, 1975).
1975). However,
However, attempts to manipulate
manipulate
the
the course
course of natural
natural sex inversion by
sex inversion by the
the administration
administration of of steroids
steroids have
have
been ineffective
ineffective (Tang(Tang et al. 1974; Chan et al.
al.,, 1974; al.,, 1977).
1977). These studies are
examined in detail in C hapter 4, this
Chapter this volume.
The achievement
achievement of functional
functional sex inversion in severalseveral gonochorist
gonochorist spe spe-
cies remains the most compelling evidence for steroid involvement in the
sex differentiation
differentiation of fish. fish. However,
However, as Reinboth (1970) (1970)has noted, it is not
possible to rule out a pharmacological
possible pharmacologicalrather than physiological
physiological action in this
process.
process. No studies
studies have examined
examined the mode of action of the steroids steroids in the
induced sex inversion
inversion of teleosts.
teleosts. In this regard,
regard, Vannini et al. al. (1975)
(1975)exam
exam-
ined the influence
influence of of actinomycin
actinomycin D D and
and puromycin,
puromycin, inhibitors
inhibitors of DNA DNA-
dependent RNA transcription RNA-dependent protein synthesis
transcription and RNA-dependent synthesis re re-
spectively,
spectively, on testosterone-induced sex sex inversion
inversion in Rana dalmatina
dalmatina tad tad-
poles.
poles. Both
Both antibiotics
antibiotics suppressed testosterone action. action. Vannini
Vannini and co-work
co-work-
ers
ers also
also reported that testosterone-induced sex sex inversion
inversion isis linked with RNA
synthesis.
synthesis. They
They havehave proposed that that the mechanisms
mechanisms of testosterone
testosterone action
action
involve
involve the derepression of latent male male genes
genes in the nuclei
nuclei of somatic
somatic tissue
tissue
236
HUNTER AND M .. DONALDSON
EDWARD M DONALDSON
which
which inin turn
turn causes
causes them
them to
to proliferate
proliferate as
as gonadal
gonadal medullary
medullary tissue.
tissue. Evi
Evi-
dence for the direct action of steroids and their specific
specific receptor molecules
on DNA and, thereby, transcriptional processes has been established
(O'Malley
(O’Malley and Schrader, 1976).
1976). Further, no studies have examined the po po-
tential sites of genetic control of hormonal action, specifically
specifically the partially
common biosynthetic pathway of the sex sex steroids or sex-specific
sex-specific receptor
sites on undifferentiated germ cells.
Therefore, additional supportive evidence for Yamamoto's
Yamamoto’s conclusions
conclusions
was confined to the specificity of sex steroids as exogenous sex inductors, the
very low effective dosage of sex steroids,
steroids, and the selective incorporation of
sex steroids into the differentiating gonad. However, based on similar evi
sex evi-
dence, later studies
studies have added uncertainty rather than support to Yamamo
Yamamo-
to's
to’s (1969)
(1969) hypothesis of a sex-steroid-inductor model.
Yamamoto based the specificity of sex-steroid action on the absence of
paradoxical
paradoxical effects and inactivity of corticoids as sex inducers in Oryzias
latipes. To date,
latipes. date, no studies have reported effective sex inversion using cor cor-
ticoids, although other chemicals such as N,N-dimethylformamide
N,N-dimethylformamide can modi modi-
fy gonadal differentiation (van den Hurk and Slof, Slof, 1981).
1981).
More recent studies have reported paradoxical
paradoxical effects
effects of
of sex steroid ad
ad-
ministration. Gresik and Hamilton (1977)
(1977) citing unpublished work by J. B.
Hamilton and D. D. Kantor report that the injection of eggs containing XX
or XY Oryzias latipes embryos with either methyltestosterone, estrone ace ace-
tate, or the synthetic progestin ethynodiol diacetate at low concentrations
favors testicular differentiation. High concentrations favor ovarian differ differ-
entiation. They also report that paradoxical
paradoxical sex inversion is more difficult to
achieve in YY
YY individuals than in XY individuals. Paradoxical
Paradoxical feminization in
Hemihaplochromis multicolor fry reared in
the cichlids was first observed in Hemihaplochromis
water containing testosterone propionate or methyltestosterone
methyltestosterone (Muller,
(Muller,
1969).
1969). Subsequently,
Subsequently, the addition of these steroids at 500 j.Lg/1 kg/l was also
found to have a paradoxical effect in other cichlids including Chichlasoma
Chichlasoma
heudeloti, and
biocellatium, Tilapia heudeloti,
biocellatium, and Oreochromis mossambicus (Hack (Hack-
mann, 1974).
1974). The effect was again demonstrated in Oreochromis mossam mossam-
bicus fed methyltestosterone
methyltestosterone at 1000
1000 mg/kg diet for a period of 50 days after
hatching
hatching (Nakamura,
(Nakamura, 1975).
1975). In
In genetic
genetic females,
females, oogenesis
oogenesis progressed,
progressed, alal-
though at a slower rate than control ovaries. The ovarian cavity was formed
paradoxically
paradoxically in genetic males. Following the end of treatment, these fish
developed intersexual gonads. Advanced spermatogenesis was observed in
the inner periphery of the efferent ducts. Maturing oocytes and a definite
ovarian
ovarian cavity
cavity were
were observed
observed on
on the outer
outer side
side of
of the
the ducts.
ducts. In
In the
the same
same
study, administration of methyltestosterone at 50 mg/kg diet resulted in
gonadal masculinization. Nakamura (1975)(1975) noted that, in contrast to his ob-
5.
5. HORMONAL
HORMONAL SEX
SEX CONTROL AND
CONTROL A N D ITS
ITS APPLICATION
APPLICATION TO
TO FISH CULTURE 237
FISH CULTURE 237
servations, Reinboth (1969) (1969) had reported that in Hemihaplochromis multi multi-
color a long-term treatment at extremely high androgen dosages (30-50 (30-50 g/kg
diet)
diet) was required
required to cause gonadal masculinization, but a short-term treat treat-
ment at similar dosages resulted in gonadal feminization. He suggested
interspecies differences as a possible explanation but acknowledged that the
major reason for these variations was unknown. More recently, paradoxical paradoxical
actions ofof testosterone and ethynyltestosterone have been reported in rain rain-
bow trout (V. (V. ]J . Bye,
Bye, 1980,
1980, personal communication) and channel catfish,
Zctalurus punctatus (Goudie
Ictalurus (Goudie et al. al.,, 1983).
1983).
Recent work with the amphibians has demonstrated that care must be
taken in the interpretation of paradoxical steroid action. action. Chieffi
Chief3 (1965)
(1965) noted
the paradoxical effects found in urodele gonads following following high dosages of
androgens. For example,
example, in Pleurodeles waltlii, waltlii, testosterone propionate in in-
duced complete feminization at all dosages administered (Gallien, (Gallien, 1950).
1950).
Chieffi
ChiefE concluded
concluded that that the
the nonspecific
nonspecific actionaction of
of the steroids
steroids argued
argued against
against
their putative roles as sex inducers. The administration of of estrogens to sever
sever-
al ranid species results in feminization,
feminization, intersexuality, or masculinization
with increasing dosages (Padoa, (Padoa, 1942; 1942; Gallien, 1941). Hso et al.
1941). Hsii al. (1978a,
(1978a,b)b)
have
have also
also demonstrated
demonstrated that that high
high dosages
dosages of of estradiol
estradiol result
result in in paradoxical
paradoxical
masculinization of Rana catesbeiana tadpoles. However, this treatment also
inhibits
inhibits Ll5-3[3-hydroxysteroid
A5-3P-hydroxysteroid dehydrogenase,
dehydrogenase, aa key key enzyme
enzyme in in ovarian
ovarian
steroidogenesis.
steroidogenesis. Hso Hsu andand co-workers
co-workers suggested
suggested thatthat if
if the paradoxical
paradoxical action
action
of
of this steroid arose
arose through an an inhibition
inhibition of of ovarian
ovarian steroidogenesis,
steroidogenesis, the the
paradoxical
paradoxical effects would lend
effects would lend support
support to to the
the steroid-inductor
steroid-inductor theory.theory.
The data from the limited number of studies employing steroidogenic
inhibitors
inhibitors or or antiandrogens
antiandrogens have have not not supported
supported specific
specific steroid
steroid action.
action.
Cyproterone acetate is the most commonly used antiandrogen in studies
with fish.
fish. In mammals, it has been shown to be a potent inhibitor of endoge endoge-
nous or exogenous testosterone (Hamada (Hamada et al. al.,, 1963).
1963).The antiandrogen has
been partially effective in blocking the development of secondary sexual
characteristics
characteristics in in the
the three-spined
three-spined stickleback
stickleback (Rouse
(Rouse et al. al.,, 1976),
1976), the
the Indi
Indi-
an
an catfish,
catfish, Heteropneustes fossilis fossilis (Sundararaj
(Sundararaj and and Nayyar,
Nayyar, 1969),1969), and
and the
the
guppy, reticulata (Smith,
guppy, Poecilia reticulata (Smith, 1976).
1976). Schreck
Schreck (1974),
(1974), citing
citing the unun-
published
published workwork of of Irons
Irons and
and Schreck,
Schreck, reported
reported aa possible
possible sex sex inversion
inversion in in
male
male Oryzias latipes fed fed the
the antiandrogen,
antiandrogen, cyproterone
cyproterone acetate,
acetate, at at 50-500
50-500
mg/kg
mg/kg dietdiet for
for 12
12 weeks.
weeks. Hopkins
Hopkins et al. al. (1979)
(1979) fed
fed cyproterone
cyproterone acetateacetate inin
combination with various natural and synthetic estrogens to Oreochromis Oreochromis
au reus (Tilapia
aureus aurea) fry.
(Tilapia aurea) fry. The
The results indicated that
results indicated that rather
rather than
than potentiating
potentiating
the effect of of the estrogens it actually lessened their effectiveness. Rastogi
and
and Chieffi
Chief3 (1975)
(1975)demonstrated
demonstrated that that the
the same antiandrogen
antiandrogen did not not inhibit
inhibit
the
the masculinizing
masculinizing effectseffects of
of testosterone
testosterone propionate
propionate or or lll-ketotestosterone
l-ketotestosterone
238
238 GEORGE
GEORGE A.
A. HUNTER AND EDWARD
EDWARD M DONALDSON
in the swordtail Xiphophorus helleri even though it inhibited the binding of of

the steroid to testosterone-sensitive target tissues.
The mechanism of action of cyproterone acetate in the lower vertebrates
is not clear. Chieffi al. (1974)
Chief3 et al. (1974) administered cyproterone acetate to Rana
esculenta tadpoles. They hypothesized that if the androgens were indeed the
esculenta
sex inductors, the administration of cyproterone acetate would result in
competition for androgen-receptor
androgen-receptor sites and subsequent abnormal testicular
development.
development. Instead Instead the
the ovaries
ovaries were masculinized. Chieffi
were masculinized. Chief3 and
and co-workers
co-workers
suggested that these results indicated
suggested that these results indicated that that the sex
sex inductors were not
inductors were not struc
struc-
turally similar to sex steroids.
steroids. However, Hsii et al. (1979)
Hsu al. (1979) have also demon
demon-
strated
strated that
that cyproterone
cyproterone acetate
acetate administered
administered inin the rearing
rearing water
water atat 1500
1500
IJ.g/l for a 7 -month period can transform
pg/l for a 7-month period can transform ovaries of ovaries of Rana catesbeiana into
catesbeiana into
testes. They suggested that the masculinizing effect of the antiandrogen was
attributable to inhibition of ovarian steroidogenesis, specifically A5_3f3- A5-3p- and
possibly 17f3-hydroxysteroid
l7a-hydroxysteroid dehydrogenase. They also hypothesized that
this inhibition of young ovaries would result initially in ovarian degeneration
and subsequent masculinization of the gonads with prolonged treatment.
Further, HsiiHsu and co-workers maintain that these results support the steroid steroid-
inductor theory.
The observation ofparodoxical steroid action also suggests the possibility
of a dominant-neutral
dominant-neutral sex mechanism of dimorphism. Studies involving the
culture of isolated mammalian and avian cortex and medulla suggest that
sexual dimorphism occurs as a result of the presence or absence of a single
sexual
dominant sex. sex. In the absence of the dominant sex the neutral sex develops
(McCarrey and Abbott, 1979). 1979). As a general rule, the dominant sex is correcorre-
lated with heterogamety (Jost, Gost, 1965).
1965). Hackmann and Reinboth (1974) (1974) noted
that paradoxical sex inversion in the lower vertebrates occurs only in one
direction. Specifically
Specifically in the urodeles and cichlids, high doses of androgen
result
result in in feminization,
feminization, butbut inin the
the families
families Ranidae and Hylidue estrogen
and Hylidae estrogen
treatment results in masculinization. Hackmann and Reinboth proposed that
the evidence from the cichlids could support a hypothesis in which the
female hormone
hormorie induces the female sex type while the absence of the hor hor-
mone
mone promotes
promotes the development
development of of male
male sex
sex type.
type. The
The evidence
evidence from
from the
breeding experiments conducted by Hackmann and Reinboth (1974) (1974) with
Hemihaplochromis multicolor indicated male heterogamety. Therefore, the
Hemihaplochromis
association between heterogamety and the dominant sex observed in birds
association
and
and mammals
mammals could could not
not be
be supported.
supported.
Similarly, the results presented by Gresik and Hamilton (1977) (1977) and van
den Hurk and Slof (1981) (1981) suggested a dominant-neutral
dominant-neutral sex hypothesis in
which interrupted testicular development results in the formation of an
ovary
ovary.... Van den Hurk and Slof (1981) (1981) obtained feminization in in rainbow trout
5.
5. HORMONAL SEX
SEX CONTROL
CONTROL AND
AND ITS
ITS APPLICATION
APPLICATION TO
TO FISH
FISH CULTURE 239
by administering progesterone in the rearing water at 300 �gll pg/l over the
period
period of of sexual
sexual differentiation.
differentiation. Van Van den den Hurk
Hurk and Slof suggested
and Slof suggested that that inin
addition to a possible direct feminizing effect of progesterone, there also also
exists
exists the
the possibility
possibility that
that progesterone
progesterone may may actact as
as aa steroid-blocking
ster'oid-blocking agent agent ofof
the
the androgenic
androgenic pathway,
pathway, resulting
resulting in in ovarian
ovarian differentiation.
differentiation. An An indirect
indirect
action
action of of the
the progestins
progestins via via aa blockage
blockage of of androgen
androgen biosynthesis
biosynthesis has has been
been
previously
previously described
described in in mammals
mammals by by Gower (1975). However,
Gower (1975). However, Van Van denden Hurk
Hurk
and
and Slof
Slof also
also noted
noted that
that progesterone
progesterone has has not
not been
been demonstrated
demonstrated to to affect
affect sex
sex
differentiation in Oryzias latipes (Yamamoto (Yamamoto and Matsuda, 1963). 1963). Recently,
van
van den Hurk and
den Hurk and Lambert
Lambert (1982)(1982) have
have also
also reported
reported thatthat progesterone
progesterone does does
not
not influence
influence sexsex differentiation
differentiation in in rainbow
rainbow trout
trout when
when administered
administered in in the
the
diet.
diet. The evidence
evidence produced
produced by van van den Hurk et al.
den Hurk (1982), demonstrating
al. (1982), demonstrating
the
the steroidogenic capabilities of
steroidogenic capabilities of rainbow
rainbow trouttrout testes
testes but
but notnot ovaries
ovaries at at the
the
time
time ofof sex
sex differentiation,
differentiation, also also supports
supports aa dominant-neutral
dominant-neutral sex sex hypothesis.
hypothesis.
The limited evidence from these studies is clearly not sufficient sufficient to deter
deter-
mine
mine whether
whether aa dominant-neutral
dominant-neutral system system of of sexual
sexual dimorphism is is present in in
fish. Presumably the unitary origin of the teleost gonad has prevented dis
fish. dis-
sociation-recombination
sociation-recombination experiments experiments which which would
would provide
provide moremore substantial
substantial
evidence for a dominant-neutral
dominant-neutral sex hypothesis. The discrete male and
female
female territories
territories inin hermaphroditic
hermaphroditic species species may provide an
may provide an ideal
ideal vehicle
vehicle forfor
such
such an an examination.
examination.
Hishida
Hishida (1962,
(1962, 1965)
1965)by by demonstrating
demonstrating the the selective
selective incorporation
incorporation of of sex
sex
steroids into
steroids into the
the developing
developing gonads, gonads, provided
provided Yamamoto
Yamamoto with with considerable
considerable
support
support for his hypothesis.
for his hypothesis. Administration
Administration of of 4-[ 14Cltestosterone
14C]testosteronepropionatepropionate
to
to larval
larval Oryzias latipes resultedresulted in in the
the accumulation
accumulation of of the labeled
labeled steroid
steroid
only
only byby the
the actively
actively differentiating
differentiating gonads gonads (Hishida, 1962). In
(Hishida, 1962). In aa later study,
later study,
Hishida (1965)
(1965) fed
fed larval
larval Oryzias latipes 16-[ 16-[14C]estrone
14C]estrone and diethylstilbes
and diethylstilbes-
trol
trol l-[14Clmonoethyl).
l-[14C]monoethyl). A 4- 4- to
to lO-fold
10-fold concentration
concentration of of the
the steroids
steroids was was
found
found in in developing
developing gonads,
gonads, againagain indicating
indicating active
active accumulation
accumulation of of the
the
steroids.
steroids. Conversion
Conversion of of estrone
estrone to to estradiol
estradiol was also demonstrated.
was also demonstrated. Further,Further,
based
based on on recovered counts, the
recovered counts, the effective
effective oral
oral dosages
dosages forfor 100%
100% sex sex inversion
inversion
were
were calculated
calculated to to be 1.8 1. 8 X X 10 - 2�g estrone
10-2pg estrone and and 1. 1.11 X X 10 - 2 �g pg didi-
ethylstilbestrol.
ethylstilbestrol. Based
Based on on this
this evidence,
evidence, Yamamoto
Yamamoto (1969) (1969) asserted
asserted thatthat the
the
levels
levels ofof steroids
steroids required
required for for manipulation
manipulation of of sex
sex differentiation
differentiation were were within
within
the
the physiological
physiological capabilities
capabilities of of the
the species.
species. Further
Further evidence
evidence that that phys
phys-
iological
iological levels
levels of
of steroid
steroid can can influence
influence sexual
sexual differentiation
differentiation was was provided
provided by by
Satoh
Satoh (1973).
(1973). InIn this
this study,
study, trunk
trunk regions
regions of of newly hatched Oryzias latipes
newly hatched
fry
fry were
were transplanted
transplanted into into the
the anterior
anterior chamber
chamber of of the
the eyes
eyes of of adult
adult fish.
fish. The
The
gonads
gonads of genetic females
of genetic females transplanted
transplanted into into male hosts did
male hosts did not
not differentiate
differentiate
into
into anan ovary,
ovary, but
but did
did form
form spermatogenic
spermatogenic cells. cells. Satoh
Satoh suggested
suggested that that the
the
results supported the
results supported the actions
actions of of androgens
androgens as as andro-inducers.
andro-inducers. AlthoughAlthough the the
HUNTER AND EDWARD M .. DONALDSON
M DONALDSON
results
results provide
provide strongstrong supportive
supportive evidence,
evidence, they they dodo notnot conclusively
conclusively demon
demon-
strate a role for the steroids in primary gonadal
strate a role for the steroids in primary gonadal differentiation. differentiation.
Studies
Studies correlating
correlating the development of
the development of steroidogenic
steroidogenic and and differentiating
differentiating
tissue
tissue or identifying steroid synthesis in sexually differentiating gonads
or identifying steroid synthesis in sexually differentiating gonads areare
rare.
rare. Dildine (1936) (1936)first
first noted
noted the correlation
correlation between
between the the development
development of of aa
stromal
stromal region
region and and the initiation
initiation of of male
male influences
influences on on the
the sex
sex differentiation
differentiation
in
in Poecilia reticulata. More recently, Takahashi (1975a) observed
reticulata. More recently, Takahashi ( 1975a) observed that,that, in
in
embryonic
embryonic Poecilia reticulata, the appearance
appearance of aggregations of stromal
of aggregations of stromal
cells
cells in
in the
the gonadal
gonadal hilus
hilus 18 18 days
days after
after the lastlast parturition
parturition are are indicative
indicative of of
testicular differentiation. Oral administration of methyltestosterone methyltestosterone to grav grav-
id
id females
females results
results in in somatic
somatic aggregation
aggregation in in the hilar
hilar region
region of of embryonic
embryonic
ovaries.
ovaries. Females
Females affected
affected in in this
this manner
manner contain
contain developing
developing oocytes
oocytes andand
malelike
malelike stromal
stromal aggregations
aggregations in in the hilar
hilar region.
region. Within
Within 20 daysdays ofof birth
birth the
ovaries completely degenerate
ovaries completely degenerate and and areare replaced
replaced by by testes
testes which
which display
display
precociously
precociously differentiating
differentiating spermsperm ducts,ducts, testicular
testicular interstitium,
interstitium, and and aa con
con-
comitant
comitant initiation
initiation of of spermatogenesis.
spermatogenesis. The period period thatthat is most sensitive
is most sensitive toto
change
change in in the
the developing
developing ovaries
ovaries is is at 18 days
at 18 days after
after the last
last parturition,
parturition, andand itit
is synchronous with the period of stromal aggregation in the hilar region of
developing testes.testes. Takahashi concluded that masculinization of the somatic
element
element is is essential
essential for for functional
functional sex sex inversion
inversion of of females,
females, and and that
that the
the
somatic
somatic differentiation
differentiation may may occur occur priorprior to to germinal
germinal differentiation.
differentiation.
Nakamura
Nakamura (1978) (1978) hashas reported
reported similar
similar stromal
stromal aggregations
aggregations in in the
the hilar
hilar re
re-
gion
gion prior
prior to to male
male differentiation
differentiation in in Oryzias latipes and and the
the mosquito
mosquito fish fish
Gambusia af finis. However,
affinis. However, Satoh Satoh and and Egami
Egami (1972)(1972) diddid not
not observe
observe sex sex
differences
differences in in the
the histological
histological structure
structure of of the
the gonadal
gonadal somatic
somatic tissue
tissue prior
prior to
to
gametogenesis in Oryzias latipes.
Yoshikawa
Yoshikawa and and Oguri
Oguri (1979)
(1979) reported
reported that that inin Oryzias latipes the the differ
differ-
entiation of somatic cells into interstitial cells always precedes the transfor transfor-
mation
mation fromfrom spermatogonia
spermatogonia to to spermatocytes.
spermatocytes. Further, Further, interstitial
interstitial cells
cells are
are
always
always found in the vicinity of germ cells in meiosis, suggesting that intersti intersti-
tial
tial cells
cells areare responsible
responsible for for the
the differentiation
differentiation of germ germ cells.
cells. However,
However, in in
the
the same
same species,
species, Satoh
Satoh ((1974)
1974) reported
reported the appearance
appearance of steroidogenic
steroidogenic
cells
cells after
after the onset of
the onset of gonadal
gonadal sex sex differentiation.
differentiation. Similarily,
Similarily, van
van denden Hurk
Hurk
al. (1982)
et al. (1982)foundfound no steroid-synthesizing structures
no steroid-synthesizing structures in in indifferent
indifferent gonads
gonads of of
rainbow
rainbow trouttrout 50 50 days
days postfertilization
postfertilization.. In this study,
In this study, steroidogenic
steroidogenic (Leydig)
(Leydig)
cells
cells were detected in differentiated testes at 100 100 days postfertilization.
Takahashi
Takahashi and and Iwasaki
Iwasaki (1973)
(1973) provided
provided the the first
first examination
examination of of the onset
onset
of
of steroidogenesis
steroidogenesis in in the
the differentiating
differentiating teleostteleost gonad.
gonad. These
These researchers
researchers
detected
detected �5-3�-hydroxysteroid
A5-3P-hydroxysteroid dehydrogenasedehydrogenase activity activity in in gonadal interstitial
gonadal interstitial
cells in Poecilia reticulata 7 days
cells in days postpartum.
postpartum. The The enzyme
enzyme activity
activity was de
was de-
tected concurrent with
tected concurrent with thethe multiplication
multiplication of spermatogonia
spermatogonia and and differentia-
differentia-
5.
5. HORMONAL
HORMONAL SEX
SEX CONTROL AND
CONTROL A N D ITS
ITS APPLICATION
APPLICATION TO
TO FISH
FISH CULTURE
CULTURE 241
tion
tion of
of cells
cells in
in the
the testicular
testicular hilus
hilus into
into the
the sperm
sperm ductduct analogues
analogues and and testic
testic-
ular stroma. The enzyme
ular stroma. enzyme was was detected
detected in in the cells
cells of
of the
the stroma
stroma butbut not
not inin
the
the sperm ductduct analogues. Further, enzyme
analogues. Further, enzyme activity
activity could
could notnot be
be detected
detected in in
fish
fish sampled
sampled at at 33 or
or 5 days
days postpartum.
postpartum. Takahashi
Takahashi and and Iwasaki
Iwasaki concluded
concluded
that
that the enzyme
enzyme activity
activity appears
appears and increases simultaneously
and increases simultaneously with with thethe dif
dif-
ferentiation
ferentiation of of the
the testicular
testicular duct
duct system rather rather than
than the the germ
germ cells.
cells. Re
Re-
cently, van den Hurk et al. al. (1982)
(1982) have conducted in vitro assays of Salmo
gairdneri gonadal
gairdneri gonadal homogenates
homogenates at 50, 100,
at 50, 100, and
and 200
200 days
days postfertilization.
postfertilization.
The
The histologically
histologically indifferent
indifferent gonads
gonads at at 50 days
days postfertilization
postfertilization contained
contained
3J3-hydroxysteroid
3P-hydroxysteroid dehydrogenase,
dehydrogenase, 5, 4-isomerase, and
5,4-isomerase, and l7a-hydroxylase,
l7au-hydroxylase,in in-
dicating
dicating thethe capacity
capacity to to synthesize
synthesize progestins.
progestins. Developing
Developing testes testes atat 100
100 days
days
postfertilization
postfertilization also also contained
contained 17a,17a, 20-desmolase
20-desmolase and and 11J3-hydroxylase,
11P-hydroxylase, in in-
dicating
dicating thethe capacity
capacity to to synthesize
synthesize androgens.
androgens. However,
However, sex sex differentiation
differentiation
had
had commenced
commenced between between 50 and and 100
100 days
days postfertilization.
postfertilization. Therefore,
Therefore, it it was
was
not possible to determine whether these androgen-synthesizing
not possible to determine whether these androgen-synthesizing capabilities capabilities
initiated
initiated or or occurred
occurred as as aa result
result ofof testicular development. Both
testicular development. Both testes
testes andand
ovaries
ovaries possessed 17P-hydroxysteroid dehydrogenase at 200 days postfertil
possessed 17J3-hydroxysteroid dehydrogenase at 200 days postfertil-
ization. Further, the
ization. Further, the ovaries
ovaries possessed
possessed aromatase,
aromatase, indicating
indicating the the capability
capability
to
to synthesize estrogen. Therefore, although androgen- or estrogen-syn
synthesize estrogen. Therefore, although androgen- or estrogen-syn-
thesizing capabilities could not be demonstrated demonstrated in indifferent gonads, a
capability
capability for for progestin
progestin synthesis was was noted. Van Van den
den Hurk
Hurk and and Slof
Slof (1981)
(1981)
had
had previously demonstrated that progesterone, administered at the time of
previously demonstrated that progesterone, administered at the time of
sex
sex differentiation,
differentiation, results
results inin gonadal feminization.
feminization. As As they
they note,
note, the the ab
ab-
sence of
sence of estrogen-synthesizing
estrogen-synthesizing capabilities
capabilities in in differentiated
differentiated gonads
gonads 100 100 days
days
postfertilization
postfertilization is is substantial
substantial evidence
evidence against
against aa steroidal
steroidal ovarian
ovarian inductor.
inductor.
In
In aa later
later study,
study, vanvan den
den Hurk
Hurk andand Lambert
Lambert (1982)(1982)demonstrated
demonstrated the the conver
conver-
sion
sion ofof 11J3-hydroxyandrostenedione
lip-hydroxyandrostenedione to to 11-ketoandrostenedione
11-ketoandrostenedione using using testic
testic-
ular
ular homogenates
homogenates from from 100-day-old
100-day-old rainbow
rainbow trout.
trout. Therefore,
Therefore, the the presence
presence
of
of 1llp-dehydroxysteroid
1 J3-dehydroxysteroid dehydrogenase
dehydrogenase was was demonstrated.
demonstrated. Further Further admin
admin-
istration
istration of of 11 J3-hydroxyandrostenedione at
llp-hydroxyandrostenedione at 60
60 mg/kg
mg/kg for for 88 weeks
weeks fromfrom first
first
feeding
feeding resulted
resulted in in an
an all
all male
male population.
population. Van den Hurk
Van den Hurk and and Lambert
Lambert
concluded
concluded that that this steroid is
this steroid is endogenous
endogenous to to rainbow
rainbow trout
trout andand responsible
responsible
for
for the
the initiation
initiation of of testicular
testicular development.
development.
D. Summary
Summary
No
No single
single model
model ofof sex
sex determination
determination and
and differentiation
differentiation may
may be recon
recon-
ciled
ciled with
with all
all the
the present
present evidence
evidence from
from the fish. The
the fish. The sex-determining
sex-determining mech
mech-
anism
anism is assumed to
is assumed to have
have aa genetic
genetic basis.
basis. Although
Although thethe majority
majority of
of fish
fish do
do not
not
have cytologically distinguishable sex chromosomes, both male and female
digametic systems
digametic systems have been demonstrated.
have been demonstrated. Nonheterosomal
Nonheterosomal chromosomal
chromosomal
GEORGE A. HUNTER AND EDWARD M. DONALDSON
AND EDWARD DONALDSON
sex-determining systems have also been reported for some species. Even
within species with well developed heterosomal systems,
systems, sex determination
has been found to be labile to extrinsic factors.
factors. Of the c).Irrently available
cprrently
models for sex determination, the polygenic system described by Kallman
(1965)
(1965) and Yamamoto (1969)
(1969) appears to fit most closely the available data.
Sfnmarily,
Similarily, the mechanism of sex differentiation in fish has not been
determined. The sex-specific
sex-specific H-Y antigen system found in higher verte verte-
brates has been reported in several fish species.
species. Whether it plays a signifi
signifi-
cant role in the process of of Se1(
sex differentiation
daerentiation in fish remains to be deter
deter-
mined. The dual-sex-inductor model of d sex differentiation continues to
dominate the conceptual approach to research in fish. fish. However, debate
continues over the ability of a unitary primordium, reported for the teleosts,
to elaborate separate inducers. Evidence supporting Yamamoto's
Yamamoto’s (1969)
(1969) hy
hy-
pothesis that the sex steroids are in fact the presumptive inducers remains
inconclusive.
inconclusive. The steroids are capable of influencing sex determination in
the majority
majority ofof gonochoristic
gonochoristic teleost
teleost species, but
but have
have been
been applied
applied with
with
variable success to hermaphroditic species.
species. The evidence for steroid action
based on the identification of steroid activity, the juxtaposition of steroido
steroido-
genic tissues to differentiating germ cells, and the effects of anti steroidal
antisteroidal
compounds remains equivocal. However, regardless of whether the action of
steroids is pharmacological or physiological,
physiological, steroids can play a decidedly
influential role in the process of sex differentiation. Therefore, they are a
valuable
valuable tool
tool for
for both
both further
further exploration
exploration of
of the
the process
process of
of sex
sex differentiation
differentiation
and
and the
the manipulation
manipulation of of gonadal
gonadal sex
sex for
for purposes
purposes of
of fish
fish culture.
culture.
III. HORMONAL SEX CONTROL

III. HORMONAL CONTROL
Although there are numerous studies concerned

concerned with the application of of
hormones to control gonadal sex, most of of them have remained within the
framework for effective treatment
treatment described by Yamamoto (1969).
(1969). Based on
his work with Oryzias Zutipes, Yamamoto suggested that, for effective treat
Oryzius latipes, treat-
ment, the species-speciijc
species-specific optimal dosage of a particular steroid should be
administered from the stage of the undifferentiated gonad through the time
of morphological differentiation. Although the majority of studies have con
con-
firmed these criteria as excellent general rules, exceptions have been re re-
ported. Further, within these criteria considerable scope for variation exists.
exists.
Therefore, to facilitate consideration of the various components of these
studies a generalized model is presented
presented (Fig.
(Fig. 1).
1).
Hormonal sex-control studies consist of of three chronologically
chronologically ordered
phases: management, treatment,
treatment, and evaluation. Within the management
phase, both the species and the gonadal sex appropriate to attaining manage-
5.
5. HORMONAL
HORMONAL SEX
SEX CONTROL AND
CONTROL A N D ITS
ITS APPLICATION
APPLICATION TO
TO FISH
FISH CULTURE
CULTURE 243
MANAGEMENT
MANAGEMENT
DESIRED
ESIRED GONADAL
OONADAL SEX
SEX
AND
AND CHOICE
CHOICE Of
Of
STRATEGY
STRATEGY
-DIRECT
-DIRECT
- INDIRECT
DPMENrl
PARAM£TERS
INFLUENCES
-ROUTE
-ROUTE OF
OF
ADMINISTRATION
ADMINISTRATION
-DOSAGE
-DOSAGE
�DURATION
-DURATION AND
AND
TIMING
TIMING
EVALUATION
I I
rn
GENOTYPE
I
PHENOTYPE
I 1 ,
EVALUATION
PROGENY
Fig. 1. Schematic
Fig. 1. Schematic model
model of
of hormonal
hormonal sex-control
sex-control studies.
studies.
ment
ment objectives
objectives are
are chosen.
chosen. The
The choice
choice ofof gonadal
gonadal sex
sex also
also includes
includes selection
selection
of
of the optimal strategy
the optimal strategy for
for achieving
achieving it.it. The treatment phase
The treatment phase involves
involves the
the
choice
choice ofof the appropriate hormone and
appropriate hormone method of
and method of application
application within
within the
the
constraints
constraints ofof the
the gonadal
gonadal and
and somatic
somatic development
development of of the species.
species. These
These
latter
latter constraints
constraints are
are in
in turn
turn influenced
influenced by by environmental
environmental conditions.
conditions. The
The
final
final phase includes the evaluation
phase includes evaluation of results from
of results from either
either treated
treated fish
fish or
or their
their
progeny.
progeny. Where
Where necessary,
necessary, these
these results
results are
are used
used to
to reformulate
reformulate objectives
objectives
and techniques in
and techniques in the
the management
management and and treatment
treatment phases.
phases.
A. Management
Management
1. SPECIES
1. SPECIESCHOICE
CHOICE
The establishment
establishment ofof management
management objectives
objectives is
is clearly
clearly interactive
interactive with
with
species
species choice.
choice. The initial
initial purpose
purpose of
of hormonal
hormonal sex-control
sex-control studies
studies was
was to
to
elucidate
elucidate the
the specific
specifb role
role of
of hormones
hormones in the process
in the process of sex differentiation.
of sex differentiation.
Further,
Further, the
the inter-
inter- and
and intraspecific
intraspecific matings
matings of
of sex-invert�
sex-inverted and
and untreated
untreated
244 GEORGE A.
GEORGE A. HUNTER
HUNTER AAND E DWARD M
N D EDWARD DONALDSON
M.. DONALDSON
provided a means for examining basic sex-determining mechanisms. The

fish provided
species chosen
species chosen for
for this
this research
research were
were primarily
primarily from
from the
the Cyprinodontidue
Cyprinodontidae
and Poeciliidue
and for reasons
Poeciliidae for reasons that
that included
included ease
ease of
of handling
handling andand breeding,
breeding, the
presence of of a marked sexual dimorphism, and the presence of of sex-linked
(Yamamoto, 1953).
genetic markers (Yamamoto, 1953).
initiation of
The initiation of applied
applied studies
studies has
has naturally
naturally involved
involved aa greater
greater empha-
empha
sis on
sis on species
species which
which are
are already
already ofof economic
economic importance
importance and and accessible
accessible for
for
treatment. However,
treatment. However, much much ofof the
the relevant research on
relevant research on various
various treatment
treatment
parameters has
parameters has been
been conducted
conducted using
using the
the same
same species
species asas in
in fundamental
fundamental
investigations. The primary concern of of these studies has been the optimiza-
optimiza
tion of
tion of treatment
treatment procedures
procedures and
and the
the development
development of of alternate
alternate strategies
strategies for
for
the production of of fish with
with a particular
particular gonadal sex. For the purpose of of this
discussion the absence of of phenotypic
phenotypic sex is included, i.e., i. e. , sterility as a
gonadal sex
gonadal sex type.
type.
2. G
2. ONADAL SEX
GONADAL SEX
In general, aa particular
In general, particular gonadal
gonadal sex is chosen
sex is chosen to enhance the
to enhance the value
value of
of
individual
individual fish because
because ofof sex-related
sex-related morphological, physiological, or
morphological, physiological, eth
or eth-
ological
ological characteristics. Further, the
characteristics. Further, the reproductive potential of
reproductive potential the popula
of the popula-
tion as a whole may be modified. Reproductive potential may be enhanced
by increasing the proportion of of females.
females. Reduction or elimination of of this
potential
potential may
may be achieved
achieved byby the production
production of sterile or
of sterile or monosex
monosex popula
popula-
tions.
tions.
The ability to control gonadal sex also allows for genetic advancement
through the creation of of inbred lines.
lines. This may be achieved by either the
production of synchronously maturing hermaphroditic fish (Jalabert Oalabert et al.
al.,,
1975)
1975) or the production of monosex genetically homozygous
homozygous individuals by
gynogenetic techniques. A A portion of these individuals may be sex inverted
by hormonal treatment and mated with their untreateduntreated gynogenetic siblings
at maturity. Repetition of this cycle over several generations results in a
rapid
rapid inbreeding
inbreeding effect
effect (Nagy al.,, 1981;
(Nagy et al. al.,, 1981;
1981; Streisinger et al. 1981; Donald
Donald-
son and Hunter,
Hunter, 1982a).
1982a).The various applications of genetic sex-control tech tech-
niques and their usefulness to breeding programs are discussed in Chapter
8, this volume.
volume.
a. Male and Female Production. Two strategies may be employed to
a.
produce monosex male and female populations. The first involves
involves the direct
application of steroids to juvenile fish which results in the redirection of sex
sex
differentiation to the desired gonadal sex.
sex. A second,
second, indirect method in in-
volves the sex inversion of homogametic individuals which are are reared to
maturity. The
The gametes from these sex-inverted homogametic individuals are
joined with the gametes
gametes of untreated homogametic fish.
fish. If the process of sex
5.
5. HORMONAL
HORMONAL SEX
SEX CONTROL AND ITS
APPLICATION TO
TO FISH CULTURE 245
FISH CULTURE 245
determination in the target species is bound to a heterosomal system, system, the

progeny should all be homogametic and, therefore, of the same sex. sex. The
monos ex group produced may then be used as a reservoir of known homo
monosex homo-
gametic individuals to be sex reversed and used to perpetuate the monosex
stock. The use of these known homogametic individuals eliminates the ne ne-
cessity for further progeny testing.
A special case of the latter approach would allow for the production of a
monosex heterogametic population. In this case, heterogametic individuals
(XY or ZW)
ZW) are sex inverted and mated with untreated heterogametic indi indi-
viduals. Approximately 25% of the progeny should be either YY or WW,
viduals. WW,
depending on the viability of these individuals. If viable, these individuals
could be raised to maturity and mated with untreated homozygous XX or ZZ
individuals resulting to the production of monosex heterozygous progeny.
Yamamoto
Yamamoto (1964a, b, 1975)
(1964a,b, 1975)has discussed the viability ofYY
of YY individuals in
the medaka and the goldfish.
goldfish. In the latter, the mating of untreated and sexsex-
inverted XY individuals results in a male-female
male-female ratio of 3:1
3:l indicating com
com-
plete YY viability. However, in Oryzias latipes the y RyR zygote, where R
YRYR
represents full xanthic coloration, is rare. However, y Ryr and yryr
YRYr YrYr indi
indi-
viduals, where r represents scanty coloration are common.common. The rarity of the
y RyR zygote has been attributed to the presence of an inert section on the
YRYR
y
YRR chromosome which is usually lethal in the duplex condition. Fineman et
al. (1974)
al. (1974) have examined the viability of the yryr YrYr individual in the white
strain of the medaka. The life expectancy of these individuals was found to be
intermediate between that of the normal male XY and female XX. The
viability ofYY
of YY individuals appears to be high in coho salmon, Oncorhynchus
Oncorhynchus
kisutch (Hunter
(Hunter et al. al.,, 1982a),
1982a), and somewhat lower in Hemihaplochromis
multicolor (Hackmann and Reinboth, 1974) 1974) and in Salmo gairdneri (John
(John-
stone al.,, 1979a).
stone et al. 1979a).
The
The direct
direct strategy
strategy has
has the advantage
advantage that
that the
the desired
desired phenotypic
phenotypic adults
adults
are produced in the same generation as the treatment. In addition, any
gonadal sex may be produced. The major disadvantage of this approach has
been the
the variability
variability ofof treatment
treatment success
success.. A
A further
further disadvantage
disadvantage occurs
occurs
when the objective is to increase the proportions of a particular sex type
which must be part of a breeding population. One-half of a population which
has been sex inverted will have the genome of the opposite sex. sex. These fish,
mated with untreated
untreated fish will produce offspring in altered sex ratios. For
example, male coho salmon have been demonstrated to be heterogametic.
Direct
Direct treatment
treatment withwith estradiol
estradiol produces
produces all-female
all-female groups.
groups. However,
However, the
the
progeny from the XY females when mated with normal males (XY) produce
offspring in a 3:1
offspring 3:l male-female
male-female ratio (Hunter et al. al.,, 1982a).
1982a). Because one of
the objectives of treating Pacific salmon is to increase the number of females
suitable for broodstock, such a treatment may not be used. Also, this ap-
246 GEORGE
GEORGE A.
A. HUNTER AND
HUNTER A N D EDWARD
EDWARD M. DONALDSON
M. DONALDSON
proach
proach would
would notnot be appropriate
appropriate where
where aa desired
desired production
moduction trait trait was
was genet
genet-
ically
ically sex
sex linked.
linked. Anderson
Anderson and and Smitherman
Smitherman (1978)
(1978) sex sex inverted
inverted Oreo
Oreo-
chromis aureus and
chromis aureus and Oreochromis niloticus fry
Oreochromis niloticus fry with
with ethynyltestosterone.
ethynyltestosterone. The The
growth
growth rates
rates between
between the the two
two sex-inverted
sex-inverted groups
groups were
were notnot significantly
significantly
different.
digerent. However,
However, both
both sex-inverted
sex-inverted groups
groups grew
grew at at aa significantly slower
significantly slower
rate
rate than
than aa normal
normal male
male control
control group produced by
group produced by manual
manual selection.
selection. TheThe
suboptimal
suboptimal growth
growth ofof the
the sex-inverted
sex-inverted groups
groups was attributed to
was attributed to the
the presence
presence
of
of the
the female
female genotype.
genotype.
The last disadvantage
disadvantage concerns
concerns the marketing
marketing of fish which have been
treated with steroids.
steroids. Steroid
Steroid treatments are typically administered to juve- juve
nile
nile fish years
years prior
prior to
to consumption.
consumption. Additionally,
Additionally, thethe amounts
amounts of of steroid
steroid
used are smallsmall and their half-lives
half-lives are short. Johnstone et al. (1978)
short. Johnstone (1978) have
reported that the half-life
half-life of estradiol
estradiol in l-year-old
1-year-old rainbow trout is less less than
12
12 hr. Similar results have been obtained with the same steroid in coho
salmon alevins (G. A. Hunter, E
alevins (G. E.. M
M,. Donaldson,
Donaldson, G. van der Kraak, Kraak, and I. I.
Baker,
Baker, unpublished).
unpublished). Fagerlund and McBride (1978) (1978) and Fagerlund and
Dye (1979)
(1979)have examined
examined the respective depletion of [3H]testosterone
[3H]testosterone and
17at-1,2-[3H]methyltestosterone
17a-1,2-[3H]methyltestosterone from yearling coho salmon.salmon. These studies
indicated that the steroids
steroids were rapidly taken into the blood and concen concen-
trated in the gonads.
gonads. Methyltestosterone was eliminated more slowly than
testosterone.
testosterone. The concentration
concentration of methyltestosterone 10 10 days from the last
administration
administration was found to be 0.01% of the dietary concentration. concentration. The
authors concluded that long before the time of
authors concluded of harvest the levelslevels of exex-
ogenous steroid
ogenous steroid level
level would have decreased to nondetectable levels. levels. It is
evident that the biological
biological basis for concern is small enough to be dis dis-
regarded especially
especially when the high concentration
concentration of of endogenous sex steroids
steroids
present in maturing salmon salmon at the time of harvest are considered (Schmidt
considered (Schmidt
and Idler, 1962).
1962). However,
However, marketing concerns concerns or legislative
legislative restrictions
restrictions
may require the use of of indirect strategies
strategies where possible.
possible.
The indirect strategy
strategy should theoretically
theoretically result in the reliable produc produc-
tion of
of monos
monosex ex groups.
groups. However,
However, this approach relies on the inheritance of of
sex as a Mendelian trait through a heterosomal heterosomal system.
system. As As previously de de-
scribed heterosomal
heterosomal systems
systems are incompletely
incompletely developed in some some species
species ofof
fish.
fish. Therefore,
Therefore, this approach may not be appropriate for use in these spe spe-
cies especially
especially where high levels of treatment effectiveness
effectiveness are required.
There
There areare several
several other
other disadvantages
disadvantages to to this
this approach.
approach. First,
First, the
the production
production
of fi sh with the desired gonadal sex requires more than one generation.
fish generation.
Second,
Second, aa means
means of
of identifYing
identifyingthe the homogametic
homogametic sex-inverted
sex-inverted adults
adults oror their
their
gametes
gametes must be available.
available. Johnstone et al. a1. (1979b)
(1979b) and V. J. Bye (personal
(personal
communication,
communication, 1980) 1980) noted that genetically female Salmo gairdneri when
masculinized
masculinized with androgens
androgens do not develop a functional functional sperm duct al al-
though they yield functional
functional sperm when dissected.
dissected. Such a characteristic
characteristichas
5.
5. HORMONAL
HORMONAL SEX CONTROL AND
SEX CONTROL AND ITS
ITS APPLICATION
APPLICATION TO
TO FISH
FISH CULTURE
CULTURE 247
provided aa valuable guide for

valuable guide for the
the selection
selection ofof sex-inverted
sex-inverted female
female adults.
adults.
However,
However, in in species
species that
that do
do not
not have
have sex-linked
sex-linked genetic
genetic markers
markers or or mor
mor-
phological
phological anomalies,
anomalies, progeny
progeny testing
testing is
is required.
required. Donaldson
Donaldson and and Hunter
Hunter
(1982a)
(1982a)outlined
outlined several
several effective
effective strategies
strategies for
for the
the production
production and
and identifica
identifica-
tion of gametes that only contain one gonosome type (X) (X) in species, such as
the Pacific
Pacific salmon
salmon which
which exhibit
exhibit aa XX-XY
XX-XY heterosomal
heterosomal system
system (Fig.
(Fig. 2). In
2). In
this situation, effective use may be made of cryopreservation techniques to
store gametes while
store gametes while progeny
progeny tests tests are
are conducted (see Chapter
conducted (see Chapter 6, 6, this
this vol
vol-
ume). Similar screening procedures have been outlined for the tilapias (Shel (Shel-
al.,, 1978;
ton et al. 1978; Jensen and Shelton, 1979) 1979) and Salmo species (Johnstone
(Johnstone et
al.,, 1979b).
al. 1979b).
The
The identification
identification of of gametes
gametes from from sex-inverted
sex-inverted homogametic
homogametic adults adults is
is
not necessary when the primary objective is merely to increase the propor propor-
tion
tion ofof the
the homogametic sex sex type.
type. ForFor example, in in the Pacific
Pacific salmon
salmon an an
increase
increase in the proportion
in the proportion of of homogametic
homogametic females
females is
is desirable
desirable for
for increasing
increasing
the
the rate
rate of
of stock
stock enhancement.
enhancement. In In such
such situations
situations the
the use
use of
of gametes
gametes fromfrom
both masculinized females
both masculinized females andand normal
normal males
males resulting
resulting in
in the
the production
production of of
75%
75% female
female offspring
offspring isis suitable.
suitable. The need need for
for progeny
progeny testing
testing may
may also
also be
eliminated
eliminated by by the
the use
use of
of gynogenetic
gynogenetic techniques
techniques inin species
species which
which areare female
female
homogametic
homogametic (Stanley
(Stanley et al. 1975; Donaldson
al.,, 1975; Donaldson and and Hunter, 1982a). The
Hunter, 1982a). The high
high
mortality usually associated with gynogenetic techniques impedes their di di-
rect
rect use
use for
for the
the production
production of of monosex
monosex groups.
groups. However,
However, gynogenetic
gynogenetic indiindi-
viduals
viduals maymay be sex sex inverted
inverted and and their
their gametes
gametes used
used for
for the breeding
breeding of of
monos
monosex ex groups.
groups.
h.
b. Sterilization. Numerous
Numerous studies
studies have
have reported
reported the the inhibitory
inhibitory effects
effects
of
of steroid
steroid treatment
treatment atat high
high dosages
dosages or
or long
long durations
durations on on both
both gonadogenesis
gonadogenesis
and
and gametogenesis
gametogenesis (Schreck
(Schreck 1974).
1974). However,
However, relatively
relatively fewfew have
have attempted
attempted
deliberate
deliberate hormonal
hormonal sterilization.
sterilization. TheThe majority
majority ofof these
these deliberate
deliberate attempts
attempts
have
have been
been confined
confined to
to the
the economically
economically important
important species.
species. Further,
Further, with
with the
the
notable
notable exception
exception of Eckstein
Eckstein and and Spira
Spira (1965),
(1965), who
who sterilized
sterilized the the gonads
gonads ofof
Oreochromis au reus with
aureus with stilbestrol
stilbestrol diphosphate,
diphosphate, all all deliberate
deliberate attempts
attempts atat
sterilization have employed
sterilization have employed androgens.
androgens.
Yamamoto
Yamamoto (1958),
(1958), working
working withwith the medaka,
medaka, first
first demonstrated
demonstrated that that
when androgens are used at dosages beyond those those required to achieve mas mas-
culinization,
culinization, aa proportional
proportional increase
increase inin percentage
percentage sterilized
sterilized fishfish results.
results.
Several
Several studies
studies have
have demonstrated
demonstrated that that the
the duration
duration of of treatment
treatment is is also
also of
of
importance. OralOral treatment of coho coho salmon
salmon at three
three dosages and three three dura
dura-
tions
tions of
of treatment
treatment demonstrated
demonstrated that that longer
longer durations
durations of treatment
treatment as as well
well as
as
higher
higher dosages
dosages result
result in
in aa higher
higher production
production of of sterile
sterile fish
fish (C. A. Hunter
( G . A. Hunter and
and
E
E.. M.
M . Donaldson,
Donaldson, unpublished).
unpublished). Van Van denden Hurk
Hurk and and Slof
Slof (1981)
(1981) similarly
similarly
observed
observed thatthat the
the administration
administration of of methyltestosterone
methyltestosterone to to juvenile
juvenile Salmo
rTiz--1
ALEVINS I
-
ANDROGEN
ALEVINS]
ANDROGEN
ALEVINS
R U E XX
PHENOTYPIC
PHENOTYPK .
I
MALE XY ALL
ALL fEMALE XX MALES
I ANDROGEN
FEMALE XX
FEMALE XX MALE ' 1
11MALE:l FEMALE
FEMALE TERMINATED
TERMINATED XX
;--
PHENOTYPIC
PHENOTYPK
MALES
MALES
MILT
MILT
X ... V
- fERT ILIZATION Of
FERTILIZATW
NORMAL
NORMAL OVA OVA
OF SUBSAMPLE
WEAMPLE
SEX
SEX RATIO
RATIO
XUY xx DETERMINATION
MTERMINATON
XY
XY XY
XY XX , FERTlLlZAnON Of
xx x----
X
xx xx
XX
+
XX
-+
'---r-
XX
J
NORMAL OVA
CRYOPRESERVATION TERMINATED
TERMINAED
I- X W Y
X /
Fig. 2.
Fig. 2. Alternative
Alternative strategies
strategies for
for the
the production
production of
of 100%
100% x milt
milt from
from Pacific salmon
Pacific salmon by
by two-stage
two-stage androgen
androgen treatment
treatment with
with test
test cross
cross (-),
(-), byby
single
single androgen-treatment
androgen-treatment cryopreservation
cryopreservation and
and test
test cross
cross (-
(- - -),
-), by
by radiation
radiation gynogenesis
gynogenesis combined
combined with
with androgen treatment (-._)
androgen treatment (--) (from
(from
Donaldson
Donaldson and
arid Hunter,
Hunter, 1982a).
1982a).
5. HORMONAL
5. HORMONAL SEX
SEX CONTROL
CONTROL AND
AND ITS
ITS APPLICATION
APPLICATION TO
TO FISH
FISH CULTURE
CULTURE 249
gairdneri at 3
gairdneri 3 pg/l
J,Lg/I in the aquarium water for 8 weeks posthatching was as
effective in producing sterile fish as treatmenttreatment at 300 J,Lg/I
pg/l for a 4-week
period. In the same study, study, treatment for a 4-week period at dosages from 3 3
to 300 pg/l
J,Lg/I indicated a dose-dependent production of sterile fish. Van den
Hurk and Slof noted that sterility was not induced when treatment was
begun at 43 days postfertilization coinciding with gonadal sex differentiation.
However, this time period also coincides with yolk-sac yolk-sac absorption and initia-
initia
tion of
of feeding. Therefore, the method of of administration ofof this steroid may
have influenced its effectiveness.
effectiveness. The influences of route of steroid admin admin-
istration on effective dosage are discussed further. further.
Application of androgens to juveniles resulting in the sterilization of of
gonads and the maintenance of of this gonadal state to adulthood have been
demonstrated in Oryzias latipes (Yamamoto, (Yamamoto, 1975),
1975), Salmo gairdneri (Ja (Ja-
labert et al.al.,, 1975;
1975; Yamazaki,
Yamazaki, 1976),
1976), Oncorhynchus kisutch (Hunter et al. aZ.,,
1982a),
1982a), and Oncorhynchus tshawytscha
tshawytscha (Hunter al.,, 1983).
(Hunter et al. 1983).
The precise mechanism of hormonally induced sterilization remains to be
determined.
determined. McBride and Fagerlund (1973) (1973) reported that treatment of 3-4- 3-4-
month-old
month-old coho salmon fry with a diet containing 10 10 mg methyltesto
methyltesto-
sterone/kg diet for a period of of 20-32
20-32 weeks resulted in sterilization of of testes,
but had little or no effect on ovaries.
ovaries. Hirose and Hibiya (1968)
(1968) administered
4-chlorotestosterone to yearling rainbow trout and found testicular degrada degrada-
tion, restraint of male germ-cell differentiation at the spermatogonium stage,
tion,
and prevention of yolk deposition in the oocytes. oocytes. Ashby (1965)
(1965) also
also reported
that oocytes, once differentiated, were highly resistant to steroid influence. influence.
Hirose and Hibiya and Ashby attributed the results to a negative-feedback
inhibition of of gonadotropin release. In this regard, Yamamoto (1975) (1975) has
reported that hypophysectomy of of juvenile medaka inhibits the transforma
transforma-
tion of spermatogonia to spermatocytes. On the one hand, testes implanted
into fish which had previously been sterilized by hormonal treatment under under-
went spermatogenesis, indicating that treatment did not adversely affect the
hypothalamus or hypophysis. On the other hand, Billard et al. (1981) dem
al. (1981) dem-
onstrated total inhibition of testicular development in adult 2-year-old rain rain-
bow trout fed 0.5 0.5 mg/kg methyltestosterone or estradiol and partial inhibi inhibi-
tion with 0.50.5 mg/kg testosterone fed during the period of spermatogenesis
aune-November).
(June-November). During this period, there was no noticeable change in
plasma gonadotropin relative to controls other than the prevention of the
gonadotropin rise in September.
September. These results suggest that steroid inhibi inhibi-
tion occurs at the level of the gonad.gonad. Whether sterilization is attributable to
feedback inhibition or direct action on the gonads, especially at very early
stages
stages of development, remains to be determined.determined.
al. (1976)
Katz et al. (1976)have hypothesized that the sterilization of Oreochromis
niloticus gonads by adrenosterone added to the rearing water at 5000 5000 J,Lg/1
pg/l
250 GEORGE
GEORGE A.
HUNTER AND EDWARD M. DONALDSON
M. DONALDSON
for
for aa 3-month-period
3-month-period involves
involves inhibition
inhibition of
of gonocyte
gonocyte migration
migration through
through the
splanchopleura
splanchopleura which
which they suggest may
they suggest may be hormonally
hormonally mediated.
mediated. They
They obob-
served
served that
that fry
fry treated
treated in
in the aforementioned
aforementioned manner
manner were
were sterile
sterile at
at the end
end
of
of treatment.
treatment. However,
However, at at 88 months,
months, 92%92% of
of the
the expected
expected number
number ofof male
male
fish
fish contained
contained testes,
testes, but
but no
no fish
fish had
had ovarian
ovarian tissues. Katz
Katz and
and co-workers
co-workers
suggest that
that the steroid action
action initially
initially blocked
blocked the migration
migration of of the
the
gonocytes,
gonocytes, thereby preventing
preventing germinal tissue development.
development. Ovarian
Ovarian tissue
tissue
was
was completely
completely destroyed,
destroyed, therefore,
therefore, repopulation
repopulation ofof gonocytes
gonocytes inin part
part byby
mitotic
mitotic division
division of
of resting cells
cells occurred
occurred only
only in
in the testes.
testes.
Largely because of the variable effectseffects of androgen administration on
differentiated oocytes, the most effective strategy for hormonal sterilization
requires the administration of steroid treatments during the period of sex
differentiation at higher dosages and longer durations than those required
for masculinization.
Several alternate methods for producing sterile fish have been proposed
including induced polyploidy, a surgery-induced autoimmunity, and chemi chemi-
cal or radiation treatments. These techniques have been reviewed for tele tele-
osts with special reference to the grass carp (Stanley,
(Stanley, 1979, 1981) and the
1979, 1981)
salmonids
salmonids (Donaldson and Hunter, 1982a). 1982a). The most promising of these
techniques, induced polyploidy, is examined in detail in Chapter 8, this
volume. The induction of triploidy appears to inhibit ovarian development to
a much greater extent than testicular development. As a result, the use of
hormonally produced all-female Salmo gairdneri has been suggested as as a
first step to producing sterile groups of this species (Lincoln and Hardiman,
1982).
1982).
The major advantage of hormonal sterilization is the ability to achieve
effective
effective treatment
treatment with genetic males and females.
females. Therefore, the establish
establish-
ment of a monosex-treatment population is not required. The disadvantages
of the technique include the application of steroids at an early stage of
development to fish destined for human consumption and the length of
treatment required.
B. Treatment
Treatment
Within this phase the appropriate hormone and method of application

are chosen. The objective of these choices is to ensure
ensure that the undifferenti
undifferenti-
ated gonad is exposed to the hormone at a sufficient dosage and duration of
treatment to redirect the course of sex differentiation. The choice of the
desired gonadal sex,
sex, made in the management phase, and the strategy to
obtain it determine whether androgens or estrogens are used. However, the
choice of a specific
specific steroid must involve considerations of route of admin-
5.
HORMONAL SEX AND ITS
APPLICATION TO
TO FISH
FISH CULTURE
CULTURE 251
251
istration, steroid dosage, treatment timing, and duration. These factors are
in turn interactive with the gonadal and somatic development of the target
species,
species, which are influenced by environmental parameters.
1. ROUTE
1. ROUTE OF
OF ADMINISTRATION
ADMINISTRATION
The choice
choice of
of aa particular
particular route
route of
of administration
administration isis clearly
clearly influenced
influenced byby
the
the development
development of of the target
target species.
species. The
The two
two most
most popular
popular routes
routes have
have
been the addition of the steroid either to the rearing water or to the diet; the
choice
choice usually depends on
usually depends on whether
whether the species is
the species is capable
capable of
of feeding
feeding (Table
(Table
II).
11). Using an innovative adaptation of these approaches, Dzwillo Dzwillo (1962)
(1962) and
Takahashi
Takahashi (1975a,
(1975a,b)b) achieved functional masculinization of embryonic gup gup-
pies by androgen treatment of gravid females either in the rearing water or
in
in the
the diet,
diet, respectively. Other routes
respectively. Other routes of
of administration
administration have
have included
included sub
sub-
cutaneous
cutaneous implantation (Egami, 1955;
implantation (Egami, 1955; Okada,
Okada, 1962),
1962), intraperitoneal
intraperitoneal injec
injec-
tions
tions (Castelnuovo,
(Castelnuovo, 1937;
1937; Eversole,
Eversole, 1939,
1939, 1941;
1941; Berkowitz,
Berkowitz, 1941;
1941; Vallowe,
Vallowe,
1957;
1957; Okada, 1964),
1964), injection into eggs (Hishida,
(Hishida, 1962),
1962), and implantation of
steroid-containing silastic capsules (Jensen et al. al.,, 1978).
1978). The use of these
alternate
alternate routes
routes have
have been
been limited
limited and,
and, with
with the
the exception
exception of of the
the latter,
latter, used
used
only for experimental purposes. Therefore, further discussion in this section
is be confined to the mor more � common routes of administration.
administration.
2.
2. DOSAGE
DOSAGE
Early
Early research
research onon the
the medaka
medaka indicated
indicated that,
that, over
over the
the effective
effective range
range ofof
dosages,
dosages, the
the level
level of
of controlled
controlled sex
sex differentiation
differentiation achieved
achieved was
was dose
dose depen
depen-
dent
dent (Yamamoto,
(Yamamoto, 1969,1969, 1975).
1975). Using
Using genetically
genetically all-male
all-male (XY) Oryzias
0yzias lati
Zati-
pes, a proportional increase in the percentage of females occurred between
pes,
oral estrone dosages of 10-25 mg/kg diet. diet. At 50 mg/kg diet, 100%100%female
fish
fish were
were produced
produced (Yamamoto,
(Yamamoto, 1959b).
1959b). The
The response
response ofof genetically
genetically all
all
female
female Oryzias latipes Zatipes to methyltestosterone was slightly different
(Yamamoto,
(Yamamoto, 1958).
1958). At oral dosages between 5 and 25-50 25-50 mg/kg diet a propro-
portional increase in the percentage of males occurred, reaching a maximum
level somewhat less than 100%. 100%.The 100% 100%level was not achieved because of
the
the production
production ofof aa few
few sterile
sterile fish.
fish. At higher
higher dosages
dosages of
of 50-300
50-300 mg/kg,
mg/kg, the
the
percentage of steriles
steriles increased to 100%.100%. Yamamoto
Yamamoto concluded that meth meth-
yltestosterone
yltestosterone both suppresses
suppresses gonadogenesis
gonadogenesis in bothboth sexes
sexes and also
also induces
sex
sex inversion of genetic females. At higher dosages the former effect effect is
intensified.
intensified. For both estrogens and androgens subthreshold dosage dosage levels
levels
result in the production of intersex gonads.
The
The result of
of several studies
studies have indicated that this dosage-effect
dosage-effect rela
rela-
tionship may not be as as straightforward as as proposed by Yamamoto.
Yamamoto. The parapara-
doxical
doxical effects
effects reported
reported in in various
various cichlid
cichlid species
species have
have already
already been
been men-
men-
Table n
Table Ll
Hormonal
Hormonal Sex-Control
Sex-Control Studies---Economically
StudiesEconomically Important
Important Species
Species
Species
Speciff Steroid Dosage
Dosage Duration and timing Treatment effect
effect Reference
Reference
Cicblids:
Cicblids: Use
Use of
ol Androgens
O.
0. aureus
aurcu Testosterone 50-1000 ",gil 5-6 week starting.
starting, 4--5
&5 week Variable
Variable eff
ects involuted and
effects Eckstein and Spira (1965)
(1965)
Methyltestosterone
Methyltestosterone posthatching unaff
ected gonads
unaKected gonads
Ethynyltestosterone
Ethynyltestosterone l1HiO mg/kg diet 18
18 days,
days, 6
6days/week
daydweek lOr
for 33 30-60
30-60 mg/kg produced 98-
mgkg produced 98- Guerrero (1975)
(1975)
Methyltestosterone
Methyltestosterone weeks
Weeks 100% Males, f
1Ul% Males, emale hetero-
female
g;unety
gamety demonstrated
demonstrated
Ethynyltestosterone
Ethynyltestostemne 30 mglkg
30 mgkg diet 32
32 days
days Monosex
Monosex males Sanieo
Sanicu (1975)
(1975)
Etbynyltestosterone
Ethynyltestosterone 60
60 mglkg
mgkg diet 21-28
21-28 days
days 98.9-100%
98.%100% Males
Males produced Shelton et
et al.
01. (1981)
(1981)
O.
0.niloticus
uilorinrp Methyltestosterone
Methyltestosterone 40
40 mglkg
mgntg diet 60
60 days
days Increased males;
males; ffemale
emale homo- Jalabert
Jalabert et
et al.
ol. (1974)
(1974)
g;unety
gamety demonstrated
demonstrated
Methyltestosterone
Methyltestosterone 15-50 mg/kg
1550 mgkg diet 42 days
days Increased males Guerrero and Abella (1976)
(1976)
to
Andrenosterone SOOO
5OOo ",
PgII
d 3
3 months postfertilization
postfertilization No germ cells apparent
apparent at 33 Katz
Katz et
et al.
al. (1976)
(1976)
<:It months, 40%
40% had testes at 88
to
months
Ethyltestosterone
Ethyltestosterone 30-00
3 l M O mglkg
mgkg diet 25-59
w 9 days
days 100%
100% Males
Males produced Tayamen and Sbelton (1978)
and Shelton (1978)
Methyltestosterone
Methyltestosterone
Methyltestosterone
Methyltestosterone 5 mglkg
mgntg diet 28 or 42 days
28 100%
100% Males
Males produced Owusu-Frimpong
Owusu-Frimpong and Nijjbar
Nijjhar
(1981)
(1981)
Methyltestosterone
Methyltestosterone S50-100
1 0 0 mg/kg
mgkg diet 30 days
days from capture 100% Males produced
100% Males Nakamura and lwabashi
Iwahashi
(1982)
(1982)
T.
T. :r.ill
Zinu ii Ethyltestosterone
Ethyltestosterone 50
50 mglkg
mgkg diet 40
40 days
days No sex inversion Guerrero (1976b)
(1976b)
Methyltestosterone
Methyltestosterone 50-100 mglkg
5o-100 mgkg diet 20 days
days starting
starting 10
10 days
days post- No sex inversion;
inversion; oogenesis
oogenesis Yoshikawa and Oguri (1978)
(1978)
hatching inhibited
Methyltestosterone
50 mglkg
mgkg diet 45
45 days
days 100%
100% Males
Males produced Wodiwode (1977)
(19m
T.macrochir
T. macmchir Methyltestosterone
40 mglkg
mgkg diet 2
2 months 100%
100s Sterilization
Sterilization Jalabert et
d l. (1974)
aol. (1974)
O.
0.rnossambicus
moasorn6icus Methyltestosterone
Methyltestosterone 1!h50
1- mg/kg
m&g diet 69
69 days
days Males produced at 10-40
100% Males 1 W Clemens
Clemens and Inslee (1968)
(1968)
mglkg;
mgkg; ffemale
emale homog;unetic
homogametic
Methyltestosterone
mgkg diet 19
19 days
days starting
starting 7
7 days
days post- 100%
100% Males
Males produced
produced Nakamura (1975)
(1975)
hatching
1000
loo0 mglkg
m&g diet 19
19 days starting 77 days
days post- Ineffective
Ineffective
hatching
hatching
Methyltestosterone
30 mgkg diet 14-28
1428 days
days 69-98% Males
6%98% Males produced
produced Guerrero (1976a)
(1976a)
50 mglkg diet
50 mgkg 30 days
days 100% Males produced
100s Guerrero (1976b)
(1976h)
Methyltestosterone
mgkg diet Fry (9-10
(%I0 mm) for 42 days 100%
1004b Males produced Anonymous
Anonymous (1979)
(1979)
30 mglkg
mgkg diet 42 days 81-&5%
8145% Males produced
pmduced Anonymous
Anonymous (1979)
(1979)
ll-Ketotestosterone
1I-Ketotestosterone 200
MX) mglkg
mgkg diet 19
19 days starting 7 days post- 100% males produced
produced Nakamura (1981al
(19Xla)
hatching
Methyltestosterone
Methvltestosterone 50
50 mglkg
mgkg diet 19
19 days starting 77 days post- 100% Males produced
produced Nakamura (19810)
(198la)
batching
hatching
10 ...!¥1
10 cpn 100%
100% Males produced
produced Nakamura (1981a)
(1981a)
Cichlids:
Cichlids: Use of Estrogens
O.
0. au reus
oureus Stilbestrol 50-1000
5 C L ...!¥1
Pg/l
~ ~ 5--6
56 weeks starting, 4-5
4-5 weeks At 50
50 and 100
100 ...
!¥1 sterility
pg/l Eckstein and Spira (1965)
(1965)
Slilbestrol-dipbosphate-
Stilbestml-diphosphate- posthatching induced
Diaethyldioxyslilben-diphosphate
Diaethyldioxystilben-diphosphate
Estriol 30-120
&120 mglkg
mgkg diet 21
21 or
or 35 days No sex inversion Jensen (1976)
(1976)
}
Estrone
Estrune 30-120
S 1 2 0 mglkg
mgkg diet 21
21 or 35 days No sex inversion
Estradiol 30-120
&120 mglkg
mgkg diet 21 or 35 days No sex inversion
Ethynylestradiol
Diethylstilbestrol
Diethylstilbestml
Estradiol 1
With, or without,
without
cryproterone
cryproterone
acetate
Ethynylestradiol with methallibure
and cryproterone
methallibure
cryproterone acetate
25-200
%ZOO mglkg
mgkg diet
Cryproterone acetate at
100 mglkg
100
mgkg diet
100 mglkg
100
m&g diet
100 m!¥kg
mdkg diet
35 or 56
35
42
56 days
42 days
6()...M
M%
90%
Females produced
% produced
90% Females produced

produced
Hopkins (1977)
(1977)
(1979)
Hopkins et al. (1979)
to
w 100
100 m!¥kg
mdkg diet
c:R
Z:
,., O.
0.niloticus
niIoticus Diethylstilbestrol
Diethylstilbestrol 25 and 100
100 mglkg
mgntg diet 25--59
2.559 days 62--90%
62-!Wh Females produced
produced Tayaman
Tayaman and Shelton (1978)
(1978)
Estrone
Estrune 100 and 100 mglkg
mgkg diet
Ethynylestradiol
Ethyn ylestradiol 20-00
2 W mglkg
mgkg diet 20 days starting
starting 10
10 days post- No sex inversion Yosbikama (1978)
Yoshikama and Oguri (1978)
hatching Some involuted gonads at 40
Some 40
and 60 mglkg
mgkg
O.
0. mossambicus
mossambicus Ethynylestradiol 50 mglkg
mgkg diet 19
19 days from 6 days post- 100%
1- Females produced Nakamura
N b u r a and Takahashi
hatching (1973)
(1973)
Salmon:
Salmon: Use
Use of
of Androgens
Androgens
Salnw
salmo sp.
4-Chlorotesterone
4-Chlomtesterone 1.0-12.5
1.&12.5 mg injection
injection 6
6 30 days starting at age 1 year Suppression of testicular
testicular differ- Hirose and Hibiya (1968)
(1968)
S. gairdneri
S.
times entiation and
and yolk
yolk deposition
Methyltestosterone
Methyltestosterone 1S-W
15-60mg/kg
mdkg diet 5 months starting 1 month 58%
58% Males and 12%
12% steriles ef al.
Jalabert et
Jalabert al. (1975)
(1975)
posthatching produced
produced
Methyltestosterone
50 mglkg
mgkg diet 5 months starting 11 month Testes devoid of
of germ cells Yamazaki (1976)
(1976)
posthatching
mgkg diet 7 months starting 1 month 87.1% Yamazaki
Yamaza!ii (1976)
(1976)
posthatching
(continued)
(continued)
Table ll-Continued
II4ontinued
Species
Species Steroid
Steroid Dosage
Dosage Duration
Duration and
and timing
timing Treatment
Treatment effect
effect Reference
Reference
S. gairdneri
S. gairdwri (cont.)
(cont.) Methyltestosterone
Methyltestosterone 250 jLg/I plus 3 mg/kg Two
Two 2-hr
2-hr immersion
immersion of
of eyed
eyed 100% Sex
100% Sex inversion
inversion Simpson
Simpson (1976)
(1976)
diet and
and alevins, 90 days
alenns, 90 days from
from
6rst
first feeding
feeding
3 mg/kg diet 90 days
90 days from
from 6rst
first ffeeding
eeding 100% Sex
Sex inversion
inversion
Methyltestosterone 250 jLg/I plus 3 mg/kg Two
Two 2-hr
2-hr immersion
immersion of
of eyed
eyed 83% Males
83% Males and 17% sterile
and 17% sterile Johnstone
Johnstone et al., (1978,
ei al., (1978,
diet eggs
eggs and
and alevins 90 days
alevius 90 days produoed
produced 1979a)
1979a)
from
from 6rst
first ffeeding
eeding Demonstration
Demonstration of
of ffemale
emale homo-
homo-
gamety
wety
Methyltestosterone
Methyltestosterone 1-10 mg/kg
1-10 mgkg diet
diet 58 days
58 days 60-68%Males
00-68% Males produoed,
produced. dem-
dem- Okada
okada et
et oJ.
nl. (1979)
(1979)
oustration
onstration of
of ffemale
emale homo-
home
gamety
wety
to Methyltestosterone
Methyltestosterone 3 mg/kg
3 m&g diet
diet 90 days
days 94% Male
94% Male V. J.
V. J. Bye
Bye (personal
(personalcommu-
cummu-
� nication,
nication, 1980)
1980)
Methyltestosterone
Methyltestostemne 25 mg/kg
mgkg diet
diet l000"
Ccdays
1ooo" days (9-12"C) 110 days
(S12"C) 110 days 60%
60% Sterility
Sterility noted
noted V.
V. J.J. Bye
Bye (personal
(personalcommu-
cummu-
nication,
nicition, 1980)
1980)
Methyltestosterone
30 mgkg diet
diet 2.9%
2.9% Mature as 2
Matnre as 2 year
year olds
olds Harbin
Harbin et
et oJ.
al. (1980)
(1980)
versus
versus 40%
40% control
cuniml
Methyltestosterone
Methyltestostemne 3-300 jL
FgfIll 28 or 56 days
or 56 days immersion
immersion Sterility,
Sterility, high
hi& mortality
mortality van
van den Hurkand
den Hurk and Slof
Slof (1981)
(1981)
Methyltestosterone 0.5 mg/kg diet
mg&g diet 6 months
months Oune-:>N'ovetnber)
Uuu4ovember) Gonadal
Gonadal inhibition
inhibition BUIard
Billard et al. (1981)
et oJ. (1981)
56 days 10046 Males produoed
100% van
van den Hurk and
lambed
Ill\-Hydroxyandrostenedione
Up-Hydroxyandmstenedioue 60 mg/kg
60 m&g 56 days produced
(1982)
(1982)
6.0 mg/kg
mgkg 56 days
56 days 94-99% Males produoed
9449% Males produced
0.66.0mwkg
0.1Hi.0 mglkg
s.
S. StJlor
scrlar Methyltestosterone 250
250 jLg
14 Il plus 30 mg/kg
PIUS 30 mg&g Two
Two 2-hr immersions of
2-hr immersions of eyed
eyed No
No female
female elements
elements Simpson
Simpson (197�1976)
(1975-1976)
diet eggs and
eggs and alevins
alevins for 120 days
for 120 days
Methyltestosterone
mgkg dietdiet 120 days
days Sterile
Sterile Johnstone et
Johnstone et al. (1978)
01. (1978)
mwkg diet
3 m& diet 90 days
days Male or
100% Male
100% Or sterile
sterile Johnstone et al.
(1978)
trotla
S. truffa
S. Testosterone 50-60 P
EXMa jLgfIll 1 1 1 days starting 170 days
111 41
41% Male, 35% Female
% Male, Female Ashby (1957)
Ashby (1957)
postfertilization 6 days/week
postfertiliz;ition daydweek
for months starting
for 3 months starting 7
months
months postfertilization
postfertilization
Testosterone
Testosterone 67 jLg/I 24%Indeterminant
24% Indeterminant testicular Ashby
Ashhy (1965)
(1965)
inhibition
inhibition
solevlinus sp.
Salevlinus sp.
S. nIl11lOycush
S. Mmoycush Testosterone propionate 700
700 mglkg
mg/kg diet 245-290"C
2452!jO'C days
days starting
starting at
at 7 Males,
Males, 33 ffemales
emales produced
produced Wenstrom
Wenstriim (1975)
(1975)
125O"C
1250°C days
days
Oncorhynchus
Oncorhynchus sp.
sp.
O.
0. kisutch
kisutch Methyltestosterone
Metbyltestosterone 1-50
1 5 0 mg'kg
mgkg diet 42
42 days
days starting 8--9
a9 months
months Severe
Severe reduction
reduction in
in spermato-
spermato- McBride
McBride and
and Fagerlund
Fagerlund
posthatching
posthatching gonia
gonia (1973)
(1973)
Methyltestosterone 0.2-10 mg'kg
mgkg diet 504
504 days starting 3-4
days starting %4 months
months Severe
Severe gonadal
gonadal inhibition
inhibition in
in Fagerlund
Fagerlund and
and McBride
McBride
po,thatching
posthatching group
group fed
fed 10
10 mg'kg
mgkg (1975)
(1975)
Methyltestosterone
Methyltestosterone 25-400
25-400 .,.gll
pg/l plus Two
Two 2-hr immersions
immersions of
of eyed 94-100% Sterile
9p100% Goetz e/
Goetz et al.
d.(1979)
(1979)
eggs
eggs and alevins
alevins
u)mg'kg
20 mgkg diet 70 days
Methyltestosterone
400 .,.gil
Pg/l Two 2-hr immersion of
of alevins
alevins 67%
67% Males produced
produced G.
G. A. Hunter and
A. Hunter E. M,
and E. M.
Donaldson
Donaldson (unpublished
(unpublished
data)
data)
Methyltestosterone
Methyltestosterone 400 .,.gil plus Two 2-hr
2-hr immersion of
of either 83-93%
@%SO% Sterile G. A. Hunter
C . A. and E.
Hunter and E. M
M.,
eyed eggs
eggs oorr alevins
alevins Donaldson
(unpublished
data)
data)
10 mg'kg diet 3 months
Methyltestosterone
Metbyltestostemne 4OO .,.gil plus Two
Two 2-hr immersion of
of alevins
alevins 82-92% Males
Males produced
produced G.
G. A. Hunter and
A. Hunter and E.
E. M.
M.
I<> Donaldson
(unpublished
<:It
<:It data)
data)
1
1 mg'kg
mg&g diet, or 21-63
2 1 4 3 days
days 22-68%
22-689 Males
Males produced
produced
�9
3-9 mglg
mg/g diet 22-78%
22-788 Sterile
Methyltestosterone
Methyltestosterone 400 pg/l
100 or 400 .,.gil plus 10 Two
Two 2-hr of eyed
2-hr immersion of 70--94% Sterile at age 3 years
7&%% e/ ol.
Hunter et
Hunter al. (198%)
(19820)
mg'kg
mgkg diet eggs and alevins for 90 days
Methyltestosterone
Methyltestosterone 200 .,.gil PIUS
ml 4 plus of eyed
Two 2-hr immersion of 97.7% Sterility at age 3 years
97.7% Donaldson and Hunter
eggs
eggs and alevins (1982b)
(1982b)
mg'kg diet
10 mgkg diet 84
84 days
O. tshawytscha
0. tshowytscho Methyltestosterone
Methyltestosterone 1.0 mgkg
0.2 or 1.0 mg/kg diet 28-84
D344 days No reduction of spermatogonia
reduction of spermatogonia McBride and Fagerlund
(1973)
(1973)
Methyltestosterone
Methyltestosterone 400 .,.giliter plus
400 pg/liter 2-hr immersions of alevins
Two 2-hr 83-93% Males;
83-938 Males; demonstration of
demonstration o e/ al. (1983)
f Hunter et
'
�9 mg'kg diet
S 9 mgkg 21-63
2 1 4 3 days female homogamety
female
O. keto
0. O. gorbusha
ketu and 0. gorbusho Methyltestosterone
Methyltestosterone 50-100 mgkg
5C-100 mg'kg diet 1 year
14 days at age 1 year of spermatogonia
Degeneration of spermatogonia Yamazaki
Yam& (1972)
(1972)
(continued)
(continued)
ll-Continued
Table II--Continued
Species Steroid Dosage
Dowe Duration
Duration and
and timing Treatment effect Reference
Reference
Salmon: Use of
Snlmw: of Ertmgens
Estrogens
Salmo sp.
Sdmo sp.
S. gairdneri
S. gairdnen Estrone I�IOO mg/kg
1&100 mgkg diet 3&-124 days posthatching
&I24 79--94% Females produced;
7-1 Okada (1973)
Okada (1973)
growth
high mortality, gmwth
depression
Estrone �120 mg/kg diet beginning 1
5 months beginning 1 month 30% intersex
54% Females, 30%
54% Jalabert et d.
Jalabert aI. (1975)
postbatching
posthatching produced
produced
Estradiol
Esmdiol 250 ...WI plus Two Zhr
2-hr immersions of
of eyed 100% Females produced
100% produced Simpson (1976)
eggs and alevins 30
eggs or 56
30 nr
days from feeding
from feeding
mg/kg diet
20 mgkg 15 days 69% Females produced
69% produced
Estradiol mg/kg diet
20 mgkg diet feeding
30 days from first first feeding 100% Female, growth depres- Johnstone et al. (1978)
of male
sion; demonstration of et of.
Johnstone ei al. (1979h)
(1979b)
l<>
cn
CIt
K) heterogamety
hetemgamety
C>
m S. malor
S. r�alar Estradiol 250 ...WI plus Two 2-hr immersion of
of eyed
eyed produced
100% Females produced (1976)
Simpson (1976)
eggs and alevins
20 mg/kg diet 21 days from
21 from first feeding
feeding produced
100% Females produced (1978)
S.
S. trotta
trutta Estradiol 50 or 3OO ...WI 70
70 or 111
11
1 days
days (56O"�oC
(5WG8EE'C High dosage produced
produced 7 Ashby (1957)
(1957)
days)
days) starting 170
170 days post- females and 3 males
females
fertilization (85O"C
fertilization (850°C days)
days)
Salvelinus
Sdcelinus sp.
sp.
S. narooycu.sh
S. namaycush Estradiol 12 mg/kg 24&-29O"C
2&2WC days 8 Females and 2 males Wenstrom
Wenstr6m (1975)
(1975)
produced
produced
SS.. fontinalis
fontinalis 20 mg/kg 60
60 days ffrom
rom first
6rst ffeeding
eeding 99%
99% Females produced
prcduced Johnstone et al.
Johnstone al. (1979a)
(1979a)
OncorhynchUfil
Oncorhqnchw sp.
0. kisutch
O. k*utch Estradiol 25-400 ...WI plus Two or six 2-hr immersion of
of Nine of
of 10
10 groups >95%
>95% Goetz et
ef al.
al. (1979)
(1979)
eyed eggs, two or
or seven im- female; low dosage group
female;
mersion of
of alevins 60%
60% ffemale,
emale, increased mor-
tality
tality and growth
gruwtb depression
depression
10
10 mg/kg
mgkg diet 70 days
Estradiol 100
100 or 400
400 ...
p.4
WI plus Two 2-hr immersions
immersions of
of eyed
eyed 86-100%
&100% Females at maturity, Hunter et aI.
d.(1982a)
(19824
eggs and alevins demonstration of male
heterogamety
5 mg/kg
m& diet 90 days
90 days
Estradiol
Esbadiol 400 ...g11 Two
Two 2-hr immersions
immersions of
of alevins
alevins 100% Females produced
produced G. A. Hunter, E. M. Don-
G. Don-
aldson,
aldson, and 1.
I. Baker
(unpublished)
Estradiol
Esbadiol 200 ...
ml g11 plus
Ppn PIUS Two 2-hr immersions of
of eyed >99%
>SS% Female at maturity Donaldson and Hunter
eggs and alevins (1982b)
(1982b)
5 mg/kg
mglkg diet
diet 84
84 days
0.tshawyucha
a. ishnuytschn 4OO
400 ...
g11 plus
Ppn Plus Two 2-hr immersions of
of 96-100%
S6100% Females produced C. A. Hunter and E. M.
G.
Donaldson
Donaldson (unpublished)
(unpublished)
2 or 5 mg/kg
mgkg diet 21-63
2143 days
5 m",kg
mglkg diet 24--S4
2&84 days
O.
0. masou
nlasm Estradiol 0.25-200
0..2.5200 ...
g11
ppn 18
18 days starting 5 days
days post- All female at 0.5-5
female 0 . 5 5 ...
gII; very
ppn, Nakamura (198I a)
(1981a)
hatching high mortality
mortality at 10-200
1&200 ...",1
pgll
Carp, Use
Carp: of Androgens
Use of Aodmgens
CterwpharyntuUm
C i e w p h q d n idella
idelln Methyltestosterone
Methyltestosterone 30 or 60
60 mg/kg
mdkg diet 14-@
1463 days from
from 7
7 days post-
po5t- No effect
effect Stanley and Thomas (1978)
(1978)
hatching
hatching or 28 days starting
28-112
S 1 1 2 days postbatching
posthatching
Methyltestosterone
Methyltestosterone Silastic
Silastic capsules 20.6-
20.6 303
303 days
days from 195
195 days
days post- Growth
Growth depression; 5
5 fish steT-
ster- Jensen ef
ef al.
al. (1978)
(1978)
31.0 ...",day
&day hatching; 192 rom 309
192 days ffrom 309 lie;
ile: ovarian
ovarian development
days lXlsthatching
posthatching suppressed
� Methyltestosterone
Methyltestosterone 60-120 mg'kg
&120 mgkg 255
95.5 or 410 days from 110 days
from 110 Growth and gonadal inhibition Shelton and Jensen (1979)
(1979)
'"
--1 posthatching
Methyltestosterone
Silastic capsules 18.7
18.7 303
303 days from
from 195
195 days post- Reduced germ cells Shelton
Sheltou and Jensen (1979)
(1979)
...
",day
PgldaY hatching
Methyltestosterone
Silastic capsules
capsules 14.9-
14.k 189-461
18!+461 days from
fmm 309 days 0-31.
W1.7% 7% Females produced
produced Shelton and Jensen (1979)
(1979)
18.4
18.4 ...
g/day
pg/day posthatching
Methyltestosterone
Methyltestosterone Silastic capsules 16.6
16.6 500
500 days from 319 days post-
h m 319 No sex inversion;
inversion; 17.4%
17.4% inter- Shelton and Jensen (1979)
(1979)
...
P&Yg/day hatching, fish stunted 123
fish 123 sex
mm
Methyltestosterone
Silastic capsules 12.5
12.5 460
460 days from 55
55 days, 128
128 mm
mm 18.5
18.5 Male,
Male. 33.3
33.3 intersex, 29.6
29.6 Shelton and Jensen
Jeusen (1979)
(1979)
...
g/day
PgldaY length sterile, and 18.5% reduced
reduced
ovaries
ovaries produced
Cyrpinus carpio
carpi0 Methyltestosterone
100 mi¥kg
mg/kg diet 4-
4- to 36-day
*day periods
periods between
between 8 71.4-S8.9%
71.483.% Males
Males produced; Nagy et al.
al. (1981)
(1981)
and
and 98 days
days posthatching 13.3-28.6%
13.&28.6%undifferentiated
undi5erentiated
258 GEORGE
GEORGE A.
A. HUNTER AND
HUNTER A N D EDWARD M. DONALDSON
EDWARD M. DONALDSON
tioned. Further, methyltestosterone

methyltestosterone treatment at very high dosages has led
to decreases in masculinizing potency without sterilization. Okada et al.
(1981) orally administered methyltestosterone
(1981) methyltestosterone for 88 weeks to genetically all all-
female Salmo gairdneri at dosages ranging from 0.01 0.01 to 500 mg/kg diet. No
inversions were observed in groups fed 0.010.01 mg-0.
mg-0.11 mg/kg diet. At 0.5, 11.0, . 0,
5.0, and 1O. O mg/kg diet 84.4,
10.0 84.4, 69. 2, 42.0, and 24.
69.2, 0% males were recorded.
24.0%
At dosages higher than 50.0 mg/kg diet, less than 7.0% males were re re-
corded. Intersex gonads were observed at dosagesdosages from 0.5 to 10. 10.0 0 mg/kg
diet.
diet.
Also related
related to steroid treatment
treatment are various deleterious effects
effects usually
associated with high steroid dosages. These effects
effects include gonadal suppres
suppres-
sion and paradoxical steroid actions, which were previously described, high
mortality (Ashby,
(Ashby, 1957;
1957; Yamamoto, 1958,
1958, 1961;
1961; Yamamoto
Yamamoto and Matsuda,
1963;
1963; Clemens and Inslee, 1968;1968; Goetz et al.
al.,, 1979;
1979; Okada et al.
al.,, 1979;
1979; van
den Hurk and Slof,
Slof, 1981),
1981), and growth depression especially with estrogen
administration (Ashby, 1957; Okada, 1973;
(Ashby, 1957; 1973; Goetz et al.
al.,, 1979;
1979; Johnstone et
al.,, 1979b;
al. 1979b; Shelton and Jensen, 1979).
1979). Therefore, it is evident that the
choice of dosage is critical to treatment
treatment efficiency.
efficiency. Several interrelated fac fac-
tors influence the optimal dosage level of a particular steroid including its
biological activity, which may be related to its origin, the route of of admin
admin-
istration, the target species, and the duration of treatment.
treatment. This latter factor
is of considerable importance and is considered separately.
a. Biological Activity. Although a wide range of natural and synthetic

a.
steroids have been used successfully
successfully to induce sex inversion, their individual
biological activities vary. Studies on the medaka reviewed
reviewed by Yamamoto
(1969)
(1969) remain the best demonstration of relative steroid potencies. Taking
advantage of the sex-linked color genes of the d-r R strain of Oryzias latipes in
d-rR
which females xrxr are white and males are orange-red xry R, researchers
xryR,
were able to determine the 50% sex-inversion level following oral admin admin-
istration of various androgens and estrogens. In general, the synthetic an an-
drogens were more potent than those of natural origin. The most potent
synthetic androgen was 19-nor-ethynyltestosterone,
19-nor-ethynyltestosterone, which produced 50%
sex inversion at a dosage of 1. 0 mg/kg diet. Less potent were fluox
1.0 fluox-
ymesterone 1.2 1. 2 mg/kg, ethynyltestosterone
ethynyltestosterone 3.4 mg/kg, methylandrosten
methylandrosten-
ediol
edio17.87.8 mg/kg, methyltestosterone
methyltestosterone 15
15 mg/kg, and testosterone propionate
560 mg/kg. The natural androgens included androstenedione 500 mg/kg and
androsterone 580 mg/kg.
mglkg. Using similar methods, Hishida and Kawamoto
Kawamoto
(1970)
(1970)demonstrated that ll-ketotestosterone
11-ketotestosterone is the most potent of the natu
natu-
ral occurring androgens at 110 110 mg/kg. Similarly,
Similarly, the synthetic estrogens
hexesterol, euvastin, and ethylestradiol achieved 50% sex inversion at 0.4,
0. 8, and 1.7
0.8, 1.7 mg/kg, respectively (Yamamoto
(Yamamoto,? 1969).
1969). To achieve 50% sex
5.
5. HORMONAL
HORMONAL SEX
SEX CONTROL AND ITS
CONTROL AND APPLICATION TO
ITS APPLICATION TO FISH
FISH CULTURE
CULTURE 259
inversion, the naturally occurring estradiol, estrone, and estriol required

dosages of 5. 8, 20, and 130
5.8, 130 mg/kg diet. White et aZ. al. (1973),
(1973), observing a
similar hierarchy of estrogen potency in the rat, suggested that the results
were to be expected because both estrone and estriol are the metabolic
products of estradiol. Similarly, l11-ketotestosterone
l-ketotestosterone has been demonstrated
to be much more potent than testosterone in inducing sex inversion in
Poecilia reticulata (Takahashi, 1975c).
(Takahashi, 1975~).
Yamamoto
Yamamoto (1969)(1969) and Hishida and KawamotoKawamoto (1970)
(1970)have suggested that
the higher potency of orally administered synthetic steroids is in part at at-
tributable to their resistance to degradation in the digestive tract. In consid consid-
ering the potencies of various steroids, the extremely low effective dosages
of 16-[14C]estrone
16-[14C]estrone determined by Hishida (1965) (1965) should be noted.noted. For this
steroid
steroid administered
administered at at aa level
level of
of 44.
44.66 mg/kg
mg/kg diet
diet and resulting in
and resulting in 84%
84% sexsex
inversion, the effective dosage at the gonadal level was calculated to be 1.5 l. 5
x
x 10 - 2 J.Lg.
lop2 pg. Therefore, it is evident that the effective quantities of of steroid at
the
the site
site of
of action
action are
are low
low relative
relative toto those
those which must be administered.
which must administered.
Several
Several studies
studies have
have demonstrated
demonstrated the the influence
influence of of the
the route
route ofof admin
admin-
istration on steroid potency. Hishida (1965) (1965)reported a 10-fold
10-fold increase in the
potency
potency of of estrone
estrone when
when administered
administered intraperitoneally
intraperitoneally versus
versus orally
orally toto
Oryzias Zatipes fry.
O y z i a s latipes fry. Takahashi (1974)
(1974) observed that when administered orally
to juvenile Poecilia reticulata, the androgenic potency of the naturally occur occur-
ring
ring l11-ketotestosterone
l-ketotestosterone was was lowlow relative
relative toto synthetic
synthetic steroid
steroid meth
meth-
yltestosterone. The equivalent
equivalent dosages for the two steroids were 200 mg/kg
and 20 mg/kg, respectively. However, when the steroids were added to the
rearing water
water ofof the same
same species,
species, lll-ketotestosterone
l-ketotestosterone treatment
treatment resulted
resulted in in
100%
100% functional masculinization at 10-25 10-25 J.Lg/I,
pg/l, but methyltestosterone
methyltestosterone was
ineffective at this dosage (Takahashi,
(Takahashi, 1975c).
1975~).
In general, optimal steroid dosage appears to be species specific. specific. Howev
Howev-
er, comparisons of relative effective dosages using available data are difficult
because of variability of treatment protocol. Further, observations for a
particular
particular species are often based on a limited number of of dosage levels.
Where comparisons may be made, the variation in effective dosage may be
large.
large. For example, similar levels of sex inversion have been achieved in
genetically all-female populations of Oryzias latipes Zatipes (Yamamoto,
(Yamamoto, 1958) 1958) and
SaZmo gairdneri (Okada et al.
Salmo al.,, 1981)
1981) that were administered methmeth
yltestosterone at 25 mg/kg diet for 49-56 49-56 days or 0.5 mg/kg diet for 60 days,
respectively. Similarly, Yamamoto and Kajishima (1969)
respectively. (1969) obtained 100% 100% sex
inversion
inversion in in goldfish
goldfish fed estrone
estrone at at 100 mg/kg diet
100 mg/kg diet for
for aa period
period of of 60
60 days
days
from first feeding. A similar level of effective treatment (94%) was obtained
treatment (94%)
by
by Okada
Okada (1973)
(1973) with SaZmo gairdneri fed the same
with Salmo same steroid
steroid at 10 mg/kg for
at 10 for aa
period
period of of 58
58 days. InIn general,
general, the
the optimal
optimal treatment
treatment dosages
dosages forfor the
the salm
salm-
on ids are
onids are somewhat
somewhat lowerlower than
than those
those for
for other
other groups
groups (Table
(Table II).
11).
260 GEORGE A.
GEORGE A. HUNTER AND EDWARD
HUNTER AND EDWARD M
M .. DONALDSON
DONALDSON
3. TIMING AND
3. TIMING AND DURATION
DURATION
The criteria established by Yamamoto (1969)(1969) for effective steroid treat
treat-
ment requires that treatment
treatment be initiated prior to the onset of normal sex
differentiation
differentiation and continued
continued until the time of morphological differentiation.
Failure to comply with this criterion results in ineffectual treatment. Indeed
the majority of studies have indicated a specific
specific period over which treatment
must be continued. However, several studies have demonstrated treatments
of
of shorter periods of time which are fully effective. Therefore, the relation
relation-
ship between the initiation and completion of sex differentiation and the
timing and duration of effective treatment proposed by Yamamoto
Yamamoto may be a
conservative estimate for many species.
Treatment is ultimately dependent on an interval of of time in the course of of
gonadal development when the gonads are labile to hormonal influences.
The presumption has been that the steroids, acting as initiators of differ differ-
entiation, should be administered at a time synchronous with natural differ differ-
entiation. Therefore, histological
histological techniques have been employed to deter- deter
mine the initiation and completion of sex differentiation. The identification
of the labile period, by definition, requires the application of steroids at
various times and durations within and outside of the period of of histologically
histologically
observable differentiation.
differentiation. Indicators such as age or size ofof fish may then be
correlated with the boundaries of of the labile period.
a.
a. Determination of of the Period ofof Sexual Dqferentiation. Gonadal sex
Sexual Differentiation.
differentiation has two distinct but highly interactive and interdependent
processes: gonadogenesis, which is the formation of the structural and sup sup-
portive elements of the gonads, and the gametogenesis, which is the forma forma-
tion of the gametes. As previously discussed, the physiological
physiological mechanisms
governing these processes are as yet undetermined. The underlying premise
of hormonal treatment
treatment is that the steroids are or have the ability to mimic the
natural sex inductors. Logically,
Logically, treatment should be started coincident with
the release of the natural inductors and, therefore, sometime prior to the
first histologically signs of sex differentiation. In fact,
histologically observable signs fact, many stud-
stud
ies have concerned themselves with gonadal differentiation in fish. fish. Howev-
Howev
er,
er, few have examined the initial period of differentiation (Harrington,
1974).
1974). The paucity of of studies and a general lack of understanding
understanding of the
mechanisms involved has resulted in considerable difficulty in establishing
criteria suitable for the identification
identification of the initiation of differentiation.
Gametogenesis, either oogenesis or spermatogenesis may be regarded as an
indisputable sign of sex
differentiation. However, the subtlety of the earliest
changes in the formation of the gametes has made their individual identifica
identifica-
tion difficult.
difficult. The dubious identification of oogonia and spermatogonia with with-
out the benefit of somatic indicators has been stressed repeatedly (Reinboth,
1972,
1972, 1982;
1982; Harrington, 1974).
1974).
5.
5. HORMONAL
HORMONAL SEX CONTHOL A
SEX CONTROL AND
N D ITS
ITS APPLICATION
APPLICATION TO
TO FISH CULTURE 261
FISH CULTURE
As a result, investigators have relied heavily on a variety of of somatic and

germinal features to demark early differentiation. Stromsten (1931) (1931) has listed
aa variety
variety ofof such
such features
features used to to identify
identify sexsex differentiation
differentiation in in Carassius
auratus. These
auratus. These features include the form and general appearance of the
gonad, the means of attachment of the gonads to the wall of the coelomic
cavity, the natme
nature and disposition of the cells of the stroma and germ cells,
the structure
structure of the nuclei, the presence or absence of a nucleolus, nucleolus, and the
vascularization of the gonad. Persov (1975) (1975) reports that toward the end of the
indifferent period, the direction of development of of the gonads of the Salmo
species may be determined by the position of the germ cells in the gonad. gonad.
When they are concentrated on the lateral side and large capillaries are on
the medial side, ovarian development is taking place. If the germ cells are
ventral to the large capillaries,
capillaries, testicular development is taking place. place. The
latter
latter assertion agrees with
assertion agrees with the
the data
data presented
presented by by Ashby (1957).
(1957). Shelton
Shelton and
and
Jensen
Jensen (1979)
(1979) reported
reported that anatomical changes
that anatomical changes in in the
the ovary
ovary involving
involving
changes
changes inin its
its shape
shape and
and development of of aa second
second point
point ofof attachment
attachment to to the
the
peritoneum are indicators of ovarian development in the grass carp, silver
carp (Hypothalmichthys molitrix), and the goldfish.
(Hypothalmichthys molitrix), goldfish.
The gonads have traditionally been considered undifferentiated during
the migration of the primordial germ cells and their subsequent mitotic
divisions.
divisions. However, several authors have reported the possibility of using
differential germ cell numbers as indicators of early sexual sexual differentiation.
differentiation.
The
The potential
potential of of this
this approach
approach for
for the
the vertebrates,
vertebrates, in in general,
general, waswas reviewed
reviewed
by Hardisty (1967).
(1967). The available literature
literature did not permit him to clarify clarify
whether differences in germ cell number were attributable to differences in
the number of prim oridaI germ cells or an earlier or more rapid proliferation
primoridal
in the germ cells of one sex. sex. However, more recent studies have indicated
that a proliferation of oogonia shortly before the initiation of meiotic activity
appears generalized for most teleosts (Nakamura, (Nakamura, 1978).
1978).
In the medaka, this rapid mitotic activity occurs either shortly before
(Onitake,
(Onitake, 1972)
1972) oror after
after hatching
hatching (Quirk
(Quirk and and Hamilton,
Hamilton, 1973).
1973). InIn several
several
cichlids, mitotic activity of the gonia occurs simultaneously with the initia initia-
tion
tion of
of somatic
somatic growth
growth leading
leading toto the
the formation
formation of of the
the ovarian
ovarian cavity.
cavity. These
These
events occur at 10-16 10-16 days posthatching at 20°C 20°C in Oreochromis mossam mossam-
bicus (Nakamura,
(Nakamura, 1978; 1978; Nakamura and Takahashi, 1973), 1973), 10-15
10-15 days
posthatching at 30°C 30°C in Tilapia zillii (Yoshikawa
(Yoshikawa and Oguri, 1977), 1977), and 15 - 16
15-16
days posthatching at 331°C
days 1°C in Oreochromis aureus (Dutta, (Dutta, 1979).
1979). In the latter
species, formation of the ovarian cavity was previously reported at 30 days
posthatching (Eckstein and Spira, 1965). 1965). However, these fish were 10-12 10-12
mm
mm atat 30 days
days compared
compared withwith 16-18
16-18 mm mm at at 15-16
15-16 days
days reported
reported byby Dutta
Dutta
(1979).
(1979). Growth related factors may account for the variance in observations. observations.
In the salmonids,
salmonids, based on increases in the number of of cysts of
of premeiotic
gonia, female differentiation was first observed 28-25 28-25 days posthatching at
HUNTER AND EDWARD M .. DONALDSON
M DONALDSON
8°C
8°C in Oncorhynchus masou (Nakamura,
(Nakamura, 1978),
1978), 17-35
17-35 days posthatching at
same temperature in Oncorhynchus keta (Nakamura,
the same (Nakamura, 1978),
1978), 103-131
days posthatching at 1°_6°C1"-6"C in Salvelinus leucomaenis (Nakamura,
Salvelinus leucomaenis (Nakamura, 1982),
1982),
and 35 days (378°C
(378°C days) posthatching (Lebrun al.,, 1982)
(Lebrun et aI. 1982) in Salmo
gairdneri. In the latter species, Takashima et al.
gairdneri. al. (1980)
(1980) reported that based
on the chromosomal arrangement and single nucleolus observed in some
germ cells primary
primary oocytes could be tentatively identified at 67 days from
fertilization at 11. 2°C (swim-up fry).
11.2"C fry). At slightly higher water temperatures
temperatures of
111°-130C,
1o_13°e, van den Hurk and Slof (1981)(1981)reported cysts of oogonia
oogonia in meiotic
prophase at the swim-up
swim-up fry stage but only 45-55 45-55 days postfertilization,
16-19 days posthatching. Ashby (1957) (1957) reported an increased number of of
oogonia 169 days postfertilization in Salmo trutta reared at 50_8°C. 5"-8"C.
The more easily detectable ovarian characteristics have been the pre pre-
ferred indicators of the initiation of gonadal differentiation. However, effec effec-
tive indicators of testicular differentiation have been reported. Nakamura
(1978)
(1978)suggested that because spermatogonia remain quiescent for a variable
period of time following
following the initiation of oogenesis, somatic differentiation is
the preferred indicator of testicular differentiation. He cited as indicators of
testicular differentiation formation of the efferent duct in Oncorhynchus
masou, Oncorhynchus keta, and Salvelinus leucomaenis, differentiation of
Salvelinus leucomaenis,
blood ''capillaries
capillaries near the efferent duct in these species and in Carassius
auratus, and aggregations of stromal cells in the hilar region of Gambusia
auratus,
af finis and Poecilia reticulata.
affinis reticulata. In Oreochromis mossambicus,
mossambicus, formation of
the ovarian cavity and the efferent duct analoges in the gonadal stroma,
indicating female and male differentiation, respectively, occur at age 20 days
concurrent with the first meiotic activity of the female germ cells. These
results indicate that testicular somatic differentiation can be demonstrated at
a relatively early stage.
stage.
Various
Various studies employing histological examination have provided exten exten-
sive evidence for the species-specific nature of sex differentiation.
differentiation. In most
gonochorist species examined, differentiation is initiated shortly after hatch hatch-
ing. However, extreme variations may occur.
ing. occur. In the cyprinodont Poecilia
Poecilia
reticulata, ovarian and testicular differentiation, based on pre meiotic ac
premeiotic ac-
tivity of oogonia and stromal aggregations in the hilus of presumptive testes,
occurs 12 and 8 days prior to parturition, respectively. In the grass carp,
ovarian differentiation based on anatomical changes was initiated between
age 50 and 75 days. Oogonial nests were not evident until 94-125 94-125 days and
perinuclear oocytes did not appear until 240-405 240-405 days.
days. Shelton and Jensen
(1979),
(1979), citing Emelyanova, report that in the silver carp, Hypothalmichthys
molitrix, cytological differentiation of the gonads was not completed until
150-180 days posthatching.
Of additional interest is the suggestion first presented by Yamamoto Yamamoto
5.
HORMONAL SEX CONTROL AND
AND ITS APPLICATION TO
ITS APPLICATION TO FISH CULTURE 263
FISH CULTURE
(1959a,
(1959a, 1962)
1962) and Yamamoto and Matsuda (1963) (1963) that the process of sex
differentiation
differentiation proceeds
proceeds in in an
an anterior
anterior to to posterior
posterior gradient
gradient in in the
the medaka.
medaka.
Yamamoto
Yamamoto (1962) suggested that
(1962) suggested that this
this gradient
gradient of of differentiation
differentiation could
could explain
explain
the
the occurrence
occurrence of of intersex
intersex gonads
gonads in in fish
fish administered
administered estrogen
estrogen treatments
treatments
of
of insufficient
insufficient dosage
dosage or or duration.
duration. Presumably,
Presumably, the the anterior
anterior portion
portion of of the
the
gonad undergoes testicular differentiation influenced by the natural sex inin
ducer, but the
ducer, but the posterior
posterior portion
portion of of the
the gonad
gonad undergoes
undergoes ovarian
ovarian differentia
differentia-
tion
tion influenced
influenced by by the exogenous estrogens.
the exogenous estrogens. In In the amphibians,
amphibians, Witschi
Witschi
(1967)
(1967) described a similar anterior to posterior gradient of differentiation in
laevis. Witschi
Xenopus laevis. Witschi and and Dale
Dale (1962)
(1962) reported
reported that that aa 2-day
2-day estrogen
estrogen
treatment
treatment of of male
male frog
frog larvae
larvae of of various
various ages
ages resulted
resulted in in aa gonad
gonad consisting
consisting of of
testicular
testicular and ovarian tissue.
and ovarian tissue. The ovarian
ovarian mode
mode shifted caudally with
shifted caudally with increas
increas-
ing
ing age
age of
of larvae.
larvae. Later,
Later, numerical
numerical examination
examination of of testicular
testicular and
and ovarian
ovarian
differentiation in Oryzias latipes by Yoshikawa Yoshikawa and Oguri (1979, (1979, 1981),
1981), re
re-
spectively, did not support Yamamoto's Yamamoto’s hypothesis. Yoshikawa and Oguri
reported
reported aa random
random distribution
distribution of of developing
developing interstitial
interstitial and
and germinal
germinal eleele-
ments. In the later study, they reported that the right ovary differentiated
before the left.
left. Although numerical analysis analysis does
does not support a gradient of
differentiation in Oryzias laUpes,latipes, the presence of a cephalocaudal gradient of
spermatogenesis, oogenesis, and the formation of the ovarian cavity has been
reported
reported in in the
the cichlid Tilapia zillii (Yoshikawa
cichlid Tilapia (Yoshikawa and and Oguri,
Oguri, 1977).
1977). A similar
similar
gradient
gradient in in the development
development of of the
the ovarian
ovarian cavity
cavity was was reported
reported for for
Oreochromis mossambicus (Nakamura
Oreochromis mossambicus (Nakamura and and Takahashi,
Takahashi, 1973).
1973). However,
However,
Johnstone
Johnstone et al. al. (1978)
(1978)observed
observed no no distinct
distinct pattern
pattern in in hermaphroditic
hermaphroditic rain rain-
bow
bow trout
trout gonads
gonads following
following administration
administration of of either
either estradiol
estradiol or or meth
meth-
yltestosterone.
yltestosterone. The The question
question of of whether
whether aa gradient
gradient of of differentiation
differentiation is is com
com-
mon
mon within
within the teleosts
teleosts remains.
remains. However,
However, these these studies
studies dodo demonstrate
demonstrate aa
variable
variable timing
timing of of differentiation
differentiation at at the level
level of of individual
individual germinal
germinal and and
somatic
somatic cells,
cells, the gonad
gonad as as aa whole
whole andand the
the individual
individual within
within aa population.
population.
Therefore, the determination of treatment timing, timing, treatment duration, and
the assessment of treatment effectiveness based on histological histological analysis
analysis must
account for this variability. This is particularly important where highly effec effec-
tive treatments are required or the treatment objective is to create simul simul-
taneously maturing intersex gonads.
b.
b. Determination of Period. Although histological
of the Labile Period. histological examina
examina-
tion may provide visual guides to the initiation and completion of sex sex differ
differ-
entiation, exact
exact delineation of the hormone-labile period requires the ad ad-
ministration of hormones. Only by this means can the timing and duration of
an
an effective
effective treatment
treatment regime
regime be established.
established.
Studies involving
involving steroid
steroid administration have demonstrated that that similar
to the histologically
histologically observable period of sexsex differentiation, the effective
effective
A N D EDWARD
EDWARD M .. DONALDSON
M DONALDSON
treatment
treatment period
period varies
varies between
between species.
species. InIn several
several species,
species, effective
effective es
es-
trogen treatment must be started with the initiation of mitotic activity of the
primordial germ cells before sex differences are observable. In Oreochromis
mossumbicus, the
mossambicus, the intention
intention of
of mitotic
mitotic activity inin the
the germ cellscells is
is reported
reported to to
occur at 8- 10 days after hatching (Nakamura and Takahashi,
8-10 Takahashi, 1973).
1973). Meiotic
activity did not occur until age 20 days. In the same study, an effective
period
period ofof ethynylestradiol
ethynylestradiol treatment
treatment starting
starting 66 days
days posthatching and and lasting
lasting
for
for 19
19 days
days was demonstrated. However,
was demonstrated. However, treatment
treatment exclusively
exclusively withinwithin the
the
indifferent
indifferent period 6-15 days
period 6-15 days was
was without
without effect.
effect. Similarly,
Similarly, Nakamura
Nakamura (1978)(1978)
reported
reported thethe first
first mitotic
mitotic activity
activity of
of germ
germ cells
cells in
in Oncorhynchus masou musou at at
14-28
14-28 days posthatching. He subsequently demonstrated an effective es es-
tradiol treatment period
period between 55 and 23 days posthatching.
The
The association
association between
between successful
successful estrogen
estrogen treatment
treatment and and thethe earliest
earliest
mitotic
mitotic activity
activity does
does not
not appear
appear toto be universal
universal within
within the teleosts.
teleosts. InIn the
the
guppy, ovarian differentiation,
differentiation, based on developing oocytes, oocytes, has been re re-
ported
ported 14 14 days
days following
following the
the preceding
preceding parturition,
parturition, 12 12 days
days before
before birth
birth
(Takahashi,
(Takahashi, 1975a,b).
1975a,b). However, Takahashi
Takahashi (1975d)
(1975d) was able to obtain com com-
plete
plete feminization
feminization of of genetic
genetic males
males byby administering
administering ethynylestradiol
ethynylestradiol at at 125
125
mg/kg diet for 30 days days after birth.
Effective androgen treatment has been demonstrated to be synchronous
with somatic sex differentiation in the guppy. guppy. Testicular differentiation in
reticulutu, based on aggregations of stromal cells in the gonadal
Poecilia reticulata,
hilus, has been reported 18 18days following the last parturition, 88 days days prior to
birth (Takahashi,
(Takahashi, 1975a,b).
1975a,b). Takahashi (1975a)
(1975a) was able to demonstrate that
the oral administration of methyltestosterone
methyltestosterone at 400 mg/kg diet to gravid
guppies 13-15 days after the preceding parturition, i.i.e., e. , from 13- 11 days
13-11 days
before the birth until the time of birth, resulted in the development of
stromal
stromal aggregations in the gonadal hilus of treated females. females. Within 20 days days
of birth, these fish
fish developed testes.
testes. Previously, Dzwillo
Dzwillo (1962,
(1962, 1966)
1966) had
demonstrated that immersion of gravid females in water containing meth meth-
yltestosterone resulted in masculinization of the offspring.
offspring. In the later study,
the steroid was administered at 3-4 3-4 mg/l for only 24 hr between 8-12 8-12 days
prior to parturition. Takahashi
Takahashi (1975a)
(1975a) suggested that these results indicated
an androgen-sensitive period of embryonic ovaries at about 88 days days prior to
birth, corresponding to the development of the stromal aggregations aggregations in the
hilus of the normal male testis.testis. Further,
Further, he attributed
attributed the successsuccess of the
preparturition methyltestosterone treatment to its action in initiating soma soma-
tic differentiation in the hilar region.
Similar
Similar to the oral
oral administration of estrogen, Takahashi
Takahashi (1975c)
(1975~) was able
to obtain complete masculinization of juvenile guppies postpartum by
adding
adding 111-ketotestosterone
1-ketotestosterone to the rearing water at 25-50 25-50 JLg/1pg/l for 3535 days
following
following birth. Inhibition of spermatogoenesis
spermatogoenesis and sperm sperm duct formation
5.
5. HORMONAL
HORMONAL SEX
SEX CONTROL AND ITS
FISH CULTURE
CULTURE 265
was observed in some some individuals. Therefore, it is apparent that treatment at

the initiation of differentiation is important in some species. species. However, in
other species the developing gametes maintain their sexually bipotent na na-
ture for a considerable period and may be influenced by hormonal treatment
over several discrete intervals.
intervals. An excellent example of this latter case is the
study by Nagy et aZ. al. (1981)
(1981) on Cyprinus carpio.
carpio. Using gynogenetic all all-
female groups of carp, these researchers were able to demonstrate that at
25°C,
25°C sex inversion could be obtained by oral administration of meth meth-
yltestosterone in any of several overlaping 36-day periods initiated between
8 and 80 days posthatching.
Effective treatments of very short duration have been obtained in several
species.
species. The 24-hr treatment
treatment used by Dzwillo (1966)(1966) to masculinize embry
embry-
onic guppies has been described previously. Hackmann and Reinboth (1974) (1974)
masculinized Hemihaplochromis mutlicolor by immersing 14- 14- to 16-day-old
16-day-old
juveniles for 42-44
42-44 hr in water containing methyltestosterone
methyltestosterone propionate at
500 .... g/l. Feminization was also achieved with a similar duration and route of
pg/l.
administration using estradiol butyrl acetate between 11.5 11.5 and 1616 days
postspawning. Coho salmon have been feminized by immersion for two 2-hr
periods, 4 and 11 11 days posthatching in water containing estradiol at 400 .... g/l
pg/l
(G.
(G. A. Hunter, E. E. M
M.. Donaldson, and I. I. Baker, unpublished). Typically the
treatment duration for other cichlids, primarily the genus Oreochromis and
the salmonids, genus Salmo have been for much longer durations (Table II). 11).
One possible explanation for the success of of unusually short treatment
treatment
periods is is that the gonads are exposed to the steroid for periods of time much
longer than the treatment
treatment itself. Several factors including steroid dosage,
rate of uptake and retention could influence the actual period of of gonadal
exposure relative to treatment duration. duration. Unfortunately, few studies have
examined these factors. In a recent investigation, G. G . A. Hunter and co co-
workers (unpublished)
(unpublished) immersed newly hatched coho salmon alevins for 2.4
hr in water containing labeled estradiol at concentrations of of either 0.2, 2.0,
20, or 200 .... gll. Preliminary results indicate that the uptake of the steroid is
pg/l.
directly related to concentration. Further, at the maximum sample period of
16
16 days posttreatment, similar percentages of the maximum uptake of la- la
beled estradiol were present at all dosage levels.
These results suggest that an inverse relationship may exist between
dosage and duration of an effective immersion treatment.treatment. This could explain
the requirement for very high immersion dosages in treatments of very short
duration. This could also explain the similar effectiveness of estradiol treat treat-
ment applied at 0.5-5 0.5-5 ....
pg/lg/l for 18
18 days starting 5 days posthatching in On On-
corhynchus masoum s o u (Nakamura,
(Nakamura, 1981a)
1981a)and a 400 .... g/l treatment applied in 2-
pg/l
hr immersions at 4 and 11 11 days after hatching (G.(G. A. Hunter, E. M. Donald
Donald-
son,
son, and I. Baker, unpublished).
unpublished). It is also interesting that Nakamura (1982) (1982)
266 GEORGE
GEORGE A.
EDWARD M DONALDSON
observed that the 18-day

18-day treatment at 10-200
10-200 J.Lg
pg estradiolll
estradiol/l resulted in a
very high mortality. Goetz et al. al. (1979)
(1979) observed no mortalities at 200 and
400
400 J.Lg
pg estradiolll
estradiol/l when confined to two 2-hr treatments in the eyed egg and
alevin stages. However, high mortalities were observed when the total num num-
ber of 2-hr immersions was increased to 13, 13, seven of which were in the
alevin stage.
The question remains whether a similar relationship may be found with
treatments involving the oral administration of steroids. Hishida (1965) (1965) ex
ex-
amined the retention of 18-[14C]estrone
18-[14C]estrone fed to juvenile medaka from the 6- 6-
mm to U-mm
ll-mm stages (1 (1 month). He found that a high proportion of free
estrogen remained in fish as late as as the 16-mm
16-mm stage, and he concluded that
the metabolic clearance mechanism for free estrogen was not established at
this stage. Therefore, it appears that steroids may be retained following oral
treatment. However, no information on the relationship between steroid
dosage and rate of uptake is available for the oral treatment of juvenile fish. fish.
The duration of steroid treatment
treatment is of comparable importance to and
may be interactive with steroid dosage for the effective sterilization of
gonads.
gonads. Takahashi (1977)
(1977) administered dosages of 11.0-2.0 . 0-2.0 g methyltesto
methyltesto-
sterone/kg diet to juvenile Poecilia reticulata for 35 35 days following
following birth.
Treatment resulted in nearly complete sterilization of both ovaries and test test-
es. Following hormone withdrawal, only the testes, although severely de
es. de-
pressed, were capable of full recovery. This capability was lost when the
treatment was extended to cover 70 days followingfollowing birth. G. A. Hunter and
E. M . Donaldson (unpublished)
E. (unpublished) treated
treated coho salmon, Oncorhynchus
kisutch, fry, which had been previously immersed as alevins in water con con-
taining methyltestosterone, with a diet containing the steroid at 1, 1, 3,
3, or 9
mg/kg for either 3,3 ,66,, or 9 weeks. Increases in duration of treatment at either
3
3 or 9 mg/kg diet resulted in increased percentages of sterile fish. fish.
c. Environmental Influences. The regulatory mechanism which deter

c. Environmental deter-
mines the timing and duration of hormone sensitivity in gonochorist species
remains to be determined.
determined. However, the labile period is assumed to be
associated with specific
specific developmental events and processes. Therefore,
several researchers have suggested that factors affecting metabolic rate and
growth (e.
(e.g.
g.,, temperature,
temperature, culture density, and feeding regimes) may influ influ-
ence the timing and duration of this period.
The few studies which have examined this area indicate that growth as
indicated by size is a reliable criterion for the initiation of sex differentiation.
This appears to be especially true for species such as the grass carp (Shelton
and Jensen, 1979)
1979) and the European eel, Anguilla anguilla (Bieniarz
(Bieniarz et al.
al.,,
1981),
1981),in which differentiation occurs a relatively long time after hatching. In
5.
ITS APPLICATION TO FISH CULTURE
CULTURE 267
the latter study, differentiation commenced when eels ranging from 11.5 . 5 to 6
years and reached 14-3514-35 cm in length.
length. Further, the results indicated that
gonadal sex differentiation was not age dependent,
dependent, but was partially corre
corre-
lated with body length.
Size was also a more reliable indicator of the onset of sex differentiation
in Oreochromis uureus, a species
Oreochromis aureus, species which displays a relatively early differ
differ-
entiation (Dutta,
(Dutta, 1979).
1979). In this study,
study, juveniles were reared at either 31°e
31°C or
21°e.
21°C. A faster growth rate was reported at the higher temperature.
temperature. Ovarian
differentiation, characterized by meiotic and mitotic activity and develop develop-
ment of the ovocoel,
ovocoel, occurred at 15- 18 mm length and 14-15
15-18 14-15 days postpost-
hatching at the high temperature,
temperature, and 14-18
14-18 mm length and 24-2724-27 days of
age at the low temperature.
temperature. Testicular development, characterized by mito mito-
tic activity of spermatogonia and development of the stromal lumina, oc oc-
curred
curred at 17-18
17-18 mm length and 19-20 19-20 days of age at 31°e,
31"C, and 16-20
16-20 mm
length and 29-30
29-30 days of age at 21°C.21°C. Irrespective of rearing conditions,
when fish attained a particular length range (14-18 mm), gonadal differentia
(14-18 mm), differentia-
tion was evident. Fish that were small for their age retained undifferentiated
gonads.
gonads. However, a minimum age related to growth was reported because
few fish reached the critical size of 14-20
14-20 mm length before age 14-1514-15 days.
Dutta (1979)
(1979) concluded that body length was better than age as an indicator
of gonadal sex differentiation.
differentiation.
al. (1981)
Shelton et al. (1981)examined the effects that factors influencing growth
rate could have on the ethynyltestosterone-induced
ethynyltestosterone-induced sex inversion of
aureus. In this study, a decreased growth rate was observed in
Oreochromis aureus.
Oreochromis
fry reared at 21°e
21°C compared with 300e.30°C. However, treatment durations of 16 16
and 19
19 days and 21-28
21-28 days were, respectively, ineffective and effective in
producing monosex male populations at both temperatures. Shelton and co co-
workers concluded that the duration of treatment within a particular age
span was more critical to treatment effectiveness than factors affecting affecting
growth.
growth. This does not specifically
specifically exclude the relevance of size to treatment.
In the 19-day
19-day treatment 33 33 and 26% of the fry were shorter than 18 18 mm at
21°
21" and 300e,
30"C, respectively. Only 3.3% 3.3% of the fish treated for 21 21 days were
shorter than 18 18 mm, with the shortest being 14 14 mm long.
long. Similar results
were obtained in groups reared at high density (2600 (2600 fry 1m 2) to reduce
/m2)
growth rate. Therefore, it appears that, that, within the range of conditions ap ap-
plied in this study, duration of treatment can be used reliably as criteria for
successful
successful treatment.
To gynogenetic all-female common carp, Nagy et al. ul. (1981)
(1981)administered
four overlapping 36-day methyltestosterone
methyltestosterone treatments initiated between
8-80
8-80 days posthatching resulting in 71.4-88 . 9% sex inversion.
71.4-88.9% inversion. The re re-
mainder of the fish
fish were female or contained undifferentiated gonads. How-
268 GEORGE
GEORGE A.
A. HUNTER
HUNTER AND
AND EDWARD
EDWARD M
M .. DONALDSON
DONALDSON
ever,
ever, using a similar dosage
dosage on fish of7-21
of 7-21 mm, 13-39
13-39 mm, and 19-57
19-57 mm
length,
length, Nagy
Nagy and
and co-workers
co-workers were
were able
able to
to obtain
obtain 90,
90, 100,
100, and
and 90%
90% males,
males,
respectively.
Therefore, it is
is evident that both size
size and age may be used effectively
effectively as
as
indicators of developmental stage when tuned to individual species characcharac-
teristics and rearing conditions.
conditions. In genera such as Oncorhynchus,
Oncorhynchus, which
differentiate prior
prior to
to feeding,
feeding, age
age either relative
relative to
to developmental
developmental markers
markers
such as hatching or with respect to the temperature-development
temperature-development relation
relation-
ship (degree
(degree days)
days) may be used effectively.
effectively. In species which differentiate at a
much later date, size criteria may be of more value.
C.
C. Evaluation
Evaluation
The
The evaluative phase involves
involves a quantification of treatment effectiveness
egectiveness
relative to management objectives.
objectives. In most studies, hormones are admin
admin-
istered to a group of genetic males and females. Following treatment, histo histo-
logical examination is used to assess
assess the proportions of gonadal sex types
present. The occurrence of a high proportion of one gonadal sex type is
usually indicative of successful sex inversion. Results that do not demon demon-
strate a clear preponderance of one gonadal sex following treatment may be
statistically
statistically compared with a control population. However, statistical nonsig
nonsig-
nificance is not conclusive evidence that
nificance that sex inversion has not occurred.
Sanieo
Sanico (1975)
(1975) treated Oreochromis aureus with estrone obtaining an ob ob-
served sex ratio not significantly
significantly different from unity. Later, Liu (1977)
(1977)
reported
reported that of these fish produced 100%
that one of 100%male offspring
offspring indicating that
sex inversion had occurred. Further, progeny testing may be required to
exclude the possibility of of sex-related mortality within treated groups
(Yamamoto, 1953).
(Yamamoto, 1953).
An alternate approach, which simplifies
simplifies the analysis
analysis of
of treatment, in
in-
volves the use ofof genetically monosex fish produced either by the mating of of
sex-inverted homogametic fish or by gynogenesis. Any deviation from the
monos
monosex ex gonadal type may be assumed to be a result of of treatment. This
approach eliminates both concerns about possible differential mortality and
the need for progeny testing to confirm sex inversion.
IV. ECONOMICALLY
IV. ECONOMICALLY IMPORTANT
IMPORTANT SPECIES
SPECIES
With
With the growing importance ofof hormonal sex-control techniques to fish
of the studies involving the economically
culture practices, an examination of
concerned with
important species is in order. This discussion is primarily concerned
5. HORMONAL
HORMONAL SEX
SEX CONTROL
CONTROL AAND ITS APPLICATION
N D ITS APPLICATION TO
TO FISH
FISH CULTURE
CULTURE 269
species from two major groups which are important to culturists. These are
the cichlids, genus Oreochromis, known colloquially as tilapias and the salm-
salm
onids, notably the genus Oncorhynchus and the genus Salmo. Salmo. Mention is
also made of of the recent research conducted in two cyprinids Ctenophayn-
Ctenopharyn
godon idella and Cyprinus carpio. These groups do not representrepresent the only
economically important species to which sex-control techniques have been
applied. Many of of the Poecilliidae and C yprinodontidae previously discussed
Cyprinodontidae
are valued by pet culturists for particular sex-related characteristics such as
body color and morphology. Further, these techniques have recently been
applied to the turbot, Scophthalamus maximus. In this species, the admin-
Scophthalamus maximus. admin
istration of estradiol or methyltestosterone added to the weaning diet at 5 or
3 mg/kg diet, respectively, for 700°C
700°C days (58
(58 days)
days) induces 100%
100%sex inver
inver-
sion. Methyltestosterone administered at 25 mg/kg weaning diet for 1000°C
sion. 1000°C
days (12°C) 100% sterility (Bye,
(12°C) induces 100% (Bye, 1982).
1982). As previously described,
Chen et al.
al. (1977)
(1977)have achieved early sex inversion in the only commercially
important protogynous hermaphrodite Epinephelus tauvina. tauuina. Goudie et al.
al.
(1983)
(1983) have achieved 100%100% feminization of channel catfish with estradiol or
ethynyltestosterone
ethynyltestosterone at dosages of 6-600 6-600 mg/kg administered for 21 days
following
following yolk sac absorption.
A. Cichlids
Cichlids
Central to the application of hormonal sex-control techniques to the

tilapias
tilapias is the desire to control breeding. In the late 1950s 1950s and early 1960s,
1960s,
numerous studies indicated that various cichlid species would be ideal for
warm-water culture. Favorable culture attributes include rapid growth using
warm-water culture.
plankton
plankton as as aa food
food source,
source, adaptability
adaptability to
to brackish
brackish or
or salt
salt water,
water, and
and high
high
reproductive potential (Hickling,
(Hickling, 1963).
1963). However, when held in ponds, the
early maturation and high fecundity of these species rapidly results in over over-
crowding of the ponds with fish fish too small for consumption.
consumption. Alternative solu
solu-
tions
tions to
to this
this problem,
problem, including
including high-density
high-density culture,
culture, polyculture
polyculture with
with aa
piscivore,
piscivore, cage rearing, repetitive harvesting, and monosex monosex culture, have
been reviewed by Jensen (1976). (1976).
The
The treatment used to achieve the monosex monosex condition must be very
effective,
effective, because the presence of the opposite sex sex at even very low levels
will
will result in overbreeding.
overbreeding. Anderson and Smitherman (1978) (1978)reported that
excessive
excessive breeding occursoccurs when females are are present at levels as
as low as 5%.
5%.
Approaches to to the production of of monos ex populations have included hand
monosex
sorting,
sorting, which
which is is time
time consuming
consuming andand expensive,
expensive, the
the interspecific
interspecific mating
mating of
of
homogametic individuals, which has has produced inconsistent results,
results, and the
use
use of hormonal sex sex control.
control.
GEORGE A. AND
HUNTER A ND E DWARD M.
EDWARD M. DONALDSON
DONALDSON
1.
1. ANDROGEN
ANDROGENTREATMENT
TREATMENT
Within monosex culture the production of
of males has been preferred. The
faster growth rate of male tilapia has been reported by several researchers
Gensen, 1976). Further, the growth rate of female tilapia is reduced at
(Jensen, 1976).
maturity (Hickling,
(Hickling, 1960).
1960).
a. Oreochromis aureus. Eckstein and Spira (1965)
a. (1965) conducted the first
experimental application of androgens to Oreochromis aureus.aureus. In this study,
testosterone and methyltestosterone were administered to the rearing rearing water
at concentrations of 50-1000
50-1000 IJ.g/I
pg/l for a period of 5-6
5-6 weeks starting at 4-54-5
weeks posthatching. These researchers had previously recorded the initial
appearance of the genital ridge at 10-11 days posthatching with the first
distinctive characteristic of the ovaries,
ovaries, a longitudinal groove occurring at 30
days. Sex differentiation based on germinal and somatic characteristics oc oc-
curred at 7-8
7-8 weeks. The effect of the androgens was variable with some
individuals having nearly involuted
involuted gonads and others being largely un un-
affected. Unfortunately, Eckstein and Spira did not specify the exact propor
propor-
tions of each gonadal sex type.
Guerrero (1975)
(1975)was the first to achieve sex inversion in this species using
the synthetic androgens ethynyltestosterone and methyltestosterone at 15, 15,
30, and 60 mg/kg diet for 18 days (6 (6 days/week
daydweek for 3 weeks). Ethynyltesto
Ethynyltesto-
sterone at 60 and 30 mglkgmg/kg produced 100 100 and 98%
98% males, respectively.
Methyltestosterone at 30 mg/kg also produced 98% 98% males. However, this
steroid was found to be less effective at 60 mg/kg and 15 mg/kg producing 85
and 84%
84% males, respectively. Treatments did not affect growth or survival.
However, degeneration of the germinal epithelium and proliferation of the
connective tissue was observed in the 60 mg/kg methyltestosterone group. group.
Males from treated groups were mated with untreated females and the
progeny analyzed. Nine families from these matings had male-to-female
ratios of 1:2-3 . 1 indicating female heterogamety. Therefore, Guerrero was
1:2-3.1
able to suggest the possibility of using the indirect method to produce monomono-
sex male fish in this species. The attempts to produce all-male stocks by the
indirect method are discussed later in this section.
section.
Recently, the pendulum has swung back in favor of the production of
monosex
monosex males by the direct method.
method. This return to the direct method is
primarily attributable to the potential inconsistencies in breeding sex-invert
sex-invert-
ed individuals because of the previously described complexity of the sex sex-
determining mechanism evident in some species of tilapia. In this regard,
Shelton and Jensen (1979)
(1979) reported that, of 100
100 Oreochromis aureus es- es
trogen-induced females mated with untreated males, 41 spawns were ob ob-
tained. Of these spawns, only five were 100% 100%male, one was 95% 95% male, one
100%
was 100% female, and the rest had a 1:1 male-female
1:l male-female ratio.
5.
5. HORMONAL
HORMONAL SEX
SEX CONTROL
CONTROL AND
A N D ITS
ITS APPLICATION
APPLICATION TO
TO FISH CULTURE 271
FISH CULTURE 271
Attempting
Attempting to to standardize
standardize treatment
treatment forfor production
production of of monosex
monosex malesmales by
by
the
the direct
direct method,
method, Shelton
Shelton et al.al. (1981)
(1981)examined
examined the the effects
effects of
of temperature,
temperature,
stocking
stocking density,
density, and
and feeding
feeding regime
regime on on treatment
treatment success.
success. Based
Based onon Guer
Guer-
rero's
rero's (1975)
(1975) successful
successful treatment
treatment aa dosage
dosage level
level of
of 60 mg mg ethynyltes
ethynyltes-
tosterone/kg
tosteronelkg diet
diet was
was used
used forfor all
all tests.
tests. The
The dietary
dietary treatment
treatment was was admin
admin-
istered either at a fixed
fixed intake of 12% 12%body weight per day or to satiation. satiation.
Stocking
Stocking densities
densities of
of 160
160 andand 2600 fry/m
fiy/m2 2 were
were examined
examined at at each
each feeding
feeding
regime.
regime. Treatment
Treatment durations
durations werewere between
between 16 16 and
and 28 days.
days. Temperatures
Temperatures of of
either
either 21°
21" or
or 30°C,
30°C, stocking
stocking density,
density, andand feeding
feeding regime
regime did
did not
not appear
appear to
to
affect
affect treatment because 100%-male
treatment because 100%-male groups
groups were produced in
were produced in all
all but
but the
the low
low
density,
density, 12%
12% feed ration,
ration, lowlow temperature
temperature groupgroup which
which attained
attained 98.9%
98.9%
males.
males. Based
Based on
on these
these results,
results, anan optimal
optimal treatment
treatment for for this
this species
species was
was
proposed
proposed consisting
consisting ofof aa minimum
minimum treatment
treatment of 21 days,
of 21 days, stocking
stocking densities
densities
up
up to
to 2600 fry/m 2, within
fry/m2, within aa temperature
temperature rangerange of of 21-30°C and and aa feeding
feeding
ration
ration of
of 12-15%
12-15% body
body weight.
weight.
mossambicus. Clemens
b. Oreochromis mossambicus.
b. Clemens and Inslee (1968)
and Inslee (1968)demonstrated
demonstrated
the
the first
first successful
successful masculinization
masculinization withinwithin the
the tilapias, specifically Oreo
tilapias, specifically Oreo-
mossambica). For
chromis mossambicus (Tilapia mossambica). For 6969 days,
days, fry
fry were
were fedfed aa dietdiet
containing methyltestosterone
containing methyltestosterone at 10-50 mg/kg
at 10-50 mg/kg diet.
diet. At
At dosages
dosages of of 00 and
and 50
mg/kg,
mg/kg, the the groups
groups matured
matured as males and
as males and females
females with
with sex
sex ratios
ratios ofof 1:3.6
1:3.6and and
1:4.4,
1:4.4, respectively.
respectively. Treatment
Treatment at at 10,
10, 30,
30, and
and 4040 mg/kg
mg/kg resulted
resulted in in 100%
100%
males
males butbut one
one fish
fish in
in the group
group treated
treated atat 20 mg/kg was
20 mg/kg was female.
female. However,
However, it it
is
is notable
notable that that the sex
sex ratios
ratios were based on
were based 8-22 fish
on 8-22 fish from
from an an original
original 100
treated
treated fish. Groups receiving
fish. Groups receiving similar
similar treatments,
treatments, butbut placed
placed in in larger
larger tanks,
tanks,
which
which provided
provided sample
sample sizes
sizes of
of 79-128,
79-128, hadhad male-to-female
male-to-female ratios ratios ofof 11:1.6
: 1 . 6 to
to
1:5. 1. The
1:5.1. The discrepancy
discrepancy between
between results
results was
was attributed
attributed to to an
an increased
increased natu natu-
ral
ral food
food supply
supply available
available toto the
the fish
fish in
in the
the larger
larger tanks.
tanks. The
The matings
matings of of 77 of
of24 24
males
males from
from experimental groups resulted
experimental groups resulted inin all-female
all-female offspring indicating
offspring indicating
that sex inversion
that sex inversion hadhad occurred
occurred and and that
that the
the female
female is is homogametic.
homogametic. Clem- Clem
ens and
ens and Inslee
Inslee suggested
suggested that,
that, although
although the results did not
results did not indicate
indicate aa clearly
clearly
superior dosage
superior dosage level,
level, the 30 mg/kg
mg/kg level
level was
was promising
promising for for further
further study.
study.
Subsequently, successful masculinization was was obtained
obtained by treatments
using 50 mg/kg
using mg/kg diet
diet of methyltestosterone for
of methyltestosterone for 19 days starting 7 days
days starting days
(Nakamura, 1975),
posthatching (Nakamura, 1975), thereby demonstrating a much reduced reduced ef- ef
fective duration
fective duration of of treatment.
treatment. Nakamura
Nakamura and and Takahashi
Takahashi (1973)(1973) had had pre- pre
demonstrated that the labile period is from 6 to 25 days of
viously demonstrated of age. In this
study, aa dosage
study, dosage ofof 1000 mg/kg
mg/kg forfor the
the same
same period
period of of time
time waswas ineffective.
ineffective.
Guerrero (1976a)
Guerrero (1976a) obtained
obtained 69, 93, and and 98%
98% male
male groups
groups treated
treated with with 30
mg/kg methyltestosterone for 14, 14, 21, or 28 days posthatching, respectively.
Based on the absence of of juveniles in ponds previously stocked with fry
treated with
treated with 50 mg mg ethynyltestosterone/kg
ethynyltestosterone/kg diet diet for
for 40 days,
days, Guerrero
Guerrero
272 GEORGE
GEORGE A.
A . HUNTER AND EDWARD
EDWARD M DONALDSON
(1976b)
(1976b) concluded that 100%
100% sex inversion had been achieved.
achieved. The growth
of treated fish was also superior to controls. Recently, dihydrotestosterone,
testosterone propionate, and methyltestosterone
methyltestosterone have been tested on
mossambicus fry.
Oreochromis mossambicus fry. Treatment with 60 mg/kg methyltesto
methyltesto-
sterone for 66 weeks achieved 100%
100% sex inversion,
inversion, and the 30 mg/kg diet
treatment
treatment only
only produced
produced 81-85%
8 1 4 5 % males
males (Anonymous,
(Anonymous, 1979).
1979).
Nakamura (1981)
(1981) has reported the first successful
successful sex inversion in this
species using the naturally occurring androgen, 11-ketotestosterone.
ll-ketotestosterone. The
effective
effective treatment
treatment required
required a dose of 200 mg/kg diet for 1919 days starting 7
days posthatching.
c. niloticus. Jalabert et ai.

c. Oreochromis niloticus. (1974) first applied meth
al. (1974) meth-
yltestosterone treatment
treatment to juvenile Oreochromis niioticus.
niloticus. The treatment
treatment
consisted of the oral administration of the steroid at 40 mg/kg diet for 2
months from first feeding.
feeding. Comparison by Chi-square indicated a higher
proportion of males in several groups. groups. Sex
Sex inversion was confirmed by the
mating of several treated males with untreated females which resulted in
100%
100% female progeny, and the demonstration of female homogamety. Guer Guer-
rero and Abella (1976)
(1976)were able to demonstrate higher percentages of males
with dosages of methyltestosterone at 15, 15, 30,
30, or 50
50 mg/kg diet for 6 weeks.
Katz et ai.
al. (1976)
(1976)have provided the only deliberate attempts to sterilize
O. niloticus based primarily on the early work with Oreochromis aureus
0. niioticus
(Eckstein and Spira, Spira, 1965)
1965) and Oreochromis mossambicus
mossambicus (Clemens
(Clemens and
Inslee, 1968).
1968). Katz et ai.al. (1976)
(1976) incubated fertilized Oreochromis
Oreochromis niloticus
eggs
eggs in water containing 500-5000
500-5000 J..L g/1 progesterone, cyproterone acetate,
pg/l
androstenedione, testosterone, adrenosterone, methyltestosterone, 11 11 hy
hy-
droxy-17a-methyltestosterone,
droxy-17a-methyltestosterone, and fluoxymetesterone,
fluoxymetesterone, 500-1000
500-1000 J..L g/1 11-
Fg/l 11-
ketotestosterone, 125-5000
125-5000 J..L g/1 estrone and estradiol, and 200-5000
pg/l 200-5000 J..L g/1
pg/l
diethylstilbestrol diphosphate.
diphosphate. All treatments resulted in very high mortality
with the exception of those involving
involving adrenosterone. Fish treated at 5000
J..L
g/1 with this steroid were found to be sterile at the end of the 3-month
p,g/l
treatment period. The gonads of these fish were described as either epi epi-
thelial flaps
flaps hanging from the dorsal peritoneal lining (5.4%)(5.4%)or hollow ducts
composed of connective tissue and blood vessels (involuted(involuted gonads).
gonads). Howev
Howev-
er,
er, when sampled at 88 months, 40% of the population had developed testes,
4.4%
4.4% had involuted gonads,gonads, and no gonadal
gonadal development
development waswas noted in the
remainder.
Tayamen and Shelton (1978) (1978)were the first to produce all-male popula
popula-
tions
tions of Oreochromis niioticus.
niloticus. Ethynyltestosterone and methyltestosterone
were
were orally
orally administered at either 30 30 or 60
60 mg/kg diet for either 25, 35, or 59
25,35, 59
days.
days. The high dosagedosage of each steroid produced all-male
all-male populations during
all
all treatment periods. The 30 30 mg/kg dosage produced all-male
all-male populations at
5. HORMONAL
HORMONAL SEX
SEX CONTROL AND ITS
FISH CULTURE
the 35-
35- and 59-day treatment periods. Recently, Owusu-Frimpong and Nij Nij-
jhar (1981)
(1981) applied a 50 mg methyltestosteone/kg diet treatment to newly
hatched fry
fi-y for 28 or 42 days resulting in the production of all-male popu
popu-
lations.
Recently, Nakamura and Iwahashi (1982) (1982) achieved complete masculi
masculi-
nization by the administration of 50 or 100100 mg/kg dosages of methyltesto
methyltesto-
sterone for a period of 30 days, beginning the day of capture from the adult
holding pond. Similar treatments begun 55 or 10 10 days after capture resulted
in the production of intersex gonads. In most cases these intersex gonads
were ovarian in the anterior region and testicular in the posterior region.
d.
d. Tilapia zillii. The first attempt to masculinize populations of Tilapia
Tikpia zUlii.
zillii was conducted by Guerrero
zUlu Guerrero (1976b)
(1976b) concurrent with his treatment
treatment of
Oreochromis mossambicus. The treatment involved administration of 50 mg
ethynyltestosterone/kg diet for 40 days, and was effective in Oreochromis
mossambicus but not in Tilapia zillii. Guerrero
Guerrero (1976b)
(1976b) attributed this dis
dis-
crepancy to incorrect timing or dosage of treatment, which he suggested
may be different for the bottom-spawning Tilapia zUlu zillii compared with the
mouth-brooding Oreochromis mossambicus. However, data was not avail avail-
able to confirm this hypothesis. Yoshikawa and Oguri (1977) (1977) examined the
zillii via histological
period of sex differentiation in Tilapia zUlii histological techniques. On
the basis of
of germ cell numbers at 15 15 days posthatching, they were able to
distinguish between future ovaries that contained many germ cells cells (some of
which were in meiotic prophase) and testes that contained fewer germ cells cells
(none
(none of which were in meiotic prophase). Following this investigation,
Yoshikawa and Oguri administered an oral treatment of methyltestosterone
methyltestosterone
at 50,
50, 100,
100, and 200 mg/kg diet for 20 days starting 10 10 days posthatching. The
treatment resulted in inhibition of oogenesis and a proliferation of somatic
elements, but did not result in sex inversion (Yoshikawa
(Yoshikawa and Oguri, 1978).
1978).
The researchers attributed
attributed treatment failure to the high dosage levels used.
However, Woiwode (1977) (1977) achieved 100%
100% sex inversion following oral ad ad-
ministration of 50 mg/kg of methyltestosterone for 45 days posthatching,
suggesting that treatment timing and duration may not have been optimal in
the previous study. However, these results are difficultdifficult to reconcile with
those of Guerrero (1976b).
(1976b). Given similar treatment duration and method of
administration, the greater effectiveness of the methyltestosterone treat treat-
ment is unexpected. Ethynyltestosterone has been demonstrated to to be of
greater potency than methyltestosterone (Yamamoto,
(Yamamoto, 1969).1969).
e. Cichlids. Several
e. Other Cichlids. Several other species have
have been examined,
examined, although
less
less intensively.
intensively. Hackmann (1974)
(1974) reported the paradoxical
paradoxical feminizing ac
ac-
tion
tion of testosterone propionate and methyltestosterone added at 500
500 f.Lgll
pg/l to
the rearing water of Oreochromis mossambicus, Tilapia heudeloti,
heudeloti, and
GEORGE A. AND
HUNTER A N D EDWARD
EDWARD M.
M . DONALDSON
DONALDSON
biocellatum. Jalabert et al.

Cichlasoma biocellatum. al. (1974)
(1974) examined the effects
effects of
of 40
mg/kg diet of methyltestosterone
methyltestosterone for 2 months on Tilapia macrochir concur
concur-
rent
rent with
with his examination
examination of
of Oreochromis niloticus. In the
the latter
latter species sex
sex
inversion was achieved. In Tilapia macrochir, although external male sec sec-
ondary characteristics were induced, the gonads were completely sterilized.
Jalabert et al.
al. (1971)
(1971) had previously indicated male homogamety in Tilapia
macrochir. Hackmann and Reinboth (1974)
macrochir. (1974) demonstrated the masculiniza
masculiniza-
tion of Hemihaplochromis multicolor by immersion of the fry for 42-45
42-45 hr in
water containing 50 mg testosterone propionate/I.
propionate/l.
2.
2. ESTROGEN
ESTROGEN TREATMENT
TREATMENT
The production of
of monosex female tilapias was advocated by Bardach et
al. (1972)
al. (1972) as a means of
of alleviating the destructive nest-building activities of
males.
a. aureus. As with the androgens, the estrogens, specifi
a. Oreochromis aureus. specifi-
cally synthetic stilbestrol and stilbestrol-diphosphate-diaethyldioxystilben
stilbestrol-diphosphate-diaethyldioxystilben-
diphosphate, were first applied to Oreochromis au reus by Eckstein and
aureus
Spira (1965).
(1965).The purpose of this study was sterilization for culture purposes.
The estrogens were applied to the rearing water at 50-1000 50-1000 fJ.g/1
pg/l for 5-6
5-6
weeks starting at 4-54-5 weeks of age. At estrogen concentrations higher than
200 fJ.g/I,
pg/l, mortalities were very high. At the dosages of 50 and 100 fJ.g/1 pg/l a
powerful
powerful inhibition
inhibition of gonadogenesis
gonadogenesis occurred,
occurred, resulting,
resulting, in
in most
most cases,
cases, in
in
complete sterility.
Based on the work of Guerrero (1975),
(1975),Jensen (1976)
(1976)tested the effective
effective-
ness of estriol, estrone,
estrone, and estradiol in inducing sex inversion in genetically
male Oreochromis aureus, which could then be used to produce all-male
progeny. In this study, these naturally occurring estrogens were admin- admin
istered to fry at 30,
30, 60,
60, or 120
120 mg/kg diet for either 21
21 or 35 days posthatch
posthatch-
ing. All treatments proved ineffective. This lack of success was attributed to
ing.
either insufficient dosage or duration of treatment.
treatment. Following the work of
Jensen, Hopkins (1977)
(1977) orally administered the two synthetic estrogens,
ethynylestradiol and diethylstilbestrol, and the naturally occurring estradiol
alone or in combination with the antiandrogen cyproterone acetate. Steroid
concentrations were 25-200
25-200 mg/kg diet, and the antiandrogen concentration
was held at 100
100 mg/kg diet.
diet. Treatment durations were either 35 or 56 days
posthatching. Ethynylestradiol at 25 or 100 100 mg/kg with the antiandrogen
produced 60 and 63% 63% females, respectively, but diethylstilbestrol alone
resulted in 64%
64% females. In a later experiment, Hopkins et al. al. (1979)
(1979) was
able to achieve 90%90% female groups by a 42-day oral administration of eth- eth
ynylestradiol and methallibure each at 100 100 mg/kg diet with or without
cyproterone acetate at 100 mg/kg. Hopkins and co-workers suggested that
5.
APPLICATION TO
TO FISH
FISH CULTURE
CULTURE 275
the anti androgen may have lessened the effectiveness of treatment because
antiandrogen
of
of its
its additional
additional antiestrogenic,
antiestrogenic, progestational,
progestational, and
and androgenic
androgenic effects.
effects.
mossambicus. Nakamura
b. Oreochromis mossambicus.
b. Nakamura and
and Takahashi
Takahashi (1973) used the
(1973)used
oral
oral administration
administration of
of ethynylestradiol
ethynylestradiol at
at 50 mg/kg
mg/kg diet
diet to
to determine
determine the
optimal period of steroid treatment in Oreochromis mossambicus.
mossambicus. Histologi
Histologi-
cal examination indicated that the primordial gonads formed 8-10 8-10 days
posthatching with the histologically discernable initiation of sex differentia
differentia-
tion occurring at 20 days of age. They were thereby able to achieve complete
feminization with a treatment which lasted for 19 19 days from 6 days of age.
The discrepancy
discrepancy between
between their
their determination
determination of the
the time
time of
of sex
sex differentia
differentia-
tion
tion and
and that
that of
of Clemens
Clemens and Inslee (1968)
and Inslee (1968) (35-48 days
days posthatching)
posthatching) was
was
attributed to the morphological criteria used.
c. niloticus. Tayamen and Shelton (1978)
c. Oreochromis niloticus. (1978) orally admin
admin-
istered diethylstilbestrol at 25 and 100
100 mg/kg diet and estrone at 100
100 and 200
mg/kg diet for 25-35
25-35 and 59 days to newly hatched fry. The estrogen treat
treat-
ments produced between
between 62 and 90%
90% female groups, indicating that, com
com-
pared with Hopkin's (1977) aureus, estrogen
(1977) treatment of Oreochromis aureus, estrogen-
induced sex inversion was much more easily attained in Oreochromis
niloticus.
niloticus.
d.
d . Tilapia zillii. Yoshikawa
Tilapia zillii. Yoshikawa and and Oguri
Oguri (1978)
(1978) administered
administered oral oral treat
treat-
ments
ments ofof ethynylestradiol
ethynylestradiol atat 20,
20, 40,
40, and
and 60 mg/kg
mg/kg diet
diet for
for 20 days starting 10
days starting 10
days
days posthatching
posthatching to to Tilapia zillii fry.
Tilapia zillii fry. Treatment
Treatment diddid not
not result
result in
in sex
sex inver
inver-
sion.
sion. The
The estrogen
estrogen inhibited
inhibited spermatogenesis
spermatogenesis at at 20 mg/kg
mg/kg andand at
at 40 and
and 60
mg/kg
mg/kg resulted
resulted in
in the production
production of of few fish containing
few fish containing involuted
involuted gonads.
gonads.
e. Cichlids. Monosex
e. Other Cichlids. Monosex female
female groups
groups of
of Hemihaplochromis
Hemi-haplochromismulti
multi-
color were obtained by Hackmann and Reinboth (1974) (1974) by addition of 250
IJ.g/1
kg/1 estradiol
estradiol butyryl
butyryl acetate
acetate to
to the
the rearing
rearing water
water for
for 42-45
42-45 hr
hr between
between U.5
11.5
and 16 days postspawning. The estrogen was effective in inducing sex inver inver-
sion over a wider period of time than the androgens, testosterone propionate
and methyltestosterone.
A summary
summary of of these
these studies
studies isis presented
presented in
in Table
Table II.
11. The
The direct
direct admin
admin-
istration of androgens
androgens is clearly the favored strategy for producing monosex
male
male populations. However, the value of the indirect indirect strategy for producing
monosex
monosex male populations in species species such as
as Oreochromis aureus, which
Oreochromis aureus,
have
have male homogamety, remains to be determined.determined. As yet, the isolation of
'
YY males
males from the matings or sex-inverted XY males
of 'sex-inverted males and untreated males,
similar to that
that conducted
conducted by Yamamoto
Yamamoto (1965)
(1965) on Oryzias latipes,
latipes, has not
been attempted.
attempted. In species such as mossambicus and Oreo
as Oreochromis mossambicus Oreo-
chromis niloticus, the sperm from these YY males could theoretically be
chromis niloticus,
used
used to to sire
sire monosex
monosex male
male populations.
populations.
A N D EDWARD
EDWARD M.
M . DONALDSON
DONALDSON
B.
B. Salmonids
Salmonids
Diverse objectives are associated with the hormonal sex control of salm salm-
on and trout (Goetz et al. 1979; Johnstone et al.
al.,, 1979; 1978, 1979a;
al.,, 1978, 1979a; Bye and
Lincoln, 1981; al.,, 1982a;
1981; Hunter et al. 1982a; Donaldson and Hunter, 1982a).1982a). This
diversity is evident within the two major strategies employed in salmonid
culture. The first, pen rearing, involves the culture of the fish in enclosures
throughout the lifetime of the fish.
fish. The second, ocean ranching, involves the
rearing ofjuvenile salmon for release into the ocean environment and subse subse-
quent harvest of these fish as adults on their anadromous migration. Hunter
al. (1982a)
et al. (1982a)have summarized the potential applications of sex-control tech tech-
niques to both strategies. Often, with pen rearing, the lack of a reliable
source of gametes requires the maintenance of large numbers of non- non
marketable female broodstock. The ability to produce monosex female
groups could dramatically reduce the number of fish required to produce the
necessary egg take, thereby reducing the costs of broodstock maintenance.
Further, all-female groups are preferred in the culture of rainbow trout, trout,
because males have the propensity to mature precociously and adult males
have poor growth rates, poor food conversion efficiencies,
efficiencies, and poor survival
when grown in seawater (Bye (Bye and Lincoln, 1981).
1981).
Sexual
Sexual maturation restricts the period over which the fish may be mar mar-
keted because of the deterioration of flesh quality and mortalities attributa
attributa-
ble to bacterial and fungal infections associated with the maturational pro pro-
cesses. The production of sterile fish would allow for the year-round
marketing of adult-sized fish of high flesh quality. The production of either
female or sterile fish would alleviate the problem of sexual maturation before
attainment of full adult size.
size.
The ocean-ranching culture strategy has problems associated with sexual
maturation and reproduction analogous to the pen-rearing approach. Al Al-
though broodstock are not held for the full life cycle, costs are associated
with the number of fish held for broodstock that must be allowed to mature
sexually and are subsequently of a lower market value. value. Further, the number
of ova which can be obtained determines
determines the hatchery smolt output.
output. There
There-
fore, the production of a high proportion of females would both reduce the
escapement required to meet hatchery objectives allowing a greater number
of fi sh to be caught and provide a means of
fish of rapidly increasing the production
of smolts.
smolts. An increase in the proportion of females is desirable in species in
which the roe is harvested and marketed separately.
The harvest
harvest of ocean-ranched stocks
stocks is also restricted in time by normal
sexual maturation. In species such as the chum salmon, Oncorhynchus keta,
the present harvest strategy, involving terminal fisheries, results in the
harvest of primarily sexually mature fish which are of relatively low market
5.
HORMONAL SEX CONTROL AN
A ND
D ITS APPLICATION TO
FISH CULTURE
CULTURE 277
value. The production of sterile fish of of high flesh quality would dramatically
increase the value
increase value of the
the catch.
catch. The
The production
production of sterile fish,
of sterile fish, which
which dodo not
not
undergo the normal anadromous migration, would extend the active fishing
season and allow alternative harvest strategies.
strategies.
The
The prevention
prevention of the anadromous
anadromous migration
migration would
would eliminate
eliminate the neces
neces-
sity for a "one-shot"
“one-shot” harvest and would allow greater flexibility in the estab estab-
lishment
lishment ofof exploitation
exploitation rates,
rates, thereby
thereby assisting
assisting the
the management
management of of mixed
mixed-
stock
stock fisheries. Furthermore, by extending the effective life span of the fish
the
the possibility
possibility of producing
producing larger
larger fish
fish exists.
exists. An
An increase
increase inin the size
size of
of fish
fish
for harvest would provide benefits to the recreational fishery in the form of
trophy-sized
trophy-sized fish,
fish, and
and to
to the
the commercial
commercial fishery
fishery through
through anan increase
increase in
in total
total
weight harvested (A higher value per unit weight is placed on large fish). fish).
This
This approach
approach requires
requires the
the use
use of stocks
stocks which
which remain
remain inin or
or return
return to
to waters
waters
accessible to
accessible to the fishery. Loss
fishery. Loss of full
full growth potential as a result of
growth potential as a result of pre
pre-
cocious male development
development is also a problem for the ocean-ranching strategy.
Again,
Again, the production of female or sterile fish would alleviate this problem.
1. ANDROGEN
1. ANDROGENTREATMENT
TREATMENT
a.
a. Salmo gairdneri. The first study involving the use of androgens in
rainbow trout was designed to determine the growth-promoting and go go-
nadal-suppressing activity of 4-chlorotestosterone
4-chlorotestosterone acetate. The steroid was
injected into l-year-old fish at dosages of 1.0-12.5 mg/fish in each of 6
(Hirose and Hibiya, 1968).
injections over 30 days (Hirose 1968). Treatment suppressed
both yolk deposition and testicular
testicular differentiation. Hirose and Hibiya sug sug-
gested this suppression was a result of a blocking of of gonadotropin release.
Recently, Billard et al. (1981) produced gonadal inhibition in adult trout by
al. (1981)
feeding a diet containing methyltestosterone or estradiol at 0.5 0.5 mg/kg during
the period of spermatogenesis Gune-November).
(June-November). Treatment during the peri peri-
od of spermiation (November-February)
(November-February) was ineffective.
The
The first application of steroids during sexual differentiation was con con-
ducted
ducted by
by Jalabert
Jalabert et al. (1975). In
al. (1975). In this
this study,
study, young
young fry
fry were
were administered
administered
methyltestosterone orally at dosages of 15, 30, and 60 mg/kg diet for 5
15, 30,
months
months starting
starting 1 month
month posthatch.
posthatch. Examination
Examination at at 2 years
years indicated
indicated aa com
com-
bined total of 58% male, 12% 18% female, and 12%
12% sterile, 18% 12% intersex fish.
fish.
Working with Horai masu,
Working maw,a variant of rainbow trout, Yamazaki (1976) (1976) re
re-
ported that the administration of methyltestosterone at 50 mg/kg diet for a
period
period identical to that used by Jalabert et al. (1975) resulted in the produc
al. (1975) produc-
tion of gonads containing richly vascularized connective tissue devoid of
germinal
germinal material at age 2 years. However, of three fish which survived to 3
years
years of
of age,
age, two
two had
had testes
testes with
with spermatogonial
spermatogonial cysts
cysts and
and one
one had
had aa
threadlike ovary with a small number of yolkless ooytes. In the same study,
278 GEORGE
GEORGE A.
HUNTER AND M. DONALDSON
EDWARD 111. DONALDSON
Yamazaki
Yamazaki (1976)
(1976)reported that treatment with methyltestosterone at 11 mg/kg
diet
diet starting
starting 11 month
month posthatch
posthatch and and lasting
lasting for
for 7 months
months resulted
resulted in in the
the
production of 87. 87.1%1% males. These
These results agree with Yamamoto's (1969) (1969)
assertation regarding the masculinizing action of androgens at low dosages
and
and sterilizing action at high dosages.
dosages.
The first production of of androgen-induced
androgen-induced monos monosex ex male Salmo gairdneri
was
was reported
reported by by Simpson
Simpson (1916).
(1976). Treatments
Treatments involved
involved the
the oral
oral administra
administra-
tion
tion of
of methyltestosterone
methyltestosterone at at 33 mg/kg
mg/kg diet
diet for
for 90 days
days from
from first feeding
feeding with,
with,
or without, previous immersions for 2-hours of the eyed-egg and alevin
stages in water containing the steroid at 250 J.Lg/l. pg/l. Immersion treatments
treatments
alone
alone were
were ineffective.
ineffective. Reduction
Reduction of of the
the duration
duration ofof oral
oral treatment
treatment to to 30
30 days
days
resulted
resulted in the production of 85% 85% males at 16 months. However, in most of
the
the male fish reported by Simpson, large areas of the gonads were devoid of
germ
germ cells andand contained
contained hypertrophied
hypertrophied connective
connective tissue.
tissue. Johnstone
Johnstone et al. al.
(1978)
(1978) reported that following
following identical treatments, all animals examined at
up to 12
12 months contained the thin filiformfiliform gonad typical of males. However,
in 17%
17%of these fish the complete gonad was composed of hypertrophied hypertrophied and
sterile connective tissue areas. In another experiment, in which the oral
treatment of 33 mg/kg was reduced to 30 days, 79% 79% males were produced.
Subsequently, milt from these mature males was used to fertilize normal ova
Gohnstone
(Johnstone et al.al.,, 1979a).
1979a). Of the males having mature testicular tissue, the
milt from sixsix could be expressed normally, but eight others had absent or
occluded system ducts.ducts. Sperm from a total of 10 males was used to fertilize
normal ova.
ova. One of these pairings resulted in the production of 100% 100%
females, indicating female homogamety in this species.
In the same year, Okada et al. al. (1979)
(1979)also demonstrated female homo homo-
gamety in Salmo gairdneri.
gairdneri. In this study, fry were fed a diet containing 1, 1, 5,
5,
or 10 mg methyltestosterone/kg diet for 58 days resulting in the production
of 60,
60, 80,
80, and 88%
88%males, respectively. Mortality ranged from 55.3 55.3to 68.7%
68.7%
in these groups. Two of 11 11 males containing morphologically abnormal testes
sired nearly 100%
100%female offspring when mated with untreated untreated female rain rain-
bow or steelhead trout.
trout, The few males found in these groups were attributed
to contamination from other groups. The subsequent subsequent production of an F F,2
generation with a normal male-female
male-female ratio indicated that the F F,1 generation
was genetically normal.
normal.
The
The observations of abnormal testicular morphology associated with sex sex-
inverted female rainbow trout has been confirmed by V.I. V.J. Bye (personal
communication, 1980; 1980; Bye and Lincoln,
Lincoln, 1981).
1981). Bye applied an oral meth meth-
yltestosterone treatment at 33 mg/kg mg/kg diet for 1000°C
1000°Cdays at 111.2"C
1 . 2°C (90
(90 days)
days)
resulting in the production of 94% 94% males.
males. At age 2 years, 55% 55% of these fish
could not be stripped.
stripped. On examination they were found to have abnormal
testes with absent or occluded sperm sperm ducts.
ducts. At age 33 years, 70%70%of the fish
5.
FISH CULTURE
that
that could
could notnot be stripped
stripped had had hermaphroditic
hermaphroditic gonads gonads consisting
consisting of of ductless
ductless
testes with an anterior cap of ovarian tissue. The progeny of 14 14 of 16
16 such
males sired all-female progeny. progeny. All fish that could be stripped had normal
tests which later sired progeny in a normal 11:1 : 1 male-to-female ratio. The
duration of methyltestosterone administration necessary to produce mono mono-
sex
sex males
males without
without steriles
steriles waswas 700°C days days at at 9°-12°C
9"-12"C with with feeding
feeding forfor 10
hrlday
hr/day (V. (V. J. Bye,
Bye, personal
personal communication,
communication, 1980). 1980). ByeBye also
also reported
reported thatthat
attempts
attempts to induce sterility with oral methyltestosterone treatment have
been largely unsuccessful.
unsuccessful. Methyltestosterone when given at 25 mg/kg diet
for
for 1000°C
1000°C daysdays inconsistently
inconsistently produces
produces 60% 60% sterility.
sterility. Harbin
Harbin et al. (1980)
(1980)
also
also attempted
attempted to to induce
induce sterilization
sterilization in in rainbow
rainbow trouttrout byby administering
administering
methyltestosterone
methyltestosterone orally orally atat 30 mg/kg
mg/kg diet
diet for
for 110 days
days from
from first
first feeding.
feeding. At At
age
age 2 years,
years, 22.5%
. 5% ofof the fish
fish were
were found
found toto bebe mature.
mature. TheThe remainder
remainder had had
filiform gonads with
filiform gonads with increased
increased vascularization
vascularization and and connective
connective tissue.
tissue. Serum
Serum
testosterone
testosterone levelslevels of of treated
treated fish fish were
were significantly
significantly lower (0.34 ±
lower (0.34 5 0.08
0.08
ng/ml) compared
ng/ml) compared with with controls
controls (14.6
(14.6 ± k 3.63
3.63 ng/ml).
ng/ml). Treated
Treated fishfish displayed
displayed
none
none of of the
the secondary
secondary sexualsexual characteristics
characteristics associated
associated with
with maturation,
maturation, pre pre-
sumably
sumably because
because of of the
the low
low levels
levels ofof androgen
androgen present.
present.
In
In aa subsequent
subsequent study,study, van van den
den Hurk
Hurk andand Slof (1981) achieved
Slof (1981) achieved steriliza
steriliza-
tion
tion in
in rainbow
rainbow trouttrout byby adding
adding methyltestosterone
methyltestosterone to to the
the rearing
rearing water
water 3
times
times per
per week
week at at 3-300
3-300 pg/lfJ.g/I for
for 28-56
28-56 days
days posthatching.
posthatching. They They determined
determined
that
that sex
sex differentiation
differentiation takestakes place
place 45-55
45-55 daysdays postfertilization.
postfertilization. Hatching
Hatching
occurred
occurred around
around 26-2926-29 daysdays postfertilization.
postfertilization. The The highest
highest percentages
percentages of of
steriles
steriles were
were obtained
obtained with with treatments having the
treatments having the longest
longest duration
duration or or the
the
highest dosage.
highest Sterility was
dosage. Sterility was not induced when
not induced when the the treatment
treatment was begun 43
was begun
or
or 57 days
days postfertilization,
postfertilization, althoughalthough thethe percentage
percentage of males increased.
of males increased.
Treatments
Treatments were were accompanied
accompanied by by growth
growth depression
depression and and increased
increased mor mor-
tality.
tality. Recently,
Recently, van van den Hurk and
Lambert (1982)
(1982)reported
reported thethe production
production
of
of 100 malemale groups
groups of of rainbow
rainbow trouttrout following
following treatment
treatment with with 1llp-hy-
1 13-hy
droxyandrostenedione
droxyandrostenedione at at 60 mg/kg
mg/kg dietdiet for
for 56 days
days from
from first
first feeding.
feeding. Treat
Treat-
ment with
ment with 1llp-hydroxyandrostenedione
1 13-hydroxyandrostenedione at at 6 mg/kg
mg/kg or or with
with methyltesto
methyltesto-
sterone
sterone at at 0.
0.66 oror 6.0 mg/kg produced
6.0 mg/kg produced 94-99% males. males.
h. Salmo Salar.
b. Simpson (1976)
Salar. Simpson (1976) first
first applied
applied androgens
androgens to to Salmo salar
for
for the
the purpose
purpose ofof producing
producing sex-inverted
sex-inverted females,
females, which
which could
could then
then be used
used
to
to sire
sire all-female
all-female salmon
salmon for
for pen
pen culture.
culture. InIn this
this study,
study, eyed
eyed eggs
eggs and
and alevins
alevins
were
were immersed
immersed in in water
water containing
containing methyltestosterone
methyltestosterone at at 250
250 fJ.g/I
pg/l for
for two
two 2-
2-
hr
hr periods.
periods. The fry fry were
were subsequently
subsequently fed fed aa diet
diet containing
containing methmeth-
yltestosterone
yltestosterone at at 30 mg/kg
mg/kg diet
diet for 120 days.
for 120 No female
days. No female elements
elements were
were found
found
in
in the
the gonads
gonads upup to
to 9
9 months
months posttreatment.
posttreatment. Johnstone
Johnstone et al.al. (1978)
(1978)analyzed
analyzed
aa group
group ofof similarly
similarly treated
treated Salmo salar. These fish
salar. These fish contained
contained sterile
sterile filiform
filiform
gonads
gonads atat 66 months.
months, InIn another group, methyltestosterone
another group, methyltestosterone was was administered
administered
280 GEORGE A.
GEORGE A. HUNTER
HUNTER AND
AN D EDWARD M.. DONALDSON
EDWARD M DONALDSON
orally at 3 mg/kg diet for 90 days after first feeding producing 100%
100%males or
sterile fish.
fish.
c. trutta. Ashby (1957)
c. Salmo trotta. (1957) reared Salmo trutta in water containing
Salrno trotta
50-60 HI days starting 170
JJ.g/I testosterone for 111
50-60 pg/l 170 days postfertilization
(8500-1738°C days). Of 29 fish examined,
(850"-1738°C days). examined, 41%
41% were females, 35% 35% were
males based on the presence of vasa deferentia, and 24%
24% containing neither
oocytes or vasa deferentia. The gonads of these latter fish were composed
primarily of connective tissue with very few germ cells. Ashby (1965)
(1965) again
administered testosterone in the rearing water at 67 JJ.g/I,
pg/l, 8 hr/day, 6 days
per week for 3 months starting 7 months postfertilization. Although some
ovarian inhibition was reported, testicular inhibition was far more pro pro-
nounced including the complete elimination of somatic and germinal tissue.
d. Salvelinus species. Wenstrom (1975)

d. (1975) fed testosterone propionate at
700 mg/kg diet to lake trout, Salvelinus namaycush, for either 245°
245"or 290°C
290°C
days starting at 1250°C
1250°C day. Of the 10 fry examined, 7 were male and 3 were
female.
e. Oncorhynchus kisutch. Coho salmon,

e. salmon, Oncorhynchus kisutch, has
been by far the most intensively studied of of the Oncorhynchus species.
species. Early
experiments involved the administration of androgens after completion of of
sex differentiation. McBride and Fagerlund (1973)
sex (1973) assessed the anabolic
effects
effects of orally administered methyltestosterone
methyltestosterone at 1, 1, 10,
10, or 50 mg/kg diet
on coho salmon with an average weight of 33.79 . 79 g for up to 42 days.
days. By the
end of 28 days,
days, a marked reduction in spermatogonia had occurred in the
groups fed 10 or 50 mg/kg. At the end of 42 days the testes were nearly
devoid of germ cells. The effect was more pronounced in fish receiving the
higher dosage.
dosage. No distinct alterations were noted in the ovary.
ovary. No effect was
observed on the gonads of fish fed the 11 mg/kg dosage. In a later study,
Fagerlund and McBride (1975)
(1975) again tested the anabolic activity of meth meth-
yltestosterone on younger fish having an average weight of of 0.78
0.78 gm. These
fish were administered 0.2,
0.2, 11.0,
. 0, or 10 mg/g diet for up to 504 days. After 56
days, clear degenerative changes were observed in the gonads of those fish
administered
administered the 10 mg/kg diet. By the end of 140 140 days no spermatogonia
could be observed in the fish in this group. By the end of 224 days the
gonads consisted of thickened connective tissue. At 140 140 days a small number
of fish in the group receiving the 10 10 mg/kg dosage were transferred to a
control diet for 364 days. Of the 24 males examined at the end of this period,
14
14 were sterile and 10 had reduced spermatogonia. Of the fish fed the 11
mg/kg for 504 days, 2 of of 13
13 appeared to be sterile, 3 had reduced sper sper-
matogonia, and 8 appeared unaffected. These results clearly suggested the
possibility of producing male or sterile coho salmon by the administration of of
5.
APPLICATION TO
TO FISH CULTURE 281
FISH CULTURE 281
methyltestosterone. With this objective and using a modification of the pro pro-
cedures
cedures described by Simpson (1975- 1976) for Salmo gairdneri, Goetz et al.
(1975-1976) al.
(1979)
(1979) conducted a study to determine the effects effects of methyltestosterone
treatment at the time of sex differentiation. Sex differentiation of coho salm- salm
on and other Oncorhynchus species has been reported to occur earlier than
that of the SaZmo (Persov, 1975).
Salmo species (Persov, 1975). An initial examination of sex differ
differ-
entiation indicated that, based on the appearance of of perinuclear
perinuclear oocytes,
differentiation was first discernable at 880°C 880°C days, 49 days posthatching
(Goetz et al. 1979). Therefore, treatments were initiated prior to and in
al.,, 1979). in-
cluding first feeding. In this study, eyed eggs were each administered 2 or 6
immersions of 2 hr duration in waterwater containing the steroid at 25-400
25-400 /-Lg/l.
pg/l.
Alevins were administered either 2 or 7 immersions of of 2 hr each. Fry were
orally administered the steroid at 20 mg/kg diet for 70 days. All groups
treated
treated in this manner were found to be 100% 100% sterile, with the exception of
the group administered the 25 mg/kg oral treatment which was 94% 94%sterile.
A group receiving the oral treatment alone was found to be 52% 52% sterile,
24.5%
24.5% intersex, and 23.5%
23.5% female.
In subsequent experiments (G. (G. A. Hunter and E E.. M. Donaldson, un un-
published data),
data), the oral dosage was reduced to 10 mg/kg administered with,
or without, prior immersions at 400 pg/l /-Lg/I in the eyed-egg and alevin stages.
Immersion in the alevin stage alone resulted in 67% 67% males and no sterile
fish. In groups administered the dietary treatment, 83-97% steriles were
fish.
produced. In order to ascertain the effect of dosage and duration of dietary
treatment,
treatment, groups of coho were administered an initial immersion treatment
in the alevin stage followed by dietary treatments
treatments at 1, 1, 3, or 9 mg/kg for a
period of 21, 42, or 63 days days.. The highest production (82-92%)
(82-92%) of males
occurred in groups receiving the 11 mg/kg dosage.
dosage. The percentage of steriles
increased with higher dosages or longer treatment durations. durations. Of the 13
males that were reared to maturity in this study and mated mated with untreated
females, one sired all-female progeny, indicating female homogamety in the
species.
Hunter et al. (1982a)
(1982a) examined the development of two groups of coho
salmon treated by immersion at 100 or 400 /-Lg/I pg/1 methyltestosterone in the
eyed-egg and alevin stage followed by dietary treatment for 90 days. At
maturity the high- and low-dosage groups contained 94 and 70% 70%sterile fish.
These sterile fish continued to live and grow for 2.5 2.5 years after the matura
matura-
tion and death of the control group.
group. The fish remaining at the end of this
period contained gonads composed entirely of connective tissue. During this
period, these fish maintained the silvery appearance characteristic of imma imma-
ture fish (Fig. 3). This study first demonstrated the potential
(Fig. 3). potential advantages of of
hormonal sterilization to the culture of Pacific salmon. In a recent field trial
of the sterilization technique, 40,000 coho salmon were administered two two 2-
282 GEORGE A.
GEORGE A.'· HUNT
HUNTER
E R AND
AND EDWARD M.. DONALDSON
EDWARD M DONALDSON
Fig. 3. Comparison
Fig. Comparison of
of three
three sterile
sterile (top)
(top) and
and two
two female
female 3-year-old
3-year-old coho
coho salmon
salmon during
during the
the
period
period of
of normal
normal maturation
maturation (December).
(December).
hr immersions in water containing methyltestosterone

inethyltestosterone at 200 fLg/I
pg/l in both
the eyed-egg and alevin stages followed
followed by an 84-day
S4-day dietary treatment at 10
10
mg/kg (Donaldson
(Donaldson and Hunter, 1982b).
1982b). A subsample was held in a seawater
net pen to determine survival, growth, and treatment success.
success. The remain
remain-
ing fish,
fish, which had been nose tagged and fin clipped, were released into the
5.
5. HORMONAL
HORMONAL SEX
SEX CONTROL AND
CONTROL A N D ITS APPLICATION TO
rrs APPLICATION TO FISH
FISH CULTURE
CULTURE 283
283
ocean in May, 1980.

1980. Analysis
Analysis of the fish held in the net pens indicated that
97. 7% of the fish were sterile. The sterile fish survived at a similar rate,
97.7% rate, but
grew at a slower rate compared to a monosex female group treated at the
same time. However, the sterile group continued to grow through the peri peri-
od in which the females matured
matured and ceased to grow.
grow. The steriles attained a
carcass weight similar to the females by the end of the normal spawning
season.
season. Furthermore, the sterile fish continued to grow the following
following year,
and the all-female group reached the end of their life cycle after the spawn
spawn-
ing season.
season. No precocious males returned to the hatchery in the fall of 1980.
1980.
Untreated fish released at a similar time would have been expected to return
in the fall of 1981;
1981; however, no sterile fish returned to the hatchery at this
time. Approximately 18 fish from the methyltestosterone-treated
methyltestosterone-treated group did
return. All contained morphologically abnormal but maturing testes. To date
sterile fish have contributed to the commercial and recreational fisheries in
both 1981
1981 and 1982 and are expected to continue this contribution until
normal ageing and death occur in the absence of sexual maturation.
f. tshawytscha. The earliest report of
f. Oncorhynchus tshawytscha. of the actions ofof an
an-
drogens on the gonads of chinook salmon, Oncorhynchus tshawytscha, is
that of McBride and Fagerlund (1973). They administered 0,
Fagerlund (1973). 0, 0.2,
0.2, or 11.0
. 0 mg
methyltestosterone/kg
methyltestosterone/kg diet to chinook fry having an average weight of 0. 78
0.78
gm for either 28,
28, 56,
56, or 84 days. The gonads of fish treated
treated for 84 days were
visibly swollen,
swollen, but no reduction in spermatogonia could be detected. Ov Ov-
aries were not affected.
affected.
More recently, the effect of dietary administration of of methyltestosterone
at various dosages and durations of treatment have been examined (G. (G. A.
Hunter and E E.. M. Donaldson, unpublished data). data). In the study by Hunter
and Donaldson, chinook alevins were administered two immersions of 22 hr
each in water containing methyltestosterone at 400 j.Lg/l. pg/l. The fish were
subsequently given oral treatments with methyltestosterone at 3 or 9 mg/kg
diet for periods of 3, 6,
6, or 9 weeks. All but one treatment resulted in a high
(83-98%)
(83-98%) percentage
percentage of of males.
males. InIn the
the 9 mg/kg-9
mg/kg-9 weekweek treatment
treatment group,
group,
43%
43% were males and 57% 57% appeared sterile at 8 months posthatching. At
maturity,
maturity, 59 males
males from
from these
these groups
groups were
were mated
mated with
with untreated
untreated female
female
chinook
chinook or
or coho
coho salmon.
salmon. Greater
Greater than
than 92%
92%female
female progeny
progeny were
were observed
observed in in
21 families, and 8 families had 100%
21 100%females.
females. These results indicate female
homogamety in this species
species (Hunter et al. al.,, 1983).
1983).
g. gorbuscha. Robertson (1953)
g . Oncorhynchus keta and Oncorhynchus gorbuscha. (1953)
identified the period of germ cell maturation in chum salmon salmon (On
(On-
corhynchus keta) as occurring from 1414 days prehatching to 42 days post
post-
hatching at 50-6°C
5"-6"C with the first appearance of primary oocytes at 55 days
posthatching. Nakamura (1978)
(1978) reported sex differentiation in chum species
A N D EDWARD M. DONALDSON
EDWARD M. DONALDSON
at 35 days posthatching at 8°C.

8°C. The only report which has examined the
effect of androgens on the gonads of these species
species is that of Yamazaki
Yamazaki (1972).
(1972).
In this study 1-year-old
l-year-old chum and pink salmon (Oncorhynchus
(Oncorhynchus gorbuscha)
gorbuscha)
were orally administered methyltestosterone
methyltestosterone at 50-100
50-100 mg/kg diet for 2
weeks. Treatment resulted in the degeneration of spermatogonia and atresia
of ova.
ova.
2. ESTROGEN
2. ESTROGEN TREATMENT
TREATMENT
a.
a. Salmo gairdneri. The earliest recorded studies employing estrogens
Sulmo gairdneri.
for the control of sex differentiation in rainbow trout were those of Padoa
(1937,
(1937, 1939).
1939). These studies involved the immersion of juvenile Salmo
gairdneri (irideus) in water containing estrone. These treatments were
gairdneri (irideus)
largely without effect. Padoa (1939)
(1939) determined that sex differentiation of of
rainbow trout takes place shortly after yolk-sac absorption is complete, dur dur-
ing the period of first feeding. The occurrence of sex differentiation in Salmo
guirdneri shortly after first feeding has been confirmed by Okada (1973),
gairdneri (1973),
Takashima et al. al. (1980),
(1980), and van den Hurk and Slof (1981).(1981). Okada (1973)
(1973)
orally administered estrone at 10, 10, 50, 100100 mg/kg diet for 58-12458-124 days
posthatching. The percentage of females obtained ranged from 79 to 94%. 94%.
Treatment was accompanied by high mortalities and a dose-dependent inhi inhi-
bition of growth.
al. (1975)
Jalabert et al. (1975)also orally administered estrone at dosages of of30,
30, 60,
and 120
120 mg/kg diet for 55 months beginning 11 month posthatching. When
examined at age 2 years, the combined groups contained 54% 54%females, 30% 30%
intersex, 10%
10%males, and 6% 6% sterile fish. al. noted the possibility
fish. Jalabert et al.
that self-fertilizing hermaphroditic
hermaphroditic trout could provide a tool for producing
highly homozygous strains of fish for breeding purposes.
Simpson (1976)
(1976)first reported the production of 100%
100%female trout by the
administration of estradiol to the rearing water at 250 pg/1 J.Lg/l for 2-hr periods
twice in both the eyed-egg and alevin stages, followed by oral administration
at 20 mg/kg diet for 30 or 56 days from first feeding. Treatment for 15 days
resulted in the production of 69% 69% females. The treatment
treatment was repeated by
Johnstone et al.al. (1978)
(1978) with similar results. In this study, one field experi
experi-
ment, involving the oral administration of 20 mg/kg for 30 days without
previous immersion, resulted in a 100% 100% female group indicating that the
treatment of eyed eggs and alevins was not essential. Oral treatment treatment for
shorter periods of time resulted in the production of small numbers of of in
in-
tersex and male fish. Some variability occurred between laboratory and field
trials. Treatment for 30 days in the laboratory resulted in the production of
89% females compared with 100%
89% 100%female groups achieved in a similar field
trial. Because the temperature regimes were similar, the discrepancy be-

Fish Physiology Ii (242-303)

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5

Studies involving experimental sex manipulation by hormones in fish

ence treatment success.

Within the Pisces, a wide range of reproductive strategies

Among the vertebrates the evolutionary tendency has been to aggregate

ence or absence of the whole Y or W chromosome determines

amphibia, sex steroids

in the swordtail Xiphophorus helleri even though it inhibited the binding of of

III. HORMONAL SEX CONTROL

Although there are numerous studies concerned

provided a means for examining basic sex-determining mechanisms. The

determination in the target species is bound to a heterosomal system, system, the

provided aa valuable guide for

Within this phase the appropriate hormone and method of application

90% Females produced

tioned. Further, methyltestosterone

a. Biological Activity. Although a wide range of natural and synthetic

inversion, the naturally occurring estradiol, estrone, and estriol required

As a result, investigators have relied heavily on a variety of of somatic and

was observed in some some individuals. Therefore, it is apparent that treatment at

observed that the 18-day

c. Environmental Influences. The regulatory mechanism which deter­

Central to the application of hormonal sex-control techniques to the

c. niloticus. Jalabert et ai.

biocellatum. Jalabert et al.

d. Salvelinus species. Wenstrom (1975)

e. Oncorhynchus kisutch. Coho salmon,

hr immersions in water containing methyltestosterone

ocean in May, 1980.

at 35 days posthatching at 8°C.

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c. Environmental Influences. The regulatory mechanism which deter