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
CONTROL AND ITS APPLICATION
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.
A. HUNTER AND EDWARD
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.
5. HORMONAL SEX CONTROL
HORMONAL SEX AND ITS
CONTROL AND ITS APPLICATION
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
Methyltestosterone 50
50 mglkg
mgkg diet 45
45 days
days 100%
100% Males
Males produced Wodiwode (1977)
(19m
T.macrochir
T. macmchir Methyltestosterone
Methyltestosterone 40
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
Methyltestosterone 50 mglkg
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
Methyltestosterone 30 mglkg
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)