GENETIC MANIPULATION IN FISH

SEX DETERMINATION AND SEX RATIO MANIPULATION

With few exceptions, individual broods of fish will contain both males and females.
 In different species, this may take the form of gonochoric (separate male and female sexes) or
hermaphroditism (ovarian and testicular tissues present in the same individual, either
consecutively or synchronously).
 For aquaculture or stocking, it may be desirable to use only one sex due to differences in
growth rate or characteristics concerned with maturity and reproduction.
 Sex ratio can be manipulated either directly (using steroid hormones to alter the sex ratio in fry
during sexual differentiation) or indirectly (manipulating the sex determination system of brood
stock so that selected fish produce monosex offspring).

1. SEX DETERMINING MECHANISMS

 Most cultured fish species are gonochoristic (i.e. salmonids, cyprinids, cichlids).
Gonochoric is separated into 2 types: differentiated and undifferentiated.
 In differentiated gonochorism, the indifferent gonad partially develops into an ovary-like
structure before developing into either ovary or testis.
 In undifferentiated gonochorism, the indifferent gonad develops directly into ovary or
testis. Most teleost are of the undifferentiated type, i.e. eel, rainbow trout, guppy.
 Although identifiable sex chromosomes are relatively are in fish, several species have
sex determining systems which operate in similar fashion to the XX female: XY male
chromosomal system found in humans and other mammals. Sex is determined by two
forms (X or Y) of a single genetic factor found in one chromosomal location.
 As the fish are diploid, each fish has two such chromosomes: the male is heterogametic
(XY; produces two types of gametes, X- or Y-bearing) while the female is homogametic
(XX; produces only one type of gamete, X-bearing).
 The 3rd possible genotype, YY, is not normally found in wild populations but may be
induced experimentally in some fish species.
 The heterogametic / homogametic system (WZ female/ZZ male) which is typical of birds
is also found in some fish species, i.e. Blue tilapia (O. aureus).
 Hermaphroditism is found in several species of cultured marine fish.
 Groupers are protogynous hermaphroditism (immature female X male).
 Serranidae is a protandrous or synchronous hermaphrodites.
 Sea bream is a protandrous hermaphrodite (immature male X female)

2. SEX REVERSAL
 A variety of natural and synthetic hormones have been used in sex reversal
experiments, i.e. methyltestosterone, estrogens.
 Dietary administration has been the most widely used method.
 Immersion treatments have allowed treatment of elevens before feeding commences,
which is most effective in salmonids.
 Silastic implants containing methyltestosterone have been used for species which will
not accept a prepared diet and can’t be treated successfully by immersion treatments at
an early age, i.e. grass carp, silver carp.
 Sex reversal treatments are normally carried out in small fish and any residues are
eliminated long before treated fish reach market size.
 The use of steroid hormones directly in the production of monosex or sterile fish for
consumption is controversial; it is permitted in some countries but not in others.
A. Sex reversal in tilapia
a) Hormonal sex reversal of tilapia fry is commercially used in many
countries, i.e. Thailand, Philippines.
b) There are two reasons for wanting monosex male fry for on growing:
1. Males are considerably bigger than females.
2. if mixed-sex populations are grown in farming environments,
reproduction will occur which will result in overstocking and
reduction in the harvest weight of the original fish.
B. Hormonal sex reversal in some fish species
 a) Using sex reversal and gynogenesis to investigate sex determination
and to manipulate sex ratio. Indirect monosex production relies on sex
determination system and sex reversal.
b) Monosex fish produced by indirect method is used in all-female rainbow
trout production in UK and all-female silver barb production in Thailand.
C. YY-male production in O. niloticus

CHROMOSOME MANIPULATION

A. Genome
 Genome refers to the chromosome complexity of a given individual. Genome
manipulations result in the alteration of the chromosome sets. extra set (3n, 4n,
polyploid);
 Replacement with a duplicate set of one and the same individual (gynogenesis,
androgenesis).
B. Spermatogenesis
 The cells in the germinal epithelium of the testis undergo certain changes leading to the
development of male gametes or sperm cells.
 Only the primodial germ cells or gonocytes emerge as the male gametes.
 A mature sperm cell consists of a haploid set of chromosomes.

C. Oogenesis
 The formation of ovum or egg.

D. Polyploidy
 Nearly all species of fish are diploid. During gamete formation (meiosis), the diploid
chromosomes first duplicate, exchange some genetic material by recombination
(crossing over) between chromatids, then separate by two equal divisions to give the
final haploid gamete (egg or sperm).
 The ovulated, unfertilized egg has to undergo the second meiotic division and results in
the loss of one haploid complement of egg chromosomes in the form of the 2nd polar
body.
 The remaining set (the female pronucleus) then fuses with the male pronucleus from the
sperm.
 Interference with the second meiotic division or extrusion of the 2nd polar body can
result in triploidy (2nd polar body is not loss and the two sets of maternal chromosomes
combine with the male pronucleus to give a triploid egg. In fish, the agents used for
inducing triploidy are usually physical: heat, cold or pressure shock depending on the
species.
 Triploid fish can be advantageous in a number of situations.
 The 3n females are effectively sterile in nearly all cases and so can be used where
suppression of reproduction is important.
 The 3n males do produce sperm but these will not give rise to viable fry if they fertilized
eggs.
 The loss of condition and growth rate observed in maturing diploids is not found in
female triploids.
 In some cases, poor survival rates in hybrid crosses can be significantly increased by
inducing triploidy in the hybrid.
 In Molluscs, the time scale of meiotic divisions differs from that of finfish.
 The unfertilized egg still has to go through both meiotic divisions when it is released.
 The possibilities for ploidy manipulation are thus wider.
 In molluscs the most widely used treatments for ploidy manipulation are chemical, with
cytochalasin B being the most common agent.
 It is also possible to interfere with the first mitosis in fish using the same physical agents.
 In mitosis, the chromosomes duplicate and then split equally: as the cell also divides this
process results in two daughter cells with identical chromosome complements to the
single parent cell.
 A pressure or temperature shock can be used to prevent the first cell division in a
normal diploid egg, resulting in duplication of the chromosome number (tetraploidy)
 D.1 Induced polyploidy
a) The enhancement of genome by the addition of one or more set(s) of
chromosomes to the normal diploid genome. 3n can be induced through the
retention of second polar body in the normally developing zygote by giving early
shock treatments. 4n can be induced by blocking the first cleavage or mitotic
division in the normally developing zygote by giving late shock treatments.

D.2 Natural polyploidy

b) Polyploidy occurs in nature in some fish species due to chromosomal
translocation, or hybrid between very distantly related species.
c) Polyploid common carp and trout (chromosomal translocation).
d) 3n hybrid between grass carp x bighead carp.

D.3 Tetraploidy
a) Tetraploidy has been induced in the rainbow trout by researchers in the USA and
Europe.
b) 4n rainbow trout are viable and fertile.
c) 4n fish produce eggs and sperm which are 2n, thus a cross between a 4n fish and
a 2n fish will produce 3n offspring without the need to use shocks to induce
triploidy.

D.4 The optimization of triploidy induction in finfish

a) Physical agents (heat, cold or pressure) rely on interfering with precise stages in
the fertilized egg to induce triploidy.
b) Four factors are crucial to achieving consistent results in different batches of eggs.
c) The incubation temp. of the eggs between fertilization and treatment must be held
constant to avoid fluctuations in developmental rate.
d) The time at which the treatment begins.
e) The duration of the treatment.
f) The intensity of the treatment.
g) Optimize shock treatments by fix the pre-treatment incubation temp. and vary the
combinations of other parameters.
h) Cold shocks seem to be preferred for cyprinids.
i) Heat shocks appear to be more efficient than cold shocks in tilapia species.
j) Pressure shocks have not been tested in a wide variety of species, but appear
promising where they have been used.
k) Suboptimal shocks frequently induce aneuploidy, a condition where the embryo
has a chromosome count intermediate between diploidy and triploidy, this is due to
incomplete retention of the set of chromosomes which would normally be lost as
the 2nd polar body.

E. Methods of ploidy identification

I. Karyotyping: Preparation of metaphase chromosome spreads and counting of
individual karyotypes.
A. Colchicine treatment and fixation of fish embryos
 Place the embryos in a solution of 0.002-0.005% colchicine in water for 4 -
6 hrs. Transfer the embryos to a chilled 0.7% NaCl solution.
 Cut off the tail region, and transfer to another petri dish containing distilled
water, leave in this solution for 8-12 min.
 Transfer the tissues from the distilled water to fixative (3 parts methanol: 1
part acetic acid).
 Leave in the fixative for 30 min. then replace the fixative with fresh fixative.
Use 2-4 ml fixative for 10-12 small embryos. Store up to 6 weeks at 4C.
B. Colchicine treatment and fixation of fish
 Place small fish to swim in a 0.01-0.1% colchicine solution for 6-7 hrs.
 Larger fish should be injected intraperitoneally with 25-100 g colchicine per
gram of body weight.
 Kill the fish and dissect out the gill arches or scales or fin edges from fish,
cutting into small (0.5 cm) pieces.
 Place the tissues individually in 10 times their volume of 0.4% KCl solution
for 20-30 min. Fix in at least two changes of fixative (3 parts ethanol: 1 part
 acetic acid) for 30 min. each. Fixed tissues can be stored for at least 1
month at 4C.
C. Slide preparation
 Remove individual embryos or small pieces of tissue from the fixative with
fine forceps.
 Blot off fixatives with tissue and place the embryos in 2-3 drops of 50%
acetic acid in well slides.
 Using a glass rod with a flat, ground end, grind the tissue for 1 min. Wash
off with 2 drops of 50% acetic acid.
 Leave for 10 min. Place glass slides on a hotplate at 45C.
 Spread cell suspension onto a warmed glass slide by using a capillary
tube and dropped onto the slide from a height of 40 cm.
 Leave for 10 seconds and then remove from the hotplate and leave to dry.
D. Slide staining
 Stain slides in 4-10% Giemsa stain (diluted in 0.01M phosphate buffer pH
7.0) for 20 min.
 Wash slides in distilled water to remove excess stain.
 Leave to dry. Place slides in xylene for 10 min.
 Remove and leave to dry.
 Mount slides permanently using cover slips and DPX or similar mountant.
 Scan the mounted slides under x100 magnification.

OREOCHROMIS KARYOTYPES
A. 2n and 3n O. niloticus karyotypes
B. 2n and 4n O. niloticus karyotypes

II. Blood cell measurements

 Remove a small blood sample and place 1 drop on a glass slide. Smear the drop
along the slide using the edge of another slide.
 Leave to dry.
 Stain the slides in Wright’s blood stain for 2 min. Transfer to a 1:1 mixture of
Wright’s stain: Sorensen’s buffer for 3 min., then rinse in distilled water.
 Leave to dry. Mount using cover slips and DPX or similar.
 Using a graticule to measure the major (length) and minor (width) axes of the
nuclei of at least 20 cells per slide.
 Calculate the nucleus volume: Nucleus volume = 4/3 ab2 (a=major axis/2;
b=minor axis/2)

III. Quantization of the DNA content using Flow Cytometry

Determine the ploidy status by Flow Cytometry with DAPI staining.

THE PROPERTIES OF TRIPLOID FISH

 Increase in cell size of 3n fish. Most juvenile 3n fish are not significantly different in size from
control 2n sibs.
 Reduced gametogenesis in 3n fish, much more marked in 3n females than 3n males.
 Mature 3n male fish generally have slightly or significantly reduced gonadosomatic indices
(GSI’s) but do produce sperm.
 Such sperm are aneuploid, variable in sperm head size and frequently abnormal.
 Eggs from a 2n female fertilized with sperm from a 3n male will give rise to aneuploid, inviable
embryos which will die early in development.
 3n male salmonids are able to “home” to the site of their birth, with a rate of return equal to
that of 2n. 3n male tilapia can spawn with 2n females. 3n females generally have very small
ovaries with only a few immature oocytes.
 3n fish exhibit good growth and condition over the spawning period comparing with 2n fish.

APPLICATIONS OF TRIPLOIDY
A. Rainbow trout farming
a) A small proportion of rainbow trout production in Scotland consists of all female triploids.
b) The majority of rainbow trout are “portion-sized” fish for consumption: triploids tend to be
used where larger fish are grown on past first maturity for consumption or angling.
 c) In other countries with higher proportions of sea cages, the percentage of all-female
triploids is correspondingly higher.

B. Atlantic salmon farming

a) All-female culture can reduce the percentage of fish maturing early (males tend to
mature earlier than females).
b) Triploidy can be used to completely suppress maturation in all-female stocks.
c) Pressure shocks appear to give a more reliable triploid yield than heat shocks and
commercial – scale pressure vessels capable of treating 2 litres of eggs (50,000 eggs
per hour) are in use, giving 90% triploid yield relative to controls.

C. Grass carp
a) Induced triploidy (cold shock) and monosex female stocking of grass carp into the USA
as a weed control agent offer ways of introducing this species with minimal risk of
reproduction.

D. Triploid hybrids
a) In salmonids, triploid hybrids have often been shown to survive better during early
development than diploid hybrids.
b) An intergeneric triploid hybrid, the “tiger” trout (female brown trout and male brook trout,
and triploidization using heat shock) has increased the survival ration from a few
percent to about 50% of the brown trout.
c) The hybrids (2n and 3n) are sterile.

GYNOGENESIS
Gynogenesis is a technique to produce diploid fish in which both sets of chromosomes are of
maternal origin. Restoration of diploidy in 1n zygotes may occur spontaneously or by chemical,
thermal, pressure shock treatments.

A. Natural gynogenesis: Gynogenesis occurs in nature in some fish species.

 Family Poecilidae i.e. Poecilia formosa.
 Family Cyprinidae i.e. Carassius auratus gibelio.
 Family Pleuronectidae.

B. Induced gynogenesis
 Gynogenesis can be artificially induced by eliminating/denaturing the genetic material
(DNA) of the sperm through irradiation either by UV or Gamma rays and activating the
eggs with the irradiated milt.
 Diploidy is restored by giving either thermal or hydrostatic pressure shock treatments.
Effective tool for producing inbred lines of fish in a much shorter time (1 generation in
mitotic gynogens, or 4-5 generations in meiotic gynogens, while 10-12 generations via
sib-mating). A tool for producing all-females where females are homogamety.
 Gynogenesis has been used in the study of sex determination and gene recombination
and as a tool for inbreeding and the production of clonal lines.

C. The process of gynogenesis has 2 stages:

 The fertilizing sperm will make no genetic contribution to the embryo, which will result
in a haploid embryo.
 Diploidization of haploid gynogenetic egg.
 If egg is shocked at the 2nd meiotic division, it will produce a “meiotic” gynogenetic, or
“Meiogyne”. If egg is shocked at the 1st cell division, it will produce a “mitotic”
gynogenetic, or “mitogyne”.
1. Meiotic gynogenesis
 Induced by administering early shock treatment to the activated egg with
UV-irradiated milt and retaining the second polar body.
 The meiotic gynogens may be heterozygous or homozygous.
2. Mitotic gynogenesis
 Induced by administering late shock treatment to the activated eggs
through the blocking of the first cleavage or the first mitotic division in the
developing zygote.
 The mitotic gynogens are homozygous.
THE ELIMINATION OF THE PATERNAL GENOME

Treating sperm before fertilization with agents such as ultraviolet light (UV), gamma rays or
chemical mutagens. UV is generally the preferred treatment: although gamma rays are more
penetrative into sperm solutions, they can result in fragments of paternal chromosomes which pass
into the embryo, while chemical agents may be persistent as well as producing chromosome
fragments.

APPROACH TO OPTIMIZE SPERM IRRADIATION WITH UV

Identification of a saline which can reversibly immobilize sperm (sperm are immotile when
diluted with the saline, but reactivate with water). Standardization of sperm concentration for
irradiation. The concentration should be relatively low, but still adequate to give maximal fertilization
rates.
Nile tilapia: 107-108 sperm/ml
Barb: 8x108 sperm/ml

UV IRRADIATION AND ASSESSMENT OF ITS EFFECTS.

A short wave (254 nm) single UV source (lamp or tube) is normally adequate for irradiation of
small volumes in petri dishes. The surface area should be a few millimeters deep. Agitation of the
petri dish during irradiation will ensure even irradiation.
Survival rates of haploid gynogens generally follow a pattern known as the pseudo – Hertwig
curve, where an initial decline at low doses is followed by an increase to a peak and then a decline to
zero. The peak represents the optimum UV dose for the conditions used and all embryos produced at
this dose should be haploid.

THE RESTORATION OF DIPLOIDY IN GYNOGENESIS

Meiotic gynogenetics are produced using the same shocks as are used for triploidy: the only
difference between the two techniques is the irradiation of the sperm in gynogenesis. Survival rates
are generally lower than for triploidy due largely to increase homozygosity.
High variation in survival rate is observed between species, between females and sometimes
between batches of eggs from the same female.
Yields of mitotic gynogenetics are generally very low: inbreeding is complete (the two haploid
sets of chromosomes are identical as they came from mitosis) and optimization of the shock
parameters is more difficult due to the low survival rates and the wide range of shock application
times.

EFFECTS OF GYNOGENESIS
 The primary effect of gynogeensis is INBREEDING.
 For MEIOGYNES, inbreeding is partially due to recombination in meiosis.
 After several generations of meiotic gynogeensis, loci at the ends of chromosome arms will still
be at least partly heterozygous, while loci near the centromeres will rapidly become
homozygous.
 Meiotic gynogenesis will not give highly inbred lines if used alone.
 MITOTIC GENESIS gives completely inbred individuals in one generation dur to the
combination of two identical chromosome sets which arise from duplication of the maternal
genome in the first mitosis.
 A further generation of gynogeensis from a female mitogyne would form the basis for a clonal
line.
 The FERTILITY OF FEMALE MITOGYNES is markedly reduced in some species, which may
make this process extremely difficult.
 MALE MITOGYNES are not so strongly affected and most are fertile.
 SEX RATIO is affected by gynogenesis. Inheritance of sex-determination genes from only the
female parent has effects on the sex ratio in many cases.
 Gynogenesis has become an important tool in the study of sex determination systems in fish.
 INBREEDING AND CROSSING between inbred lines is widely utilized in crop plants.
 The INBRED LINES are poor performers and may be difficult to maintain.
 The F1 HYBRID LINES (crosses between the inbred lines) are favored because they are
genetically uniform and also fairly heterozygous.
 This tends to give very uniform characteristics in the crop, making harvesting and processing
easier.