Seminar

1.0 INTRODUCTION

Demand for fish is soaring worldwide and it appears unlikely that the increasing demand can be

met through increased natural harvest. There is international recognition that many of natural

oceans and freshwater fisheries are being harvested to their limit (Lakra and Ayyappan, 2003).

Currently, the quantity of animal protein harvested from global aquatic sources via the capture

of natural fish populations is at maximum sustainable yield. Many major fish stocks are

showing precipitous declines in productivity due to overfishing and further increases are not

anticipated under the current global conditions and environment (Dunham et al., 2001). Wild

fish stocks have been heavily fished or overfished, which has resulted in a noticeable levelling

of fish landings at around 60 million tonnes, with harvest from oceans unlikely to expand

(Hardy, 1999). From a global perspective, the total supply of fish food (excluding China) has

been growing at a rate of 2.4% since 1961 while the human population has been growing at

1.8% p.a. (NEPAD, 2005). The Food and Agriculture Organization (FAO) predicts a 36%

increase in the world population, with only a 30% increase in production from aquaculture and

fisheries (Dunham, 2004). Hardy (1999) predicts a 55 million tons shortage in demanded

seafood products by 2025 resulting from levelled wild catch and increasing demand.

Thus aquaculture remains the last hope for providing enough fish for the world, being the

cheapest source of animal protein (Ayoola and Idowu, 2008). Aquaculture can help to meet the

increasing demand, and biotechnology can make a great contribution to improve aquaculture

yields (Lakra and Ayyappan, 2003). By 2025, aquaculture will have to increase by 350% to

cover the impending shortage (Hardy, 1999). With increased demand for aquacultured foods has

come a need for more efficient production systems (Dunham, 2004). The development of

improved fish seed stocks that can contribute to increased fish production is seen as one of the

key solutions to meeting the future food demands of the growing world population (Hammed et

al., 2010).

[1]

Biotechnology is one tool that holds much promise towards addressing these aquacultural

problems. Biotechnology has opened a new window for development of genetic resources in

aquaculture. Genetic technologies can be utilized in aquaculture for a variety of reasons, not just

to improve production but also marketability, culturability and the conservation of natural

resources (Moses et al., 2005). Genetics can greatly contribute to production efficiency,

enhancing production and increasing sustainability. Resource utilization can be greatly

improved and impediments to sustainability, such as slow growth of fish, inefficient feed

conversion, heavy mortality from disease and the associated use of chemicals, loss of fish from

low oxygen levels, inefficient harvest, poor reproduction, can all be diminished by utilizing

genetically improved fish (Dunham, 2004). Understanding and manipulating the molecular and

hormonal aspects of reproduction, development and growth, together with gene cloning and

transfer technologies, will result in full control of those processes, leading to optimal

performances and improved phenotypes (Zohar, 2008).

Since the early 1980s, research in aquaculture and fisheries genetic biotechnology has steadily

grown, and now research in this area is extremely active. Cultured fish are being improved for a

multitude of traits, including growth rate, feed conversion efficiency, disease resistance,

tolerance of low water quality, cold tolerance, body shape, dress-out percentage, carcass quality,

fish quality, fertility and reproduction and harvestability (Dunham, 2004). The main vision of

aquaculture biotechnology is to achieve improvements of aquacultural stock, preservation of

genetic resources, disease diagnosis, and control of microbial/microalgal genetic engineering

(Nwokwa, 2012).

[2]

2.0 LITERATURE REVIEW

The use of modern biotechnology to enhance production of aquatic species holds great potential

not only to meet demand but also to improve aquaculture. Genetic modification and

biotechnology also holds tremendous potential to improve the quality and quantity of fish reared

in aquaculture (Ayoola and Idowu, 2008). Biotechnology has the potential to enhance

reproduction and the early developmental success of culture organism (Food and Agriculture

Organization, 2000).

The several areas of aquaculture where biotechnology can be applied include production of high

quality fish seedling. The potential areas of biotechnology in aquaculture include the use of

synthetic hormones in induced breeding, production of monosex, uniparental and polyploidy

population, molecular biology, transgenic fish, gene banking, improved feeds and health

management and development of natural products from marine organisms (Pandian and

Koteeswaran, 1998).

2.1 ARTIFICIAL PROPAGATION

Availability of quality fish seed of the candidate species in adequate quantity is one of the most

important factors for a sustainable and profitable fish farming, which involves a number of

management practices in the maintenance of quality brood fish in adequate number. However,

for the majority of species, spawning and seed supply is seasonal, thus multiple spawning

species are often preferred for successful aquaculture (Mair, 2002). Artificial propagation

methods constitute a major practicable means of providing enough quality seed for rearing in

confined enclosure such a fish ponds, reservoirs and lakes (Charo and Oirere, 2000). Fish

culture today is hardly possible without the artificial propagation of fish seeds of preferred

cultivable fish species. Apart from being able to obtain quality seed, the artificial propagation

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technique can also be used to develop strains superior to their ancestors by the methods of

selective breeding and hybridization (Akankali et al., 2011).

The most successful method of artificial reproduction in catfish is by induced breeding through

hormone treatment followed by artificial fertilization and incubation of fertilized eggs and the

subsequent rearing to fingerlings (Ndimele and Owodeinde, 2012). Hormonal stimulation

allows year-round production of gametes and fry of economically valuable species.

Domestication of species for aquaculture is necessary and the number of domesticated aquatic

species is still rising rapidly. Hormone therapy is applied to improve and control of reproductive

cycles during the domestication (Muhammet et al., 2013).

Gonadotropin releasing hormone (GnRH) is the most used now in the induced breeding of fish

and marked commercially throughout the world (Alok et al., 2000). The induced breeding of

fish is now successfully achieved by the development of GnRH technology (Lakran and

Ayyappan, 2003). GnRH is the key regulator and central initiator of reproductive cascade in all

vertebrates (Bhattacharya et al., 2002). It is a decapeptide and was first isolated from pig and

sheep hypothalami with the ability to induce pituitary release of luteinising hormone (LH) and

follicle stimulating hormone (FSH) (Schally et al., 1973). Salmon GnRH is now profusely used

in fish breeding and marked commercially under the name of “Ovaprim” throughout the world.

In fact, most of the economically important culturable fish in land locked water do not breed

until the hormone induces them. Ovaprim is administered intramuscularly at 0.5ml per kg of

fish (Ndimele and Owodeinde, 2012).

2.2 HYBRIDIZATION

Enhanced growth rate is the most desirable trait for stock improvement in aquaculture.

Increased heterozygosity from hybridization has resulted in improved growth and other

desirable characters in a variety of species such as developmental compatibility, food

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conversion efficiency, and oxygen metabolism (Danzmann et al., 1985). Hybridization attempts

to produce fish that combines valuables traits from more than one species or high heterosis

(hybrid vigour) (Aluko, 1993). Hybridization is aimed to evolve a hybrid or strain of superior

quality than the parent species. A hybrid between white bass (Morone chrysops) and the striped

bass (Morone saxatilis) is called sunshine bass; it exhibits faster growth and has many good

culture characteristics than either parent under captive culture system (Smith, 1988). Crosses of

the black crappie x white crappie (Pomoxis nigromaculatus x Pomoxis annularis) stocked in

small ponds and impoundments were reported to grow faster (positive heterosis) than

conspecific parents (Hooe et al., 1994).

Plate 1: Hybrid sunfish which grows faster than either parent

Hybridization can also be used to produce single sex groups of fish when the sex determining

mechanisms in the parental lines are different. An example is the hybridization of Nile tilapia,

Oreochromis niloticus and the blue tilapia, Oreochromis aureus produces an all-male progeny

which controls unwanted reproduction (Ayoola and Idowu, 2008). The hybrid between striped

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bass (Morone saxatilis) and yellow bass (Morone mississipiensis) produced 100% females with

excellent survival and growth (Wolters and DeMay, 1996).

Many inter-specific hybridizations result in sterility thus functioning as reproductive isolating

mechanism to prevent the permanent mixing of genes from two species. The more distantly

related the two species, the greater the 1ikelihood their hybrid being sub-viable or sterile

(Aluko, 1993). Production of sterile animals may be advantageous to diminish unwanted

reproduction or to improve growth rate and avoiding energy loss due to prolific breeding. The

tiger trout, a hybrid between brown trout (Salmo trutta) and brook trout (Salvelinus fontinalis) is

sterile with good growth rate and therefore is useful for stocking areas where reproduction is to

be very limited (Rahman et al., 2013).

Table 1: Hybridization to produce monosex population in Tilapia

Crosses (female × male) Results

Oreochromis niloticus ×Oreochromis variabilis

Oreochromis nigra × Oreochromis urolepis hornorum

Oreochromis vulcani × Oreochromis urolepis hornorum

Oreochromis vulcani × Oreochromis aureus

98-100% male progeny

Oreochromis niloticus × Oreochromis urolepis hornorum

Oreochromis niloticus × Oreochromis aureus

100% male progeny

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Source: Fuentas-Silva et al. (2013).

2.2.1 GENETIC MECHANISM OF HYBRIDIZATION

The genetic explanation of hybridization particularly in tilapia is the multiple sex determining

mechanism. Chen (1969) has reported that both XY and WZ sex determining mechanisms exist.

Males are the heterogametic sex XY and females the homogametic sex XX for species such as

Oreochromis niloticus and Oreochromis mossambicus (Wohlfarth and Hulata, 1983). Females

are the heterogametic sex WZ and males the homogametic sex ZZ in Oreochromis aureus and

Tilapia hornorum. When homogametic males of say Oreochromis wawa (ZZ) are hybridized

with homogametic males XX it produces an all-male progeny (ZW). When heterogametic males

XY are hybridized with heterogametic females WZ, four combinations of sex chromosomes,

XW, XZ, YW and YZ and produced in equal proportion. The maleness chromosome from

either species is dominant to the femaleness chromosome, so when heterogametic males are

hybridized with heterogametic females, 75% of the progeny are males. Only the XW genotype

results in females (Aluko, 1993).

2.2.2 USEFULNESS OF HYBRIDIZATION

Elimination of unwanted reproduction in ponds by production of single sex or sterile

populations (Ayoola and Idowu, 2008).

Heterosis for growth rate, ability of hybrids to be more harvestable than their parent

species (Aluko, 1993).

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Hybridization is not only a preferred method of genetic improvement but also a potential

tool for stock improvement through transmitting desirable traits in inferior parents

(Rahman et al., 2013).

2.3 MONOSEX CULTURE

Monosex populations of fish are desirable in aquaculture for a variety of reasons. Some species

of fish mature at small sizes and young ages prior to the desired time of harvest. This can

decrease production because unwanted reproduction results in crowding of the fish and higher

densities than intended in the culture pond as well as wasted energy from the sexual activity of

the stocked fish (Dunham, 2004). This is a major problem particularly in tilapia which can

decrease production because precocious maturity always results in overcrowding and stunted

growth in the culture pond.

In aquaculture, one sex is often more desirable for the market than the other due to sexual

dimorphism. Sexual dimorphism for growth occurs in most cultured fishes for flesh quality and

carcass yield (Aluko, 1993). Stone (1981) demonstrated that male Oreochromis niloticus grows

2.5 and 2.2 times faster than females when grown in cages mixed and separately respectively.

All-male progeny would be beneficial for catfish culture since they grow 10–30% faster than

females, depending upon strain of catfish (Smitherman and Dunham, 1985). All-female

populations are desirable in salmonids because of the more rapid growth of females, the early

sexual maturity and associated slow growth of males and the poor flesh quality of males,

especially early maturing males (Hulata, 2001). Faster growth of one sex is presumably a result

of a combination of genetic and hormonal factors, although competition and suppression of one

sex and magnification of initial size differences are alternative explanations for sexually

dimorphic growth. Thus, monosex culture of the faster growing sex can increase production.

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Plate 2: Sexual dimorphism for size in Nile tilapia, Oreochromis niloticus. The male

is the larger individual (Dunham, 2004).

2.3.1 METHODS OF MONOSEX CULTURE

Monosex culture can be achieved by:

Manual sexing

Mechanical separation

Sterilization

Hormonal sex reversal

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2.3.1.1 MANUAL SEXING

Some species of fish can be easily sorted into males and females. The colours can be

sufficiently differentiated to serve as reliable sex indicators; the structure of the anal papilla may

be used; the opening of the oviduct is distinguishable in the female and is not present in the

male. Turning the fish over, it is possible to look at the secondary sex organs (shaped like a

small cone) located behind the anus. For example, male tilapia has a simple papilla, while the

female have a slightly wider organ with a wide opening to allow eggs to eject during mating

(Fuentes-Silva et al., 2013).

Plate 3: Sexual dimorphism in Tilapia, male (left) and female (right).

Manual separation of the sexes requires the least amount of technology for monosex culture, but

it is extremely wasteful, tedious and inefficient (Lovshin and Da Silva, 1976). While it is an

easy technique, it is extremely laborious and human accuracy varies from 80 to 90%, which

leads to the presence of females in the pond. Additionally, it is stressful for the fish (Penmann

and McAndrew, 2000). Also, the fingerlings must be grown to a size large enough to visually

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determine sex before manual separation, resulting in half the fingerling production being wasted

since the slower-growing sex is culled unless the slower-growing sex is utilized in separate

ponds, when prevention of reproduction is the primary consideration (Popma, 1987). Moreover,

this technique may be useful in small populations, but in commercial practice their use increases

the cost of skilled labour and increases the risk of human error, leading to uncontrolled

reproduction (Fuentes-Silva et al., 2013). Therefore, this method is rarely used (Penmann and

McAndrew, 2000).

2.3.1.2 MECHANICAL SEPARATION

An alternative method to hand-sexing is mechanical separation by size grading, which can

separate most of the fish by sex. Some of the same disadvantages exist, as half the initial

fingerling production is wasted. If elimination of reproduction in early-maturing species in

production ponds is the primary goal, grading will probably result in too many mistakes to

adequately separate the sexes to eliminate reproduction.

2.3.1.3 STERILIZATION

Sterilization is an alternative to prevent unwanted reproduction in ponds. Sterilization might

also promote growth, change the behaviour of the fish, alter body composition or improve

carcass yield. Sterilization can potentially be accomplished through surgery, immunology or

with chemicals.

Surgical sterilization

Donaldson and Hunter (1982) indicated that surgical sterilization was successful in

salmonids. However, Laird et al. (1978) reported that the problems associated with this method

include labour and the necessity of complete testicular removal to prevent development of

secondary sexual characteristics. Surgical sterilization has not been successful in fish and does

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not allow permanent sterilization because fish have the ability to regenerate gonadal tissue. Both

testes and ovaries of grass carp fully regenerate and function even after the surgery is conducted

on sexually mature fish (Underwood et al., 1986). Testicular regeneration occurs in both adult

blue catfish and channel catfish, although it is slow and may take several years for complete

regeneration. Ictaluriid catfish with regenerated testes produce viable sperm (Bart, 1994).

The obvious disadvantages of surgical sterilization include large labour expenditures and the

possibility of mortality, which make it impractical on a large scale. Although tilapia, grass carp,

salmonids and catfish all have high survival after gonadectomy (Bart, 1988; Bart and Dunham,

1990), the labour expenditure does not allow the use of this technique for large-scale

commercial production. Surgical sterilization might be feasible for applications when only a

few, valuable fish need sterilization (Dunham, 2004).

Chemical sterilization

Chemosterilization has shown some potential, although, as with the other sterilization

procedures, the effects have been temporary (Stanley, 1979). Hoar et al. (1967) found that,

within 1 week after cessation of methallibure treatment, spermatogenesis resumed. Yamazaki

(1976) has reported that overdoses of hormones have led to sterilization in fish.

Chemosterilization can be done by the use of chemicals such as methallibure and cyproterone

acetate. These chemosterilants cause delayed spawning, reduced mating and total fry production

(Hanson and Manion, 1978).

Methallibure may act by blocking production or release of gonadotrophins at one or more sites

(Malven et al., 1971). This compound acts as a chemical hypophysectomising agent by

interrupting the production and release of gonadotrophins (Pandey and Leatherland, 1970).

Methallibure has prevented spermatogenesis and testicular steroidogenesis in sea perch,

Cymatogaster aggregata (Wiebe, 1968), blocked gonadal differentiation and suppressed

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spermatogenesis in guppies, Poecilia reticulata (Pandey and Leatherland, 1970). This drug also

reduced gonad weight and maturation of gonads in both sexes of goldfish, sea perch and

stickleback, Gasterosteus aculeatus (Hoar et al., 1967).

2.4 SEX REVERSAL

The production of single sex groups of fish can be accomplished by manipulation of the

developing gametes and embryo (Food and Agriculture Organization, 2014). Monosex

populations can be produced by direct hormonal sex reversal. The use of sex control techniques

to influence characteristics of economically desirable teleost species is becoming an important

management tool to increase aquaculture production (Lakran and Ayyappan, 2003). Sex

reversal is a method of controlling reproduction in fish and ultimately improving the rate of

growth.

Hormonal or endocrine control involves the treatment of fish with sex steroids during the early

phase before sex differentiation starts. The process of sex differentiation in teleost is protracted

and labile rendering the hormonal induction of sex reversal possible in gonochoristic and

hermaphroditic species (Lakran and Ayyappan, 2003). The principle behind this method lies on

the fact that at the stage when the fish larvae are said to be sexually undifferentiated (right after

hatching up to about 2 weeks or up to the swim-up stage), the extent of the androgen (male

hormone) and the oestrogen (female hormone) pre-sent in a fish is equal (Fuentes-Silva et al.,

2013). Although the male or female genotype is established at fertilization, phenotypic sex

determination occurs later in development. The artificial elevation of the appropriate sex

hormone is sufficient to overcome the natural hormone or gene product during the period of

sexual differentiation and to dictate the sex of the individual (Dunham, 2004).

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2.4.1 METHODS OF SEX REVERSAL

Sex reversal can be accomplished by administering the exogenous hormones in various ways

which include:

By bath: Coho salmon have been sex-reversed to femaleness by bathing the

embryos in 25 μg/l of 17β-oestradiol, followed by oral administration of 10 mg/kg

17β-oestradiol to the fry (Goetz et al., 1979). López et al. (2007) also obtained

92.6% of males using 1.8 mg methyl testosterone/L, with an immersion of 4 hours,

between 10 and 14 days after fertilization.

In feed: In the majority of species, sex reversal has been accomplished by feeding

the young fish hormone-treated feed (Shelton et al., 1978). Tilapia have been sex-

reversed to all-maleness by feeding 10–60 mg 17α-methyltestosterone/kg feed for

21–35 days post swim-up (Popma, 1987).

Through implants: Grass carp were sex-reversed to all-maleness with silastic

implants of 17-methyltestosterone in the abdominal cavity (Boney et al., 1984). Sex

reversal was accomplished when the implants are placed in the fish at 85mm body

length and the hormone is released until the fish reach about 200mm. The

androgenic hormone was released from the implant over a 60-day interval

encompassing the period of sex determination for grass carp (Shelton, 1986).

2.4.2 TYPES OF HORMONES

Different steroids have been used over the years to induce sex reversal even if 17α-

methyltestosterone is the most common (Pandian and Varadaraj, 1990) for Oreochromis

mossambicus; 17α-ethynyltestosterone (Shelton et al., 1981), trenbolone acetate (Galvez et al.,

1996), and mibolerone (Torrans et al., 1988) with Oreochromis aureus; and fluoxymesterone

with Oreochromis niloticus (Phelps et al., 1992).

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2.4.3 SEX REVERSAL FROM FEMALES TO MALES

Sex reversal to all-maleness in tilapia is now a routine throughout most of the world in both

industrialized and developing countries. Feeding swim-up fry 10-60mg 17α-

methyltestosterone/kg feed for 21–35 days results in populations with 95–100% males

(Muhaya, 1985). Cagauan et al. (2004) evaluated sex reversal of Nile Tilapia Oreochromis

niloticus by immersing the eggs in different concentrations of 17α-methyl testosterone. Highest

percent male of 91% was attained at 800 μg/L hormone concentration at 96-h immersion time

com-parable with the 88 to 89% in 400 to 600 μg/L hormone concentration at the same

immersion time.

2.4.5 SEX REVERSAL FROM MALES TO FEMALES

Several oestrogenic compounds have been used to produce monosex female populations. The

hormone 3-oestradiol has been one of the most efficacious compounds, and oestrone and

ethynyloestradiol can also be used for feminization (Yamazaki, 1983). Coho salmon have been

sex-reversed to femaleness by bathing the embryos in 25 μg/l of 17β-oestradiol, followed by

oral administration of 10 mg/kg 17β-oestradiol to the fry (Goetz et al., 1979). Immersion of

alevin, Oncorhynchus masou, with 0.5–5 mg/l of 17β-oestradiol produced 100% females

(Nakamura, 1981).

2.4.5 Y-Y SUPERMALE TILAPIA

The genetic males, that are physically female, can be mated with normal males to produce a

group of all-male tilapia that grow faster and have less unwanted matings than a group of

mixed-sex tilapia (Dunham, 2004). The all-male offspring of this process have two male

chromosomes and these fish can be used in turn as brood stock for subsequent generations. A

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big advantage of this technique is that all-male populations can be produced in those subsequent

generations without the use of any hormones (Food and Agriculture Organization, 2014).

Fry

↓

Hormonal feminisation

↓

XY phenotypic females

↓

Eggs

↓

Activation with irradiated sperm

↓

Heat shock to suppress meiosis II

↓

Gynogens: 50% XX females and 50% YY males

↓

Cull all females, remaining fish are YY supermales

Figure 1. Method for the production of Y-Y supermale tilapia.

2.4.6 DETERMINING FACTORS

Several factors influence the effectiveness of sex reversal in fish, including

Species of fish: Grass carp are difficult to train to accept artificial feed, limiting options

to accomplish sex reversal. However, sex reversal of grass carp from genetic females to

phenotypic males is effective utilizing silastic implants of methyl testosterone (Boney et

al., 1984).

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Genetics: All attempts to produce all-male populations of channel catfish with

androgens failed. In fact, administration of testosterone to channel catfish fry results in

populations skewed towards femaleness. Apparently, biofeedback systems of channel

catfish react to the elevated levels of androgen by digesting and converting the excess

androgen to oestrogen-like compounds, ultimately elevating oestrogen levels and sex-

reversing genetic males to phenotypic females (Davis, et al., 2000).

Type and dosage of hormone: Sex reversal by oral administration of feed incorporated

with methyl testosterone is probably the most effective and practical method for the

production of all male Tilapia (Fuentes-Silva et al., 2013). Sex reversal by immersion of

Oreochromis niloticus eggs in 17 α-methyl testosterone (MT) gave a 91%

masculinization in 800 μg/l at 96-hr IT and about 88-89% masculinization in 400 and

600 μg/l at 96-hr IT (Cagauan et al., 2004).

Duration of treatment and timing of treatment: Timing and duration of hormone

treatment are extremely important. Popma (1987) learned that tilapia fry as large as 14–

15mm can be effectively sex-reversed, and that the duration of the treatment was also

important, not in terms of absolute time but in terms of the growth of the fry. Treatments

of 28 days duration yielded higher percentages of males than 21-day treatments. A large

percentage of females was found in populations where some of the fry were 12mm or

less when treatment was terminated; however, most of these females were sex-reversed

if the treatment was not terminated until all the fry reached 13–14mm. Gale et al. (1996)

demonstrated that fry immersion for just three hours in 17α-methyldihydrotestosterone

on two days resulted in masculinisation of Nile Tilapia with a success rate of 93%.

2.5 POLYPLOIDY

Chromosome sex manipulation techniques to induce polyploidy (triploidy and tetraploidy) have

been applied extensively in cultured fish species (Pandian and Koteeswaran, 1998). These

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techniques are important in the improvement of fish breeding as they provide a rapid approach

for gonadal sterilization, sex control, improvement of hybrid viability and clonation (Lakra and

Ayyappan, 2003). By manipulating ploidy, sterile, unisex or highly homozygous cohorts of

animals can be produced (Beaumont et al., 2010).

The polyploid state refers to individuals with extra sets of chromosomes. The normal and most

common chromosome complement is two sets (diploid). Triploidy refers to individuals with

three sets of chromosomes and tetraploidy refers to individuals with four sets. Hexaploids have

six sets, and aneuploids have at least a diploid set with one or more additional chromosomes,

but not a full complement, to the set.

A potential benefit of polyploidy is positive changes in carcass traits (Dunham, 1990). Reduced

gonadal development leads to less waste in processing. The flesh quality of triploid rainbow

trout females is improved relative to diploid females because post-maturational changes are

prevented (Bye and Lincoln, 1986).

2.5.1 TRIPLOIDY

Induced triploidy is widely accepted as the most effective method for producing sterile fish for

aquaculture and fisheries management (Lakran and Ayyappan, 2003). Inducing triploidy is the

only practical means in which to sterilize large numbers of fish without using of potentially

harmful chemicals or radiation (Benfey, 1989). In this method, the main aim is to produce

sterile fish by using normal spermatozoa. It is through the triploidization technique that

sterilization can be achieved by administration of an environment shock shortly after post

fertilization (Kizak et al., 2013). The sterility of triploids would be desirable for species such as

tilapia, where excess reproduction may occur in production ponds (Lakra and Ayyappan, 2003).

Therefore, degradations due to sexual maturation are overcome by triploidy technique (Piferrer

et al., 2009).

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Culture of triploid fish can be advantageous for several reasons. The potential of increased

growth, increased carcass yield, increased survival and increased flesh quality are the main

culture advantages. At the onset of sexual maturity, reduced or inhibited gonadal development

may allow energy normally used in reproductive processes to be used for growth of somatic

tissue (Thorgaard and Gall, 1979). Triploids would reach a larger size than diploids because of

their larger cell size (Dunham, 2004). Taniguchi et al. (1986) have reported increased growth

rate in triploid fish compared to their normal diploid siblings. This increased growth rate can be

a result of lack of sexual development since the growth rate of fish slows as they approach

sexual maturity or increased cell size.

Triploid induction can also allow production of otherwise non-viable or sub-viable diploid

hybrids probably because of the presence of a balanced maternal chromosome set in triploids

that is not present in diploid hybrids. Diploid Oreochromis niloticus female × Tilapia rendalli

male hybrid embryos experience near 100% mortality. However, this hybrid combination is

viable when triploidy is induced (Chourrout and Itskovich, 1983). Several salmonid hybrids are

non-viable in the diploid state but are viable when triploidy is induced. This has allowed

production of otherwise unviable rainbow trout × coho salmon triploid hybrids with increased

resistance to infectious haematopoietic necrosis virus (Parsons et al., 1986) and osmoregulatory

ability. Survival of the tiger trout, a brown trout female crossed with a brook trout male, is

raised from 5 to 34% when triploidy is induced (Scheerer et al., 1986).

2.5.1.1 ADVANTAGES OF TRIPLOIDY

They are all sterile and are useful for stocking natural water bodies where population

control is desired.

They grow faster at and after sexual maturity.

Triploid hybrids may be more viable.

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2.5.1.2 INDUCTION OF TRIPLOIDY

Triploidy can be induced by exposing eggs to physical or chemical treatment shortly after

fertilization to inhibit extrusion of the second polar body. Triploid fish are expected to be sterile

because of the failure of homologous chromosomes to synapse correctly during the first meiotic

division.

Methods of triploidy induction include:

Temperature shock (hot or cold).

Hydrostatic pressure shock.

Chemicals such as colchicine, cytochalasin-B or nitrous oxide.

Crossing tetraploids and diploids.

Although hot and cold shocks can produce good results in warm- and cold-water fish, hot

shocks seem to produce better results in cold-water fish and vice-versa. Another physical shock

method commonly used is to place eggs into a pressure chamber and subject them to pressure of

up to 9000 lb in–2 (≈ 60 megapascals or 600 bar; normal SCUBA tanks are pressurized to

around 3000 lb in–2) (Beaumont et al., 2010). The application of thermal treatment (heat shock)

is easier to administer than pressure shock and is suitable for large-scale production of triploid.

Optimal heat shocks generally give sufficient yields of triploids in rainbow trout (Thorgaard et

al., 1981), Atlantic salmon (Johnstone, 1985) and Chinook salmon (Utter et al., 1983).

2.5.2 TETRAPLOIDY

Tetraploids have a balanced set of chromosomes, which can result in viability and fertility.

Tetraploidy in fish is commonly produced by disrupting the first cleavage with thermal or

hydrostatic pressure shocks in eggs fertilized with normal sperm. Viable tetraploids have been

produced by these methods in a number of fish species (Pandian and Koteeswaran, 1998).

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Theoretically, the progeny of matings between tetraploids and diploids should be 100% triploid.

If the tetraploids are fertile, replacement of 4N brood stock is accomplished by mating

tetraploids with each other to produce the next generation of tetraploids. Induced tetraploids can

be useful for mass production of triploid fish by mating them with normal diploids. Tetraploid

breeding lines are of potential benefit to aquaculture by providing a convenient way to produce

large numbers of sterile triploid fish through simple crosses between tetraploids and diploids

(Guo et al., 1996).

2.5.2.1 INDUCTION OF TETRAPLOIDY

Tetraploid induction involves fertilizing eggs with normal sperm and exposing the diploid

zygote for physical or chemical treatment to suppress the first mitotic division (Lakran and

Ayyappan, 2003). Allowing karyokinesis, while blocking cytokinesis, produces tetraploids.

Similar techniques – temperature shocks and hydrostatic pressure – are used to induce both

triploidy and tetraploidy, but of course later in embryonic development for tetraploids (Rezk,

1988). Again, pressure treatments appear to be more consistent than temperature shocks for

producing both triploidy and tetraploidy (Bury, 1989).

2.5.3 DETERMINING FACTORS

The success of treatments to induce polyploidy depends on:

Time of initiation of the shock: The best time for initiation of the shock varies widely

among different species, but is related to the rate of development and, specifically for

triploidy, the timing of the second meiotic division and, for tetraploidy, the timing of the

first mitotic division (Dunham, 2004). Research conducted by Xu and Chen (2009) on

Japanese flounder indicated that extrusion of the 2nd polar body occurs between 3 and 4

min at 16°C. Thus, the optimum time to inhibit 2nd polar body extrusion is 3 min post-

fertilization.

[21]

The magnitude of the shock: The proportion of eggs that respond to shock depends

upon the shock magnitude. Of the physical shocks, heat and cold are the easiest to

administer and consist of a sudden raising or lowering of the temperature by 5–10°C

from ambient (Beaumont et al., 2010).

Duration of the shock: The most effective shock for triploidy induction is 3 min after

fertilization for 25 min. This treatment produced the highest survival rate, one of the

lowest abnormality rates, and all-triploid fry in Japanese flounder (Xu and Chen, 2009).

Genetics: Strain or family effects may have a bearing on polyploid induction efficiency.

The strain of rainbow trout may react differently for different temperature shocks to

induce triploidy (Anders, 1990). A heat shock of 30°C was required to produce 100%

triploidy in a domestic strain of rainbow trout, whereas 28°C was required for the same

result in a wild strain (Solar and Donaldson, 1985).

Quality of the gametes: The quality of gametes can affect the efficiency of polyploidy

(Palti et al., 1997). When viability of bighead carp eggs, Hypophthalmichthys nobilis,

was above 59%, high rates of triploidy were produced, but, when the viability was less

than 40%, no triploids were produced (Aldridge et al., 1990).

2.6 ANDROGENESIS AND GYNOGENESIS

2.6.1 ANDROGENESIS

Androgenesis is the process by which a progeny is produced by the male parent with no genetic

contribution from female. This is the production of viable progeny with all paternal inheritance

as the DNA of the egg is inactivated by radiation (Nwokwa, 2012). Induction of androgenesis

can produce all male population in fish which would have commercial application in

aquaculture. It can also be used in generating homozygous lines of fish and in the recovery of

lost genotypes from the cryopreserved sperms. Androgenetic individuals have been produced in

a few species of cyprinids, cichlids and salmonids (Bongers et al., 1994).

[22]

2.6.1.1 INDUCTION OF ANDROGENESIS

Androgenesis is a developmental process, facilitating the inheritance of an exclusively paternal

genome. It involves two steps:

Elimination or inactivation of the egg’s genome.

Monospermic or dispermic activation of embryonic development by the haploid or

diploid gamete

The first treatment is the deactivation of the female genome by UV or gamma rays. Ultraviolet

irradiation is preferred for simplicity and safety, but also because it demerizes the DNA rather

than fragmenting it. Egg activation with untreated spermatozoa then requires diploidization of

the haploid zygote by some form of shock to interrupt the first mitotic division (Shelton, 2000).

The second employs diploid sperm - the gonad product of a tetraploid male - to fertilise the

irradiated egg and diploid embryos are produced without further treatment (Beaumont et al.,

2010).

Due to irradiation damage suffered by eggs, homozygous expression of lethal genes and damage

inflicted by thermal shock treatment to suppress the first mitotic cleavage, survival of the

androgenotes is very low. However, efforts have been made to improve the survival of the

intraspecific androgenotes (Kirankuma and Pandian, 2004). For instance, Thorgaard et al.

(1990) excluded the thermal shock treatment for restoration of diploidy in genome-eliminated

eggs of rainbow trout, Oncorhynchus mykiss, by using diploid sperm for activation, and

significantly improved the survival of the androgenotes.

[23]

Figure 2: Induction of androgenesis.

2.6.2 GYNOGENESIS

Gynogenesis is the process of animal development with exclusive maternal inheritance.

Gynogenesis involves the parthenogenetic development of an egg or the stimulation of an egg

by a genetically inactive spermatozoon. All-female inheritance is accomplished by activating

cell division with irradiated sperm and then restoring diploidy to the developing zygote (Aluko,

1993). Irradiation breaks or destroys the DNA in the sperm, so there is no paternal contribution

to the zygote, but the sperm is still motile and can penetrate the egg and activate cell division.

2.6.2.1 INDUCTION OF GYNOGENESIS

Irradiation rather than Y-irradiation (which may produce chromosome fragments from donor

sperm) is employed to destroy the DNA in the sperm (Aluko and Awopetu, 1992). However,

[24]

gamma irradiation has the advantage of being more penetrating than UV rays. Also, UV rays are

only effective when the milt is diluted and spread in a thin layer (Chourrout, 1986). Chemical

mutagens, such as dimethylsulphate, can also inactivate large volumes of sperm; however, this

procedure also produces supernumerary chromosome fragments (Chourrout, 1986).

Gynogenesis is induced by fertilizing the eggs with irradiate spermatozoa. This is followed by

the retention of the polar body. Retention of the polar body is accomplished with temperature

shocks or pressure treatments. The treatment is applied shortly after sperm penetration prior to

extrusion of the polar body. The most effective time for these shocks varies among species

(Thompson and Purdom, 1986). If the first cell division is blocked, a single diploid cell results.

Gynogens produced by this technique - mitotic gynogens or mitogynotes - are 100%

homozygous since a single set of chromosomes is duplicated (Dunham, 2004). A way of

producing diploid gynogens is to block the extrusion of the second polar body; then the diploid

gynogen has two sets of chromosomes, both of maternal origin. This type of gynogen is referred

to as a meiotic gynogen, or meiogen, since it was produced by blocking the second meiotic

division.

2.6.3 DETERMINING FACTORS

Factors influencing the effectiveness of androgenesis and gynogenesis include:

Shock type and intensity.

Duration and time of application.

Temperature: Because the rate of development is inversely temperature dependent,

either the pre-shock incubation temperature must be standardized or the shock time

must be calibrated to account for the temperature effect (Shelton, 2000).

[25]

Figure 3: Induction of meiotic gynogenesis – blocking the extrusion of the second

polar body.

2.7 TRANSGENICS

Transgenics involve the transfer of genes from one species into another species, in this case,

fish. These traits may include improvement of growth rates, larger size, more efficient feed

conversion into muscle and control of sexual maturation (Elzaeems, 2004). Researchers are

trying to develop fish which are larger and grow faster, more efficient in converting their feed

into muscle, resistance to disease, tolerant of low oxygen levels in the water and tolerant to

[26]

freezing temperatures (Ayoola and Idowu, 2008). Transgenic technology provides a means by

which such a quantum leap in production is possible (Hew and Fletcher 2001). The

identification, isolation and reconstruction of genes responsible for desirable traits, and their

transfer to brood stock, offer powerful methods of genetic/phenotypic improvement that would

be difficult, if not impossible to achieve using traditional selection and breeding techniques

(Devlin, 1997).

Transgenics may be defined as the introduction of exogenous gene/DNA into host genome

resulting in its stable maintenance, transmission and expression. The technology offers an

excellent opportunity for modifying or improving the genetic traits of commercially important

fishes, molluscs and crustaceans for aquaculture (Lakran and Ayyappan, 2003). It is a short cut

to achieving genetic change for fast growth, disease resistance, tolerant to low level of dissolved

oxygen in the water and fish resistant to freezing temperature (Ude et al., 2006).

Plate 4: Transgenic loach (top 2) as compared to their non-transgenic counterpart

(bottom).

[27]

The first transgenic fish was produced by Zhu et al. (1985) in China, who claimed the transient

expression in putative transgenics, although they gave no molecular evidence for the integration

of the transgene. The technique has now seen successfully applied to a number of fish species.

Dramatic growth enhancement has been shown using this technique especially in salmonids

(Devlin et al., 1994). Some studies have revealed enhancement of growth in adult salmon to an

average of 3-5 times the size of non-transgenic controls, with some individuals, especially

during the first few months of growth, reaching as much as 10-30 times the size of the controls

(Devlin et al., 1994; Hew et al., 1995). Therefore, researchers have developed new strains of

transgenic fish which naturally produce just the right amount of growth hormone to speed their

growth. Such fish is more cost-effective since they would produce higher levels of growth

hormone on their own, and they would pass this trait to their offspring (Dunham, 2004).

An increased resistance of fish to cold temperatures has been another subject of research in fish

transgenics for the past several years (Fletcher et al., 2001). Coldwater temperature is a stressor

to many fishes and few are able to survive water temperatures much below 0-1oC. This is often

a major problem in aquaculture in cold climates. Interestingly, some marine teleosts have high

levels (10-25 mg/ml) of serum antifreeze proteins (AFP) or glycoproteins (AFGP) which

effectively reduce the freezing temperature by preventing ice-crystal growth (Lakran and

Ayyappan, 2003). The isolation, characterization and regulation of these antifreeze proteins

particularly of the winter flounder, Pleuronectes americanus, has been the subject of research

for a considerable period in Canada. The introduction of AFPs to gold fish also increased their

cold tolerance, to temperatures at which all the control fish died (Wang et al., 1995). Similarly,

injection or oral administration of AFPs to juvenile milkfish and tilapia led to an increase in

resistance to a 26 to 13oC drop in temperature (Wu et al., 1998). The development of stocks

harbouring this gene would be a major benefit in commercial aquaculture in countries where

winter temperatures often border the physiological limits of these species (Lakran and

Ayyappan, 2003).

[28]

2.7.1 INDUCTION OF TRANSGENESIS

Transgenesis is done by:

Microinfecting freshly fertilized eggs with a fish-growth hormone linked to a

suitable fish promoter.

Electroporation, which involves transferring the genetic material or DNA into fish

embryos through the use of an electric current.

A foreign gene can be transferred into fish in vivo by introducing DNA either into embryos or

directly into somatic tissues of adults (Sudha et al., 2001). Direct delivery of DNA into fish

tissues is a simple approach, providing fast results and eliminating the need for screening

transgenic individuals and selecting germ line carriers. Gene transfer and expression following

intramuscular direct injection of foreign DNA into skeletal muscles of fish has been achieved

(El-zaeems and Assem, 2004). By microinfecting into freshly fertilized eggs a fish- growth

hormone gene, linked to a suitable fish promoter, transgenic fish with remarkable growth rates

have been obtained (Hew and Fletcher, 2001).

Electroporation involves the use of series short electrical pulses to permeate cell membranes,

thereby permitting the entry of DNA molecules into embryos (Nwokwa, 2012). This system

allows for the ease of integrating the DNA into the embryo up to 20%. The overall rate of DNA

integration in electroporation may be equal or slightly above that of microinjection and the

amount of time required to handle large numbers of eggs in electroporation is way less than

needed in microinjection (Chen et al., 1998). In recent research, gene has been transferred by

electroporation of the sperm rather than the embryo. Electroporation is, therefore, considered as

an efficient and versatile massive gene transfer technology.

[29]

3.0 CONCLUSION

Aquaculture production increases but the question remains whether the industry grows in a

sustainable manner and fast enough to meet the future projected demand. The expansion and

intensification of aquaculture will no doubt be further boosted by the application of

biotechnology. The application of artificial propagation has allowed for control and all year

round production of fish in the culture environment. With hybridization technique,

heterozygosity is increased, and fast growing, disease resistant and/or sterile fish that combine

valuable traits from both parents have been produced. Polyploidy gives fish with more than the

diploid number of chromosomes and larger sizes than the diploid ones, especially the sterile

triploids. In androgenesis and gynogenesis, only one parent contributes to the DNA of the

progeny and this helps in producing a monosex progeny which is desirable in some fish species.

The newest technology, transgenics, permits the incorporation of a specific gene into fish eggs

or embryos. With this technology, disease resistant, fast growing, high feed converting fish can

be produced.

[30]

4.0 RECOMMENDATIONS

With the various possibilities and potentials of biotechnology application in aquaculture, the

following recommendations can be made:

Genetic improvement of cultured fish and shellfish increases productivity and turnover

rate and results in better use of resources and reduces production cost. Therefore, it

should be given higher priority by government, NGOs and commercial organizations.

Research on genetically improved aquatic organisms should continue because of their

potential benefits (especially in developing countries); however, much greater

understanding of potential environmental impacts is necessary.

Along with increasing production of aquatic food products, biological techniques should

be applied to increase productivity and improve product quality.

Insignificant portion of funding has been devoted to biotechnology and aquaculture in

Africa. Additional donor support for research in key areas of biotechnology and

aquaculture is therefore, necessary..

Development of biotechnology will need to be shared among different disciplines and

stakeholders. In this regard, educating of decision-makers in food safety and

biotechnology regulation will be also very important.

To realize the full potential of the transgenic fish technology in aquaculture, several

important scientific breakthroughs are required. These include:

i. More efficient technologies for mass gene transfer.

ii. Suitable promoters to direct the expression of transgenes at optimal levels

during the desired developmental stages.

iii. Identified genes of desirable traits for aquaculture and other applications.

iv. Safety and environmental impacts of transgenic fish.

[31]

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