Inbreeding

In fish farming, if proper care is not taken the fishes can breed with their close relatives or same parental generation which may cause early mortality of fish, poor growth rates and other genetical abnormalities. This phenomenon is called inbreeding or homozygosis and the offspring is called homozygote. Homozygosis is the condition when two genes at particular locus are with same allele. Inbreeding causes the reduction of desirable traits and some times may cause fertility. If a farm based on limited number of population of brood stocks the progeny over long periods can face inbreeding problems.

The advantage of inbreeding:

Sometime the inbreeding is not encouraged and has some advantageous point also. Production of inbreed lines are very use full in improvement of stock. Production of inbreed lines have following advantages.

· To produce pure lines of fish
· Pure lines of strains are used for perfect hybridization of fish to obtain favourable heterosis, monosex.
· Pure lines help in gene mapping
· To determine

1. Phenotypical variations
2. Extent of inbreeding depression
3. chromosomal makeup to the fish

Cross Breeding

Crossbreeding is the solution of inbreeding depression, because the fishes are allowed to breed with different breed verities, strains or genotypes of farmed species. To improve the stocks artificial selection techniques are applied. This is called selective breeding. From heterozygosis the stocks can be improved and the aims of cross breeding to achieve –
· Better growth rate
· Better desired qualities
· Better FCR value
· Increase the survival rate and lowered the early stage of mortality

Gynogenesis

It is the process to produce individuals from maternal chromosomes only eventually to obtain homozygosity. Gynogenesis in fish farming is used to form inbred lines to achieve proper hybridization and selective breeding.

Methods:

Sperm nucleus is inactivated prior to fertilization by use of X-Rays, chemicals – dimethyl sulphate.
Upon fertilisation the resulting diploid individuals retain the second polar body nucleus (maternal) besides the egg nucleus, because the eggs are exposed to sub lethal temperature shocks before or after fertilization which suppress the meiotic divisions of eggs i.e. ensuring the non reduction of nuclear components.

In India Gynogenesis are tried on Indigenous as well as Exotic carps. Eggs of rohu are fertilised with irradiated sperms of Catla and then exposed to cold 12°C and heat 39°C shocks to obtain gynogenetic rohu.

Need for Genetically Selected Fish

The fishes from mass selection are not found to be successful for faster growth rate, or with better FCR value. In other hand in captive condition there is the chance of inbreeding. So we need genetically improved stock for following purposes –
Selective breeding and formation of improved species with better growth rate, low mortality and resistance to the diseases
To obtain better fertilisation rate
To achieve better fertilisation rate
To achieve better FCR value
To achieve better flesh content and shape of the fish

Selection Methods:

· The fishes are to be selected from different rivers to avoid close relatives of the fishes. Reservoirs should be avoided, because there is a chance of inbreeding.
· Genetically superior and pure lines are used for this purpose
· Mutant fish with superior character are used
· The brooders from different parental families much have deeper bodies with desirable qualities, like small heads, greater fatty make up.
· The population can be separated from original sources from at least two generations
· The brooders should have fastest growth rate, grater disease tolerance capacity and adaptable to extreme environment
· By chromosomal manipulation – Gynogenesis or Androgenesis
· Hormonal manipulation – Feminisation or Masculisation
· By genetic engineering – trangenesis or mutation

The fishes are selected by following criteria –

1. Body and development of fins
2. Better growth rate and better FCR value
3. Better disease tolerance capacity
4. Fatty make up (larger size and heavier growth rate)
5. Greater longevity
6. Higher fecundity
7. Greater adaptability in different stages and temperature variations
8. Hardier etc

Sex Control of Fish

Sex control is the technique to produce desired sex by various ways. The species achieved faster growth rate with better FCR. These are economically important and prolific breeding or over crowding due to prolific breeding can be avoided. The methods are as follows –

Feminisation ----------------- Hormonal treatment
----------------- Back cross with sex reversed male and normal female

Masculization ---------------- Hormonal Treatment

Contracept ---------------------- Drugs/Chemicals/Hormones

Sterilisation -------------------- Drugs/Chemicals/Hormones
-------------------- Hormonal Treatment
--------------------- Heat & Cold Shock/Pressure Shock
--------------------- X-ray

The role of Hormones in sex reversal –

Sex reversal has been achieved by hormonal control methods. The hormone which is used in feminisation is estradiol – 17-ß, ethyl estradiol. The hormones are used in early maturation stages administered by food and the undifferentiated gonads are transferred to the female ovary. Dose and treatment period is specific to the single species as for example 20g estradiol is used per kg of food, with 120 days treatment period for salmon fry for 100% feminisation. The female hormone which is use in early developmental stages in male fishes can change the male to female phenotypically, but genetically it would be XY.

For synthetic hormone oestrogen, one has to take care and should avoid high level of dose because it may cause liver damage and mortality of fry.

In case of masculisation the 17-α Methyl testosterone is used in early maturation period of fish. It can convert the female fish into male fish phenotypically, but genetically it would be XX. Generally much higher dose of androgen can be used in two ways – I. by food, II. Bathing of larvae in hormone treated water. In case of salmon fry the dose of hormone in food is about 3mg/kg and for Tilapia it is 20-30mg/kg of food. The androgen is also growth stimulating hormone.

Transgenic Fish

A transgenic fish is one which carries one or more than one foreign genes. The foreign genes are selectively incorporated by micro injection into the egg with a view to produce transgenic fish.

The progress was made in genetic engineering to isolate eukaryotic genes in 1970s and by 1980s. However, the technique was applied to the fish much later; nevertheless some significant progress was made which has potential of application in fisheries. More than a dozen of fish is produced by 1989.

Fish transgenies are difficult because of tough egg-chorion which impedes microinjection. A prior puncture or use of micro pile (an opening in the egg surface for sperm entry during fertilisation) has to be made for microinjection. The micropile is made by or by using trypsin digestion. The gene can be transferred by electroporation (exposing the egg chorion in an electric shock for a fraction of second) or by retroviral injection. The foreign gene then transferred into the nucleoplasm or the cytoplasm. In case of retroviral injection, the genes are first incorporated into the viral genome, and then through the virus the gene are transferred into the host by injection. However in case of fish the method is also not tough as because the fertilisation and embryonic development is external.

Application:

· The fish of superior quality or desired traits are produced by this process. Giant sized fish or super fish can be produced. This can be achieved by incorporating the growth promoting genes – bovine growth hormone gene or human growth hormone gene. Example in China, the giant loaches are made by growth hormone gene of human.
· The transgenic Atlantic salmon are given with anti-freeze protein gene of polar flounders. This was done to promote to make resistance power of salmon in polar region and habit of salmon can be extended to Polar Regions also.

Future goal:

The technique has great promises in future to make desirable traits of various farm fishes. Such dream fish of fast growth rate withy fatty make up, greater longevity in all environmental condition, omnivorous feeding habit with higher fecundity, greater adaptability, more resistance to disease, biocides and pollutants, lack of bones and other undesirable features can be made in future. Aquarium fish trade can also be rise by using this technique. Various beautiful coloured fish can be made by this technique.

Sterile Fish

Sterility is the loss of reproductive power due to any reason leads better growth and food conversion efficiency. A huge energy is utilised for gonadal maturation and production of gametes in fish. If the sexual maturation is stopped the fish can grow more rapidly. The all foods supplied in that case are transferred to the flesh and no wastage of food or energy for gonadal maturation can be seen in sterile fish. Not only this, sterility avoids prolific reproduction and over crowding of fish. Sterile fish can be produced in various ways –

Induction of autoimmunity of gonads
Extirpation of gonads
Chromosomal manipulation for production of polyploidy
Hybridization
Hormonal sex reversal to super males and super females of giant sized fish
Non hormonal chemicals which can sterile fish
Exposure to UV rays or radioactive substances

Among the above mentioned process hormonal sterility is applied in case of sterile fish production where insufficient application of male hormone or female leads partial change of male to female or female to male and can produce sterile fish. The method is very easy but not acceptable in all countries because hormone treated fish is bad for human consumption.

Non hormonal chemicals like methallibure or cyproterone acetate. Both these are chemosterilants can reduce Gonadotropin production and lower androgen secretion, but do not prevent the gonad maturation. These drugs will have to be administered to the young fry in their diet; hence the utility in commercial purpose is doubtful.

Hybridization of some species can produce sterile fish which is very easy, economical and no doubt to human consumption.

Exposure of fish or fry in UV rays or any other radio active radiation cause gonadal destruction leads to the sterility of fish. The method needs well equipped laboratory with very much experienced workers. The production of such type of sterile fish is not encouraged in commercial purpose.

Polyploidy (Broiler Fish): It is one of the methods to produce sterile fish most easily. The triploid can be achieved experimentally by chromosomal manipulation brought about the suitable exposure to thermal or hydrostatic pressure shock at early stages of development. The mechanism involved to prevention of second polar body from the eggs.

Triploid can produce by a cross between the female common carp and rohu are sterile by following way:

By intergeneric mating between a diploid female of one species (any IMC, for example) and triploid male of another species (common carp)

By subjecting the fertilised eggs involving the egg of one species and sperm of another, to the action of antibiotic cytochalasin or colchicines which are mitotic inhibitor & so disrupt the first cleavage mitosis. By subjecting the normally fertilised eggs, involving artificial insemination of egg of one species (common carp) by the sperm of another species (rohu), to heat cold or hydrostatic pressure shocks. The shocks suppress the release of 2nd polar body (i.e. early metaphase of meiosis II).

Mono Sex Culture

Mono-sex culture is based on the culture of fish by producing all males or all females depending upon the sex which have better food conversion ratio and growth rate. Sex of fish genetically is determined by the sex chromosomes (X, Y, Z, or W). The male determining gene M is present on any of the three X, Y and W. in XY mechanism, the females are XX and males are XO. Some species have ZZ female and ZW male. In platy fish there are 3 sex chromosomes – X, Y and W; XX, WX and WY are some combinations.

However for all male or female productions the flowing procedures are maintained –

The sex of fish is identified before maturity and male and females are separated. The process is laborious. Desired quantity of male or females are not produced by that process.

Experimental hybridization in Tilapia can produce monosex stock. Inter specific and intra specific mating yields monosex male stocks as follows

♂ T, macrohir × ♀ T. nilotica
♂ T. hornorum × ♀ T. mossambica
♂ T. mossambica × ♀ T. nilotica
♂ T. mossambica (African) × ♀ T. mossambica (Malaysian)

Treatment with sex hormones: It is another easiest way, when male sex hormone methyl testosterone is administered through feeding in early developmental stages of female fish. The genotype female (XX) then transferred to phenotype male (XX). If such sex reversed male are crossed with normal female, the progeny will be 100% female. The gonads of fish (teleost) are undifferentiated at early stages of maturity and it can be triggered to produce male or female gonads by that process.

Sex reversed male (XX) × Normal female (XX)
↓
Female (XX) ……. F1

The hormone treated sex reversed male are generally not fit for human consumption. But F1 progeny is normal female and suitable for human consumption. But sometimes the culturist does not produce F1 progeny to produce only male population. In case of Tilapia the little amount of methyl testosterone (15-60mg/kg of food) is administered. The drug is given for 30-50 days of life, during which gonadal differentiation takes place. The uses of estrogenic steroids are not successful. However the production of all females has been attempted in salmon and trout. Oral administration of 17-ß estradiol at 20mg/kg of food is given to the juvenile trout & salmon up to 60 days resulted sex reversal of males to females. In pacific salmon (Onchorhynchus spp.), the immersion of young fish in drug as well as feeding appears to be necessary for sex reversal.

Necessity of Monosex Culture:

Some time one sex of certain species has better growth rate and food conversion efficiency. To culture that sex (male/female) monosex culture is essential. For example we can say that the male Tilapia grows faster than female, then the culture of male is beneficial in case Tilapia.

When the fecundity of certain species is very much high and if they can breed in captivity without any inducing agent, there is the possibility of overcrowding of fish, which leads to stunted growth (due to the prolific reproduction e.g. Tilapia).

The production of monosex fish is easier than the production of sterile fish, so in commercial purpose generally monosex cultured is mostly prefer.

Example of Monosex Production in Nature:

According to recent report, the flat fish population in the estuary of British River Tyne, a heavily polluted river, was found more than 50% of males possessing abnormal testes. The tendency of their sex change from male to female was probably due to the female hormones. But the actual reason and mechanism of the action is not known.

Hybridization of Fish

It is the technique of breeding of fishes between two species or genera which ordinarily do not breed.

Hybridisation in nature:

Most fishes release their eggs and sperms in water and fertilisation are external. Fish hybridizes more frequently than tetrapodes so fertilization of closely related species which leaves in same water bodies, are common. Reservoir is the most important area where natural hybridization occurs frequently than rivers, because the area is not too large as rivers and scarcity of certain species with preponderance of others. Naturally hybridized fishes are found in following families – Esocidae, Catastomidae, Cyprinidae, Salmonidae, Poecillidae etc of about 56 families.

Technique of Hybridization:

The hybridization is done actually by inducing the virgin fishes or small aged group fishes. The hybridized fish possess intermediate character of two species. This type of hybridization is also known as diploid hybridization. This hybridized fishes are capable to produce new fish up to F2 progeny. Inter specific and Inter generic both type of hybridization is done in India.

Inter specific hybridization – Inter specific hybrids are generally producing by mating between two different species in same genus. In India mating female kalbasu and Labeo rohita is highly successful. Over 94% fertilisation was obtained. The growth rates of hybrids are superior to the parent Kalbasu. It attains maturity in two years. The hybrids also can be bred by hypophysation and can be obtained F2 generation. However the fishes are selected by following process –

The brooders are selected in first maturity generally virgin.
One pair of males (rohu) and one pair of females are injected to induce them prior to breeding by hypophysation technique.
The breeders are kept separated for some times and then they are released in breeding hapa with suitable breeding conditions.

The inter generic hybridization – In this method male and female are generally selected from different genera and produced by above mentioned methods. In India, successful hybrids are as follows –

Parent ♂ Parent ♀ Hybrids
Catla catla Labeo rohita Rohu-Catla
Catla catla Labeo calbasu Catla-Kalbasu
Catla catla Cirrhinus mrigala Catla-Mrigal
Labeo rohita Cirrhinus mrigala Rohu-Mrigal

Inter generic hybrids between Catla and Rohu attains full maturity in 3yrs and they also be induced to breed. Crossing between Rohu and Mrigal is more successful and 90% fertilisation is done and hybrids attain full maturity in 2yrs and showed intermediate character. Mrigal-Kalbasu hybrids attain full maturity within 2-3yrs. These hybrids are also capable to produce new ones. These are –

Parent ♂ Parent ♀ Hybrids
Catla catla Mrigal-Kalbasu Catla-Mrigal-Kalbasu
Labeo calbasu Mrigal-Kalbasu Rohu-Mrigal-Kalbasu

These hybrids are matured within one year.