What are its applications for reproductive control in fish production?

The control of seasonal production and reproduction in farm animals have become major research goals. The applications of biotechnology to fish farming and ornamental fish production are numerous and valuable in both economic (food production, aquarium trade) and environmental terms (conservation of natural biodiversity for endangered species and protection of natural biodiversity from escapee domesticated strains). With the growing demand for fish products, biotechnology can help in the development of high quality, economical produce, thereby reducing pressure on natural populations.

In terms of fish reproduction for aquaculture, this means development of methods for the production of good quality gametes, independent of season, producing progeny with specific desired characteristics (good growth rate, flesh quality etc), meeting consumer demand. This technology should also contribute to the preservation and dissemination of genetic improvements.

Biotechnology may be used to:

• Control the sex of fish (e.g. produce single-sex populations)
• Delay the age of first sexual maturation (puberty)
• Control the reproductive cycle (in terms of gametogenesis and spawning)
• Support the genetic improvement of stocks, to produce strains more resistant to disease, displaying better growth rates (and food conversion ratios), more tolerant to stress and temperature changes, providing a product with an improved nutritional value, taste, texture and appearance.
• Improve management of gametes (short and long-term storage, transportation, development of media for in-vitro fertilisation)
• Conserve endangered or economically important strains via selective breeding programmes and management of captive broodstock (spermatogonial transplantation, cryopreservation of gametes)
• Produce sterile farmed animals to prevent the impact of escapees on the local biodiversity
• Assist in providing the aquarium trade with ornamental fish

Which methods are currently practiced?

In-vitro fertilisation
This is the fertilisation of fish oocytes with manually/artificially collected sperm and ovules. Since the 1990's, research has permitted the development of several media (for dilution, to prevent spermatozoa activation, to preserve gamete fertilizing capability, for in-vitro gamete maturation...), thereby the success rates of the fertilisation process.

A dilution medium for washing oocytes, Ova-fishÒ (IMV technologies), prior to in-vitro fertilisation, is currently available, which enables an in-vitro hatching rate success of 60-70% (for rainbow trout). Ovafish solution removes debris from the eggs, such as blood, urine and mucus, which may otherwise affect fertilisation success. Another medium, ActifishÒ (IMV technologies), has been developed to improve the fertilising capability of fish sperm. Actifish is a fertilisation medium that can be used for fresh, conserved or frozen semence. Another medium, MaturFishÒ (IMV technologies), has also been developed to promote sperm maturation. All these media have been specifically developed for the trout, but other examples exist for numerous species, including those of marine origin.

Long and short term preservation of gametes

Cryopreservation enables the long-term preservation of sperm of individuals of interest (disease resistant phenotypes etc.), for a virtually unlimited period of time. This is useful for many reasons, for example: to develop breeding programmes independent of maturation period; to market standard quality sperm; to provide a year round supply of gametes; and to preserve a genetic stock of both commercially important strains as well as those species threatened by extinction in the wild. Research has permitted the development of several dilution mediums to protect sperm during freezing. However, freezing technology has very limited uses.

Oocytes cannot currently be frozen without losing their viability. However, research is in progress on the vernalisation of oocytes, this is very low temperature storage, without freezing, and could preserve the oocytes for several months.

Different media and temperatures have also been investigated for the short-term storage of both eggs and sperm. For example, studies have shown that trout eggs can be stored at 12°C for 3 days in coelomic fluid with no detrimental effect on egg quality.

Sex control

The control of fish sex could be useful where one sex displays advantageous characteristics, such as larger adult size, production of high-value caviar (sturgeon), faster growth rate, or higher age at first sexual maturation (thereby avoiding degradation of flesh quality and certain bacterial infections). Sex control is currently only practiced commercially for salmonids, tilapia and hiramé.

Monosex populations of the most advantageous sex may be produced either by direct sex control via steroid treatment of the fish destined for consumption (masculinisation by administration of androgens; feminisation by administration of estrogens); or by genetic control and steroid treatment of broodstock (indirect hormonal treatment, gynogenesis, androgenesis, hybridisation); or by control of external factors (temperature, density etc.).

Genetic sex regulation may be achieved by:

• Crossing sex-reversed adult broodstock (administering androgens to produce "neo" males and estrogens to produce "neo" females; a "neo" fish is one which is genetically male or female, but phenotypically of the opposite sex) with normal males or females to produce single-sex progeny. Within Europe, only indirect androgen treatment of broodstock is permitted (i.e. production of all female progeny). This has obvious environmental advantages over the hormonal treatment of whole populations of pre-differentiated fish.
• Gynogenesis, or the fertilisation of oocytes with inactivated sperm (irradiated) with normal sperm, to eliminate the female chromosomes and produce all female progeny when the genotype is XX/XY. This method is more successful than androgenesis, but not widely used commercially.
• Androgenesis, or the fertilisation of inactivated oocytes (irradiated) with normal sperm, to eliminate the female chromosomes and produce all male progeny when the genotype is ZZ/ZW. In practice this method is not very successful and therefore not used commercially.
• Hybridisation of two different species from the same genera, producing single-sex progeny. However, the resulting offspring do not always display a satisfactory growth rate.

(Note: control of external factors, such as temperature for some gonochoristic fish - which do not change sex during their lifetime, such as tilapia or seabass; and social stimuli for hermaphrodites, such as seabream - which change sex during their lifetime, is a "gentle" method for controlling sex, but it is not yet commercially viable as it is not sufficiently reliable).

Control & synchronisation of spawning

The spawning season may be controlled by the manipulation of species-dependent environmental factors, such as photoperiod and temperature. These methods are routinely used to produce eggs in salmonid and some marine species such as seabass, turbot and seabream.

Spawning events may also be induced and spermiation enhanced by administration of gonadotropin releasing hormone analogs (GnRHa or LHRHa), hcG (human chorionic gonadotropin) or fish pituitary extracts. For example, within Europe, a synthetic GnRH analog, GonazonÒ (Intervet), is officially approved as an inducer of ovulation, showing good success rates in salmonids for both ovulation and subsequent fertilisation. In some other species, such as cyprinids, more potent inducers are used (hCG or Carp Pituitary Extract). Other speciality pharmaceutical products such as OvaprimÒ have been developed, which combine a GnRH analog with domperidone, a potent inhibitor of the dopaminergic system. Fish farmers may also control production by speeding up or slowing down embryonic development by temperature and / or feeding practices.

Control of puberty 

Puberty may also be delayed by using different photoperiod regimes, for example in Atlantic salmon production. Puberty may also be completely eliminated, at least in females, by induction of sterility by triploidy. The application of antireceptor vaccinations, which have specific antagonistic effects of gametogenesis, is also in development.

Control of fertility

The production of reproductively sterile fish (triploids) is very useful in responsible fish farm management and limits the genetic risk associated with the escape of domesticated fish into the wild. These farmed populations cannot interbreed with wild populations and therefore do not pose a threat to the natural biodiversity. In some species sterile fish reach a larger size without the complications of sexual maturity and the consequent reduction in flesh quality, which is desirable in sectors of the fish farming industry where large fish are most valuable. Generally, triploids grow more slowly than diploids. Triploidy is also not 100% successful (98% success rate), and as it is not feasible to test individual fish to check whether or not they are triploid, there will always be a certain number of individuals within the stock that are not triploid and are thus capable of reproducing. It should also be remembered that if the aim is to produce small sized fish (e.g. up to 200g), sexual maturity is not a problem as the fish will not attain maturity before they have reached their marketable size.

All female triploid offspring may be produced by the indirect use of externally administered hormones and by pressure / temperature treatment of eggs for a few minutes, post fertilisation. Within the European Union, it is forbidden to market hormone-treated fish for human consumption (see "legislation" page for further details). Methyltestosterone may be used to sex-reverse broodstock females (XX) to neo-males (only sperm containing the X chromosome). These neo-males (XX) are then crossed with normal females (XX). Induction of triploidy in the resulting zygotes will produce all-female triploid offspring (XXX). This is currently practiced on rainbow trout and perch. However, current research focuses on the development of alternative, more environmentally sensitive methods that are also species specific.

Female triploids are always sterile, they do not undergo gonad development; however triploid males are only functionally sterile, they develop gonads and undergo puberty, but their sperm does not give rise to viable progeny.

Development of gene transfer technologies for fish reproduction

Gene transfer technology has been applied to aquaculture species to obtain sterile fish by preventing the expression of GnRH. Further information can be obtained from the following papers:

• Wei Hu et al. 2007. Antisense for gonadotropin-releasing hormone reduces gonadotropin synthesis and gonadal development in transgenic common carp (Cyprinus carpio). Aquaculture 271: 498-506.
• Uzbekova, S. et al. 2000. Transgenic rainbow trout expressed sGnRH-antisense RNA under the control of sGnRH promoter of Atlantic salmon. J. Mol. Endocrinol. 25 (3): 337-350.

Principal further research requirements

• Optimisation of species-specific hormonal treatments, in terms of the development of rapidly metabolised molecules that do not release biologically active catabolites into the environment (e.g. possible use of recombinant fish gonadotrophins for inducing gonadal development).
• Development of alternative methods to exogenous hormones based on genetic selection, environmental variable manipulation (e.g. photoperiod and temperature) and gene transfer technology, for fertility and sexual cycle control.
• Genetic basis of reproduction, for selection criteria / gene markers for broodstock companies
• Improved knowledge on important steps in fish reproductive cycles (sex control, puberty, gametogenesis and spawning)
• Improved knowledge on the determinants of gamete quality (e.g. surrogate broodstock technology)
• Research into reducing the impact of fish farms on the environment (in terms of the genetic integrity of the wild population, the discharge of hormones and antbiotics into the environment etc.)
• In terms of cryopreservation, a better knowledge of sperm membrane biology is needed. An improved understanding is required of the mechanisms regulating tolerance to freezing (for sperm and oocytes), as well as the formulation of improved protocols.
• Conservation of genetic resources and regeneration of populations of valuable animals or endangered species (e.g. spermatogonial transplantation).
• Development of generic methods for the domestication of new species

This factsheet was prepared with the expert assistance of scientists Alexis Fostier, Ana Gomez, Jean-Jacques Lareyre and Pierrick Haffray.