Sexual Development in Fish, Practical Applications for Aquaculture

Transcription

1 Review Article Sex Dev 2009;3: DOI: / Received: November 3, 2008 Accepted: January 14, 2009 Sexual Development in Fish, Practical Applications for Aquaculture A. Cnaani a B. Levavi-Sivan b a Department of Poultry and Aquaculture, Institute of Animal Science, Agricultural Research Organization, Bet-Dagan, b Department of Animal Sciences, Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University of Jerusalem, Rehovot, Israel Key Words All-female All-male Monosex Puberty Reproduction control Sexual dimorphism Sterility Abstract Aquaculture is one of the fastest rising sectors of world food production. Hundreds of fish species are cultured, providing an affordable, high quality food source. Two aspects of sexual development are critically important for the continued improvement of cultured fish stocks: sexual dimorphism and control of reproduction. In this paper, we review the main methods used to control sex determination in fish and their application in some of the most widely cultured species. Specifically, we review the techniques available for the production of all-male, all-female, and sterile populations. Techniques for endocrinological control of reproduction are also discussed. Copyright 2009 S. Karger AG, Basel The process of species domestication for use in modern agriculture involves managing all life stages, especially the control of reproduction. The ability to control reproduction provides the opportunity to manage the number and timing of offspring for commercial purposes. However, unlike terrestrial farm animals, fish species display a variety of sex determination and developmental processes, with biologically complex variables and mechanisms. Compared to mammals, these pathways are a relatively flexible process and can be affected and modified by various factors [Devlin and Nagahama, 2002]. While the diversity of sexual developmental systems and the plasticity of the related biological pathways give the aquaculture industry some unique management tools, the interplay of genetic and environmental influences on sexual developmental control of fish can be elusive. Many fish of commercial interest to the aquaculture industry exhibit reproductive dysfunctions. These dysfunctions, also presented by many wild animals held in captivity, result from the lack of the appropriate natural spawning environment, and/or may be related to one or more sites along the brain-pituitary-gonadal (BPG) axis [Zohar, 1989; Yaron, 1995; Ohta et al., 1997]. Reproductive problems are usually more serious in female broodstocks. Gaining control over fish reproduction is vital for different aspects of fish culture. For example, it is difficult to obtain fry from some captive fish species, while in others the challenge is to synchronize spawns for efficient management. Additionally, ovary maturation in species in which fish eggs are a marketed product requires an effective plan to monitor and manipulate sexual development. Producing viable offspring from captive broodstock permits domestication and genetic improvement to proceed and is a prerequisite for successful early rearing and efficient production. Fax karger@karger.ch S. Karger AG, Basel /09/ $26.00/0 Accessible online at: Avner Cnaani Department of Poultry and Aquaculture Institute of Animal Science, Agricultural Research Organization Bet-Dagan (Israel) Tel , Fax , avnerc@volcani.agri.gov.il

2 There are several traits of high economical value that are related to the fishes sexual development. Sexual dimorphisms of economical importance are observed in growth rate, time and age of maturation, body shape, and carcass composition in some edible fish species. Additionally, sexual differences in color pattern and fin shape are important in some ornamental fish species. Even when there are no significant differences between sexes in those economically important traits, it may be advantageous to culture monosex stocks in order to avoid unwanted or uncontrolled reproduction [Lutz, 2001; Dunham, 2004]. An important consideration for the control of sexual determination and development of many aquatic fish species is an understanding of the variety and complexity of these systems. There has been a tendency among aquaculture geneticists to explain mechanisms of sex determination by formulating simplistic sex determination models, with only 1 or 2 factors that fit the results of mating trials. However, these simplistic portraits of Mendelian inheritance are often inadequate [Lutz, 2001]. In species with complex and multifactorial systems, phenotypic sex can be viewed as a threshold trait, influenced by major and minor genetic and environmental factors. Nevertheless, simplified conceptual systems such as the male or female heterogametic model can often provide a framework that will be sufficient for the practical control of sex determination. During the past 3 decades, several modern technologies have matured from an experimental phase and been incorporated into the aquaculture industry [Hulata, 2001]. In this review we will present traditional and newly emerging techniques used to control and manipulate sexual development in cultured fish and briefly review their applications on some of the more widely cultured species. M o n o s e x Cu l t u r e Methods for Monosex Production Sorting. Manual sorting of the sexes requires the least amount of technology for monosex culture and was applied effectively in several places. However, this technique is laborious and wasteful, as fingerlings must be grown to a size large enough to determine sex before manual separation and culling of the slower-growing sex. The process itself is a stressful experience for the fish, prone to mistakes, and in many cases results in relatively high mortality and low success compared to other methods [Hickling, 1963; Dunham, 2004]. Interspecific Hybridization. Hybridization between different species may result in monosex populations, skewed sex ratios, and sterilization [Bartley et al., 2001]. The phenomenon of all-male hybrids has been observed in sunfishes (family Centrarchidae) [Ricker, 1948], but the best known examples are in tilapias (family Cichlidae). Early models to explain the occurrence of all-male hybrids were Mendelian, with a female having an XX genotype being crossed with a male having a ZZ genotype and bearing a maleness-determining gene [Hickling, 1960]. Later studies demonstrated that mating results are inconsistent, probably due to a polygenic mechanism and alleles of the sex-determining genes not yet fixed in the studied species [Wohlfarth and Wedekind, 1991; Cnaani et al., 2008]. Most of the research on the genetic basis and commercial potential of monosex hybrids has been carried out in tilapias as many Oreochromis hybrids are characterized by skewed sex ratios [Wohlfarth and Hulata, 1983; Wohlfarth and Wedekind, 1991; Wohlfarth, 1994]. In the family Moronidae an all-female population was obtained by crossing different bass species. However, the mechanism producing such a biased sex ratio is not yet known [Wolters and DeMay, 1996; Bartley et al., 2001]. Hormonal Treatment. Many fish species are responsive to hormonal induction of sexual development and determination. In many cultured species, sex is genetically established at fertilization. However, phenotypic sex determination may occur later in development, and the timing of sex determination varies among species. The treatment of sexually undifferentiated fry by administration of hormones has been shown to work well in a wide range of species. Production of monosex populations by direct hormonal treatment requires elucidation of the labile period of sexual differentiation during which the fish are susceptible to hormonal masculinization or feminization [Dunham, 2004]. Hormonal treatments are commonly applied in gonochoristic species but have also been successfully administrated in a few hermaphroditic species, where they are used to provide broodstocks. Dozens of different steroids have been used for sex-reversal in fish. The most commonly applied androgen is 17 -methyltestosterone (MT), which has been tested in most of the cultured fish species to induce masculinization, and the estrogens 17 - estradiol [E2] and diethylstilbestrol [DES] that are used to induce feminization [Pandian and Sheela, 1995; Piferrer, 2001; Devlin and Nagahama, 2002]. Hormones are usually administrated via dietary supplementation by dissolving the steroid in alcohol prior to mixing with the diet. Immersion techniques have also Sexual Development in Aquaculture Sex Dev 2009;3:

3 been applied successfully, and in some species they appear to be more efficient than the dietary administration. The labile period for sex reversal is variable among species, depending on the sexual differentiation pathway [Shelton, 2006]. Generally, sex reversal treatments are applied early during embryogenesis in Salmonidae, while post-hatching treatment is more effective in Cichlidae and Cyprinidae. Injections and implants have been less successful, and their use is limited to some hermaphrodites and to species with late sexual maturation [Dunham, 2004]. Chromosomal Manipulations. In fish species that have external fertilization, pre-embryonic events such as insemination, 2nd polar body extrusion, and 1st mitotic cleavage can be manipulated to induce parthenogenesis and different levels of ploidy. Gynogenesis and androgenesis are the terms describing maternal and paternal uniparental reproduction. The nuclear content of either the sperm or egg is destroyed by UV or gamma irradiation, and the treated gamete can be then fused with an untreated egg or sperm to form a haploid embryo. Haploid embryos can be subsequently made diploid by inhibition of either the 2nd meiotic division or the 1st mitotic cell division. After blocking the 1st cell division, the resulting individuals carry only the duplicated set of chromosomes inherited from the untreated egg or sperm donor and are fully homozygous [Pandian and Koteeswaran, 1998; Komen and Thorgaard, 2007]. Such chromosome-set manipulations may lead to production of all-male, all-female, or potentially sterile populations. Combinations of ploidy induction and hormonal sex reversal in gynogens render the scope for generating broodstock able to produce all-male or all-female populations [Gomelsky, 2003]. Broodstock Development. Sex reversal can be difficult to achieve in some species, while in others it is not always effective enough for commercial use. Marketing of hormonally treated sex-reversed fish can also be a problem as health and food safety regulations disallow them in several countries due to public health and environmental concerns. One way to get around these problems would be to combine a method for sex reversal with a breeding scheme that aims to develop a broodstock that will produce monosex fry. Various breeding strategies utilizing sex reversal and breeding, progeny testing, gynogenesis, and androgenesis can lead to the development of all-male or all-female populations that are homozygous XX, YY, or ZZ and sometimes are called super-male or superfemale [Dunham, 2004]. Mating of sex reversed fish with untreated fish can yield monosex populations when the desired sex is the homogametic one. This is the case for the production of all-male blue tilapia (Oreochromis aureus), a female heterogametic (WZ-ZZ) species [Melard, 1995], and for the production of all-female rainbow trout (Oncorhynchus mykiss), which is a male heterogametic (XX-XY) species [Bye and Lincoln, 1986]. However, this breeding scheme requires progeny testing, as only half of the treated fish are neo-females (with ZZ genotype) in the case of hormonally feminized blue tilapia, or neo-males (with XX genotype) in the case of hormonally masculinized rainbow trout. An alternative method uses gynogenesis to produce all-female broods, followed by sex-reversal of these gynogenetic fry through the administration of masculinizing hormones. The final step will be breeding the sex-reversed gynogenetic fish (neo-males with XX genotype) with normal females. This method eliminates the need for progeny testing to determine the genetic sex of phenotypic males [Gomelsky, 2003]. When the desired sex is a heterogametic one, a more complex breeding scheme is required, aiming to produce the genotype that does not occur naturally, such as YY individuals which, if viable and fertile, could be mated to normal females to produce an XY all-male population. In species such as the Nile tilapia (Oreochromis niloticus), it is possible to produce these novel individuals homozygous for the sex-determining chromosome (designated as YY). Sex reversing hormonal treatment would be used as a 1st step, with progeny testing to identify feminized neo-females (with an XY genotype) that have a 3: 1 male:female ratio when mated with normal XY males. Only one third of the male offspring resulting from neo-females has the YY genotype. They should be identified through a 2nd progeny testing after mating with normal females to produce all-male progeny [Beardmore et al., 2001]. This is a laborious three-generation breeding program that requires several years and considerable growing space, a procedure more suited to larger research institutions. The number of generations required to produce YY males can be shortened by the use of chromosome-set manipulations. Although not yet demonstrated on a commercial scale, YY males were directly produced from a normal (XY) O. niloticus male by androgenesis followed by progeny testing [Ezaz et al., 2004]. Another way to improve this breeding program is by using sex-linked DNA markers. Fish are chosen for progeny testing according to identified sex-linked markers in the tested family, thus significantly reducing the number of fish to be tested in each generation and increasing the proportion of the required genotype in each generation [A. Cnaani, M. Ron, G. Hulata, unpublished work]. 166 Sex Dev 2009;3: Cnaani /Levavi-Sivan

4 Al l-ma l e P ro du c t ion Tilapia. Tilapias (Oreochromis spp.) are the first group of fish species in which male monosex culture became a common practice [Hickling, 1963]. All-male populations are preferred due to their faster growth, but it is more important to prevent uncontrolled early reproduction that results in unknown biomass and large size differences in the ponds when both sexes are present [Wohlfarth and Hulata, 1983; Beardmore et al., 2001]. Tilapia show a large degree of sexual dimorphism compared to most cultured fish species, and sexes can be identified on the basis of external genital morphology at a relatively small fish size. Hand sexing at a size of approximately 10 g is painstaking and inefficient but was widely applied until the 1960s [Hickling, 1963]. Today, this technique is rarely used as more advanced and profitable methods have been developed. Unlike edible fish from other families, tilapias have a variety of sex determination systems, with male or female homogametic species and also some more complex patterns of sex determination [Cnaani et al., 2008]. Due to these different sex determination systems and the ability of interspecific hybridization in tilapia, some of the crosses between species result in all-male populations [Wohlfarth and Hulata, 1983]. Following the first reports on all-male tilapia hybrids [Hickling, 1960; Fishelson, 1962], the commercial culture of these hybrids has been initiated in various countries. There are several different crosses between tilapia species that gave all-male hybrid fry, but the cross between female Nile tilapia (O. niloticus) and male blue tilapia (O. aureus) drew the most interest and resulted in widespread dispersal of these 2 species [Wohlfarth and Hulata, 1983]. Although an efficient and reliable method, it is currently used only in a few farms in Israel and Taiwan, producing all-male hybrid fry from interspecific crosses such as O. niloticus! O. aureus. The need for constant management of the broodstocks, keeping the 2 parental species separated and avoiding contamination with hybrids, was a major obstacle for the long term use in developing countries [Lovshin, 1982]. The establishment of an easy, cheap, and relatively efficient androgen sex reversal procedure in tilapia was another reason for abandoning production of interspecific hybrids as a major method for all-male production [Wohlfarth, 1994]. In contrary to the currently limited use of interspecific hybridization to produce all-male progeny, hormonal sex reversal is being widely used. Once the technique was scaled-up from experimental to farm use, it has been established as a practicable technology in developing countries. Today, a significant proportion of the tilapia industry is using a protocol based on the use of the synthetic androgen MT incorporated into the fry diet at the beginning of the first feeding stage. This direct sex reversal has become increasingly criticized, and although oral administration techniques are apparently safe for the consumer, the environmental impact from uneaten food and metabolites may be a greater problem. In the USA, sex reversal of tilapia is a licensed procedure, while in the European Community the direct use of hormones is banned [Penman and McAndrew, 2000]. More environmental-friendly materials should be developed in order to continue the use of direct sex reversal [Golan et al., 2008]. The development of hormonal sex reversal led to breeding programs aimed at producing viable and fertile males that have a YY genotype. The process of obtaining these YY males was described above and has been applied by a number of research groups. Further advance in this broodstock development has been the sex reversal of some YY offspring using estrogens to produce YY neofemales. Once these fish are available, crosses between YY males and YY neo-females give 100% YY male offspring and enable easy renewal of broodstock [Penman and McAndrew, 2000; Beardmore et al., 2001]. Catfish. All-male progeny would be beneficial for channel catfish (Ictalurus punctatus) production, as males grow 10 30% faster than females, depending upon the strain (with some strains not showing any difference) [Goudie et al., 1994]. Unlike most other fish, in channel catfish it is problematic to change genetic females into functional phenotypic males. All-female populations can easily be produced by oral administration of estrogens, but all attempts to produce all-male populations by androgenic treatment failed and sometimes even resulted in sex ratios skewed towards femaleness [Davis et al., 2000]. It is still possible to hormonally sex-reverse channel catfish into females, breed them with normal males, and isolate YY males through progeny testing. These YY males can be mated with normal XX females to produce allmale XY progeny. However, the production of channel catfish YY broodstock was held back due to severe reproduction problems of YY females, and the large scale progeny testing needed to identify YY males in order to maintain the broodstock is currently not economically feasible [Dunham et al., 2001]. Guppy. Guppies (Poecilia reticulata) are a widely cultured species for the ornamental tropical fish market. Guppies include dozens of strains, distinct from each other mainly by the males fin shapes and colors. Due to Sexual Development in Aquaculture Sex Dev 2009;3:

5 their sexual dimorphism, male guppies are of much higher value than females, and there is an economic advantage in skewing the sex ratio towards the males. Guppies have a male heterogametic sex determination system (XX-XY). By using endocrine sex reversal followed by progeny testing it was demonstrated that YY females can be produced, resulting in all-male progenies when mated with normal or XX males [Kavumpurath and Pandian, 1993]. The survival of YY males and females is low, and obtaining them is a time-consuming process. Additionally, because of the relatively short life span of broodstock and the low number of fry (compared to other fish species), it seems that induced sex reversal is not economically feasible. Indeed, to the best of our knowledge, this method was not implemented in the guppy aquaculture industry. Masculinization of fry using androgens will lead to an all-male population. However, there are also side effects as early sexual maturation, growth inhibition, and morphological abnormalities, making this procedure impractical. Administration of androgens to adult fish shortly before marketing is sometimes used by commercial farms to enhance colors of males and females [A. Cnaani, personal observations], but this effect is not permanent, and the enhanced colors decline within few weeks. Al l-fe m a l e P ro du c t ion Trout and Salmon. In salmonid fishes, all-female populations are preferred because males mature earlier at a smaller size and have lower flesh quality than females. Male heterogametic sex determination was found in all salmonid species [Davidson et al., 2008], and feminization can be achieved either directly through hormonal treatment or indirectly through sex-reversed parents [Piferrer, 2001]. In many salmonid species, steroid treatments are performed by egg immersion around the time of hatching. It has been suggested that these treatments are effective because the administrated steroid is accumulated in the yolk, which is of a considerable volume in salmonids and may act as a reservoir that provides a continued steroid supply to the developing alevins during the labile period leading to sex differentiation [Piferrer, 2001]. Protocols for the production of monosex all-female populations were developed in the 1970s and 1980s for Atlantic salmon (Salmo salar), rainbow trout (Oncorhynchus mykiss), Coho salmon (O. kisutch), and Chinook salmon (O. tshawytscha) [Donaldson, 1996]. The use of sex-reversed fish as broodstock is widely employed, and all-female populations are the majority of the rainbow trout industry in Europe and the US, as well as the Chinook salmon industry in Canada [Bye and Lincoln, 1986; Dunham, 2004]. In Japan, there is a commercial production of all-female triploid trout and other salmonid species [Arai, 2001]. Common Carp. Common carp (Cyprinus carpio) is the most widely cultured fish in the world, especially in Asia and Eastern Europe. Carp is another group of species in which all-female populations would have economical advantage due to their faster growth and the value of eggs in some countries. In the Israeli aquaculture industry, allfemale offspring are produced in a government research station and released to commercial farms. These allfemale populations are produced by hormonally sexreversing XX gynogenetic females into males and using these males for breeding [Gomelsky et al., 1994; Rothbard, 2006]. As compared to other methods, genetic production of all-female progenies by sex reversing gynogenetic females with methyltestosterone to produce XX neo-males and crossing them with normal females is the most practical for aquaculture. Renewal of the broodstock can be easily done by treating an all-female progeny with androgens, resulting again in XX neo-males [Gomelsky, 2003]. All-female carp cultures resulted in a reduction of variation in harvest size and in about 7 8% yield improvement in commercial farms compared to mixed-sex cultures, with females being 15% heavier than males [Cherfas et al., 1996]. A slightly lower effect was found in experiments conducted in the Central European conditions of the Czech Republic [Kocour et al., 2003, 2005]. However, the economical advantage of rearing allfemale carp in this region is still doubtful due to technical difficulties and the expenses involved in production and renewing the neo-male broodstock. Hybrid Striped Bass. The most widely used hybrid striped bass is produced by crossing female white bass (Morone chrysops) with male striped bass (M. saxatilis) for a cross named sunshine bass. During the 1st year, males appear to grow faster than females; however, females grow faster during the 2nd year and are usually heavier when the fish are harvested [Davis and Ludwig, 2004]. Monosex culture of hybrid striped bass is not a common practice in aquaculture, although in recent years there has been an increasing effort to obtain broodstock to produce monosex populations. This move aims to take advantage of the females better growth and to avoid limitation on transfer and introduction of exotic species. Several studies were conducted to develop and calibrate hormonal sex reversal in hybrid striped bass, with both MT for masculinization and E2 for feminiza- 168 Sex Dev 2009;3: Cnaani /Levavi-Sivan

6 tion [Gomelsky et al., 1999; Davis and Ludwig, 2004]. Hormonal sex reversal of male striped bass to female, combined with progeny testing to identify XX males, may be a useful method to produce all-female populations. Gynogenetic fish were also produced from white bass [Gomelsky et al., 1998], and hormonal masculinization of these fish should produce XX neo-males that can be used as broodstock to produce all-female progeny. An all-female hybrid was obtained by crossing striped bass females with yellow bass (M. mississippiensis) males for a cross named paradise bass. However, in a comparative study for production characteristics, the mixed-sex sunshine bass was superior, and no production parameter for the all-female paradise bass indicated that there is an advantage for their commercial culture [Wolters and DeMay, 1996]. Eel. Freshwater eels (genus Anguilla) enter rivers and streams as sexually undifferentiated glass eels and develop into males or females before migrating back to sea as silver eels. Although heteromorphic sex chromosomes have been identified in some species (e.g., A. anguilla, A. japonica, and A. rostrata ), sexual development is labile, and there is strong environmental influence on sex determination in these species [Davey and Jellyman, 2005]. While females grow faster than males and reach higher mass and market value, the majority of eels develop into males under aquaculture conditions. Wild eels are subjected to commercial and recreational harvesting, and there is a significant concern about the sustainability of eel fisheries worldwide. Females are at particular risk due to their longer duration in freshwater and greater size [Dekker, 2003]. Manipulation of sex ratios in favor of females has the potential of increasing production in aquaculture facilities and support natural populations that are currently under fishing pressure [Davey and Jellyman, 2005]. Natural and synthetic sex steroids can have strong feminizing effects on sexually undifferentiated eels, and sex steroids are readily administered orally to captive eels. Possible resistance by consumers to the use of synthetic hormone treatments in aquaculture may make the use of naturally occurring hormones, such as in gonads of other species, preferable. Reduction of eel stocking density and limitation of interference and social stress may also promote the development of females, although this may not be an economically viable strategy [Davey and Jellyman, 2005]. For wild eels, transferring individuals from areas of high to low density, such as lakes with limited natural recruitment, resulted in high proportions of females and increased growth [Wickström and Westin, 1996]. Silver Barb. The silver barb (Barbonymus gonionotus) is an important species in freshwater aquaculture and inland fisheries in Thailand and the neighboring countries. Females grow faster than males, an advantage in countries where the ovaries are also consumed. All-female gynogenetic fish were hormonally masculinized, and most of these neo-males produced all-female progenies. Allfemale batches grew faster and had better survival than mixed-sex in pond culture [Pongthana et al., 1999]. Monosex female silver barbs are cultured on a commercial scale in Thailand and Bangladesh. In Thailand, neomale broodstocks perform well in hatcheries, which now commercially supply monosex female fingerlings [Dunham et al., 2001]. Atlantic Halibut. In Atlantic halibut ( Hippoglossus hippoglossus) there is an advantage for using all-female populations in aquaculture as females reach market size sooner than males due to their faster growth and later maturity [Bjornsson, 1995]. A protocol for the production of diploid gynogenetic fish as a means for producing allfemale populations in aquaculture has been developed for this species [Tvedt et al., 2006]. However, the low survival of gynogenetic diploids is a major obstacle for using it as a direct means for all-female production on a commercial scale. Direct sex-reversal was demonstrated by feeding postlarval halibut formulated diets supplemented with either MT or E2 for 45 days [Hendry et al., 2003]. The successful masculinization of genetic females is the first step towards indirect feminization through the crossing of these neo-males (phenotypic male with XX karyotype) with normal females. However, this or the use of indirect feminization in commercial scale has not been reported. European Sea Bass. European sea bass (Dicentrarchus labrax) females grow 20 50% faster than males, and there is an interest to develop all-female monosex populations for the aquaculture industry [Saillant et al., 2001]. However, recent efforts to understand the sex determination mechanism have been unsuccessful. Progeny analyses following steroid sex-reversal and gynogenesis also were not effective [Zanuy et al., 2001], and no heteromorphic sex chromosomes or sex-linked markers have been found. However, there are noticeable parental effects on progeny sex ratio. There is evidence that sex determination in European sea bass may be influenced by multifaceted genetic and environmental interactions involving light, salinity, ph, and temperature [Ron, 2006]. Sexual Development in Aquaculture Sex Dev 2009;3:

7 Production of Sterile Fish Many fish species have been moved outside of their native range and some of them have been widely dispersed. Species like common carp, rainbow trout, and tilapias were among the first to be introduced throughout the world, but in times when there was little environmental awareness. In the last few decades there is a growing environmental and ecological concern regarding the introduction of exotic species and their effects on indigenous species and ecosystems. The application of techniques for reproductive control in aquaculture was a precursor to the use of these procedures on introduced exotic species [Shelton and Rothbard, 2006]. There is a relatively large experience in producing triploid cyprinid fish that have reduced reproductive capacity, mostly by inducing autotriploidy. For that purpose there is an advantage to produce triploid females (XXX) over triploid males (XXY) as the former are completely sterile and lack gonads, while males have testicular fragments that occasionally produce viable spermatozoa [Shelton, 2006]. The main purpose of introducing grass carp (Ctenopharyngodon idella) in many countries has been to control aquatic vegetation. Concerns over the introduction of an exotic species were reduced by applying techniques to prevent unwanted reproduction. The use of sterile triploid grass carp has become a common practice in US aquaculture and fisheries management [Rothbard et al., 2000] even though in some rare cases triploid grass carp were found to maturate [Benfey, 1999]. Black carp (Mylopharyngodon piceus) is a snail eating fish that, despite the success in production of sterile fish, its marketing, and introduction, was blocked because of ecological concerns in the US and a lack of funding for the biological control of bilharziasis in African countries [Rothbard, 2006]. An alternative use of sterile fish is for controlling the population of an already introduced exotic species. Extinction of a population can be achieved as a result of shifting the sex ratio caused by the introduction of large quantities of sterile males. This approach has been applied primarily in the control of insects, but a similar strategy has been applied toward the control of sea lamprey (Petromyzon marinus), a serious pest since its introduction into the Great Lakes. This project has obtained promising results so far [Twohey et al., 2003], but among other things, sterilization techniques (currently chemosterilization by bisazir injection) should be improved and become more cost effective [Bergstedt and Twohey, 2007]. Many of the introduced exotic species are highly fecund and capable of producing high numbers of offspring. Therefore, even a low percentage of successful mating involving normal males may be enough to sustain the population, and the number of sterile males needed to overwhelm the fertile ones would be too high from a practical standpoint. Instead of attempting to reduce the total number of progeny by using sterile males, a more efficient strategy could involve shifting the sex ratio of the progeny to eliminate one sex from the population. This will cease further reproduction, as new generations will have only one sex [Gutierrez and Teem, 2006]. Although changing the sex ratio of the population would seem to be difficult to achieve, a precedent for the reduction of a fish population as a result of altered sex ratios has been suggested for the Chinook salmon in the Colombia River [Nagler et al., 2001]. Gutierrez and Teem [2006] developed a theoretical model for the elimination of an introduced Nile tilapia population through the release of YY females. With the tilapia life span and frequent spawning intervals, the timeframe for extinction is estimated in the range of decades. This strategy employs an available technology for manipulation of sex determination, but it requires a long time commitment and allocation of resources. Hybridization between species often results in offspring that are sterile or with low reproductive capacity, having problems with gonad development [e.g., Hulata et al., 1980, 2004]. Hybrids between natural tetraploid and diploid cyprinid species result in sterile triploid offspring. However, some of the offspring have only reduced fertility and are still able to reproduce, while a small portion of the progeny are diploid and could be fully fertile [reviewed by Bartley et al., 2001]. The sunshine bass (hybrid of white and striped bass) is generally sterile, but some percentages of the offspring are capable of reproduction, as was found in rivers in Georgia and South Carolina [Avise and Van den Avyle, 1984]. Thus, although the general concept of species leads us to think that interspecific hybrids will be sterile, the plasticity of reproductive mechanisms in fish is diminishing the practical use of interspecific hybridization for sterilization. Puberty and Gamete Development While sex determination mechanisms are highly variable among fish, later stages of sexual development such as endocrinological pathways involved in gonad development and maturation are more conserved. More detailed 170 Sex Dev 2009;3: Cnaani /Levavi-Sivan

8 information is available on the molecular and cellular biology of the gonad development than on early stages of sexual differentiation. Thus, the following sections will focus more on describing reproductive biology pathways which are relevant for aquaculture. Testis and Spermatogenesis Spermatogenesis, the formation of sperm that is highly adapted for delivering its genes to an egg, is a complex developmental process. It begins with the mitotic proliferation of spermatogonia, then proceeds through 2 meiotic divisions followed by spermiogenesis, during which the haploid spermatids develop into spermatozoa. Spermatozoa then undergo maturation, obtaining the ability to fertilize. Morphologically and physiologically, the spermatogenetic cycle can be divided into the following stages: spermatogonial stem cell renewal, spermatogonial proliferation toward meiosis, 2 meiotic divisions, spermiogenesis, and sperm maturation [Miura and Miura, 2003]. Spermatogonial mitosis can be categorized by spermatogonial stem cell renewal and spermatogonial proliferation toward meiosis [Clermont, 1972]. Mitosis of eel spermatogonial stem cells was promoted by the implantation of E2, but was suppressed by tamoxifen (an antagonist of estrogen), indicating the importance of estradiol during the renewal of spermatogonial stem cells. When gonadotropin (GTH) is secreted from the pituitary, spermatogonial mitosis switches from stem cell renewal to proliferation toward meiosis. GTH does not act directly on germ cells but rather through the gonadal biosynthesis of 11- ketotestosterone (11-KT), which is the major androgen in teleosts [Miura, 1991; Billard, 1992]. Spermatozoa acquire the ability of motility during their passage through the sperm duct. Sperm maturation, the phase during which nonfunctional gametes develop into mature spermatozoa, involves only physiological, not morphological, changes. Ovary and Oogenesis Similar to primordial follicle development in mammals, primary oocyte growth in fish begins with the onset of meiosis and subsequent meiotic arrest in the diplotene stage of the first prophase. The oocytes are then completely enveloped by a monolayer of presumptive granulosa cells, and a thin theca cell layer and epithelial sheath are added to the surface, forming the basic follicle structure [Patino and Sullivan, 2002]. As the follicle develops, the nucleus of the oocyte increases in size, and numerous ribosome-producing nucleoli appear around its periphery ( perinucleolus stage) [Wallace and Selman, 1990]. Initiation of secondary growth is signified by the appearance and accumulation of cortical alveoli. Vitellogenesis (yolk incorporation) marks the final phase of secondary growth during which dramatic follicle growth occurs as the oocyte sequesters vitellogenin, a hepatically derived yolk protein precursor, from the bloodstream [Patino and Sullivan, 2002]. During vitellogenesis, estradiol secreted by the ovary regulates the synthesis of vitellogenins and choriogenins in the liver. When vitellogenesis is complete, there is a steroidogenic shift in which the production of testosterone and estradiol by the ovary is usually reduced, while the production of 17,20 -dihydroxy-4-pregnen-3-one (DHP) is dramatically enhanced, leading to meiotic maturation [Yaron and Levavi-Sivan, 2006]. The rise in DHP during this phase is responsible for induction of the final maturation of oocytes. The LH and FSH secretion in fish is stimulated by several hypothalamic agents, with GnRH being the central stimulator and dopamine being the central inhibitor [Yaron and Levavi-Sivan, 2006]. Through binding to membrane receptors, FSH stimulates follicular development in the ovary, while LH stimulates processes leading to final oocyte maturation and ovulation. Sex Change in Hermaphroditic Fish Sex reversal, the transformation of an individual from one sex to the other in adulthood, is recognized in hermaphroditic fish. It has long been evident that estrogens are the key factors driving female development during the process of sex reversal. In the protandrous (begins life as a male and then changes into a female) anemone fish ( Amphiprion spp.), 11-KT was detected in the sex-reversing fish, and mature females had a higher level of E2, which indicated that the change in gonadal steroidogenesis is associated with sex reversal [Godwin and Thomas, 1993]. In the protandrous black porgy (Acanthopagrus schlegelii), high levels of plasma E2 during the prespawning and spawning seasons were correlated with natural sex change [Chang et al., 1994]. In the protandrous gilthead seabream (Sparus aurata), treatments with E2 induced various changes, including the development of the ovarian parts in the ambisexual gonads and the inhibition of testicular development [Condeca and Canario, 1999]. Moreover, it was shown that differential expression of the cyp19a1a gene is associated with sex reversal and that LH regulates the expression of cyp19a1a during the process of sex reversal [Wong et al., 2006]. Sexual Development in Aquaculture Sex Dev 2009;3:

9 Induced Reproduction Aquaculture requires control over the entire life cycle of the farmed organism, including its reproduction. 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. Methods for hormonal spawning induction of fish were already in use as early as 1930 when Houssay injected some viviparous fish with extracts of pituitary glands freshly removed from other species of fish and observed that the females underwent ovulation [Houssay, 1930]. In some species, captivity is affecting spermatogenesis and male fertility, and hermaphrodite fish present several reproduction challenges in different life stages. Spawning Induction In many fish, hormonal manipulation is required for induction of ovulation and spawning. Currently, there are several known methods for induction of spawning in fish that can be divided into 2 main categories: The Hypophyseal Approach. The hypohyseal approach is based on injecting pituitary hormones, mainly LH, to fish in order to stimulate final oocyte maturation and ovulation. Mammalian GTH, especially human chorionic gonadotropin (hcg), is purified from the urine of pregnant women. hcg, which is commonly used mainly for marine species, is often given in a single dose that ranges between 100 and 4,000 IU/kg body weight [Zohar and Mylonas, 2001]. However, hcg may generate an immune response in the recipient fishes [Levavi-Sivan et al., 2004]. In more recent years the use of ground piscine pituitaries and pituitary extracts to induce spawning in fish was widely used. Treatment has been standardized to a small priming dose (10 20%) and a larger resolving dose (100%) given h apart. Effective doses range from 2 to 10 mg of pituitary per kg body weight of the recipient fish [Yaron, 1995]. There are various drawbacks of the hypophysation methods, including the potential for diseases transmission from donor fish to recipient broodstocks and the fact that the pituitary extract will be effective only on fish from the same species or on closely related species. The Hypothalamic Approach. The hypothalamic approach manipulates the physiology of the pituitary by employing GnRH superactive analogs with or without dopamine antagonists. GnRH and GnRHa were effective in inducing ovarian development, final oocyte maturation (FOM), and ovulation in doses ranging from 1 to 15 g/kg GnRH or 1 to 100 g/kg GnRHa [Donaldson, 1996]. In the early 1980s it was found that dopamine in some fish acts at the pituitary level by inhibiting the basal release of gonadotropin and attenuating the action of GnRH on the gonadotrophs [Chang et al., 1984]. Different fish species vary in the degree of their dopaminergic inhibition. In fish with strong dopaminergic inhibition the simultaneous removal of dopamine inhibition and an increase in GnRH release constitute one of the neuroendocrine events leading to the preovulatory LH surge and spawning. Accordingly, the widely adopted method of spawning induction in these fish relies on the simultaneous application of a potent dopamine receptor (DA-R) antagonist and GnRH superactive analog [Yaron, 1995; Yaron et al., 2003]. GnRH analogs given in slow-release devices can alleviate problems arising from repeated handling and stress. Indeed, such implants have been devised, where the GnRHa is mixed with cholesterol or a nondegradable copolymer of ethylene and vinyl acetate (EVAC) or biodegradable microspheres using co-polymers of lactic acid and glycolic acid (LGA). The implants are injected intramuscularly, proportional to the weight of the spawning fish [Zohar and Mylonas, 2001]. There are several advantages of the hypothalamic approach. First of all, the high potency of GnRH analogs in small amounts ( g/kg) is sufficient for inducing LH release and synthesis. Secondly, GnRH does not generate any immune response in the recipient fishes, and as a synthetic peptide, there is no risk of transmitting diseases. Induction of Sperm Production Many fish exhibit reproductive dysfunctions when reared in captivity. Most commonly, males produce small volumes of milt or milt of low quality [Billard, 1989]. Long-term treatment with GnRHa via delivery systems has proven effective in enhancing milt production in fish and offers certain advantages over the use of acute treatments with either GTH preparations or GnRHas. Production of sperm with very high density of spermatozoa can be a serious problem in cultured fish, especially in flatfish broodstocks. For example, in the European plaice (Pleuronectes platessa), milt from wild fish during the spawning season has a spermatocrit of 60% and flows readily upon stripping. On the contrary, male fish maintained in captivity produce milt with abnormally high 172 Sex Dev 2009;3: Cnaani /Levavi-Sivan

10 spermatocrit in excess of 85%, which is also very viscous and sticky. This milt does not mix well with water and, obviously, cannot fertilize eggs. A similar observation was made in captive Atlantic halibut, where GnRHa delivery systems induced thinning of the milt, making it appropriate for use in artificial fertilization procedures. Conversely, milt from control fish had very high viscosity and spermatocrit [reviewed by Zohar and Mylonas, 2001]. Fish Breeding and Biotechnology The failure of fish to undergo final oocyte maturation, ovulation, spermiation, and spawning can be the result of the lack of LH secretion from the pituitary. Consequently, most spawning induction related research and development efforts have focused on the use of exogenous GnRHs to trigger the release of LH and the chain of events leading to final gonadal development. However, it is also evident that the endocrine system upstream from the pituitary is impaired in captive fish. The next generation of spawning induction technologies will thus be based on: (1) Recombinant fish hormones produced by different expression systems that would be applied for the induction of gonadal development in aquaculture fishes [Levavi-Sivan et al., 2008]. (2) GnRH neurons are the final common conduit through which the brain regulates the secretion of pituitary gonadotropins, and hence, all of reproduction. Converging evidence supports the notion that kisspeptinkiss1 receptor signaling directly regulates GnRH secretion in mammals [Seminara and Crowley, 2008], and recently this has been shown to be true also in fish [Biran et al., 2008]. S u m m a r y The ultimate goal of controlling sexual development in cultured fish is to increase the profitability of aquaculture operations. Aquaculture production involves a large number of different species, each with their own distinct biological characteristics that influence their culturing practices, with species having different traits of economical value. Individual species are adapted to specific aquatic environmental conditions; thus, biological technologies developed for a particular species are not directly transferable to other species. It can be anticipated that knowledge and technologies developed for model fish species aiming to answer basic science questions will be adapted for cultured species, and that the number of fish species in which control of sexual development will be a routine management procedure will continue to increase. Acknowledgment We would like to thank Gideon Hulata and Abe Tucker for their useful comments on early versions of this manuscript. References Arai K: Genetic improvement of aquaculture finfish species by chromosome manipulation techniques in Japan. Aquaculture 197: (2001). Avise J, Van den Avyle M: Genetic-analysis of reproduction of hybrid white bass! striped bass in the Savannah river. T Am Fish Soc113: (1984). Bartley D, Rana K, Immink A: The use of interspecific hybrids in aquaculture and fisheries. Rev Fish Biol Fisher 10: (2001). Beardmore J, Mair G, Lewis R: Monosex male production in finfish as exemplified by tilapia: applications, problems, and prospects. Aquaculture 197: (2001). Benfey TJ: The physiology and behavior of triploid fishes. Rev in Fish Sci 7: (1999). Bergstedt R, Twohey M: Research to support sterile-male-release and genetic alteration techniques for sea lamprey control. J Great Lakes Res 33: (2007). Billard R: Endocrinology and fish culture. Fish Physiol Biochem 7: (1989). Billard R: Reproduction in rainbow trout sex differentiation, dynamics of gametogenesis, biology and preservation of gametes. Aquaculture 100: (1992). Biran J, Ben-Dor S, Levavi-Sivan B: Molecular identification and functional characterization of the kisspeptin/kisspeptin receptor system in lower vertebrates. Biol Reprod 79: (2008). Bjornsson B: The growth pattern and sexual maturation of Atlantic halibut (Hippoglossus hippoglossus L.) reared in large tanks for 3 years. Aquaculture 138: (1995). Bye V, Lincoln R: Commercial methods for the control of sexual-maturation in rainbowtrout (Salmo gairdneri R. ). Aquaculture 57: (1986). Chang CF, Lee MF, Chen GR: Estradiol-17 associated with the sex change in protandrous black porgy, Acanthopagrus schlegelii. J Exp Zool 268: (1994). Chang JP, Peter RE, Nahorniak CS, Sokolowska M: Effects of catecholaminergic agonists and antagonists on serum gonadotropin concentrations and ovulation in goldfish evidence for specificity of dopamine inhibition of gonadotropin secretion. Gen Comp Endocrinol 55: (1984). Cherfas N, Gomelsky B, BenDom N, Joseph D, Cohen S, et al: Assessment of all-female common carp progenies for fish culture. Isr J Aquacult-Bamid 48: (1996). Clermont Y: Kinetics of spermatogenesis in mammals: seminiferous epithelium cycle and spermatogonial renewal. Physiol Rev 52: (1972). Sexual Development in Aquaculture Sex Dev 2009;3: