The Progressive Fish-Culturist 59:1 18-128, 1997 American Fisheries Society 1997

Manipulations of the Reproductive System of Fishes by Means of Exogenous Chemicals

REYNAEDO PATiNO Texas Cooperative Fish and Wildlife Research Unit, U.S. Geological Survey Texas Tech University, Lubbock, Texas 79409-2120, USA Abstract Environmental control of reproductive activity of captive fish is feasible (or potentially feasible) but, with few exceptions, is currently impractical for most species. Therefore, chemical methods of manipulating reproductive activity continue to be widely used in fish production operations worldwide. However, the control of fish reproduction in captivity cannot be exercised without regard to adequate environmental conditions, which can differ markedly for different species. This review provides a synopsis of relevant aspects of fish reproductive physiology and addresses current and promising future chemical methods of sex control, gonadal recrudescence, and spawning. Most research on the control of reproduction in fishes has focused on female physiology because ovarian development and maturation are easily disturbed by environmental stressors. Control of sex ratios by steroid treatment has become a well-established technique for several fish species, but the technique continues to be problematic in some cases. Final gonadal growth and spawning usually can be achieved by implant treatment with gonadotropin-releasing hormone analogs (GnRHa), which in some species have to be applied in combination with dopamine antagonists to enhance responsiveness to GnRHa However, efforts to accelerate gonadal recrudescence and maturational competence by chemical means have yielded mixed results, reflecting a relative lack of understanding of the basic physiological and biochemical mechanisms controlling these processes. The potential benefits of using reproductive pheromones to manipulate gonadal development and spawning has been demonstrated in a few species, but further research is needed to determine whether this technique is applicable to fish culture. Because a reliable supply of young fish is critical for the expansion and diversification of fish culture operations, the use of chemicals in combination with adequate environmental conditions to control gametogenesis and spawning in fishes will continue to be an important tool for the fish culturist. The ability to manipulate the reproductive system of fishes and enhance the reliable supply of fingerlings is relevant to fish culture in general, but is particularly important to the expansion and diversification of intensive fish culture operations (e.g., Bromage 1995). Control over the timing of gamete production allows broodstock spawning to be confined to the most appropriate period of the year for hatchery management and operations or to be staggered throughout the year to allow multiple spawnings and maximum use of facilities. In this manner, hatchery operation costs can potentially be minimized and fish production can be maintained or even enhanced. Control of sex ratios or fertility are other important management tools in aquaculture. For example, in some species, one sex may be of greater commercial value than the other due to sex-specific phenotypic traits, such as higher growth rates (in the food fish market) or body form or coloration (in the ornamental fish market). Also, for species that reach sexual maturity before they reach market size, culture of monosex or infertile populations can enhance overall production rates by reducing the

energy that otherwise would be spent in reproductive activity and redirecting it into somatic growth. The timing of "natural" (i.e., not chemically induced) gonadal development as well as the sex ratio in some captive fishes can be controlled by the judicious manipulation of the appropriate environmental factors (Bromage 1995; Strussmann and Patino 1995). The greatest advantage of using environmental methods for reproductive manipulation of food fish, is that chemical use can be avoided. Thus, both the issue of human health impacts and concerns about drug permitting and regulations are alleviated. However, exclusively environmental control of sexual development and activity has been accomplished with relatively few species. This approach also can require considerable investments in hatchery infrastructure and operations, which makes it impractical in most situations (see also Shelton 1989). Therefore, in general, the most practical and reliable methods of reproductive control currently available for fishes consist of the use of exogenous chemicals that affect various aspects of the reproductive system. Chemical methods of reproductive modification are the main subject of the present review, but where appropriate, nonchemical methods are also briefly discussed. An understanding of the actions of chemicals on fish reproduction requires basic knowledge of the reproductive system. Therefore, relevant aspects of the organization and regulation of reproductive processes are described first. Applied aspects of reproductive biology are subsequently discussed, including the use and action of chemicals in the control of sex ratios, gonadal recrudescence, and spawning.

Basic Reproductive Biology

Environmental Physiology As in other vertebrates, reproductive processes in fishes are controlled by endogenous biological rhythms as well as environmental cues (Munro 1990). Environmental cues important for reproduction consist of ultimate factors (e.g., water quality, food availability, predation) which, within an evolutionary context, have determined when and where breeding occurs, and proximate cues (e.g., photoperiod and temperature and their directional changes, appearance of spawning substrate, pheromones), which serve to entrain reproductive processes to environmental cycles. Thus, environmental cues that control reproduction in fishes include physical, chemical, and biological factors. Environmental cues are detected by receptors in sensory organs, such as the eyes, olfactory organ, pineal gland, and are then transduced by the central nervous system into physiological signals that elicit appropriate responses from specific targets in the body. The most important pathway of communication between the central nervous system and the sex organs (gonads) is the endocrine system. Thus, endocrinological research of the brainpituitary-gonadal axis has played a preeminent role in efforts to understand reproductive processes in fishes (Reading and Patiho 1993). Because fishes are poikilothermic vertebrates, temperature is one environmental cue that can also directly affect reproductive processes at levels other than the central nervous system. Brain-Pituitary Function The pituitary gland, located at the base of a region of the brain known as the hypothalamus, produces the gonadotropin hormones or gonadotropins (GtH). The pituitary cells that produce GtH are the gonadotrops. With some exceptions (Van Oordt and Peute

1983), hypothalamic control of gonadotrop activity in teleosts is exerted via direct nervous innervation (Peter et al. 1990). One potent stimulatory factor from the hypothalamus is gonadotropin-releasing hormone (GnRH). In some teleosts, dopamine acts as gonadotropin release inhibitory factor (GRIF) by inhibiting GnRH-induced GtH release (Goos et al. 1987; Peter et al. 1991). However, in other species, dopaminergic inhibition of GtH release is either weak or nonexistent (Copeland and Thomas 1990; Carrillo et al. 1995; Zohar et al. 1995). Pituitary GtH production seems to be under positive as well as negative feedback control by gonadal steroids, and positive feedback is the dominant mode of control in juvenile fish and during gonadal recrudescence (Crim and Evans 1983; Trudeau et al. 1991). Like most other vertebrates, teleosts produce two types of pituitary GtH, which are referred to as GtH-I and GtH-II; these hormones are believed to be functionally homologous to tetrapod follicle-stimulating hormone and luteinizing hormone, respectively, because the former seems to be primarily involved in follicular growth and the latter in maturation (Swanson 1991). Gonadal Function Ovary.Among vertebrates, ovarian function is usually more sensitive to adverse environmental conditions than is testicular function. Thus, from the standpoint of animal agricultural production, understanding female reproductive physiology is generally of greater relevance. This biased interest has resulted in relatively greater research efforts invested towards understanding ovarian physiology. The basic unit of the ovary is the ovarian follicle (Patiflo and Takashima 1995). The ovarian follicle essentially consists of a central germ cell (oocyte) and surrounding somatic cells (follicle cells). A monolayer of follicle cells, the granulosa cells, envelops the oocyte and makes direct cytoplasmic contact with it via intercellular channels (gap junction channels). Granulosa cells also make gap junctional contacts with each other. The granulosa cell-oocyte complex is not supplied with blood vessels; therefore, gap junction channels are believed to play important roles in nutrient trafficking during oocyte growth and in the intercellular exchange of chemical signals, including maturational signals (York et al. 1993; Yoshizaki et al. 1995). A basement membrane and an outer, heterogeneous layer of theca cells encloses the granulosa cell-oocyte complex. Ovarian follicle cells produce steroid hormones. Estradiol-17,B is an important steroid produced by the growing ovarian follicle. This steroid leaves the follicle via blood vessels supplying the theca cell layer and is transported to the liver, where it induces the production of vitellogenin. Vitellogenin is transferred to the ovary via circulation, where it is taken up by the oocyte and deposited as yolk. During the final stages of follicular development, follicle cells acquire the ability to produce maturation-inducing steroid (MIS), which, depending on the species, may be 17a,20~-dibydroxy-4-pregnen-3-one (DHP)or17a,20,13,21-trihydroxy-4-pregnen-3-one (20,B-S). The maturational action of MIS is local. It binds to a receptor on the surface of the oocyte and triggers oocyte maturation (Nagahama 1987; Redding and Patiflo 1993). Estradiol-17g production seems to be under the physiological control of GtH-I, whereas MIS production is controlled by GtH-II (Swanson 1991). The acquisition of oocyte maturational competence is an important yet, until recently, largely ignored event of late follicular development. Maturational competence is defined as the ability of the intrafollicular oocyte to respond to MIS stimulation. This competence precedes MIS production and is under the control of GtH (possibly GtH-II; Patino and Thomas 1990a; Redding and Patiflo 1993), perhaps via follicular production of insulinlike growth factors (Kagawa et al. 1994). In fishes and amphibians, maturational
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competence is associated with increased concentrations of oocyte MIS receptors, which presumably enhance oocyte sensitivity to MIS (Patiflo and Thomas l990b; Thomas and Patiflo 1991; Liu and Patiflo 1993). In Atlantic croaker Micropogonias undulates, maturational competence is also accompanied by the activation of gap junction protein (connexin) genes and the enhancement of granulosa cell- oocyte gap junction contacts (York et al. 1993; Yoshizaki et al. 1994). Research with nonpiscine vertebrates has indicated that follicle cell-oocyte coupling seems required for the transduction of maturational signals (e.g., Patiflo and Purkiss 1993). Testis.Most teleost testes are of the lobular type. Namely, they contain lobules or blind-ended sacs that enclose the germinal epithelium. Male germ cell development (spermatogenesis) can be divided into three general phases: meiosis (chromosome reduction divisions), spermiogenesis (sperm formation), and spermiation (sperm release) (Patiflo and Takashima 1995). Meiosis and spermiogenesis occur within spermatocysts inside the lobule. Spermatocysts contain the developing spermatocytes and Sertoli cells. The latter serve to support, regulate, and nourish the spermatocytes. Premeiotic (secondary spermatogonial) and meiotic cell divisions are incQmplete; thus, spermatocytes remain joined by intercellular bridges during meiosis and spermiogenesis. The resulting cellular syncytium is thought to provide the means for the developmental synchrony observed among all germ cells within a given spermatocyst. Mature spermatozoa are released from spermatocysts into the sperm duct system at spermiation. Leydig cells are usually found in the interstices between the lobules. These steroidogenic cells produce androgens and other steroids. Androgens are necessary to support spermatogenesis (Miura et al. 1991). An important androgen produced by the teleost testis is 11- ketotestosterone. In salmonids, GtH-I levels are elevated during the earlier stages of spermatogenesis; thus GtH-I may be the physiological GtH that controls androgen production (Planes and Swanson 1995). As in the ovary, MIS (DHP in the species studied) is also produced by the testis. This maturational steroid may play a role in spermiation and in the acquisition of sperm motility (Miura et al. 1992). At the time of spermiation in salmonids, GtH-II levels in blood become higher than those of GtH-I; GtH-II also has higher MIS-inducing activity (Planes and Swanson 1995). Therefore, GtH-II seems to be the physiological hormone that controls (via MIS) the release of sperm. Sex Differentiation Although many cases of hermaphroditism have been documented in teleost fishes, most species are gonochoristic. Namely, the gonadal sex of the individual is established during early development and is stable thereafter. However, the direction of gonadal sex differentiation in gonochoristic fishes may be defined not only by the genotype of the individual but, as is being discovered in an increasing number of species, also by the environment (Strussmann and Patiflo 1995). Temperature is one environmental factor that can affect sex differentiation in fishes. In atherinid fishes, for example, high or low water temperatures at the appropriate time during hatchling development yield a preponderance of males or females, respectively (Conover and Fleisher 1986; Strussmann and Patiflo 1995). In channel catfish Ictalurus punctatus, high- temperature treatment (34C) between days 10 and 24 after fertilization can skew the sex ratio towards females (Patiflo et al. 1996). Temperature can also affect the sex ratio in several tilapia species, but the results obtained have been inconsistent between studies and between species (Mair et al. 1990; Baroiller et al. 1995a, l995b). Some of the inconsistencies observed with the tilapias may be explained by the different experimental temperature ranges used in the various studies;
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also, it is possible that the response to temperature can vary even among different strains of the same species (see Strussmann and Patiflo1995). The specific genes that ultimately direct gonadal sex differentiation in fishes have not been identified. However, it appears that the timing and pattern of steroid production during ontogenesis are important elements of this process (Reading and Patiflo 1993). Bimodal patterns of body steroid content (Feist et al. 1990) and of steroidogenesis (Fitzpatrick et al. 1993) have been reported in salmonids at the time of sex differentiation. Also, numerous studies have shown that steroid treatment during a critical period of gonadal sex differentiation can yield monosex, or primarily monosex, populations (typically, androgens produce males and estrogens produce females) (Schreck 1974; Hunter and Donaldson 1983; Shelton 1989). Expression of aromatase, the enzyme that produces estrogens, and the consequent production of estradiol- 17,B seem to be of physiological relevance in directing the onset of ovarian differentiation in salmonids (Piferrer et al. 1994). Moreover, in tilapias, treatment of genotypic female fry with antiestrogens results in testicular development (Hines and Watts 1995). Thus, ovarian differentiation may be a steroid (estrogen) receptor-mediated event. Interestingly, estradiol-17' also seems to be involved in the natural induction of gonadal sex reversal (male-to-female) in a protandrous hermaphrodite, the black porgy Acanthopagrus schlegeli (Chang et al. 1994). An androgen receptor with high affinity to masculinizing steroids has been recently described in the gonads of adult teleosts (Fitzpatrick et al. 1994). If the presence of this receptor also can be confirmed in the indifferent gonads, it would indicate the possibility that natural or exogenous androgen-induced testis formation is also a steroid (androgen) receptormediated event. Finally, the available evidence indicates that the timing of gonadal sex differentiation, but not its direction, may be influenced by pituitary hormones (Van Winkoop et al. 1994). Pheromones and Reproduction Pheromones are chemical signals released into the environment by an individual to elicit a response from other individuals of the same species. The presence of sex pheromones has been established in several teleost families (Stacey et al. 1994). Sex pheromones are those pheromones involved in the communication between nonspecific individuals that relate to some aspect of their reproductive function. Research on sex pheromones in fishes has been primarily directed toward the physiology of spawning behavior. Also, most knowledge available in this regard is derived from research performed with goldfish Carassius auratus (Sorensen et al. 1990; Stacey et al. 1994). In this species, DHP seems to serve a dual role. This steroid not only induces final gonadal maturation in both sexes (see earlier discussion), but it is also released into the water by the female to induce spermiation in males. Moreover, as females begin the subsequent process of ovulation, their ovaries also produce prostaglandins. These chemicals are then released into the water to elicit spawning behavior in males. The response of males to these pheromones involves detection of the chemical signals by the olfactory epithelium and transduction by the brain into endocrine signals (see earlier discussion). Thus, it appears that female sex pheromones act to synchronize final maturation and spawning between male and female goldfish. Additional steroidal (and some nonsteroidal) compounds have been proposed as final maturation pheromones in various fish species. Pheromones that act at earlier stages of gonadal development also appear to be present in fishes. For example, male pheromones that induce ovarian follicle development in females have been reported in the African catfish Clarias gariepinus (Van Weerd and
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Richter 1991) and blue (=threespot) gourami Trichogaster trichopterus (Degani and Schreibman 1993). Like the final maturation-inducing pheromones, the gonadal growthinducing pheromones may act at the level of the central nervous system (olfaction). However, the chemical nature of these pheromones is unknown. Applied Reproductive Biology Control of Sex Ratios Chemical treatments.In many gonochoristic teleosts, gonadal sex can be easily manipulated if the appropriate treatment is performed before their gonads become irreversibly committed to either sex. Although various nonsteroidal compounds have been used to induce sex change in teleosts (Piferrer et al. 1994; Hines and Watts 1995), in general the most reliable results have been obtained with steroid treatment. However, sex control by steroid treatment is not fully effective in some species. In channel catfish, for example, steroid-induced masculinization of genotypic females has been difficult to achieve (Goudie et al.1983; Davis et al. 1990, 1991; see also below). Synthetic steroids usually have greater potency than natural steroids, either because they have greater intrinsic activity or because they are degraded at a slower rate. Steroid treatment normally should begin before the gonads show histological signs of differentiation (Yamamoto 1969), which occurs within the first few days or weeks of hatching in most of the species studied. The duration of the treatment needed to achieve maximum effect varies with the species and must be empirically determined. In salmonids, steroid exposures as short as 2 h have been successfully used to induce sex change (Piferrer et al. 1994; Feist et al. 1995). Methods of treatment normally involve the immersing of eggs or fry into solutions containing steroids (particularly for species in which natural sex differentiation occurs before feeding) or the feeding of diets containing steroids. The synthetic androgen,17a-methyltestosterone (MT) has been commonly used to transform genetic females into functional males in various species. More recently, mibolerone (Torrans et al. 1988), 17a- methyldihydrotestosterone (MDHT) (Piferrer et al. 1994), trenbolone acetate (TBA) (Galvez et al. 1995), and other synthetic androgens have proven to be generally more effective than MT. However, there seems to be some speciesspecific differences in androgen effectiveness. In channel catfish, for example, MT, MDHT, and a variety of other synthetic androgens failed to masculinize genetic females (Davis et al. 1990, 1991), whereas TBA treatment seemed to produce monosex male populations (Galvez et al. 1995). Compounds that inhibit aromatase enzyme activity (Piferrer et al. 1994) or estrogen action (Hines and Watts 1995) also have been used to produce malebiased populations, presumably by inhibiting estrogen production or receptor binding, respectively. Male monosex populations are desirable in commercial tilapia culture operations in order to eliminate or reduce unwanted reproduction. Thus, androgen treatment of tilapia fry has become routine in several countries (Shelton 1989; MacIntosh and Little 1995). Functional feminization of genetic males has been accomplished by treatment with the natural estrogen, estradiol-17, or with several synthetic estrogens, such as ethynylestradiol or diethystilbestrol (Hunter and Donaldson 1983). In some species, such as channel catfish, certain androgens can produce paradoxical feminization of genetic males (Goudie et al. 1983; Davis et al. 1990, 1991) and thus can be used for the production of female populations.

Steroid treatment can also be used to produce infertile populations, which provides an alternative to the use of monosex populations for the purpose of inhibiting reproductive development and activity. Normally, treatments for inducing infertility involve steroid dosages and exposure periods greater than those required for functional sex change (Hunter and Donaldson 1983). Nonchemical treatments.There are options for the production of monosex or sterile food fish populations that avoid direct treatment with steroids. For example, crosses between sex-reversed females carrying a male genotype (XY) with normal XY males yield progeny containing 25% males with a YY genotype. If these YY males are then crossed with normal XX females their entire progeny will consist of untreated, normal XY males. Indeed, this particular scheme has been achieved with channel catfish (Davis et al. 1991). Chromosome- set manipulations (androgenesis, gynogenesis, polyploidy; Thorgaard 1995) and interspecific hybridization (Hunter and Donaldson 1983; Zohar 1989) also can be used to produce monosex or sterile populations. However, all of these nonchemical options present some practical problems. For example, strategies involving steroid treatment and selective matings require several generations to implement and thus can be relatively expensive. Also, chromosome-set manipulations and interspecific hybridizations often yield low survival rates; thus their general applicability has not been established. Even in cases where interspecific hybrids are viable, the need to maintain pure inbred lines makes this strategy impractical in many hatchery situations (Zohar 1989). Manipulations of water temperature may be yet another option for the nonchemical control of sex in fishes. As already mentioned, the range of species for which the rearing temperature of fry has been shown to affect population sex ratios seems to be much broader than previously believed. This technique has worked well at the experimental level for the pejerrey Odonthestes bonariensis, a recreationally and commercially significant fish in South America and Japan. All-male as well as all-female populations can be readily obtained by temperature manipulations during the fry stage (Strussman and Patiflo 1995; Strussmann et al. 1996). However, further research is needed to judge the general effectiveness of this temperaturebased method of sex control in fishes. Control of Gonadal Development and Spawning Chemical methods for the induction of gonadal development and spawning in fishes cannot be practiced without regard to environmental influences. Thus, depending on the characteristics of their natural habitat and their breeding habits, captive fishes require defined conditions of water temperature and quality, photoperiod, nutrition, spawning substrate, and other exogenous factors in order to make them responsive to artificial maturation- or spawning-inducing stimuli. Therefore, the use of chemicals alone should not be viewed as substitute for species-specific husbandry practices. Also, off-season gamete production from seasonal breeders requires environmental adjustments such as shifts or compressions in photoperiod or temperature patterns (Bromage 1995). However, within the confines established by environmental conditions, chemical treatments can be very useful to manipulate or remedy reproductive processes or failures in otherwise healthy fishes. Induction of maturation and spawning.One of the earliest and most important success stories on the use of chemicals to manipulate fish reproduction was the development in the early 1930s of the technique known as hypophysation (Houssay 1931). This technique involves the injection of pituitary gland (hypophysis) suspensions or extracts into broodfish to stimulate gonadal maturation and spawning. However, by current standards
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hypophysation is a crude and unreliable technique because its effectiveness depends on the maturational condition of the donor fish pituitaries (in terms of their GtH-II content, which can vary widely). Calibrated carp pituitary extract has been commercially available in Israel for use in the culture of cyprinid species, but ongoing research there aims to examine the advantages of replacing this modified hypophysation procedure with current GnRH technology (Rothbard and Yaron 1995; see below). Nevertheless, the relatively low cost of hypophysation makes it a popular technique worldwide for the artificial induction of fish spawning (Zohar 1989; Bromage 1995). Intensive fish culture systems usually require methods of induced spawning that are more reliable than the original hypophysation technique. This need has led to considerable research to identify alternative chemical treatments. Human chorionic GtH (hCG), partially purified fish GtH-II, and more recently, GnRH are among the preparations that have been tested. However, hCG can cause immunoreactions in the recipient fish, thus reducing or even eliminating the effect of the hormone in subsequent injections of the same broodstock individuals. Also, the effectiveness of fish or mammalian GtH as inducers of spawning in fishes seems to be fairly species-specific. In contrast, GnRH (a decapeptide) does not seem to elicit immune responses in recipient fish and also has a broader species-spectrum of effectiveness than GtH. Moreover, current technology allows the production of sufficient amounts of synthetic, superactive GnRH analogs at costs that seem justifiable within the context of intensive fish farming. Therefore, treatment with GnRH is rapidly becoming a favorite chemical method to induce spawning in fishes (Zohar 1989; Bromage 1995). Much of the recent research on the induction of fish spawning by means of GnRH has focused on three areas: designing superactive GnRH analogs, determining the role of putative GRIF, and designing effective GnRH delivery systems (Zohar 1989; Zohar et al. 1995). The design of superactive GnRH analogs is based on the proven principle that analogs with relatively high binding affinities for the pituitary GnRH receptor and with relatively low degradation rates will have higher potency in inducing pituitary GtH release than native GnRH (Habib) and Peter 1991; Zohar et al. 1995). In cyprinids and some other fishes, endogenous GRIF (dopaminergic substances) also play an important role in the negative control of GtH release, and therefore treatment with GnRH analogs alone is unable to induce their full spawning potential. In these species, GnRH analogs normally are applied in combination with dopamine antagonists, such as domperidone or pimozide (Peter et al. 1988). However, dopamine antagonist cotreatment is unnecessary in those species such as sciacnid fishes (Copeland and Thomas 1990), sea bass Dicentrarchus labrax (= European bass Morone labrax) (Carrillo et al. 1995), and gilthead seabream (=gilthead bream) Sparus auratus (Zohar et al. 1995) that lack significant dopaminergic inhibition of pituitary GtH release. In fact, in sea bass pimozide had an adverse effect on GnRH-induced spawning (Carrillo et al. 1995). There are two basic delivery systems for spawninducing hormones: those that produce acute (bolus) target tissue exposures to the hormone and those that yield a more sustained or chronic exposure. Currently, GnRH (and GtH) are most commonly delivered by bolus injections into the fish. However, in many species multiple injections of hormones (two or more) are usually necessary to achieve satisfactory spawning results (Zohar 1989). In this regard, it seems likely that the first (priming) injection is needed to induce final differentiation of the ovarian follicle, including the acquisition of maturational competence, and that subsequent (resolving) injections induce MIS production and the consequent maturation and ovulation of the oocyte (see also Patiflo and Thomas 1990a). Even in cases where maturational competence is not of concern, if the target species is one

that normally spawns several times over a prolonged breeding period then multiple injections may still be necessary to achieve full spawning potential. A major drawback of multiple hormone injections is that some species are unable to cope with the stress associated with repeated handling. Handling stress can cause gonadal regression and, in some cases, even death of the broodstock. One way to avoid excessive handling of the broodstock is by using bolus feeding strategies rather than injections. For example, dietary administration of GnRH analogs has been used successfully to induce spawning of spotted seatrout Cynoscion nebulosus (Thomas and Boyd 1989), and oral delivery of GnRH analogs also seemed to be effective in common carp Cyprinus carpio, rainbow trout Oncorhynchus mykiss, and the African catfish Clarias gariepinus (Breton et al. 1995). Also, feeding a diet containing GnRH analog to spotted seatrout at 3-5- week intervals resulted in multiple spawnings over their natural breeding period without any noticeable declines in fecundity, fertility, or embryo survival (P. Thomas and others, University of Texas Marine Science Institute, unpublished data). This hormone feeding technique minimizes the handling of fish to a single ovarian biopsy for the initial selection of appropriate female broodstock. Although the applicability of this technique to commercial aquaculture needs further study, the results obtained to date show promise (see also Solar et al. 1990; McLean et al. 1991). The need for multiple treatments with GnRH analog can be eliminated with the use of sustained delivery systems. Cholesterol or cholesterol-cellulose implants containing GnRH analog have yielded good results with various warmwater and coldwater species, and more recently, a number of biodegradable polymers also have been successfully tested (e.g., Zohar et al. 1995; Mylonas et al. 1995). In a recent study (Mylonas et al. 1995), GnRH analog was loaded on slow-release microspheres made of biodegradable polyanhydride and injected into striped bass Morone saxatilis and At [antic salmon Salmo salar. This single treatment resulted in successful ovulation in females and higher sperm production in males of both species. Another interesting but largely untested possibility for the chemical control of maturation and spawning in fishes concerns the use of reproductive pheromones. For example, the steroid DHP produced by female goldfish controls not only the onset of ovarian maturation in females but also spermiation in males (see Basic Reproductive Biology). Therefore, if added to water, this steroid could be used to control the timing or amount of sperm production. Indeed, preliminary tests have confirmed the feasibility of this procedure to increase male fertility in common carp and goldfish (Zheng et al. 1995). However, more research is necessary before the applicability of this procedure to commercial aquaculture can be properly evaluated. Induction of gonadal growth (recrudescence) and maturation.Failure of the gonads to develop and reach maturational competence also can be of concern in fish culture operations, especially when it relates to efforts to advance puberty and maturation of broodstock. However, relatively little research has been performed on chemical methods to accelerate gonadal recrudescence. Unlike the case of final maturation, it appears that neither bolus nor chronic treatments with GnRH analog alone can effectively induce early gonadal growth (Garcia 1989; Schreck et al. 1995). However, as mentioned earlier, gonadal steroids have a positive influence on the production of pituitary GtH in immature and recrudescing fishes. Therefore, several studies have used sustained-release system to test the effects that steroids, in combination with GnRH analog, have on gonadal development. For example, implants of GnRH analog and testosterone accelerated sexual development in rainbow trout (Crim and Evans 1983) and striped mullet Mugil cephalus (Tamaru et al. 1989).
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However, androgen analogs such as MT have provided mixed results. In sea bass (=barramundi perch) Lates calcarifer, for example, implants of MT alone suppressed oocyte development, but when given in combination with GnRH analog, they had no effect on the latter's ability to induce early maturation (Garcia 1990). In striped mullet, MT given with GnRH inhibited ovarian development in relation to untreated controls or to fish treated with GnRH analog and testosterone combined (Tamaru et al. 1989). Overall, the information available on the effectiveness of steroids to accelerate gonadal growth is promising; however, it is not possible at this time to make generalizations about the applicability of this technique to aquaculture. Combinations of environmental phase shifts and hormone treatment have been successfully used to accelerate recrudescence and spawning or to stagger spawning activity throughout the year in some species, such as the sing), (=stinging catfish) Heteropneustes fossilis (Alok et al. 1994) and gilthead seabream (Zohar et al. 1995). Finally, the discovery of ovarian recrudescenceinducing pheromones of male fish origin (Van Weerd and Richter 1991; Degani and Schreibman 1993) suggests that these pheromones may be useful to enhance early ovarian growth. Tests of this potential technique will become possible once the pheromones are identified. Conclusions Considerable advances in chemical methods to control fish reproduction have been made since the inception of the hypophysation procedure over 60 years ago. Factors that have supported the development of reproductive technology in fishes include an improved knowledge of their basic reproductive processes (including environmental influences) and the recent advances in GnRH analog pharmacology and delivery systems. Also, the control of sex ratios by steroid treatment has become a fairly simple and consistent procedure in several species. However, the reliable supply of fry continues to be a major obstacle to the expansion and diversification of fish culture operations. Although there are close to 25,000 extant fishes, of which about 1,000 have been cultivated in some form (Bromage 1995), adequate techniques of reproductive management exist for only a very small percentage. Relevant aspects of female reproductive development that are still poorly understood include the physiological and biochemical mechanisms that regulate gonadal recrudescence and maturational competence. Therefore, although environmental methods to induce spawning should be used when available and practical, it seems evident that continued research and development of chemical methods will be necessary to achieve captive spawning in the vast majority of aquacultural fishes. Acknowledgments Funding from U.S. Department of Agriculture, grant 91-37203-6680, and from Texas Higher Education Coordinating Board, grant 003644-060, contributed to the development of this manuscript. Valuable criticisms of an early draft by Martin S. Fitzpatrick, Izhar Khan, and Nick C. Parker are greatly appreciated. General information on teleost pheromones and GnRH slow-release methods was provided by Peter Sorensen and Yonathan Zohar, respectively, and is also appreciated. The Texas Cooperative Fish and Wildlife Research Unit is jointly supported by the U.S. Geological Survey, Texas Tech University, the Texas Parks and Wildlife Department, and the Wildlife Management Institute. This is Technical Report T9-735 of the College of Agricultural Sciences and Natural Resources, Texas Tech University.
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