Fish Physiology and Biochemistry vol. 7 nos 1-4 pp 395-405 (1989) Kugler Publications, Amsterdam/Berkeley

Endocrinology and fish farming: Aspects in reproduction, growth, and smoltification Y. Zohar National Centerfor Mariculture, Israel Oceanographicand Limnological Research, Eilat, Israel Keywords: endocrinology, fish, farming, reproduction, spawning, sex control, growth, smoltification

Introduction The close links between the development of fish farming and the science of endocrinology are schematically demonstrated in Fig. 1 which indicates three of the major goals in fish farming. The first is to reproduce a particular species in captivity. This involves controlling spawning, increasing fecundity, altering sex ratio, and in some cases, advancing puberty. The second is to achieve high survival rates. Again, reproduction is involved since the timing of spawning might be related to survival rates of larvae and fingerlings. In some fish species, especially marine pelagic spawners (e.g., sea bass, sea bream, mullet), the spawned eggs and the newlyhatched larvae are extremely small and the larval phase is critical in terms of survival. High survival rates in some salmonids depend on successful smoltification. Understanding and controlling metabolism will, no doubt, contribute to improving both larval growth and smoltification. The third goal is to accelerate the growth of the farmed fish. For this one has to be able to manipulate gametogenesis and produce monosex or sterile fish populations. Interfering with metabolism and increasing appetit might also result in accelerated growth rate. In salmonids, smoltification and transfer to seawater are of major importance for obtaining an optimal growth rate. All the above processes, which are related to the principal goals in fish farming, are controlled by a wide range of hormones. Fig. 1 lists some of the

hormones which have been used or proposed for use in improving reproductive success, survival rate and growth rate. Understanding the basic hormonal mechanisms which control the processes of reproduction, growth, metabolism, osmoregulation, and smoltification underlies the development of technologies and therapies to overcome hormonal failures and to improve performances of farmed fish. This paper summarizes the "Workshop on Aquaculture" held at the First International Symposium on Fish Endocrinology, and discusses the contribution of basic studies in fish endocrinology to the development of bio-technologies applicable in fish farming. Aspects of three fields of major importance to fish farming are reviewed: reproduction, growth, and smoltification. Reproduction Induction, synchronization, and phase shifting of ovulation and spawning In most farmed fish, a regular egg supply depends on the manipulation of ovulation and spawning. In many species, exposure of the fish to suboptimal environmental conditions results in atresia or vitellogenic oocytes; the processes of maturation, ovulation, and spawning have to be induced. In others, these processes have to be synchronized to enable predictable egg supply. Phase-shifted spawning time will allow year-round production of eggs and fingerlings.

396

ENDOCRINOLOGY AND FISH FARMING GnRH, GHRH

-

-

I

GnRH

GH

CATECHOLAMIES

INSUUN

EISUUN

GEH

PROLACTN

TRH, TSH

STERODS T T 3 '4

STERODS

SRF

T T 3 '4

SOMATOMEDINS

oF r

f

IREPRODUCTIVE SUCCESS

SPAWNING FECUNDITY SEX RATIO PUBERTY

rF

VI IF I

T

T

F I rF

HIGH SURVIVAL RATE

RAPID GROWTH ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

SPAWNING TIME LARVAL GROWTH SMOLTIFICATION METABOLISM

SEX RATIO STERILITY METABOLISM APPETITE SMOLTIFICATION

Fig. 1. Fish farming is dependent upon success in three areas - reproduction, survival and growth (indicated in the boxes). This requires control of physiological processes and parameters indicated below. Some of the hormones which are involved in their regulation or have been used to improve related performances in fish farming are indicated above.

Environmental manipulation of broodfish is currently used to phase shift spawning time in a variety of fish species. The hormonal approach is presently more feasible for the induction and synchronization of ovulation and spawning in commercial hatcheries. A variety of hormones of the brainpituitary-gonadal axis have been used for these purposes and continue (Lam 1982; Donaldson and Hunter 1983). Pituitary extracts and human chorionic gonadotropin have been the most frequently used agents in commercial farms. Calibrated pituitary extracts (with gonadotropin content determined by radioimmunoassay and bioassay) of different fish species are commercially available for prices of US$1 to US$2/kg body weight (BW) of fish. The high degree of species specificity of fish gonadotropins (GtH) and the expenses involved in obtaining purer preparations of GtH led to the search for alternative hormonal therapies to manipulate ovulation and spawning. Increasing evidence suggests that the lack of final oocyte maturation,

ovulation and spawning in farmed fish is the result of a failure to release GtH (Zohar 1988a, b). The use of GtH releasing hormones (GnRH) for the induction of a GtH ovulatory surge and of ovulation and spawning seems to represent the most efficient therapy since: 1. GnRH and its analogs stimulate the secretion of endogenous GtH; they can be synthesized and obtained in a pure form; they exhibit a low degree of biological species specificity. Mammalian GnRH preparations, commercially available, can be used in a variety of fish. GnRH and its analogs are small peptides which are non-immunogenic and practical application requires low dosages (a few tg/kg) which is economically advantageous (Sigma price for [D-Ala 6 -Pro9 - NET]-LHRH is around US$.02//4g). GnRH-based spawning induction therapy is currently employed in a variety of commercially important fish species (Donaldson and Hunter 1983; Zohar 1988a, b), such as salmonids, cyprinids, mullet, milkfish, seabream, sea bass, plaice, flounder,

397 catfish, sablefish, and herring. This therapy is very effective in inducing ovulation in salmon females held in seawater net pens (W. Dickhoff, comment in this workshop). The further development of GnRH-based spawning induction technology depends on research efforts in three major fields: the interactions between GnRH and the gonadotropin release inhibitory factor (GRIF) in the regulation of GtH release and ovulation, structure-activity relationship of GnRH and its analogs and, the mode of GnRH administration to the fish. These are discussed below. Since the demonstration that dopamine acts as a gonadotropin-release inhibitory factor in goldfish (Peter et al. 1986) a dual regulation of GtH release by GnRH and dopamine has been proposed for carp, Chinese loach, Chinese bream, African catfish, European eel, coho salmon, and tilapia. The efficiency of the GnRH spawning-induction therapy might thus depend, at least in some species, on the removal of the inhibitory dopamine effect on GtH release. In such species, GnRH analogs induce only a moderate GtH surge but do not result in ovulation. Dopamine antagonists, such as pimozide and domperidone, potentiate the effect of GnRH analogs on GtH release and on ovulation. The combination of the fish GnRH analog [D-Arg 6-Pro9 NET]-salmon-GnRH (sGnRHa) and domperidone is currently being used in the major Chinese hatcheries to induce spawning in carp species. A spawning induction kit which includes these two components is commercially available from Syndel Laboratories (Canada). However, in a wide range of teleost species, including such in which dopamine has been shown to exert an inhibitory effect on GtH release (different salmonids, gilthead seabream, Chinese loach), ovulation and spawning are successfully induced by a low dose (1-20 jg/kg BW) of a GnRH analog (GnRHa) alone. The possibility of using dopamine antagonists in such fish in order to further reduce the GnRHa dose to be injected needs to be evaluated both from the physiological and the economic points of view. It seems that the intensity of the dopaminergic inhibitory effect on GtH release varies among teleost species. In fish in which this effect is dominant, such as cyprinids, it has to be neutralized in order to enable GnRHa induction of ovulation. In others,

administration of a low dose of GnRHa alone overcomes the dopaminergic inhibition and successfully induces ovulation and spawning. Much research effort has recently been devoted to the selection of superactive GnRH analogs to induce spawning. In the goldfish (Peter et al. 1985), carp and Chinese loach (Lin et al. 1988), the piscine GnRH analog [D-Arg 6 -Pro9 NET]-sGnRH was shown to be in vivo the most potent in terms of GtH release. This analog was also shown to have the highest affinity to goldfish and catfish pituitary receptors (Habibi et al. 1987; de Leeuw et al. 1988), to have increased affinity to plasma binding protein in goldfish (Huang and Peter 1988) and to be highly resistant to enzymatic degradation by the pituitary, kidney, and liver of Sparus aurata (Goren et al. 1987, Zohar et al. 1989). However, despite these characteristics in some fish such as the Atlantic salmon (L.W. Crim, comment in this workshop) and Sparus aurata(Zohar et al. 1989), this sGnRHa was no more potent than analogs of mammalian GnRH in inducing GtH release and ovulation. Moreover, there are indications that differences in in vivo potencies of GnRH analogs might be overcome by a moderate increase in their injected dose. Therefore, one can question whether there is a need to use piscine GnRH analogs or the existing mammalian GnRH analogs. The latter are commercially available at lower prices and are superactive in all tested fish species, in some cases more so even than analogs of sGnRH. GnRH analogs, which are resistant to enzymatic degradation, have prolonged biological half lives. However, when injected to fish at doses to induce ovulation, they rapidly disappear from the circulation (30-60 min in goldfish: Sherwood and Harvey 1986; 1-2h in seabream: A Goren and Y. Zohar, unpublished data). The GtH surge caused by a single injection of a GnRHa lasts approximately 48h in goldfish, trout, and seabream which might be insufficient to induce ovulation in females that had not yet reached final stages of vitellogenesis. Moreover, in fish with asynchronous ovarian development, which are frequent spawners, a single injection of GnRHa might induce only partial ovulation and spawning, as is the case in the gilthead seabream, Sparus aurata. In contrast to mammals, the phenomenon of

398 desensitization of pituitary GtH response to chronic administration of GnRH does not exist in teleosts, at least not at the periovulatory period. Therefore, sustained administration of GnRH analogs offers a feasible solution to overcome the short bioactive life of the peptides. Controlled release GnRH delivery systems have been proven efficient in inducing constantly elevated GtH secretion and in inducing, accelerating, or synchronizing spawning in a variety of fish species: rainbow trout (Crim et al. 1983), Atlantic salmon (Crim and Glebe 1984), goldfish (Sokolowska et al. 1984), milkfish (Lee et al. 1986), sea bass (Harvey et al. 1985), and seabream (Zohar 1988b). When needed, the controlled-release delivery systems can be used to administer a mixture of products: GnRH, dopamine antagonists, thyroid hormones, etc. In some fish, such as certain groupers, sexual maturity is attained at the age of 5 to 6 years. Only then can induction of spawning be considered. Advancing puberty in such fish might contribute to their broodstock management. Evidence that gonadal steroids are involved in puberty (Crim et al. 1982) and that a combined treatment with steroids and GnRH triggers precocious gonadal development (Crim and Evans 1983; Magri etal. 1985) is indicative of the future possible hormonal advancement of puberty in farmed fish. One of the major considerations in developing a spawning-induction treatment is the timing of the injection of the hormone. Presently, females are selected for treatment on the basis of their overall appearance (coloration, shape of the abdomen, etc.) or of their oocyte developmental stages as determined by the examination of an ovarian biopsy. However, these parameters are not always good indicators of female responsiveness. In fact, the change in the ovarian steroidogenic potential leading to the follicular capacity to produce the maturational-inducing steroid takes place only at the very end of vitellogenesis (Fostier and Jalabert 1986). These considerations led Fitzpatrick et al. (1987) to search for a hormonal indicator to predict the sensitivity of female coho salmon to GnRHa treatment; they found that plasma testosterone can serve as such an indicator. Similar studies on additional fish species will no doubt contribute to in-

OBJECTIVES FOR THE PRODUCTION OF MONOSEX POPULA TIONS: 1. Prevent wild spawning 2. Farm the faster-growing sex 3. Farm the most resistant sex OBJECTIVES FOR THE PRODUCTION OF STERILE POPULATIONS: 1. Achieve a better somatic growth 2. Prevent post-spawning mortalities in salmonids 3. Improve fisheries of salmonids 4. Prevent deterioration of flesh quality at breeding 5. Prevent increased sensitivity to stress/disease at breeding 6. Prevent wild spawning 7. Enable stocking fish in new ecological territories.

Fig. 2. The main objectives for the production of monosex or sterile populations in fish farming.

crease the efficiency of hormonal treatment to induce ovulation and spawning. As indicated above, a failure to release GtH underlies the lack of final oocyte maturation, ovulation, and spawning which characterize many farmed fish. This failure of the GnRH function is not yet understood. Studying the molecular basis of GnRH synthesis and release and the regulation of these processes will lead to fundamental and applied contributions to the field of fish reproductive endocrinology.

Sex control The objectives for the production of monosex and sterile fish populations in fish farming and sea ranching are summarized in Fig. 2. Interspecific hybridization is one option to obtain monosex or sterile fish. However, its success under farming conditions is limited. The other options are the manipulation of the phenotypic sex of the fish by treatments with sex steroids or of the genotypic sex by chromosome set manipulations. Use of steroids In many fish species, especially tilapias, monosex populations are obtained by exposing all fish to

399 steroids, usually applied with the food. This practice is referred to as "the direct method". The major drawback of the direct method is related to the large scale manipulation of steroids which it invo,¢ves. Estrogen-induced feminization in salmonids is achieved by feeding fish with pellets which contain estrogen at doses ranging from 3 to 120 mg/kg for periods of a few weeks to a few months (Hunter and Donaldson 1983). In tilapias, androgen-induced masculinization is induced by feeding the fish with a diet containing 10 to 100 mg of steroids/kg for periods of 21 to 70 days. The exposure of all marketable fish stocks to such a treatment requires the handling of large quantities of feed which contain high dosages of steroids. These diets are applied to fish in large water volumes for long periods. In addition, in most cases synthetic stable steroids are used. Although some studies have shown a rapid elimination of the steroids from the fish body, they do not measure possible metabolites of the administered steroids. Also, the danger of steroid contamination of the water and the fish farm facilities is ignored. The indirect method of steroid treatment presents an excellent alternative for the production of monosex fish populations. This method produces a limited number of sex inversed homogametic individuals by steroid treatment. These individuals are used to cross with untreated homogametic fish resulting in genotypic monosex progeny. Fish destined for human consumption are not treated with steroids and exposure to steroids can be carried out with optimal care at isolated, relatively small facilities. In Canada and in the United Kingdom the sperm produced by sex-inversed genotypic females ("female milt") is used commercially to fertilize eggs of normal females to produce all-female populations of chinook salmon (Donaldson 1986) and rainbow trout (Bye and Lincoln 1986), respectively. This approach can be very useful in tilapia species with male homogamety such as Oreochromis aureus. However, to date, efforts to sex-invert genotypic male tilapias into phenotypic females by estrogen treatment have had limited success. To facilitate the management of steroidal sex control, to increase its efficiency and to reduce the risk involved, more intensive research efforts

should be invested towards studying the nature of sex inducers in fish, and the labile period of sex differentiation. Information on steroids present in the gonad at the time of sex differentiation might be of great value when determining the steroid to be used for sex control, and the timing of its administration. Since such information is limited, most studies on steroid sex control are based on Yamamoto's (1969) hypothesis that in fish androgens and estrogens are male and female sex inducers, respectively. However, the study by Van den Hurk and Lambert (1982) suggests that in rainbow trout estrogens are not the gynoinducers and that ovarian development is promoted by the absence of enzymes which produce androinducers (androgens). Data from cases in which estrogenic feminization is not successful, such as in tilapias, might thus reflect the fact that the estrogens are not the gynoinducers. In such cases, another approach might be considered to induce feminization - the use of blockers for steroidogenic enzymes which produce the androinducers, such as for 11/3 hydroxylase in the trout. Further progress in steroidal sex control is clearly dependent on more research on steroidogenic potential of the fish gonad at the time of sex differentiation. Studies on steroid sex manipulation face the problem of determining the optimal period for steroid application. Criteria used for identification of gonadal differentiation vary considerably among studies. As a result, in many experimental studies or commercial trials, fish are exposed to relatively high doses of steroids for long periods. A precise determination of the labile period of sex differentiation might largely facilitate and shorten the steroidal treatment, and reduce the effective dose. Such an approach was adapted by Piferrer and Donaldson (1987) who found in coho salmon that a single 2h immersion in estradiol 173 efficiently induced feminization when administered between 8 days pre-hatch and 13 days post-hatch; a similar single 2h immersion in 17a-methyltestosterone efficiently induced masculinization when administered between 6 and 13 days post-hatch. The maximal sensitivities to steroids occurred before gonadal sex differentiation could be distinguished

400 histologically. Similar studies should be conducted on additional fish species which are candidates for sex control. Chromosome set manipulation Gynogenetic females were produced in a number of teleost species which have female homogamety, including salmonids, cyprinids, and cichlids. Exposing gynogenetic females to androgens results in the production of XX males, which are used to cross normal females to obtain all-female populations. In species with male homogamety, androgenesis followed by estrogenic sex inversion of the males can be applied. This approach should be developed for the cichlid Oreochromis aureus. In salmonids, triploid females (XXX) are sterile and do not show any endocrine evidence of a reproductive cycle (Lincoln and Bye 1987; Benfey et al. 1987). Such triploids do not develop secondary sexual characteristics; they grow faster and reach larger sizes as compared to normal fish. Triploid males, however, show similar hormonal profiles to diploid males, develop testis, show secondary sexual characteristics, and do not grow faster. All-female triploid rainbow trout are produced by fertilizing normal eggs with sperm of genotypic females (XX sperm) and retaining the second polar body (Lincoln and Scott 1983). An elegant alternative for the direct triploidization is to cross tetraploid females (XXXX) with XX males. In order to obtain female tetraploids, normal eggs have to be fertilized with "female milt" before the suppression of the first mitosis. The above considerations suggest that the most promising approach for the induction of monosex or sterile fish population is the combination of chromosome set manipulation and steroid treatment for the production of the desired broodstock (masculinized females, tetraploid females, etc.). A simple insemination will then give rise to the progeny required for stocking. The technologies to be developed and the phenotype and genotype to be produced should be individually considered for each fish species according to its homogametic sex and according to its reproductive physiology.

Growth and development Improvement of growth rates and developmental success have been major concerns in fish farming. Basic and applied interest in growth and development have been approached for the larval and adult phases of the fish's life cycle. In some fish of commercial importance, mainly in marine pelagic spawners, larval growth and development are the major bottlenecks for successful farming. The size of the newly-hatched larvae, in particular the size of their mouth and upper part of their digestive tract, is too small in relation to the smallest available rotifers, which serve as their first food. Also, such larvae present developmental abnormalities, such as non-inflated swim bladders and skeletal deformations. As a consequence, survival rates of larvae are low. This is the case in seabream, sea bass, striped bass, milkfish, mullet, and others. Artificial acceleration of larval growth and improvement of larval developmental success might increase survival rates and contribute significantly to the successful farming of the fish of interest. For many years, thyroid hormones, which are believed to be involved in larval development, metamorphosis, and growth, have been shown by T.J. Lam's group to improve performances and survival rates in a variety of fish species: tilapia, carp, guppy, gouramy, milkfish, and grouper. In all these species, the thyroid hormones were applied by immersing the larvae in triiodothyronine (T3) or thyroxine (T4) solutions for periods ranging from a few days to a few weeks. The upscaling of such a technique is not practical and alternative methods for the administration of the hormones should be considered. The incorporation of T3 or T4 in microencapsulated feed is now being evaluated (T.J. Lam, comments in this workshop). A promising alternative for the administration of thyroid hormones originated in studies showing that newly fertilized eggs of salmonids (Tagawa and Hirano 1987; Sullivan et al., this volume) and striped bass (Brown et al. 1987) are relatively rich in T3 and T4. These hormones are believed to be of maternal origin, which is in agreement with T3

401 binding to estradiol 173 induced proteins in the female goldfish (MacKenzie and Ray 1989). During larval development and yolk sac absorption, the level of thyroid hormones decrease progressively, which reflects their utilization in the absence of their synthesis. Only later on, concurrent to the onset of thyroid function, does the level of the thyroid hormones rise (Sullivan et al. 1987; Tagawa and Hirano 1987; Brown et al. 1988). These findings suggest that thyroid hormones required for early development are transported from the brood females into the oocytes. This led to attempts at increasing the levels of thyroid hormones in eggs and larvae by their administration to the brood females, together with the hormone used for the induction of ovulation and spawning (HCG or GnRH). This was done in the striped bass (Brown et al., this volume) and in rainbow trout (Sullivan et al., this volume). In the striped bass, elevation of T3 content of the eggs was induced by injection of the hormone to brood females (S.B. Brown, comment in this workshop). The resulting larvae exhibited a faster growth and increased rates of swimbladder inflation and of survival, compared to controls. In commercial hatchery conditions, T3 treatment of the females resulted in fourfold increase in fingerling survival. These original results open new possibilities for hormonal improvement of larval survival, applicable on a large scale. Adult phase A variety of hormones have been shown to be implicated in fish growth: growth hormone (GH), prolactin, GtH, thyroid hormones, insulin, steroids, and others. These hormones regulate a wide range of processes which are related to growth: food intake, ingestion, absorption, assimilation, metabolism, and excretion. GH, thyroid hormones, insulin, and steroids have been used to improve the efficiency of those processes which result in an increased appetite, a better food conversion rate, and faster growth (Donaldson et al. 1979; McBride et al. 1982; Higgs et al. 1982; Matty 1986). Only the use of anabolic steroids gained commercial importance. In most cases, synthetic androgens are fed to the fish for a period of a few weeks. As mentioned previously, the use of steroids

might contaminate the fish farm facility and thus represent a risk for the consumer. Also, prolonged steroid treatment of the adults, such as practiced for acceleration of growth, might have some deleterious side effects: accelerated gonadal development, change in skin coloration, increased sensitivity to pathogens, skeletal deformation, damage to liver and kidney or digestive tracts, and a change in flesh quality. An alternative approach should thus be used for improving growth rate. One approach which has been considered is the manipulation of GH secretion. The possibility that GnRH functions also as a GH releasing factor (goldfish: Marchant et al. 1987) should be further studied and may serve as a basis for the development of therapies for the acceleration of growth in farmed fish. To date, the most promising agent to be used for growth acceleration in fish farming is GH. GH of different origins enhances appetite, conversion rate and growth in a variety of fish species (Donaldson et al. 1979). It is a readily degradable protein and its use in fish farming does not involve long-term contamination risk. The commercial production of recombinant vertebrate GH and the growthpromoting effect of these molecules in fish (Gill et al. 1985) make its use economically feasible. Recently, rainbow trout recombinant GH was produced and characterized (Agellon et al. 1986) and was shown to be potent in accelerating growth in that species (Agellon et al. 1988a). A major problem in the use of GH for growth acceleration in fish farming is related to its mode of administration. In earlier studies, feeding the hormone to the fish was excluded; since it is a polypeptide, the GH was supposed to be digested in the alimentary canal before its absorption in the intestine. Thus the hormone was mostly applied by either injection, implant, or via the water (immersion). However, all these modes of administration are difficult to upscale to commercial fish farming. Recent studies showing that feeding high doses of GH enhances growth (Degani and Gallagher 1985) and that the fish gut can absorb a small proportion of intact, biologically active recombinant GH (McLean et al., this volume) opens again the option of applying the GH via the food. The economic

402 feasibility of this option should be evaluated, as it might require large quantities of the hormone. Protecting the GH from digestion at the stomach might increase the efficiency of the feeding option. The recent isolation and characterization of the fish GH gene (Agellon et al. 1988b; Du and Hew, this volume) introduced the exciting possibility of producing transgenic fish enriched with the gene encoding for GH. Efforts presently invested in understanding the molecular and neuroendocrine basis of growth hormone function will no doubt lead in a few years to the establishment of a technology for the production of fast-growing fish to be used in commercial farming.

Smoltification Raising the fresh water parr phase of salmon involves high hatchery expenses. Some salmonid species (Atlantic salmon, coho salmon) require more than one year to complete smoltification. The need to produce saltwater-viable smolts in the shortest possible time has led to intensive research on the mechanisms involved in acquiring seawater adaptability and their regulation. The applied purpose of this research is to establish indices for the completion of smoltification and for the timing of seawater release, and to develop technology to accelerate smoltification. The acquisition of sea water tolerance involves many processes in a range of physiological systems: gills, kidneys, digestive tract, liver, skin, muscle, and others. These are regulated by a variety of hormones, amongst which are GH, prolactin, thyroid hormones, and steroids. The prediction of saltwater viability of smolts has been based on morphological (size, coloration), behavioral, metabolic, enzymatic (gill Na+ / K + ATPase and succinic dehydrogenase activity), biochemical (body composition) and physiological (salinity tolerance) indices (Langdon 1985). However, these criteria are not always precise and do not always coincide with the peak of smoltification (Folmar and Dickhoff 1981; Langdon 1985). For many years, thyroid and steroid hormones have been monitored during the process of smoltifi-

cation. The recent development of homologous radioimmunoassays for salmonid GH and prolactin has led to the establishment of the complete endocrine profile which accompany smoltification. Treating presmolts with the above hormones added information on their relative functions. However, the precise cause-effect relationships between the different hormones and the processes which are associated with smoltification are not yet completely understood. Out of all the studied parameters which vary during smoltification, only the kinetics of T4 concentration in the fresh water coho salmon was suggested to be a useful index for the commercial growers, of the optimal timing of smolt transfer to seawater (Folmar and Dickhoff 1981). However, it is evident at present that more information is required on the endocrine and physiological mechanisms involved in smoltification, before the optimal smolts can be precisely identified. As there is a size threshold to smoltification (Langdon 1985), growth promoting substances, especially anabolic steroids, have been used in attempts to accelerate the acquisition of seawater adaptability. However, although anabolic steroids enhanced growth of salmon parr, in some cases they inhibit smoltification (McBride et al. 1982; Higgs et al. 1982). These data, and earlier-mentioned problems involved with the use of steroids in fish farming, exclude the steroidal option in acceleration of smoltification. Since thyroid hormones are known to be involved in stimulating both growth and smoltification, they have been used to accelerate the acquisition of sea water tolerance in a variety of salmonids (Dickhoff and Sullivan 1987). The results are controversial: enhancement of different processes associated with smoltification and of seawater survival was obtained or not depending on the hormone used (T3 or T4), its dose, timing and duration of application. Studies showing a stimulatory effect of GH on smoltification (Miwa and Inui 1985) point out the possibility of using recombirant GH to advance this process. The events associated with smoltification are probably regulated by a variety of hormones acting in synergism, which should be further investigated. It might be too simplistic to

403 assume that a one-hormone treatment will efficiently accelerate smoltification. As smoltification is closely synchronized with the environment (Langdon 1985), manipulation of the environmental factors might represent a better approach for its advancement.

Concluding remarks The present paper attempts to demonstrate the close association between basic and applied research in fish endocrinology. The development and intensification of fish farming is dependent upon success in controlling and manipulating some major physiological functions such as reproduction, development, growth, and osmoregulation. This will be possible only once a deep understanding of the environmental and hormonal regulation of these functions is acquired. Basic research into fish physiology and endocrinology will lead to the development of endocrine technologies designed to improve the management and yield of fish farming. Special care should be given to eliminate any possible risks for the farmer, consumer, or potential market.

Acknowledgements I would like to express my deep gratitude to the following persons, for contributing their ideas and data to the workshop: Dr. C. Brown, Dr. W.W. Dickhoff, Dr. E.M. Donaldson, Dr. T.J. Lam, Dr. P.Y. LeBail, Dr. H.R. Lin, Dr. R.E. Peter, Dr. P. Prunet and Dr. C. Schreck.

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