15.2 Reproductive cycles and environmental cues
15.3 The Hypothalamus
15.4 The pituitary gland
15.5 The gonadotropic hormones
15.6 The ovary
15.7 Hormonal regulation of vitellogenesis
15.8 Hormonal regulation of oocyte maturation and ovulation
15.9 The testis
15.10 Hormonal regulation of spermatogenesis and spermiation
15.11 Feedback actions of gonadal steroids
15.12 Preservation of gametes
15.13 Use of sex steroids in sex reversal
15.14 The pineal
The foregoing review briefly summarizes the salient features of the reproductive physiology of teleost fishes including cultivated species. Areas where basic research input is necessary to fill gaps in our knowledge as also those that are of immediate relevance to increasing our ability to breed cultivated fishes in captivity are highlighted in this section.
The single most important desideratum for successful propagation of cultivated fishes through aquaculture is the availability of pure seed of uniform age, size and quality and free of diseases, parasites and pests at the site of culture ponds. These strict requirements are seldom fulfilled where the seed is collected from the rivers, estuaries or other natural sources. The problem has been mitigated to some extent with the advent of the hypophysation technique for spawning cultivated species. Nevertheless, even in countries where hypophysation is extensively practised, the fish seed so produced is often grossly inadequate to meet the growing requirements and has to be supplemented with collections from the wild. The principal thrust of basic research in the immediate future should, therefore, be directed towards reducing our dependence on wild collections of seed. The problem can be tackled simultaneously at two different levels. The first task is to raise sufficient number of broodstock in captivity, and the second is to perfect the techniques of hypophysation to remove, as far as possible, the elements of uncertainty and improve the chances of success. However, any breakthrough in achieving these objectives will be possible only if basic information on the reproductive physiology of cultivated as well as laboratory fishes is available not only in areas that are of immediate relevance to aquaculture but also in those that may have no apparent bearing on aquaculture. It is also vitally important to realize that much of the existing information on the fundamental principles of reproductive physiology has emerged from work on fishes that have no economic or commercial importance but have served as excellent laboratory models all over the world to solve several problems of piscine reproductive physiology. The need for promoting coordination between laboratory research and field investigation cannot be overemphasized. A multidisciplinary experimental approach involving the participation of scientists with diverse specializations is strongly recommended since only then can meaningful results be obtained in a short span of time.
The basic principles of reproductive physiology of teleost fishes transcend species barriers, and it is, therefore, possible to discern certain trends that help in developing common working hypotheses for a number of fishes. However, environmental cues that initiate, reproductive processess to culminate in spawning during the most favourable season of the year differ in the finer nuances from one species to another and from one latitude to the next even within a species (see review of the work in section 2). A line, of investigation that must occupy a very high priority in the context of mass propagation of juveniles is the accurate delineation of the environmental factors such as photoperiod (daylength), temperature, rainfall and other biotic and abiotic factors, which promote gametogenesis in every cultivated species., This is admittedly a difficult task, for in nature, many of the factors are interlinked. For instance, if nature both daylength and temperature increase or decrease almost contemporaneously, hence difficult to dissociate. Only under controlled conditions of the laboratory can the effects of these two factors be. dissociated to permit a proper evaluation of the effects of each factor on various phases of gametogenesis. Modern biochemical techniques may be used for measurements of plasma levels of gonadotropin and sex steroids to draw more meaningful conclusions. Clearly an interdisciplinary approach is called for.
Charts of reproductive cycles in the context of seasonal changes in environmental factors of as many cultivated species as possible have to be prepared not only in their native areas but also in the introduced areas.
Once the environmental variables (synchronizers or zeitgebers), that promote gonadal recrudescence under natural and experimental conditions are identified, the fish can be subjected to a suitable combination of individually studied environmental factors to accelerate gonadal development. Some of the obvious advantages of this line of study are: (i) advancement in sexual maturity by reduction in generation time, or conversely, postponement of sexual maturity to increase somatic growth, (ii) extension of the breeding season and reduction in the time interval between successive spawnings to get seed throughout the year, and (iii) repeated inbreeding leading to possible domestication of the species.
It would be scientifically, and in the long run commercially rewarding, if cultivated species with a wide distribution north and south of the equator are studied to determine whether gonadal recrudescence is dependent on photoperiod or temperature or both. If so, it should be determined how near the equator they would be operative.
Each species of fish has evolved to function with maximum efficiency within a particular range of temperatures which it actively selects from those available. It is important to determine if thermal preferenda of cultivated fishes coincide with the optimal temperatures conducive to spermatogenesis in males and vitellogenesis in females. If information on thermal preference is available, culture of a particular species may be recommended in waters which attain a range of temperatures optimum for growth and reproduction. Gonadal maturation in fact takes place much more rapidly at the preferred temperature than at other temperatures (see section 2). An interesting study can be planned to understand the relationship between temperature and reproductive activities for the same species at different latitudes.
Studies on thermoperiod relationship with gonadal weight gain must be given a high priority since gonadal weight gain can be stimulated or inhibited depending on the time of day when the thermocycle is commenced (see section 2). This chronobiological approach is of considerable importance to aquaculture and may hold the key to producing precociously gravid fish. Timed application of heat by releasing or recirculating thermal waters into fish ponds can help optimize growth and reproduction.
Work reviewed in section 2 shows that environmental factors favouring gonadal recrudescence are entirely different from those stimulating maturation and ovulation.
The role of dissolved gases, solute concentration in rain water, soil contributions, aquatic vegetation, smell factor, and algal metabolites in inducing spawning of freshwater fishes is poorly understood. Algal blooms present before and after monsoon rains have to be studied and those that show wide fluctuations in abundance may be cultured in the laboratory and algal metabolites identified. Results of water analysis and identification of algal metabolites in pond and river waters may throw light on environmental deficiencies in confined waters vis-à-vis flowing waters. Such investigations call for collaborative efforts among limnologists, physiologists and microbiologists.
It is now established that the fish hypothalamus acts as a link between the environment and the endocrine system whose activity it controls through production of releasing or inhibiting factors at different times to modulate the reproductive cycle. Even though the hypothalamo-hypophysial complex has been studied in a number of cultivated species using the bulk staining techniques, stereotaxic atlases of the hypothalamic nuclei are not yet available except in goldfish, killifish and to some extent in rainbow trout (see section 3). Thus, critical studies involving selective destruction or stimulation of specific hypophysiotropic centres in the brain are not yet possible in most fishes. It is principally for this reason. that the relative contribution of the nucleus lateralis tuberis (NLT), the nucleus preopticus (NPO) and other areas of the brain in the regulation of gonadotropic activity of the pituitary is not clearly understood. Whether the NLT is the source of stimulatory as well as inhibitory factors involved in the regulation of the reproductive functions of the pituitary is not elucidated. The role of prostaglandins in the inhibitory mechanism is also not clear and further work is necessary.
To date, only the synthetic mammalian luteinizing hormone-releasing hormone (LH-RH) has been tried for initiating gonadotropic functions in fishes (see section 3). This peptide acts on the pituitary and brings about the release of conspecific gonadotropin which in turn acts on the gonads. Most of the superactive analogues of LH-RH synthesized by pharmacologists have to be screened for maturation and ovulation activities in cultivated fishes.
A high priority area of research with vast potentiality in aquaculture is to understand the nature of gonadotropin-releasing factor (GRF) in cultivated fishes. Isolation and characterization of GRF of fishes, though an arduous task, is well worth pursuing in view of its potential in spawning cultivated fishes. The piscine GRF molecule, once identified can be synthesized for large-scale application in aquaculture.
The potentialities of such studies are enormous. If highly potent analogues of piscine GRF are synthesized, they could be used to induce spawning in fishes bypassing the strict environmental prerequisites for the preovulatory gonadotropin surge. Also, since the injected GRF would induce gonadotropin release from the recipient's pituitary, the many problems associated with the conventional hypophysation technique (see section 8) would be solved to a very great extent. If a breakthrough in this direction is to be achieved, it would involve collaborative efforts of biochemists, pharmacologists, and fish physiologists as well as the assistance of pharmaceutical industry for mass production of the active principle.
It is anticipated that application of synthetic GRF for induction of maturation and spawning of cultivated fishes will eventually replace the existing techniques of hypophysation. However, till such time as the appropriate GRF molecules are identified, synthesized and made commercially available on a global scale, there is a vital need for improving and perfecting the existing methods of hypophysation.
The area of research suggested here falls within the realm of basic research. Bulk-staining of the hypothalamo-hypophysial complex may be resorted to in an effort to understand the morphological relationship between the various neurosecretory nuclei in the hypothalamus and the pituitary.
Many pituitary cells in the teleost fishes are directly innervated by axons arising from the nucleus lateralis tuberis or nucleus preopticus and this phenomenon is unique among the vertebrates. In some cases, the axons are in contact with the perivascular spaces surrounding groups of cells (see section 3). This can be resolved only by studying the innervation as well as vasculature of the pituitary cell types by electron microscopy.
The economics of using fish gonadotropins, extracted from carps or other universal donors, for large-scale spawning of cultivated fishes is still debatable. There is, however, a possibility of future synthesis of these gonadotropins, once their amino acid sequence is worked out. Fractionation of the pituitary glands obtained from cultivated fishes has to be undertaken to determine if separate vitellogenic and maturational hormones exist. The purified hormones can then be tested in suitable models to determine the gonadotropin-sensitive phases in gametogenesis in both sexes.
The identification and purification procedures depend largely upon the availability of sensitive and specific bioassays. The ideal situation is to develop bioassay techniques using the same species to avoid species specificity problems. The bioassays used thus far have been summarized in the foregoing review (see section 8). It would be. extremely worthwhile to develop standard bioassays for testing the vitellogenic and maturational activities of various conspecific gonadotropic preparations.
The other, more practical use for purified gonadotropins, would be in detecting plasma levels of gonadotropins during various phases of the breeding cycle by radioimmunoassay. Yet another advantage, which is even more significant to aquaculture, is to develop an easy, reliable and inexpensive test for determining the gonadotropic content of pituitary extracts used in hypophysation. This test should be so simple that within a few minutes the fish farmer can estimate the gonadotropic content of the extract. Antibodies developed against purified gonadotropin (maturational) could be mixed with a suitable dye that would develop colour on addition of a known quantity of the extract containing gonadotropin; the intensity of the colour should be proportional to the concentration of the maturational hormone. The colour developed could then be compared with a set of standard colours with calibration for gonadotropic potency. Such tests should be developed for all the cultivated species that are spawned through hypophysation.
The poor results often encountered in hypophysation work are due to many factors (see review in section 8). The effectiveness of the injected pituitary extract is greatly influenced by ambient temperature; higher the temperature faster is the uptake of the hormone from the site of injection and shorter the half-life of the hormone and vice versa. It is, therefore, highly desirable that carefully planned experiments are conducted at different temperatures to measure plasma levels of gonadotropin following injections of gonadotropin or pituitary extract in cultivated fishes that are spawned through hypophysation.
Another related study concerns the determination of the time of the day when the gonads are most sensitive to gonadotropin which may vary with the species. If this information is available, it might eventually lead to reduction in the cost of aquaculture since the required dosage of the injected extract would be less when the gonad is most sensitive.
A pituitary bank has been established by the Aquaculture Development and Coordination Programme (ADCP) of FAO in Rome to cater to the needs of hypophysation research in developing countries. Pituitary glands from uneconomic fish species and from edible frogs may be collected for hypophysation work to reduce the cost of pituitary production and thus the propagation of cultivated fishes.
The results of hypophysation will dramatically improve if safe and reliable methods are evolved for sampling ovarian oocytes. Eggs obtained by stripping or by inserting a catheter into the urogenital opening may be studied under magnification. Diameters may be determined for all stages of vitellogenesis and also for signs of follicular atresia. An atlas for oocyte development through the various stages of vitellogenesis mentioning the external appearance, colour and diameters should be prepared for each species.
The etiology and histophysiology of follicular atresia and the functions of "corpus luteum" and "corpus atrecticum" during oogenesis are poorly understood, and further investigation may be profitable especially in the maintenance of captive broodstock. Histoenzymological and electron microscopic studies coupled with those involving uptake of labelled precursors is. different compartments of the ovarian follicle may throw light on this problem.
The role of sex steroids in reproductive behaviour is an area where basic research input is necessary. The. action of sex steroids on the kidney in producing sex attractants in cultivated fishes is an interesting area of study. The sex attractant or pheromone, which may be species specific should be extracted and chemically identified. Such pheromones may be of potential value in fish culture as well as in basic studies on spawning behaviour in fishes.
Under favourable environmental conditions, the captive broodstock of most cultivated fishes undergoes vitellogenesis. However, a few species that have been introduced to areas away from their native habitat do not always become optimally gravid in the new environment. This may possibly be due to absence or deficiency of vitellogenic hormone(s) in the fish in the exotic habitat. Whether or not vitellogenesis can be induced in such fishes by exogenous administration of hormones can be determined only if the hormonal profile in the vitellogenic female in the native habitat is worked out.
Basic studies have to be initiated to understand the stimulatory actions of piscine (vitellogenic and. maturational) and mammalian gonadotropins on various ovarian compartments during vitellogenesis. The role of sex steroids and their possible synergism with pituitary, thyroid and interrenal hormones in induction of vitellogenic and maintenance of yolky oocytes also needs to be elucidated.
A high priority should be given for the development of a simple, reliable, quick and inexpensive method for ascertaining the stage of ovarian development in the broodstock. An immune logical approach is well worth following for which vitellogenin, the egg-yolk precursor present in the blood, has to be isolated and antibodies raised against it. The antibodies could then be used to develop a simple test for detection of vitellogenin in small quantities of fish plasma. Radioimmunoassay techniques can be developed for precise estimation of vitellogenin in small plasma volumes. If the vitellogenic content can be correlated with the diameter of egg, it would prove very meaningful. Such studies should be extended to all cultivated species.
In most fishes, postspawning feeding leading to deposition of lipids and proteins in tissues and fat bodies is utilized for vitellogenin synthesis during the following vitellogenic period. In this context, the role of estrogen, anabolic androgens, prolactin and thyroxine in the vitellogenic process is not properly understood and merits further study. Dosages for estrogen supplementation have to be carefully worked out for various fishes, age groups and stages of sexual maturity, since excessive administration may lead to sterility due to negative feedback action (see section 11). Development of special diets for brood-stock that are rich in lipids, calcium, protein and other components of yolk material would be beneficial.
Research on several aspects of oocyte maturation and ovulation have to be initiated to improve the chances of success in hypophysation. The negative results often obtained in hypophysation can sometimes be traced to erroneous assessment of the maturity of oocytes of the recipient females. The need for development of better methods for ascertaining the vitellogenic stages of oocytes has already been highlighted.
In view of the expense and labour involved in isolating and purifying fish gonadotropins, their use in large-scale spawning of cultivated fishes may be limited even where the starting material in the form of trash fish or marine catfish is relatively abundant and inexpensive. Even the conventional technique of hypophysation with crude pituitary homogenate is becoming expensive owing to rising costs of quality pituitary material and its processing and transportation to fish culture centres. Concerted attempts have, therefore, to be made to search for newer, better and less expensive spawning agents.
Since oocyte maturation and ovulation is regulated by three levels of control mechanisms viz., the hypothalamus, the. pituitary and the ovary, it would be worthwhile to initiate research to understand the regulatory mechanisms at each of the. three levels (see sections 15.3 and 15.5).
Challenging the pituitary by LH-RH or its more potent analogues would be the closest to the normal situation since the releasing hormone would act on the pituitary r-o bring about the release of conspecific gonadotropin which in turn promotes the formation of the species specific maturation-inducing steroid (see section 15.3). Since this situation is ideal, top priority should be given to this type of research.
Work reviewed earlier has shown great promise for the use of C21 steroids, which are the terminal hormones that initiate maturational changes at the level of the oocytes. Such Steroids, commercially synthesized from plant sterols, are relatively inexpensive and have a long shelflife. An area of research which should be accorded a very high priority is the assessment of the scope of applicability of steroid hormones in replacing protein hormones in fish culture. It is equally important to study the long-term effects of steroid hormones on the fecundity of fish and growth of fish fry and the residual activity of steroids, if any, in treated fishes.
In recent years, use of in vitro techniques for studying the effects of hormones on cultured oocytes has proved invaluable because it permits rapid and simultaneous screening of a large number of test substances on oocytes obtained from a small number of fishes. The techniques, however, necessitate formulation of suitable isoosmotic culture media for each species. Thus, suitable media for culturing oocytes have to be formulated to test the action of various gonadotropic and steroid hormones on in vitro Oocyte maturation and ovulation. Also for each species under study, the oocyte stages involved in maturation and ovulation may be studied and delineated so that some landmark stages can be identified for subsequent assessment of the in vivo effects of hormones on oocyte maturation and ovulation. Further, if maturation occurs without ovulation, some other hormone or principle such as prostaglandins or proteolytic enzymes may be involved in inducing ovulation.
Knowledge of plasma steroid profiles in gravid fishes before spawning and after gonadotropin stimulation may throw light on steroid combinations that inhibit maturation vis-à-vis those that favour this process. Thorough studies on in vitro oocyte maturation have to be conducted to test the relative effects of plasma steroids that appear in vivo following gonadotropin injection as well as those of conversion products formed by ovaries from steroid precursors. Possibly, the ovarian steroid metabolites may sensitize oocytes so that they can respond better to gonadotropin or to other steroids of ovarian or interrenal origin.
In addition, a large number of steroids, not necessarily native to the fish, may be tested for oocyte maturation-inducing activities. This will help in finding a steroid or combination of steroids which may be much more potent than those synthesized by the fish.
In vivo studies using steroids or combinations of pituitary extracts and steroids may be tried for timing of the final stages of maturation and ovulation and procedure standardized for aquacultural application.
Where and how steroids act on oocytes are poorly understood and research is needed in these areas.
Other substances such as copper salts and a wide range of antiestrogens such as clomiphene, tomaxifen and related substances have to be tried for inducing maturation and ovulation in cultivated fishes. Their potential use in inducing spawning in cultivated fishes has to await future research developments.
Some of the suggested areas of research are basic while others have great value in improving the success of hypophysation.
The steroidogenic potential of the testis at various stages of the breeding cycle should be studied using labelled precursors not only to understand the transformation products but also to determine the substrate preference in relation to the stage of sexual maturity. Temperature optima for sex steroid formation is another area that merits further Study.
Plasma levels of steroids should be determined both on circadian and circannual bases and correlated with spermatogenic stages and development of secondary sexual characters and sex accessories.
The role of sex steroids in regulating the activities of the sex accessories such as seminal vesicles and even kidney, which may be involved in the production of milt as well as sex attractants, is another important area of study (see section 9). Here too, many pituitary hormones such as growth hormone, prolactin and thyrotropin may synergize with androgens in regulating the secretory activities of sex accessory glands.
The role of sex steroids in regulating the appearance of secondary sexual characters and breeding behaviour has to be studied. Estimation of sex steroid levels may help in the determination of the sex of fish even during the nonbreeding season.
The metabolism of sex steroids and role of steroid conjugates has been studied only in a few species. Steroid conjugation facilitates its release into the medium as a sex attractant or pheromone. This work will be highly rewarding especially in spawning of cultivated fishes.
Studies are required to determine the effects of piscine and mammalian gonadotropins as well as sex steroids on the initiation and maintenance of Spermatogenesis. The synergistic role of other hormones such as growth hormone, prolactin, and thyroxine in influencing Spermatogenesis merits further study. Temperature optima for Spermatogenesis have to be determined for the cultivated species if acceleration of Spermatogenesis is required (see section 2).
Studies involving measurement of plasma levels of gonadotropin and various androgens during Spermatogenesis and spermiation may be initiated to gain an insight into the hormonal levels associated with the various testicular events.
A high priority area of research pertains to the evaluation of the quality of spermatozoa in terms of motility and survival in species where fertilization of hand-stripped eggs is the normal practice. Evaluation of sperm quality may be made in association with the constituents of the milt such as spermatocrit values, protein and mineral concentration, and androgens, to mention a few important parameters. These lines of investigations are important areas for further research and would have wide application in aquaculture.
The recent work in this area (see section 11) shows that gonadal steroids exercise both negative and positive feedback actions on gonadotropin secretion. Since some steroids, nonsteroidal antiestrogens and related compounds, exercise a positive feedback action on gonadotropin release, it is worthwhile to start studies to determine if any of them could be used as an ovulating agent.
Except where special diets are available, cultivated fishes are routinely fed on mixtures of cereal, bran, oil cakes, other grains and slaughter-house wastes. The nature of oil cakes varies in different regions according to local availability. Some varieties of oil seeds are very rich in natural estrogens. It is not unlikely that impairment of fertility might occur as a consequence of inclusion of certain varieties of oil cakes in the fish diet. Instances where presence of certain substances in the diet result in sterility in live-stock are known.
The review on preservation of gametes (see section 12) reveals that even though only a few of the cultivated species have been investigated, the feasibility of this technique has been fully demonstrated. Preservation of spermatozoa and ova is essential if the programme of selective breeding and hybridization has to succeed.
Since the males sometimes become sexually active earlier than females, it is necessary to preserve spermatozoa for fertilizing the ripe ova obtained later in the season.
Short-term versus long-term preservation of spermatozoa may be investigated to establish the relative advantages. Suitable media must be evolved for increasing the fertilizing capacity of sperm. The technique and methods of collection of milt and cryopreservation have to be standardized. The constituents of milt and spermatocrit values have to be determined for each species. The nature of diluents and cryoprotectants and their ratios as well as techniques of freezing, storage and thawing must be investigated thoroughly for each species. The quality of cryopreserved spermatozoa has to be evaluated.
Cryopreservation of ova has to be extended to other species. The technique of storage of spermatozoa and fertilized ova at near-zero temperature has great potential use in fish breeding and requires further research.
Sperm and ova banks can be established to safeguard rare and endangered species as also individuals with desirable qualities. Techniques such as gynogenesis and polyploidy, that manipulate chromosomes of cultivated fishes, have application in genetics and selective breeding and merit further study.
The specificity of sex steroids as exogenous inducers versus endogenous inducers is not clearly determined and needs further work.
Even though monosex or sterile populations of fishes such as tilapia have been produced, there is little information on the influence of steroids on the genotype. Methods of steroid administration and the stage in the life cycle when steroid treatment is to be initiated have to be standardized to get consistent results.
The advisability of human consumption of steroid-treated fish has to be determined by studying the residual activity of steroids in treated fish.
Sex inversion in cultivated hermaphroditic fishes by steroid treatment is an important area of research which has great potential in aquaculture.
The participation of the pineal organ in the overall scheme of endocrine control of gonadal activity is very poorly understood in fishes in general (see section 14). Its pro-gonadal and antigonadal effects are governed by so many variables, both in experimental design as well as those inherent in the fish itself, that only under strictly controlled laboratory conditions can its true physiological significance be understood. However, it is unanimously accepted that the pineal acts as a photoreceptor in almost all fishes studied and exercises both inhibitory and stimulatory effects on gonads (see section 14). It may, therefore, be important to take this organ into consideration in planning photoperiod-thermoperiod experiments involving induction of precocious gonadal recrudescence or postponement of gonadal regression.