Constantinos C. Mylonas

Endocrine manipulations of spawning in cultured fish : from hormones to genes

Abstract Almost all fish reared in captivity exhibit some form of reproductive dysfunction. In females, there is often failure to undergo final oocyte maturation, ovulation and spawning; while in males milt production may be reduced and of low quality. These dysfunctions are due to the fact that fish in captivity do not experience the conditions of the spawning grounds, and as a result there is a failure of the pituitary to release the maturational gonadotropin, luteinizing hormone (LH). Reproductive hormones have been utilized since the 1930s to stimulate reproductive processes and induce ovulation/spermiation and spawning. The first methods employed freshly ground pituitaries collected from reproductively mature fish, which contained gonadotropins (mainly LH) and induced steroidogenesis and gonadal maturation. Eventually, purified gonadotropins became available, both of piscine and mammalian origin, e.g., carp or salmon gonadotropin, and human chorionic gonadotropin. In the 1970s, spawning induction methods begun employing the newly discovered gonadotropin-releasing hormone (GnRH), which induces the secretion of the fishs own gonadotropin from the pituitary, thereby overcoming the endocrine failure observed in captive broodstocks. Development of highly potent, synthetic agonists of GnRH (GnRHa) constituted the next generation of hormonal manipulation therapies, and created a surge in the use of hormones to control reproductive processes in aquaculture. The most recent development is the incorporation of GnRHa into polymeric sustained-release delivery systems, which release the hormone over a period of days to weeks. These delivery systems alleviate the need for multiple treatments and induce (a) long-term elevation in sperm production and (b) multiple spawning in fish with asynchronous or multiple-batch group-synchronous ovarian physiology. Based on the recent discovery of GnRH multiplicity in fish and the increasing understanding of its functional significance, new GnRH agonists can be designed for more potent, affordable and physiologically-compatible spawning induction therapies. Future strategies for improved spawning manipulations will be based on understanding the captivity-induced alterations in the GnRH system, and on new approaches for their repair at the level of GnRH gene expression and release.

Broodstock management and hormonal manipulations of fish reproduction.

Control of reproductive function in captivity is essential for the sustainability of commercial aquaculture production, and in many fishes it can be achieved by manipulating photoperiod, water temperature or spawning substrate. The fish reproductive cycle is separated in the growth (gametogenesis) and maturation phase (oocyte maturation and spermiation), both controlled by the reproductive hormones of the brain, pituitary and gonad. Although the growth phase of reproductive development is concluded in captivity in most fishes-the major exemption being the freshwater eel (Anguilla spp.), oocyte maturation (OM) and ovulation in females, and spermiation in males may require exogenous hormonal therapies. In some fishes, these hormonal manipulations are used only as a management tool to enhance the efficiency of egg production and facilitate hatchery operations, but in others exogenous hormones are the only way to produce fertilized eggs reliably. Hormonal manipulations of reproductive function in cultured fishes have focused on the use of either exogenous luteinizing hormone (LH) preparations that act directly at the level of the gonad, or synthetic agonists of gonadotropin-releasing hormone (GnRHa) that act at the level of the pituitary to induce release of the endogenous LH stores, which, in turn act at the level of the gonad to induce steroidogenesis and the process of OM and spermiation. After hormonal induction of maturation, broodstock should spawn spontaneously in their rearing enclosures, however, the natural breeding behavior followed by spontaneous spawning may be lost in aquaculture conditions. Therefore, for many species it is also necessary to employ artificial gamete collection and fertilization. Finally, a common question in regards to hormonal therapies is their effect on gamete quality, compared to naturally maturing or spawning broodfish. The main factors that may have significant consequences on gamete quality-mainly on eggs-and should be considered when choosing a spawning induction procedure include (a) the developmental stage of the gonads at the time the hormonal therapy is applied, (b) the type of hormonal therapy, (c) the possible stress induced by the manipulation necessary for the hormone administration and (d) in the case of artificial insemination, the latency period between hormonal stimulation and stripping for in vitro fertilization.

Use of GnRHa-delivery systems for the control of reproduction in fish

The most commonly observed reproductivedysfunctions in cultured fish are theunpredictability of final oocyte maturation(FOM) in females, and the diminished volume andquality of sperm in males. Gonadotropin-releasing hormone agonists (GnRHa)have been used extensively in order tostimulate the release of pituitary luteinizinghormone (LH) required to induce FOM, ovulationand spermiation. Because multiple hormonaltreatments are often necessary for a successfulresponse, fish must be monitored and handledextensively, which is labor intensive,stressful to the fish and can often result inbroodstock mortalities. To ameliorate thisproblem, sustained-release delivery systems forGnRHa have been developed during the last twodecades and have been increasingly applied incontrolling reproduction of a variety ofcultured fish. Solid implants of cholesterolor poly[ethylene-vinyl acetate], andbiodegradable microspheres ofpoly[lactide-glycolide] or poly[fatty aciddimer-sebasic acid] release GnRHa for a periodof time (from a few days to many weeks.) GnRHa-delivery systems do not causedesensitization of the pituitary gonadotrophsin fish, and by stimulating a sustainedelevation of plasma LH they induce the naturalprogression of plasma steroid increasesassociated with FOM and spermiation. Thismethod has been used with very encouragingresults in females of more than 40 culturedspecies and has been effective in inducing FOM,ovulation or spawning in fish with synchronous,group-synchronous and asynchronous ovariandevelopment. In males, GnRHa-delivery systemshave been tested in more than 20 species,producing significant increases in miltproduction for up to 5 weeks. Future researchshould focus on the optimization of thistechnology in terms of (a) using the mostpotent GnRHa, (b) identifying the mostappropriate GnRHa release kinetics according tothe reproductive biology of different species,and (c) determining minimum effective doses. Developments in these areas will greatlyenhance the effectiveness and efficiency ofGnRHa-delivery systems, while at the same timereducing their cost thus making them moreaffordable to the aquaculture industry.

A Genetic Linkage Map of the Hermaphrodite Teleost Fish Sparus aurata L.

The gilthead sea bream (Sparus aurata L.) is a marine fish of great importance for fisheries and aquaculture. It has also a peculiar sex-determination system, being a protandrous hermaphrodite. Here we report the construction of a first-generation genetic linkage map for S. aurata, based on 204 microsatellite markers. Twenty-six linkage groups (LG) were found. The total map length was 1241.9 cM. The ratio between sex-specific map lengths was 1:1.2 (male:female). Comparison with a preliminary radiation hybrid (RH) map reveals a good concordance, as all markers located in a single LG are located in a single RH group, except for Ad-25 and CId-31. Comparison with the Tetraodon nigroviridis genome revealed a considerable number of evolutionary conserved regions (ECRs) between the two species. The mean size of ECRs was 182 bp (sequence identity 60–90%). Forty-one ECRs have a known chromosomal location in the pufferfish genome. Despite the limited number of anchoring points, significant syntenic relationships were found. The linkage map presented here provides a robust comparative framework for QTL analysis in S. aurata and is a step toward the identification of genetic loci involved both in the determination of economically important traits and in the individual timing of sex reversal.

Preparation and Administration of Gonadotropin-Releasing Hormone Agonist (GnRHa) Implants for the Artificial Control of Reproductive Maturation in Captive-Reared Atlantic Bluefin Tuna (Thunnus thynnus thynnus)

Abstract Mature migrating Atlantic bluefin tuna (Thunnus thynnus thynnus) were captured in the Mediterranean Sea with a purse seine and reared in floating cages for 2 to 3 years. During the natural spawning period (June–July) of two consecutive years, fish were randomly implanted underwater with a controlled-release delivery system (implant) loaded with gonadotropin-releasing hormone agonist (GnRHa), in order to induce final oocyte maturation (FOM), ovulation/spermiation, and spawning. At the time of sampling, males were significantly larger than females (ANOVA, P < 0.001), having a mean (± SE) fork length and body weight of 190 ± 3 cm and 122 ± 5 kg, compared to 176 ± 3 cm and 94 ± 4 kg of females, respectively. All fish were reproductively mature, with their age ranging between 5 and 12 years and males being a year older, on average. After GnRHa implantation, fish were monitored for spawning and the release of eggs, and were sacrificed at different times after hormone treatment in order to examine the progressive effect of the treatment on gonad maturation. The in vitro GnRHa release from the produced implants was maximal during the first 2 d, with a mean (± SE) release of 525 ± 166 μ g GnRHa implant−1 day−1. The plasma GnRHa profile in vivo reflected the release in vitro, and statistically significant elevations in plasma GnRHa levels were measured until 7 d after treatment (ANOVA, P < 0.01). The underwater implantation procedure was improved between 2004 and 2005, requiring an average (± SD) of 3.1 ± 1.4 min for each fish, and was 64 and 84% successful in 2004 and 2005, respectively. There were no differences between the histological appearance of the testes of GnRHa-treated and control males, and almost all of them contained intra-testicular spermatozoa. However, the proportion of spermiating control males (n = 17) was only 12% compared to 26% for the GnRHa-implanted males (n = 19). Also, there were no differences between controls and GnRHa-implanted fish in sperm concentration, initial spermatozoa motility, or duration of forward motility, which ranged between 29.02–48.54 × 1010 spermatozoa ml−1, 58–63% and 8–9 min, respectively. Final oocyte maturation (FOM) and post-ovulatory follicles (POFs) occurred in 63% and 88%, respectively, of the GnRHa implanted females (n = 16), compared to 0% and 21%, respectively, of the control females (n = 14). In addition, two GnRHa-implanted females in 2005 were found to be ovulated at the time of sacrifice, and their eggs were fertilized in vitro with sperm from spermiating males, which resulted in viable embryos and larvae. Finally, although spawning was not observed, fertilized eggs were collected from the cages. Larvae produced from these eggs were identified as Atlantic bluefin tuna, demonstrating that the present GnRHa implantation method can be used to induce FOM, ovulation/spermiation, and spawning in captive-reared Atlantic bluefin tuna.

Sex-related changes in estrogen receptors and aromatase gene expression and enzymatic activity during early development and sex differentiation in the European sea bass (Dicentrarchus labrax)

The present study addresses the role of aromatase and estrogen receptors in sex differentiation and development. With this purpose, a sea bass female- and a male-dominant group were obtained by successive size gradings since in this species females are already larger than males at the time of sex differentiation. Changes in cyp19a and cyp19b gene expression and enzymatic activity were monitored by a validated real-time PCR and a tritiated water assay, respectively, during early development and sex differentiation. Changes in mRNA expression of estrogen receptors, both erb1 and erb2, were also assessed during this period. Results show clear sex-related differences in cyp19a gene expression and enzymatic activity in gonads, with females exhibiting significantly higher levels than males at 150 days post hatching (DPH), when histological signs of sex differentiation were evident. cyp19b gene expression and activity in brain were detectable during early ontogenesis at 50 DPH but no clear sex-related differences were observed. Both erb1 and erb2 showed higher gene expression levels in testis than in ovaries around 200-250 DPH, corresponding with the time of testicular differentiation and precocious male maturation, but no sex-related differences were found in the brain. Together these results indicate that in the European sea bass high expression levels of cyp19a are associated with ovarian differentiation and thus cyp19a can be considered as a suitable molecular marker of ovarian differentiation. However, the involvement of cyp19b in sex differentiation cannot be concluded. In addition, the higher levels of erb1 and erb2 in males versus females during sex differentiation, coinciding with precocious male maturation in the sea bass, suggest an important role for these receptors in testicular development and maturation.

Vitellogenin, steroid plasma levels and spawning performance of cultured female Senegalese sole (Solea senegalensis).

The Senegalese sole (Solea senegalensis) is a high value market flatfish, which aquaculture is compromised by severe reproductive problems; these are mostly found in soles hatched and raised in captivity (F1 generation). To gain knowledge on the reproductive dysfunctions observed in cultured (F1) Senegalese sole, this work aimed at developing a specific vitellogenin (VTG) ELISA, for the measurement of plasma VTG levels in this species. Profiles of VTG were correlated with those of sexual steroids and spawning performance of an F1 broodstock, during three consecutive years. The Senegalese sole VTG (ssVTG) was purified by precipitation with MgCl(2)-EDTA and anion-exchange chromatography and showed a molecular mass of 172 kDa, by SDS-PAGE. Specific antibodies were obtained and used to develop a competitive ELISA, which had a sensitivity of 3.6 ng ml(-1), and inter- and intra-assay coefficients of variation of 9.5% (n=29) and 6.7% (n=12), respectively. Annual profiles of plasma VTG showed a major peak at pre-spawning, and a second minor rise around autumn, which mirrored plasma profiles of both estradiol (E(2)) and testosterone (T) levels. Spontaneous spawning occurred every year in the spring season, but no fertilized eggs were obtained. In conclusion, this study described, for the first time, the purification and development of a sensitive and specific ELISA for Senegalese sole VTG. The endocrine and spawning data suggested that F1 female broodstock showed normal VTG and steroid releasing profiles in captivity with occurrence of spontaneous spawning, but no fertilization of the eggs was recorded.

Atlantic Bluefin Tuna (Thunnus Thynnus) Farming and Fattening in the Mediterranean Sea

The Atlantic bluefin tuna (Thunnus thynnus) is one of the tunas with the highest commercial value and it is supporting the capture-based tuna aquaculture industry in the Mediterranean Sea. This is a seasonal activity and it involves the capture of fish from the wild and their rearing in sea cages for periods ranging between 3 months to 2 years. Short-term rearing is done mainly to: (a) achieve a greater body fat percentage and (b) obtain a better price by not flooding the market in the brief fishing period. Due to the increasing fear of a collapse of the fishery, the International Commission for the Conservation of Atlantic Tunas currently reduced the total allowable catches for 2010 to 13,500 mtn from 32,000 mtn previously. Therefore, there is great interest in establishing a proper and sustainable tuna aquaculture industry. This necessitates the development of specific technologies for tuna aquaculture that will not rely on captured individuals from the wild, as it is practiced today. This article reviews the methods used for the farming and fattening of the species in the Mediterranean Sea, and the current status of the efforts at controlling reproduction in captivity.

Spawning induction of individual European sea bass females (Dicentrarchus labrax) using different GnRHa-delivery systems

The present study evaluated the effect of different delivery systems loaded with gonadotropin-releasing hormone agonist (GnRHa) on spawning induction, and egg and larval quality in female European sea bass (Dicentrarchus labrax). Mature females (mean±S.E.M. oocyte diameter=905±10 μm) were treated with [d-Ala6, Pro9 NEt]-mGnRHa using GnRHa-loaded microspheres, Mc (60 μg kg−1 body weight, bw) GnRHa-loaded implants, EVAc (50 μg kg−1 bw) or received a single injection of GnRHa in saline, IN (15 μg kg−1 bw). Controls (C) were injected with saline. Females were allocated individually in 2-m3 tanks with two GnRHa-treated males and were allowed to spawn without further handling. All GnRHa treatments induced spawning 3 days after treatment (d.a.t.), whereas control fish did not spawn during the 21-day experimental period. GnRHa microspheres and implants induced up to four consecutive spawns in one female, whereas GnRHa-injected fish spawned only once. Egg quality seemed relatively lower in all GnRHa-treated groups compared to spontaneously spawning fish (natural spawns, NAT group) maintained in large populations (80 fish in 15-m3 tanks), possibly due to both the isolation of individual females in separate tanks and the stress from handling. Mean egg quality of the first spawns was variable, but similar in all GnRHa treatments (6.6–18.2% buoyancy; 21.7–27.4% hatching). In subsequent spawns, the Mc-treated females produced eggs of higher buoyancy than the EVAc group (20.5±6.0% vs. 2.87±1.14%), and similar to the first spawn of the GnRHa-injected group. Our results indicate that, compared to a single GnRHa injection, GnRHa-loaded microspheres can induce multiple spawns in European sea bass, without a negative effect on egg quality.

Endocrine regulation and artificial induction of oocyte maturation and spermiation in basses of the genus Morone

Studying the process of final oocyte maturation (FOM) and spermiation in striped bass (Morone saxatilis), it was determined that the failure to undergo FOM and produce normal amounts of milt in captivity was due to low plasma levels of luteinizing hormone (LH) during the spawning period. As a result, the expected changes in the plasma profiles of the steroids involved in FOM and spermiation do not take place. Based on the hormone profiles and the associated histological examinations, FOM can be separated into an early phase (germinal vesicle (gv) migration) and a late phase (germinal vesicle breakdown). The progestogen 17,20β,21-trihydroxy-4-pregnen-3-one (17,20β,21-P) was identified as the maturation-inducing steroid (MIS), but the high levels of 17,20β-dihydroxy-4-pregnen-3-one (17,20β-P) present during late FOM, suggest a role for this steroid also. In wild striped bass, spermiation was associated with increases in LH and unchanged plasma levels of 17,20β,21-P and 17, 20β-P. In culture, spermiation in both striped bass and white bass (M. chrysops) proceeds under undetectable levels of LH, 17,20β,21-P and 17,20β-P. GnRHa-induced spermiation, however, was associated with increases in plasma LH and 17,20β-P, but not 17,20β,21-P. Treatment of females with GnRHa-delivery systems induced long-term increases in plasma LH and the appropriate changes in sex-steroid hormones. In white bass, GnRHa-delivery systems induced two successive spawns within 3 days from treatment, and in a commercial production facility induced ovulation of 90% of the females, producing 294,500 eggs kg−1 with 81% fertilization. Similarly treated striped bass underwent FOM and ovulation within 10 days after treatment, while tank spawning resulted in the production of 170,000 eggs kg−1 with 47% fertilization success. In male fish, GnRHa-delivery systems induced significant elevations of expressible milt for up to 20 days, resulting in a fourfold increase in production of spermatozoa, without affecting sperm density, motility or fertilization %.