Silvia Zanuy

Control of puberty in farmed fish

Puberty comprises the transition from an immature juvenile to a mature adult state of the reproductive system, i.e. the individual becomes capable of reproducing sexually for the first time, which implies functional competence of the brain-pituitary-gonad (BPG) axis. Early puberty is a major problem in many farmed fish species due to negative effects on growth performance, flesh composition, external appearance, behaviour, health, welfare and survival, as well as possible genetic impact on wild populations. Late puberty can also be a problem for broodstock management in some species, while some species completely fail to enter puberty under farming conditions. Age and size at puberty varies between and within species and strains, and are modulated by genetic and environmental factors. Puberty onset is controlled by activation of the BPG axis, and a range of internal and external factors are hypothesised to stimulate and/or modulate this activation such as growth, adiposity, feed intake, photoperiod, temperature and social factors. For example, there is a positive correlation between rapid growth and early puberty in fish. Age at puberty can be controlled by selective breeding or control of photoperiod, feeding or temperature. Monosex stocks can exploit sex dimorphic growth patterns and sterility can be achieved by triploidisation. However, all these techniques have limitations under commercial farming conditions. Further knowledge is needed on both basic and applied aspects of puberty control to refine existing methods and to develop new methods that are efficient in terms of production and acceptable in terms of fish welfare and sustainability.

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.

Development of broodstock diets for the European Sea Bass (Dicentrarchus labrax) with special emphasis on the importance of n−3 and n−6 highly unsaturated fatty acid to reproductive performance

Commercially fabricated diets allow greater control over the composition of biochemical components and reduce the risks of disease introduction, which are significant concerns when using the wet fish diets commonly used for most farmed marine broodstocks. However, satisfying the dietary lipid requirements of marine broodstock using artificial diets has proved difficult, particularly with respect to their highly unsaturated fatty acid (HUFA) composition. Two groups of mature sea bass, each divided between three replicated tanks, were fed two dry pelleted diets over a 2-year period, encompassing two spawning seasons. The first diet contained a good quality Northern Hemisphere meal and oil; the second differed only in the source of oil, which was substituted with tuna orbital oil (TOO). The use of TOO in the dry pelleted formulation allowed the manipulation of n−3 and n−6 HUFA in the resulting eggs, specifically arachidonic acid (20:4 n−6; AA), eicosapentaenoic acid (20:5 n−3; EPA) and docosahexaenoic acid (22:6 n−3; DHA). The results showed that dietary manipulation of these HUFA could improve levels and ratios of AA, EPA and DHA which were transferred to the resulting eggs with improvements in early survival and hatching success repeated over successive spawning seasons. The dry diet containing TOO facilitated comparable reproductive performance to the wet fish diet (Boops boops) which has previously been considered the most effective broodstock diet. The improvements in reproductive performance are discussed in relation to the proportion of these HUFA with respect to each other in total egg lipid and the phospholipid classes phosphatidylcholine (PC), phosphatidylinositol (PI) and phosphatidylethanolamine (PE) and to their potential impact on eicosanoid formation. Finally, this study has shown that a commercially fabricated diet can be successfully used as sensitive investigative tool for aquaculture research.

Evidence for two distinct KiSS genes in non-placental vertebrates that encode kisspeptins with different gonadotropin-releasing activities in fish and mammals.

Kisspeptins, the products of KiSS-1 gene, have recently emerged as fundamental regulators of reproductive function in different mammalian and, presumably, non-mammalian species. To date, a single form of KiSS-1 has been described in mammals, and recently, in several fish species and Xenopus. We report herein the cloning and characterization of two distinct KiSS-like genes, namely, KiSS-1 and KiSS-2, in the teleost sea bass. While KiSS-1 encodes a peptide identical to rodent kisspeptin-10, the predicted KiSS-2 decapeptide diverges at 4 amino acids (FNFNPFGLRF). Genome database searches showed that both genes are present in non-placental vertebrate genomes. Indeed, phylogenetic and genome mapping analyses suggest that KiSS-1 and KiSS-2 are paralogous genes that originated by duplication of an ancestral gene, although KiSS-2 is lost in placental mammals. KiSS-1 and KiSS-2 mRNAs are present in brain and gonads of sea bass, medaka and zebrafish. Comparative functional studies demonstrated that KiSS-2 decapeptide was significantly more potent than KiSS-1 peptide in inducing LH and FSH secretion in sea bass. In contrast, KiSS-2 decapeptide only weakly elicited LH secretion in rats, whereas KiSS-1 peptide was maximally effective. Our data are the first to provide conclusive evidence for the existence of a second KiSS gene, KiSS-2, in non-placental vertebrates, whose product is likely to play a dominant stimulatory role in the regulation of the gonadotropic axis at least in teleosts.

Seasonal changes in plasma levels of gonadal steroids of sea bass, Dicentrarchus labrax L.

Levels of plasma testosterone (T) and 11-ketotestosterone (11-KT) in males and plasma 17 beta-estradiol (E2), 17 alpha-20 beta-dihydroxy-4-pregnen-3-one (17 alpha,20 beta-diOH-P), and T in females were assayed by radioimmunoassay at monthly intervals throughout the sexual cycle of sea bass (Dicentrarchus labrax L.). 17 alpha,20 beta-DiOH-P was maintained at low levels (below 1 ng/ml) throughout the year, even during the spawning period (January-March). A bimodal seasonal pattern of plasma testosterone was observed. Plasma T and E2 levels became significantly increased in December (advanced gametogenesis period) and then showed further increases during January and February (first half of the spawning period) in parallel with the growth of the vitellogenic oocytes. Multiple spawnings of individual females were also observed during the spawning period affecting the relative fecundity of the eggs. A possible role of E2 on this behavior is discussed. In males, both plasma T and 11-KT initially increased in November and then showed further increasings during the rest of the period of gametogenesis (December) to reach their peak levels in the first half of the spawning period (end of January). These increased and sustained higher levels of plasma steroids coincided with the presence of spermiating males. A second peak of plasma testosterone appeared at the end of the postspawning period-beginning of the pregametogenesis period (May-June) both in males and females and their possible role with the preparation of the gonad for the next reproductive cycle is discussed.

Immunohistochemical localization of three different prepro-GnRHs in the brain and pituitary of the European sea bass (Dicentrarchus labrax) using antibodies to the corresponding GnRH-associated peptides

The distribution of the cells expressing three prepro‐gonadotrophin‐releasing hormones (GnRH), corresponding to salmon GnRH (sGnRH), seabream GnRH (sbGnRH), and chicken GnRH‐II (cGnRH‐II) forms, was studied in the brain and pituitary of the sea bass (Dicentrarchus labrax) by using immunohistochemistry. To circumvent the cross‐reactivity problems of antibodies raised to GnRH decapeptides, we used specific antibodies generated against the different sea bass GnRH‐associated peptides (GAP): salmon GAP (sGAP), seabream GAP (sbGAP), and chicken‐II GAP (cIIGAP). The salmon GAP immunostaining was mostly detected in terminal nerve neurons but also in ventral telencephalic and preoptic perikarya. Salmon GAP‐immunoreactive (ir) fibers were observed mainly in the forebrain, although sGAP‐ir projections were also evident in the optic tectum, mesencephalic tegmentum, and ventral rhombencephalon. The pituitary only receives a few sGAP‐ir fibers. The seabream GAP‐ir cells were mainly detected in the preoptic area. Nevertheless, sbGAP‐ir neurons were also found in olfactory bulbs, ventral telencephalon, and ventrolateral hypothalamus. The sbGAP‐ir fibers were only observed in the ventral forebrain, innervating strongly the pituitary gland. Finally, chicken‐II GAP immunoreactivity was only detected in large synencephalic cells, which are the origin of a profuse innervation reaching the telencephalon, preoptic area, hypothalamus, thalamus, pretectum, posterior tuberculum, mesencephalic tectum and tegmentum, cerebellum, and rhombencephalon. However, no cIIGAP‐ir fibers were detected in the hypophysis. These results corroborate the overlapping of sGAP‐ and sbGAP‐expressing cells in the forebrain of the sea bass, and provide, for the first time, unambiguous information on the distribution of projections of the three different GnRH forms expressed in the brain of a single species. J. Comp. Neurol. 446:95–113, 2002.

Daily rhythms of insulin and glucose levels in the plasma of sea bass Dicentrarchus labrax after experimental feeding

Significant and inverse circadian rhythms are demonstrated in glucose and plasma insulin in fish fed a natural diet. The highest glucose levels are found during the light period, around feeding time, and the insulin level peaks during the dark period. As a possible cause for the insulin rhythmicity, the daily variations in several plasma amino acids are considered. The feeding times could be a training factor for metabolic rhythms, which are maintained even during a fast of 7 days. The differences in the compositions of the diets could be responsible for the lack of circadian rhythms in fish fed on a commercial diet.

Induction of triploidy and gynogenesis in teleost fish with emphasis on marine species

The induction of triploidy and gynogenesis by chromosome set manipulation has traditionally been studied more intensively in freshwater than in marine fish. In the last years, however, several studies have applied these manipulations in about a dozen marine species, including mainly sparids, moronids and flatfishes. This paper focuses on the methodologies used to induce, verify, and assess performance of both triploids and gynogenetics of these marine species. Since many of them are batch spawners and have small and fragile eggs and larvae, peculiarities relating to broodstock management, gamete quality and mortality assessment during early larval stages are also taken into account. However, data show that if handling is correct and the treatments are optimized, triploid and gynogenetic rates of 100% can be easily achieved. Survival of triploids with respect to the controls is about 70–80%, whereas in gynogenetics it is generally low and more variable, depending on the species considered. In the marine fish investigated so far, triploidy has not resulted in significantly higher growth rates. On the other hand, the induction of gynogenesis has resulted in the production of both all-female and mix-sex stocks. Throughout the paper, special reference is made to the European sea bass (Dicentrarchus labrax L.), a species of both basic and applied interest, for which a comprehensive study has been carried out on the induction, verification and performance of triploids and gynogenetics.

Differential expression of three different prepro‐GnRH (gonadotrophin‐releasing hormone) messengers in the brain of the european sea bass (Dicentrarchus labrax)

The expression sites of three prepro‐gonadotrophin‐releasing hormones (GnRHs), corresponding to seabream GnRH (sbGnRH: Ser8‐mGnRH, mammalian GnRH), salmon GnRH (sGnRH: Trp7Leu8‐mGnRH), and chicken GnRH‐II (cGnRH‐II: His5Trp7Tyr8‐mGnRH) forms were studied in the brain of a perciform fish, the European sea bass (Dicentrarchus labrax) by means of in situ hybridization. The riboprobes used in this study correspond to the three GnRH‐associated peptide (GAP)‐coding regions of the prepro‐GnRH cDNAs cloned from the same species (salmon GAP: sGAP; seabream GAP: sbGAP; chicken GAP‐II: cIIGAP), which show little oligonucleotide sequence identity (sGAP versus sbGAP: 42%; cIIGAP versus sbGAP: 36%; sGAP versus cIIGAP: 41%). Adjacent paraffin sections (6 mm) throughout the entire brain were treated in parallel with each of the three anti‐sense probes and the corresponding sense probes, demonstrating the high specificity of the hybridization signal. The results showed that both sGAP and sbGAP mRNAs had a broader expression in the olfactory bulbs, ventral telencephalon, and preoptic region, whereas cIIGAP mRNA expression was confined to large cells of the nucleus of the medial longitudinal fascicle. In the olfactory bulbs, both the signal intensity and the number of positive cells were higher with the sGAP probe, whereas sbGAP mRNA‐expressing cells were more numerous and intensely stained in the preoptic region. Additional isolated sbGAP‐positive cells were detected in the ventrolateral hypothalamus. These results demonstrate a clear overlapping of sGAP‐ and sbGAP‐expressing cells in the forebrain of the European sea bass, in contrast to previous reports in other perciforms showing a clear segregation of these two cell populations. J. Comp. Neurol. 429:144–155, 2001.

The effect of modifications in photoperiod on spawning time, ovarian development and egg quality in the sea bass (Dicentrarchus labrax L.)

Abstract Under simulated natural conditions female sea bass ( Dicentrarchus labrax ) in eastern Spain (latitude 40° N and longitude 0°) spawned over a 6–8-week period in February and March, with each female spawning on more than one occasion. Generally, male fish began spermiation up to 2 months before, and continued for at least 1 month after the spawning period of the female fish. Exposure of fish to 1 month of long days (LD 15 9 ) from either 2nd May (Group C), 3rd June (Group D) or 3rd July (Group E) in an otherwise constant short day (LD 9 15 ) photoperiod regime, speeded up the rates of maturation, thus increasing the proportions of oocytes entering exogenous vitellogenesis during October and November, and also brought forward the timings of ovulation and spawning. In contrast, constant long days from 2nd May (Group B) delayed maturation and spawning time by 2–3 months. Fish maintained under constant short days throughout the experiment (Group A) spawned up to 6 weeks in advance of the control fish, suggesting that endogenous timing mechanisms may operate in this species. Spawning occurred naturally in all the experimental and control fish without the necessity for induction with pituitary or hypothalamic hormones. For the control fish, egg quality (defined as the proportion of eggs spawned which were floating and viable), hatching rate and survival to first feeding (both expressed as percentages of the numbers of floating or good quality eggs) averaged 78.3±7.7% (mean±s.e), 84.8±3.3% and 54.6±20% respectively. With the exception of the eggs and fry from Group B, these measures of quality and survival appeared unaffected by the photoperiod treatments. The eggs and fry produced by the fish maintained under long days (Group B) towards the end of their spawning period (i.e. in May), showed much more variable egg quality and survivals through hatch and first-feeding, possibly because of the higher sea water temperatures which prevailed at the time of their delayed spawning. Fecundities expressed as numbers of eggs per kg of post-spawning fish weight ranged from 247–305 000/kg for Groups A, C, D and E and the controls, but were significantly reduced in Group B fish. Egg sizes, which ranged from 1.147–1.176 mm in diameter, were unaffected by the photoperiodic manipulations of spawning time. It is concluded that artificial control of daylength can improve the supply of eggs and fry for commercial on-growing without any adverse effects on the fecundity of the broodstock or the quality and survival of the eggs and fry.