Manuel Carrillo

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.

Current knowledge on the melatonin system in teleost fish.

Melatonin is a much conserved feature in vertebrates that plays a central role in the entrainment of daily and annual physiological rhythms. Investigations aiming at understanding how melatonin mediates the effects of photoperiod on crucial functions and behaviors have been very active in the last decades, particularly in mammals. In fish a clear-cut picture is still missing. Here we review the available data on (i) the sites of melatonin production in fish, (ii) the mechanisms that control its daily and annual rhythms of production and (iii) the characterization of its different receptor subtypes, their location and regulation. The in vivo and in vitro data on melatonin effects on crucial neuroendocrine regulations, including reproduction, growth, feeding and behavioral responses, are also reviewed. Finally we discuss how manipulation of the photic cues impact on fish circannual clock and annual cycle of reproduction, and how this can be used for aquaculture purposes.

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.

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.

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.

Comparative insights of the kisspeptin/kisspeptin receptor system: Lessons from non-mammalian vertebrates

Kisspeptins, the peptide products of the Kiss1 gene, were initially identified in mammals as ligands of the G protein-coupled receptor 54 (GPR54; also termed Kiss1R) with ability to suppress tumor metastasis. In late 2003, the indispensable role of kisspeptins in the control of reproductive function was disclosed by the seminal observations that humans and mice carrying inactivating mutations of GPR54 displayed hypogonadotropic hypogonadism. Since then, numerous experimental studies, conducted initially in several mammalian species, have substantiated the roles of kisspeptins as essential players in the physiologic regulation of key aspects of reproductive maturation and function, including the timing of puberty onset, the dynamic control of gonadotropin secretion via stimulation of GnRH neurons, the transmission of the negative and positive feedback effects of sex steroids, the metabolic regulation of fertility and the control of reproductive function by environmental (photoperiodic) cues. Notably, while studies about kisspeptins in non-mammals appeared initially to lag behind, significant efforts have been devoted recently to define the genomic organization and functional characteristics of kiss/kisspeptins and gpr54 in different non-mammalian species, including fish, reptiles and amphibians. These analyses, which will be comprehensively revised herein, have not only substantiated the conserved, essential roles of kisspeptins in the control of reproduction, but have also disclosed intriguing evolutionary aspects of kisspeptins and their receptors. Such comparative approaches will be instrumental to fuel further studies on the molecular regulation and physiological roles of kisspeptins, thus helping to unveil the complex biology of this system as indispensable regulator of the reproductive axis in a wide diversity of animal species.

Effects of Polyunsaturated Fatty Acids and Prostaglandins on Oocyte Maturation in a Marine Teleost, the European Sea Bass (Dicentrarchus labrax)

Abstract The effects of the polyunsaturated fatty acids (PUFAs), arachidonic acid (AA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and prostaglandins (PGs) on oocyte maturation were investigated in a marine teleost, the sea bass (Dicentrarchus labrax). Follicle-enclosed postvitellogenic, preovulatory oocytes were cultured in vitro and maturation was verified by assessing volume increase, lipid droplet coalescence, yolk clarification, and germinal vesicle migration and breakdown. Human chorionic gonadotropin was administered as the maturation-inducing gonadotropin (GTH) and was capable of inducing maturation in a time- and dose-dependent manner. Free AA induced maturation in a dose- and time-dependent manner and enhanced GTH-induced maturation, while EPA, DHA, and oleic acid were ineffective. Maturation induced by GTH was significantly suppressed by a phospholipase A2 blocker, suggesting that mobilization of AA was involved in GTH-induced maturation. Moreover, EPA and DHA exhibited a significant, dose-dependent attenuation of GTH-induced maturation. Maturation induced by GTH was inhibited in the presence of a cyclooxygenase inhibitor, indomethacin, and this inhibition was reversed by addition of AA, PGE2, or PGF2α. PGE2 and PGF2α alone were both effective stimulators of maturation, while PGE1 and PGE3 were ineffective. The effect of PUFAs on oocyte maturation in vitro were corroborated with studies in vivo. Oocytes were obtained from females fed a commercial, PUFA-enriched diet (RD) and maturational behavior was compared with oocytes from females fed a natural diet (ND) with a higher EPA content and n-3:n-6 ratio. Although no significant difference was observed in the rate of spontaneous oocyte maturation, a higher percentage of GTH-induced maturation and lower percentage of atresia were observed in RD oocytes. Moreover, while basal PGE production from oocytes from both groups was the same, RD oocytes produced significantly higher levels of PGE in the presence of hCG. The results from this study provide evidence for the participation of AA metabolism in GTH-induced oocyte maturation, and suggest that other PUFAs and PGs may play important roles in the induction of maturation in a marine teleost.