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Senegalese sole was one of the earliest identified candidate species with high potential for aquaculture diversification in the south of Europe.íts culture has been possible, and commercially attempted, for several decades, but intensive production has been slow to take off. This has been explained mostly by serious disease problems, high mortality at weaning, variable growth and poor juvenile quality. However, a strong and sustained research investment that started in the eighties has led to a better understanding of the requirements and particularities of this species. More recently, better management and technical improvements have been introduced, which have led to important progress in productivity and given a new impetus to the cultivation of Senegalese sole. As a result, the last 5 years have marked a probable turning point in the culture of sole towards the development of a knowledge-driven, competitive and sustainable industry. This review will focus on the main technical improvements and advances in the state of knowledge that have been made in the last decade in areas as diverse as reproductive biology, behaviour, physiology, nutritional requirements, modulation of the immune system in response to environmental parameters and stress, and characterization and mitigation of the main disease threats.ít is now clear that Senegalese sole has important particularities that differentiate it from other current and candidate marine aquaculture species, which bring about important challenges, some still unsolved, but also notable opportunities (e.g. a nutritional physiology that is better adapted to dietary vegetable ingredients), as will be discussed here. © 2014 Wiley Publishing Asia Pty Ltd.

It is known that cryopreservation promotes cellular damage that could compromise sperm quality. In sperm, seminal plasma provides the major defense against oxidative stress. However, dilution in the extender before cryopreservation reduces the co ncentration of antioxidants in seminal plasma, diminishing sperm protection against reactive oxyge n species (ROS). In previous studies of our group it was demonstrated that this type of damage could be in part counteracted by the addition of antioxidants into the extenders. Extenders suppleme nted with vitamins C or E, improved sperm motility of cryopreserved sperm (Martínez-Páramo et al., 2012), and taurine and hypotaurine, besides improving motility, reduced DNA fragmentati on (Martínez-Páramo et al., 2013). However, the antioxidant supplemented extenders did not redu ce lipid peroxidation or improved sperm viability. Taking into account that broodstock diet plays an important role in gametogenesis and gamete quality (Izquierdo et al., 2001), in this wo rk, a nutritional approach to enhance the sperm antioxidant defense was attempted. Therefore, three experimental diets were tested in European seabass and gilthead seabream with the aim of reinf orcing the sperm antioxidant system before and after cryopreservation. The supplements chosen were vitamins, selenium and zinc, because of their antioxidant capacity and important role in sp ermatogenesis.

Precocious maturation is a common feature in European sea bass males which occurs just before reaching the marketable size, reducing growth and feed conversion and representing an important problem that reduces the commercial value of fish. The sperm produced by precocious sea bass males could be useful for reproductive purposes since these males could be selected for breeders and used earlier as a new broodstock. However the characterization of sperm quality in these males during the reproductive season is fundamental in determining its possible use in reproduction or cryopreservation. Thus, in the present work, sperm from precocious European sea bass (Dicentrarchus labrax) males was characterized to determine the effect of seasonality on parameters influencing quality throughout the reproductive period. Several characteristics associated with sperm quality were determined: motility, cell concentration, sperm pH and osmolality, cell viability, lipid peroxidation (malondialdehyde-MDA), phospholipid, cholesterol and fatty acid composition of the plasma membrane. The results demonstrated seasonal variation in some of the parameters tested, the quality of samples being relatively poor at the beginning of the spawning season, corresponding with the highest content of MDA and the lowest motility parameters. Cell viability, phosphatidylcholine and phosphatidylethanolamine, polyunsaturated fatty acids n-3 and saturated fatty acids did not show significant differences during the reproductive period. However, monounsaturated fatty acids and polyunsaturated fatty acids n-6 showed a reduction at the end of the reproductive period. Parameter correlations showed that motility was negatively correlated with lipid peroxidation (-. 0.394, p. <. 0.05), cholesterol/phospholipid ratio (-. 0.449, p. <. 0.05) and osmolality (-. 0.478, p. <. 0.01). Our results demonstrated that lipid peroxidation could explain the seasonal variation in sperm quality, since the highest malondialdehyde val

Low sperm volume and quality is pointed out as a major reason for the low fertilization rates observed in Senegalese sole (Solea senegalensis) broodstocks both wild and F1. R ecent studies by our group have shown that the poor and variable sperm quality is related to the high sensibility of plasma membrane to seawater during fertilization. Diet enrichment in fatty acids and cholesterol could change the composition of the sperm membrane and the addition of antioxidants would protect sperm to peroxidation.Therefore, in the present work we tested specific diets enriched with DHA, selenium and Vit. E, aiming to improve sperm sole quality.

Cryopeservation is a technique with undoubted interest, not only for fish farming but also for the conservation and genetic improvement of fish stocks.

Some of the Senegalese sole (Solea senegalensis) broodstock reproductive constraints are related to sperm quality. Although they present two defined spawning season (spring and autumn), males gave semen during all the year thus an exhaustive annual sperm analysis is important to determine the seasonal changes in semen quality. Sampling was performed monthly during one year, analyzing different cellular parameters to better understand sperm quality limitations obstructing sole mass production. The percentage of progressive motile cells and their linear velocity showed a decrease from March (beginning of the first spawning season) to July (when the highest temperatures were observed), followed by a slight increase in August and October (second spawning season). DNA fragmentation values showed highest values between the two spawning seasons and decreased to the end of the year. The percentage of apoptotic cells was lowest in March (beginning of the first spawning season) and the highest in November. The percentage of cells resistant to seawater exposure presented two peaks related with both spawning seasons. There was a tendency for the semen to attain a quality peak between the beginning and the middle of the first spawning season (March–May), followed by a pronounced decrease, achieving the lowest values during the months with the highest temperature. Also, the different males present in the broodstocks reach their sperm quality peak at different times, which will result in an unequal contribution for the next generation.

Earthen ponds are the main production system for seabass and seabream in Portugal and in Southern Spain, in particular in Cádiz province. Different farms use various levels of stocking densities and pond sizes, but in general these are semi-intensive systems covering large areas with ponds ranging from one to several hectares and production levels from 0.5 to 6 Kg/m3 (mostly below 2 kg/m3) at the end of the production cycle.

Earth ponds are the main production system for seabass and seabream in Portugal and in Spain, in particular in the Cádiz province. Different farms use various levels of intensification and pond size, but in general these are semi-intensive ponds covering large areas, ranging from one to several hectares, and with production ranging from 0.5 to 1.5 Kg/m2 at the end of the production cycle. Production costs in this farming system are higher compared to intensive cage farms, and its economic sustainability depends on product differentiation and optimization of production.

The effect of dietary taurine supplementation on growth performance, metamorphosis success and amino acid metabolism of Senegalese sole (Solea senegalensis) larvae was investigated. These parameters were assessed in larvae fed control and taurine supplemented microcapsules during the pelagic phase. Subsequently, a similar evaluation was carried out in newly settled larvae fed upon Artemia, in order to verify the effect of earlier dietary taurine supplementation in larvae reared under improved feeding conditions. Results showed that dietary taurine supplementation did not affect larval growth performance and metamorphosis during the pelagic phase. However, by the end of the trial, Senegalese sole previously fed taurine supplemented microcapsules had a significantly higher growth performance and metamorphosis completion success than larvae fed control microcapsules. These differences were likely related to the improvement of feeding conditions upon settlement, which probably helped revealing the positive effects of earlier dietary taurine supplementation on Senegalese sole performance. Additionally, Senegalese sole may have benefited from taurine antioxidant properties during metamorphosis, since larval antioxidant defences may saturate at this stage. Furthermore, results from metabolic trials have shown that dietary taurine supplementation significantly increased amino acid retention in Senegalese sole larvae when a concomitant increase of taurine body levels was found. Therefore, an increase in larval growth potential and metamorphosis success was observed under dietary taurine supplementation and these results may help understanding why dietary taurine supplementation has been reported to simultaneously increase taurine body levels and growth performance in other fish species, leading to a better comprehension on the role of taurine during fish development.

Phenylalanine is the precursor of tyrosine, which is involved in the synthesis of several molecules with key roles in the regulation of metabolism and growth, stress response and pigmentation. In this study, three experimental diets were tested: an amino acid (AA) balanced diet supplemented with phenylalanine, another supplemented with phenylalanine and tyrosine and a non-supplemented AA balanced diet. Rotifers were enriched with liposomes encapsulating free AA in order to obtain a balanced AA profile. The experimental diets resulted in similar larval survival, growth, enzyme activities of AA catabolism and nitrogen excretion in all treatments. High levels of skeletal deformities were registered and significant differences were found between the control and the phenylalanine treatment for the percentage of vertebral compressions in the trunk region of the vertebral column (30% in the control and 5% in the phenylalanine group). A significantly higher survival to a temperature stress test was found for larvae fed the diet supplemented with phenylalanine and tyrosine. The results suggest that supplementation of phenylalanine/tyrosine in fish diets may be useful in order to reduce skeletal deformities and mortalities caused by stress. The present study confirms that AA requirements may be sufficient for covering growth and survival but insufficient to cover other metabolic processes.

Sperm quality seems to be one of the reasons for the reproduction constraints faced by Senegalese sole (Solea senegalensis) aquaculturists. Previous studies in this species indicated that the sperm quality of individuals kept in culture varies throughout the year and that different sperm subpopulations can be identified in ejaculates according to the motility pattern of spermatozoa. Aiming to better understand factors affecting sole sperm quality in captivity, sperm of 11 males was assessed during the reproductive season using different parameters: motility characteristics using CASA analysis; cell plasma membrane resistance to seawater hyperosmolarity; DNA fragmentation with single-cell gel electrophoresis; and early apoptosis, labeled with Annexin-V FITC. Computer-assisted sperm analyses motility data were treated using multivariate analysis to identify the presence of different spermatozoa subpopulations according to their motility pattern. Four distinct sperm subpopulations were obtained: Subpop1, which includes fast linear spermatozoa; Subpop2, made up of fast nonlinear spermatozoa; Subpop3, which includes slow linear spermatozoa; and Subpop4, which contains slow nonlinear spermatozoa. The sperm subpopulation structure varied with time after activation and with male. Low cell resistance to the seawater hyperosmotic conditions was noticed. The Annexin-V assay allowed the identification of an apoptotic population ranging from 6% to 20%. A high percentage of cells (64.1%) showed a DNA fragmentation level below 30%, but these values varied significantly between males. DNA fragmentation appears to be related to cell membrane resistance to hyperosmotic conditions faced by the cells when in contact with seawater. This condition seems to modulate the composition of the motile sperm population and performance after activation. This phenomenon could be related to the spermatozoa maturation process.

The Senegalese sole is a marine pleuronectiform that naturally occurs in Southern Europe and Mediterranean region where it is being produced in aquaculture, in particular in Portugal and Spain. The aim of this study was to assess the quality of hatchery-reared larvae in comparison with those reared in the wild, and determine to which extension wild growing larvae are also affected by skeletal deformities. The main structures affected included those forming the axial skeleton, the caudal fin complex and both anal and dorsal fins, with the most prevalent anomalies affecting caudal vertebrae and arches. Hatchery-reared fish presented a higher incidence of deformities (79%) compared with the 19% observed in wild specimens. In wild postlarvae collected in Autumn no deformities were observed. This work clearly shows that wild Senegalese sole present less skeletal deformities than those hatchery-reared during larval stages, indicating a selective mortality of wild deformed fish and/or an effect of aquaculture-related rearing conditions in the development of skeletal deformities in sole.

Despite the recent progress in the production of inert diets for fish larvae, feeding of most species of interest for aquaculture still relies on live feeds during the early life stages. Independently of their nutritional value, live feeds are easily detected and captured, due to their swimming movements in the water column, and highly digestible, given their lower nutrient concentration (water content>80%). The present paper reviews the main types of live feeds used in aquaculture, their advantages and pitfalls, with a special emphasis on their nutritional value and the extent to which this can be manipulated. The most commonly used live feeds in aquaculture are rotifers (Brachionus sp.) and brine shrimp (Artemia sp.), due to the existence of standardized cost-effective protocols for their mass production. However, both rotifers and Artemia have nutritional deficiencies for marine species, particularly in essential n-3 highly unsaturated fatty acids (HUFA, e.g., docosahexaenoic acid and eicosapentaenoic acid). Enrichment of these live feeds with HUFA-rich lipid emulsions may lead to an excess dietary lipid and sub-optimal dietary protein content for fish larvae. In addition, rotifers and Artemia are likely to have sub-optimal dietary levels of some amino acids, vitamins and minerals, at least for some species. Several species of microalgae are also used in larviculture. These are used as feed for other live feeds, but mostly in the ‘green water’ technique in fish larval rearing, with putative beneficial effects on feeding behaviour, digestive function, nutritional value, water quality and microflora. Copepods and other natural zooplankton organisms have also been used as live feeds, normally with considerably better results in terms of larval survival rates, growth and quality, when compared with rotifers and Artemia. Nonetheless, technical difficulties in mass-producing these organisms are still a constraint to their routine use. Improvements in inert microdiets w

Amino acids are the building blocks for growth and the major energy source during fish larval stages. Deficient amino acids can be supplemented in the diets, overcoming problems such as low growth rates and skeletal deformities. In this study, three experimental diets were tested: a balance diet supplemented with lysine, a balance diet supplemented with tryptophan and a control with no supplementation. Trials were conducted with Diplodus sargus larvae from 1 to 25 days after hatching (DAH). A microencapsulated diet was introduced at 15 DAH in co-feeding with live feed and from 20 DAH larvae were fed only this diet. The effect of the supplemented diets was assessed in terms of survival, growth rate, skeletal deformities, ammonia excretion and activity of amino acid catabolism enzymes. The results showed a similar survival in all treatments. However, larvae given tryptophan supplementation had a lower weight on 25 DAH. No significant differences were found in ammonia excretion, frequency or type of deformities or enzymatic activity. Tryptophan and lysine supplementation failed to improve larval growth, survival or larval quality.

Fish larvae present high amino acid requirements due to their high growth rate. Maximizing this growth rate depends on providing a balanced amino acid diet which can fulfil larval amino acid nutritional needs. In this study, two experimental microencapsulated casein diets were tested: one presenting a balanced amino acid profile and another presenting an unbalanced amino acid profile. A control diet, live feed based, was also tested. Trials were performed with larvae from 1 to 25 days after hatching (DAH). Microencapsulated diets were introduced at 8 DAH in co-feeding with live feed and at 15 DAH larvae were fed the microencapsulated diets alone. Results showed a higher survival for the control group (8.6 ± 1.3% versus 4.2 ± 0.6% and 3.2 ± 1.8%) although dry weight and growth were similar in all treatments. The proportion of deformed larvae as well as the ammonia excretion was lower in the group fed a balanced diet than in the unbalanced or control groups (38.3% deformed larvae in control, 30% in larvae fed unbalanced diet and 20% on balanced diet group). Furthermore, larvae fed the microencapsulated diets presented higher docosahexaenoic acid and arachidonic acid levels. This study demonstrates that dietary amino acid profile may play an important role in larval quality. It also shows that balanced microencapsulated diets may improve some of the performance criteria, such as skeletal deformities, compared to live feeds.

[EN] Live preys commonly used in fish larval rearing seem to be imbalanced in terms of amino acids. Manipulation of their amino acid composition is difficult, but the use of microencapsulated diets allows this manipulation. This study analysed the effect of amino acid supplementation, in order to compensate for dietary amino acid imbalances, on growth and survival of gilthead seabream (Sparus aurata) larvae. Larvae were reared until 32 days after hatching, in a closed recirculating water system (19 °C), using only live food (rotifers and Artemia). Thereafter, larvae were fed Artemia or one of the experimental microencapsulated diets: supplemented with indispensable amino acids (IAAsup diet), or supplemented with dispensable amino acids (DAAsup diet). Experiment lasted for 10 days. Dietary supplementation with indispensable amino acids resulted in A/E ratios [(each indispensable amino acid) × (total indispensable amino acids)−1 × 1000] more similar to the ones of larval seabream and in higher IAA:DAA ratios than in the DAAsup diet. Survival was similar in larvae fed the IAAsup diet (75%) or Artemia (87%), but was significantly lower in larvae fed the DAAsup diet (52%). Larvae from all treatments more than doubled their average dry weight during the experimental period. Final dry weight was similar in larvae fed both microcapsules, but these were lighter than larvae fed with Artemia. Relative growth rate (RGR) and total biomass production tended to be higher in larvae fed the IAAsup (RGR = 9% day−1) than the DAAsup diet (RGR = 7.5% day−1) and only in this last treatment these parameters were significantly lower than in larvae fed with Artemia. Therefore, dietary supplementation with indispensable amino acids resulted in a more balanced dietary amino acid profile, which significantly increased survival. Further studies introducing microdiets earlier in the development seem necessary in order to optimise growth., [FR] Les proies vivantes utilisées dans les élevages larvaires semblent déséquilibrées en terme d'acides aminés. Manipuler la composition en acides aminés est difficile mais l'usage d'aliments encapsulés le permet. Cette étude analyse l'effet d'une supplémentation en acides aminés sur la croissance et la survie des larves de daurade royale (Sparus aurata) afin de compenser un déséquilibre. Les larves sont élevées jusqu'à 32 jours après éclosion en circuit fermé (eau à 19 °C), en utilisant des aliments vivants uniquement (rotifères et Artemia). Les larves sont ensuite nourries avec des Artemia ou bien avec des aliments microencapsulés expérimentaux, supplémentés en acides aminés soit indispensables (régime IAAsup) soit non-indispensables (régime DAAsup). La durée de l'expérience est de 10 jours. La supplémentation en acides aminés indispensables conduit à des ratios A/E [(chaque AA indispensable) × (total en AA indispensables)−1 × 1000] plus proches de ceux de la larve de dorade et des ratios IAA/DHA plus élevés que ne le fait la supplémentation en acides aminés non-indispensables. Le taux de survie est similaire chez les larves nourries avec le régime IAAsup (75 %) ou Artemia (87 %) mais inférieur significativement chez les larves soumises au régime DAAsup (52 %). Le poids sec moyen de ces larves, quel que soit leur régime alimentaire, a doublé durant la période expérimentale. Le poids sec est similaire chez les larves nourries de microcapsules mais moins élévé chez celles nourries d'Artemia. Le taux de croissance relative (RGR) et la production de biomasse totale tendent à être plus élevés chez les larves nourries en IAAsup (RGR = 9 % jour−1) que celles nourries en DAAsup (RGR = 7,5 % jour−1). C'est seulement dans ce dernier traitement que les paramètres sont plus faibles significativement que chez les larves nourries d'Artemia. Ainsi, les supplémentations alimentaires en acides aminés indispensables peuvent augmenter la survie. D'autres études, introduisant des mi

Cryopreservation of fish embryos requires an optimal distribution of cryoprotectants inside all embryo compartments. Traditional techniques for the incorporation of cryoprotectants (CPAs) have failed to protect all fish compartments, especially the yolk sac which has been considered the principal point of embryo chilling sensitivity. In the present study, microinjection was used to incorporate cryoprotectants into the yolk sac of gilthead seabream (Sparus aurata) embryos at tail bud stage. The effect of microinjection viability, cryoprotectant toxicity and chilling resistance was evaluated through the hatching rate. Larval survival at first feeding was also determined in microinjection viability and cryoprotectant toxicity studies. Permeabilized seabream embryos were microinjected with 2.35 nl dimethyl sulfoxide (Me2SO), methanol (MeOH), ethylene glycol (EG) (5 M, 10 M and pure) or sucrose (10% and 15%). In a second experiment, 29.5 nl and 154.0 nl of the highest concentration of each cryoprotectant were used in the same embryo stage. To test the effect of microinjected cryoprotectants on embryo chilling resistance, 29.5 nl of pure Me2SO or 15% sucrose was microinjected into the yolk sac of tail bud stage embryos and then at a later stage, (tail-bud-free), were exposed to 3 M Me2SO solution at − 10 °C for 30 min. Our results showed that microinjection technique did not affect the viability of tail bud stage embryos as is shown by the high hatching and survival rates. Hatching and larval survival rate at first feeding were not affected with any of the CPAs tested, showing percentages higher than 75% and 90%, respectively, when embryos were microinjected with a smaller quantity of cryoprotectant. Sucrose was the cryoprotectant better tolerated at higher concentration and volume. Cryoprotectant concentration inside the yolk higher than 1.18 M for Me2SO, 1.5 M for EG and 2 M for methanol decreased the hatching rate. Microinjection allowed the delivery of high concentrat

A key to success in the culture of marine fish species is the mass production of high quality fry, a process largely dependent on successful first feeding and normal development and growth of fish larvae. In this regard it is important to examine the structural and functional development of the endocrine system (pituitary, thyroid, interrenal glands) during early ontogeny of marine fish. Particularly, the thyroid hormones, thyroxine (T4) and triiodothyronine (T3), influence numerous metabolic processes, such as growth, differentiation, metamorphosis, reproduction, respiration, migratory behaviour, central nervous system activity, seasonal adaptation, etc. Therefore the aim of this study was to describe the development of the thyroid gland and the ontogeny appearance of the thyroid hormones in Solea senegalensis larvae by means of histological and immunohistochemical techniques. The first thyroid follicle was present at 4 days-post-hatch (dph) coinciding with first feeding. During metamorphosis (12–20 dph) the follicles increased in both number and size, and by 30 dph presented the same characteristics as that seen in adult fish. Tissue immunostaining of both thyroid hormones decreased during the endogenous larvae development to nearly undetectable levels at the completion of yolk-sac absorption. During larvae exogenous phase, T3 and T4 immunostaining was first detected by 6 dph and an increase of specific staining for both hormones was detected between 12 and 20 dph, during metamorphosis phase.

Epidermal, branchial and digestive mucous cells, and the gastric glands of larvae/postlarvae (from hatching until 45 days posthatching) of three fish species (two teleostean and a chondrostean) were investigated using conventional histochemical methods (periodic acid schiff -PAS-, diastase-PAS; alcian blue pH 0.5, 1 and 2.5) in order to distinguish neutral and acidic (carboxylated and sulphated) glycoconjugates, as well as bromophenol blue reaction for identification of proteins. Additionally, the presence and distribution of sugar residues in the oligosaccharide side chains of glycoconjugates were investigated using horseradish peroxidase (HPR)-conjugated lectins (Con A, DBA, WGA and UEA-I). Most mucous cells (digestive, epidermal and branchial) of Siberian sturgeon, Acipenser baeri, sea bream, Sparus aurata and Senegal sole, Solea senegalensis larvae were PAS- and alcian blue- (pH 2.5 and 0.5) positive, with small variations between organs/tissues and species. Bromophenol blue reaction (general proteins) was positive in a minority of the mucous cells, usually in those cells which were PAS-negative. Proteins rich in sulphydryl (-SH) and/or disulphide (-S-S-) groups related with the glycoprotein nature of the glycoconjugates present in mucous cells were also observed. Epidermal, branchial and digestive mucous cells of all studied larvae did not contain glycogen or lipids. Con A lectin staining was negative in all mucous cells types of sea bream and sole, but oesophageal mucous cell of sturgeon were reactive to different lectin reactions, suggesting the presence of mannose -Man- and/or glucose -Glc-, L-fucose -Fuc- ; N-acetyl-D-galactosamine -GalNAc-, as well as N-acetyl-D-glucosamine- GlcNAc - and/or sialic acid -NANA- residues. Digestive mucous cells of all studied larvae were positive to WGA and DBA lectins. Epidermal and branchial mucous cells of sea bream and sole were Con A, DBA and UEA-I unreactive. However, mucous cells of sturgeon larvae were stained with UE