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Microalgae are one of the most promising groups of functional ingredients for inclusion in microdiets for Senegalese sole (Solea senegalensis) larvae, supplying components with anti-oxidant effects that may be important during an accelerated stage of development where excessive reactive oxygen substance production leading to oxidative stress may occur. The diatom Phaeodactylum tricornutum complies with such potential since it is rich in fucoxanthin, a carotenoid with demonstrated antioxidant activity. This study aimed at determining the effect of dietary P. tricornutum supplementation on the growth performance, survival and levels of oxidative stress of Senegalese sole postlarvae. Four dietary treatments were tested: a commercial microdiet, used as control, and three experimental variants where P. tricornutum was included (1) whole cells at 5%-WP5, (2) broken cells at 5%-BP5 and (3) broken cells at 10%-BP10. At the end of the trial, no significant differences were found in growth performance of larvae fed diets where P. tricornutum was included at 5% (WP5 and BP5) and the commercial control. However, larvae from the BP10 treatment had a significantly higher dry weight than larvae from the remaining treatments. Moreover, the experimental diets had no significant effects on oxidative stress. Nonetheless, lipid peroxidation levels were significantly higher in smaller larvae fed diets where P. tricornutum whole cells were included, relatively to the commercial control. The results of the present study demonstrate that P. tricornutum broken cell biomass has the potential for inclusion in weaning microdiets for Senegalese sole, increasing larval growth performance at an inclusion level of 10 %. European Union European Commission [613588] info:eu-repo/semantics/publishedVersion

Senegalese sole (Solea senegalensis) production presents several nutritional challenges, making this species a good candidate to study the dietary potential of bioactive compounds. Since proper nutrition plays a fundamental role in fish biology, it is necessary to further investigate species-specific and well-balanced diets in order to improve Senegalese sole juvenile farming. Algae have antioxidant properties, high-quality dietary protein, and are a source of bioactive compounds. This study evaluates the effects of dietary microalgal inclusion in both health status and growth performance of Senegalese sole post-larvae. Individuals 41 days after hatching (DAH) were randomly distributed among 12 tanks and four experimental diets were randomly distributed by triplicate groups. A basal diet served as CTRL and the experimental diets were formulated to include 3% of each of the algal biomass (CHLO, Chlorella sp. from heterotrophic production; PHAEO, Phaeodactylum sp.; and NANNO, Nannochloropsis sp.). At 50 DAH, 20 post-larvae/tank were collected and homogenized for analysis of immune and oxidative status, and at 61 DAH the total length, dry weight, and survival were assessed. No changes were observed in survival and total length of individuals, post-larvae fed NANNO, and CHLO dietary treatments increased dry weight at 61 DAH compared with those fed CTRL. While post-larvae immune status was apparently not altered by dietary treatments at 50 DAH, the total glutathione content decreased in fish fed PHAEO and CHLO dietary treatments compared to control diet. The observed results on improvement of growth performance without adverse effects on the immune status and decrease of endogenous total glutathione point to the fact that Nannochloropsis sp., Phaeodactylum sp., and Chlorella sp. could work as potential candidates for inclusion in microdiets for Senegalese sole. VALORMAR project - Compete 2020 [POCI-01-0247-FEDER-024517]; Lisboa 2020; CRESC Algarve 2020; Portugal 2020; European Union through European Regional Development Fund; Foundation for Science and Technology (FCT)Portuguese Foundation for Science and TechnologyEuropean Commission [UIDB/04423/2020, UIDP/04423/2020]; FCT, PortugalPortuguese Foundation for Science and TechnologyEuropean Commission [SFRH/BD/108243/2015, PD/BDE/129262/2017, IF/00197/2015] info:eu-repo/semantics/publishedVersion

Senegalese sole (Solea senegalensis) is a highly valuable flatfish species targeted for aquaculture diversification in Southern-European countries. Although its adaptation to captivity was foreseen in the 1970´s, its commercial production at a relevant industrial scale is extremely recent. One of the factors causing this delay in commercialisation was a severe bottleneck occurring during weaning, where incipient larval growth performances and survivals were impairing farming during the first decades of farming. However, a significant evolution has recently occurred for Senegalese sole weaning, with larval growth rates, survivals and quality drastically increasing, and good results becoming more reproducible. These progresses were only possible following significant R&D efforts leading to increased knowledge on larval nutritional physiology, weaning strategies, feeding practices and microdiet formulation and production. To this end, this work reviews the advancements that contributed for the evolution occurring in Senegalese sole weaning during the last decades. This overview shows that, for instance, while in the year 2000 larval weaning would only be achieved at around 60 days after hatching (DAH), with fish achieving 1 g (wet weight basis) at around 120 DAH, nowadays it is possible to perform an early weaning starting immediately after settling (15 DAH) and achieving 1 g at 65 DAH. Future perspectives on Senegalese sole weaning should target an optimisation of microdiet nutritional and physical properties to a further adequacy for the pelagic larvae, where the predatory capacity is reduced and lower particle sizes lead to fast and high nutrient leaching. Nevertheless, this work shows that early life-feed replacement is a reality for Senegalese sole, although it remains uncertain if it will ever occur at the onset of exogenous feeding.

Larvae and early juveniles of fast-growing fish species show tremendous growth potential, leading to higher requirements for protein, high-unsaturated fatty acids, and other nutrients. Several commercial weaning diets with relatively high success have been developed for low/moderate-growing species; however, additional challenges are outlined to meet growth potential and energy requirements of such fast-growing species. The objective of the present study was to evaluate two commercial microdiets for marine fish, one having simultaneously a very high protein and high lipid contents (HIGH), and another (MOD) with a high protein and moderate lipid content, in a growth performance trial with greater amberjack post-larvae from 33 to 78 days after hatching. Moreover, histological preparations of liver, anterior and posterior intestine were assessed for hepatic and intestinal lipid inclusions quantification and gut epithelial brush height measurement. Activities of the digestive enzymes: pepsin, trypsin, chymotrypsin, lipase and amylase were also analyzed. Post-larvae fed HIGH microdiet exhibited higher final weight and lower feed conversion ratio that those fed on MOD microdiet. Liver displayed a higher level of lipid inclusions for the MOD diet than for HIGH diet. Moreover, enterocytes of posterior intestine presented a much higher level of supranuclear vacuoles for the HIGH diet compared to MOD diet. The lower Trypsin/Chymotrypsin ratio observed at the end of the experiment in larvae fed on MOD diet may indicate a deficiency in protein of this diet. Together, these results support that larvae of greater amberjack, and likely other fast-growing marine fish species, require high protein–high lipid microdiets. The use of microdiets developed targeting slower growing marine species may lead to sub-optimal performances in fast-growing larvae., This study was undertaken by Project WEANTUNA supported by Eurostars (E! 7994) and co-financed by FEDER through PO Algarve 21 in the framework of QREN 2007-2013 (30200).

Summary The objectives of this study were to determine the effects of a tailor made purified diet (PUR) supplemented with phosphatidylcholine (PC) or phosphatidylethanolamine (PE) on the reproductive performance, gamete quality, embryo development and growth as well as skeletal malformations of the larvae in zebrafish, Danio rerio. The PUR diet was tested against a commercial diet as a negative control (CD). For each diet, one experimental group of eight adult zebrafish (three males, five females) was maintained in 3-L tanks in a water recirculating system under continuously controlled conditions (photoperiod 14 h light: 10 h dark; water flow rate 7.3 L h−1; 28°C; pH 7.5; 700 μS). Two trials were performed to confirm the results. The CD treatment resulted in the highest final weight of the breeders (0.60 ± 0.18 g); however, the fish failed to spawn. The PC supplemented diet promoted a reasonable final weight of the breeders (0.48 ± 0.10 g) similar to the CD and with a mean hatching rate of 93.80 ± 6.9%. Nevertheless, the PC treatment induced the highest rate of skeletal malformations in progeny (82.40 ± 4.1%), which could be related to the low larval survival at 28 days post-fertilization (dpf) (48.00 ± 11.3%). In comparison to the control groups, broodstock fed the PE diet showed the best results in sperm quality, which was revealed by higher total and progressive motility and higher velocities than sperm from males fed the CD and PUR diets. Furthermore, the PE group produced significantly higher egg diameters (1.20 ± 0.05 mm) when compared to the PUR diet (1.14 ± 0.03 mm). This study highlights the importance of phospholipids in zebrafish gamete quality, and that supplying PE in broodstock diets can improve the reproductive performance of zebrafish.

The present study reveals the first characterization of the plasma melatonin rhythms of the meagre (Argyrosomus regius) under aquaculture conditions. Melatonin levels were monitored during a 24 h cycle under a photoperiod of 16 L:8D and under constant darkness (DD), respectively to characterize the daily rhythm of this indoleamine and to test its endogenous origin. Besides, to identify which light intensities are perceived as night or day by this species, the degree of inhibition of nocturnal melatonin production caused by increasing intensities of light was tested (3.3, 5.3, 10.5, and 120 μW/cm2), applying 1 h light pulses at Mid-Dark. The result for melatonin daily rhythm in plasma showed a typical profile: concentration remained low during all daytime points, increasing greatly during dark points, with maximum values at 16:00 and 22:00 h, zeitgeber time. Under DD conditions, the plasma melatonin profile persisted, with a similar acrophase but with a lower amplitude between subjective day and night periods, indicating this rhythm as being endogenously driven. Moreover, meagre seemed to be very sensitive to dim levels of illumination during the night, since an intensity of just 3.3 μW/cm2 inhibited melatonin production. However, only the pulse of 5.3 μW/cm2 caused a melatonin drop till daytime concentrations. Thus, the threshold of light detection by the pineal organ was suggested as being located between 3.3 and 5.3 μW/cm2. Such results are an added value for this species biology knowledge, and in consequence to its adaptation to aquaculture conditions, allowing the improvement of culture husbandry protocols.

Senegalese sole farming is gaining “momentum” and the development of an optimised diet is pivotal to ensure its competitive and sustainable commercial culture. The lower fish performance often observed when replacing large amounts of dietary fishmeal by plant protein sources may result from an unbalanced supply of selected nutrients, such as taurine, which is absent in plants but abundant in fish. However, taurine is highly soluble in water and sole has a passive feeding behaviour. Therefore, the aim of this study was to test different forms of taurine inclusion in plant protein-based diets, in order to guarantee a reduced leaching of this nutrient to the environment and to ensure a reliable delivery at fish tissues. Two basal diets were formulated: a fishmeal-rich (FM) and a low fishmeal (PP85) diet, in which vegetable protein sources replaced 85% of marine-derived proteins. Based on the PP85 formulation, diets were supplemented with: crystalline l -taurine (FreeTau), encapsulated taurine microparticles (EncTau), or delayed-release taurine microcapsules (DRTau). Leaching experiments were performed for each diet and post-prandial taurine kinetics was analysed in plasma samples of Senegalese sole tube-fed with the experimental diets. Results showed that taurine losses in the FreeTau diet increased exponentially after 5 min of water immersion, reaching 100% after 15 min. Taurine encapsulation significantly reduced taurine losses after 15 min to 25% (EncTau diet) or 40% (DRTau diet). Taurine was rapidly absorbed in FM and FreeTau diets, attaining a peak in plasma at approximately 1 h after feeding. Taurine kinetics was quite different in fish fed with the encapsulated diets, as plasma levels were maintained elevated during an extended period of time. Maximal levels in diet EncTau and DRTau were attained at 1 and 6 h after feeding, respectively, demonstrating the successful production of sustained and delayed-release microcapsules. Taking into account its passive feeding behaviour, this study indicates that taurine should be previously encapsulated in diets for Senegalese sole. Depending on the objective of the study and on the feeding strategy adopted, encapsulated or delayed-release taurine microparticles should be used.

In diet formulation for fish, it is critical to assure that all the indispensable amino acids (IAA) are available in the right quantities and ratios. This will allow minimizing dietary AA imbalances that will result in unavoidable AA losses for energy dissipation rather than for protein synthesis and growth. The supplementation with crystalline amino acids (CAA) is a possible solution to correct the dietary amino acid (AA) profile that has shown positive results for larvae of some fish species. This study tested the effect of supplementing a practical microdiet with encapsulated CAA as to balance the dietary IAA profile and to improve the capacity of Senegalese sole larvae to utilize AA and maximize growth potential. Larvae were reared at 19 A degrees C under a co-feeding regime from mouth opening. Two microdiets were formulated and processed as to have as much as possible the same ingredients and proximate composition. The control diet (CTRL) formulation was based on commonly used protein sources. A balanced diet (BAL) was formulated as to meet the ideal IAA profile defined for Senegalese sole: the dietary AA profile was corrected by replacing 4 % of encapsulated protein hydrolysate by CAA. The in vivo method of controlled tube-feeding was used to assess the effect on the larvae capacity to utilize protein, during key developmental stages. Growth was monitored until 51 DAH. The supplementation of microdiets with CAA in order to balance the dietary AA had a positive short-term effect on the Senegalese sole larvae capacity to retain protein. However, that did not translate into increased growth. On the contrary, larvae fed a more imbalanced (CTRL group) diet attained a better performance. Further studies are needed to ascertain whether this was due to an effect on the voluntary feed intake as a compensatory response to the dietary IAA imbalance in the CTRL diet or due to the higher content of tryptophan in the BAL diet.

Senegalese sole (Solea senegalensis) is a highly-valuable flatfish species with interest for diversification of aquaculture in Mediterranean countries. Unlike other fish species known to have a high dietary DHA requirement during the larval stage, several studies have suggested dietary DHA could play a minor role for Senegalese sole during the early developmental stages. This suggestion was recently supported by studies that indicated an ability of Senegalese sole to biosynthesise DHA from its precursors during the larval period. However, it is still uncertain if Senegalese sole post-larvae can effectively grow on low dietary DHA levels during weaning. Moreover, recent studies focusing on the juvenile phase of Senegalese sole have suggested a possible impairment of this species to efficiently digest or absorb high dietary lipid levels. Yet, if a reduction of dietary lipid levels would affect a normal development of Senegalese sole during the early stages remains to be established. Therefore, this study aimed at determining if a reduction of dietary DHA and lipid levels would affect Senegalese sole performance and lipidme-tabolism duringweaning and post-larval period. In this study, Senegalese sole post-larvae were reared according to four dietary treatments: low lipid levels (treatments LFAT and LFAT_DHA) and moderate lipid levels (MOD-FAT and MODFAT_DHA). DHA levels in LFAT_DHA and MODFAT_DHA diets were higher than in LFAT and MOD-FAT diets, respectively. Subsequently, effects of experimental diets on Senegalese sole post-larval growth, survival, fatty acid composition and histopathological condition of intestinal and liver tissues were evaluated. Results showed that feeding on diets with low DHA and lipid levels significantly reduced the growth performance of Senegalese sole post-larvae. However, at the end of the experimental period, no significant differences were observed for post-larval survival in response to dietary treatments. In overall, this study disadvises that weaning diets for Senegalese sole post-larvae should contain low DHA and lipid levels, as a reduction of these levels may compromise post-larval growth performance.Statement of relevance: Determining essential fatty acid requirements in marine fish larvae is of utmost importance for the aquaculture industry, contributing to fulfil the growth potential of farmed species. In the case of Senegalese sole, a highly valued species farmed in Mediterranean countries, it may have the capacity to biosynthesise DHA from its precursors during the larval stage. However, the inclusion of DHA-rich oils in weaning diets is costly, considerable savings could be achieved by replacing DHA-rich oils by common fish oils in weaning diets for this species. Furthermore, Senegalese sole has also been shown a poor ability to deal with high dietary lipid levels during the juvenile stage. However, no studies have addressed this capacity in Senegalese sole larvae. This study comprises an evaluation on the effects of reducing dietary DHA and lipid levels during Senegalese sole weaning by integrating an assessment of these effects on post-larval growth, survival, fatty acid composition and histopathological condition. (C) 2016 Elsevier B.V. All rights reserved. Project SOLEAWIN (310305/FEP/71)

23 páginas, 3 figuras, 4 tablas., 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., This work was supported by project POCI/CVT/60176/2004—SULFAAR, financed by POCI 2010 (FCT, Portugal), which is co-financed by FEDER. Wilson Pinto and Cláudia Aragão benefited from grants SFRH/BD/27889/2006 and SFRH/BPD/37197/2007 (FCT, Portugal), respectively. Manuel Yúfera contribution was funded by Consejería Innovación, Ciencia y Empresa—Junta de Andalucía (Spain) which is co-financed by FEDER (project P06-AGR-01697). This publication benefits from participation in LARVANET COST action FA0801.

Trabajo presentado en la IV International Meeting on Marine Research (IMMR 2014), celebrada en Peniche (Portugal) el 10 y 11 de julio de 2014., The improvement of micro diets, for fish larvae, has been a major challenge in past years; however some problems remain without a resolution. Besides its tremendous growth rates, at early developmental stages, fish larvae present an immature digestive system and a progressive metamorphic process that suggest special nutritional requirements. It is therefore essential to provide fish larvae with easy and/or quickly metabolized protein sources, like protein hydrolisates, high bioavailable forms of vitamins and trace minerals. The low molecular weight of such compounds, in association to the high surface area of the micro-particles, makes them highly soluble and prone to water leaching losses. These nutrient losses, not only makes these nutrients unavailable to the larvae, but originate an important increase of organic matter in the rearing tanks, leading to detrimental water quality criteria. The development of high-performing micro-particulate diets for fish larvae requires a delicate balance between an adequate quantitative and qualitative nutritional supply and good water stability of the feeds, without compromising the palatability and the digestive breakdown. A series of binders, protein-based (e.g. fish gelatin, wheat gluten, spray-dried plasma) and carbohydrate-based (gums, alginates, pectins, starches, dextrin, polyols) were incorporated at graded levels (1, 2.5 and 5%) in a standard formulation for seabream larvae. Diets were manufactured by a proprietary low-shear extrusion process and afterwards processed to obtain a 200-400 micron fraction. All diets were analyzed for the following criteria: water leaching of nitrogenous compounds, turbidimetry and particle water stability. An additionally approach relied on the use of various encapsulation techniques (spray-drying, fluid-bed drying and vacuum coating) to incorporate trace minerals (Zn and Mn) and amino acids (taurine, lysine and methionine). Technological results show that the beneficial effects towards nutrient leaching and physical stability of the micro-pellets after immersion in water are highly dependent on binder type and less markedly on binder dose. Through the use of selected binders (e.g. guar gum and gelatin) in association to a spray-drying encapsulation process, we were able to significantly alter the release profile of Zn, Mn and taurine. Some of these prototypes were also tested in vivo with gilthead seabream larvae and clearly suggest that microencapsulation technologies that allow a controlled release profile of nutrients result in beneficial effects to larval growth and quality criteria. The results of several specific scenarios will be presented., This work has been partly funded under projects 13380-MICALA and 23000-ZEBRAFEEDS co-financed by FEDER through PO Algarve 21 in the framework of QREN 2007-2013, and by the EU seventh Framework Programme by the ARRAINA project No. 288925: Advanced Research Initiatives for Nutrition & Aquaculture.

This study evaluated the effect of dietary taurine supplementation on the growth performance and methionine metabolism of gilthead seabream larvae. For this purpose, a growth experiment was carried out in which seabream larvae were fed upon rotifers supplemented with “blank” or taurine-enriched liposomes. A complementary trial was also done in which seabream larvae fed with live prey were subsequently tube-fed a solution containing a L-[U-14C] methionine with or without a taurine supplement. Results from the growth experiment showed that rotifers were successfully enriched with taurine, but no effects were observed on larval growth performance, survival or amino acid composition. Furthermore, dietary taurine supplementation did not result in an increase of larval taurine levels, a factor that may have been determinant for the absence of effects observed on growth performance. In the tube-feeding trial, results showed that dietary taurine supplementation led to an increase of methionine retention in larvae. These findings suggest the existence of an active taurine biosynthesis pathway for gilthead seabream during the larval stage. Hence, gilthead seabream may not be dependent on dietary taurine to maintain the taurine body pool, since it may convert taurine from methionine if required. Taken together, the results from this study indicate that dietary taurine supplementation does not seem to enhance the larval growth performance in fish species able to biosynthesise taurine during this stage, which seems to be the case of gilthead seabream. However, this study also showed that in these species, dietary taurine supplementation may ultimately affect larval metabolism by increasing methionine availability for several important physiological purposes, contributing to a better understanding on the role of taurine during the early life stages of fish development.

Aromatic amino acids (AA, phenylalanine and tyrosine) are the precursors of thyroid hormones, which regulate metamorphosis in all vertebrates. In fish, this is a critical developmental stage where physiological requirements for aromatic AA may increase. Therefore, it is possible that dietary aromatic AA supplementation may be beneficial to accomplish a successful transition from larvae to the juvenile stage. This work aimed to assess the effect of dietary aromatic AA supplementation on tyrosine metabolism in species with an accentuated (Senegalese sole, Solea senegalensis) and a less marked (gilthead seabream, Sparus aurata) metamorphosis. For this purpose, either species were fed Artemia and subsequently received or not an aromatic AA supplement through tube-feeding, together with a L-[U-14C] tyrosine marker, throughout metamorphosis. Tyrosine was elected as a tracer since the metabolic fate of this aromatic AA is directly linked to the production of thyroid hormones and metamorphosis. Results showed that dietary phenylalanine supplementation did not increase tyrosine retention in Senegalese sole larvae during metamorphosis, suggesting that this species may not be able to biosynthesise tyrosine from phenylalanine at a sufficient rate to supply its physiological requirements until after metamorphosis. Furthermore, dietary tyrosine supplementation effectively increased tyrosine availability in Senegalese sole body fluids at metamorphosis, most likely for coping with metamorphosis-related processes, such as production of thyroid hormones. Therefore, dietary tyrosine supplementation may be beneficial for Senegalese sole during metamorphosis. On the other hand, results indicated that gilthead seabream larvae do not seem to require an additional supply of aromatic AA during metamorphosis, probably because the physiological requirements for tyrosine do not increase during this developmental stage. The different results observed for Senegalese sole and gilthead seabream are probably related to the complexity of metamorphosis that each species undergoes and to the needs for production of thyroid hormones, which seems to affect aromatic AA requirements during this critical stage of development. These findings may be important for physiologists, fish nutritionists and for the flatfish aquaculture industry.