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European eel, Anguilla anguilla, larval culture faces a bottleneck during the transition to exogenous feeding. To stimulate gut-priming, in the present study, prebiotics (AgriMOS, mannan-oligosaccharides, and beta-(1,3 and 1,6)-poly-D-glucose), probiotics (Bactocell, Pediococcus acidilactici), and synbiotics (AgriMOS + Bactocell) were administered to European eel larvae during the endogenous prefeeding stage. Eel larvae were reared in 2 L incubators with an initial stocking density of similar to 200 larvae/L. Each treatment (pre-, pro-, synbiotics, and control), represented by 3 replicated incubators, was connected to a separate recirculating aquaculture system. The gut-priming agents were introduced directly into the rearing water. Results revealed increased mortality when larvae were introduced to synbiotics and impaired growth in connection to probiotics and synbiotics. Larvae receiving prebiotics showed similar survival and growth to larvae reared without gut-priming agents. The immune gene expression revealed a lag phase between maternally inherited protection (c3, igm, and il10) and the gradual buildup of the larvae's own immune system (il1 beta, irf7). The lack of treatment-related immune (c3, igm, il10, il1 beta, and irf7) and stress/repair (hsp70, hsp90) responses revealed an immature immuno-readiness. Digestion (try, ctra, ctrb, tgl, and amyl), food intake (cck), and appetite (ghrl)-related genes were expressed at basal levels already on 4 days post-hatch, which combined with phenotypic plasticity of the appetite-regulating ghrelin (ghrl), indicated a prospective adaptive capability towards earlier maturation of the larval digestive capacity. Overall, we contemplate that the application of gut-priming agents in water has merit; however, as no beneficial effect was observed, we conclude that the regimen applied is not recommendable in the present form and needs to be customized for future eel larval culture. As such, water management strategies and rearing options need to be further explored to establish prefeeding and feeding regimens, targeting optimized culture conditions, and the production of healthy eel offspring. Grant agreement nos. 7076-00125B (ITSEEL) and the ENV-Fund info:eu-repo/semantics/publishedVersion

The sustainability of the Aquaculture industry relies on optimising diets to promote nitrogen retention and maximise fish growth. The aim of this study was to assess how different dietary formulations influence the bioavailability and metabolic fate of distinct amino acids in gilthead seabream juveniles. Amino acids (lysine, tryptophan, and methionine) were selected based on their ketogenic and/or glucogenic nature. Seabream were fed practical diets with different protein (44 and 40%) and lipid contents (21 and 18%): 44P21L, 44P18L, 40P21L, and 40P18L. After three weeks of feeding, the fish were tube-fed the correspondent diet labelled with 14C-lysine, 14C-tryptophan, or 14C-methionine. The amino acid utilisation was determined based on the evacuation, retention in gut, liver, and muscle, and the catabolism of the tracer. The metabolic fate of amino acids was mainly determined by their nature. Tryptophan was significantly more evacuated than lysine or methionine, indicating a lower availability for metabolic purposes. Methionine was more retained in muscle, indicating its higher availability. Lysine was mainly catabolised, suggesting that catabolism is preferentially ketogenic, even when this amino acid is deficient in diets. This study underpins the importance of optimising diets considering the amino acids’ bioavailability and metabolic fate to maximise protein retention in fish. info:eu-repo/semantics/publishedVersion

Commercial diets for tilapia juveniles contain high levels of plant protein sources. Soybean meal has been utilised due to its high protein content; however, soy-based diets are limited in methionine (Met) and require its supplementation to fulfil fish requirements. dl-Methinone (dl-Met) and Ca bis-methionine hydroxyl analogue (MHA-Ca) are synthetic Met sources supplemented in aquafeeds, which may differ in biological efficiency due to structural differences. The present study evaluated the effect of both methionine sources on metabolism and growth of Nile tilapia. A growth trial was performed using three isonitrogenous and isoenergetic diets, containing plant ingredients as protein sources: DLM and MHA diets were supplemented on equimolar levels of Met, while REF diet was not supplemented. Hepatic free Met and one-carbon metabolites were determined in fish fed for 57 d. Metabolism of dl-Met and MHA was analysed by an in vivo time-course trial using 14C-labelled tracers. Only dl-Met supplementation significantly increased final body weight and improved feed conversion and protein efficiency ratios compared with the REF diet. Our findings indicate that Met in DLM fed fish follows the transsulphuration pathway, while in fish fed MHA and REF diets it is remethylated. The in vivo trial revealed that 14C-dl-Met is absorbed faster and more retained than 14C-MHA, resulting in a greater availability of free Met in the tissues when fish is fed with DLM diet. Our study indicates that dietary dl-Met supplementation improves growth performance and N retention, and that Met absorption and utilisation are influenced by the dietary source in tilapia juveniles.

To enhance fish general health, feeds can be supplemented with health-promoting additives, reducing the need to use chemotherapeutics. Incorporation of marine algae biomasses in aquafeeds has been shown to improve fish immune status by enhancing innate immune response. This study evaluated the effects of Phaeodactylum tricornutum incorporation in feed by two different processes, either as freeze-dried biomass or broken cell wall biomass, on fish health status and performance. Triplicate groups of gilthead seabream juveniles (13.3 ± 0.3 g) were either fed a control diet (CTRL) with an extreme (i.e., 0% fishmeal), nutritionally balanced, formulation, or two experimental diets formulated as the CTRL with 1% inclusion of the microalga P. tricornutum at the expense of wheat meal: BC diet contains P. tricornutum broken cells and WC diet microalgae whole cells. After 2 and 12 weeks of feeding, blood was collected for hematological procedures, whereas plasma and mucus were sampled for immune parameters. Head-kidney, liver, and white skeletal muscle were also collected for gene expression measurements. No major differences were observed in hematological nor plasma humoral parameters after 12 weeks irrespective of dietary treatment. Arrays of 29–31 genes were analyzed in the different tissues, revealing an early dietary effect (2 weeks) in a tissue-specific pattern. In the liver, the major effect was found in the GH/IGF axis and in muscle there was a late downregulation of myostatin (mstn) gene, mainly due to WC diet, even though all fish had similar growth performance. Regarding the head-kidney, BC diet led to alpha-2-macroglobulin (a2m) gene upregulation. Also, the same treatment showed increased mucus alternative complement pathway and bactericidal activity at 2 and 12 weeks, respectively. Hence, it seems that BC diet has a potential stimulatory effect that might be relevant as a prophylactic measure before a predictable stressful event., This work has been funded under the EU FP7 by the MIRACLES project No. 613588: Multi-product Integrated bioRefinery of Algae: from Carbon dioxide and Light Energy to high-value Specialties and by national funds through FCT - Foundation for Science and Technology within the scope of UIDB/04423/2020 and UIDP/04423/2020. B. Reis, L. Ramos-Pinto, B. Costas, and S. Engrola were supported by the Portuguese Foundation for Science and Technology (PD/BDE/129262/2017, PD/BDE/114436/2016, IF/00197/ 2015, and IF/00482/2014, respectively).

Methionine is a limiting amino acid (AA) in fish diets, particularly in those containing high levels of plant protein (PP), and is key in the immune system. Accordingly, outcome on the fish immune mechanisms of methionine-deficient and methionine-supplemented diets within the context of 0 % fishmeal formulation, after a short and prolonged feeding period, was studied in European seabass (Dicentrarchus labrax). For this, seabass juveniles were fed a (i) fishmeal-free diet, meeting AA requirements, but deficient in methionine (MET0·65); (ii) as control, the MET0·65 supplemented with l-methionine at 0·22 % of feed weight (CTRL); (iii) two diets, identical to MET0·65 but supplemented at 0·63 and 0·88 % of feed weight of l-methionine (MET1·25 and MET1·5, respectively); and (iv) a fishmeal-based diet (FM), as positive control. After 2 and 12 weeks of feeding, blood and plasma were sampled for leucocyte counting and humoral parameter assays and head-kidney collected for gene expression. After 2 weeks of feeding, a fishmeal-free diet supplemented with methionine led to changes in the expression of methionine- and leucocyte-related genes. A methionine immune-enhancer role was more evident after 12 weeks with an increased neutrophil percentage and a decreased expression of apoptotic genes, possibly indicating an enhancement of fish immunity by methionine dietary supplementation. Furthermore, even though CTRL and FM present similar methionine content, CTRL presented a reduced expression of several immune-related genes indicating that in a practical PP-based diet scenario, the requirement level of methionine for an optimal immune status could be higher.

The dietary protein to energy ratio (P/E) has proven to influence protein utilization and/or growth in several fish species. This study intended to unravel the bioavailability and metabolic fate of lysine and methionine in gilthead seabream (Sparus aurata) juveniles fed plant diets with different P/E ratios. Seabream juveniles were fed two isonitrogenous diets (45% crude protein) differing in crude lipids (20 and 14%): LowP/E ( P / E ratio = 20.0 mg protein k J − 1 ) and HighP/E ( P / E ratio = 21.4 mg protein k J − 1 ). After three weeks, fish ( 11.6 ± 4.3 g ) were tube-fed the respective diet labelled with 14C-protein (L-amino acid mixture), 14C-lysine, or 14C-methionine. Protein, lysine, and methionine utilization were determined based on the proportion of 14C-amino acid evacuated, retained in the free or protein-bound fraction of liver and muscle, or catabolized. This study revealed that a decrease in P/E ratio resulted in lower amino acid evacuation (p p < 0.05 ) due to a simultaneous higher retention of lysine and methionine, without affecting the overall protein retention. Methionine catabolism was significantly lower than lysine or protein independently of the P/E ratio ( p < 0.05 ), reinforcing that this amino acid is preferentially spared for metabolic functions and not used as energy source. In contrast, increasing the dietary P/E ratio decreased lysine catabolism and increased its availability for growth. The bioavailability and metabolism of individual amino acids should be considered when optimizing P/E ratios in diets for gilthead seabream juveniles. Formulating diets with optimum P/E ratios will improve diet utilization and fish performance.

Egg biochemical composition is among the main factors affecting offspring quality and survival during the yolk-sac stage, when larvae depend exclusively on yolk nutrients. These nutrients are primarily embedded in the developing oocytes during vitellogenesis. In aquaculture, assisted reproduction procedures may be applied enabling gamete production. For the European eel (Anguilla anguilla), reproductive treatment involves administration of pituitary extracts from carp (CPE) or salmon (SPE) to induce and sustain vitellogenesis. In the present study, we compared the influence of CPE and SPE treatments on offspring quality and composition as well as nutrient utilization during the yolk-sac stage. Thus, dry weight, proximal composition (total lipid, total protein), free amino acids, and fatty acids were assessed in eggs and larvae throughout the yolk-sac stage, where body and oil-droplet area were measured to estimate growth rate, oil-droplet utilization, and oil-droplet utilization efficiency. The results showed that CPE females spawned eggs with higher lipid and free amino acid contents. However, SPE females produced more buoyant eggs with higher fertilization rate as well as larger larvae with more energy reserves (estimated as oil-droplet area). Overall, general patterns of nutrient utilization were detected, such as the amount of total lipid and monounsaturated fatty acids decreasing from the egg stage and throughout the yolk-sac larval stage. On the contrary, essential fatty acids and free amino acids were retained. Notably, towards the end of the yolk-sac stage, the proximal composition and biometry of surviving larvae, from both treatments, were similar.

In the present work, recent studies that contributed for improving dietary protein in practical microdiets for Senegalese sole larvae are revised, in an attempt to overcome some of the current problems in this species larviculture, such as the difficult early adaptation to inert diets and highly variable growth rates. Different diet formulation strategies were employed in an attempt to improve Senegalese sole larvae capacity to utilize and deposit protein throughout metamorphosis, to further maximize growth potential: (1) increasing indispensable amino acids (IAA) content; (2) meeting the ideal IAA profile by adjusting the dietary AA profile to the larval body AA profile; and (3) decreasing the complexity (molecular weight) of dietary protein to increase its digestibility. Either manipulating the quality or the complexity of dietary protein had impact on the larvae capacity to utilize protein and direct it for growth. Sole larvae seem to be able to adapt their digestive functions and metabolic capacity to dietary protein. Moreover, increasing the dietary IAA/DAA ratio and changing the degree of hydrolysis of dietary protein affected the expression pattern of muscle growth related genes, with consequences on muscle cellularity and potential for growth. The expression of DNA methyltransferases was altered in response to changes in dietary protein. The novelty of such information in fish may trigger further studies on the effect of dietary protein on the epigenetic regulation of growth. In conclusion, optimal protein quality for Senegalese sole seems to change during larval development. Whereas the inclusion of a moderately hydrolysed protein comes up as a promising way to improve growth in early larval stages, larger peptides and intact protein seem to be more suitable to sole post-larvae and young juveniles. Therefore, these results suggest that dietary protein fraction formulation of microdiets for Senegalese sole should be adapted to each developmental stage, what has important consequences for practical larval microdiets formulation and feeding protocols.

Simple Summary The production of marine fish larvae is still recognized to have high mortality rates, so to achieve a more sustainable and competitive aquaculture industry it is essential to develop high-quality larvae. Therefore, this work evaluates if dietary supplementation of natural antioxidant curcumin enhances fish larvae robustness, hence improving growth performance and health status of the farmed fish. For that, two doses of curcumin were assessed and showed that curcumin can modulate larvae condition, digestive capacity, and antioxidant status throughout development. These results bring new perceptions of the effects of dietary curcumin in marine fish larvae that until now have not been studied. Moreover, this data suggested that curcumin can be a suitable feed additive contributing to the development and optimization of microdiets for fish larvae. Abstract The larval stage is highly prone to stress due to the ontogenetic and metabolic alterations occurring in fish. Curcumin inclusion in diets has been shown to improve growth by modulating oxidative status, immune response, and/or feed digestibility in several fish species. The aim of the present work was to assess if dietary curcumin could promote marine fish larvae digestive maturation and improve robustness. Gilthead seabream larvae were fed a diet supplemented with curcumin at dose of 0 (CTRL), 1.5 (LOW), or 3.0 g/Kg feed for 27 days. From 4 to 24 days after hatching (DAH), no differences were observed in growth performance. At the end of the experiment (31 DAH) LOW larvae had a better condition factor than CTRL fish. Moreover, HIGH larvae showed higher trypsin and chymotrypsin activity when compared to CTRL fish. LOW and HIGH larvae were able to maintain the mitochondrial reactive oxygen species production during development, in contrast to CTRL larvae. In conclusion, curcumin supplementation seems to promote larvae digestive capacity and modulate the oxidative status during ontogeny. Furthermore, the present results provide new insights on the impacts of dietary antioxidants on marine larvae development and a possible improvement of robustness in the short and long term.

Simple Summary Oxidative stress has a direct impact on the welfare of fish, affecting growth performance and health status. Natural plant extracts present a high antioxidant capacity, due to a diversity and abundant content of polyphenols. Thus, the aim of this work was to identify if plant extracts, such as curcumin, green tea, and grape seeds, can promote oxidative status, and ultimately, enhance the growth and physiological stress response of postlarvae. Our results showed that plant extracts can improve the growth and oxidative status of the fish. Moreover, they may help fish to cope under stressful conditions. Dietary formulations with natural supplements may be a viable strategy to improve fish robustness during early life stages, and can therefore contribute to the development of aquafeeds and promote the sustainability of aquaculture production. Abstract Plant extracts are known for their high content and diversity of polyphenols, which can improve fish oxidative status. A growth trial with Senegalese sole postlarvae (45 days after hatching) fed with one of four experimental diets—control (CTRL), and supplemented with curcumin (CC), green tea (GT), and grape seed (GS) extracts—was performed to assess if supplementation could improve growth performance and oxidative status. At the end of the growth trial, postlarvae were submitted to a thermal stress to assess their robustness. Sole growth was improved by CC and GS diets when compared to those fed the CTRL. CC and CTRL postlarvae presented the lowest oxidative damage (lipid peroxidation and protein carbonylation values). Stress-related biomarkers (heat shock protein 70 and glutathione-S-transferase) decreased in CC fish compared to those fed the CTRL diet, which might be due to a direct antioxidant capacity. In contrast, oxidative damage increased in GT and GS sole reared in standard conditions. However, after a thermal stress, GT and GS diets prevented the increase of protein carbonylation content and the decrease of antioxidant glutathione, depending on exposure time. Overall, dietary supplementation with natural extracts modulated oxidative status and stress response after a short/long-term exposure to temperature.

Background Oxidative stress has a direct impact on fish production, affecting both growth and health status. Plant based extracts, such as those from green tea, curcumin and grape seeds, are known for their abundant content and diversity of polyphenols. These bioactive compounds have a high antioxidant capacity making such extracts good additives to include in fish diets potentially improving the oxidative status of fish, and therefore enhancing growth and stress resistance of farmed fish.A growth trial with Senegalese sole postlarvae (45 days after hatching) fed with four experimental diets, a control (CTRL) and three supplemented with natural antioxidants, namely curcumin (CC), green tea (GT) and grape seed (GS) extracts, was performed during 25 days to check if these supplements could improve growth performance and fish oxidative status. Moreover, a thermal stress was applied at the end of the growth trial, to assess the effect of these supplemented diets in stress resistance of sole postlarvae.ResultsSole growth was improved by the dietary inclusion of CC and GS compared to CTRL. Postlarvae from CC and CTRL present the lowest values of oxidative damage (lipid peroxidation and protein carbonylation). Moreover, the fish fed CC showed a decrease in stress related biomarkers (heat shock protein 70 and glutathione-S-transferase) compare to CTRL, which might be due to direct antioxidant capacity. In contrast the supplementation of GT and GS increased the content of oxidative damage in sole reared in standard conditions. However, after a thermal stress exposure both GT and GS treatment seem to prevent the increase of protein carbonylation content and the decrease in the levels of antioxidant glutathione in sole, depending on the time of exposure. ConclusionsOverall, dietary supplementation with these natural extracts modulates oxidative status and stress response after a short/long term temperature increase. The incorporation of curcumin seems to be a safe additive to include in sole diets, enhancing growth and oxidative status. The supplementation of the studied doses of green tea and grape seed extracts in fish diets needs further research as they may act as pro-oxidant depending of the culture conditions.

Gilthead seabream (Sparus aurata) is vulnerable to low water temperature, which may occur in the Southern Europe and Mediterranean region during Winter. Fish are poikilothermic animals, therefore feed intake, digestion, metabolism and ultimately growth are affected by water temperature. This study aimed to evaluate growth performance, feed utilisation, nutrient apparent digestibility, and nitrogen losses to the environment in gilthead seabream juveniles reared under low temperature (similar to 13 degrees C). Three isolipid and isoenergetic diets were formulated: a diet similar to a commercial feed (COM) that contained 44% crude protein and 27.5% fishmeal, and two experimental diets with a lower protein content of 42% (ECO and ECOSup). In both ECO diets fishmeal inclusion was reduced (10% in ECO and 7.5% in ECOSup diet) and 15% poultry meal was included. Additionally, the ECOSup diet was supplemented with a mix of feed additives intended to promote fish growth performance and feed intake. The ECO diets presented lower production costs than the COM diet, whilst incorporating more sustainable ingredients. Gilthead seabream juveniles (+/- 154.5 g initial body weight) were randomly assigned to triplicate tanks and fed the diets for 84 days. Fish fed the ECOSup diet attained a similar final body weight than fish fed the COM diet, significantly higher than fish fed the ECO diet. ECOSup fed fish presented significantly higher hepatosomatic index than COM fed fish, most likely due to higher hepatic glycogen reserves. The viscerosomatic index of ECOSup fed fish were significantly lower compared to COM fed fish, which is a positive achievement from a consumer's point of view. ECOSup diet exhibited similar nutrient digestibility than the COM diet. Moreover, feeding fish with the ECO diets resulted in lower faecal nitrogen losses when compared to COM fed fish. The results suggest that feeding gilthead seabream with an eco-friendly diet with a mix of feed additives such as the ECOSup diet, promoted growth and minimised nitrogen losses to the environment. Nutritional strategies that ultimately promote feed intake and diet utilisation are valuable tools that may help conditioning fish to sustain growth even under low temperatures. European UnionEuropean Commission [727315]; Portuguese Foundation for Science and Technology (Ministry of Science and Higher Education, Portugal) [DL 57/2016/CP1361/CT0033]

Welfare in aquaculture is a pressing topic. One of the main measures to improve the life of farmed fish is implementing environmental enrichment, which can include the addition of structural covers to rearing tanks. Here we test the effects of tank covers (fully covered, 50 % covered and uncovered) in an all-male population (N = 75 tagged individuals: 5 individuals per tank, 5 tanks per treatment) of farmed Nile tilapia using behavioural and physiological welfare indicators. Video recordings were performed over 21 days to evaluate overall swimming (swim bursts, freezing on the bottom or hovering in the water column) and aggression events (bites and chases). At the end of the experiment, blood was collected to assess circulating cortisol. Results showed a clearly lower welfare in the partially covered tanks regarding behavioural and physiological variables. The present study highlights the need to correctly study 1) the ethology of the farmed species and 2) the enrichment strategy to be implemented, before any measure is taken. While environmental enrichment is undoubtedly a much needed area of research and a promising solution to improve the welfare of farmed fish, a proper ethological assessment must be made a priori to avoid detrimental effects of poorly devised approaches. UID/Multi/04326/2020; IF/00482/2014/CP1217/CT0005; DL 57/2016/CP1361/CT0033 info:eu-repo/semantics/publishedVersion

Microalgae, small-sized algae, have been studied as a natural freshwater and marine resource for a number of economically relevant applications, including animal feed. This chapter unveils the currently known dietary microalgae effects on the production and meat quality of livestock (ruminants, pigs, poultry, and rabbits) and aquaculture species. Microalgae are classified into four major groups: diatoms (Bacillariophyceae), green (Chlorophyceae), golden (Chrysophyceae), and blue-green (Cyanophyceae) algae. Microalgae are mainly composed of proteins, carbohydrates, lipids, vitamins, minerals, and bioactive compounds, such as carotenoids. They have a variable nutrient composition, depending on several factors such as species, and production conditions (e.g., nutrient availability), enzyme utilization, and biomass status (whole or defatted algae meal). Research evidence so far has shown that the microalgae could successfully replace fish meal and fish oil in diets for fish and improve growth and meat quality in pigs, poultry, and rabbits. In ruminants, dietary inclusion of marine microalgae has improved the milk and meat fatty acid profile. Microalgae have various bioactive compounds that have been shown to enhance defense activity and health status in several animal species, confer tissue protection and antioxidant effects, and affect pigmentation in fish. The nutritional value and major effects of algal biomass used in animal feeds are compiled and discussed.

Fish and other marine animals have a unique and intimate interaction with their surrounding environment. Diet type or trophic level play significant roles in modulating species digestive physiology. However, little is known about how the trophic niche influences digestive activity and gut microbiota in scombrids species. The aim of the present study was to analyse and describe the digestive physiology of the skipjack tuna (Katsuwonus pelamis) and the Atlantic bonito (Sarda sarda) as bioindicator of the feeding ecology and trophic niche for both species in the Gulf of Cádiz (Spain). For that, fish proximate composition, pH of digestive organs and digestive enzymes activity levels were analysed in 10 individuals of each species to gain insight into the digestive physiological adaptations of the two species of scombrids. In addition, intestinal microbiota composition was determined for the skipjack tuna. The integration of the results suggested a clear trophic niche segregation between both species. Stomach pH was associated in both species with infrequent feeding events. Body proximate composition and digestive lipases activity patterns pointed to a high predominance of lipids in the Atlantic bonito diet, suggesting oily fish as main prey. On the other hand, results supported the skipjack tuna as a highly opportunistic feeder with a more varied diet, including fish but also crustaceans as preys. The gut microbial community in the latter species is dominated by Firmicutes and Tenericutes at phylum level, and by Mycoplasma, Blautia and Dorea at genus level. The present study contributes to advance the knowledge on the feeding behaviour and physiology diversity in fish species as a result of adaptation to a particular habitat.

Oxidative stress has a direct impact on fish production, affecting both growth and health status. Plant based extracts, such as those from green tea, curcumin and grape seeds, are known for their abundant content and diversity of polyphenols. These bioactive compounds have a high antioxidant capacity making such extracts good additives to include in fish diets potentially improving the oxidative status of fish, and therefore enhancing growth and stress resistance of farmed fish. A growth trial with Senegalese sole postlarvae (45 DAH) fed with four experimental diets, a control (CTRL) and three supplemented with natural antioxidants, namely curcumin (CC), green tea (GT) and grape seed (GS) extracts, was performed during 25 days to check if these supplements could improve growth performance and fish oxidative status. Moreover, a thermal stress was applied at the end of the growth trial, to assess the effect of these supplemented diets in stress resistance of sole juveniles.Sole growth was improved by the dietary inclusion of CC and GS compared to CTRL. Juveniles from CC and CTRL present the lowest values of oxidative damage (lipid peroxidation (LPO) and protein carbonylation (PC)). Moreover, the fish fed CC showed a decrease in stress related biomarkers (heat shock protein 70 (HSP70) and glutathione-S-transferase (GST)) compare to CTRL, which might be due to direct antioxidant capacity. In contrast the supplementation of GT and GS increased the content of oxidative damage in sole reared in standard conditions. However, after a thermal stress exposure both GT and GS treatment seem to prevent the increase of PC and decrease of antioxidant glutathione (GSH) in sole and depending on the time of exposure. In conclusion, dietary supplementation with these natural extracts modulates oxidative status and stress response after a short/long term temperature increase. The incorporation of curcumin seems to be a safe additive to include in sole diets, enhancing growth and oxidative status. The supplementation of the studied doses of green tea and grape seed extracts in fish diets needs further research as they may act as pro-oxidant depending of the culture conditions.

This data article is associated with the research article "Evaluating the impact of methionine-enriched diets in the liver of European seabass through label-free shotgun proteomics". Here it is described the data obtained from proteomic analysis of 36 European seabass juveniles (3 fish x 3 replicate tanks) after 18 days of feeding with experimental diets containing four inclusion levels of methionine (Met): 0.77%, 1%, 1.36% and 1.66% Met (w/w). We analysed this dataset and compared it with that obtained during the long-term feeding period i.e., 85 days. Fish liver proteins were digested with trypsin and purified peptides were analysed by LC-MS/MS. Proteins were identified with at least two peptides at 0.1% Decoy false discovery rate (FDR). In this dataset, we present the analysis of the differential abundant proteins (DAP) with significant differences across treatments after 18 days of feeding (One-Way ANOVA, p < 0.05). Treatment's comparisons were also performed between the 18- and 85-days feeding trials through Two-Way ANOVA (p < 0.05). MS/MS raw data are available via ProteomeXChange with identifiers PXD019610 and 10.6019/PXD019610 (18-days dataset); and PXD019622 and 10.6019/PXD019622 (85-days dataset). This dataset corresponds to fish sampled after 18-days of experimental trial and is made available to support the study conducted in the afore-mentioned article, by performing the analysis during a short-term period of feeding. The data presented may be further used in other nutritional studies e.g., addressing hepatic changes mediated by Met. ALG-01-0247-FEDER-3520/ UIDB/04326/2020/ IF/00482/2014/CP1217/CT0005 info:eu-repo/semantics/publishedVersion

Methionine is a limiting amino acid (AA) in fish diets, particularly in those containing high levels of plant protein (PP), and is key in the immune system. Accordingly, outcome on the fish immune mechanisms of methionine-deficient and methionine-supplemented diets within the context of 0 % fishmeal formulation, after a short and prolonged feeding period, was studied in European seabass (Dicentrarchus labrax). For this, seabass juveniles were fed a (i) fishmeal-free diet, meeting AA requirements, but deficient in methionine (MET0·65); (ii) as control, the MET0·65 supplemented with l-methionine at 0·22 % of feed weight (CTRL); (iii) two diets, identical to MET0·65 but supplemented at 0·63 and 0·88 % of feed weight of l-methionine (MET1·25 and MET1·5, respectively); and (iv) a fishmeal-based diet (FM), as positive control. After 2 and 12 weeks of feeding, blood and plasma were sampled for leucocyte counting and humoral parameter assays and head-kidney collected for gene expression. After 2 weeks of feeding, a fishmeal-free diet supplemented with methionine led to changes in the expression of methionine- and leucocyte-related genes. A methionine immune-enhancer role was more evident after 12 weeks with an increased neutrophil percentage and a decreased expression of apoptotic genes, possibly indicating an enhancement of fish immunity by methionine dietary supplementation. Furthermore, even though CTRL and FM present similar methionine content, CTRL presented a reduced expression of several immune-related genes indicating that in a practical PP-based diet scenario, the requirement level of methionine for an optimal immune status could be higher.

Fish meal replacement by plant-protein sources is a priority in aquaculture feeds. Within this framework, dietary supplementation with essential amino acids (EAA), as tryptophan (TRP), is strategic to ensure that the individual nutritional needs are met, besides promoting enhanced immunological status. The purpose of this study was to examine the beneficial effects of TRP incorporation in plant-protein source diets on fish growth performance and nutritional status. We tested diets with 20% lower (LTRP) and 27% higher (HTRP) of the putative requirements of TRP for seabream (Sparus aurata) and assessed its impact on fish physiology and liver metabolism and proteome. After 12 weeks, growth performance, body proximate, hepatic composition and liver metabolic profiling were similar between diets. Nevertheless, liver proteome analysis indicated a higher accumulation of proteins involved in acute-phase responses, typically triggered by infection, inflammation or trauma, in fish fed with HTRP diet as compared with those fed with LTRP. The overall results obtained suggest a potential beneficial effect of TRP supplementation in terms of immune stimulation, without compromising growth or feed intake. Moreover, proteomics and metabolic profiling demonstrate to be valuable tools in this endeavour. Significance: Nutritional needs are hard to assess in aquaculture fisheries, and many times controversial depending on the methodology employed. The estimated amino acid requirements depend on both fish species and stage development, making it extremely hard to standardise. On the other hand, the substitution of fish-based to plant-based protein sources diets towards a sustainable aquaculture, may imbalance these requirements, being necessary further studies to assess the impact on fish growth and development. Finally, the incorporation of crystalized amino acids such as TRP into diets aims global better performance both at fish health/immune condition and growth development. This work focused on the potential beneficial effects of TRP supplementation into diets with a plant-based protein source, addressing the effects on the liver metabolism and proteome, and on growth performance of Gilthead seabream juveniles, a species with special relevance and economical importance in the Mediterranean region. The present study by employing proteomics together with metabolic profiling shows that TRP supplementation at the tested doses, does not compromise growth performance, and seems to stimulate the immune system. Our findings can contribute to the development of new feed formulations for Gilthead seabream species, therefore, reinforcing the resilience and competitiveness of the on-growing aquaculture industry and impact directly the sustainability of living resources with the decrease of the fisheries needs to fulfil the human search for quality proteins consume. European Union through FEDER [ALG-01-0247-FEDER3520] European Union through COMPETE 2020 [ALG-01-0247-FEDER3520] European Union through CRESC Algarve 2020 [ALG-01-0247-FEDER3520] Portuguese Foundation for Science and Technology (FCT)Portuguese Foundation for Science and Technology [UIDB/04326/2020] Portuguese Foundation for Science and Technology (FCT) through COMPETE Programme Portuguese Foundation for Science and Technology (FCT) through Operational Human Potential Programme info:eu-repo/semantics/publishedVersion

Aquaculture is one of the most thriving animal production sectors, and Nile tilapia (Oreochromis niloticus) farming represents 8% of total finfish culture. However, the industry sustainability depends on the development of cost‐effective and environmental friendly feeds. This study aimed to reduce dietary protein levels in diets for juvenile tilapia and to minimize diet environmental impact while maximizing biological efficiency. A growth trial was performed using five isoenergetic plant protein‐based diets with decreasing levels of crude protein: 360, 340, 320, 300 and 280 g/kg diet (D360, D340, D320, D300 and D280, respectively). Dietary protein utilization was assessed by metabolic trials using a radiolabelled amino acid mixture. Tilapia in all treatments showed similar growth performance and feed intake. Feed conversion ratio was significantly higher in fish fed the D280 than the D360 diet, while no differences were found for other treatments. Protein retention was significantly higher in tilapia fed the D300 than the D360 diet. Amino acid catabolism increased in fish fed the D360 diet, though without significant differences in muscle amino acid retention. This study demonstrates that dietary protein levels can be reduced to 300 g/kg diet without hindering tilapia growth and feed conversion ratio, while reducing environmental nitrogen losses. FCT: UID/Multi/04326/2019/ IF/00482/2014/CP1217/CT0005/ DL57/2016/CP1361/CT0033. info:eu-repo/semantics/publishedVersion

The relatively easy access to fish worldwide, alongside the increase of aquaculture production contributes to increased fish consumption which result in higher prevalence of respective allergies. Allergies to fish constitute a significant concern worldwide. beta-parvalbumin is the main elicitor for IgE-mediated reactions. Creatine, involved in the muscle energy metabolism, and ethylenediamine tetraacetic acid (EDTA), a calcium chelator, are potential molecules to modulate parvalbumin. The purpose of this study was to test creatine (2, 5 and 8%) and EDTA (1.5, 3 and 4.5%) supplementation in fish diets to modulate beta-parvalbumin expression and structure and its allergenicity in farmed European seabass (Dicentrarchus labrax) while assessing its effects on the end-product quality. Fish welfare and muscle quality parameters were evaluated by plasma metabolites, rigor mortis, muscle pH and sensory and texture analysis. Proteomics was used to assess alterations in muscle proteome profile and metabolic fingerprinting by Fourier transform infrared spectroscopy was used to assess the liver metabolic profile. In addition, IgE-reactivity to parvalbumin was analysed using fish allergic patient sera. Metabolic fingerprinting of liver tissue revealed no major alterations in infrared spectra with creatine supplementation, while with EDTA, only absorption bands characteristic of lipids were altered. Comparative proteomics showed up regulation of (tropo) myosin and phosphoglycerate mutase 2 with Creatine supplementation. In the case of EDTA proteomics showed up regulation of proteins involved in cellular and ion homeostasis. Allergenicity seems not to be modulated with creatine or EDTA supplementation as no decreased expression levels were found and IgE-binding reactivity showed no quantitative differences. 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.

European aquaculture is an industry with a high sustainability profile contributing to the supply of safe seafood. However, several diseases can affect farmed fish and it is imperative to find alternatives for chemotherapeutic treatments when disease outbreaks occur. Maintenance of health through nutrition is well-establish in modern animal farming, and amino acids (AA) are promising candidates as functional additives to improve fish health. Therefore, the goal of this research is to provide a better understanding of the influence of tryptophan supplementation on nutritional condition and immune mechanisms in fish. Triplicate groups of fish (13.3 ± 0.3g) previously fed with a fishmeal-based diet were either fed a control diet with an extreme formulation (0% fishmeal) but meeting the AA requirements (CTRL), or the SUP diet, formulated as the CTRL with an increase in tryptophan (TRP) content. After 2 and 13 weeks of feeding, head-kidney (HK), liver (L) and white skeletal muscle (WSM) were collected for gene expression, whereas plasma was suited for humoral immune parameters. A holistic approach using transcriptomic, humoral and zootechnical parameters was undertaken. The expression of 29–31 genes for WSM, L or HK confirms an effect due to the treatment across time. A two-way ANOVA analysis revealed that 15–24 genes varied significantly depending on the tissue, and the multivariate analysis by means of PLS-DA explained (R) and predicted (Q) with four components up to 93% and 78% of total variance, respectively. Component 1 (R = 50.06%) represented the time effects, whereas components 2 (24.36%) and 3 (13.89%) grouped fish on the basis of dietary treatment, at early sampling. The HK results in particular suggest that fish fed SUP diet displayed an immunostimulated state at 2 weeks. No major differences were observed in plasma humoral parameters, despite an increase in antiprotease and peroxidase activities after 13 weeks regardless of dietary treatment. These results suggest that tryptophan supplementation may improve the seabream immune status after 2 weeks. Hence, the use of functional feeds is especially relevant during a short-term feeding period before a predictable stressful event or disease outbreak, considering that these putative advantageous effects seem to disappear after a 13 weeks feeding period., This work was partially supported by Projects ALISSA (reference ALG-01-0247-FEDER-3520), UID/Multi/04423/2019, IF/00197/2015 and IF/00482/2014, financed by Portugal and the European Union through FEDER, COMPETE 2020 and CRESC Algarve 2020, in the framework of Portugal 2020, and through the COMPETE and Operational Human Potential Programmes and national funds through Fundação para a Ciência e a Tecnologia (FCT, P ortugal). L. Ramos-Pinto, S. Engrola and B. Costas were supported by FCT, Portugal (PD/BDE/114436/2016, IF/00482/2014 and IF/00197/2015, respectively).

Methionine is the first limiting AA in fish diets, particularly in those containing high levels of plant protein sources (e.g. soybean). It is also important to highlight that methionine presents a key role in the immune system of many vertebrate animal models, including fish. info:eu-repo/semantics/publishedVersion

Metabolic programming refers to the induction, deletion, or impaired development of a somatic structure or "setting" of a physiological system by an early life stimulus operated at a critical period during development. Ghrelin is the only known orexigenic gut hormone, is an acylated peptide that acts as an endogenous ligand specific for growth-hormone secretagogue-receptor. The aim of the present work was to evaluate if an in ovo ghrelin administration could positively influence the zebrafish performance in the long-term and to gain insight on the mechanisms associated to ghrelin regulation of food intake during the larval phase. Food intake, growth potential, protein metabolism, expression of target genes involved in ghrelin, feeding behaviour regulation and locomotor activity were assessed in zebrafish (Danio rerio) larvae at 25 days post-fertilization. Elevated levels of acylated ghrelin in zebrafish eggs did not result in increased growth or food intake. Differences in mRNA expression between larvae fasted for 16 h before and 1 h after feeding were found for igf1ra, gh1 and pomca. Moreover, ghrelin treated larvae showed higher swimming activity, indicating that the peptide may have an important role on foraging activity. The present study addressed for the first time the effects of an early stimulus of ghrelin during the embryonic stage of zebrafish, however, further studies are needed to clarify the metabolic pathways affected by the early stimulus as well as focus on the effects on metabolic regulation of energy balance through lipid and carbohydrate metabolism. FCT/MCTES (Portugal)Portuguese Foundation for Science and Technology [PTDC/CVT/102481/2008, UID/Multi/04326/2013] FCT - European Social Fund [IF/00482/2014/CP1217/CT0005] Operational Programme Human Potential Foundation for Science and Technology of Portugal (FCT)Portuguese Foundation for Science and Technology

The present study is focused to elucidate the main characteristics of the digestive function of this carnivorous fast-growing fish living at high temperatures. With this aim, we have examined the effects of an increased temperature from 30 to 34 °C on the daily pattern of gastrointestinal pH, enzymatic proteolytic digestive activity and the feed transit time in early juveniles of cobia (Rachycentron canadum), a species living in tropical and subtropical waters with an increasing aquaculture production. Fish were fed two meals a day. Gastric luminal pH was permanently acidic (mean pH values: 2.76–4.74) while the intestinal pH increased from neutral/slightly acidic to slightly alkaline when the digesta was present, with an increasing alkalinity from proximal to distal intestine (mean pH values: 6.05 to 7.69). The temperature did not affect the gastric pH but a slightly higher acidity was induced in the intestine at 34 °C. Pepsin activity showed a daily rhythm at 30 °C with maximum in the middle of the light period, while at 34 °C some hourly changes coinciding with feed adding without a clear daily trend during the 24-h period were observed. The trypsin activity exhibited a daily rhythm at both temperatures with an increase after morning feeding to reach a maximum several hours later. Average pepsin activity during the daily cycle was slightly higher at 34 °C (6.1 and 7.3 U mg−1 BW at 30 and 34 °C respectively), but values were significantly different only at 8 and 24 h after the morning meal. Similarly, the trypsin activity was significantly affected by the temperature only at 8 and 16 h after the morning meal, but daily activity averages were similar (1.20 and 1.29 U g−1 BW at 30 and 34 °C respectively). The partial transit rates of the first meal in the stomach for each period inter-samplings were higher during the first 4-h period and decreased progressively along the rest of the 24-h cycle at both temperatures, but no significant differences were detected at 30 °C. In addition, the transit was notably faster at 34 °C particularly during the first 8 h after feeding, with rates between 100 and 65% of total volume displaced (intake or released) during each 4-h period. In the intestine the transit rate was relatively constant and similar at both temperatures during 12 h after feeding. Then the rates remained very low during the following 12 h. Residence time of the first meal was longer at 30 than at 34 °C, particularly in the stomach (12 h:02 min vs 4 h:54 min respectively). In the intestine the difference was not so large (8 h:18 min vs 6 h:24 min respectively). In a parallel study under same conditions, cobia reared at 30 °C grew faster and showed a more favorable feed conversion ratio than those at elevated temperature (34 °C). The present results indicate that at 34 °C, a subtle increase of proteolytic activity cannot compensate for the faster gut transit rate. Therefore, 30 °C is more appropriate temperature for the early on-growing of cobia because at higher temperatures the digestion efficiency decrease being one of the causes for a lower growth., The project WISEFEED received funding by the European Union's H2020 programme (Marie Skłodowska-Curie grant No 691150). Additional funding from project EFISHDIGESTAGL2014-52888 (MINECO, Spain + FEDER/ERDF contribution). S.E. acknowledges a Foundation for Science and Technology of Portugal (FCT) investigator grant IF/00482/2014/CP1217/CT0005 funded by the European Social Fund, the Operational Programme Human Potential and FCT. This work also received national funds through FCT through project UDI/Multi/04326/2013 and Norwegian Agency for Development Cooperation NORHED, No. QZA-0485 SRV-13/0010.

New and more efficient methods to sustainably intensify Aquaculture production are essential to attain the seafood demand for direct human consumption in the near future. Nutrition has been identified as one strategy of early exposure that might affect animal early development and later phenotype. This strategy may have positive consequences in the modulation of fish digestive physiology, which will correlate with higher performance outputs. Thus, improving fish digestive efficiency will lead to higher productivity and lower biogenic emission from aquaculture facilities, minimising the impact on the environment while increasing the biological efficiency. An innovative in ovo nutritional modulation technique based on low-frequency ultrasounds was used to enhance the transport of amino acids across the embryo membranes. An early stimulus with either arginine or glutamine, both involved in gut maturation, was applied in zebrafish (Danio rerio) embryos at 3.5 hours post-fertilization (hpf). At 22 days post-fertilization (dpf), growth performance, digestive enzyme activities and gut microbiota composition were analysed to evaluate the larval nutrition-induced metabolic plasticity and the effects on fish digestive efficiency. Results showed that fish survival was not affected either by the sonophoresis technique or amino acid supplementation. Final dry weight at 22 dpf was statistically higher in larvae from glutamine treatment when compared to the control even with lower trypsin activity, suggesting a higher nutrient digestion capacity, due to a slightly modulation of gut microbiota. Higher arginine supplementation levels should be tested as strategy to enhance growth at later developmental stages. In conclusion, this study demonstrated the efficiency of sonophoresis technique for in ovo nutritional modulation and suggests that in ovo glutamine supplementation might promote growth at later developmental stage through a positive microbiota modulation.