Exploring first-feeding diets for European eel larval culture: Insights at morphological, nutritional, and molecular levels.

Closing the life cycle of European eel (Anguilla anguilla) in captivity is targeted to provide a sustainable, year-round supply of juveniles for aquaculture. Present focus is on the nutritional requirements during the larval first-feeding period. In this study, three experimental diets were tested on hatchery-produced European eel larvae from the onset of the first-feeding stage commencing 10 days post hatch (dph) until 28 dph. Larval mortality was recorded daily, while sampling was conducted at regular intervals to record larval biometrics and analyze the expression of genes related to digestion, appetite, feed intake and growth. Two periods of high mortality were identified: the first appeared shortly after introduction of feeds (10-12 dph), while the second occurred 20-24 dph, indicating the "point of no return". This interpretation was supported at the molecular level by the expression of the gene encoding the "hunger hormone" ghrelin (ghrl) that peaked at 22 dph in all dietary trials, suggesting that most larvae were fasting. However, in larvae fed diet 3, ghrl expression was downregulated beyond 22 dph, which indicated that those larvae were no longer starving at this stage, while upregulation of genes encoding the major digestive enzymes (try, tgl, and amyl2a) advocated their healthy development. Moreover, for larvae fed diet 3, the expression of those genes as well as genes for feed intake (pomca) and growth (gh) continued to increase towards 28 dph. These results together with the registered highest survival, largest dry weight increase, and enhanced biometrics (length and body area) pointed to diet 3 as the best-performing. As a whole, this first-feeding study represents a landmark being the first to document European eel larval growth and survival beyond the point of no return, providing novel insights into the molecular development of digestive functions during the first feeding stage.

Fish Processing and Digestion Affect Parvalbumins Detectability in Gilthead Seabream and European Seabass.

Consumption of aquatic food, including fish, accounts for 17% of animal protein intake. However, fish consumption might also result in several side-effects such as sneezing, swelling and anaphylaxis in sensitized consumers. Fish allergy is an immune reaction to allergenic proteins in the fish muscle, for instance parvalbumin (PV), considered the major fish allergen. In this study, we characterize PV in two economically important fish species for southern European aquaculture, namely gilthead seabream and European seabass, to understand its stability during in vitro digestion and fish processing. This information is crucial for future studies on the allergenicity of processed fish products. PVs were extracted from fish muscles, identified by mass spectrometry (MS), and detected by sandwich enzyme-linked immunosorbent assay (ELISA) after simulated digestion and various food processing treatments. Secondary structures were determined by circular dichroism (CD) after purification by anion exchange and gel filtration chromatography. In both species, PVs presented as α-helical and ß-sheet structures, at room temperature, were shown to unfold at boiling temperatures. In European seabass, PV detectability decreased during the simulated digestion and after 240 min (intestinal phase) no detection was observed, while steaming showed a decrease (p < 0.05) in PVs detectability in comparison to raw muscle samples, for both species. Additionally, freezing (−20 °C) for up to 12 months continued to reduce the detectability of PV in tested processing techniques. We concluded that PVs from both species are susceptible to digestion and processing techniques such as steaming and freezing. Our study obtained preliminary results for further research on the allergenic potential of PV after digestion and processing.

Metabolic Fate Is Defined by Amino Acid Nature in Gilthead Seabream Fed Different Diet Formulations.

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.

Alternative Proteins for Fish Diets: Implications beyond Growth.

Aquaculture has been challenged to find alternative ingredients to develop innovative feed formulations that foster a sustainable future growth. Given the most recent trends in fish feed formulation on the use of alternative protein sources to decrease the dependency of fishmeal, it is fundamental to evaluate the implications of this new paradigm for fish health and welfare. This work intends to comprehensively review the impacts of alternative and novel dietary protein sources on fish gut microbiota and health, stress and immune responses, disease resistance, and antioxidant capacity. The research results indicate that alternative protein sources, such as terrestrial plant proteins, rendered animal by-products, insect meals, micro- and macroalgae, and single cell proteins (e.g., yeasts), may negatively impact gut microbiota and health, thus affecting immune and stress responses. Nevertheless, some of the novel protein sources, such as insects and algae meals, have functional properties and may exert an immunostimulatory activity. Further research on the effects of novel protein sources, beyond growth, is clearly needed. The information gathered here is of utmost importance, in order to develop innovative diets that guarantee the production of healthy fish with high quality standards and optimised welfare conditions, thus contributing to a sustainable growth of the aquaculture industry.

Metabolic and nutritional responses of Nile tilapia juveniles to dietary methionine sources.

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.

Effect of creatine and EDTA supplemented diets on European seabass (Dicentrarchus labrax) allergenicity, fish muscle quality and omics fingerprint.

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. ß-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 ß-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.

Exploring the Biotechnological Value of Marine Invertebrates: A Closer Look at the Biochemical and Antioxidant Properties of Sabella spallanzanii and Microcosmus squamiger.

Sabella spallanzanii and Microcosmus squamiger were profiled for proximate composition, minerals, amino acids, fatty acids (FA), carotenoids, radical scavenging activity on the 2,2-diphenyl-1- picrylhydrazyl (DPPH) radical, oxygen radical absorbance capacity (ORAC) and iron and copper chelating properties. Microcosmus squamiger had the highest level of moisture and crude protein, S. spallanzanii was enriched in crude fat and ash. Both species had similar levels of carbohydrates and energy. There was a prevalence of arginine and glycine in S. spallanzanii, and of taurine in M. squamiger. The most abundant minerals in both species were Na, Ca, and K. The methanol extract of S. spallanzanii had metal chelating properties towards copper and iron, while the methanol extract of M. squamiger was able to chelate copper. M. squamiger extracts had similar ORAC values. Fucoxanthinol and fucoxanthin were the major carotenoids in the M. squamiger dichloromethane extract. Saturated FA were more abundant than unsaturated ones in methanol extracts, and unsaturated FA prevailed in the dichloromethane extracts. Palmitic acid was the predominant FA in methanol extracts, whereas eicosapentaenoic (EPA) and dihomo-γ-linolenic acids were the major compounds in dichloromethane extracts. Low n-6/n-3 ratios were obtained. Our results suggests that both species could be explored as sources of bioactive ingredients with multiple applications.

Dietary Curcumin Promotes Gilthead Seabream Larvae Digestive Capacity and Modulates Oxidative Status.

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.

Dietary Natural Plant Extracts Can Promote Growth and Modulate Oxidative Status of Senegalese Sole Postlarvae under Standard/Challenge Conditions.

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.

Characterization and comparison of the digestive physiology of two scombrids, Katsuwonus pelamis and Sarda sarda, in the Gulf of Cádiz.

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.

Egg nutritional modulation with amino acids improved performance in zebrafish larvae.

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.

Data on European seabass fed with methionine-enriched diets obtained through label free shotgun proteomics.

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.

Evaluating the impact of methionine-enriched diets in the liver of European seabass through label-free shotgun proteomics.

Plant protein sources play an essential role in aquaculture by reducing the use of fish meal to sustainable levels, although further supplementation is needed to fulfill fish nutritional requirements. This work addressed fish growth performance and proteome changes to dietary methionine in European seabass juveniles. A dose-dependent response to methionine (Met) was observed on fish growth consistent with proteomic analyses, suggesting Met requirement ≥0.9% (w/w). Fish fed at 0.77% (w/w) exhibited reduced growth and an enrichment in proteins involved in cellular homeostasis. Proteomics data suggest an optimal nutritional status at 1.36% Met (w/w), together with putative beneficial effects on the immune system up to 1.66% Met (w/w). The response to dietary Met involved the convergence of different metabolic and signalling pathways implicated in cell growth and immune response e.g., mTOR, Hedgehog or the T Cell receptor signalling, coupled with a fine-tuning regulation of amino acid metabolism and translation.

Dietary Antioxidant Supplementation Promotes Growth in Senegalese Sole Postlarvae.

Somatic growth is a balance between protein synthesis and degradation, and it is largely influenced by nutritional clues. Antioxidants levels play a key role in protein turnover by reducing the oxidative damage in the skeletal muscle, and hence promoting growth performance in the long-term. In the present study, Senegalese sole postlarvae (45 days after hatching, DAH) were fed with three experimental diets, a control (CTRL) and two supplemented with natural antioxidants: curcumin (CC) and grape seed (GS). Trial spanned for 25 days and growth performance, muscle cellularity and the expression of muscle growth related genes were assessed at the end of the experiment (70 DAH). The diets CC and GS significantly improved growth performance of fish compared to the CTRL diet. This enhanced growth was associated with larger muscle cross sectional area, with fish fed CC being significantly different from those fed the CTRL. Sole fed the CC diet had the highest number of muscle fibers, indicating that this diet promoted muscle hyperplastic growth. Although the mean fiber diameter did not differ significantly amongst treatments, the proportion of large-sized fibers (>25 µm) was also higher in fish fed the CC diet suggesting increased hypertrophic growth. Such differences in the phenotype were associated with a significant up-regulation of the myogenic differentiation 2 (myod2) and the myomaker (mymk) transcripts involved in myocyte differentiation and fusion, respectively, during larval development. The inclusion of grape seed extract (GS diet) resulted in a significant increase in the expression of myostatin1. These results demonstrate that both diets (CC and GS) can positively modulate muscle development and promote growth in sole postlarvae. This effect is more prominent in CC fed fish, where increased hyperplastic and hypertrophic growth of the muscle was associated with an upregulation of myod2 and mymk genes.

Dietary methionine supplementation improves the European seabass (Dicentrarchus labrax) immune status following long-term feeding on fishmeal-free diets.

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.

How tryptophan levels in plant-based aquafeeds affect fish physiology, metabolism and proteome.

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.

Dietary tryptophan supplementation induces a transient immune enhancement of gilthead seabream (Sparus aurata) juveniles fed fishmeal-free diets.

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 ±â€¯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 (R2) and predicted (Q2) with four components up to 93% and 78% of total variance, respectively. Component 1 (R2 = 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.

The role of dietary methionine concentrations on growth, metabolism and N-retention in cobia (Rachycentron canadum) at elevated water temperatures.

This study determined impacts of dietary methionine concentrations at two temperatures on growth, feeding efficiency and N-metabolites in juvenile cobia. Methionine concentrations of the experimental diets were deficient (M9; 9 g/kg), sufficient (M12; 12 g/kg) and surplus (M16, 16 g/kg). Water temperature was normal (30°C) or elevated (34°C). Twenty cobia in triplicate tanks were fed the experimental diets for 6 weeks. Both methionine and temperature affected cobia's growth and feeding efficiency. Cobia fed M9 performed lower than the fish fed M12 and M16 diets. Additionally, cobia reared at 34°C performed poorer than at 30°C, probably due to lower voluntary feed intake in the fish reared at 34°C. Protein efficiency ratio and protein productive value in cobia fed M9 diet were less than M12 or M16 diets. This was confirmed with the improved retentions of indispensable amino acids (AAs). No interactions between methionine and temperature were observed in growth and protein accretion. At 30°C, CF improved, while HSI and VSI declined upon methionine supplementation levels. Of which an interaction between temperature and methionine was present. Plasma, muscle and liver free AA and N-metabolites were affected by methionine and temperature. Furthermore, temperature affected cobia's lipid class composition, resulting in increased phospholipids and cholesterol at 34°C.

Does a ghrelin stimulus during zebrafish embryonic stage modulate its performance on the long-term?

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.

Partition and metabolic fate of dietary glycerol in muscles and liver of juvenile tilapia.

This study investigated the effect of dietary glycerol on the metabolism of juvenile tilapia (Oreochromis mossambicus) and to determine its metabolic fate. The experimental diets contained 0% (Group CON), 5% (Group G5) and 15% glycerol (Group G15) and were fed for 40 d to apparent satiation, three times a day. For the metabolism trials, six fish from each treatment were randomly chosen and tube-fed with five pellets labelled with 14C-glycerol [14C(U)] in order to evaluate the absorption, catabolism, retention and partition of glycerol in muscle and liver. Group G5 presented the highest 14C-glycerol retention and the lowest catabolism, with no significant differences between Groups CON and G15. In Group CON, the highest percentage of 14C was incorporated in muscle lipids; with no significant differences between Groups G5 and G15. Furthermore, no treatment effects were found for hepatic 14C-lipid and for 14C in hepatic and muscle non-lipid extract. In the non-lipid and non-protein fraction, the highest radioactivity was measured in livers of Group G5, however no significant differences were found for this fraction between Groups CON and G15 in liver and for all treatments in muscle. The results of the present study can have practical implications in diet formulations for tilapia and for other aquaculture species with similar feeding pattern since juvenile tilapia are able to metabolise dietary glycerol into lipids, protein and/or carbohydrates and to use it as energy source.