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Stimulation of the fish immune system using immunostimulants is an environmentally friendly strategy to minimize bacterial outbreaks in aquaculture. Different biological and synthetic immunostimulants can enhance non-specific innate immune responses by directly activating immune cells. An example are Bacillus spp., known for their immunostimulatory effects, although the exact mechanisms by which Bacillus spp. offer protection against diseases remains to be elucidated. Furthermore, most studies have focused on Bacillus spp. cells, while the immunostimulant effect of their extracellular metabolome, known to harbour biologically important metabolites, including antimicrobial molecules, has been scarcely evaluated. Here, we evaluated the in vitro immune-modulatory properties of extracellular extracts of three Bacillus spp. strains (B. subtilis FI314, B. vezelensis FI436 and B. pumilus FI464), previously isolated from fish-guts and characterized for their in vitro and in vivo antimicrobial activity against a wide range of fish pathogens. Bacillus spp. extracellular extracts did not affect immune cells viability, but remarkably increased pathogens' phagocytosis when seabream head-kidney leukocytes were challenged with Vibrio anguillarum and Edwardsiella tarda. All extracts significantly increased the engulfment of bacterial pathogens 1 h post-infection. Cells stimulated with the extracellular extracts showed an up-regulation of the expression of immune-relevant genes associated with inflammation, including IL-1β, IL-6, and COX-2. In cells challenged with E. tarda, FI314 extracellular extract significantly increased the expression of IL-1β, IL-6, and COX-2, while FI436 and FI464 significantly increased IL-6 expression. The results of this study revealed that the extracellular molecules from Bacillus spp. fish isolates improved the in vitro response of gilthead seabream immune cells and are thus promising candidates to act as immunostimulants, helping fish fight diseases.

This study assessed the effect of supplementation of low-fishmeal (FM) diets with taurine in white seabream (Diplodus sargus) juveniles. Four isoproteic (36% CP) and isolipidic (18% CL) diets were formulated to include 15%FM or 5%FM, supplemented or not with 1% of Taurine (TAU). Twelve groups of 15 fish (IBW = 58 g) were fed each diet to near satiation for 10 weeks. Growth performance, feed intake, and feed efficiency were not affected by dietary FM level. Nevertheless, dietary TAU supplementation increased growth performance and feed efficiency. Nitrogen and energy retention were improved by both dietary FM level and TAU supplementation. Plasma cholesterol, lactate, and bilirubin did not differ among dietary treatments, but dietary TAU supplementation decreased plasma glucose and triglycerides levels. Decrease of FM level reduced total bile salts while TAU supplementation had the opposite effect. No differences were noticed in innate immune parameters assessed. Overall, results indicate that white seabream can be fed with diets almost devoid of FM, and that dietary TAU supplementation increases growth performance and feed efficiency. Moreover, results suggest that dietary TAU was limiting both in 15% and 5% FM diets.

A growth trial was conducted to evaluate the effect of dietary plant-protein replacement by corn distillers dried grains with solubles (DDGS), and of exogenous carbohydrases supplement (Natugrain®TS, BASF) in gilthead seabream (Sparus aurata) juveniles. For that purpose, a basal diet was formulated with 35% of fish meal and 20% soybean meal (SBM) as main protein sources. Two other diets were formulated incorporating 15 or 35% of DDGS, replacing 37.5% or 100% of SBM (diets DDGS15 and DDGS35, respectively). Another diet was formulated similar to diet DDGS35 and supplemented with 0.1% of a commercial non-starch polysaccharidases complex, Natugrain®TS, BASF (CHOase; diet DDGS35ENZ). Triplicate groups of fish (IBW = 15 ± 1 g) were fed the experimental diets for 67 days at 22 °C. At the end of the trial, growth performance, voluntary feed intake, feed efficiency, protein and energy retention were not affected by dietary DDGS incorporation. Dietary CHOase supplementation also did not affect growth performance, but increased feed efficiency, nitrogen and energy retention. Dietary inclusion of DDGS tended to decrease production cost (€ per kg of fish), and dietary CHOase supplementation further reduced production cost, which was significantly lower with diet DDGS35ENZ than with the basal diet. Whole-body composition, hepatosomatic and visceral indexes were not affected by the dietary inclusion of DDGS, though a trend was noticed for a decrease of whole-body lipid, energy, and visceral index with the increase dietary DDGS. Plasma glucose, protein, albumin, and globulins levels were similar among diets, whereas plasma triglycerides increased, and cholesterol decreased with the increase of dietary DDGS. Hepatic glycolytic enzymes activities (hexokinase and glucokinase) were similar among groups, while gluconeogenic (fructose biphosphatase) activity, GDH and ASAT activities decreased with the increase of dietary DDGS. The hepatic activity of oxidative stress-related enzymes catalase and superoxide dismutase were not affected by dietary treatments, but G6PDH and GR activities decreased, and liver lipid peroxidation increased with dietary DDGS level. Dietary CHOase supplementation decreased overall lipid peroxidation. Overall, results of this study indicate that total replacement of SBM by DDGS in diets for gilthead seabream did not compromise growth performance or feed utilization efficiency, while supplementation of DDGS-based diet with exogenous carbohydrases improved feed utilization efficiency and economic efficiency ratio.

Distillers' dried grains with solubles (DDGS) has low tryptophan (Trp) relatively to the branched-chain amino acids (BCAA) levels, and this may reduce transport of Trp through the blood-brain barrier due to competition for the same transport carrier. This may affect synthesis and release of serotonin, with negative consequences in stress tolerance. In the present study, it is hypothesized that a Trp/BCAA unbalance in high DDGS diets may impair the capacity of gilthead seabream (Sparus aurata) juveniles to cope with chronic stress induced by high stocking density. Three DDGS-based diets (30%DDGS+13%FM) were formulated and supplemented with Trp at 0, 0.13, and 0.25% of the diet and tested in triplicate, at two initial stocking densities (5 and 16 kg m−3), in a 2 × 3 total randomized factorial design. The growth trial was performed with 12 g fish and lasted 64 days. Irrespective of the diet, high stocking density reduced growth performance and feed intake, but not feed efficiency. Plasma protein, triglycerides, and cholesterol levels; whole-body lipid, hepatosomatic index, and liver glycogen; hepatic activity of key-enzymes of glycolysis and lipogenesis were also reduced. Moreover, plasma glucose level and hepatic activity of key-enzymes gluconeogenesis were increased. Irrespective of stocking density, diets supplementation with Trp did not affect growth and feed efficiency, but increased hepatic lipase activity and reduced liver lipids, plasma triglycerides and cholesterol levels, and hepatic activity of key-enzymes of amino acid catabolism. Moreover, dietary Trp supplementation restored plasma glucose levels of fish kept at high stocking density to levels similar to that of fish kept at low stocking density. Overall, present results indicate that high stocking density reduced growth performance without affecting feed efficiency of gilthead seabream. Dietary Trp supplementation did not counteract the negative effect of stocking density on growth performance but seemed to mitigate stress response of gilthead seabream juveniles kept at high stocking density.

This study aims to understand the interactive effects of high dietary vegetable oils (VO) and carbohydrate (CH) content in European seabass cellular, humoral, and molecular innate immune responses to an acute stress. For that purpose, European sea bass juveniles (74.0 ± 1.5 g) were fed four diets differing in lipid source (fish oil (FO) or a blend of vegetable oils (VO)) and carbohydrate content (0% (CH–) or 20% (CH+) gelatinized starch). Nine fish per dietary treatment were sampled after 73 days of feeding and used as control whereas the remaining fish were subjected to an acute stress (netting, 1 min air exposure, and transfer to smaller tanks). Those fish were then sampled after 1 h and considered the stressed group. In the present study, dietary VO incorporation affected fish humoral immune parameters with a decrease of haematocrit, plasma peroxidase activity, and head-kidney mc2r and gr1 mRNA expression levels. Regardless of dietary treatment, higher cortisol levels were accompanied by an increase of several haematological, cellular, and humoral parameters in response to an acute stress. Dietary VO incorporation led to lower neutrophil numbers, plasma antiproteases activity, and head-kidney cox2 expression level in stressed fish. Fish fed CH+ diets showed a reduction of head-kidney mc2r expression levels in response to stress, while plasma cortisol remained unchanged, plasma NO decreases and antiproteases activity increased. In fish fed the VO diets, CH+ led to a decrease of plasma bactericidal activity compared to that of group CH-, while the opposite pattern was observed for plasma peroxidase. In conclusion, dietary substitution of FO by VO in CH– diet negatively affected the immune response in both undisturbed and stressed fish, while the effect of dietary CH incorporation in VO diet was not clear.

Macroalgae are promising ingredients for aquafeeds, but their recalcitrant polysaccharide structure limits their wide use. To disrupt this structure, different physical (ultra-sounds, autoclave, microwaves), chemical (acid, alkaline), and biotechnological (solid-state fermentation, SSF; SSF followed by sequential hydrolysis, SSF-SH) treatments were carried out in Ulva rigida. The chemical composition, morphological microstructure, release of reducing sugars, soluble protein, and phenolic compounds were evaluated in the final products. All treatments increased U. rigida protein content, while lipid content increased after autoclave, ultra-sound, microwave, and SSF treatments. SSF-SH treatment was more effective in disrupting the cellulose and hemicellulose fractions, resulting in a higher reducing sugars release. The alkaline treatment was more efficient in reducing lignin content and increasing phenolic compounds, antioxidant activity, and soluble protein of U. rigida. The most promising products were then included at 5% in diets for European seabass (Dicentrarchus labrax) juveniles. Crude, ultra-sound, alkaline, and SSF-SH-treated U. rigida reduced fish growth, while SSF-treated U. rigida resulted in similar growth as the positive control (fish meal-based diet). Feed intake was reduced in all experimental groups, but the feed efficiency of fish fed SSF-U. rigida diet and the protein efficiency ratio of fish fed crude, alkaline, and SSF-treated U. rigida were higher than the positive control. The highest total alkaline proteases activity was achieved with the crude U. rigida diet, while no differences were observed in fish fed the positive control, ultra-sound, and SSF diets. Overall, of the processing methods tested, the most efficient was SSF, which improved feed efficiency of European seabass juveniles fed U. rigida-including diet without affecting growth performance., This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/ 04469/2020 unit, the BioTecNorte operation (NORTE-01-0145-FEDER000004), funded by the European Regional Development Fund under the scope of Norte 2020 – Programa Operacional Regional do Norte, the R&D&I project “Development of innovative sustainable protein and omega-3 rich feedstuffs for aquafeeds, from local agro-industrial byproducts”, reference POCI-01-0145-FEDER-030377, funded by European Regional Development Fund (ERDF) and FCT. Helena Fernandes was supported by the PhD grant with the reference SFRH/BD/131219/2017, funded by the Portuguese Foundation for Science and Technology (FCT). Nicole Martins was supported by the PhD grant with the reference SFRH/BD/137919/2018, funded by the Portuguese Foundation for Science and Technology (FCT)., info:eu-repo/semantics/publishedVersion

To evaluate the effects of feeding frequency (FF) and dietary protein/carbohydrate (P/CH) ratios on appetite regulation of gilthead seabream, two practical diets were formulated to include high protein and low carbohydrate (P50/CH10 diet) or low protein and high carbohydrate (P40/CH20 diet) content and each diet was fed to triplicate groups of fish until visual satiation each meal at a FF of 1, 2, or 3 meals per day. Feed intake and feed conversion ratio were higher in fish fed 2 or 3 meals than 1 meal per day and in fish fed the P40/CH20 than the P50/CH10 diet. The specific growth rate was only affected by FF, being higher in fish fed 2 or 3 meals per day than 1 meal per day. Expression of the cocaine-amphetamine-related transcript, corticotropin-releasing hormone, ghrelin receptor-a (ghsr-a), leptin, and neuropeptide y in the brain, cholecystokinin (cck) in the intestine, and leptin and ghrelin in the stomach was not affected by FF or dietary P/CH ratio. This is the first time that ghrelin cells were immune-located in the stomach of gilthead seabream. Fish fed 3 meals per day presented lower cck expression in the brain than those fed twice per day and higher hepatic ghsr-b expression than those fed once per day. Fish fed P40/CH20 diet presented higher hepatic leptin expression than those fed P50/CH10 diet. In conclusion, present results indicate that feeding a P40/CH20 diet at 3 meals a day seems to decrease the satiation feeling of gilthead seabream compared to fish fed higher P/CH ratio diets or fed 1 or 2 meals a day.

The effect of methionine and taurine supplementation of plant protein based diet for meagre (Argyrosomus regius) juveniles was assessed on growth performance and metabolic hepatic enzymes activity. For that purpose, four isolipidic (180.0 g kg−1 crude lipids) and isoproteic (420.0 g kg−1) practical diets were formulated containing 820.0 g kg−1 of protein from plant origin and 180.0 g kg−1 from fishmeal. A 2 × 2 factorial arrangement of treatments was used with methionine at 7.5 and 10.0 g kg−1 dry diet, supplemented or not with taurine at 10.0 g kg−1 dry diet. The feeding trial was conducted with fish of 50.0 ± 0.6 g mean initial body weight and lasted for 65-day. Dietary taurine supplementation improved growth and feed utilization while methionine supplementation had no effect. Diets did not affect whole-body and liver composition. No interaction was observed between the supplemented amino acids. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities increased only with taurine supplementation, while glutamate dehydrogenase (GDH) activity was unaffected by dietary treatment. Fructose-1,6-bisphosphatase (FBPase) activity was affected both by methionine and taurine supplementation. In conclusion, dietary methionine supplementation was inefficient to overcome the decreased taurine level in plant-based diets and thus, taurine should be incorporated in plant protein-based diets, as an efficient nutritional strategy for the mitigation of growth limitations of meagre fed diets with high levels of plant feed ingredients. Moreover, as growth performance was not improved by increasing dietary methionine this suggests that under our experimental conditions meagre requirements are met with 7.5 g kg−1 dietary methionine.

Available online 10 February 2022, This work aimed to evaluate the potential of a carbohydrases-rich extract produced by SSF of BSG to enhance the nutritional value of plant-based diets for European seabass (Dicentrarchus labrax) juveniles. BSG was fermented by A. ibericus (MUM 03.49) to obtain an aqueous enzymatic extract (1178 and 8317Ug1 cellulase and xylanase activity, respectively). A control (50% protein; 18% lipids) diet was formulated with 55% DM of plant-feedstuffs (PF), and four other diets were formulated similarly to the control but including 0.4% and 0.8% DM of lyophilized extract (BSG-extract), corresponding to 4000 and 8000U of cellulase kg1 of diet, directly included in the diet (BSG4 and BSG8 diets) or used to pre-treat the PF mixture of the control diet (PreBSG4 and PreBSG8 diets). Pre-treatment of PF with BSG-extract increased three-fold the dietary reducing sugars content and two-fold the antioxidant activity than the control diet. Cellulase and xylanase activities were highest in diets including BSG-extract directly added than pre-treated PF diets. These diets were fed to European seabass (IBW=22±1g) for 66days. Dietary supplementation with BSG-extract, mixed in the diet or used as a pre-treatment of PF, did not affect growth performance and whole-body composition (p0.05) of European seabass but increased feed efficiency (up to 18%; p, The authors thank UNICER, a soft-drink company (Porto, Portugal), for providing the brewer's spent grain used in this study. This work was supported by the project "SPO3- Development of innovative sustainable protein and omega-3 rich feedstuffs for aquafeeds, from local agro-industrial by-products" [reference POCI-010145-FEDER030377] funded by European Regional Development Fund (ERDF) and Portuguese Foundation for Science and Technology (FCT) and by strategic funding of FCT Technology through the projects UIDB/04423/2020 and UIDP/04423/2020. The H.F. was supported by FCT, MCTES, FSE, and UE grant [reference SFRH/BD/131219/2017] under the North Portugal Regional Operational Program (NORTE2020)., info:eu-repo/semantics/publishedVersion

Probiotics benefits in fish farming have been usually inferred appraising the effects observed on the host and not through the direct assessment of probiotic dynamics in the host gut microbiota. To overcome this gap, quantitative PCR (qPCR) can be a powerful approach to study the bacterial dynamics in fish gut microbiota. The presented work proposes four B. licheniformis-specific DNA markers and details a qPCR method to track putative probiotics B. licheniformis on fish gut. The four B. licheniformis-specific DNA markers - BL5B (hypothetical protein BL00303), BL8A (serA2), BL13C (rfaB) and BL18A (ligD) - were selected and validated by PCR and multiplex-PCR with 20 B. licheniformis isolates and a broad range of non-target bacteria. To assess the dynamics of B. licheniformis in the digesta of farmed fish, a qPCR was validated using markers BL8A and BL18A and calibration curves obtained for both markers with digesta samples spiked with B. licheniformis cells showed a high correlation (R2 > 0.99) over 6 log units (CFU/reaction), and a limit of detection (LOD) as low as 247 CFUs/reaction. Furthermore, the consistent qPCR repeatability and reproducibility underline the specificity and reliability of the qPCR proposed. Ultimately, the possibility to monitor the dynamics of B. licheniformis probiotics in the gut microbiota of farmed fish might be instrumental to optimize best practices in aquaculture.

A growth trial was conducted to determine the dietary taurine requirement for European sea bass juveniles fed plant feedstuffs-based diets. Eight isoproteic (45% crude protein) and isolipidic (18% crude lipid) diets were formulated containing a mixture of plant feedstuffs and fish meal (corresponding to 80% and 20% of total dietary protein, respectively) and taurine (Tau) levels ranging from 0.2 to 1.7% (diets 0.2Tau; 0.3Tau; 0.4Tau; 0.5Tau; 0.7Tau; 0.9Tau; 1.2Tau; 1.7Tau). Triplicate groups of 12 fish (IBW = 55 g) were fed each diet for 10 weeks. Dietary Tau level up to 0.7% improved growth performance while maximum feed efficiency and protein efficiency ratios were obtained with a dietary Tau level of 1.2%. Whole-body lipid content of fish fed the 1.2 and 1.7% Tau diets was lower than in the other groups, while whole-body protein and ash contents, and hepatosomatic and visceral indexes were not affected by diet composition. Daily growth index, nitrogen retention, and feed efficiency were fitted to Tau levels using different models. The five-saturation kinetics model was selected as the more parsimonious model, and based on this model the dietary Tau requirement for European sea bass juveniles under the present experimental conditions was estimated to be 0.47–0.50% DM.

Many studies have assessed the effects of incorporation of plant feedstuffs in fish diets on growth performance, whereas few studies have addressed the effects of fish meal replacement by plant protein sources on fish immune parameters. Thus, the aim of this study was to evaluate the effects on immune response of different inclusion levels of carob seed germ meal (CSGM) as partial replacement for fish meal in diets for meagre (Argyrosomus regius) juveniles. Fish were fed four experimental diets with increased CSGM inclusion levels [0% (control), 7.5% (CSGM7.5), 15% (CSGM15) and 22.5% (CSGM22.5)]. After 1, 2, and 8 weeks of feeding fish were sampled to determine haematological profile and several humoral parameters in plasma and intestine. Results showed that dietary inclusion of CSGM did not negatively affect the immune parameters of meagre. In addition, total numbers of red and white blood cells, as well as thrombocytes, lymphocytes, monocytes, and neutrophils counts were not affected by dietary treatments. All parameters evaluated in plasma were unaffected by dietary CSGM inclusion after 1 and 2 weeks of feeding, with only the haemolytic complement activity showing an increase in fish fed diets with CSGM after 1 week and in fish fed CSGM22.5 diet after 2 weeks. Regarding the innate immune parameters analysed in the intestine, it could be highlighted the increase in alkaline phosphatase and antiprotease activities in fish fed the diet with the higher inclusion of CSGM at 8 weeks. Overall, results suggest that high dietary CSGM inclusion do not compromise immune status or induce an inflammatory response in meagre juveniles.

The aim of this study was to determine the potential of corn distillers dried grain with solubles (DDGS) to partially replace fishmeal (FM) in practical diets for turbot. For that purpose, a control diet was formulated to include 40% FM and a mixture of plant protein ingredients (soybean meal, corn gluten, and wheat gluten). Three other diets were formulated based on the control but with 10, 17.5, or 25% of DDGS replacing FM. Diets were tested in triplicate, in an 84-days growth trial with juveniles of 29 g initial body weight. Feed intake was not affected by diet composition, but growth and feed efficiency linearly decreased with the increase of dietary DDGS level. Whole-body dry matter and protein contents were not affected by diet composition, but lipid and energy content were higher in fish fed the control diet than the 17.5DDGS and 25DDGS diets and the 25DDGS diet, respectively. The apparent digestibility coefficients (ADCs) of protein and amino acids were similar among diets, while the ADCs of energy decreased with the increase of dietary DDGS level. Digestive amylase and lipase activities in posterior intestine were lower in fish fed the 17.5DDGS and 25DDGS diets than the control diet, while proteases activity was not affected by diet. No differences among dietary treatments were observed on plasma glucose, but plasma total protein, albumin, triglycerides, and cholesterol were lower in fish fed the DDGS diets. Activity of key enzymes of glycolysis, gluconeogenesis, and lipogenesis was not affected by diet composition, but activity of alanine aminotransferase increased with the increase of dietary DDGS. Moreover, oxidative status of liver and intestine was not affected by dietary treatments, but susceptibility to oxidative stress was higher in the intestine than in the liver. Overall, it is concluded that replacing FM by DDGS in practical diets for turbot juvenile reduced growth performance and impaired overall nitrogen and energy metabolism.

The establishment of well-balanced diets that meet nutrient requirements is important to optimize a large-scale production of new aquaculture species. This is the case of meagre (Argyrosomus regius), a promising new aquaculture species, with great potential owing to its high growth rate, feed efficiency and easy adaptation to captivity. Knowledge on the nutritional requirements of this species is still scarce, namely regarding essential fatty acids, which are required to sustain growth, development, immune status and survival. A feeding trial was performed with meagre fingerlings (2.8 g ± 0.23) testing 5 increasing dietary n-3 LC-PUFA levels (0.8, 1.4, 2.0, 2.6 and 3.6% DM) with the purpose of evaluating the n-3 LC-PUFA requirements for fast growth of meagre fingerlings. Meagre reflected very high specific growth rates (4.1 to 4.6%) and low feed conversion ratios (0.7 to 0.8), thus highlighting its great potential for aquaculture production. Fish fed 0.8% n-3 LC-PUFA showed the lowest growth, which was significantly improved by increasing the dietary n-3 LC-PUFA levels up to 2.0–2.6%. DHA and ARA were preferentially retained over EPA in whole fish body. Fish fed 0.8% n-3 LC-PUFA showed an up-regulation of fads2 and elovl5 relative gene expressions. Thus, meagre seems to have active Δ6 desaturases and Elovl5, but their activities being insufficient to produce DHA and EPA from PUFA precursors to sustain fast growth, at least under the experimental conditions tested. Young meagre shows a typical marine requirement for n-3 LC-PUFA, estimated to be, at least, 2.0% DM of the diet.

A trial was conducted to evaluate the effect of dietary exogenous enzymes supplementation on apparent digestibility of macronutrients, energy and amino acids, and on digestive enzymes activity and gut microbiota of turbot (Scophthalmus maximus) juveniles fed diets rich in plant feedstuffs. For that purpose, a control diet was formulated to contain circa half protein from fishmeal and the other half from plant feedstuffs (dried distillers' grains with solubles, soybean meal, corn gluten, and wheat gluten). Two other diets were formulated similar to the control but including exogenous enzyme complexes: Synergen® (Alltech; product of solid state fermentation of Aspergillus niger) or Natugrain®TS (BASF; non-starch polysaccharides degrading enzymes complex). Both exogenous enzyme complexes increased the apparent digestibility coefficient (ADC) of dry matter, while the ADC of protein, lipid, and energy were only increased with Natugrain®TS. Moreover, the ADC of methionine, isoleucine, and aspartic acid were increased with both exoenzymes supplementation, while the ADC of lysine and glycine were increased with Synergen® and the ADC of arginine and threonine were increased with Natugrain®TS. Dietary supplementation with Synergen® or Natugrain®TS did not affect intestinal pH but increased the activity of lipase and protease in the posterior intestine, while amilase activity was increased only with Natugrain®TS. Microbiota was also affected by both exoenzymes complexes, increasing its richness and diversity. Present results indicate that diet supplementation with exogenous enzymes complexes, Synergen® or Natugrain®TS, may be a feasible strategy to enhance the digestibility of plant feedstuff-rich diets for turbot juveniles.

A growth trial was performed to assess the interactive effects of high dietary levels of arachidonic acid (ARA) or docosahexaenoic acid (DHA), and carbohydrates on growth performance, feed utilization, proximate composition, plasma metabolites, and intermediary metabolism. Four isoproteic (47% crude protein) and isolipidic (18% crude lipids) diets were formulated for gilthead sea bream juveniles (47.5 g of initial mean body weight) to include low or high gelatinized starch (5 and 20% starch; LS and HS diets, respectively) and different dietary ARA/DHA content (2.3/0.3 and 0/2.6; ARA and DHA diets, respectively). Dietary ARA/DHA content did not affect growth performance or feed utilization. However, dietary starch inclusion increased feed efficiency, protein efficiency ratio, and energy retention (% intake). Dietary ARA/DHA profile did not affect the whole-body composition, but dietary starch increased whole-body dry matter, lipid, and gross energy content and decreased protein content. Low dietary ARA/DHA ratio increased plasma total protein and total lipids, triglycerides, and very-low-density lipoprotein cholesterol (VLDL) and decreased high-density lipoprotein cholesterol (HDL) and low-density lipoprotein cholesterol (LDL), independently of the dietary starch level. On the other hand, in LS diets, total cholesterol levels were lower in fish fed the diet rich in DHA than those fed with ARA. HS diets increased glucose, total protein and lipids, triglycerides, and VLDL plasmatic values, and decreased HDL. HS diets increased the carbohydrates intermediary metabolism and lipogenic enzymes. ARA and HS increased fatty acid desaturase 2 (FADS2) and decreased elongation of very long-chain fatty acid 5 protein (ELOVL5) in the liver, and only HS decreased ELOVL5 in the intestine. The muscle fatty acid content followed the dietary fatty acid profile, but ARA, EPA, and DHA content were increased in LS diets. In conclusion, few interactions between ARA or DHA and dietary digestible carbohydrates were found. The results indicate that at least in the presence of high ARA levels the n-3 LC-PUFA requirements were fulfilled with 0.5% EPA + DHA and gilthead sea bream tolerates well dietary inclusion of 20% starch.

Immune responses relies on an adequate provision of multiple nutrients that sustain the synthesis of key effector molecules. These needs are depicted in the already reported increase of circulating free amino acids in fish under stressful conditions. Since aquaculture and the inherent fish welfare are an emergent call, the immunomodulatory effects of amino acids on gut- and skin-associated lymphoid tissues of the European seabass (Dicentrarchus labrax) were studied under unstressed conditions and after an inflammatory insult. To achieve this goal, fish were distributed in duplicate tanks (fifteen fish per tank) and were fed for 14 days with methionine or tryptophan-supplemented diets at 2× dietary requirement level (MET and TRP, respectively) or a control diet meeting the amino acids requirement levels (CTRL). Afterwards, samples of skin and posterior gut were collected from 6 fish per dietary treatment for the assessment of the immune status while the remaining animals were intraperitoneally-injected with inactivated Photobacterium damselae subsp. piscicida and subsequently sampled either 4 or 24 h post-injection. The immune status of both mucosal surfaces was poorly affected, although a tryptophan effect was denoted after bacterial inoculation, with several immune-related genes up-regulated in the gut at 4 h post-injection, which seems to suggest a neuroendocrine-immune systems interaction. In contrast, skin mucosal immunity was inhibited by tryptophan dietary supplementation. Regarding methionine, results were often statistically non-significant, though increasing trends were denoted in a few parameters. Overall, dietary methionine did not significantly affect neither gut nor skin immunity, whereas tryptophan supplementation seems to induce modulatory mechanisms that might be tissue-specific.

Made available in DSpace on 2022-04-29T08:29:32Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-10-15 Recent research has provided new mechanistic insights that amino acids (AA) coordinate protein synthesis by expressing the evolutionarily conserved target of rapamycin complex 1 (mTORC1) signaling pathway genes. An 8-week feeding trial was conducted to evaluate the impacts of experimental diets (420 g kg−1 crude protein; 15 MJ g kg−1) with protein-bound AA (Control), crystalline amino acids (CAA) supplemented at 60 g kg−1 and CAA at 120 g kg−1 on growth performance, AA retention, and mTORC1 signaling pathway (mTOR, mLST8, and Raptor) genes in Nile tilapia, Oreochromis niloticus fingerling (n = 360; 4.3 ± 0.1). At the end of feeding trial, the time-course postprandial plasmatic lysine and methionine were determined by collecting blood samples of fish immediately after feeding and at 60, 120, 240, 480 and 960 min. Each diet was randomly distributed into eight replicates groups of 12 fish and hand-fed, six times a day until apparent satiety for eight weeks. Compared with fish fed CA120 diet, body weight gain (BWG), feed intake (FI), feed efficiency (FE), protein retention efficiency (PRE) and retention of six essential and five non-essential AA parameters were improved in fish fed Control and CA60 diets. Furthermore, fish fed CA60 diet showed significantly lower whole-body lipids and higher crude protein than those fed other diets, whereas humidity and ash were not affected by dietary treatments. Plasmatic lysine and methionine linearly increased in fish fed Control diet. However, time-course plasmatic lysine and methionine were affected along with quadratic effect, and plasmatic lysine and methionine peaked at 461 and 496 min in fish fed CA60 diet, being reduced at 364 and 405 min in fish fed CA120 diet, respectively. The mechanistic insights of well-balanced AA coordinating protein synthesis in fish fed Control and CA60 diets was identified through a similar response on relative gene expression of mLST8 and mTOR genes, but higher than that in fish fed CA120 diet. Overall, this study validated CAA up to 60 g kg−1 without adverse effects on fish growth performance. However, a high CAA level at 120 g kg−1 depressed growth performance and may be considered to produce industrial-scale feeds for precision tilapia feeding. Statement of relevance: This study revealed new mechanistic insights that amino acids regulate protein synthesis through genomic approaches by regulating relative gene expression of mTORC1 signaling pathway. The data is relevant to the formulation of well-balanced amino acids precision tilapia feeding. PPZ/UEPG – Animal Science Post Graduation Program State University of Ponta Grossa Ichthus Unlimited Department of Morphology Institute of Bioscience of Botucatu São Paulo State University Graduate Program in Environment and Regional Development University of Western São Paulo CIIMAR Interdisciplinary Center for Marine and Environmental Research Department of Biology Faculty of Sciences Porto University Department of Animal Science State University of Ponta Grossa Department of Morphology Institute of Bioscience of Botucatu São Paulo State University

[EN] A digestibility trial was conducted to evaluate the effect of fishmeal (FM) replacement with meat and bone meal on diets digestibility, digestive enzymes activity, and microbiota modulation in gilthead seabream (Sparus aurata) juveniles fed diets (45% CP; 20% CL) including 0, 50, and 75% of protein from MBM (diets MBM0; MBM50; MBM75). The ADC of protein was high and unaffected by dietary MBM level, whereas the ADC of energy was higher with diet MBM50 than with MBM0. The ADC of essential amino acids was also high and not affected by diet composition, except for the ADC of phenylalanine and tyrosine, which were lower in diet MBM75 than in the other diets. Pepsin, trypsin, chymotrypsin, lipase, and total alkaline proteases activities were all unaffected by diet composition. Dietary inclusion of MBM modulated gastrointestinal tract microbiota, decreasing the average number of operational taxonomic units and microbial richness. Dietary MBM inclusion promoted an increase of Vibrio, Bacillus, and Mycobacterium genera, whereas colonization by Staphylococcus and Corynebacterium decreased. Overall, present results indicate that up to 75% of FM protein can be replaced by MBM protein in diets for gilthead seabream juveniles without major adverse effects on diet digestibility and digestive function. However, gastrointestinal microbiota was modulated, and further research should be conducted to evaluate the impact of gastrointestinal microbiota modulation on immune and health status of gilthead seabream., This research was partially supported by the Strategic Funding UID/Multi/04423/2013 through national funds provided by FCT - Foundation for Science and Technology and European Regional Development Fund (ERDF), in the framework of the programme PT2020. C.R. Serra was supported by grant SFRH/BPD/101038/2014, from FCT.

This study aimed to evaluate the effects of dietary methionine (Met) and taurine (Tau) supplementation on growth performance, feed utilization, and oxidative status of European sea bass juveniles. For that purpose, a 12-week growth trial was performed to test four isolipidic (18%) and isonitrogenous (6.7%) practical diets, containing 82% of the protein from plant origin and 18% from fish meal. Diets were formulated to have a Met level below (LMet) or above (HMet) requirements and were supplemented or not with 1% Tau (LMetTau and HMetTau). No dietary Met or Tau effects were observed on growth, feed utilization, and whole-body composition. Increasing dietary Met level led to increased catalase (CAT) and glutathione peroxidase (GPX) activities in the liver and total (tGSH) and reduced (GSH) glutathione in the intestine, while decreasing glutathione reductase (GR) and GPX activities in the intestine. Most of these effects were only observed in fish fed diets not supplemented with Tau. Dietary Tau supplementation decreased intestinal tGSH, GSH and oxidised (GSSH) glutathione content, the activity of GPX both in liver and intestine, and glucose-6-phosphate dehydrogenase (G6PDH) activity in the liver. Dietary Tau supplementation also affected hepatic CAT and intestinal GR activities, but effects were dependent of the dietary Met level. Overall, European sea bass seems to cope well with a plant-protein rich diet without Met or Tau supplementation. Both dietary Met and Tau were shown to modulate fish antioxidant response but without altering the oxidative damage level.

The aim of this study was to evaluate the beneficial effects of dietary arginine supplementation on growth performance, urea biosynthesis, and N excretion of juvenile gilthead sea bream ( Sparus aurata ) fed fish meal and plant-feedstuffs based diets. For that purpose, five isonitrogenous (45% crude protein) diets were formulated as follows: two diets were fish meal-based and contained 3.0 and 4.0% arginine; three diets were plant feedstuff-based and contained 1.6, 3.0, and 4.0% arginine. Triplicate groups of gilthead seabream juveniles (24 g) were fed each diet for 10 weeks. At the end of the trial, growth performance of fish fed the fish meal-based diets was higher than that of the other groups. Dietary arginine content did not affect growth performance. Ammonia excretion was not affected by diet composition, while urea excretion was directly related with arginine intake. Carbamyl phosphate synthase activity was not detected in the liver, while arginase activity increased and ornithine carbamyl-transferase decreased with dietary arginine content. Overall, an excess of dietary arginine had no beneficial effects on growth performance and feed utilisation of gilthead seabream juveniles fed plant feedstuffs-based diets. A functional urea cycle was not detected in the liver of gilthead sea bream juveniles.

The nutritional value of whole wheat, corn, rye and barley was evaluated for European sea bass. Four isoproteic and isolipidic diets containg 30% of each whole cereal meal were formulated. A growth trial and a digestibility trial were conducted and fish performance was evaluated in terms of growth, feed intake, feed utilization, macronutrients digestibility, liver composition, glycaemia and cholesterolemia levels and gut histomorphology. Growth and feed utilization ranged from 1.43 to 1.46 and from 0.55 to 0.58, respectively, and were similar among experimental groups. Whole body and liver composition, hepatosomatic and visceral indexes were also not affected by dietary composition. Corn diet revealed the lower dry matter (62.9) and starch (55.5) apparent digestibility coefficients. Protein digestibility in fish fed the rye diet was the lowest of all groups (91.3) and differed from that of wheat diet group (93.2), which might be due to antinutritional factors present in this feedstuff. Plasmatic glucose levels suggested a slower absorption rate in fish fed barley and corn diets. No signs of enteropathy related to the whole cereal meals were observed. Among cereals tested, rye seemed to be the less tolerated by sea bass, although all diets were well accepted and sea bass performed equally well with all diets.

A trial was conducted to evaluate fish meal (FM) replacement with meat and bone meal (MBM; 53% CP, 15% CL, 27% Ash) in diets for gilthead seabream (Sparus aurata) juveniles. Three extruded experimental diets were formulated (45% CP; 20% CL) to include 0, 50 and 75% of protein from MBM (diets MBM0; MBM50; MBM75). Triplicate groups of seabream (IBW = 25 g) were fed these diets to satiety for 12 weeks. Growth performance and feed efficiency were similar with the diets MBM0 and MBM50, but were lower with diet MBM75, while the opposite was true for feed intake. Whole-body composition was not affected by diets composition except for crude lipid and energy content, which were lower with the diet MBM75. Protein and essential amino acids retention were unaffected by diet composition, while energy retention was lower with the diet MBM75. In terms of economic efficiency, diets with MBM resulted in a lower production costs, with the lowest economic conversion ratio (€ kg− 1 fish produced) being obtained for the MBM diets while the maximum economic profit (€ kg fish− 1) was obtained for diet MBM50. Overall, up to 50% of FM protein can be replaced by MBM protein in diets for gilthead seabream juveniles, without compromising growth performance, feed utilization, and nutrient retention. Statement of relevance Replacing 50% of fishmeal protein improved economic return.

Inclusion of prebiotics in aqua feeds, though a costly strategy, has increased as a means to improve growth. Still, its effects on health improvement are not fully disclosed. Regarding their immunestimulatory properties, research has focused on carbohydrates such as fructooligosaccharides and xylooligosaccharides demonstrating their modulatory effects on immune defences in higher vertebrates but few studies have been done on their impact on fish immunity. Replacing fish meal (FM) by plant protein (PP) sources is a current practice in the aquaculture business but their content in antinutrients is still a drawback in terms of gut well-functioning. This work intends to evaluate the short-term effect (7 or 15 days feeding the experimental diets) on juvenile European seabass (Dicentrarchus labrax) immune status of dietary i) replacement of FM by PP sources; ii) prebiotics supplementation. Six isoproteic (46%) and isolipidic (15%) diets were tested including a FM control diet (FMCTRL), a PP control diet (PPCTRL, 30 FM:70 PP) and four other diets based on either FM or PP to which short-chain fructooligosaccharides (scFOS) or xylooligosaccharides (XOS) were added at 1% (FMFOS, PPFOS, FMXOS, PPXOS). The replacement of FM by PP in the diets induced nitric oxide (NO) and lysozyme production, while immunoglobulins (Ig), monocytes percentage and gut interleukin 10 (IL10) gene expression were inhibited. Dietary scFOS supplementation inhibited total bactericidal activity and neutrophils relative percentage regardless protein source and increased plasma NO and thrombocytes percentage in fish fed FM-based diets, while monocytes percentage was increased in PPFOS-fed fish. XOS supplementation down-regulated immune gene expression in the gut while it partly enhanced systemic response. Inconsistency among results regarding FM replacement by PP-based ingredients exposes the need for further research considering both local and systemic responses. Distinct outcomes of prebiotic supplementation were highlighted reflecting sight-specific effects with no clear interaction with protein source.

This study aimed to evaluate the effect of including 10%, 20% and 30% of mealworm (Tenebrio molitor, TM) larvae meal (diets TM10, TM20 and TM30, respectively), replacing 25%, 50% and 75% of fishmeal (FM) in a isoproteic (50%) and isolipidic (19%) FM-based diet (CTR) for meagre (Argyrosomus regius). The diets were fed for 9 weeks to triplicate groups of fish with 18.0 g initial body weight and results were evaluated for growth performance; whole-body composition; amino acid (AA) catabolism and digestive enzymes activity; nutrients and chitin digestibility. A digestibility trial was also simultaneously conducted for 48 days. Growth and feed utilization parameters decreased linearly as dietary TM inclusion level increased, while mortality, hepatosomatic and visceral indexes, and AA catabolism enzymes activity were unaffected by the dietary treatments. Whole-body protein was reduced in fish fed TM20 and TM30 diets, with the latter group also having lower energy content than fish fed the CTR diet. The apparent digestibility coefficients (ADC) of dry matter, energy, protein, and AA (except leucine, valine and phenylalanine), decreased as dietary TM inclusion level increased. The lower ADC of protein was related to a linear decrease in trypsin activity. Lipase and total alkaline protease activities were also lower in fish fed the TM30 diet than in the other groups. A quadratic response of total alkaline protease with the increase of dietary TM meal was similar to that observed in the intensity of zymograms bands corresponding to proteases other than trypsin or chymotrypsin-like. Chitin ADC was null and no chitinolytic activity was found in the intestine. Overall, meagre presented a limited capacity to utilize TM, with a 10% TM dietary inclusion already resulting in significant impairment of fish digestive capacity and growth performance, while higher TM dietary levels further negatively affecting fish whole-body composition.

A study was undertaken to evaluate the effect of dietary fishmeal (FM) replacement with Hermetia illucens prepupae larvae meal (HM) on oxidative stress indicators and fillet quality traits during shelf-life of European seabass (Dicentrarchus labrax) juveniles. HM was used replace 0%, 15%, 30% and 45% of dietary FM, corresponding to an inclusion level of 0%, 6.5%, 13%, and 19.5% of HM. After a feeding period of 62 days, fillet quality traits were analysed immediately after sampling (T0) and after three days of storage at +2/4 °C (T3). At the end of the feeding trial, hepatic lipid peroxidation and activities of superoxide dismutase decreased, catalase activity decreased at intermediary HM inclusion levels, while glutathione peroxidase and glutathione reductase activities were unaffected with the dietary inclusion of HM. Irrespectively of the diet, storage time did not affect the skin and fillet (dorsal and ventral regions) colour, while fillet pH was higher at the highest dietary HM inclusion level. Biometry traits, slaughter yield, fillet proximate composition, and liver fatty acid (FA) profile were not affected by dietary treatment. Fillet fatty acid profile was also unaffected by diet or storage time, except total C18-FA that decreased at the two intermediary levels of HM inclusion, n-6 PUFA that was higher in fillets from fish fed the HM-based diets, and total n-3 PUFA that decreased with the increase of dietary HM level at T3. The n-3/n-6 ratio was not affected by the diets but decreased with the refrigeration time. Docosapentaenoic acid (DPA; 22:5n-3) decreased with the storage time, irrespectively of the dietary treatment. Fillet lipid peroxidation increased with storage time but decreased with the dietary inclusion of HM. Overall, dietary replacement of fish meal by HM up to 19.5% (22.5% of total dietary protein) did not affect the fillet quality traits and shelf-life of European seabass while reducing fillet lipid oxidation.

This study aimed to evaluate the effect of replacing fish meal by poultry by-product meal (PBM) in actual commercial standard diets for gilthead seabream juveniles on growth performance, diet digestibility, digestive function, intestinal microbiota, and provide an economic analysis of using PBM in practical diets. Six isoproteic and isolipidic diets were formulated including increasing levels (from 0 to 37.5%) of PBM in replacement of fish meal, corresponding to a fish meal protein replacement in the diets up to 83%. Each diet was randomly assigned to triplicate groups of fish (IBW 63 g) and the growth trial lasted 70 days. No differences between groups were observed on growth performance, voluntary feed intake, feed efficiency, nitrogen utilization, and whole-body composition. Digestive enzymes activity and the ADC of protein, phosphorus, and energy were also not affected by dietary treatment. Regarding gut microbiota, fish fed diet PBM22.5 showed higher species richness and diversity of digesta microbiota, but no differences between groups were observed in mucosa OTUs, richness, and diversity indices. The cost of diet formulation decreased with the dietary replacement of FM by PBM, highlighting the potential of poultry by-products inclusion in commercial diets. Overall, this study indicates that dietary FM could be replaced at least up to 83% with PBM in diets for gilthead seabream juveniles without affecting growth performance and feed utilization while improving the economic efficiency of these diets.

The aim of this study was to assess the effect of dietary taurine (Tau) in intermediary metabolism, oxidative status, and Tau biosynthesis capacity in European seabass juveniles. For that purpose, four diets (45% protein and 18% lipid) were formulated with increasing levels of Tau (0.2, 0.5, 0.7, and 1.2% of DM) and fed to triplicate groups of fish (IBW 55g) until apparent visual satiety for 10 weeks. Total bile acid content in the liver increased as dietary Tau supplementation increase, while the opposite occurred in the plasma. Plasma glucose, total cholesterol, HDL, and LDL cholesterol decreased, while total and indirect bilirubin increased with the increase of dietary Tau level. The activity of the key intermediary enzymes measured decreased with the increase of dietary Tau up to 0.7%. Regarding the antioxidant-related enzymes, dietary Tau supplementation decreased hepatic catalase, glutathione peroxidase, glutathione reductase, and superoxide dismutase activities with the maximum effects being observed as dietary Tau increased from 0.2 to 0.5%. Overall, there was a decreasing trend of hepatic lipid peroxidation with the increase of dietary Tau. This study is the first to demonstrate that in European seabass juveniles the capacity of expressing cysteine dioxygenase, an enzyme involved in the cysteine sulfinate pathway for Tau synthesis, although its expression was not affected by Tau levels in the diet. This study indicates that dietary Tau levels affect intermediary metabolism, namely carbohydrates and lipidic metabolism, and improve the antioxidant status of European seabass juveniles. Furthermore, although CDO expression was detected, European seabass seems unable to regulate its activity, these results supporting the essentiality of Tau for this species.

"Available online 3 August 2020", Oilseed cakes have potential for new applications as substrates for solid-state fermentation (SSF), to increase their nutritional value by increasing its polyunsaturated fatty acids (PUFAs) or protein content. In this sense, it was performed a screening of oilseed cakes to be used as substrates for the production of PUFAs by Mortierella alpina Peyronel MUM 9412. Of all by-products tested, linseed cake (LSC) was the oilseed cake that achieved the highest production of total PUFAs: 153.09 ± 2.25 mg/g. Overall, the PUFAs and protein contents of fermented LSC increased 33% and 11%, respectively. Further, supplementation of the rapeseed cake with linseed oil, prior to the SSF, proved to increase the PUFAs production in about 26%. This study demonstrated the potential of SSF for improving linseed and linseed cakes nutritional composition and the positive effect of linseed oil as inductor to improve the PUFAs production by M. alpina., José Manuel Salgado was supported by grant CEB/N2020 – INV/01/ 2016 from Project “BIOTECNORTE - Underpinning Biotechnology to foster the north of Portugal bioeconomy” (NORTE-01-0145-FEDER000004). This work was supported by the project “Development of innovative sustainable protein and omega-3 rich feedstuffs for aquafeeds, from local agro-industrial by-products”, reference POCI-01-0145- FEDER-030377, funded by European Regional Development Fund (ERDF). This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2019 unit and BioTecNorte operation (NORTE-01- 0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte., info:eu-repo/semantics/publishedVersion

Leptin, ghrelin, and insulin influence lipid metabolism and thus can directly affect adipose tissue characteristics, modulating the organoleptic quality of aquaculture fish. The present study explored gilthead seabream (Sparus aurata) cultured preadipocytes development, and the regulation of adipogenesis by those three hormones. Preadipocytes presented a fibroblast-like phenotype during the proliferation phase that changed to round-shaped with an enlarged cytoplasm filled with lipid droplets after complete differentiation, confirming the characteristics of mature adipocytes. peroxisome proliferator-activated receptor-γ (pparγ) expression was higher at the beginning of the culture, while fatty acid synthase and 3-hydroxyacyl-CoA dehydrogenase gradually increased with cell maturation. The expression of lipoprotein lipase-like, lysosomal acid lipase (lipa), fatty acid translocase/cluster of differentiation-36 (cd36), and leptin receptor (lepr) were not affected during cell culture development; and undetectable expression levels were observed for leptin. Concerning regulation, leptin inhibited lipid accumulation significantly reducing pparγ and cd36 gene expression, both in early differentiating and mature adipocytes, while ghrelin decreased the expression of pparγ in the early differentiating phase but did not reduce intracellular lipid content significantly. Additional insulin past the onset of adipogenesis did not affect lipid accumulation either. In conclusion, at present culture conditions leptin has an anti-adipogenic function in differentiating preadipocytes of gilthead seabream and continues exerting this role in mature adipocytes, while ghrelin and insulin do not seem to influence adipogenesis progression. A better understanding of leptin, ghrelin, and insulin impact on the adipogenic process could help in the prevention of fat accumulation, improving aquaculture fish production and quality.

A study was undertaken to evaluate the effects of supplementation of high soybean meal (SBM)/low fish meal (FM) based diets for white seabream ( Diplodus sargus ) juveniles with Natugrain® TS, a nonstarch carbohydrases (NSPase) enzyme complex. Four isoproteic (36% CP) and isolipidic (18% CL) diets were formulated in a 2 × 2 factorial design with FM/SBM levels and NSPase supplementation as factors. Diets included 15%FM/32%SBM or 5%FM/42%SBM, supplemented or not with 0.04% of Natugrain® TS. Twelve groups of 15 fish (IBW = 58 g) were fed each diet to near satiation for 10 weeks. The apparent digestibility coefficients of the diets were evaluated in a different trial. Reduction of dietary FM from 15 to 5% did not affect voluntary feed intake, growth performance, feed efficiency, whole body composition, apparent digestibility coefficients, distal intestine and liver histomorphology, and digestive enzyme activities. However, dietary NSPase supplementation greatly increased overall feed, protein, and energy utilization, and this effect was higher in the low than in the high FM diet. Whole-body protein and lipid content also increased with dietary NSPase supplementation, and nitrogen and energy fecal losses were reduced. Moreover, in the low FM diet NSPase supplementation promoted an increase of amylase and lipase activities but not of protease activity. Overall, results indicate that white seabream perform well with diets almost devoid of FM. Also, NSPase supplementation to high plant feedstuff diets improves feed and nutrient utilization efficiency, and contribute to a reduction of fecal waste production. Statement of relevance Dietary supplementation with carbohydrase complexes is a possible strategy to overcome some of the drawbacks of plant based feedstuffs diets for aquaculture species, enhancing nutrient availability and alleviate digestive disorder. White seabream accepted, grew and utilized almost non-FM diets, if dully supplemented with carbohydrases. Dietary carbohydrase supplementation considerably increased feed utilization efficiency, reducing environmental waste.

The aim of this study was to evaluate the effects of short-chain fructooligosaccharides (scFOS), xylooligosaccharides (XOS) and galactooligosaccharides (GOS) on immune and hepatic oxidative status, and gut morphology of white sea bream juveniles. Four diets were formulated: a control diet with fish meal (FM) and plant feedstuffs (PF) (30FM:70PF) and three test diets similar to the control but supplemented with 1% of scFOS, XOS or GOS. Dietary prebiotic incorporation did not affect total blood cell counts, hematocrit, hemoglobin, red blood indices or differential white blood cell counts. Fish fed GOS had lower ACH50 and nitric oxide than fish fed control diet. XOS enhanced immune status through the increase in alternative complement pathway (ACH50), lysozyme and total immunoglobulin. The higher activity of glucose 6-phosphate dehydrogenase in fish fed FOS compared to the other dietary groups was the only related antioxidant enzyme affected by prebiotics in the liver. GOS ameliorated the precocious adverse effects of PF based diet on gut histomorphology, as denoted by the lower incidence of histological alterations in fish fed GOS for 15 days. In conclusion, XOS and GOS at 1% might have potential to be used as prebiotics in white sea bream juveniles.

The effects of whole cereal meals on growth, nutrient digestibility, glycaemic response and gut morphology were studied in gilthead seabream juveniles. Groups of 30 fishes (in triplicate) were fed four isoproteic (47%) and isolipidic (16%) diets including 30% of wheat, corn, barley or rye meals. Growth performance was higher in fish fed the wheat diet than the rye diet. Feed efficiency and protein efficiency ratio were higher with the wheat and barley diets than in the other groups. There were no differences in apparent digestibility coefficients (ADCs) of dry matter, protein or lipids among diets. However, ADC of corn and barley starch was lower than that of wheat and rye starch. Average plasma glucose levels were lower in fish fed the barley diet. Plasma glucose peaked 10 h after feeding in all groups except the corn group which peaked 8 h after feeding. Shorter hyperglycaemia duration was observed in fish fed the corn diet (12 h) than the other diets (> 12 h). Gut morphology was similar in all groups irrespective of the sampling point. Based on growth performance, feed utilization efficiency, ADC of starch and glycaemic response we can conclude that wheat and barley are more efficiently used than corn or rye by gilthead seabream juveniles and thus are the preferential cereal sources to be used as plant feedstuffs in diets for this species. Statement of relevance Effective growth of fish at reduced costs.

A study was undertaken to evaluate dietary glutamine supplementation effects on gilthead sea bream performance, intestinal nutrient absorption capacity, hepatic and intestinal glutamine metabolism and oxidative status. For that purpose gilthead sea bream juveniles (mean weight 13.0g) were fed four isolipidic (18% lipid) and isonitrogenous (43% protein) diets supplemented with 0, 0.5, 1 and 2% glutamine for 6weeks. Fish performance, body composition and intestinal nutrient absorption capacity were not affected by dietary glutamine levels. Hepatic and intestinal glutaminase (GlNase), glutamine synthetase (GSase), alanine aminotransferase, aspartate aminotransferase and glutamate dehydrogenase activities were also unaffected by dietary glutamine supplementation. In the intestine GlNase activity was higher and GSase/GlNase ratio was two-fold lower than in the liver, suggesting a higher use of glutamine for energy production by the intestine than by the liver. The liver showed higher catalase and glucose-6-phosphate dehydrogenase activities, while the intestine presented higher glutathione peroxidase and glutathione reductase activities and oxidised glutathione content, which seems to reveal a higher glutathione dependency of the intestinal antioxidant response. Total and reduced glutathione contents in liver and intestine and superoxide dismutase activity in the intestine were enhanced by dietary glutamine, though lipid peroxidation values were not affected. Overall, differences between liver and intestine glutamine metabolism and antioxidant response were identified and the potential of dietary glutamine supplementation to gilthead sea bream's antioxidant response was elucidated.

Vegetable oils (VO) are generally accepted as valuable alternatives to fish oils but are devoid of n-3 LC-PUFA (Long-Chain-Polyunsaturated Fatty Acids) and rich in C18-PUFA. Marine fish have limited capacity to express Δ6/Δ5 desaturases (FADS1 and FADS2) and elongases of very-long-chain fatty acids (Elovl), which severely limits their capacity to produce LC-PUFA from C18-PUFA. This study aimed to evaluate the effects of dietary essential fatty acids(EFA) ratios, arachidonic (ARA), eicosapentaenoic (EPA), and docosahexaenoic (DHA) acids on growth performance, feed utilization, whole-body composition, hepatic and intestinal activity of key-enzymes of lipid and carbohydrate metabolism, and expression of lipid metabolism-related genes. Diets were formulated to include ARA/EPA/DHA ratios of 2.0/0.2/0.1 (Diet A); 1.0/0.4/0.4 (Diet B); 0/0.6/0.6 (Diet C); and 0/0.3/1.5 (Diet D) and were fed to triplicate groups of gilthead sea bream juveniles for 56 days. Dietary EFA ratios did not affect growth performance and whole-body composition. Nevertheless, feed and protein utilization were higher with diet B than diets C or D. Except for ARA, muscle EFA profile followed that of the diets. Plasma triglycerides were higher with diet B and C than A, and plasma cholesterol decreased in fish fed the higher DHA diet. Hepatic ELOVL5 and phospholipase A2 were upregulated in fish fed diet C compared to diet A. In the intestine, ELOVL5 levels were down-regulated in fish fed diet A and D compared to fish fed diet B. Overall, results showed that besides EPA and DHA, adequate dietary ARA level needs to be provided to optimize feed utilization efficiency of gilthead sea bream juveniles.

Corn distillers dried grains with solubles (DDGS) is a co-product of corn ethanol production which biological value is dependent on technological processes and so may be highly variable among production plants. Therefore, two digestibility trials were conducted to evaluate the apparent digestibility coefficients (ADCs) of nutrients and energy of two commercial sources of DDGS and their effects on digestive enzyme activities in European seabass and meagre juveniles. DDGS digestibility was determined based on the substitution of 30% of a reference diet (45% CP; 16% CL) by each test DDGS. ADCs of protein (92–98%), most amino acids (75–99%) and lipids (82–89%) were high while that of energy (58–68%) was low, reflecting the high indigestible fiber content of DDGS. ADCs of nutrients and energy of DDGS were similar in both species, except for some amino acids (lysine, arginine, histidine, isoleucine, tyrosine and aspartic acid) which were higher for seabass than for meagre. Independent of fish species, ADCs of protein and energy were higher in DDGS1 than in DDGS2, whereas ADCs of lipids and most amino acid were not affected by DDGS source. Digestive enzymes activities were species specific: protease activity was higher in meagre while lipase and amylase activities were higher in seabass. For both species, all digestive enzymes activities were higher in mid intestine than in anterior or distal intestine. Overall, it is concluded that corn DDGS is well digested and seems to have high potential for inclusion in diets for seabass and meagre juveniles.

The effects of short-chain fructooligosaccharides (scFOS) and xylooligosaccharides (XOS) on growth, feed utilization and liver activity of key enzymes of glycolytic, gluconeogenic, and lipogenic pathways were studied in European sea bass juveniles. This is the first study about the effect of prebiotics on fish glucose metabolism and few and contradictory studies are available about prebiotic effect on lipid metabolism. Fish were fed isoproteic (46%) and isolipidic (15%) diets based on fish meal (FM diets) or plant ingredients (PP diets; 30 FM:70 PP) as main protein sources. Four other diets were formulated similar to the control diets (PPC; FMC) but including 1% scFOS or 1% XOS (PPFOS, PPXOS, FMFOS and FMXOS diets). Growth performance was higher in fish fed PPXOS diet than PPC diet. No effect of dietary prebiotics on feed efficiency was noticed. Glucokinase activity was higher in fish fed FMFOS and FMXOS diets than FMC diet. Lipogenic enzyme activities (malic enzyme, fatty acid synthetase, glucose-6-phosphate dehydrogenase) were lower in fish fed diets including XOS than in the other groups. Glycolytic (glucokinase, pyruvate kinase) and lipogenic enzyme activities were higher, and gluconeogenic (fructose-1,6-bisphosphatase) enzyme activity was lower in fish fed FM diets than the PP diets. Overall, dietary XOS decreased lipogenesis, independently of dietary protein source, and improved growth performance in fish fed PP diets. In fish fed FM diets, XOS and scFOS increased glycolytic activity. XOS seemed to have good potential to be used as prebiotic in European sea bass.

Fish are usually exposed to farm operations that can activate the stress system, which in turn diverts energy from normal metabolic processes (i.e. growth or immune defence) to deliver it into the physiological systems activated to cope with stress. The main goal of this study was to assess the effect of high stocking densities on growth performance of Senegalese sole (Solea senegalensis) juveniles reared under intensive farming conditions. The impact of high densities on plasma cortisol and metabolites, humoral immune parameters and hepatic oxidative status was evaluated. Senegalese sole juveniles (45 ± 5 g average body weight) were stocked at three different initial densities (7, 17 and 24 kg m− 2) throughout a 60 days rearing period. Final stocking densities were 13, 31 and 40 kg m− 2 with no differences in terms of growth performance. Similar levels of plasma cortisol, circulating leucocytes and metabolites and humoral innate immune parameters suggest that stocking densities do not affect the hematological and biochemical status of Senegalese sole juveniles, at least under the current experimental conditions. Overall, this study presents the possibility for Senegalese sole on-growing at high stocking densities, strengthening the needs for its intensive commercial farming.

Plant ingredients contain antinutritional factors that may affect fish growth performance and health when included in their diets. The present work aimed at studying the effect of two soy purified antinutrients, saponins and phytosterols, on growth, intestinal morphology and immune response of on-growing sea bream. Fish were fed one of seven diets for 48 days: control diet (fishmeal and fish oil based) and six experimental diets containing low (0.1%) or high (0.2%) levels of soya saponins, low (0.5%) or high (1.0%) levels of phytosterols or a combination of 0.1% saponins +0.5% phytosterols or 0.2% saponins +1.0% phytosterols. At day 15 and at the end of the trial pyloric caeca (PC) and distal intestine (DI) samples were collected for histological evaluation. At the end of the trial plasma was sampled for cholesterol analysis and DI was sampled for gene expression of immune and functional genes. Saponins and phytosterols did not affect fish growth, feed utilisation or plasma cholesterol levels. Histologically only a mild increase in the number of eosinophilic granular cells in the PC after 15 and 48 days of feeding in fish fed 0.1% saponins and 0.5% phytosterols, respectively. Gene expression of immune related genes was not affected by the presence of dietary antinutrients. Sea bream in grow-out phase showed high tolerance for saponins and phytosterols either alone or in combination, thus indicating an ability to cope with dietary plant feedstuffs containing such antinutrients, at least within the concentration range used in the present study.

The use of plant ingredients in aquafeeds for piscivorous fish species is a reality that exposes fish to a number of antinutritional factors present in plants. The present study is the first to evaluate the effect of two purified antinutrients, saponins and phytosterols, in sea bream juveniles. For that purpose, seven diets were formulated: a control diet (fishmeal and fish oil based) and six experimental diets containing low (1 g kg− 1, SapL) or high (2 g kg− 1, SapH) levels of purified soya saponins, low (5 g kg− 1, PhytL) or high (10 g kg− 1, PhytH) levels of purified phytosterols or a combination of 1 g kg− 1 saponins + 5 g kg− 1 phytosterols (SapPhytL) or 2 g kg− 1 saponins + 10 g kg− 1 phytosterols (SapPhytH). Fish were fed for 48 days in order to evaluate growth performance, feed utilization, plasma cholesterol, and gut health as assessed by histomorphological evaluation and gene expression profiling of immune and functional markers. Fish fed the diets PhytH, SapPhytL and SapPhytH showed better feed utilization and PhytH and SapPhytH showed higher protein utilization than the other groups, although this was not reflected in improved growth performance. Histomorphological analysis of the distal intestine revealed increased variation in supranuclear vacuole sizes after 48 days of feeding diets SapH, SapPhytL and SapPhytH and increased number of intraepithelial leukocytes in response to all dietary treatments except SapL and SapPhytL. Although juvenile sea bream growth was not affected by dietary inclusion of saponins and phytosterols, the results indicated some disturbances of the intestinal mucosal structure that could compromise function and/or protection from potential dietary antigens or opportunistic pathogens.

The supplementation effect of a high plant protein diet with methionine and taurine on nutrient digestibility, digestive enzymes activity, plasma and intestine bile acid content, and plasma metabolites was assessed in meagre juveniles. A control (CT) diet was formulated with 42% crude protein and 18% lipids, and including only 10% fishmeal, corresponding to 18% of the dietary protein level. Three other diets were formulated similar to the control but supplemented with 0.26% methionine (MET), 1.0% taurine (TAU), or 0.26% Met plus 1.0% Tau (MET+TAU), in a 2 × 2 factorial design. Final dietary Met levels were 0.75 and 1.0%, and Tau levels were 0.10 and 1.20%. Diets were fed to triplicate groups of 103 ± 21.2 g fish, twice a day, to apparent satiation. Fish were assembled in fiberglass tanks (60 L of water capacity), each one with feces settling column. Dietary Met supplementation increased apparent digestibility of dry matter, protein, lipids, and energy while dietary Tau supplementation increased lipids digestibility. Lipase activity was not affected by dietary methionine level, but it was higher in fish fed the taurine supplemented diet at the higher methionine level. Proteases and amylases activities were not affected by diet composition. Total bile acid in plasma and anterior intestine increased with dietary Tau supplementation. Dietary Tau supplementation also increased plasmatic levels of cholesterol, total proteins, and triglycerides. Overall, results of this study indicate that dietary methionine supplementation improved diet digestibility and that dietary taurine supplementation improved lipid digestibility and positively affected lipid metabolism in meagre.

Characterization of the major classes of digestive enzymes (amylase, protease and lipase) in the pyloric caeca and intestine of an omnivorous, white seabream (Diplodus sargus) and a carnivorous, meagre (Argyrosomus regius) fish species was assessed in response to dietary brewer's spent yeast (BSY, Saccharomyces pastorianus) supplementation. For that purpose, three isonitrogenous (50%) and isolipidic (12%) diets were formulated: unsupplemented (control) or supplemented with BSY at 1 or 2%. White seabream exhibited lower proteolytic but higher amylase and lipase activities than meagre. In white seabream, BSY administration enhanced amylase and protease activities in the pyloric caeca and the intestine and lipase activity in the pyloric caeca, while in meagre, improvement on amylase activity in the pyloric caeca was observed with the 2% BSY diet. The results of the current study indicate that white seabream has higher ability to digest non-protein energy sources than meagre and that diet supplementation with BSY improved digestive capacities in both species.

A trial was undertaken to estimate the protein requirement of sharpsnout sea bream ( Diplodus puntazzo ) and the protein sparing of dietary lipids. Ten diets were formulated to contain 5 protein levels (ranging from 15 to 55%) and two lipid levels (12 and 18%). Each diet was assigned to duplicate groups of 15 fish with a mean individual body weight of 49.3 g. A quadratic model was used to adjust weight gain and N retention (g kg ABW − 1 day − 1 ) to dietary protein levels. Based on that model, optimum dietary protein requirement was estimated to be 42.9% for maximum weight gain and 43.8% for maximum N retention, corresponding to a protein intake of 7.68 g kg ABW − 1 day − 1 . Protein requirement for maintenance was estimated to be 0.71 g kg ABW − 1 day − 1 . Dietary lipid level improved protein utilization efficiency but did not affect protein requirement. Whole-body protein content increased with dietary lipids and protein content, but no other relevant differences in body composition were noticed. Hepatosomatic index increased with dietary starch and lipid levels and was directly correlated to liver glycogen content. Diet composition affected plasma glucose clearance and cholesterolaemia but not plasma protein and triglyceride levels.

The present work aims to study the effect of a soluble non-starch polysaccharide (NSP), guar gum on white sea bream intestinal health through oxidative status and distal intestine morphology evaluation. A control diet was formulated to contain 40% crude protein, 14% crude lipids and 35% pregelatinized maize starch. Three other diets were formulated to include the same levels of all ingredients as control diet except for guar gum, which was included at 4% (diet GG4), 8% (diet GG8) or 12% (diet GG12). Compared to the control, lower catalase (CAT), superoxide dismutase (SOD) and glutathione reductase (GR) activities and higher SOD, glutathione peroxidase (GPX) and glucose-6-phosphate dehydrogenase (G6PD) activities were observed in the whole intestine of fish fed with diets GG4 and GG12, respectively. Thiobarbituric acid-reacting substances (TBARS) levels were unaffected by diet composition. No morphological alterations in the distal intestine were found among groups. Overall, data on key enzymes of the antioxidant enzymatic defense mechanism suggested that diet-induced reactive oxygen species (ROS) generation is dependent on guar gum inclusion levels and low dietary inclusion of soluble NSP (up to 4%) may have a gut oxidation stress prevention effect.

This study assessed the effect of short (one or two weeks) feed deprivation periods on compensatory growth response of gilthead seabream juveniles. A 10 weeks growth trial was performed and three different feeding protocols were tested: control group (C) fed to apparent satiation twice a day, 6 days a week, during the whole experimental period; one week or two weeks unfed groups (groups U1 and U2, respectively) and re-fed for the rest 8 weeks of the trial as the control group. Each feeding protocol was tested in triplicate groups of fish with an initial body weight of 58 g. Starvation for one or two weeks induced significant losses of body weight that, comparatively to the control group, totaled 18.5 and 30.5% for U1 and U2 group, respectively. An intense mobilization of liver lipid and glycogen was observed after 1 or 2 weeks of starvation while perivisceral lipids, eviscerated-body lipids and energy contents were only decreased in fish unfed for 2 weeks. During the subsequent re-feeding period no recovery of body weight lost was noticed in the previously unfed groups, and feed intake and feed efficiency were identical in all groups. No differences between slopes of growth trajectory between the control and unfed groups were noticed, indicating no convergence of growth of unfed groups towards that of control group. A total recovery of the whole-body composition, organ indices, liver and viscera composition was observed at the end of the trial. Overall, gilthead seabream juveniles were not able to compensate growth after the feed starvation imposed. Therefore, it can be concluded, that under the experimental conditions applied short – one or two weeks – feed restriction periods are not advisable as management strategy to be used in gilthead seabream production.

A glucose tolerance test was performed in European sea bass juveniles to evaluate the effect of a glucose load on plasma insulin and insulin-like growth factor-I (IGF-I) levels. Interaction between these hormones, plasma triacylglycerides and liver glycogen was also determined. After being fasted for 48 h, fish were intraperitoneally injected with either 1 g of glucose per kg body weight or a saline solution. Plasma glucose levels peaked at 23–25 mmol l− 1, 2–6 h after the glucose injection and fish exhibited hyperglycemia until 12 h, when plasma glucose recovered to basal levels. Plasma insulin levels did not change significantly between sampling points, but insulin level was higher in the glucose group than in the control 4–6 h and 24 h after injection. Though plasma IGF-I levels remained constant for a long time (except at time 12 h) in the glucose injected fish, at times 6–9 h, 24 h and 48 h IGF-I levels were higher in the glucose group than in the control. Glucose administration led to lower plasma triacylglyceride levels than saline solution at times 9–12 h. Although liver glycogen content was not affected by the glucose injection (except at times 4 h and 48 h), comparative to saline solution injection, there was a significant increase of liver glycogen at 6 h and 9 h. Results of this study indicate that under these experimental conditions glucose is probably not the most important stimulator of insulin release. Insulin may have contributed to the increase of IGF-I levels and to the enhancement of glucose uptake by the liver in glucose injected fish.

Digestibility studies in Senegalese sole are very scarce due to the difficulty of collecting faeces, which are semi-liquid and therefore dissolve very fast in water. Thus, in this species it is not possible to use sedimentation columns or faeces netting methods for faeces collection. Classical stripping is also not adequate due to the coiled shape of the intestine. Hence, up to now digestibility studies in this species applied the intestine dissection method for faeces collection. To overcome the inconveniences related to this methodology, namely the need to sacrifice animals, in this study we present an adaptation of the stripping method, which combines simultaneous stripping and anal sphincter stimulation and that allows faeces collection of Senegalese sole without sacrificing the animals.

A study was undertaken to establish the optimum dietary essential amino acid (EAA) profile for gilthead seabream juveniles based on the amino acid (AA) deletion method. For that purpose 11 diets were formulated to be isonitrogenous (6.72% N) and isolipidic (18%). In the control diet half of the nitrogen was provided by protein bound-AAs (fish meal) and the other half by a mixture of crystalline L-AAs. The overall AA profile of this diet was made similar to that of fish meal protein. Ten other diets were formulated identical to the control except for the deletion of 45% of a single EAA in each diet and by adjusting the nitrogen level with a non-essential AA mixture. Each diet was assigned to four groups of 22 fish (initial body weight of 4.6 g) and the trial lasted 43 days at a water temperature of 25 °C. Fish were fed by hand, twice daily, using a pair-feeding scheme. At the end of the trial the relationship between nitrogen gain and AA intake of the test and control diets was determined. Based on these data, and assuming that nitrogen retention responds linearly to dietary EAA content when a given AA is limiting, the quantity of each EAA that can be removed from the control diet without affecting nitrogen retention was computed and the ideal dietary EAA profile for gilthead seabream juveniles was estimated. Expressed relative to lysine (= 100) A/E ratios were estimated to be: arginine, 108.3; threonine, 58.1; histidine, 36.8; isoleucine, 49.7; leucine, 92.7; methionine, 50.8; phenylalanine + tyrosine, 112.3; valine, 62.6; and tryptophan, 14.6. This EAA profile correlates tightly to the whole-body EAA composition of gilthead seabream ( R 2 = 0.99; p > 0.001).