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Centro de Investigación en Sanidad Animal (CISA), Research on immunotherapeutic agents has become a focus for the treatment of fish diseases. The ability of algae to produce secondary metabolites of potential interest as immunotherapeutics has been documented. The present research intended to assess antiviral and antibacterial activities of macro- and microalgae extracts against viral and bacterial pathogens and explore their immunomodulatory potential using zebrafish (Danio rerio) larvae as a model organism. The cytotoxicity and antiviral activity of eight methanolic and ethanolic extracts from two macroalgae (Fucus vesiculosus, Ulva rigida) and two microalgae (Nannochloropsis gaditana, Chlorella sp.) were analyzed in established fish cell lines. Six extracts were selected to evaluate antibacterial activity by disk diffusion and growth inhibition assays. The three most promising extracts were characterized in terms of fatty acid composition, incorporated at 1% into a plant-based diet, and evaluated their effect on zebrafish immune response and intestinal morphology in a short-term feeding trial. All extracts exhibited in vitro antiviral activity against viral hemorrhagic septicemia and/or infectious pancreatic necrosis viruses. Methanolic extracts from F. vesiculosus and U. rigida were richer in saturated fatty acids and exhibited in vitro antibacterial action against several bacteria. Most promising results were obtained in vivo with F. vesiculosus methanol extract, which exerted an anti-inflammatory action when incorporated alone into diets and induced pro-inflammatory cytokine expression, when combined with the other extracts. Moreover, dietary inclusion of the extracts improved intestinal morphology. In summary, the results obtained in this study support the potential of algae as natural sources of bioactive compounds for the aquaculture industry., M. Monteiro, A. S. Lavrador, R. A. Santos, and F. Rangel were supported by grants SFRH/BD/114995/2016, ZEBRALGRE_BM_2019-003, SFRH/BD/131069/2017, and SFRH/BD/138375/2018, respectively, from FCT — Foundation for Science and Technology, under the POCI program. A. Couto C. Serra and P. Enes have a scientific employment contract supported by national funds through FCT. E. da Costa and M. R. Domingues are financially supported by FCT/MCTES (Portugal) through CESAM (UIDB/50017/2020 + UIDP/50017/2020), QOPNA (FCT UID/QUI/00062/2019), LAQV/REQUIMTE (UIDB/50006/2020), and RNEM (LISBOA-01–0145-FEDER-402–022125). This research was partially supported by the Strategic Funding to UID/Multi/04423/2019 (POCI-01–0145-FEDER-007621) through national funds provided by FCT under the project PTDC/CVT-WEL/5207/2014.

Aquaculture is responsible for more than 50% of global seafood consumption. Bacterial diseases are a major constraint to this sector and associated with misuse of antibiotics, pose serious threats to public health. Fish-symbionts, co-inhabitants of fish pathogens, might be a promising source of natural antimicrobial compounds (NACs) alternative to antibiotics, limiting bacterial diseases occurrence in aquafarms. In particular, sporeforming Bacillus spp. are known for their probiotic potential and production of NACs antagonistic of bacterial pathogens and are abundant in aquaculture fish guts. Harnessing the fish-gut microbial community potential, 172 sporeforming strains producing NACs were isolated from economically important aquaculture fish species, namely European seabass, gilthead seabream, and white seabream. We demonstrated that they possess anti-growth, anti-biofilm, or anti-quorum-sensing activities, to control bacterial infections and 52% of these isolates effectively antagonized important fish pathogens, including Aeromonas hydrophila, A. salmonicida, A. bivalvium, A. veronii, Vibrio anguillarum, V. harveyi, V. parahaemolyticus, V. vulnificus, Photobacterium damselae, Tenacibaculum maritimum, Edwardsiela tarda, and Shigella sonnei. By in vitro quantification of sporeformers' capacity to suppress growth and biofilm formation of fish pathogens, and by assessing their potential to interfere with pathogens communication, we identified three promising candidates to become probiotics or source of bioactive molecules to be used in aquaculture against bacterial aquaculture diseases.

The effect of partially replacing fishmeal (FM) by black soldier fly larvae meal (Hermetia meal--HM) in meagre (Argyrosomus regius) diet was evaluated for nutrient digestibility and digestive enzyme activity. For that purpose, triplicate groups of fish (18.0 ± 0.02 g) were fed during 48 days either a control diet (CTR), without HM, or one of three diets including 100, 200 and 300 g/kg of HM, replacing 17, 35 and 52% of FM, respectively. Apparent digestibility coefficients (ADCs) of dry matter, energy, protein, lysine, isoleucine, leucine, phenylalanine, alanine, glutamate, glycine and serine presented a decreasing response with increased HM inclusion. Chitin ADC was null, independent of dietary HM inclusion. Total alkaline protease activity increased while trypsin activity decreased with dietary HM inclusion. No intestinal chitinolytic activity was detected. Intestinal alkaline protease zymogram revealed nine bands with proteolytic activity against casein, with molecular weights ranging between 15 and 75 kDa. Anti-protease activity in the intestine was not affected by dietary inclusion of HM compared to the CTR diet. Overall, it is concluded that replacement of up to 17% FM with HM (100 g/kg HM inclusion level) in meagre diets has no major adverse effects in diet digestibility and digestive enzyme activity.

Made available in DSpace on 2020-12-12T02:21:51Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-01-01 Proper inclusion of carbohydrates in aquafeeds can increase protein and lipid retention preventing the catabolism of these nutrients for energy purposes. This research aimed to evaluate performance, metabolic and enzymatic indicators in pacu (Piaractus mesopotamicus) juveniles fed diets with 350 or 400 g/kg starch (S) and 190 or 210 g/kg digestible protein (P) (diets S35P19, S35P21, S40P19 and S40P21 respectively). Overall, the best growth performance was achieved with diet S35P21. Dietary protein and starch levels had no effect on feed intake, feed conversion ratio and protein efficiency ratio. The lowest whole-body protein content was recorded in fish fed the S35P19 diet, while fish fed diet S40P21 presented the highest whole-body lipid content. Regardless of dietary protein level, S40 diets led to higher plasma glucose and triglycerides levels, and higher liver glycogen content. No differences were observed on hepatosomatic index or muscle lipids content. Increasing dietary starch level led to an increase in mesenteric fat index and in liver glucokinase activity, while higher dietary protein promoted a decrease in liver lipid content. Fish fed diet S40P21 showed higher liver glucose-6-phosphate dehydrogenase activity than fish fed the S35P21 and S40P19 diets. Dietary protein and starch levels had no effect on pyruvate kinase and fructose-1,6-bisphosphatase activity. Overall, dietary starch enhanced glycolytic and lipogenic pathways but did not depress gluconeogenic pathway. The results of this study indicate that a diet with 350 g/kg starch and 210 g/kg digestible protein was the most adequate for pacu in terms of growth performance and feed utilization. Universidade Estadual Paulista (Unesp) Centro de Aquicultura da Unesp (Caunesp) Universidade Estadual Paulista (Unesp) Faculdade de Ciências Agrárias e Tecnologia (FCAT-Unesp) Universidade do Porto CIMAR/CIIMAR - Centro Interdisciplinar de Investigação Marinha e Ambiental Universidade do Porto Faculdade de Ciências Departamento de Biologia Universidade Estadual Paulista (Unesp) Faculdade de Ciências Agrárias e Veterinárias Universidade Estadual Paulista (Unesp) Centro de Aquicultura da Unesp (Caunesp) Universidade Estadual Paulista (Unesp) Faculdade de Ciências Agrárias e Tecnologia (FCAT-Unesp) Universidade Estadual Paulista (Unesp) Faculdade de Ciências Agrárias e Veterinárias

Brewers spent grain (BSG) is the largest by-product originated from the brewery industry with a high potential for producing carbohydrases by solid-state fermentation. This work aimed to test the efficacy of a carbohydrases-rich extract produced from solid-state fermentation of BSG, to enhance the digestibility of a plant-based diet for European seabass (Dicentrarchus labrax). First, BSG was fermented with A. ibericus to obtain an aqueous lyophilized extract (SSF-BSG extract) and incorporated in a plant-based diet at increasing levels (0control; 0.1%, 0.2%, and 0.4%). Another diet incorporating a commercial carbohydrases-complex (0.04%; Natugrain; BASF) was formulated. Then, all diets were tested in in vitro and in vivo digestibility assays. In vitro assays, simulating stomach and intestine digestion in European seabass, assessed dietary phosphorus, phytate phosphorus, carbohydrates, and protein hydrolysis, as well as interactive effects between fish enzymes and dietary SSF-BSG extract. After, an in vivo assay was carried out with European seabass juveniles fed selected diets (0control; 0.1%, and 0.4%). In vitro digestibility assays showed that pentoses release increased 45% with 0.4% SSF-BSG extract and 25% with Natugrain supplemented diets, while amino acids release was not affected. A negative interaction between endogenous fish enzymes and SSF-BSG extract was observed in both diets. The in vivo digestibility assay corroborated in vitro data. Accordingly, the dietary supplementation with 0.4% SSF-BSG increased the digestibility of dry matter, starch, cellulose, glucans, and energy and did not affect protein digestibility. The present work showed the high potential of BSG to produce an added-value functional supplement with high carbohydrases activity and its potential contribution to the circular economy by improving the nutritional value of low-cost and sustainable ingredients that can be included in aquafeeds. © 2021, The Author(s)., Te authors thank UNICER, a sof-drink company (Porto, Portugal), for providing the brewer’s spent grain used in this study. Tis study was supported by the project “SPO3-Development of innovative sustainable protein and omega-3 rich feedstufs for aquafeeds, from local agro-industrial by-products”, reference POCI-01- 0145-FEDER-030377, funded by European Regional Development Fund (ERDF) and Portuguese Foundation for Science and Technology (FCT) and by strategic funding of UIDB/04423/2020 by UIDB/04469/2020 unit, the BioTecNorte operation (NORTE-01-0145-FEDER-000004) through national funds provided by FCT, and under the scope of the strategic funding of UIDB/04469/2020 unit. Te H.F. and C.C were supported by grants SFRH/BD/131219/2017 and SFRH/BPD/114942/2016, respectively, from FCT, MCTES, FSE, and UE under the North Portugal Regional Operational Programme (NORTE2020). José Manuel Salgado was supported by Beatriz Galindo contract of Ministry of Education (Spain)., info:eu-repo/semantics/publishedVersion

The interest in using marine organisms as a source of novel bioactive compounds has increased recently, reinforcing the need to find efficient and cost-effective methods to extract these biocompounds. Thus, the present study has focused on evaluating four solvent systems and three extraction methods to obtain extracts of three macroalgae (Gracilaria sp., Fucus vesiculosus, and Ulva rigida) and two microalgae (Chlorella sp. and Nannochloropsis gaditana). The extracts were evaluated regarding yield, phenolic content, and potential antioxidant activity. Results showed that a lower organic solvent:water ratio increased macroalgae, but not microalgae, biomass extraction efficiency. Levels of total phenolics, ortho-diphenols and flavonoids were highly influenced by algae material and solvent system applied. Strong 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) scavenging activity was obtained with ethanol:water, 80:20 (E80:20) microalgal extracts, while no visible trend was detected for macroalgae extracts. However, methanolic extracts were the most effective in scavenging of 2,2-diphenyl-1-picrylhydrazyl (DPPH). In macroalgae, a high positive correlation was observed between phenolic content and antioxidant capacity while in microalgae, the opposite was verified, suggesting that in microalgae, phenolic compounds are not the main contributors to the observed antioxidant capacity.

Aquaculture is the best alternative to fulfil global fish demand, however it still relies heavily on fisheries-derived products for aquafeeds production. This study assesses and compares the environmental impacts of producing four experimental diets to gilthead seabream with different dietary protein (P) to carbohydrate (CH) ratios (P50/CH10 and P40/CH20). The diets were made either with fish meal (FM) or plant feedstuffs (PF) as main protein sources and fish oil (FO) or vegetable oils (VO) as lipid sources. The functional unit used was 1 kg of experimental diet. The studied boundaries included aquafeed ingredients production (S1), compound aquafeeds production under laboratory conditions (S2), and transportation between S1 and S2 locations. The present study applied the Recipe Endpoint method, hierarchist version (V1.13; Europe recipe H/A). Background data was collected from ecoinvent database and related literature. For each aquafeed ingredient used, it was accounted either the agriculture production or fishery activities, the processing unit, and transportation between the production and processing locations. Ingredient mixing and processing was done at the Marine Zoology Station (MZS) located at Porto, Portugal. It was also taken into account the road transportation of aquafeed ingredients between a commercial company and the MZS. Regardless of dietary protein source or P/CH ratio used, all diets had the same single score index. In agreement with several studies, S1 was the system with the highest environmental impact. On the other hand, S2 was the lowest environmental contributor step to all formulated diets, except for diet P50/CH10, where the lowest environmental impact was related with the aquafeed ingredients transportation to MZS. Fisheries-derived ingredients were the biggest contributors to environmental impact. In the hypothesis of replacing FO from Portuguese fisheries by-products by FO of Peruvian anchovy fisheries or by soybean oil (SBO), the environmental impact of the diets would be decreased, being the replacement by SBO the best environmental alternative. There was highlighted a tendency for PF-based diets having lower environmental impact score when compared to the FM-based diets after both hypothetical replacements. Studying the replacement of by-products FO by Peruvian anchovy FO or by SBO allowed to emphasize the importance of adequate ingredients selection for reducing the environmental impact. As limitations of the current study it is important to mention: the use of pelletization as manufacturing process and the non-valorization of fish by-products as recycled aquafeed ingredients.

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.

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.

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.

Here, we present the genome sequences of two environmental Bacillus strains with broad hydrolytic capacity toward different nonstarch polysaccharides (NSPs) that were isolated from the gut of marine fish fed NSP-rich diets. Several genes that may contribute to the NSP-degrading behavior were identified through in silico analysis.

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 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.

This study aimed at assessing the effect of short-chain fructooligosaccharides (scFOS), xylooligosaccharides (XOS), and galactooligosaccharides (GOS) on European sea bass juveniles gut microbiota. Four practical diets were formulated with fish meal (FM) and plant feedstuffs (PF) as protein sources (circa 30:70 of protein from FM:PF) and to include 1% of α-cellulose (control diet, CTR), or 1% of scFOS, XOS, or GOS (diets FOS, XOS, and GOS, respectively). Triplicate groups of fish with 79 g were fed with the experimental diets during 30 days. Gut content was sampled at days 7 and 30 for allochthonous microbiota characterization. Lactobacillus, Pseudomonas, Vibrio, and Burkholderia were the main genera found in fish gut. Gut allochthonous microbiota presented an increased number of operational taxonomic units (OTUs) and a higher Margalef index of bacterial richness in fish fed scFOS and XOS. Gut allochthonous microbial OTUs, Margalef species richness index, Shannon’s diversity index, and SIMPER similarity were unaffected by sampling day. In conclusion, scFOS and XOS modulated European sea bass gut microbial community and this effect persisted throughout time. These changes in gut microbiota composition contribute to explain the positive effects on growth performance, immune status, and lipid and glucose metabolism previously reported in European sea bass fed with scFOS and XOS.

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

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 increased demand for food fish, together with the banning of antibiotics for fish growth promotion and the greater public awareness for healthy fish production, led to an increased interest in the potential of functional feeds as health promoters. Prebiotics are functional ingredients that are used in fish, being reported to be associated with improvements in growth, feed efficiency, gut microbiota, digestive enzyme activities, gut morphology, immune status, disease resistance, intermediary metabolism, and stress responses. Those benefits may be related with the increase in gut beneficial bacteria, bacteria end-products of prebiotic fermentation, or the interaction between prebiotics and pattern recognition receptors. Due to the reported prebiotic-related positive effects in fish, a large amount of studies have been published in the last years, mainly focusing on prebiotics’ benefits to fish. Thus, the present review focuses on prebiotics’ potential as functional ingredients, focusing on their mechanisms of action. In addition, this review summarizes prebiotic effects in Mediterranean aquaculture fish, whose production has greatly increased in the last years. Finally, topics that deserve to be further explored, such as prebiotic effects related with fish age and size, and timing and duration of administration are suggested.

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.

This study aimed at evaluating the effects of short-chain fructooligosaccharides (scFOS), xylooligosaccharides (XOS) and galactooligosaccharides (GOS) on growth performance, hepatic metabolism, gut microbiota and digestive enzymes activities 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 control but supplemented with 10 g of scFOS, XOS or GOS per kilo diet, which were fed to fish during 12 weeks. Prebiotics had no effect on growth, feed efficiency or gut microbiota. Plasmatic triglycerides were lower in fish fed XOS than FOS and GOS diets. Malic enzyme activity was lower in fish fed XOS than FOS diet. Fish fed XOS diet had lower fatty acid synthetase (FAS), a key lipogenic enzyme and higher alanine aminotransferase activities. Fifteen days after the start of the trial, an enhancement of total alkaline protease, trypsin and lipase activities was observed in fish fed prebiotics, but such effect disappeared at 12 weeks. In conclusion, scFOS, XOS or GOS seem to have limited applicability in white sea bream feed.

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 taurine (tau) supplementation to low fishmeal (FM) diets on growth performance, oxidative status, and immune response of European seabass juveniles. Four isoproteic (46% crude protein) and isolipidic (19% crude lipid) diets were formulated to contain either 25 or 12.5% FM and a mixture of plant feedstuffs, supplemented or not with 1% tau. Twelve groups of 20 fish (IBW = 9.4 g) were fed each diet for 9 weeks. Reduction of dietary FM from 25 to 12.5% impaired growth performance, feed efficiency, and protein efficiency ratio but had no effect on nitrogen retention (% N intake). Independently of FM level, dietary tau supplementation improved growth performance and nitrogen retention without affecting feed efficiency. Dietary FM level reduction increased liver G6PDH activity, but did not affect lipid peroxidation or activities of redox key enzymes. Contrarily, dietary tau supplementation decreased hepatic G6PDH and GPX activities and lipid peroxidation. Gene expression COX-2 was not affected either by FM or tau levels but TNF-α increased with the reduction of FM level but not with the tau level. Dietary tau supplementation decreased Casp3 and Casp9 expression regardless of dietary FM level. Overall, this study evidenced that dietary tau supplementation improved growth performance and antioxidant response and reduced intestine inflammatory and apoptosis processes.

The macroalgae aquaculture industry has grown up in the last years, and new applications for macroalgae should be considered. In this work, sequential biological treatments as solid-state fermentation (SSF) by Aspergillus ibericus and enzymatic hydrolysis (EH) were applied to washed and unwashed Ulva rigida. SSF of unwashed macroalgae showed higher xylanase (359.8 U/g), cellulase (73.07 U/g) and β-glucosidase (14.9 U/g) activities per dry mass of macroalgae. After SSF, two strategies to carry out EH were assayed. The best process was SSF followed by EH by simply adding a buffer. The non-starch polysaccharides content was reduced by 93.2 %, achieving a glucan conversion of 98 %. In addition, the antioxidant activity was improved 2.8-fold and the protein concentration of macroalgae extracts increased from 16.9 % to 29.8 % (w/w). These biological treatments allowed to increase macroalgae value as feedstuff with potential for use in aquafeeds., Helena Fernandes was supported by PhD grant SFRH/BD/131219/ 2017, funded by the Portuguese Foundation for Science and Technology (FCT). 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 R&D&I 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) and Portuguese Foundation for Science and Technology (FCT); by the InovFeed project (ref. MAR-02.01.01-FEAMP-0111) supported by Programa Operacional Mar2020, and by the Portuguese Foundation for Science and Technology under the scope of the strategic funding of UID/BIO/04469/2019 and BioTec-Norte 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

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.

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 gut microbiomes of fish play important roles in host development, digestion and health. Evidence suggests that abiotic factors, such as diet and rearing temperature, could affect fish gut microbiota. In this study, the effect of dietary short-chain fructooligosaccharides (scFOS) on turbot intestinal health, microbiota and digestive enzymes was investigated at two rearing temperatures: 15 and 20 °C. Four practical diets were supplemented with scFOS at 0, 5, 10 and 20 g kg−1. scFOS did not affect fish performance. PCR-DGGE did not show differences in bacterial profiles between dietary treatments; however, the number of operational taxonomic units, richness and diversity were higher at 20 °C. Enzyme activities in the foregut were not affected by rearing temperature, while in the hindgut, enzyme activities were higher at 15 °C. Total alkaline protease, α-amylase and lipase activities in the foregut were higher in fish fed 20 g kg−1 scFOS. Prebiotic supplementation had no effect on hindgut α-amylase activity, while lipase activity of fish reared at 20 °C was higher in fish fed diet with 5 g kg−1 scFOS. No differences were observed in intestinal morphology. This was the first study to simultaneously evaluate the effect of dietary prebiotic level and rearing temperature on fish intestinal microbiota and health.

To assess the effect of feeding frequency on the utilization of dietary starch by white sea bream juveniles, triplicate groups of fish were fed an experimental diet (400 g kg−1 protein, 140 g kg−1 lipids and 350 g kg−1 pregelatinized maize starch) to apparent visual satiation 2, 3 or 4 times a day for 63 days. Growth performance, feed utilization efficiency, glycaemia, cholesterolaemia, plasma triacylglycerides, liver lipids and glycogen content were unaffected by feeding frequency. α-Amylase activity increased from the pyloric caeca to the posterior intestine and was higher in fish fed twice a day than in fish fed 3 or 4 times a day. Hepatic glucokinase (GK) activity decreased with the increase in feeding frequencies, whereas fructose-1,6-bisphosphatase (FBPase) activity increased. Hepatic glucose-6-phosphate dehydrogenase and glutamate dehydrogenase activities were unaffected by feeding frequency. Overall, feeding frequency did not improve white sea bream dietary starch utilization. α-Amylase, GK and FBPase activities responded to dietary starch consumed at each meal, denoting a good metabolic adaptation of the fish to the feeding conditions.

This study was the first to investigate the effect of soy antinutrients, saponins and phytosterols, in on-growing sea bass. Seven diets were formulated: a control diet (fishmeal and fish oil based) and six diets containing 1 or 2 g kg−1 levels of soya saponins, 5 or 10 g kg−1 levels of phytosterols or a combination of 1 g kg−1 saponins + 5 g kg−1 phytosterols or 2 g kg−1 saponins + 10 g kg−1 phytosterols. After a 59 days feeding trial fish were weighed, faeces collected for digestibility studies and plasma sampled for cholesterol quantification. At day 15 and 59, distal intestine (DI) samples were collected for histological evaluation and quantification of maltase (Malt), alkaline phosphatase (AlP) and leucine aminopeptidase (LAP) activities. At day 59, DI was collected for gene expression of AlP, Malt and immune related genes. Antinutrients had no effect on fish performance, digestibility, plasma cholesterol levels and digestive enzymes activities and gene expression. Only mild morphological changes were present in groups fed high levels of saponins + phytosterols. Expression of immune related genes showed no differences between groups. Overall, on-growing sea bass showed high tolerance to dietary antinutrients up to the levels tested.

The effect of short-chain fructooligosaccharides (scFOS) incorporation on growth, feed utilization, body composition, plasmatic metabolites and selected liver enzyme activities of turbot juveniles reared at winter (15 °C) and summer (20 °C) temperatures was studied. Four comparable diets were formulated to contain circa 50 : 50 fish meal and plant ingredients as protein sources. Experimental diets included increasing levels of scFOS (0, 5, 10 and 20 g kg−1). Final weight was higher at 20 °C, but thermal growth unit, feed efficiency, nitrogen and energy retention were better at 15 °C. scFOS supplementation did not affect fish growth performance. Fish reared at 15 °C had higher liver glycogen, visceral and hepatosomatic indices. Liver lipids, plasma triglycerides, total lipids, cholesterol HDL and LDL were higher in turbot reared at 20 °C. Malic enzyme, fatty acid synthetase, alanine aminotransferase and glutamate dehydrogenase activities were higher in fish reared at 15 °C. Malic enzyme was lower in turbot fed with 20 g kg−1 scFOS compared to control diet; however, fatty acid synthetase presented an increasing trend as dietary scFOS increased up to 10 g kg−1. Glutamate dehydrogenase activity was higher in fish fed the control diet. Results seem to indicate no benefits of scFOS incorporation to diets on growth performance of turbot.

This study was carried out to establish biochemical parameters with potential diagnostic value to assess the nutritional status of healthy seabass. For that purpose, triplicate groups of seabass juveniles were submitted to different feeding protocols: fed for 14 days; fed for 7 days followed by 7 days of fasting or fasted for 14 days. At the end of the trial, body, liver and viscera were randomly sampled for proximate composition analysis. Blood was also collected and the following plasma parameters were analyzed by standard clinical methods: glucose; cholesterol; triglycerides; protein; inorganic phosphorus; calcium; magnesium; alkaline phosphatase (ALP); aspartate aminotransferase; lactate dehydrogenase; creatine phosphokinase and lipase. No major effect of feed deprivation on body composition, visceral index, perivisceral and hepatic lipid content were observed, whereas hepatosomatic index and hepatic glycogen were reduced. Previous feeding conditions strongly influenced the plasma parameters in seabass. Comparatively to the fed group, plasma glucose, cholesterol and calcium levels were reduced after 2 weeks of fasting while plasma triglycerides, protein, inorganic phosphorus and ALP attained minimum levels after 1 week of fasting. Overall, enzymatic activity parameters showed higher variability than biochemistry parameters. In conclusion, during short-term starvation (

This study aimed to elucidate the effects of two soy antinutrients, saponins and phytosterols, on growth, intestinal health and function of European sea bass juveniles. Seven fishmeal-based diets were formulated: a control diet without antinutrients and six experimental diets containing low or high levels of soyasaponins (SapL and SapH, respectively), low or high levels of phytosterols (PhytL and PhytH, respectively) and low or high levels of soyasaponins + phytosterols (SapPhytL and SapPhytH, respectively). A feeding trial was conducted for 59 days after which blood was collected for plasmatic cholesterol quantification and intestinal samples were collected for histology, digestive enzymes activity and gene expression analysis. Histology was also performed at day 15. PhytL resulted in high distal intestine (DI) relative weight and decreased plasma cholesterol, while PhytH caused inflammatory changes in the DI. SapH depressed maltase and alkaline phosphatase activity in DI and the histological evaluation indicated some inflammatory changes. The SapPhytH resulted in decreased maltase activity in the DI and indications of inflammatory changes that were supported by the results on gene expression profiles. The antinutrients tested did not compromise growth, however, caused some gastrointestinal disturbance that may affect fish in the long term.

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.

The effect of dietary carbohydrate complexity on growth, feed utilization, and glycemia was studied in European sea bass juveniles. Four isonitrogenous (50% crude protein) and isolipidic (15% crude lipids) diets were formulated to contain 20% pregelatinized maize starch (PGS diet), dextrin (DEX diet), maltose (MAL diet), or glucose (GLU diet). No effect of dietary carbohydrate complexity on growth was noticed. Feed efficiency and protein efficiency ratio were lower in fish fed the GLU diet than in the other groups, whereas the opposite was observed for feed intake. Plasma glucose peaked 3–4 h after feeding in fish fed the MAL and GLU diets, whereas in fish fed the PGS and DEX diets the peak was reached 5–6 h after feeding. Peak plasma glucose concentration (13 mmol/L) was higher in fish fed the GLU diet than the other diets (9 mmol/L). Shorter hyperglycemia duration was observed in fish fed the MALT and GLU diets (6 h) than the PGS and DEX diets (10 h). Complex carbohydrates delayed plasma glucose concentration peak compared with simple sugars, whereas the opposite was observed for hyperglycemia duration. Overall, dietary maltose, dextrin, and starch were apparently better utilized as energy source than glucose by European sea bass juveniles.

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.

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).

The effects of various dietary protein levels on growth performance, whole body composition and nutrient utilization were studied in two-banded sea bream (Diplodus vulgaris), a candidate species for aquaculture. Fish (initial weight 6.1 g) were fed to satiety six iso-energetic diets, containing 5%, 12.5%, 25%, 35%, 45% or 55% of crude protein during 72 days. Fish fed 35% and 45% protein attained better growth and feed utilization than the other groups (P < 0.05). Daily growth index and feed conversion ratio were the poorest for fish fed 5% and 12.5% protein (P < 0.001), while the 25% and 55% protein groups had intermediate performance. Lipid retention increased significantly from 13.7% to 30.1% (P < 0.0001) and protein retention decreased from 35.5% to 21.3% (P < 0.01) with increasing protein levels from 12.5% to 45%. Muscle protein, lipid and energy concentrations were not significantly affected by dietary protein level. The estimated protein requirement for maintenance and maximum growth of two-banded seabream growing from 6 to 20 g were 7.5% and 35.7%, respectively. Protein requirements as calculated from body protein gain were 2.3 and 6.5 g of protein intake per kilogram body weight per day.

A trial was undertaken to estimate the protein requirement of white sea bream (Diplodus sargus). Five fish meal-based diets were formulated to contain graded levels of protein (from 60 to 490 g kg⁻¹). Each diet was assigned to triplicate groups of 25 fish with a mean individual body weight of 22 g. Fish fed the 60 g kg⁻¹ protein diet lost weight during the trial, while growth improved in the other groups as dietary protein level increased up to 270-370 g kg⁻¹. Feed efficiency improved as dietary protein level increased. Maximum protein efficiency ratio (PER) was observed with the 17% protein diet. N retention (NR) (% N intake) was not different among groups fed diets with 17% protein and above. Ammonia excretion (g kg⁻¹ABW day⁻¹) increased as dietary protein level increased, while no differences in urea excretion were noted. An exponential model was used to adjust specific growth rate and NR (g kg⁻¹ day⁻¹) to dietary protein level. Based on that model, dietary protein required for maximum retention was 330 g kg⁻¹, while for maximum growth it was 270 g kg⁻¹. On a wet weight basis, there were no differences in whole body composition of fish-fed diets with 170 g kg⁻¹ protein and above, except for the protein content, which was lower in group fed the 170 g kg⁻¹ protein diet than the 490 g kg⁻¹ protein diet. Specific activities of hepatic amino acid catabolism enzymes (glutamate dehydrogenase, alanine aminotransferase and aspartate aminotransferase) increased as dietary protein levels increased. There were no differences among groups in fatty acid synthetase and malyc enzyme but 6-phosphate dehydrogenase (G6PDH) was significantly lower in fish fed the 60 g kg⁻¹ protein diet than the 170 and 490 g kg⁻¹ protein diets.

— A 8-wk trial was performed with white sea bream, Diplodus sargus, juveniles (17.3 g initial weight) to evaluate the effect of dietary lipid levels on growth performance and whole-body composition. Six experimental diets were formulated to be isoproteic (45% protein) and to contain from 9 to 24% dietary lipid levels (dry matter basis). Fish meal and fish oil were used as the main protein and lipid sources, respectively. At the end of the trial, there were no significant differences (P > 0.05) among groups in fish performance or in whole-body composition. From the results of this trial, it is concluded that, within the range of values tested, increasing dietary lipid levels above 9% has no advantage as it does not promote growth or spare protein for growth purposes.

An experiment was performed to evaluate the growth performance and feed utilization of white sea bream juveniles (initial weight, 14 g) fed diets of cornstarch of diierent origins (normal and waxy). Four experimental diets were formulated to be isolipidic and to contain normal and waxy starch (26% or 42%) at two protein (36% and 48%) levels. The growth trial lasted 15 weeks and, at the end of the trial, there were no diierences in the growth rate among groups. At the highest dietary starch level ^ but not at the lowest level ^ the feed e⁄ciency ratio and PER were signi¢cantly lower in ¢sh fed the waxy starch diet. Protein e⁄ciency ratio and N retention (% N intake) were not aiected by starch source but were signi¢cantly higher in the diets with a lower protein content. No diierences in energy retention (% energy intake) were observed among groups. Except for the protein content, which was signi¢cantly higher in ¢sh fed diets with a high protein level, no other diierences were observed among groups in whole-body composition. Hepatosomatic Index (HSI) was signi¢cantly higher in ¢sh fed highstarch diets, but there were no diierences in visceral indices among groups. The apparent digestibility coe⁄cients of protein and energy were not aiected by the dietary starch level, but were signi¢cantly lower in diets including waxy starch. Glutamate dehydrogenase activity was higher in ¢sh fed highprotein diets, but it was not aiected by dietary starch source. Alanine aminotransferase and aspartate aminotransferase activities were not diierent among groups. Glucose 6-phosphate dehydrogenase, malic enzyme and fatty acid synthetase activities were not aiected by the dietary starch level, but were signi¢cantly lower in ¢sh fed waxy starch. The results of this study indicate that diets for white sea bream juveniles may include up to 42% starch without negative eiects on ¢sh performance. Moreover, normal starch appears to be more e⁄ciently used as an energy source than waxy starch.