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Aquaculture has been one of the fastest-growing food industry sectors, expanding at the pace of consumers' demands. To promote safe and effective fish growth performance strategies, and to stimulate environmentally friendly solutions to protect fish against disease outbreaks, new approaches are needed to safeguard fish welfare, as well as farmers and consumers interests. Here, we tested the use of cyanobacterial extracellular vesicles (EVs) as a novel nanocarrier system of heterologous proteins for applications in fish. We started by incubating zebrafish larvae with Synechocystis sp. PCC6803 EVs, isolated from selected mutant strains with different cell envelope characteristics. Results show that Synechocystis EVs are biocompatible with fish larvae, regardless of their structural composition, as EVs neither induced fish mortality nor triggered significant inflammatory responses. We establish also that cyanobacteria are amenable to engineering heterologous protein expression and loading into EVs, for which we used the reporter sfGFP. Moreover, upon immersion treatment, we successfully demonstrate that sfGFP-loaded Synechocystis EVs accumulate in the gastrointestinal tract of zebrafish larvae. This work opens the possibility of using cyanobacterial EVs as a novel biotechnological tool in fish, with prospective applications in carrying proteins/enzymes, for example for modulating their nutritional status or stimulating specific adaptive immune responses.

The present study evaluated the effects of feeding frequency (FF) and dietary protein/carbohydrate (P/CH) ratios on intestinal histomorphology, microbiota profile, and digestive and oxidative stress-related enzyme activities of gilthead seabream (Sparus aurata). To this purpose, two practical diets were formulated: one with 50% P and 10% CH (P50/CH10) and other with 40% P and 20% CH (P40/CH20). Triplicate groups of fish with9.1±0.01 gwere fed these diets for 60 days until visual satiation at a FF of 1, 2, or 3 meals per day. Distal intestine histomorphology was not affected by diet composition or FF. However, the pyloric caeca (PC) of fish fed 1 meal per day presented more gut fold height alterations than the other groups, except in fish fed diet P50/CH10 3 meals per day, where no changes was observed. Fish fed diet P40/CH20 3 meals per day also presented higher PC submucosa cellularity than the other groups. Fish fed diet P40/CH20 presented a higher number of operational taxonomic units, microbial richness, and diversity indices than fish fed diet P50/CH10. Amylase was the only measured digestive enzyme affected by the experimental conditions, presenting higher activity in fish fed diet P50/CH10 once per day. Glucose-6-phosphate dehydrogenase activity was lower in fish fed 2 meals per day than only 1. While catalase activity was lower in fish fed 2 than 3 meals per day. Glutathione reductase activity was the only measured parameter affected both by dietary P/CH ratio and FF, being inferior in fish fed once per day the P50/CH10 diet than the P40/CH20 diet and, also in the P50/CH10 diet, to fish fed 1 than those fed 3 meals per day. Overall, no major interactions was observed between dietary P/CH ratio and FF; however, a P40/CH20 diet fed 2 meals per day might be recommended for gilthead seabream juveniles.

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.

The present study aimed to evaluate the effects of feeding or feed deprivation on the orexigenic and anorexigenic responses at the central (whole brain) and peripheral (anterior and posterior intestine, stomach, and liver) system levels in European seabass. For this purpose, a group of fish (208 g) was fed a single meal daily for 8 days (fed group) and another group was feed-deprived for 8 days (unfed group). Compared to the fed group, in the whole brain, feed deprivation did not induce changes in

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.

This study aimed to determine the effects of dietary inclusion of Tenebrio molitor larvae (yellow worms) meal (TM) on meagre fish (Argyrosomus regius) whole-body fatty acids (FA) profile and hepatic and intestine oxidative status. For that purpose, fish were fed for 9 weeks a fishmeal-based diet (control) or diets including 10%, 20%, or 30% TM. With the increase in dietary TM level, whole-body oleic acid, linoleic acid, monounsaturated FA, and n−6 polyunsaturated FA (PUFA) increased while saturated FA (SFA), n−3 PUFA, n−3 long chain-PUFA, SFA:PUFA ratio, n3:n6 ratio, and FA retention decreased. Hepatic superoxide dismutase (SOD), glucose-6-phosphate dehydrogenase (G6PDH), and glutathione reductase (GR) activities increased and catalase (CAT) and glutathione peroxidase (GPX) activities decreased with dietary TM inclusion. Hepatic total and reduced glutathione were lower in fish fed 20% TM. Intestinal CAT activity and oxidized glutathione increased and GPX activity decreased with dietary TM inclusion. Intestine SOD, G6PDH, and GR activities increased and malondialdehyde concentration decreased in fish fed the diets with lower TM inclusion levels. Liver and intestine oxidative stress index and liver malondialdehyde concentration were unaffected by dietary TM. In conclusion, to avoid major whole-body FA changes or antioxidant status imbalances, it is recommended to limit TM to 10% inclusion in meagre diets.

The present study was conducted to investigate the effects of dietary fish oil replacement with a mixture of vegetable oils and probiotic supplementation on plasma biochemical parameters, oxidative stress, and antioxidant ability of Seriola dumerili. Specimens with an initial weight of 175 g were used. Four feeds were formulated with 0% (FO-100), 75% (FO-25), and 100% (FO-0 and FO-0+ with the addition of Lactobacillus probiotics) substitution of fish oil with a mixture of linseed, sunflower, and palm oils. After 109 days, no significant differences were observed in the activity of antioxidant enzymes in the liver, foregut, and hindgut, only glucose-6-phosphate dehydrogenase activity in the liver was higher in the fish fed the FO-100 diet than in those fed the FO-0 diet. No significant differences were observed in the total, reduced, and oxidized glutathione and the oxidative stress index in the liver. In addition, lipid peroxidation in the liver and red muscle values were higher in the fish fed the FO-100 diet than in the fish fed the FO-0+ diet, however, the foregut of the fish fed the FO-100 diet presented lower values than that of the fish fed the FO replacement diet, with and without probiotics. There were significant differences in cholesterol levels in the FO-100 group; they were significantly higher than those observed with the fish diets without fish oil. To sum up, fish oil can be replaced by up to 25% with vegetable oils in diets for Seriola dumerili juveniles, but total fish oil substitution is not feasible because it causes poor survival. The inclusion of probiotics in the FO-0+ diet had no effects on the parameters measured.

Tryptophan (Trp) is an essential amino acid, commercially available as a feed-grade product, and is a precursor to serotonin and melatonin, which are both important molecules in stress mitigation. Meagre have a high potential for marine aquaculture diversification but are highly susceptible to stressful conditions. This study aimed to assess the potential role of dietary tryptophan supplementation in meagre juveniles in order to minimize the deleterious effect of potential stress conditions. For this, a growth trial was performed wherein meagre juveniles were fed four isoproteic (45%DM) and isolipidic (16%DM) diets; namely, a control diet, and three diets similar to the control diet but supplemented with varying levels of tryptophan, graded according to the resulting percentage in each diet’s dry matter (Control, 0.25%Trp, 0.5%Trp, and 1%Trp), corresponding to a total dietary tryptophan of 1.06, 1.70, 2.08, and 3.24 g 16 g−1 N, respectively. Diets were tested in triplicate, and fish were fed twice a day, six days a week, for eight weeks. Five days after the end of the growth trial, a time-course blood sampling was performed at 0 h, 1 h, 3 h, 6 h, and 12 h after feeding. At the 6 h sampling point, the liver was also collected. Overall, our results indicate that 1Trp supplementation (total dietary Trp 3.24 g 16 g−1 N) may be harmful to fish, decreasing growth performance and feed utilization, although doses up to 0.5Trp do not influence these parameters. Voluntary feed intake lineally decreased with the increase in the level of dietary Trp. Whole-body lipid content decreased at the highest tryptophan inclusion, whereas no changes were observed in protein levels. After 12 h from feeding, plasma glucose levels were lower in all dietary treatments supplemented with tryptophan compared to those observed in the control. Hepatic enzyme activity of protein catabolism decreased with dietary Trp inclusion. Overall, our results suggested that while a dietary Trp level increase of up to 2.08 g 16 g−1 N did not affect growth performance and feed efficiency, both these parameters were severely compromised with a Trp level of 3.24 g 16 g−1 N.

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

To evaluate the effects of dietary protein/carbohydrate (P/CHO) ratio and feeding frequency (FF) on growth, intermediary metabolism, and economic efficiency of gilthead seabream (Sparus aurata) juveniles, two practical isolipidic (17%) diets were formulated to include high protein (50%)/ low starch (10%) (diet P50/CHO10) or low protein (40%)/ high starch (20%) (diet P40/CHO20). Triplicate groups of fish with 9.1 ± 0.01 g were fed for 60 days with these diets until visual satiation at three FF: one (9:00), two (9:00 and 17:00), or three (9:00, 13:00, and 17:00) meals per day. Dietary P/CHO ratios did not affect growth performance while feeding 2 or 3 meals per day improved fish growth. Fish fed diet P40/CHO20 had increased feed intake (FI), protein efficiency ratio (PER), and nitrogen retention (NR), and lower feed efficiency (FE), nitrogen intake (NI), and economic conversion ratio (ECR). Feeding 2 or 3 meals per day increased FI, NI, ECR, and economic profit index, and decreased FE, PER, and NR. Fish fed diet P40/CHO20 presented increased hepatic lipid and glycogen content, hepatocyte area covered by lipid vacuoles, and glucokinase (gk) gene expression, and decreased glutamate dehydrogenase expression. Fish fed 3 meals per day had decreased plasma triglycerides and total protein levels, while fish fed 2 or 3 meals per day presented decreased hepatic growth hormone receptor-i (ghr-i), gk, and fatty acid synthase gene expression. Interaction between P/CHO ratio and FF was only observed in plasmatic glucose, cholesterol, and total lipids levels, and insulin-like growth factor-1, and ghr-ii gene expression. Overall, glycogenesis, glycolysis, and economic efficiency seemed to be increased while the amino acid catabolism was reduced in fish fed the P40/CHO20 diet. Higher FF increased growth and economic efficiency, and reduced glycolysis and lipogenesis pathways. In conclusion, a diet with P40/CHO20 ratio fed twice a day appears to be the most adequate strategy regarding feed utilization and economic efficiency for gilthead seabream juveniles in order to achieve optimum sustainable aquaculture.

11 Pág. Centro de Investigación en Sanidad Animal (CISA) Centro de Biotecnología y Genómica de Plantas (CBGP), Bacillus subtilis has been documented in the past years as an effective probiotic for different aquacultured species, with recognized beneficial effects on water quality, fish growth and immune status. Furthermore, its potential as a vaccine adjuvant has also been explored in different species. In the current work, we have used B. subtilis spores as delivery vehicles for the presentation of the VP2 protein from infectious pancreatic necrosis virus (IPNV). For this, the VP2 gene was amplified and translationally fused to the crust protein CotY. The successful expression of VP2 on the spores was confirmed by Western blot. We then compared the immunostimulatory potential of this VP2-expressing strain (CRS208) to that of the original B. subtilis strain (168) on rainbow trout (Oncorhynchus mykiss) leukocytes obtained from spleen, head kidney and the peritoneal cavity. Our results demonstrated that both strains significantly increased the percentage of IgM+ B cells and the number of IgM-secreting cells in all leukocyte cultures. Both strains also induced the transcription of a wide range of immune genes in these cultures, with small differences between them. Importantly, specific anti-IPNV antibodies were detected in fish intraperitoneally or orally vaccinated with the CRS208 strain. Altogether, our results demonstrate B. subtilis spores expressing foreign viral proteins retain their immunomodulatory potential while inducing a significant antibody response, thus constituting a promising vaccination strategy., This work was supported by the Spanish Ministry of Science, Innovation and Universities (projects AGL2017-85494-C2-1-R, AGL2017-85494-C2-2-R and PID2020-113268RB-I00) and by the Comunidad de Madrid (grants 2016-T1/BIO-1672 and 2018-T2/BIO-10874). The research was also partially supported by the Strategic Funding UIDB/04423/2020 and UIDP/04423/2020 through national funds provided by the Portuguese Foundation for Science and Technology (FCT) and European Regional Development Fund (ERDF), in the framework of PT2020.

The aquaculture industry is one of the fastest-growing sectors in animal food production. However, farming of carnivorous fish strongly relies on the use of wild fish-based meals, a practice that is environmentally and economically unsustainable. Insect-based diets constitute a strong candidate for fishmeal substitution, due to their high nutritional value and low environmental footprint. Nevertheless, data on the impact of insect meal (IM) on the gut microbiome of farmed fish are so far inconclusive, and very scarce in what concerns modulation of microbial-mediated functions. Here we use high-throughput 16S rRNA gene amplicon sequencing and quantitative PCR to evaluate the impact of different IMs on the composition and chitinolytic potential of the European sea bass gut digesta- and mucosa-associated communities. Our results show that insect-based diets of distinct origins differently impact the gut microbiota of the European sea bass (Dicentrarchus labrax). We detected clear modulatory effects of IM on the gut microbiota, which were more pronounced in the digesta, where communities differed considerably among the diets tested. Major community shifts were associated with the use of black soldier fly larvae (Hermetia illucens, HM) and pupal exuviae (HEM) feeds and were characterized by an increase in the relative abundance of the Firmicutes families Bacillaceae, Enterococcaceae, and Lachnospiraceae and the Actinobacteria family Actinomycetaceae, which all include taxa considered beneficial for fish health. Modulation of the digesta community by HEM was characterized by a sharp increase in Paenibacillus and a decrease of several Gammaproteobacteria and Bacteroidota members. In turn, a mealworm larvae-based diet (Tenebrio molitor, TM) had only a modest impact on microbiota composition. Further, using quantitative PCR, we demonstrate that shifts induced by HEM were accompanied by an increase in copy number of chitinase ChiA-encoding genes, predominantly originating from Paenibacillus species with effective chitinolytic activity. Our study reveals an HEM-driven increase in chitin-degrading taxa and associated chitinolytic activity, uncovering potential benefits of adopting exuviae-supplemented diets, a waste product of insect rearing, as a functional ingredient.

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 present study aimed to evaluate the effects of feeding or feed deprivation on the orexigenic and anorexigenic responses at the central (whole brain) and peripheral (anterior and posterior intestine, stomach, and liver) system levels in European seabass. For this purpose, a group of fish (208 g) was fed a single meal daily for 8 days (fed group) and another group was feed-deprived for 8 days (unfed group). Compared to the fed group, in the whole brain, feed deprivation did not induce changes in npy, agrp1, and cart2 expression, but increased agrp2 and pomc1 expression. In the anterior intestine, feed deprivation increased cck expression, while in the posterior intestine, the npy expression increased and pyyb decreased. In the stomach, the ghr expression decreased regardless of the feeding status. The hepatic lep expression increased in the unfed fish. The present results suggest a feed intake regulation mechanism in European seabass similar to that observed in other teleosts.

This study aimed to assess the effect of Hermetia illucens meal (HM) dietary inclusion on meagre oxidative status. Thus, fish were fed a fishmeal-based diet (CTR diet) and three other diets with increasing levels of HM inclusion, namely 10%, 20%, and 30% (diets HM10, HM20, and HM30, respectively). At the end of the trial, hepatic and intestine superoxide dismutase, catalase, and glucose-6-phosphate dehydrogenase activities and malondialdehyde concentration were unaffected by the diet composition. Liver glutathione peroxidase activity was higher in the fish fed the HM20 diet than in the fish fed the CTR and HM30 diets, and glutathione reductase activity linearly increased with the dietary HM level. The hepatic total glutathione and reduced glutathione contents were significantly lower in fish fed the HM20 diet than in fish fed the CTR and HM10 diets. In the intestine, the oxidized glutathione (GSSG) content and oxidative stress index linearly increased with the increase in dietary HM level, with the GSSG content of fish fed the HM20 diet being significantly higher than of fish fed the CTR diet. In conclusion, 30% HM might be included in meagre diets without negatively affecting hepatic and intestine oxidative status.

Brewer's spent grain (BSG) is the main brewery industry by-product, with potential applications in the feed and food industries due to its carbohydrate composition. In addition, the lignocellulosic nature of BSG makes it an adequate substrate for carbohydrases production. In this work, solid-state fermentation (SSF) of BSG was performed with

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.

Nannochloropsis gaditana was tested as functional ingredient in low fishmeal diets for gilthead seabream juveniles, and its short‐term effects were evaluated in terms of intestinal morphology, digestive function, intestinal immunological and oxidative status, and intestinal microbiota. A diet with 850 g/kg of the protein provided by plant feedstuffs and 150 g/kg provided by fishmeal was used as control, and three other diets identical to the control were supplemented with 5, 7.5 and 15 g/kg N. gaditana meal and fed to 56.6 g fish for 37 days. At the end of this period, intestinal mucosa integrity and digestive capacity (luminal enzyme activity and absorption) were not altered by dietary microalgae supplementation. Intestinal transcript levels of key markers of inflammation (IL‐1β and TNF‐α) and of key enzymes involved in redox homeostasis (CuZnSOD, MnSOD and catalase) were also similar across experimental groups. However, microbiota richness increased with dietary supplementation of microalgae, and such modulation requires further investigation, particularly regarding its potential effects on disease resistance at short and long term.

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.

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

Fishery production, considering both capture and aquaculture, is a major animal protein source for humans [...]

Two trials were conducted to evaluate the effects of soyabean meal replacement by maize distillers dried grains with solubles (DDGS) in diets for pacu juveniles. Five diets were formulated with 0, 100, 200, 300 and 400 g of DDGS/kg diet replacing up to total dietary soyabean meal. In trial 1, the experimental diets were fed to five groups of fish to evaluate the apparent digestibility coefficients (ADC). In trial 2, four groups of fish were fed each experimental diet for 100 d to evaluate the effects of these diets on digestive enzyme activity, intestine oxidative stress and intestine morphology. The ADC of DM and energy was reduced with dietary DDGS inclusion, while the ADC of lipids was increased, and no differences were observed for the ADC of protein. Independent of dietary treatment, pH increased from anterior to the distal intestine with dietary DDGS inclusion. Digestive enzyme activities were higher on anterior than the distal intestine. Dietary DDGS decreased lipase, amylase, chymotrypsin and trypsin activities, while no differences were observed for total protease activity. Intestine glucose-6-phosphate dehydrogenase was reduced in fish fed the DDGS diets, while catalase activity increased. Lipid peroxidation was lower in fish fed DDGS diets than the control. Intestine histomorphology improved with dietary DDGS inclusion. Overall, the negative effects of soyabean meal could be decreased by dietary replacement with maize DDGS which may have a prebiotic effect, improving intestine health.

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.

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.

Aquaculture is among the industries growing at the fastest rate in the world. This industry has been recognized to play a critical role in food production for a continuously expanding world population. However, despite various technological innovations and improvements in production techniques, this sector is still associated with misperceptions and negative opinions hampering its implementation and wide consumption of its products. The integrated multi-trophic aquaculture (IMTA) concept was developed as a way to increase the sustainability of intensive aquaculture systems, using an ecosystem-based approach. In this study, following this sustainable aquaculture concept, a closed recirculation IMTA system, at laboratorial scale, was developed and tested with the simultaneous production of fish, sea urchin and seaweed for 70 days. Based on this proof of concept, a hands-on experimental activity was developed to teach and communicate recent scientific advances in environmental sustainability and value of aquaculture products to young students and the general public. This experimental activity was tested and evaluated with students (n = 60) of basic and high-school (secondary) learning cycles. A quantitative assessment was carried out through a short questionnaire provided to the students before and after the experimental activity. After the experimental activity, a qualitative assessment was also performed through questions expressed without preconceived categories or hypotheses. Results indicated that the overall frequency of students who consider the ocean to be “very important” and “extremely important” increased from 68 to 81% after performing the experimental activity. Moreover, the percentages of correct answers to the questions related to IMTA concepts also increased significantly after the experimental activity. In the discussion of the experimental activity results, the students stated that they appreciated the opportunity to develop a hands-on experimental activity, which allowed them to increase their knowledge and obtain information on aquaculture and the quality of its products.

This study aimed to evaluate the effect of dietary protein source (fishmeal, FM; or plant-feedstuffs, PF) and dietary protein/carbohydrate (P/CH) ratio on gilthead seabream appetite regulation and intermediary metabolism. Additionally, the effect of sampling 5 h after feeding (AF) compared to 24 h AF was also evaluated. Four isolipidic diets were formulated having as major protein sources FM or PF (20% FM and 80% PF), and P/CH ratios of 50/10 or 40/20, being the pregelatinized maize starch the main carbohydrate source (diets FM-P50/CH10; FM-P40/CH20; PF-P50/CH10; PF-P40/CH20). Diets were fed until satiation to 140 g gilthead seabream for 41 days. The expression of appetite regulation genes was assessed at 5 and 24 h AF, while other evaluated parameters were assessed only at 5 h AF. Liver leptin expression was higher at 5 h AF, and brain leptin receptor (lepr) expression was higher at 24 h AF. Brain expression of cocaine- and amphetamine-regulated transcript (cart), leptin and ghrelin receptor (ghrr)-a and liver ghrr-b were also affected by sampling time, but the effects were dependent of the diet provided. FM-based diets promoted the expression of brain cart and leptin (at 24 h AF), and liver growth hormone receptor (ghr)-ii, and increased plasma cholesterol and total lipids levels. Fish fed the PF-based diets had higher liver glycogen content, number and size of adipocytes, and expression of hepatic leptin (at 24 h AF), fatty acid synthase, glucokinase, and target of rapamycin. Regarding dietary P/CH ratio, fish fed the P50/CH10 diets presented higher feed efficiency, plasma triglycerides, and expression of intestine cholecystokinin (at 5 h AF), liver ghrr-b (at 24 h AF), glutamate dehydrogenase and ghr-ii. The protein efficiency ratio, hepatosomatic and visceral indices, plasmatic glucose level, and brain lepr expression (at 5 h AF) were higher in fish fed the P40/CH20 diets. The majority of appetite regulation related-genes were not affected by the use of PF-based diets, while the higher dietary CH seemed to lead to a shorter satiety sensation. PF-based diets promoted liver lipid deposition, hypocholesterolemia, and the activation of glycogenesis pathway, while higher CH content induced an increase in plasma glucose that appeared to be stored as lipids. In conclusion, PF-based diets with up to 20% of CH can be used in gilthead seabream without compromising growth performance and FI, and only slightly modifying appetite and metabolic parameters.

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.

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.