Targeting the Mild-Hypoxia Driving Force for Metabolic and Muscle Transcriptional Reprogramming of Gilthead Sea Bream (Sparus aurata) Juveniles

Reduced oxygen availability generates a number of adaptive features across all the animal kingdom, and the goal of this study was targeting the mild-hypoxia driving force for metabolic and muscle transcriptional reprogramming of gilthead sea bream juveniles. Attention was focused on blood metabolic and muscle transcriptomic landmarks before and after exhaustive exercise. Our results after mild-hypoxia conditioning highlighted an increased contribution of lipid metabolism to whole energy supply to preserve the aerobic energy production, a better swimming performance regardless of changes in feed intake, as well as reduced protein turnover and improved anaerobic fitness with the restoration of normoxia. On-growing juveniles of gilthead sea bream were acclimated for 45 days to mild-hypoxia (M-HYP, 40-60% O-2 saturation), whereas normoxic fish (85-90% O-2 saturation) constituted two different groups, depending on if they were fed to visual satiety (control fish) or pair-fed to M-HYP fish. Following the hypoxia conditioning period, all fish were maintained in normoxia and continued to be fed until visual satiation for 3 weeks. The time course of hypoxia-induced changes was assessed by changes in blood metabolic landmarks and muscle transcriptomics before and after exhaustive exercise in a swim tunnel respirometer. In M-HYP fish, our results highlighted a higher contribution of aerobic metabolism to whole energy supply, shifting towards a higher anaerobic fitness following normoxia restoration. Despite these changes in substrate preference, M-HYP fish shared a persistent improvement in swimming performance with a higher critical speed at exercise exhaustion. The machinery of muscle contraction and protein synthesis and breakdown was also largely altered by mild-hypoxia conditioning, contributing this metabolic re-adjustment to the positive regulation of locomotion and to the catch-up growth response during the normoxia recovery period. Altogether, these results reinforce the presence of large phenotypic plasticity in gilthead sea bream, and highlights mild-hypoxia as a promising prophylactic measure to prepare these fish for predictable stressful events.This work was financially supported by a grant from the European Commission of the European Union under the Horizon 2020 research infrastructure project AQUAEXCEL2020 (652831) to J.P-S. Additional funding was obtained by a Spanish MICINN project (Bream-AquaINTECH, RTI2018-094128-B-I00). J.A.M.-S. received a Postdoctoral Research Fellowship (Juan de la Cierva-Formacion, Reference FJCI-2014-20,161)

Understanding how high stocking densities and concurrent limited oxygen availability drive social cohesion and adaptive features in regulatory growth, antioxidant defense and lipid metabolism in farmed gilthead sea bream (Sparus aurata)

The study combined the use of biometric, behavioral, physiological and external tissue damage scoring systems to better understand how high stocking densities drive schooling behavior and other adaptive features during the finishing growing phase of farmed gilthead sea bream in the Western Mediterranean. Fish were grown at three different final stocking densities (LD, 8.5 kg/m3; MD, 17 kg/m3; HD, 25 kg/m3). Water oxygen concentration varied between 5 and 6 ppm in LD fish to 3–4 ppm in HD fish with the summer rise of water temperature from 19°C to 26°C (May–July). HD fish showed a reduction of feed intake and growth rates, but they also showed a reinforced social cohesion with a well-defined endogenous swimming activity rhythm with feeding time as a main synchronization factor. The monitored decrease of the breathing/swimming activity ratio by means of the AEFishBIT data-logger also indicated a decreased energy partitioning for growth in the HD environment with a limited oxygen availability. Plasma glucose and cortisol levels increased with the rise of stocking density, and the close association of glycaemia with the expression level of antioxidant enzymes (mn-sod, gpx4, prdx5) in liver and molecular chaperones (grp170, grp75) in skeletal muscle highlighted the involvement of glucose in redox processes via rerouting in the pentose-phosphate-pathway. Other adaptive features included the depletion of oxidative metabolism that favored lipid storage rather than fatty acid oxidation to decrease the oxygen demand as last electron acceptor in the mitochondrial respiratory chain. This was coincident with the metabolic readjustment of the Gh/Igf endocrine-growth cascade that promoted the regulation of muscle growth at the local level rather than a systemic action via the liver Gh/Igf axis. Moreover, correlation analyses within HD fish displayed negative correlations of hepatic transcripts of igf1 and igf2 with the data-logger measurements of activity and respiration, whereas the opposite was found for muscle igf2, ghr1 and ghr2. This was indicative of a growth-regulatory transition that supported a proactive instead of a reactive behavior in HD fish, which was considered adaptive to preserve an active and synchronized feeding behavior with a minimized risk of oxidative stress and epidermal skin damage

Modulation of gilthead sea bream gut microbiota by a bioactive egg white hydrolysate: Interactions between bacteria and host lipid metabolism

This study aimed to highlight the relationship between diet, animal performance and mucosal adherent gut microbiota (anterior intestine) in fish fed plant-based diets supplemented with an egg white hydrolysate (EWH) with antioxidant and antiobesogenic activity in obese rats. The feeding trial with juveniles of gilthead sea bream (Sparus aurata) lasted 8 weeks. Fish were fed near to visual satiety with a fish meal (FM)/fish oil (FO) based diet (CTRL) or a plant-based diet with/without EWH supplementation. Specific growth rate decreased gradually from 2.16% in CTRL fish to 1.88% in EWH fish due to a reduced feed intake, and a slight impairment of feed conversion ratio. Plant-based diets feeding triggered a hyperplasic inflammation of the anterior intestine regardless of EWH supplementation.This work was supported by the EU H2020 Research Innovation Program under the TNA Program (project AE150009) at IATS-CSIC Research Infrastructure within AQUAEXCEL2020 Project (652831). This output reflects only the author’s view and the European Union cannot be held responsible for any use that may be made of the information contained herein. Additional funding was obtained by a Spanish MICINN project (Bream-AquaINTECH and RTI2018–094128-B-I00). MCP was funded by a Ramón y Cajal Postdoctoral Research Fellowship [RYC2018-024049-I/AEI/10.13039/501100011033 cofunded by the European Social Fund (ESF) and ACOND/2020 Generalitat Valenciana].Peer reviewe

Evaluation of growth performance, oxidative stress and immune response in gilthead sea bream fed with novel feed formulations

Trabajo presentado en Aquaculture Europe 2020, celebrado en modalidad virtual del 12 al 15 de abril de 2021.[Introduction]: As the aquaculture sector continues to expand while being more environmentally conscious, the development of sustainable aquafeeds is becoming increasingly important (FAO, 2020). Tolerance to the replacement of fishmeal and fish oil in feeds has been largely studied in gilthead seabream (Sparus aurata) (Gasco et al., 2018; Karapanagiotidis, Psofakis, Mente, Malandrakis, & Golomazou, 2019), and many products emerge now as potential alternatives to ingredients used in conventional formulations. A main goal of GAIN EU project is to evaluate emerging ingredients, already commercially available, using different formulation concepts that consider all fish nutritional requirements. GAIN diets are based on circularity principles, maximizing resource efficiency, while contributing towards zero waste in the agro-food value chain, feed cost-effectiveness, and having good social acceptance. The present study aims to understand the real impacts of these novel feed formulations on growth performance, nutritional condition, immunity, and oxidative status using biomarkers.[Methods]: Quadruplicate groups of gilthead seabream (Sparus aurata) were fed ad libitum with four different diets. Three of them have been designed to facilitate aquaculture eco-intensification through increased circularity and resource utilization: NOPAP - formula without terrestrial animal by-products processed animal protein; PAP - formula with terrestrial animal by-products processed animal protein; and MIX - a mixture of NOPAP and PAP. The fourth feed followed a standard commercial formulation and was used as a control diet. After a 77-day feeding trial, plasma samples were collected to evaluate humoral parameters (protease, anti-protease, bactericidal activity and IgM). Liver and head kidney tissues were collected for the simultaneous profiling of a panel of 42 (liver) or 29 (head kidney) genes, as markers of growth performance, lipid and energy metabolism, and immune and antioxidant activities by qPCR. Liver samples were also used to analyse oxidative biomarker (Lipid peroxidation and catalase).[Results]: Tested feed formulations did not affect growth performance or feed intake. However, fish fed PAP and MIX diets had a higher feed conversion ratio (FCR) and protein efficiency ratio than control and NOPAP groups. This impairment was accompanied by a decreased hepatic expression of igf-i and ghr1. NOPAP diet slightly increased innate immunity parameters, showing better results on bactericidal, IgM, and anti-protease activity, as well as a significant up-regulation of il-8 in head kidney. Fish fed with PAP diet displayed an up-regulation of pro-inflammatory genes, namely il-8 and other cytokines (il-1β, tnf-α), chemokines (ck8), and chemokine receptors (ccr3). The same pattern was found for the T-cell markers cd3x, cd4, and cd8a. The activity of the antioxidant enzyme catalase was significantly lower in fish fed with PAP and MIX diet, being a possible indication of decreased antioxidant defences. This is supported by the observed regulation of antioxidant genes (mn-sod/sod2, gpr-170, gpr-94, and gpr-75), although not statistically significant.[Discussion]: The similar performance of novel formulations and the control diet indicates that they can be considered as viable options for seabream feeds. Differences in FCR suggest that NOPAP can promote a better bioavailability and/or increased absorption of key nutrients than PAP and MIX diets. Indeed, this impairment was also evidenced by their hepatic expression pattern of markers of growth performance. In general, PAP exhibited an opposite response to the NOPAP group. NOPAP was closer to the control diet, and MIX showed intermediate values between PAP and NOPAP in almost all parameters. The markedly pro-inflammatory head kidney expression profile in PAP fish may be also indicative of an impaired response at the mucosal level. In any case, the low proportion of differentially expressed genes between the experimental diets and control (18 out of 71) constitutes an additional and indirect confirmation of their suitability.[Conclusions]: Novel feed formulations for gilthead seabream seem to be viable options for a near future. In any case, all results are related to the formulation itself and cannot be attributed to a specific ingredient alteration. More studies are necessary to understand the cost-benefit of these new formulations and their market acceptability to optimize sustainability within the current/predictable European regulatory framework.“This project was financed from the European Union’s Horizon 2020 research and innovation programme under grant agreement N° 773330 (GAIN), with additional support from Nord university (Norway) and Sparos SA (Portugal)”

Modulation of gilthead sea bream gut microbiota by a bioactive egg white hydrolysate

Trabajo presentado en Aquaculture Europe 2020, celebrado en modalidad virtual del 12 al 15 de abril de 2021.[Introduction]: A bioactive egg white hydrolysate (EWH) treated with pepsin has demonstrated potent in vitro and in vivo antioxidant and anti-inflammatory properties, improving oxidative stress and inflammation biomarkers on genetically and diet induced obese rats (Requena et al., 2017). However, the effects of protein hydrolysates and bioactive food-derived peptides on gut microbiome remain relatively poorly studied in mammals and fish in particular. Thus, the aim of this study was to unravel the main effects on fish performance, histopathological scoring and mucosal adherent gut microbiota of EWH supplementation in a fish fed a formulation with a high replacement of marine feedstuffs by alternative plant ingredients, using gilthead sea bream as a farmed fish model. [Methods]: The feeding trial lasted 8 weeks (May-July) under natural photoperiod and temperature conditions. Juvenile fish (20-24 g initial body weight, 4.8-4.9 kg/m3) were fed near to visual satiety with control (CTRL) or low fish meal (FM)/fish oil (FO) diets with/without egg white hydrolysate (EWH) supplementation (7.5%). DNA from the adherent bacteria of the anterior intestine was collected and the V3-V4 region of the 16S rRNA of each sample was amplified and sequenced by Illumina MiSeq. Taxonomic assignment was performed with a custom-made pipeline using the RDP database. Alpha diversity was calculated using Phyloseq, and beta diversity using PERMANOVA and partial least-squares discriminant analysis (PLSDA) models. Metagenome prediction and pathway analysis were performed using Piphillin.[Methods]: The feeding trial lasted 8 weeks (May-July) under natural photoperiod and temperature conditions. Juvenile fish (20-24 g initial body weight, 4.8-4.9 kg/m3) were fed near to visual satiety with control (CTRL) or low fish meal (FM)/fish oil (FO) diets with/without egg white hydrolysate (EWH) supplementation (7.5%). DNA from the adherent bacteria of the anterior intestine was collected and the V3-V4 region of the 16S rRNA of each sample was amplified and sequenced by Illumina MiSeq. Taxonomic assignment was performed with a custom-made pipeline using the RDP database. Alpha diversity was calculated using Phyloseq, and beta diversity using PERMANOVA and partial least-squares discriminant analysis (PLSDA) models. Metagenome prediction and pathway analysis were performed using Piphillin.[Results]: Daily specific growth rates (SGR) varied significantly from 2.16 in CTRL fish to 1.88 in EWH fish as a result of a reduced feed intake. A slight impairment of feed conversion ratio, from 1.03 to 1.10, was also observed. Intermediate values on growth performance parameters were reported with the low FM/FO diet without EWH supplementation. No changes in total plasma antioxidant capacity, and faecal concentrations of lactic acid and short-chain fatty acids were found among dietary groups. The dietary replacement of FM/FO triggered a hyperplasic inflammation of the anterior intestine submucosa that was not alleviated by EWH supplementation. Conversely, alterations on the staining pattern and amount of goblet cells at the level of anterior intestine were reversed in EWH fish, together with a decreased accumulation of lipid vacuoles in the epithelium of posterior intestine, a high abundance of hepatic melanomacrophage centers, and depletion of hepatocyte lipid depots until the restoration of CTRL fish values. Illumina sequencing reads were assigned to 2,117 OTUs and a significantly lower richness was found in the EWH group. Indeed, at the phylum level, Proteobacteria reached the highest proportion in CTRL and EWH fish, whereas Firmicutes were decreased and Actinobacteria increased with the replacement of FM/FO. The proportion of Actinobacteria was restored to CTRL values with the dietary EWH supplementation. Additionally, EWH triggered the highest amount of Bacteroidetes and Spirochaetes phyla. Detailed differences in microbiota composition were analysed with a statistically validated PLS-DA which clearly separated CTRL fish from fish fed low FM/FO diets along x-axis (component 1, 37.4%), whereas component 2 (43.2%) separated the low FM/FO diets with/without EWH along y-axis (Fig. 1). This analysis disclosed 165 OTUs discriminating among diets (VIP ≥ 1), with 46 OTUs representing at least the 1% in one of the groups. For these abundant bacteria, a first type of response was mediated by 17 OTUs that were increasing with the FM/FO replacement and decreasing again in EWH fish. In this group, Neisseriaceae family and species of Ralstonia, Lactobacillus, Streptococcus, Corynebacterium and Nocardioides genera were included. A group of 14 OTUs were present in high proportion in the CTRL group, but decreased in fish fed the two low FM/FO diets. In this case, the dietary plant ingredients drove the decrease of the Comamonadaceae family and Mesorizhobium, Brochotrix, Bacillus, Clostridium sensu stricto and Exiguobacterium genera. The remaining 15 OTUs increased their proportion in fish fed the EWH diet, being in a very low proportion in the other two dietary groups. This response triggered the presence of Bacteroidetes phylum, Rhodospirilalles order and Granucatella, Bradyrizhobium, Propionibacterium and Streptophyta genera. Inferred metagenome results showed two pathways corresponding to primary bile acid biosynthesis and steroid degradation consistently underrepresented in the microbiota of EWH fish when compared to the other two groups[Conclusions]: These results reinforce the central role of gut microbiota in the regulation of host metabolism and lipid metabolism in particular (Hegyi et al., 2018), supporting a main role of the EWH as an anti-obesity and satiety factor in fish as suggested in rat models of obesity. The potential use of this functional food ingredient in finishing diets, and the role of gut microbiota in tuning fillet fatty acid composition of marketable fish merits further research.This work was funded by the TNA programme (AE150009) within H2020 AQUAEXCEL2020 project (652831) to GAWP for accessing to IATS-CSIC facilities

Somatotropic Axis Regulation Unravels the Differential Effects of Nutritional and Environmental Factors in Growth Performance of Marine Farmed Fishes

The Gh/Prl/Sl family has evolved differentially through evolution, resulting in varying relationships between the somatotropic axis and growth rates within and across fish species. This is due to a wide range of endogenous and exogenous factors that make this association variable throughout season and life cycle, and the present minireview aims to better define the nutritional and environmental regulation of the endocrine growth cascade over precisely defined groups of fishes, focusing on Mediterranean farmed fishes. As a result, circulating Gh and Igf-i are revitalized as reliable growth markers, with a close association with growth rates of gilthead sea bream juveniles with deficiency signs in both macro- or micro-nutrients. This, together with other regulated responses, promotes the use of Gh and Igf-i as key performance indicators of growth, aerobic scope, and nutritional condition in gilthead sea bream. Moreover, the sirtuin-energy sensors might modulate the growth-promoting action of somatotropic axis. In this scenario, transcripts of igf-i and gh receptors mirror changes in plasma Gh and Igf-i levels, with the ghr-i/ghr-ii expression ratio mostly unaltered over season. However, this ratio is nutritionally regulated, and enriched plant-based diets or diets with specific nutrient deficiencies downregulate hepatic ghr-i, decreasing the ghr-i/ghr-ii ratio. The same trend, due to a ghr-ii increase, is found in skeletal muscle, whereas impaired growth during overwintering is related to increase in the ghr-i/ghr-ii and igf-ii/igf-i ratios in liver and skeletal muscle, respectively. Overall, expression of insulin receptors and igf receptors is less regulated, though the expression quotient is especially high in the liver and muscle of sea bream. Nutritional and environmental regulation of the full Igf binding protein 1–6 repertoire remains to be understood. However, tissue-specific expression profiling highlights an enhanced and nutritionally regulated expression of the igfbp-1/-2/-4 clade in liver, whereas the igfbp-3/-5/-6 clade is overexpressed and regulated in skeletal muscle. The somatotropic axis is, therefore, highly informative of a wide-range of growth-disturbing and stressful stimuli, and multivariate analysis supports its use as a reliable toolset for the assessment of growth potentiality and nutrient deficiencies and requirements, especially in combination with selected panels of other nutritionally regulated metabolic biomarkers

Fish meal-free diets supplemented with health promoters support optimal growth in gilthead sea bream, with benefitial changes in gene expression, intestinal microbiota and improved intestinal disease recovery

Trabajo presentado en la International Conference & Exposition Aquaculture Europe, celebrada en Funchal, Maderia (Portugal) del 04 al 07 de octubre de 2021.[Introduction]: The exponential growth of the aquaculture sector requires the development of sustainable aquafeeds with less dependence on marine products. Tolerance to fish meal (FM) and fish oil replacement in the economically important gilthead sea bream (Sparus aurata) is being extensively studied with many products emerging as alternative feed ingredients. It has been demonstrated that alternative diets influence the composition of intestinal adherent microbial populations, which have a key role on host metabolism, health and disease resistance. In addition, low fish meal diets showed an increased susceptibility to enteric parasites (Piazzon et al., 2017). Clearly, differences in diet have an impact on the overall health and metabolism of the fish and many parameters have to be taken into account when studying alternative diets for their use in aquaculture. In this study we evaluated the effect of a novel feed formulation (NoPAP SANA) with total replacement of FM by insect meal and bacterial fermentation biomass, and supplemented with the health-promoter additive SANACORE®GM (Palenzuela et al., 2020), on growth performance, gene expression, intestinal microbiota and disease resistance in gilthead sea bream.[Methods]: Tagged gilthead sea bream of mean weight 21.3 g were distributed in two open-flow tanks (160 fish/tank) and fed ad libitum during 34 days with control or NoPAP SANA diets. Twelve fish/diet were sacrificed and head kidney (HK), liver (L) and posterior intestine (PI) were taken for RNA extraction. From the same fish, the adherent bacteria of PI were collected and immediately used for DNA extraction. RNA from HK, L and PI was used to run three customized PCR-arrays including genes of interest for each tissue, with markers of performance and metabolism (L), immune system (HK and PI), epithelial integrity, nutrient transport and mucins (PI). Using the bacterial DNA, the V3-V4 region of the 16S rRNA of each individual sample was amplified and sequenced by Illumina MiSeq. After quality filtering, taxonomic assignment was performed with a custom-made pipeline using the RDP database. Alpha diversity was calculated using Phyloseq and beta diversity using PERMANOVA and PLS-DA models. Metagenome prediction and pathway analysis were performed using Piphillin. Differential gene expression and OTU presence and abundance correlations were studied using the corrplot R package. From the remaining fish, 70 fish/group were challenged with the intestinal parasite Enteromyxum leei by effluent exposure and the remaining fish were used as controls. The challenge lasted 78 days, including a non-lethal diagnosis sampling at day 40. At the end of the challenge all fish were sampled for histological and molecular diagnosis. Biometric values from all fish were taken in all sampling points.[Results]: A slight decrease in condition factor and specific growth rate was detected in the NoPAP SANA group. However, all fish grew efficiently considering gilthead sea bream standards. NoPAP SANA group showed differential expression of 17 out of 44 genes in L, two out of 29 in HK, and 4 out of 44 in PI. The bacterial composition at the PI showed no major differences in diversity or at the phylum level. However, 29 abundant (>1%) OTUs significantly changed with the diet. From these, 10 OTUs were significantly correlated with differential expression of genes in the different tissues, highlighting Pseudoxanthomonas which was positively correlated with the expression of seven L genes, or Actinomyces, significantly correlated with the expression of L and HK genes (Fig. 1). Inferred metagenome analyses revealed that the altered microbiota with NoPAP SANA diet could account for changes in 15 metabolic pathways. The intensity and prevalence of infection after the parasite challenge was not significantly different between diets. In fact, infected fish from both groups showed similar recovery rates.[Conclusions]: NoPAP SANA promoted good growth parameters and efficient conversions arising as a good alternative for a FMbased diet in gilthead sea bream diets. This diet modulated the expression of several genes in L showing the capacity to reduce lipogenesis, mitochondrial activity and the risk of oxidative stress and, at the same time, promoting an antiinflammatory gene expression profile in HK and PI. Changes were also detected in the adherent bacterial populations of PI, with significant changes of OTUs that could potentially account for significant metabolic alterations. The correlations between presence and abundance of intestinal bacteria with changes in gene expression of different tissues, together with the pathway analysis results, show that microbiota changes can have an impact on host metabolism at a systemic level, and vice versa. Clearly, the changes induced by this novel FM-free diet supported an accelerated growth with an overall feed conversion ratio close to 1 and no increased susceptibility against this intestinal parasite, as often observed in studies when replacing a FM-based diet.GAIN (EU-H2020 #773330); RYC2018-024049-I/AEI/10.13039/50110001103

A novel fish meal-free diet formulation supports proper growth and does not impair intestinal parasite susceptibility in gilthead sea bream (Sparus aurata) with a reshape of gut microbiota and tissue-specific gene expression patterns

The exponential growth of the aquaculture sector requires the development of sustainable aquafeeds with less dependence on marine products. The maximized replacement of fish meal (FM) and fish oil (FO) with plant ingredients has been extensively studied in the economically important species gilthead sea bream (Sparus aurata). Recently, major progress has been done with other alternative raw materials, though some non-pathological inflammatory response persisted with feed formulations that increased the circularity of resource utilization. In the present study, we evaluated the effects on growth performance, gene expression, intestinal microbiota and disease resistance of a FM-free diet (NoPAP SANA), based on plant ingredients, aquaculture by-products, algae oil, insect meal and bacterial fermentation biomasses as main dietary oil and protein sources, and supplemented with a commercially available health-promoting feed additive (SANACORE®GM). Juveniles of 21 g initial body weight were fed control or NoPAP SANA diets for 34 days, and head kidney, liver and posterior intestine were collected for gene expression analyses using customized PCR-arrays. Each tissue-specific PCR covered 96 genes in total and included markers of growth performance, lipid and energy metabolism, antioxidant defence, immune system, and intestinal function and integrity. From the same fish, the adherent bacteria of the posterior intestine were studied by Illumina sequencing of the V3-V4 region of the 16S rRNA. The remaining fish were challenged with the intestinal parasite Enteromyxum leei for 78 days and sampled for parasite diagnosis. Both control and NoPAP SANA fish grew efficiently considering gilthead sea bream standards. Before parasite challenge, the NoPAP SANA group showed differential expression of 17, 2 and 4 genes in liver, head kidney and posterior intestine, respectively. The intestinal bacterial composition showed no major differences in diversity or at the phylum level. However, 29 abundant OTUs significantly changed with the diet. From these, 10 OTUs were significantly correlated with differentially expressed genes in the different target tissues. Inferred metagenome analyses revealed that the altered microbiota with NoPAP SANA diet could account for changes in 15 metabolic pathways. The intensity and prevalence of infection after the parasite challenge did not significantly vary between dietary treatments, and infected fish from both groups showed similar disease outcome. Altogether, these results indicate that the NoPAP SANA diet promoted optimal growth and a healthy condition in gilthead sea bream without affecting susceptibility against the tested intestinal parasite, as often observed with alternative diets following current industry formulations.This project has received funding from the European Union's Horizon 2020 Research and Innovation Programme under grant agreement No 773330 (GAIN, Green Aquaculture Intensification). This publication reflects the views only of the authors, and the European Commission cannot be held responsible for any use which may be made of the information contained therein. Additional funding was obtained by a Spanish MICINN project (Bream-AquaINTECH, RTI2018–094128-B-I00, AEI/FEDER, UE). M.C·P was funded by a Ramón y Cajal Postdoctoral Research Fellowship (RYC2018-024049-I, co-funded by the European Social Fund & ACOND/2020 Generalitat Valenciana)

How do novel feed formulations affect growth performance, oxidative stress and immune response of atlantic salmon?

Trabajo presentado en la International Conference & Exposition Aquaculture Europe, celebrada en Funchal, Maderia (Portugal) del 04 al 07 de octubre de 2021.[Introduction]: The aquaculture industry continues to grow faster than any other sector of food production. The need to make aquaculture as sustainable and more environmentally conscious as possible is becoming clearer everyday (FAO, 2020). With this in mind, the replacement of fishmeal and fish oil in aquafeeds has been studied in Atlantic salmon (Salmo salar) (e.g., Bendiksen et al., 2011) with many products emerging as potential alternatives to conventional ones (e.g., Hodar et al., 2020). One of the main objectives of the EU project GAIN is to evaluate new ingredients that are already commercially available using different formulation concepts that consider all the fish nutritional needs. GAIN diets are based on circular economy principles and maximize resource efficiency, while contributing to zero waste in the agri-food value chain, being cost-effective feeds, and having good social acceptability. The present study aims to understand the actual effects of these novel feed formulations on growth performance, nutritional status, immunity and oxidative status.[Methods]: Quadruplicate groups of Atlantic salmon were fed ad libitum with three different diets. Two diets were developed to facilitate the eco-intensification of aquaculture through increased circularity and resource utilization (NOPAP - formula without processed animal protein - and PAP - formula with processed animal protein). The third diet was a commercial-like formulation that was used as a control. After a 96-day feeding trial, plasma samples were collected to evaluate humoral parameters (protease, anti-protease, bactericidal activity, and IgM). Liver and head kidney tissues (collected at day 45 and 96) were used for the simultaneous profiling by PCR array of a panel of 38 or 28 genes, respectively, as markers of growth performance, lipid and energy metabolism, and immune and antioxidant activities. Liver samples were also used to analyse lipid peroxidation. In addition, after 45 and 96 days, the lice count and fish welfare were also assessed by standard methods. The dorsal skin and foregut were collected at days 45 and 96 for mucosal mapping (mucous cell area, density, and barrier status).[Results]: Growth performance was adequate and comparable to commercial standards for the novel diets tested. Other parameters analysed, including those related to key performance indicators, intestinal and skin dorsal mucosal mapping, plasma innate immune defences, and lipid peroxidation in the liver did not significantly differ across diets. Regarding head kidney gene expression, at Day 45, 2 out of 28 genes in the array were differentially expressed (p<0.05). Gene expression of fish fed with novel feed formulations showed a pro-inflammatory profile evidenced by the up regulation of il-8, and a down regulation of il-10.At Day 96, the same genes continued to be differentially expressed, but gene clec1b (membrane protein) was also up-regulated. However, the rest of the analyses do not support this pro-inflammatory profile. A longer trial may bring light to some of the current results. In turn, the liver had a differential gene expression only at the second sampling point (Day96), where 4 out of 38 genes in the array were affected, including growth performance (igf2), lipid metabolism, elongases (elovl4), and energy metabolism (ucp2l and sirt1). These transcriptomic changes may be attributed to an initial response to the experimental diets. Cross-analysis of gene expression by time points and dietary treatment (two-way ANOVA) yielded only 2 out of 38 genes that had significantly different expression across treatments. The differentially expressed genes were related to growth performance (igf2) and lipid metabolism (elovl4).[Conclusions]: The novel feed formulations of the GAIN project for Atlantic Salmon seem to be viable options for the near future. In any case, all results are related to the formulation itself and cannot be attributed to a specific ingredient alteration. More studies are necessary to understand the cost-benefit of these new formulations and their market acceptability to optimize sustainability within the current/predictable European regulatory framework.This project was financed by the European Union’s Horizon 2020 research and innovation programme under grant agreement N° 773330 (GAIN), with additional support from Nord University (Norway) and SPAROS Lda (Portugal)

Client applications and Server Side docker for management of RNASeq and/or VariantSeq workflows and pipelines of the GPRO Suite

The GPRO suite is an in-progress bioinformatic project for -omic data analyses. As part of the continued growth of this project, we introduce a client side & server side solution for comparative transcriptomics and analysis of variants. The client side consists of two Java applications called "RNASeq" and "VariantSeq" to manage workflows for RNA-seq and Variant-seq analysis, respectively, based on the most common command line interface tools for each topic. Both applications are coupled with a Linux server infrastructure (named GPRO Server Side) that hosts all dependencies of each application (scripts, databases, and command line interface tools). Implementation of the server side requires a Linux operating system, PHP, SQL, Python, bash scripting, and third-party software. The GPRO Server Side can be deployed via a Docker container that can be installed in the user's PC using any operating system or on remote servers as a cloud solution. The two applications are available as desktop and cloud applications and provide two execution modes: a Step-by-Step mode enables each step of a workflow to be executed independently and a Pipeline mode allows all steps to be run sequentially. The two applications also feature an experimental support system called GENIE that consists of a virtual chatbot/assistant and a pipeline jobs panel coupled with an expert system. The chatbot can troubleshoot issues with the usage of each tool, the pipeline job panel provides information about the status of each task executed in the GPRO Server Side, and the expert provides the user with a potential recommendation to identify or fix failed analyses. The two applications and the GPRO Server Side combine the user-friendliness and security of client software with the efficiency of front-end & back-end solutions to manage command line interface software for RNA-seq and variant-seq analysis via interface environments