Under control: how a dietary additive can restore the gut microbiome and proteomic profile, and improve disease resilience in a marine teleostean fish fed vegetable diets

[Background]: The constant increase of aquaculture production and wealthy seafood consumption has forced the industry to explore alternative and more sustainable raw aquafeed materials, and plant ingredients have been used to replace marine feedstuffs in many farmed fish. The objective of the present study was to assess whether plant-based diets can induce changes in the intestinal mucus proteome, gut autochthonous microbiota and disease susceptibility of fish, and whether these changes could be reversed by the addition of sodium butyrate to the diets. Three different trials were performed using the teleostean gilthead sea bream (Sparus aurata) as model. In a first preliminary short-term trial, fish were fed with the additive (0.8%) supplementing a basal diet with low vegetable inclusion (D1) and then challenged with a bacteria to detect possible effects on survival. In a second trial, fish were fed with diets with greater vegetable inclusion levels (D2, D3) and the long-term effect of sodium butyrate at a lower dose (0.4%) added to D3 (D4 diet) was tested on the intestinal proteome and microbiome. In a third trial, the long-term effectiveness of sodium butyrate (D4) to prevent disease outcome after an intestinal parasite (Enteromyxum leei) challenge was tested. [Results]: The results showed that opposed forces were driven by dietary plant ingredients and sodium butyrate supplementation in fish diet. On the one hand, vegetable diets induced high parasite infection levels that provoked drops in growth performance, decreased intestinal microbiota diversity, induced the dominance of the Photobacterium genus, as well as altered the gut mucosal proteome suggesting detrimental effects on intestinal function. On the other hand, butyrate addition slightly decreased cumulative mortality after bacterial challenge, avoided growth retardation in parasitized fish, increased intestinal microbiota diversity with a higher representation of butyrate-producing bacteria and reversed most vegetable diet-induced changes in the gut proteome. [Conclusions]: This integrative work gives insights on the pleiotropic effects of a dietary additive on the restoration of intestinal homeostasis and disease resilience, using a multifaceted approach.This work has been carried out with financial support from the European Union under grant projects ARRAINA (FP7-KBBE-2011-288,925) to JPS, MP and VK and ParaFishControl (H2020-634429) to ASB. Additional funding has been received from Spanish Ministry of Economy and Competitiveness (MINECO) project no. AGL2013- 48560-R to JPS and ASB, and Generalitat Valenciana (PROMETEOII/2014/085) to ASB. MCP was contracted under CSIC PIE project no. 201740E013 and MINECO FPDI-2013-15741, and IE under APOSTD/2016/037 grant by the “Generalitat Valenciana”.We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI)

Serum metabolomics tells the story of disease degree in a fish enteritis model

Comunicación presentada en la 19th International Conference on Diseases of Fish and Shellfish, celebrada en Oporto (Portugal) del 9 al 12 de septiembre de 2019.[Introduction]: In animal production, enteritis is responsible for serious economic losses, being intestinal parasitism a major stress factor leading to malnutrition and lowered performance and production efficiency. The intestinal myxozoan parasite Enteromyxum leei dwells between gut epithelial cells and causes severe desquamative enteritis in gilthead sea bream (Sparus aurata) that impairs nutrient absorption causing anorexia, cachexia, growth impairment, reduced marketability and increased mortality. This study aimed to outline the gut failure produced in this fish-parasite model using a multifaceted approach and to find and validate serum non-lethal markers of gut barrier dysfunction.[Methodology]: Intestinal integrity was studied in parasitized and non-parasitized fish by immunohistochemistry with specific markers for cellular adhesion (E-cadherin) and tight junctions (Tjp-1 and Cldn3) and by functional studies of permeability (oral administration of FITC-dextran) and electrophysiology (Ussing chambers). Serum samples from parasitized and non-parasitized fish were analyzed using non-targeted metabolomics and some significantly altered metabolites were selected to be validated using commercial kits.[Results]: The expression of the tight junction proteins Tjp-1 and Cldn3 was significantly lower in parasitized fish along all the intestine, while no differences were found in E-cadherin labeling. Some parasitized fish showed a significant increase in paracellular uptake measured by FITC-dextran detection in serum. Electrophysiology studies showed a decrease in transepithelial resistance in infected animals, which showed a diarrheic profile when compared to the normal absorptive profile of the control animals. Serum metabolomics revealed 3702 ions, from which the differential expression of 20 identified compounds significantly separated control from infected groups in multivariate analyses (PLS-DA), and even separated groups by intensity of infection. Of these compounds, inosine and creatine were identified as relevant and tested with commercial kits in serum samples.[Conclusion]: This study demonstrates the loss of barrier function induced by the enteric parasite E. leei and underlines key markers to differentiate control and infected fish. The untargeted serum metabolomics approach did not reveal specific effects by the parasite, but more a profile typical of absorption dysfunction and anorexia, which are, of course, part of the disease signs.Funding: ParaFishControl H2020 project (634429), Aquaexcel2020 (652831, TNA AE10004-INTEBREAM), AGL2013-48560-R

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

Smart biosensing device for tracking fish behaviour

Biosensor technology for tracking individual challenged fish behaviour has the potential to revolutionize aquaculture, allowing farmers and breeders to orientate selective breeding towards more robust and efficient fish or improve culture conditions for a more sustainable and ethical production. The proposed solution within the AQUAEXCEL2020 EU project is a stand-alone, small and light (1 g) device (AEFishBIT), based on a tri-axial accelerometer and a microprocessor. It is externally attached to the operculum to monitor physical activity by mapping accelerations in x- and y-axes, while operculum beats (z-axis) serve as a measurement of respiratory frequency. The conducted operculum attachment protocol does not show signs of tissue damage or growth impairment in active feeding gilthead sea bream. AEFishBIT offers a wide range of new information based on individual behaviour, allowing to point out the asynchrony of movements as an indirect measure of aging and adaptability to farming environment, as well as to discriminate different coping behaviour (proactive or reactive) of gilthead sea bream challenged with low water oxygen concentrations. AEFishBIT also provides reliable information of disease outcome in fish parasitized with an intestinal myxozoan, emerging as a powerful tool for sensing the quality of the environment and improving selective breeding protocols.The study has received funding from the European Union’s Horizon 2020 research and innovation programme, GA no 652831 (AQUAEXCEL2020)

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)

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 anti-obesogenic 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. However, EWH ameliorated the goblet cell depletion, and the hepatic and intestinal lipid accumulation induced by FM/FO replacement. Illumina sequencing of gut mucosal microbiota yielded a mean of 136,252 reads per sample assigned to 2,117 OTUs at 97% identity threshold. The bacterial diversity was similar in all groups, but a significantly lower richness was found in EWH fish. At the phylum level, Proteobacteria reached the highest proportion in CTRL and EWH fish, whereas Firmicutes were decreased and Actinobacteria increased with the FM/FO replacement. The proportion of Actinobacteria was restored by dietary EWH supplementation, which also triggered a highest amount of Bacteroidetes and Spirochaetes. At a closer look, a widespread presence of Lactobacillales among groups was found. Otherwise, polysaccharide hydrolases secretors represented by Corynebacterium and Nocardioides were increased by the FM/FO replacement, whereas the mucin-degrading Streptococcus was only raised in fish fed the plant-based diet without EWH. In addition, in EWH fish, a higher abundance of Propionibacterium was related to an increased concentration of intestinal propionate. The antagonism of gut health-promoting propionate with cholesterol could explain the inferred underrepresentation of primary bile acid biosynthesis and steroid degradation pathways in the EWH fish microbiota. Altogether, these results reinforce the central role of gut microbiota in the regulation of host metabolism and lipid metabolism in particular, suggesting a role of the bioactive EWH peptides as an anti-obesity and/or satiety factor in fish.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 co-funded by the European Social Fund (ESF) and ACOND/2020 Generalitat Valenciana].Peer reviewe