Acute stress response in gilthead sea bream (Sparus aurata L.) is time-of-day dependent: Physiological and oxidative stress indicators

Since fish show daily rhythms in most physiological functions, it should not be surprising that stressors may have different effects depending on the timing of exposure. In this study, we investigated the influence of time of day on the stress responses, at both physiological and cellular levels, in gilthead sea bream (Sparus aurataL.) submitted to air exposure for 30 s and then returned to their tank. One hour after air exposure, blood, hypothalamus and liver samples were taken. Six fish per experimental group (control and stressed) were sampled every 4 h during a 24-h cycle. Fish were fed in the middle of the light cycle (ML) and locomotor activity rhythms were recorded using infrared photocells to determine their daily activity pattern of behaviour, which showed a peak around feeding time in all fish. In the control group, cortisol levels did not show daily rhythmicity, whereas in the stressed fish, a daily rhythm of plasma cortisol was observed, being the average values higher than in the control group, with increased differences during the dark phase. Blood glucose showed daily rhythmicity in the control group but not in the stressed one which also showed higher values at all sampling points. In the hypothalamus of control fish, a daily rhythm ofcorticotropin-releasing hormone(crh) gene expression was observed, with the acrophase at the beginning of the light phase. However, in the stressed fish, this rhythm was abolished. The expression ofcrh-binding protein(crhbp) showed a peak at the end of the dark phase in the control group, whereas in the stressed sea bream, this peak was found at ML. Regarding hepatic gene expression of oxidative stress biomarkers: (i)cytochrome c oxidase 4showed daily rhythmicity in both control and stressed fish, with the acrophases located around ML, (ii)peroxiredoxin(prdx) 3 and5(prdx5) only presented daily rhythmicity of expression in the stressed fish, with the acrophase located at the beginning of the light cycle and (iii)uncoupling protein 1showed significant differences between sampling points only in the control group, with significantly higher expression at the beginning of the dark phase. Taken together, these results indicate that stress response in gilthead sea bream is time-dependent as cortisol level rose higher at night, and that different rhythmic mechanisms interplay in the control of neuroendocrine and cellular stress responses

The effects of nisin-producing Lactococcus lactis strain used as probiotic on Gilthead sea bream (Sparus aurata) growth, gut microbiota, and transcriptional response.

The present research tested the effects of dietary nisin-producing Lactococcus lactis on growth performance, feed utilization, intestinal morphology, transcriptional response, and microbiota in gilthead sea bream (Sparus aurata). A feeding trial was conducted with fish weighting 70-90 g. Fish were tagged with passive, integrated transponders and distributed in nine 500-L tanks with 40 fish each. Fish were fed for 12 weeks with either a control (diet A) or experimental diets (diets B and C) in triplicate (3 tanks/diet). Extruded pellets of diets B and C were supplemented with a low (2 x 109 CFU/kg) and a high (5 x 109 CFU/kg) dose of probiotic, respectively. No significant differences were found between groups for the feed conversion ratio or specific growth rates. However, the final body weight of fish fed diet C was significantly higher than the control group with intermediate values for fish fed diet B. Histological analysis conducted using a semi-quantitative scoring system showed that probiotic did not alter the morphology of the intestine and did not trigger inflammation. With regard to the transcriptomic response, a customized PCR array layout was designed to simultaneously profile a panel of 44 selected genes. Significant differences in the expression of key genes involved in innate and acquired immunity were detected between fish fed probiotic and control diets. To analyze the microbiota associated to the feeds and the gut autochthonous microbial communities, we used the Illumina MiSeq platform for sequencing the 16S rRNA gene and a metagenomics pipeline based on VSEARCH and RDP databases. The analysis of gut microbiota revealed a lack of colonization of the probiotic in the host\u2019s intestinal mucosa. However, probiotic did modulate the fish gut microbiota, confirming that colonization is not always necessary to induce host modification. In fact, diets B and C were enriched with Actinomycetales, as compared to diet A, which instead showed a higher percentage of Pseudomonas, Sphyngomonas, and Lactobacillus genera. These results were confirmed by the clear separation of gut bacterial community of fish fed with the probiotic from the bacterial community of control fish group in the beta-diversity and PLS-DA (supervised partial least-squares discriminant analysis) analyses

Dietary supplementation of heat-treated Gracilaria and Ulva seaweeds enhanced acute hypoxia tolerance in gilthead sea bream (Sparus aurata)

Intensive aquaculture practices involve rearing fish at high densities. In these conditions, fish may be exposed to suboptimal dissolved O2 levels with an increased formation of reactive O2 species (ROS) in tissues. Seaweeds (SW) contain biologically active substances with efficient antioxidant capacities. This study evaluated the effects of dietary supplementation of heat-treated SW (5% Gracilaria vermiculophylla or 5% Ulva lactuca) on stress bioindicators in sea bream subjected to a hypoxic challenge. 168 fish (104.5 g average weight) were distributed in 24 tanks, in which eight tanks were fed one of three experimental diets for 34 days: (i) a control diet without SW supplementation, (ii) a control diet supplemented with Ulva, or (iii) a control diet with Gracilaria. Thereafter, fish from 12 tanks (n=4 tanks/dietary treatment) were subjected to 24 h hypoxia (1.3 mg O2 l-1) and subsequent recovery normoxia (8.6 mg O2 l-1). Hypoxic fish showed an increase in hematocrit values regardless of dietary treatment. Dietary modulation of the O2-carrying capacity was conspicuous during recovery, as fish fed SW supplemented diets displayed significantly higher haemoglobin concentration than fish fed the control diet. After the challenge, survival rates in both groups of fish fed SW were higher, which was consistent with a decrease in hepatic lipid peroxidation in these groups. Furthermore, the hepatic antioxidant enzyme activities were modulated differently by changes in environmental O2 condition, particularly in sea bream fed the Gracilaria diet. After being subjected to hypoxia, the gene expression of antioxidant enzymes and molecular chaperones in liver and heart were down regulated in sea bream fed SW diets. This study suggests that the antioxidant properties of heat-treated SW may have a protective role against oxidative stress. The nature of these compounds and possible mechanisms implied are currently being investigated. © 2017. Published by The Company of BiologistsThe authors are grateful to Pedro Eloi, Fernando Magalhães and Carlos Morais (ICBAS-UP) for fish maintenance. We would like to thank Kevin Tromp (WIAS-WU), Luis Pereira, Francisca Silva-Brito, and Filipa Fontinha for technical assistance during sampling and analysis (CIIMAR-UP). The authors would like to express gratitude to Rui Pereira (ALGA plus), Jorge Dias (Sparos Lda.), Jon Svendsen and Maria João Peixoto (CIIMAR-UP) for technical advic

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)

The potential of a combination of pungent spices as a novel supplement in gilthead seabream (Sparus aurata) diets to aid in the strategic use of fish oil in aquafeeds: a holistic perspective

This work studied the potential of a combination of pungent spices (capsicum, black pepper, ginger, and cinnamaldehyde) to be used as a supplement in diets of gilthead seabream (Sparus aurata; 44.1 ± 4.2 g). During 90 days, fish were fed three experimental diets with low inclusion of fish oil and containing poultry fat as the main source of lipids, supplemented with graded levels of the tested supplement: 0 (control), 0.1 (SPICY0.1%), and 0.15% (SPICY0.15%). As a result, the pungent spices enhanced the growth performance, the activity of the bile-salt-activated lipase in the intestine, and decreased fat deposit levels within enterocytes. The SPICY0.1% diet reduced the feed conversion ratio and the perivisceral fat index and lipid deposits in the liver. Moreover, the ratio of docosahexaenoic acid/eicosapentaenoic acid in fillet increased in fish fed the SPICY0.1% diet, while the hepatic levels of docosahexaenoic acid and total n-3 polyunsaturated fatty acids increased in fish fed the SPICY0.15% diet. Furthermore, there was an effect on the expression of some biomarkers related to lipid metabolism in 2-h postprandial fish (fasn, elovl6, scd1b, cyp7a1, lpl, and pparβ), and in 48 h fasted-fish fed with the SPICY0.1% diet, a regulation of the intestinal immune response was indicated. However, no significant differences were found in lipid apparent digestibility and proximate macronutrient composition. The spices did not affect biomarkers of hepatic or oxidative stress. No differences in microbial diversity were found, except for an increase in Simpson’s Index in the posterior intestine of fish fed the SPICY0.1% diet, reflected in the increased relative abundance of the phylum Chloroflexi and lower relative abundances of the genera Campylobacter, Corynebacterium, and Peptoniphilus. In conclusion, the supplementation of gilthead seabream diets with pungent spices at an inclusion of 0.1% was beneficial to enhance growth performance and feed utilization; reduce fat accumulation in the visceral cavity, liver, and intestine; and improve the fish health status and condition. Results suggest that the tested supplement can be used as part of a nutritional strategy to promote a more judicious use of fish oil in fish diets due to its decreasing availability and rising costs

Cysteamine improves growth and the GH/IGF axis in gilthead sea bream (Sparus aurata): in vivo and in vitro approaches

Aquaculture is the fastest-growing food production sector and nowadays provides more food than extractive fishing. Studies focused on the understanding of how teleost growth is regulated are essential to improve fish production. Cysteamine (CSH) is a novel feed additive that can improve growth through the modulation of the GH/IGF axis; however, the underlying mechanisms and the interaction between tissues are not well understood. This study aimed to investigate the effects of CSH inclusion in diets at 1.65 g/kg of feed for 9 weeks and 1.65 g/kg or 3.3 g/kg for 9 weeks more, on growth performance and the GH/IGF-1 axis in plasma, liver, stomach, and white muscle in gilthead sea bream (Sparus aurata) fingerlings (1.8 ± 0.03 g) and juveniles (14.46 ± 0.68 g). Additionally, the effects of CSH stimulation in primary cultured muscle cells for 4 days on cell viability and GH/IGF axis relative gene expression were evaluated. Results showed that CSH-1.65 improved growth performance by 16% and 26.7% after 9 and 18 weeks, respectively, while CSH-3.3 improved 32.3% after 18 weeks compared to control diet (0 g/kg). However, no significant differences were found between both experimental doses. CSH reduced the plasma levels of GH after 18 weeks and increased the IGF-1 ones after 9 and 18 weeks. Gene expression analysis revealed a significant upregulation of the ghr-1, different igf-1 splice variants, igf-2 and the downregulation of the igf-1ra and b, depending on the tissue and dose. Myocytes stimulated with 200 µM of CSH showed higher cell viability and mRNA levels of ghr1, igf-1b, igf-2 and igf-1rb compared to control (0 µM) in a similar way to white muscle. Overall, CSH improves growth and modulates the GH/IGF-1 axis in vivo and in vitro toward an anabolic status through different synergic ways, revealing CSH as a feasible candidate to be included in fish feed

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

Selection for growth is associated in gilthead sea bream (Sparus aurata) with diet flexibility, changes in growth patterns and higher intestine plasticity

Farmed gilthead sea bream (Sparus aurata) is able to grow efficiently with new feed formulations based on plant ingredients. Here, two experimental diets with standard and high inclusion levels of plant ingredients were formulated to assess the suited use of plant-based diets in fish with different growth genetic backgrounds. To pursue this issue, a long-term feeding trial (12-months) was conducted with fish (17 g initial body weight) of 16 families coming from the broodstock of PROGENSA project, that were grown communally in the IATS-CSIC experimental facilities. All fish in the study (2545) were PIT-tagged, and their pedigree was re-constructed with 96% success by using a SMsa1 multiplex of 11 microsatellites, which revealed the main parents contributions of 5 females and 6 males. Each diet was randomly assigned to replicate 3000 L tanks, gathering each replicate a similar family composition through all the feeding trial. Data on growth performance highlighted a strong ge- netic effect on growth trajectories, associated with enhanced growth during winter in fish selected for faster growth. No main dietary effects were found on growth rates or condition factor, and regression-correlation analyses of growth rates across families on both diets suggest that genome by diet interaction was weak, while genetic variation accounted for most of the growth phenotypic variation. Hepatosomatic index (HSI) and me- senteric fat index (MSI) of five families, covering the growth variability of the population, were regulated nu- tritionally and genetically, but without statistically significant genome by diet interactions. Fish from faster growing families showed shorter intestines after being fed the control diet, but this phenotype was masked by the enriched plant-based diet. Collectively, the results demonstrate that selection for faster growth is associated in gilthead sea bream with different growth trajectories and a high diet flexibility and intestine plasticity.Versión del editor2,04

Ghrelin and its receptors in Gglthead Sea bream: nutritional regulation

Ghrelin is involved in the regulation of growth in vertebrates through controlling different functions, such as feed intake, metabolism, intestinal activity or growth hormone (Gh) secretion. The aim of this work was to identify the sequences of preproghrelin and Ghrelin receptors (ghsrs), and to study their responses to different nutritional conditions in gilthead sea bream (Sparus aurata) juveniles. The structure and phylogeny of S. aurata preproghrelin was analyzed, and a tissue screening was performed. The effects of 21 days of fasting and 2, 5, 24 h, and 7 days of refeeding on plasma levels of Ghrelin, Gh and Igf-1, and the gene expression of preproghrelin, ghsrs and members of the Gh/Igf-1 system were determined in key tissues. preproghrelin and the receptors are well conserved, being expressed mainly in stomach, and in the pituitary and brain, respectively. Twenty-one days of fasting resulted in a decrease in growth while Ghrelin plasma levels were elevated to decrease at 5 h post-prandial when pituitary ghsrs expression was minimum. Gh in plasma increased during fasting and slowly felt upon refeeding, while plasma Igf-1 showed an inverse profile. Pituitary gh expression augmented during fasting reaching maximum levels at 1 day post-feeding while liver igf-1 expression and that of its splice variants decreased to lowest levels. Liver Gh receptors expression was down-regulated during fasting and recovered after refeeding. This study demonstrates the important role of Ghrelin during fasting, its acute down-regulation in the post-prandial stage and its interaction with pituitary Ghsrs and Gh/Igf-1 axis