La composition en acides aminés, dans le régime alimentaire des larves de daurade royale, affecte-t-elle leur performance ?

7 páginas, 2 figuras, 4 tablas.[EN] Live preys commonly used in fish larval rearing seem to be imbalanced in terms of amino acids. Manipulation of their amino acid composition is difficult, but the use of microencapsulated diets allows this manipulation. This study analysed the effect of amino acid supplementation, in order to compensate for dietary amino acid imbalances, on growth and survival of gilthead seabream (Sparus aurata) larvae. Larvae were reared until 32 days after hatching, in a closed recirculating water system (19 °C), using only live food (rotifers and Artemia). Thereafter, larvae were fed Artemia or one of the experimental microencapsulated diets: supplemented with indispensable amino acids (IAAsup diet), or supplemented with dispensable amino acids (DAAsup diet). Experiment lasted for 10 days. Dietary supplementation with indispensable amino acids resulted in A/E ratios [(each indispensable amino acid) × (total indispensable amino acids)−1 × 1000] more similar to the ones of larval seabream and in higher IAA:DAA ratios than in the DAAsup diet. Survival was similar in larvae fed the IAAsup diet (75%) or Artemia (87%), but was significantly lower in larvae fed the DAAsup diet (52%). Larvae from all treatments more than doubled their average dry weight during the experimental period. Final dry weight was similar in larvae fed both microcapsules, but these were lighter than larvae fed with Artemia. Relative growth rate (RGR) and total biomass production tended to be higher in larvae fed the IAAsup (RGR = 9% day−1) than the DAAsup diet (RGR = 7.5% day−1) and only in this last treatment these parameters were significantly lower than in larvae fed with Artemia. Therefore, dietary supplementation with indispensable amino acids resulted in a more balanced dietary amino acid profile, which significantly increased survival. Further studies introducing microdiets earlier in the development seem necessary in order to optimise growth.[FR] Les proies vivantes utilisées dans les élevages larvaires semblent déséquilibrées en terme d'acides aminés. Manipuler la composition en acides aminés est difficile mais l'usage d'aliments encapsulés le permet. Cette étude analyse l'effet d'une supplémentation en acides aminés sur la croissance et la survie des larves de daurade royale (Sparus aurata) afin de compenser un déséquilibre. Les larves sont élevées jusqu'à 32 jours après éclosion en circuit fermé (eau à 19 °C), en utilisant des aliments vivants uniquement (rotifères et Artemia). Les larves sont ensuite nourries avec des Artemia ou bien avec des aliments microencapsulés expérimentaux, supplémentés en acides aminés soit indispensables (régime IAAsup) soit non-indispensables (régime DAAsup). La durée de l'expérience est de 10 jours. La supplémentation en acides aminés indispensables conduit à des ratios A/E [(chaque AA indispensable) × (total en AA indispensables)−1 × 1000] plus proches de ceux de la larve de dorade et des ratios IAA/DHA plus élevés que ne le fait la supplémentation en acides aminés non-indispensables. Le taux de survie est similaire chez les larves nourries avec le régime IAAsup (75 %) ou Artemia (87 %) mais inférieur significativement chez les larves soumises au régime DAAsup (52 %). Le poids sec moyen de ces larves, quel que soit leur régime alimentaire, a doublé durant la période expérimentale. Le poids sec est similaire chez les larves nourries de microcapsules mais moins élévé chez celles nourries d'Artemia. Le taux de croissance relative (RGR) et la production de biomasse totale tendent à être plus élevés chez les larves nourries en IAAsup (RGR = 9 % jour−1) que celles nourries en DAAsup (RGR = 7,5 % jour−1). C'est seulement dans ce dernier traitement que les paramètres sont plus faibles significativement que chez les larves nourries d'Artemia. Ainsi, les supplémentations alimentaires en acides aminés indispensables peuvent augmenter la survie. D'autres études, introduisant des micro-aliments de façon plus précoce dans le développement, semblent nécessaires afin d'optimiser la croissance.The authors acknowledge funding by project AGL2004-06669-C02-01 (MCYT, Spain) and grant SFRH/BPD/14835/2003 (FCT, Portugal).Peer reviewe

Sustainable fish feeds: potential of emerging protein sources in diets for juvenile turbot (Scophthalmus maximus) in RAS

In Europe, turbot aquaculture has a high potential for sustainable production, but the low tolerance to fishmeal replacement in the diet represents a big issue. Therefore, this study investigated the effects of more sustainable feed formulations on growth and feed performance, as well as nutritional status of juvenile turbot in recirculating aquaculture systems. In a 16-week feeding trial with 20 g juvenile turbot, one control diet containing traditional fishmeal, fish oil and soy products and two experimental diets where 20% of the fishmeal was replaced either with processed animal proteins (PAP) or with terrestrial plant proteins (PLANT) were tested. Irrespective of diets, growth performance was similar between groups, whereas the feed performance was significantly reduced in fish of the PAP group compared to the control. Comparing growth, feed utilisation and biochemical parameters, the results indicate that the fish fed on PAP diet had the lowest performance. Fish fed the PLANT diet had similar feed utilisation compared to the control, whereas parameters of the nutritional status, such as condition factor, hepato-somatic index and glycogen content showed reduced levels after 16 weeks. These effects in biochemical parameters are within the physiological range and therefore not the cause of negative performance. Since growth was unaffected, the lower feed performance of fish that were fed the PAP formulation might be balanced by the cost efficient formulation in comparison to the commercial and the PLANT formulations. Present study highlights the suitability of alternative food formulation for farmed fish

Dietary micronutrient composition affects fillet texture and muscle cell size in Atlantic salmon (Salmo salar)

During the past 20 years, plant ingredients have taken over as the main constituents in feed for Atlantic salmon. This has changed the nutrient composition of the feeds, the bioavailability of nutrients and perhaps nutrient metabolism. Plant‐based diets also contain more anti‐nutrients. The EU‐funded project ARRAINA re‐evaluated recommendations for micronutrient supplementation to Atlantic salmon feeds, and the present study compared a diet supplemented with micronutrients according to NRC (2011) (control diet, 100% NP (nutrient package)) with a diet supplemented according to the new recommendations (New NP). Tissue concentrations of pyridoxine, pantothenic acid, niacin, vitamin C, Zn and Se were significantly higher; and Cu was lower in Atlantic salmon fed the diet with the New NP compared to the control fish. The New NP also gave a near significant effect on growth, decreased muscle firmness and increased muscle cell size, and it affected metabolism of nitrogen‐containing metabolites in the muscle. While we cannot be certain which micronutrient(s) gave these effects, the B vitamins are probable candidates, since they are mediators of intermediary metabolism and have been shown to influence some of the affected metabolites

Health status in gilthead seabream (Sparus aurata) juveniles fed diets devoid of fishmeal and supplemented with Phaeodactylum tricornutum

To enhance fish general health, feeds can be supplemented with health-promoting additives, reducing the need to use chemotherapeutics. Incorporation of marine algae biomasses in aquafeeds has been shown to improve fish immune status by enhancing innate immune response. This study evaluated the effects of Phaeodactylum tricornutum incorporation in feed by two different processes, either as freeze-dried biomass or broken cell wall biomass, on fish health status and performance. Triplicate groups of gilthead seabream juveniles (13.3 ± 0.3 g) were either fed a control diet (CTRL) with an extreme (i.e., 0% fishmeal), nutritionally balanced, formulation, or two experimental diets formulated as the CTRL with 1% inclusion of the microalga P. tricornutum at the expense of wheat meal: BC diet contains P. tricornutum broken cells and WC diet microalgae whole cells. After 2 and 12 weeks of feeding, blood was collected for hematological procedures, whereas plasma and mucus were sampled for immune parameters. Head-kidney, liver, and white skeletal muscle were also collected for gene expression measurements. No major differences were observed in hematological nor plasma humoral parameters after 12 weeks irrespective of dietary treatment. Arrays of 29–31 genes were analyzed in the different tissues, revealing an early dietary effect (2 weeks) in a tissue-specific pattern. In the liver, the major effect was found in the GH/IGF axis and in muscle there was a late downregulation of myostatin (mstn) gene, mainly due to WC diet, even though all fish had similar growth performance. Regarding the head-kidney, BC diet led to alpha-2-macroglobulin (a2m) gene upregulation. Also, the same treatment showed increased mucus alternative complement pathway and bactericidal activity at 2 and 12 weeks, respectively. Hence, it seems that BC diet has a potential stimulatory effect that might be relevant as a prophylactic measure before a predictable stressful event.This work has been funded under the EU FP7 by the MIRACLES project No. 613588: Multi-product Integrated bioRefinery of Algae: from Carbon dioxide and Light Energy to high-value Specialties and by national funds through FCT - Foundation for Science and Technology within the scope of UIDB/04423/2020 and UIDP/04423/2020. B. Reis, L. Ramos-Pinto, B. Costas, and S. Engrola were supported by the Portuguese Foundation for Science and Technology (PD/BDE/129262/2017, PD/BDE/114436/2016, IF/00197/ 2015, and IF/00482/2014, respectively)

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)”

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)

Dietary Methionine Improves the European Seabass (Dicentrarchus labrax) Immune Status, Inflammatory Response, and Disease Resistance

Methionine presents a pivotal role in the regulation of many cellular events with crucial impact on the immune system, such as in processes involved in the control of inflammation and polyamines synthesis. Accordingly, the present study aimed to assess the modulatory effects of dietary methionine on the European seabass (Dicentrarchus labrax) immune status, inflammatory response and disease resistance to Photobacterium damselae subsp. piscicida (Phdp). For this purpose, fish were randomly distributed in three independent groups (three replicates per group) and each was fed the corresponding diet: a control diet (CTRL) formulated to meet the established amino acid requirements for the species; a diet supplemented with methionine at 0.5% of feed weight relative to the CTRL diet (8.2% of methionine concentration above CTRL); and one supplemented with methionine at 1% of feed weight to the CTRL diet (11.8% of methionine concentration above CTRL). To evaluate the immune status of fish fed with each of the diets before being submitted to bacterial infection fish were sampled from each group at 2 and 4 weeks after the beginning of feeding. Non-sampled fish were injected intraperitoneally with Phdp (5 × 103 cfu/fish) at 4 weeks after initiation of feeding and the inflammatory response (at 4, 24, and 48 h post-infection) and survival (lasting 21 days post-infection) evaluated. Fish hematological profile, peripheral cell dynamics, plasma humoral immune parameters, leucocyte migration to the inflammatory focus and head-kidney gene expression were evaluated. Results show that methionine dietary supplementation improves seabass cellular immune status without evidence of activation of pro-inflammatory mechanisms. Additionally, the observed enhanced immune status provided by methionine supplementation translated into an improved immune response to infection, as higher cellular differentiation/proliferation and recruitment to the inflammatory focus, improved plasma humoral immune parameters and modulation of key immune-related genes was observed. Lastly, after a bacterial challenge, higher survival was observed in fish fed supplemented diets, ultimately corroborating the positive effect of methionine administration for 4 weeks in the cellular immune status

The Pierre Auger Observatory III: Other Astrophysical Observations

Astrophysical observations of ultra-high-energy cosmic rays with the Pierre Auger ObservatoryComment: Contributions to the 32nd International Cosmic Ray Conference, Beijing, China, August 201