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Bacillus spp. supplementation as probiotics in cultured fish diets has a long history of safe and effective use. Specifically, B. velezensis show great promise in fine-tuning the European sea bass disease resistance against the pathogenicity caused by several members of the Vibrio family. However, the immunomodulatory mechanisms behind this response remain poorly understood. Here, to examine the inherent immune variations in sea bass, two equal groups were fed for 30 days with a steady diet, with one treatment supplemented with B. velezensis. The serum bactericidal capacity against live cells of Vibrio anguillarum strain 507 and the nitric oxide and lysozyme lytic activities were assayed. At the cellular level, the phagocytic response of peripheral blood leukocytes against inactivated Candida albicans was determined. Moreover, head-kidney (HK) total leukocytes were isolated from previously in vivo treated fish with LPS of V. anguillarum strain 507. Mechanistically, the expression of some essential proinflammatory genes (interleukin-1 (il1b), tumor necrosis factor-alpha (tnfa), and cyclooxygenase 2 (cox2) and the sea bass specific antimicrobial peptide (AMP) dicentracin (dic) expressions were assessed. Surprisingly, the probiotic supplementation significantly increased all humoral lytic and cellular activities assayed in the treated sea bass. In addition, time-dependent differences were observed between the control and probiotic treated groups for all the HK genes markers subjected to the sublethal LPS dose. Although the il1b was the fastest responding gene to a significant level at 48 h post-injection (hpi), all the other genes followed 72 h in the probiotic supplemented group. Finally, an in vivo bacteria challenge against live V. anguillarum was conducted. The probiotic fed fish observed a significantly higher survival. Overall, our results provide clear vertical evidence on the beneficial immune effects of B. velezensis and unveil some fundamental immune mechanisms behind its application as a probiotic agent in intensively cultured European sea bass.

Antimicrobial peptides (AMPs) play an important role in the innate immune response of vertebrates by creating a hostile environment for any invading pathogens. Piscidins are potent teleost specific AMPs, which have a broad spectrum activity. We have identified a novel piscidin active peptide, in the greater amberjack, Seriola dumerili, that consists of 25 aa, which forms an amphipathic helix with distinct hydrophobic and positively charged regions. Following homology and phylogenetic analysis the greater amberjack piscidin was deemed to belong to the group 3 family of piscidins. Piscidin was expressed constitutively at immune sites, with transcript level highest in the spleen and gut, at an intermediate level in the gills and lowest in the head kidney. Following in vivo stimulation with PAMPs (poly I:C, LPS and flagellin) piscidin transcript level increased in gills in response to flagellin, in gut and spleen in response to poly I:C, and in head kidney in response to poly I:C, LPS and flagellin. Head kidney and spleen cells were then isolated from greater amberjack and incubated with each of the PAMPs for 4, 12 and 24 h. Piscidin expression was unchanged at 4 and 12 h post PAMP stimulation in head kidney cells but at 24 h transcript level was markedly upregulated compared to control (unstimulated) cells, especially with the bacterial PAMPs. In contrast, spleen cells upregulated piscidin expression by 4 h post stimulation with poly I:C and flagellin, and remained upregulated to 24 h with flagellin exposure, but had returned to baseline levels by 12 h using poly I:C. To determine if piscidin expression could be modulated by diet, greater amberjack were fed diets supplemented with MOS and cMOS for 30 days when transcript level was determined. It was found that MOS supplemented diets increased expression in the spleen, cMOS supplemented diets upregulated transcript levels in the gills and head kidney, whilst a diet containing both MOS and cMOS upregulated transcript in the gut, when compared to fish fed the control diet. Finally, a synthetic greater amberjack piscidin was produced and showed bacteriostatic activity against a number of bacterial strains, including both Gram positive and Gram negative fish pathogens.

The present study aimed to determine the effects of dietary inclusion of selenium (Se) in the form of either inorganic Se (sodium selenite, NaSe) or organic Se (hydroxy-selenomethionine, OH-SeMet) on gilthead seabream (Sparus aurata). Triplicate groups of 150 fish each (6.2 ± 0.04 g initial body weight) were distributed in 15 tanks and fed one of five experimental diets for 63 days. A control diet (C) without Se supplementation (0.8 mg Se/kg diet), was formulated and served as a basal diet for the other 4 experimental feeds, supplemented with either 0.2 or 0.5 mg Se/kg supplied in the form of NaSe or OH-SeMet. Fish growth performance, biochemical composition of liver and muscle and Se content, malondialdehyde (MDA) concentration in muscle and hepatopancreas, hepatocyte morphology and lysozyme activity in the serum were studied. Furthermore, expression of related hepatic genes, such as manganese superoxide dismutase (Mn-sod), catalase (cat), heat shock protein 70 (hsp70), and glutathione peroxidase (gpx) was also analyzed. After the trial, fish were exposed to an acute and chronic confinement stress. Blood samples for cortisol analysis were taken at 0 and 2 h after the acute stress and after 7 days of chronic stress. The highest growth rate was observed in fish fed OH-SeMet at a level of 0.2 mg/kg, but with no significant difference with fish fed the control diet with no-added Se. The lowest growth was observed in fish fed NaSe up to 0.5 mg/kg. Increase in dietary Se, particularly in the form of OH-SeMet, led to an increase in Se contents in liver and muscle. Furthermore, dietary inclusion of OH-SeMet, led to a significant (p In summary, supplementation of Se up to 0.2 mg/kg (1–1.1 mg/kg analyzed dietary Se), particularly in the form of OH-SeMet, led to a beneficial effect on growth, maintenance of hepatic morphology and improved protection of juvenile gilthead seabream from acute or chronic stress. Besides, OH-SeMet was found to be more effective than NaSe in protection against oxidative stress in fish muscle.

Meagre (Argyrosomus regius) is a promising new aquaculture species, however with some bottlenecks to high-scale commercial production. One of those constraints are the scarce information about its nutritional requirements, which difficult the production of optimized diets and can lead to pathological alterations in liver, such as hepatic steatosis. Furthermore, meagre production is widely affected by granulomatosis, an infectious or non-infectious disease that may compromise fish performance and appearance. The cause of non-infectious granulomatosis is still unclear, but nutritional deficiencies are potential explanations. Since essential fatty acids are nutrients required to sustain vital functions, growth, but also immune status and disease resistance, the present work evaluated, in a 30-day feeding trial, the effect of 5 dietary n-3 long-chain polyunsaturated fatty acid levels (0.8, 1.4, 2.0, 2.6 and 3.5% in dry weight of the diet) on liver health status of meagre fingerlings. Fish fed the lowest n-3 LC-PUFA diets (0.8 and 1.4%) showed a higher hepatosomatic index, as well as a higher hepatic lipid infiltration, which resulted in severe hepatic steatosis. Oleic acid and docosahexaenoic acid were the most important fatty acids driving the variability of meagre hepatic fatty acid composition and, consequently, hepatic histological alterations. Although fatty acid composition of phospholipids was less affected by the dietary n-3 LC-PUFA, meagre fed 0.8% n-3 LC-PUFA tended to present lower levels of essential fatty acids and higher levels of oleic acid in polar lipids. Moreover, the diet with the lowest n-3 LC-PUFA content (0.8%) led to a higher incidence of hepatic granulomas, suggesting a possible relation between essential fatty acid deficiency and hepatic granulomatosis in meagre. These results suggest that the inclusion of vegetable oils in diets for meagre fingerlings should be taken with careful, guaranteeing that, at least, 2.0% DW of n-3 LC-PUFA is available in feeds, in order to maintain the normal hepatic function in meagre.

Genetic selection programmes in gilthead seabream mainly focus on traits related to growth, disease resistance, skeletal anomalies, or fillet quality. However, the effect of selection for growth on the reproductive performance of seabream broodstock has not received much attention. The present study aimed to determine the effect of selection for growth traits, high (HG) or low (LG) growth, and broodstock feeding with fish oil (FO diet) or rapeseed oil (RO diet) as main lipid sources, on reproductive performance of gilthead seabream. For the first part of the spawning season (Phase I) HG and LG broodstock were fed a commercial diet and the HG broodstock produced a higher number of larvae and higher viable eggs, hatching and larval survival rates than LG broodstock, affecting egg fatty acid profiles. For the second part of the study (Phase II) broodstock were fed one of the two diets containing FO or RO. Fecundity in terms of viable eggs, hatchlings, and larvae produced, as well as fertilization rates, were improved in HG broodstock. Some fatty acids such as 18:0, 20:2n-6, 20:3n-3 or EPA/ARA were also affected by the growth selection. According to the two-way ANOVA analysis, feeding the RO diet did not significantly affect fecundity parameters, but slightly reduced fertilization and hatching rates in HG broodstock. Nevertheless, HG broodstock showed better spawning quality parameters than LG broodstock, even when they were fed the RO diet. Egg fatty acid profiles reflected diet composition, although DHA contents were not affected. In conclusion, broodstock selected for high growth had a positive effect on broodstock performance, and FO replacement by RO did not markedly affect reproduction providing that fatty acid contents were sufficient to fulfill the essential fatty acid requirements of gilthead seabream broodstock.

Systemic granulomatosis is a growing disease with a high morbidity, which affects to the majority of farmed meagre (Argyrosomus regius). The impossibility of isolating any infectious agents has hypothesized a nutritional origin of the disease. In order to try to elucidate the nutritional origin of granulomas, juvenile meagre were fed for 15 weeks with six diets containing different levels of vitamin E and C and with or without addition of vitamin K: Diet 0 (basal premix, no K, 150 mg kg‐−1 E, 20 mg kg‐−1 C), K (added 23 mg kg‐−1 K), EC (300 mg kg‐−1 E, 70 mg kg‐−1 C), KEC (23 mg kg‐−1 K, 300 mg kg‐−1 E, 70 mg kg‐−1 C), EECC (450 mg kg‐−1 E, 230 mg kg‐−1 C) and KEECC (23 mg kg‐−1 K, 450 mg kg‐−1 E, 230 mg kg‐−1 C). The diet EC significantly increased meagre growth in terms of final weight and length. Fish fed the highest levels of vitamin E and C presented lower percentage of granulomas in liver and heart than fish fed diet 0. The scored severity of granulomatosis in liver and kidney (main affected organs) tended to be lower with dietary increase of vitamin E, C and addition of vitamin K (from 1.83 diet 0 to 1.3 diet KEECC and from 0.91 diet 0 to 0.39 diet KEECC). In liver, the diet KEECC significantly increased catalase expression compared with diet 0. In kidney tnfα expression was significantly up-regulated in fish fed diet EECC and KEECC. In heart, low vitamin E and C levels (300 and 70 mg kg‐−1, respectively) significantly increased superoxide dismutase and glutathione peroxidase expression and high addition increased the expression of tnfα and cox-2 (0 or 23 mg kg‐−1 K, 450 mg kg‐−1 E and 230 mg kg‐−1 C, diet EECC and KEECC). The results show that combination of high dietary content of vitamin K and antioxidant vitamins E and C (23, 450 and 230 mg kg‐−1, respectively) influenced in the incidence of the granulomatosis, which suggests that this pathology could be mediated by nutritional factors.

The effects of two new anesthetics (essential oils) extracted from oregano (OO) and eucalyptus (EO) plants were studied in European sea bass (Dicentrarchus labrax) stress response and welfare parameters in comparison to the effects of clove oil (CO) that is one of the most used anesthetics in aquaculture. A time-course study on evolution of plasma cortisol after anesthesia was conducted. Relative expression of stress-related genes, including glucocorticoid receptor (gr), heat shock protein 70 and 90 (hsp70, hsp90) steroidogenic acute regulatory protein (star), cytochrome 11B (cyp11b1) and hypoxia inducible factor (hif) was determined in liver, gills and head kidney after anesthesia. After anesthetic use, there was a time effect on plasma cortisol concentration, with highest values registered after 2 h for all the experimental groups. EO induced a secondary increase of plasma cortisol 24 h after the use of this anesthetic. Hsps expression level in gills was high at 0 h in EO fish group, whereas hif expression increased after 2 h of exposure in CO fish. In hepatic tissue, gr expression increased after 24 h in the EO group. In head kidney tissue, expression of steroidogenesis-related genes star and cyp11b1 increased in EO fish group, whereas in the OO group, the expression of the same genes decreased at 2 h and 24 h of exposure. Although the three anesthetics generally showed similar patterns of variation of all analyzed parameters, results indicate that EO could be deleterious for welfare, whereas OO affects fish welfare to a lesser extent than CO and EO. Overall, these results show that OO could be a good anesthetic for fish based on greater effectiveness, lower optimum concentration and less impact on fish stress.

A sustainable growth of the aquaculture sector implies the use of sustainable novel raw materials as replacers of the traditional fishmeal (FM) and fish oil (FO) ingredients. This fact has led to the development of sustainable and functional diets as part of a management strategy to reduce the effects on fish growth performance and health derived from low FM/FO dietary contents. In this sense, Antarctic krill (Euphausia superba) is considered a potential candidate in dietary inclusions to potentiate fish growth and health status. In this study, European sea bass (Dicentrarchus labrax) were fed a practical diet with either a 15% fishmeal content (KM0; control diet) or the same diet substituted by 30% (KM5; 50 g KM/kg diet) or 50% (KM7.5; 75 g KM/kg diet) Antarctic krill meal (KM) for 12 weeks in triplicates. At the end of the feeding trial, growth performance, liver morphology, liver proximate composition, lipid classes and fatty acid profiles, as well as the expression of hepatic genes related with lipid metabolism were evaluated. Fish fed KM-based diets presented higher (p 0.05) biochemical composition and fatty acid profile. However, smaller hepatocellular area and lower grade of cytoplasm vacuolization as well as a better alignment around sinusoidal spaces were found. The analyses of liver lipid classes revealed a positive correlation between the level of dietary KM and the pigmented material such as astaxanthin and free fatty acid content, as well as a negative correlation with the cholesterol levels. The expression of hepatic genes studied demonstrated a downregulation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (hmgr) and delta-6-desaturase (fads2) expression levels in fish fed KM-based diets. Besides, gene expression levels of fatty acid binding protein 7 (fabp7) and lipoprotein lipase (lpl) were significantly correlated with KM dietary levels. Altogether, these results profile KM as a potential promoter of growth and liver health in European sea bass fed low fish meal and oil diets.

In this study, the anesthetic effect of two herbal oils extracted from oregano ( Origanum sp.) and eucalyptus ( Eucalyptus sp.) plants was evaluated. Two marine aquaculture species were used: European sea bass ( Dicentrarchus labrax ) of 90.91 ± 2.46 g and meagre ( Argyrosomus regius ) of 127.43 ± 3.07 g. The oil chemical composition was analyzed. The main components were carvacrol for oregano oil and 1,8-cineol for eucalyptus oil. Three doses were analyzed for each anesthetic on both species to determine the optimal dose. Clove oil, an anesthetic commercially used in the industry, was used as control one. Results pointed out that neither anesthetics caused side effects or mortality on any of the two species and that effectiveness of oregano oil was higher than that of eucalyptus oil and similar or even higher than that of clove oil depending on the species. Their optimal doses were 50 and 75 μL·L − 1 of oregano oil for meagre and sea bass, respectively, and 300 μL·L − 1 of eucalyptus oil for both species. The times for oregano oil were 49 s and 40 s for sedation stage and 3.51 min and 6.05 min for recovery stage on European sea bass and meagre, respectively. The times for Eucalyptus oil were longer. Overall, these results introduce oregano oil as a good alternative to clove oil and opens up the possibility of other plant based oils to be tested and their effectiveness as anesthetics to be proved. Statement of relavence Phenoxyethanol is the major synthetic anesthetic which has been used in Aquaculture industry. These new herbal anesthetics will be new environmental friendly, herbal anesthetics for aquaculture species. Both of them are competitive with clove oil which is commonly used as a herbal anesthetics in this industry. Therefore we think this is an important innovation for Aquaculture industry and worthy to publish these results in this journal.

Several causative factors have been proposed for the occurrence of skeletal anomalies in fish larvae, among which we quote nutritional factors, such as LC-PUFAs. This study aimed to investigate the effect of different dietary DHA and ARA level and ratio on pikeperch (Sander lucioperca) larval development and performance, digestive capacity, fatty acids composition, skeleton anomalies and molecular markers of oxidative stress status (sod, gpx, and cat), stress response (StAR, gr, pparα, hsl and pepck), fatty acid synthesis (fadsd6, elovl5), eicosanoids synthesis (pla2, cox2, lox5, pge2, and lta4h), and bone development (twist, mef2c, sox9, and alp). Pikeperch larvae were fed six microdiets containing two different dietary levels of DHA (0.5% and 3.5%) combined with three levels of ARA (1.2%, 0.6%, and 0.3%). Dietary fatty acid changes did not affect growth performance but significantly influenced enzymatic activities. A significant increase in skeletal anomalies with DHA intake increment was recorded. StAR, cox2, pla2 and hsl expression were significantly depressed in 2.5% DHA larvae. An opposite effect of dietary DHA elevation was recorded in gpx expression. Both DHA and ARA had a significant effect on pparα, gr, and pge2 expressions. Although no significant interactions were found, pge2, gr, and pparα displayed a differential pattern of expression between the different treatments. A strong association was found for the larval tissue amount of ARA and DHA with eicosanoid metabolism, stress response and skeleton anomaly related genes. These results denoted the effects of dietary LC-PUFAs on immune/stress gene regulation and their potential implication in skeleton development.

The aim of this study was to evaluate the combination of maximum replacement of fishmeal (FM) and fish oil (FO) by alternative meals (AM) and oils (AO) to develop nutritious, but more eco-friendly diets for European sea bass ( Dicentrarchus labrax ) juveniles. European sea bass of 9.8 ± 1.5 g and 9.1 ± 0.5 cm were fed nine isonitrogenous (45% crude protein) and isolipidic (21%) diets containing gradually reduced levels of FM and FO as follows (%FM/%FO): 58/15, 20/6, 20/3, 10/6, 10/3, 5/6, 5/3 and 0/0. Another diet, 0/0 + , was similar to the 0/0 but supplemented with LC-PUFA from microbial and marine sources. After 90 days of feeding, European sea bass fed the 0/0 diet showed reduced (P + diet). Reduction of FM/FO down to 5/3 did not significantly affect fish survival or condition factor, whereas reduction to 5/6 did neither affect sea bass length or feed conversion and reduction down to 10/3 did not affected body weight, DGI and perivisceral fat or visceral weight. FM reductions down to 5% significantly reduced growth rate. FO substitution by vegetable oils (VO) led to reduced growth and increased liver to body weight ratios, liver lipid deposition and hepatocellular size. In liver and muscle, FO substitution increased n-6 fatty acids (FA) and n-6/n-3 ratio and reduced saturated FA and n-3 LC-PUFA, whereas monounsaturated FA were not increased in proportion to dietary contents. Moreover, FO reduction increased the products from Δ6 desaturase and elongases, Elovl6 and Elovl5. In conclusion, the results of this study show that it is possible to reduce dietary FM and FO levels down to 10% and 3% in mostly plant-based diets without affecting growth performance of European sea bass juveniles.

The main objective of this study was to determine the effect of two feeding additives (cMOS: Actigen®, Alltech Inc., USA) and a phytogenic specifically designed for reducing ectoparasite incidence, combining garlic and labiatae herbal extracts (HERB; Delacon, Austria) on growth performance, stress and immune response of greater amberjack (Seriola dumerili) before and after an experimental parasitization with the skin ectoparasite Neobenedenia girellae, a monogenean with high incidence on Seriola cultured in sea cages. Two parallel trials were conducted in order to determine the effect of 70 days of functional diets supplementation (0.2% cMOS and 200 ppm HERB) on greater amberjack: a) Growth performance and stress resistance and b) disease resistance against N. girellae. Additionally, in order to evaluate the effects of the dietary supplements pre- and post- parasitization challenge against N. girellae, the expression of some immune-related genes was evaluated. The relative expression of Il-1β, piscidin, hepcidin, IgT, tnf-α, muc-2, cathelicidin, complement c3, cd8, and caspase-3 was determined in skin. The activity of protease and lysozyme in serum and skin mucus were also evaluated, as well as parasite load and parasite total size. The use of both functional additives did not affect fish growth performance. However, the dietary supplementation of HERB significantly (p In general, greater amberjack supplementation with cMOS and HERB at the dose used in the present study was effective for reducing stress and for increasing the skin associated lymphoid tissue (SALT) immune response against N. girellae during the infection process.

The main objective of this study was to assess the effects of graded levels of replacement of fishmeal (FM) and fish oil (FO) by terrestrial meals (TM) and vegetable oils (VO) on gut health of European sea bass (Dicentrarchus labrax) based on gut morphology, gut-associated immune system (GALT) and gut microbiota composition. Reducing the dietary FM and FO levels down to 10 and 3%, respectively did not affect the growth of European sea bass. Increase in TM and VO markedly affected anterior and posterior gut morphology and fatty acid composition, which mirrored the dietary fatty acid profile. FO replacement by VO increased lipid deposition in anterior gut lamina propria, denoting an effect in lipid metabolism and lipoprotein synthesis. In posterior gut, FM replacements by TM induced an engrossment of the submucosa, an up-regulation of the proinflammatory cytokines IL-1β and TNFα genes, a relatively high mucus production and changes in mucosal microbiota profiles. Finally, supplementation of 0%FM-0%FO diets with LC-PUFA contributed to maintain gut homeostasis in terms of GALT response and microbiota profiles alterations. Statement of relevance FM and FO appropriate dietary levels for European seabass

The generation of a variety of new therapeutic agents to control and reduce the effects of pathogen in aquaculture is urgently needed. The antimicrobial peptides (AMPs) are one of the major components of the innate defenses and typically have broad-spectrum antimicrobial activity. However, absorption and distributions of exogenous AMPs for therapeutics application on farmed fish species need to be studied. Previous studies in our laboratory have shown the properties of hepcidin as an effective antimicrobial peptide produced in fish in response to LPS and iron. Therefore, we decided to investigate the antimicrobial activity of four synthetic variants of hepcidin against Vibrio anguillarum in vitro, and using the more effective peptide we demonstrated the pathogen's ability to protect against the infection in European Sea bass. Additionally the uptake of this peptide after ip injection was demonstrated, reaching its distribution organs such as intestine, head kidney, spleen and liver. The synthetic peptide did not show cytotoxic effects and significantly reduced the accumulated mortalities percentage (23.5%) compared to the European Sea bass control (72.5%) at day 21. In conclusion, synthetic hepcidin shows antimicrobial activity against V. anguillarum and the in vivo experiments suggest that synthetic hepcidin was distributed trough the different organs in the fish. Thus, synthetic hepcidin antimicrobial peptide could have high potential for therapeutic application in farmed fish species.

Aquaculture, the fastest growing food production sector cannot continue to rely on finite stocks of marine fish as the primary source of the omega-3 (n-3) long-chain polyunsaturated fatty acids (LC-PUFA), eicosapentaenoic acid (EPA; 20:5n3) and docosahexaenoic acid (DHA; 22:6n-3), for feeds. A four-month feeding trial was conducted to investigate the impact of a de novo oil, with high levels of EPA and DHA, obtained from transgenic Camelina sativa on growth performance, tissue fatty acid profiles, and expression of lipid metabolism genes when used as a replacement for fish oil in feed for European seabass (Dicentrachus labrax). Triplicate groups of 50 juvenile fish (initial weight 16.7 ± 0.92 g) per tank were fed for 4 months with one of three isolipidic and isoproteic experimental diets consisting of a standard diet containing a commercial blend of fish oil and rapeseed oil (CFO), a diet containing transgenic Camelina oil (TCO), or a blend of fish oil and rapeseed oil with enhanced levels of EPA and DHA (EFO) formulated to match the n-3 LC-PUFA profile of the TCO feed. Final weight of fish fed the GM-derived oil was not different to fish fed either CFO or EFO. Slight lower growth performance of fish fed TCO at the beginning of the trial was related to transient reduced feed intake, possibly caused by glucosinolates in the raw Camelina sativa oil. The GM-derived oil improved the nutritional quality of the fish fillet by enhancing total n-3 PUFA levels compared to the fish fed the other two feeds, and maintained flesh EPA and DHA at the same levels as in fish fed the diets containing fish oil. The metabolic response in liver and intestine was generally relatively mild although diets TCO and EFO seemed to trigger a metabolic response consisting of an up-regulation of both β-oxidation (cpt1a) and fatty acid transport (fabp1), possibly reflecting higher levels of LC-PUFA. Overall, the present study indicated that an oil of terrestrial origin, Camelina sativa, when engineered to contain high levels of EPA and DHA can replace fish oil in feeds for European seabass with no detrimental impact on growth or feed efficiency, while also maintaining or increasing tissue n-3 LC-PUFA contents.

The main objective of this study was to evaluate the stress response of the greater amberjack, Seriola dumerili, by determining plasma and skin mucus cortisol levels, as physiological indicator of fish stress, and skin mucin-2 gene expression as indicator of skin mucin production, under three different potential stressors related to aquaculture practices: temperature, handling and fasting. For trial 1, 45 greater amberjack juveniles (19.5 ± 4.1 g) were divided in three graded rearing temperatures (17, 22 and 26 °C) and maintained for 120 days, in order to define the basal cortisol level for this species after a long-term thermal acclimation within the range described for this species. Trial 2 was conducted under two different rearing densities and two different handling protocols with 222 fish (425.02 ± 36.9 g) divided in 4 treatments by triplicate for 90 days, in order to study the effect of routine handling protocols on the selected stress markers for this species. Trial 3 used 72 fish (302.27 ± 21.4 g) divided in two experimental groups, one fed to apparent satiety and another one subjected to fasting for 10 days (triplicates for each group). After trials 2 and 3, a shallow water challenge was conducted to further study the adaptive response of greater amberjack juveniles to combined stress situations. Plasma and skin mucus were obtained for cortisol determination at 1, 24 and 72 h (trial 2) and 1, 3 and 24 h (trial 3) post stress challenge. Additionally, skin was dissected out for determining muc-2 gene expression. Long-term acclimation to temperature did not affect plasma or mucus cortisol levels and muc-2 gene expression, being basal levels of circulating plasma cortisol around 7.5 ng/mL. A high stocking density (8 kg m−3) induced an elevation (p

The complete removal of fishmeal (FM) and fish oil (FO) is required to promote the sustainable development of aquaculture and for that, fast growing high quality fish that are fed without FM and FO are necessary. Early nutritional programming may allow the production of fish better adapted to utilize diets with vegetable meals (VM) and oils (VO). The main objective of this study was to research in the potential value of fatty acids as modulators of early nutritional programming in marine fish for a better utilization of VO/VM. For that purpose gilthead sea bream (Sparus aurata) broodstock were fed four different replacement levels of FO by linseed oil (LO) and their effect on fecundity and spawn quality, egg composition, Δ-6-desaturase (Δ6D) gene expression, progeny growth performance and their growth response to a challenge with diets low in FO and FM, but high in VO and VM. The results showed that feeding gilthead sea bream broodstock with high LO diets had very long-term effects on the progeny. Thus, FO replacement by LO up to 80–100% in broodstock diets for gilthead sea bream not only reduced fecundity and spawn quality, but also growth of 45 dah and 4-month-old juveniles, as well as Δ6D gene expression. However, when the 4 month-old juveniles were fed with a low FM and FO diet, even those from broodstock fed only 60% replacement of FO by LO showed a higher growth and feed utilization than juveniles from parents fed FO. These results demonstrate the interesting potential of early nutritional programming of marine fish by broodstock feeding to improve long-term performance of the progeny. Further studies are being conducted to determine optimum nutrient levels in the broodstock diets and the molecular mechanisms implied to develop effective nutritional intervention strategies for this species. Statement of relevance This study demonstrates for the first time in fish the potential of broodstock nutrition to conduct early nutritional programming of culture fish for a better utilization of low fish meal and fish oil diets by the progeny, showing its effect not only during reproduction and larval development but also during on-growing.

The decreased availability of fish oil, traditionally used as oil source in marine aquafeeds, has lead to the search for alternatives oils. Vegetable oils (VO) are being extensively used as lipid sources in marine fish diets, inducing an imbalance on certain dietary fatty acids. Alteration on the dietary ratio of w-6/w-3 has been described to have detrimental effects on fish immunity. Senegalese sole has high susceptibility to stress and diseases, and little is known on the effects of dietary VO on its immunity. In this study, Senegalese sole juveniles were fed diets (56% crude protein, 12% crude lipid) containing linseed (100LO), soybean (100SO) or fish (100FO) oils as unique oil source. Growth, cortisol and intestinal fatty acid composition were determined after 90 days. Moreover, at the final of the experiment a stress test (5 min of net chasing) was carried out. To evaluate the effect of diets and stress on intestine immunology, expression profiles of a set of 53 immune-related genes using RT-qPCR was also performed. The use of VO did not induced changes in fish growth, but affected fatty acid profile of intestine and expression of immune-related genes. The use of SO (rich in n-6 fatty acids) induced an over-expression of those genes related to complement pathway, recognizing pathogen associated to molecular patterns, defensive response against bacteria, defensive response against viruses, antigen differentiation, cytokines and their receptors. This general over-expression could indicate an activation of inflammatory processes in fish gut. When a stress was applied, a decrease of mRNA levels of different immune-related genes with respect to the unstressed control could be observed in fish fed 100FO. However, fish fed 100LO, with a higher ALA/LA ratio, seemed to ameliorate the effects of combined effects of FO substitution plus stressful situation whereas fish fed 100SO did not show this type of response.

The inclusion of complementary antioxidative factors, such as selenium (Se), could counteract the high oxidation risk in early weaning diets high in polyunsaturated fatty acids (PUFA). The present study investigated the effects of graded levels of Se derived yeast with krill phospholipids (KPL) on skeletal development, survival, stress resistance, oxidative status and biochemical composition of seabream larvae. Seabream larvae were completely weaned at 16 dph and fed five microdiets for 30 days with different levels of Se: 2SE, 4SE, 6SE, 8SE and 12SE (1.73, 3.91, 6.41, 8.47, 11.65 mg kg− 1 dietary dry weight, respectively). Increases in Se up to 11.65 mg kg− 1 dietary dry weight significantly improved survival rate (54%) and stress resistance, but did not affect larval growth. Seabream larvae fed diets supplemented with 12SE (11.65 mg kg− 1) showed a gradual increase in this mineral according to dietary Se levels, denoting the progressive absorption of this nutrient. The degree of larval lipid oxidation, as indicated by malondialdehyde (MDA) content and antioxidant enzyme (AOE) gene expression, was significantly lower in larvae fed 8SE and 12SE diets compared to those fed 2SE and 4SE diets. Furthermore, a reactive response as a result of Se inclusion was observed by the increase in osteocalcin, osteonectin, osteopontin, alkaline phosphatase and matrix gla protein gene expression in larval tissues, suggesting a well skeletal development. These results denoted the high efficiency of Se as an antioxidant factor and the importance of the inclusion of adequate levels (11.65 mg Se kg− 1 diet) in early weaning diets.

Escapes of farmed fish into the ecosystem have recently received a marked interest from scientists for their competition with wild populations or the risk of genetic contamination. Fatty acid profiles have been proposed as bio-indicators due to the inclusion of terrestrial oils in aquafeed ingredients. This study evaluated 1) the effect of wasted food on the fatty acid content of a farm-associated fish and 2) the suitability of fatty acid profiles as a bioindicator of aquaculture-ecosystem interactions, using the bogue ( Boops boops ) as a model. This species is an opportunistic fish usually associated with–or even found in–sea farms, and with a high natural occurrence along the Mediterranean and Atlantic coasts. The results showed that fish farms had a direct effect on the condition index, muscle and whole lipid content of bogue inside or around the sea cage, and this effect disappeared completely at 3 km from the cages. Despite the fact that aquafeeds also affected the bogue fatty acid profiles by increasing linoleic and oleic acids and reducing DHA, these profiles were very similar to those of bogue sampled close to a sewage outfall, denoting that these fatty acids are not exclusively influenced by aquaculture and therefore should not be considered as good biomarkers for escapees. However, bogues influenced by aquaculture were higher in linolenic acid than those found further than 3 km from the farm or close to the sewage, suggesting this fatty acid as a better indicator of aquaculture influence.

The physiological response to stressors in fish, including hormonal profiles and associated tissue responsiveness, is less documented. The aim of this study was to evaluate feeding gilthead sea bream ( Sparus aurata ) with diets containing linseed oil (LO) and soybean oil (SO) as substitutes to fish oil (FO) and their effect on fatty acid profile of head kidney and the consequent effect on stress response to a crowding challenge. Fish were fed 8 experimental diets with different levels of substitution 0% (FO), 70% (70LO, 70SO, 20LO50SO and 50LO20SO) and 100% (100LO, 100SO and 50LO50SO) over a period of 8 months. At the end of the feeding trial, samples of head kidney were collected for biochemical analysis and the fish were challenged by a crowding test. During the challenge, samples of plasma for cortisol analysis were collected at 0 h, 2 h, 5 h, 24 h, 48 h and 1 week in order to study acute and chronic stress responses. Results showed that fish fed vegetable oils (VO) had significantly decreased ARA, EPA, DHA and n-3 HUFA, while LA, LNA and total C18 PUFAs were significantly increased. The basal cortisol levels were significantly increased in fish fed 70LO, 100LO, 50LO20SO and 50LO50SO. The physiological response to crowding was significantly affected by the diet. After 2 h of crowding, all the treatments showed higher cortisol, with fish fed 100LO had significantly the highest response registering 131.38 pg/ml. After 5 h and 24 h, plasma cortisol was reduced in all treatments except in 50LO20SO. After 48 h of crowding, the plasma cortisol was increased in all treatments with the maximum value seen in fish fed 100LO (72.12 pg/ml). These levels were decreased in fish fed FO, 70LO, 100LO and 50L050SO after 1 week of crowding, but remained higher in fish fed 70SO, 100SO, 20LO50SO and 50LO20SO. In conclusion, fish fed LO diets showed the same response pattern as the control but with higher intensity regaining the basal levels after 1 week as the control, while fish fed SO had a slow response but changed the pattern characterized by a lower response at the beginning and longer recuperation without regaining the control value even after 1 week.

The objective of this study was to determine the effect of mannan oligosaccharides derived from the outer cell wall of a select strain of Saccharomyces cerevisiae (Bio-Mos, Alltech Inc, USA) on mucus production, selected mucus immune parameters activity, gut morphology and in vivo and ex vivo gut bacterial translocation for European sea bass (Dicentrarchus labrax). Specimens were fed 4 g kg⁻¹ dietary MOS level of inclusion in a commercial sea bass diet for eight weeks. At the end of this period, anterior gut mucosal folds height, width and folds surface area were increased by MOS supplementation (P < 0.05, n = 240). Posterior gut presented shorter folds (P < 0.05, n = 240) but wider that those fed control diet (P < 0.05, n = 240) resulting in increased total surface area (P < 0.05, n = 240). For rectum, feeding MOS reduced fold length (P < 0.05, n = 240). Gut morphological analyses showed an enhancement in the number of cells secreting acid mucins by area unit, higher density of eosinophilic granulocytes (ECGs) in the mucosa for fish fed MOS together with an improvement in gut mucus lysozyme activity which could be related to the reduced in vivo and ex vivo gut bacterial translocation found. No differences were found for the skin mucus immune parameters evaluated.

Increased energy content in fish feeds has led to an enhanced fat deposition, particularly in European sea bass, concerning fish farmers. Inclusion of conjugated linoleic acid (CLA) could reduce fat deposition as in other vertebrates. To determine if dietary CLA affects fat deposition, lipid metabolism, lipid composition and morphology of different tissues, growth and selected immune parameters, European sea bass juveniles were fed 4 graded levels of CLA (0, 0.5, 1 and 2%). Growth and feed conversion were not affected by CLA, whereas feed intake was reduced (P

The post mortem degradation processes that take places in fish muscle gradually modify the initial state of freshness. Tissue degradation is accompanied by drastic myofibrillar proteolysis produced as a consequence of the activation of proteolytic enzymes. In this study, structural changes were correlated with the textural properties of sea bream (Sparus aurata) muscle during 14 days of post mortem cold storage through the immunohistochemical detection of muscle filament proteins (desmin, actin and dystrophin), as well as endoproteases (μ-calpain, m-calpain) and their endogenous inhibitor (calpastatin). Actin and desmin were detected in fish muscle as late as 10 days post mortem (dpm) while dystrophin could not be detected after 4 dpm. In contrast, labelling for both calpains and calpastatin persisted during the entire storage period. Fracturability was clearly reduced after 7 dpm, while greatest changes in hardness, gumminess and chewiness were observed during the first week of storage. Our immunohistochemical observations indicate the disappearance of cytoskeleton proteins at different times post mortem. Thus, actin and desmin persist even when the muscle tissue shows a deteriorated appearance and texture, while dystrophin vanishes soon after the death of the fish. Detachment between myofibres and the myocommata was concomitant with the loss of dystrophin and also corresponded to the time when the reduction in flesh hardness observed was most significant. On the contrary, after 4 days of storage, calpain activity remained practically unaltered in refrigerated muscle specimens.

Despite being proposed as a potential candidate for diversification of marine aquaculture, limited larval survival and the occurrence of elevated levels of skeletal deformities restrict the commercial production of red porgy. The present study was conducted to determine the effect of rotifer enrichment, particularly on DHA, on growth, survival and occurrence of skeleton deformities in this species. The study included two trials, in one the viability of commercial emulsions for rotifer enrichment was evaluated whereas in another the effect of the elevation of DHA in rotifer emulsions on the larvae was studied. No significant differences were found in growth between larvae fed different rotifers in both trials suggesting that 1.9% dw DHA is enough to fulfil the DHA requirements of red porgy larvae for maximum growth. However, a marked positive effect of rotifer DHA content supplementation on larval survival was found. A low larval survival was found when rotifers had a high DPA content, in agreement with the results obtained for other Sparids. This fatty acid was accumulated in red porgy larval tissues in high levels (0.79% dw DPA) when rotifers rich in DPA were the main source of food. Although the external appearance of the juveniles seemed to be normal, X-ray observations showed elevated levels of bone abnormalities associated, in both trials, to low DHA content in the live prey. Among different anomalies, the presence of fused vertebrae was the most frequent deformity for both rearing trials. Although DHA content in the rotifers used for feeding was the only difference a 50% reduction in the number of deformed fish for each type of deformity studied was obtained when the larvae fed higher DHA levels, denoting the important role of this FA in the prevention of deformities at the rotifer feeding stage. Further studies are needed to elucidate the importance of essential fatty acids on the development of bone deformities in fish, since the functions of HUFA are different, and their absolute levels and ratios among them can lead to very different effects in fish metabolism, including bone formation.

The use of vegetable oils in fish nutrition has been extensively studied; and recent work has focused attention on replacing fish oil with alternative fatty acid sources and their effect on the immune system. However, little is known about the effect of these oils on immune parameters such as the fish interferon system. In this study we evaluate the effect of two vegetable oils (linseed and soybean) on gilthead sea bream Mx expression and other innate immune parameters. Experimental diets were formulated where fish oil was totally replaced by vegetable oils or for a mixture of them (50% linseed and 50% soybean). Another diet prepared with pure fish oil was used as a control. Two experiments were carried out in order to evaluate growth, feed utilization, serum alternative complement pathway activity, serum lysozyme and phagocytic activity of head kidney leucocytes as well as Mx expression in the liver. In the first experiment fish were fed with experimental diets for 6 months and then, growth and feed utilization as well as immune parameters were analyzed. In the second experiment, fish from the previous feeding trial were injected with either a sub-lethal dose of Photobacterium damselae subsp. piscicida (94/99) or a synthetic dsRNA (Poly I:C) in order to stimulate an Mx response. The results show that total substitution of fish oil by vegetable oils decreased the growth of gilthead sea bream juveniles. Furthermore, both phagocytic activity and serum alternative complement pathway activity were significantly reduced by the inclusion of either vegetable oil individually in the sea bream diets, but the diet with mixed vegetable oils had no significant effect. There was no effect on serum lysozyme levels but the basal constitutive levels of Mx transcript expression in the liver were elevated in the fish fed the vegetable oil diets. The time-course of the Mx response to injection of Poly I:C was shorter in the fish fed the fish oil diet and the fish fed the diet based on a mixture of both vegetable oils showed a faster Mx response to bacterial injection. Following stimulation with Poly I:C or PDP the fish fed the vegetable oil based diets still maintained higher basal levels of hepatic Mx expression than the fish fed the fish oil diet which returned to undetectable levels.

The objective of this study was to determine the effect of two levels of inclusion of mannan oligosaccharides derived from the outer cell wall of a select strain of Saccharomyces cerevisiae (Bio-Mos, Alltech Inc, USA) on growth, feed utilization, immune status and disease resistance of European sea bass (Dicentrarchus labrax). Specimens of 35 g at initial density of 3 kg/m3 were fed during 67 days at 0 per thousand, 2 per thousand and 4 per thousand dietary MOS level of inclusion in a commercial sea bass diet. Food conversion rate, specific growth rate, whole body biochemical composition, phagocyctic index of head kidney macrophages, NBT index, lysozyme and alternative complement pathway (ACP) activities as well as gut and liver histological structure were evaluated. Growth significantly increased at both MOS dietary inclusion levels. Histological features of the liver showed lower lipid vacuolization and regular-shaped morphology of hepatocytes around the sinusoidal spaces denoting a better utilization of dietary nutrients. No differences were found on gut histological evaluation. Statistical differences (P

We characterised a population of macrophages potentially involved in the immunoregulation induced by experimental cysticercosis. Following Taenia crassiceps infection, macrophages recruited in the peritoneal cavity were isolated and co-cultured at different ratios with T cells from naïve mice previously stimulated with anti-CD3/CD28 antibodies; these macrophages inhibited naïve T cell proliferation. This suppressive effect was Interleukin (IL)-10, Interferon-gamma (IFN-gamma), and nitric oxide (NO) independent. In contrast, macrophage-T cell contact was necessary to maintain anergy of T cells. Reverse transcriptase-PCR analysis of these macrophages showed higher transcripts of IL-10, chitinases Fizz1 and Ym1, and arginase-1 compared with naïve macrophages; by contrast, IL-12p40, and inducible nitric oxide synthase (iNOS) transcripts were undetected, whereas C-C chemokine ligand 5 (CCL5) was unchanged. Analysis of the membrane molecules expressed on Taenia-induced macrophages showed an up-regulation of several markers, mainly programmed death ligand 1 (PD-L1) and PD-L2. Blockade of PD-L1, PD-L2 or their receptor PD-1, but not of another marker, eliminated their ability to inhibit T-cell proliferation. Parallel experiments using ovalbumin (OVA)-peptide as a model antigen displayed similar results. Additionally, the same mechanism appears to be functional in splenocytes of T. crassiceps-infected mice given that blockade of PD-1, PD-L1 or PD-L2 re-established their ability to proliferate in response to parasite antigens. Moreover, Taenia-induced macrophages were able to suppress a mixed lymphocyte reaction in a PD-1-dependent manner. Thus, cestode infections induce macrophages alternatively activated with strong suppressive activity involving the PD-1/PD-L's pathway.

To determine the effect of long term feeding of diets with high levels of vegetable oils gilthead seabream juveniles were fed for 7 months with several diets containing vegetable oils at two inclusion levels. Anchovy oil was the only lipid source of FO (fish oil) diet, whereas in diets 60RO, 60LO and 60SO, 60% of fish oil was substituted by rapeseed (RO), linseed (LO) or soybean (SO) oils, respectively, and 80% was substituted by linseed or soybean oils in diets 80LO and 80SO. Afterwards, all fish were fed only a fish oil diet to determine the progressive evolution of fillet fatty acid profiles, since fish muscle fatty acid composition changes by feeding vegetable oils affecting its nutritional value for human health. Results showed that substitution by vegetable oils of up to 60% fish oil in diets for gilthead seabream does not affect growth and feed utilization even after a long feeding period. However, 80% substitution of fish oil significantly reduced growth. Feeding vegetable oils reduced muscle contents of docosahexaenoic acid (DHA) and arachidonic acid (ARA) to a lower degree than their reduction in the diet, denoting their importance, reduction of eicosapentaenoic acid (EPA) in muscle being more pronounced. Moreover, re-feeding with a FO diet for 60 days effectively recovered muscle DHA and ARA contents, whereas those of EPA were not recovered even after 90 days. Linoleic acid was strongly retained even after “wash out”. Fish fillets were very well accepted by the panel of judges. Dietary soybean oil inclusion produced significantly less shiny and less hard fillets, but more juicy and adhesive than FO fillets, with a slight earthy flavour when fish were fed 80SO diet. However linseed oil inclusion only increased shininess when fish were fed 80LO diet. A significantly higher chroma value was obtained for 80LO fish fillets, although it was not noticeable to the human eye.

In the present study 75 g European sea bass were fed for 8 months with different diets (22% dietary lipid content) containing vegetable oils at two inclusion levels. The control diet (Diet FO) contained anchovy oil as the only lipid source; in diets 60RO, 60LO, and 60SO of fish oil was substituted by rapeseed, linseed or soybean oils, respectively; finally, in diet 80LO, 80% of the fish oil was substituted by linseed oil. Fish were fed to apparent satiation three times a day. All fish were individually weighed once per month. Lipid and fatty acid composition of diets and fish fillets were determined at the beginning, middle and end of the experimental period. Once the commercial size was reached, all fish were fed a 100% FO containing diet during 150 days. No significant differences were found in feed intake. Fish fed the diets containing 80% linseed oil or 60% rapeseed oil had significant ( P After 150 days of re-feeding period with a 100% fish oil diet, DHA levels were restored in those fish previously fed diets containing vegetable oils, but EPA levels remained lower when compared with fish fed 100% FO diet. Flesh content of linoleic and linolenic acids remained higher in those fish previously fed soybean and linseed oil containing diets, respectively.

The aim of the present study was to evaluate the morphological changes in the intestine of gilthead seabream fed different lipid sources. Five experimental diets containing 22% lipid and 45% crude protein were formulated: the control diet included fish oil as the only lipid source, whereas the other four diets contained linseed, soybean or rapeseed oils at two different levels of fish oil substitution, 60% or 80%. Gilthead seabream juveniles of 79 g mean body weight were fed the experimental diets for 3 months. At the end of experiment, samples of anterior intestine were taken for ultrastructural study. A morphometric study was performed to complete the morphological results. Irreversible tissue damages (necrosis or cell degeneration) could not be found in the intestinal epithelium of fish, regardless the diet fed. Microvillis were well developed, among which some absorptive vesicles were observed in fish from all diets. The major ultrastructural differences in the enterocytes of fish fed different diets were the accumulation of supranuclear lipid droplets and the formation of lipoproteins. Lipid droplet accumulation was increased by the percent of fish oil replaced. The morphometric study revealed that the cellular space occupied by lipid droplets was 2.3 times higher when fish were fed a diet containing linseed or soybean oils at a 60% replacement level than in the control fish, whereas in fish fed the vegetable oils at a 80% replacement level and rapeseed at 60% it was 5 times higher than in the control fish. In the intercellular spaces and lamina propria two different classes of lipoproteins in relation to their size were found: very low density lipoproteins (VLDL) of 30 to 85 nm in diameter and chylomicrons (QM) of over 100 nm. Fish fed a soybean oil diet, particularly at a 60% replacement level, exhibited a much higher accumulation of these granules in dilated intercellular spaces. The rest of experimental groups showed chylomicrons as the main lipoprotein. These results are discussed in relation to fatty acid compositions and cellular mechanisms involved in lipid absorption.

Commercial feeds for gilthead seabream are highly energetic, containing fish oil as the main lipid source. The steady production and raising prices of fish oil encourage the inclusion of vegetable oils in fish feeds. Fish oil could be at least partially substituted by vegetable oils in diets for marine species, being this substitution resulted in good feed utilization and maintenance of fish health, since imbalances in dietary fatty acids may alter the immunological status and stress resistance in fish. In order to evaluate the effect of vegetable oils on gilthead seabream health, fish were fed different isonitrogenous and isocaloric diets for 101 days (Experiment I) and 204 days (Experiment II). In Experiment I, diets were formulated to contain 60% of the fish oil used in the control diet (FO) as soybean oil (Diet 60SO), rapeseed oil (60RO), linseed oil (60LO) or a blend of those oils (Mix). In Experiment II, the same diets plus two which contained 80% of the fish oil as soybean oil (80SO) and linseed oil (80LO), respectively, were assayed. At the end of both experiments, basal levels of different immunological parameters were determined, including both humoral immunity (alternative complement pathway activity and serum lysozyme activity) and cellular immunity (circulating neutrophil activity and phagocytic index of head kidney macrophages). In addition, response to a confinement stress was assayed in terms of variations in plasma cortisol. The effect of dietary vegetable oils on fatty acid composition of head kidney macrophages and circulating red blood cells (RBC) was also studied. No effects of dietary vegetable oils were found in fish fed the experimental diets for a medium period. Feeding dietary vegetable oils for a long period did not affect lysozyme or neutrophil activity. However, in Experiment II, the inclusion of soybean oil reduced both serum alternative complement pathway activity (from 249 IU/ml (FO2) down to 153.8 IU/ml (60SO2)) and head kidney phagocytic activity (from 25.75% (FO2) down to 14.58% (80SO2). Inclusion of rapeseed oil reduced phagocytic activity. Fish fed vegetable oil-containing diets showed different patterns of stress response, especially those fish fed the linseed oil diets that showed a significant increase in plasma cortisol level after stress. The fatty acid composition of head kidney macrophages reflected the fatty acids content of the respective diets, but a selective incorporation of essential fatty acids into these cells was observed. The same trend was found in circulating red blood cells, indicating the important role of essential fatty acids on these cells. Sixty percent of fish oil can be replaced by a blend of different vegetable oils without affecting gilthead seabream health. However, if single vegetable oil is used to replace 60% of fish oil, fish health can be affected in terms of immunosuppression or stress resistance. Rapeseed oil affected head kidney macrophages activity, soybean oil affected serum alternative complement pathway activity and linseed oil altered stress response of fish.

Red porgy Pagrus pagrus were placed and maintained in white, grey and black background fibreglass tanks for 2 weeks. Additionally, fish kept in white and black background tanks were then subjected to crowding stress. After 2, 9, 16 and 23 days, blood samples were taken and plasma cortisol, alpha melanocyte stimulating hormone (α-MSH) and glucose values were analysed and compared with values from uncrowded fish from white or black tanks. Measurements of plasma cortisol and α-MSH in unstressed red porgy from white, grey and black tanks revealed no significant differences among the three groups. However, the results show that background colour markedly affects the in vitro interrenal sensitivity to both α-MSH and ACTH, as interrenal cells from black adapted fish had become virtually unresponsive to both secretagogues. Crowded fish on a black background showed a prominent increase in plasma cortisol after 2 days, which was followed by a recovery. In fish crowded on a white background however, the increase of cortisol was lower but was maintained through the entire experiment. Plasma α-MSH levels increased at 23 days as a consequence of crowding; this increase was also dependent on the background, being more prominent in fish placed in white background tanks. Thus, in the red porgy, a white background appears to modify the stress response, particularly in the long term.

The aim of the present study was to determine the impact of replacing at least 50% of the fish oil by alternative lipid sources in diets for rainbow trout on growth, lipid digestibility, fatty acid profiles of liver and muscle and tissue histology. Four experimental diets were formulated combining two fish oils (capelin and anchovy) with different vegetable oils (soybean, rapeseed, palm and olive) and one type of animal fat (lard), in order to obtain 60% to 80% fish oil replacement. A fifth diet prepared with pure fish oil (capelin oil) served as control. The diets were fed to apparent satiation twice a day to triplicate groups of 30 rainbow trout with an initial weight of 250 g for 64 days at 12 °C. Growth was good (TGC 3 3.7–3.9) and independent of diet. Final body weight was on average 760 g. Feed conversion ratios (FCR) ranged from 0.72 to 0.79. The only significant difference in FCR was found in the group fed an olive oil–lard combination replacing 80% of the fish oil. This diet also had a lower apparent lipid digestibility (79% vs. 91–94% for the rest of the diets). The liver and muscle fatty acid compositions were similar within groups and reflected that of the diets. But, tissue levels of 20:5 n −3 were lower and levels of 22:6 n −3 were higher than their respective dietary percentages. Histologically, a supranuclear accumulation of lipid droplets was observed in the intestinal cells of some of the groups fed diets supplemented with vegetable oils. Similarly, livers from these groups showed large amounts of lipid droplets within the hepatocytes. The present results suggest that most of the fish oil can be replaced by the alternative sources tested without compromising growth and feed utilisation. However, the histological changes observed suggest an impact of dietary lipid source on the transport and/or metabolism of fat in the fish. This requires further investigation.

This study investigates the effect of dietary vitamin E on juveniles of gilthead seabream under stressful situations, focusing on the effects on growth, haematology, some immune parameters and plasma cortisol as indicators of stress. Two sardine meal-based experimental diets, one of them supplemented with 150 mg of alpha tocopherol kg(-1) of diet (control) and another one without vitamin E supplementation (diet NE), were assayed under two different stress conditions: overcrowding as a chronic stressor (during 15 weeks) and repetitive chasing as an acute repetitive stressor. Low levels of vitamin E in the diet depleted alternative complement pathway activity [from 167.23 U ml(-1) (control fish) down to 100.99 U ml(-1)] and also nonspecific haemagglutination. Also, fish fed a non-supplemented diet showed an elevation of plasma cortisol basal levels without a stressor influence [from 3.91 ng cortisol ml(-1) plasma (control fish) up to 21.70 ng cortisol ml(-1) plasma]. Low levels of vitamin E in the diet also produced an increase of erythrocyte fragility. Under chronic stress, fish fed the vitamin E-deficient diet showed a reduction in growth and survival, and alterations in haematological parameters, such as an additional haemoconcentration in response to overcrowding when compared with control fish. Under repetitive stress, fish fed the vitamin E deficient diet showed faster elevation of plasma cortisol levels in response to stress and a lower survival rate than control fish. Production of oxygen radicals by blood neutrophils was reduced under repetitive stress in fish fed the non-supplemented diet. These results suggest that fish fed the vitamin E-deficient diet had lower stress resistance.

Macrophage aggregates (MAs) are structures in the spleen, kidney and sometimes liver of fishes which have various functions such as recycling/storing/detoxification of cellular wastes and exogenous substances. They have been also reported to be important in the specific immune response and are used as health indicators. Changes in MA density, size and pigment content have been used in national and local monitoring programs in the U.S. as indicators of contaminant exposure. However, MA number and structure can also be affected by other factors, including general stress or nutritional status of fish. An experiment was conducted to study the effects of vitamin E and n −3 highly unsaturated fatty acid (HUFA) deficiencies and stocking density on spleen and kidney MAs of gilthead seabream, one of the most important species for Mediterranean aquaculture. Fish were held at two stocking densities, high and low, and fed experimental diets. Diet NE had no supplemental vitamin E, Diet NFA was deficient in n −3 HUFA and Diet C was a control diet. Number, size and shape factor of MAs were measured using image analysis. The percentage of tissue occupied by MAs was calculated from these measurements. The results showed that high stocking density alone increased the number of splenic but not kidney MAs of fish fed the control diet. A deficiency of n −3 HUFA alone also increased the number of splenic but not kidney MAs at both stocking densities. Vitamin E deficiency alone had no significant effect on MAs in either organ. However, the combined effect of vitamin E deficiency and high stocking density increased the number and size of kidney but not splenic MAs. This study indicates that specific dietary deficiencies can influence MA accumulation and that splenic MAs may be more responsive to general stress than kidney MAs.

High stocking density is an aquaculture-related situation which causes crowding stress in fish and potentially affecting the immune system. Protection against disease can be induced through immunostimulation, enhancing defence mechanisms. Vitamins C and E are among the most important nutrients influencing the immune system, although the effect of these vitamins in the amelioration of stress in fish has not been studied in detail. An experiment was carried out to study the effect dietary supplementation of vitamin C or vitamin E on the immune system of fish under a crowding stress situation in a commercial farm. A commercial diet for this species was supplemented with vitamin C or vitamin E and these diets were assayed at high stocking density (over 40 kg m−3) and the results were compared with those from fish fed the commercial diet and held at farm conditions (20 kg m−3). Some biological and immunological parameters were studied at the end of the experimental period. Fish held at high stocking density showed lower final weight than fish held at lower stocking density. These also showed higher plasma cortisol levels (over 6 ng cortisol ml−1 plasma for all the diets assayed) than those fish held at lower density (1.13 ng cortisol ml−1 plasma). High stocking density produced higher serum lysozyme activity values in fish fed the commercial diet compared with those fish held at lower density (67.49 and 36.84 units ml−1, respectively). This effect was not observed in fish held at high stocking density but fed vitamin C and vitamin E supplemented diets (lysozyme activity: 21.97 and 19.71 units ml−1, respectively). High stocking density also appeared to decrease serum alternative complement activity. In those fish fed the vitamin E supplemented diet, levels were similar to fish held at low stocking density, suggesting the role of this vitamin in this defense mechanism.

In a previous study, several histological alterations were found in the liver of gilthead seabream fed with a diet containing 30% soybean meal (SBM). In the current study, SBM containing diets were supplemented with either potassium phosphate, zinc sulfate or phytase ( Aspergillus ficuum ), or increasing the dietary n −3/ n −6 fatty acids ratio to meet that of a fish meal based diet. Diet composition did not affect fish growth, feed efficiency (FE) or protein productive value (PER). Phosphorous supplementation significantly reduced hepatosomatic indexes (HSI), although it did not alter liver lipid content. Both phosphorous supplementation and correcting the dietary n −3/ n −6 fatty acid ratio significantly altered the lipid and protein content in fish muscle. Only these two treatments and principally the corrected dietary n −3/ n −6 fatty acid ratio, improved the liver histological alterations observed in fish fed with SBM based diets.

Two experiments were conducted to compare the effect of the partial substitution of fish meal by two different protein sources, corn gluten (CGM) and meat and bone meals (MBM) in diets for juveniles gilthead seabream. Growth, feed efficiency, protein efficiency ratio and protein productive values were not significantly affected by the source of dietary protein, although higher values were observed with diets containing increasing levels of meat and bone meal. No differences were found in fish proximate composition at the end of the experiment. Histological studies of liver tissue showed no liver alteration in fish fed diets including increasing levels of corn gluten meal. On the contrary, an increased deposition of lipids, nuclei polarization and isolated necrotic focus were found in hepatocites of fish fed diets exceeding 20% of meat and bone protein. Apparent protein digestibility (ADC) in diets containing CGM showed similar results to those obtained with the control diet. However, a significant reduction in digestibility was observed when MBM was used as partial substitute of fish meal protein. A negative correlation between dietary ash content and protein digestibility was observed. Higher amounts of nitrogen were excreted as levels of CGM and MBM increased in the diets, being significantly higher in the case of 40% substitution either with CGM or MBM protein.

The effect of fish meal quality on growth, feed efficiency, protein digestibility and fillet composition was studied in a growth experiment with gilthead seabream. The experiment included a comparison of pelleted feed with extruded feed. The fish were fed three extruded diets which varied in the quality of the fish meal used in the respective feeds: low quality fish meal, good quality fish meal, and a mixture (50%) of the two fish meal qualities. Fish meal quality was judged by protein digestibility as measured with mink as the test animal and by the content of biogenic amines. A fourth experimental group was fed a pelleted feed with the same mixed fish meal blend. The experiment started with 70 g seabream and lasted for 3 months until the fish reached about 160 g. All groups showed good growth and feed efficiency during the experimental period, with daily specific growth rates (SGR) of 0.90–1.00% and feed efficiencies (FE; fish weight gain per feed offered) from 0.58 to 0.66. The fish fed the feed with low quality fish meal showed significantly poorer feed efficiency than the other groups, and there was a significant correlation between feed efficiency obtained in seabream and the protein digestibility as measured with mink. No difference was observed among groups fed the extruded diets, for growth, fillet composition or amount of liver or viscera, although a significant correlation was obtained between SGR and protein digestibility in mink. Fillet lipid content was higher in fish fed the extruded feed, compared with those fed the corresponding pelleted diet. Protein digestibilty in seabream showed no difference among the extruded feeds, while the pelleted feed was significantly lower.