Supplementation of arachidonic acid rich oil in European sea bass juveniles (Dicentrarchus labrax) diets: effects on growth performance, tissue fatty acid profile and lipid metabolism.

The aim of this study was to evaluate the effects of increasing dietary arachidonic acid (ARA) levels (from 1 to 6% of total fatty acids) on European sea bass (Dicentrarchus labrax) juveniles' growth performance, tissue fatty acid profile, liver morphology as well as long-chain polyunsaturated fatty acids (LC-PUFA) biosynthesis, triglyceride and cholesterol synthesis and lipid transport. A diet with total fish oil (FO) replacement and defatted fish meal (FM) containing a 0.1-g ARA g-1 diet was added to the experimental design as a negative control diet. Dietary ARA inclusion levels below 0.2 g ARA g-1 diet significantly worsened growth even only 30 days after the start of the feeding trial, whereas dietary ARA had no effect on fish survival. Liver, muscle and whole body fatty acid profile mainly reflected dietary contents and ARA content increased accordingly with ARA dietary levels. Tissue eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA) levels were positively correlated among them. Hepatic lipid vacuolization increased with reduced dietary ARA levels. Expressions of fatty acyl desaturase 2 and 3-hydroxy-3-methylglutaryl-coenzyme genes were upregulated in fish fed the negative control diet compared to the rest of the dietary treatments denoting the influence of ARA on lipid metabolism. Results obtained highlight the need to include adequate n-6 levels and not only n-3 LC-PUFA levels in European sea bass diets.

Disease resistance and response against Vibrio anguillarum intestinal infection in European seabass (Dicentrarchus labrax) fed low fish meal and fish oil diets.

The aim of this study was to assess the effects of low levels of dietary fish meal (FM) and fish oil (FO) on disease resistance and gut associated lymphoid tissue (GALT) response after an experimental intestinal infection with V. anguillarum in European sea bass (Dicentrarchus labrax) For that purpose, sea bass juveniles were fed one of four diets containing combined levels of FO and FM as follows: 20%FM/6%FO, 20%FM/3%FO, 5%FM/6%FO and 5%FM/3%FO during 153 days. At the end of the feeding trial, fish were subjected to either an in vivo exposure to a sub-lethal dose of V. anguillarum via anal inoculation or to an ex vivo exposure to V. anguillarum. Additionally, inducible nitric oxide synthase (iNOS) and tumor necrosis factor α (TNFα) gut patterns of immunopositivity were studied. Growth performance was affected by dietary FM level, however ex vivo gut bacterial translocation rates and survival after the in vivo challenge test were affected by dietary FO level. After 5 months of feeding, low dietary FM levels led to a posterior gut up-regulation of interleukin-1ß (IL-1ß) and TNFα, major histocompatibility complex-II (MHCII) and cyclooxygenase-2 (COX2), which in turn reduced the gut associated lymphoid tissue (GALT) capacity of response after 24 h post infection and conditioned European sea bass capacity to recover gut homeostasis 7 days post infection. Immunoreactivity to anti-iNOS and anti-TNFα presented a gradient of increased immunopositivity towards the anus, regardless of the dietary FM/FO fed. Strong positive anti-TNFα isolated enterocytes were observed in the anterior gut in relation to low levels of dietary FM/FO. Submucosa and lamina propria immunoreactivity grade was related to the amount of leucocyte populations infiltrated and goblet cells presented immunopositivity to anti-iNOS but not to anti-TNFα. Thus, reducing FO content from 6% to a 3% by VO in European sea bass diets increases ex vivo and in vivo gut bacterial translocation rates, whereas reducing FM content from 20% down to 5% up-regulates the expression of several posterior gut inflammation-related genes conditioning fish growth and GALT capacity of response after bacterial infection.

Supplementation of arachidonic acid rich oil in European sea bass juveniles (Dicentrarchus labrax) diets: Effects on leucocytes and plasma fatty acid profiles, selected immune parameters and circulating prostaglandins levels.

The main objective of this study was to assess the effects of graded levels of dietary arachidonic acid (ARA), supplemented from alternative sources, on fatty acid composition of plasma and head kidney leucocytes of European sea bass (Dicentrarchus labrax). For that purpose, sea bass juveniles were fed four diets containing graded levels of ARA as follows: 0.5% (ARA0.5), 1% (ARA1), 2% (ARA2) and 4% (ARA4) during 60 days. At the end of the feeding trial fatty acid profiles of plasma and head kidney leucocytes were analyzed. Besides, plasma prostaglandins levels, head kidney leucocytes respiratory burst activity; peroxidase activity and phagocytic index were assayed. Reducing dietary ARA levels below 1% markedly reduced European sea bass growth performance. However, fish fed diet ARA0.5 tried to compensate this dietary ARA deficiency by a selective deposition of ARA on plasma and head kidney leucocytes, reaching similar levels to those fish fed diet ARA1 after 60 days of feeding. Nevertheless, head kidney phagocytic capacity was reduced as dietary ARA content in relation not only to variations on membrane composition but also to changes on fish basal prostaglandins levels. Results obtained demonstrated the importance to supply the necessary quantity n-6 LC-PUFA, and not only n-3 LC-PUFA levels, in European sea bass diets, in relation to not only growth performance but also immune system function.

Factors that predict survival in patients with cirrhosis considered for liver transplantation.

OBJECTIVE: To identify prognostic factors for survival at 6 and 12 months in patients evaluated for liver transplantation using Child-Pugh (CP) classification and the Model for End-Stage Liver Disease (MELD) score. METHODS: We evaluated 144 patients with cirrhosis who were candidates for liver transplantation. We excluded patients with hepatocellular carcinoma, recent liver recipients, and patients who died because of factors unrelated to liver disease. The studied variables were age and sex; prothrombin time; platelet count; albumin, cholesterol, bilirubin, creatinine, and serum sodium concentrations; CP classification and MELD score; and the presence of ascites, encephalopathy, hepatorenal syndrome, spontaneous bacterial peritonitis, and previous variceal bleeding. Data were processed using statistical software (SPSS version 13.0). RESULTS: Of the 144 patients, 27 (18.7%) did not survive because of complications of liver disease. Univariate analysis showed the most significant factors to be sex, prothrombin time, bilirubin and albumin levels; ascites, encephalopathy, CP classification, and MELD score. At Cox regression analysis, only CP classification proved to be a valid predictor of survival in our cohort. The lowest survival according to CP classification at 6 and 12 months corresponded to stage C and to MELD scores higher than 15. CONCLUSIONS: Child-Pugh classification is an independent prognostic factor for recipient survival. Stage C in the CP classification and a MELD score higher than 15 were strongly related to worse survival. Both scores must be taken into consideration for adequate evaluation of liver transplantation for candidates.

Immune stimulation and improved infection resistance in European sea bass (Dicentrarchus labrax) fed mannan oligosaccharides.

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<0.05) on the phagocytic index were denoted with the inclusion of 4 per thousand Bio-Mos group. A positive correlation was found between the levels of lysozyme and alternative complement pathway activities in blood and the level of inclusion of MOS in diets. After the feeding trial, a cohabitation challenge test and direct gut inoculation were also performed with the pathogen Vibrio alginolyticus in a ratio 3:1. Twenty-one days post-challenge the number of cohabitant fish infected in the control group reached 33% comparing with none on the 0.4 per thousand MOS group. Finally, new fish were infected with V. alginolyticus by gut canalisation. After 24h post-infection no significant difference was denoted between groups and 48 h post-infection total infected fish in the control group was twice that of the 2 per thousand and 4 per thousand MOS groups.

Effect of dietary lipids on plasma fatty acid profiles and prostaglandin and leptin production in gilthead seabream (Sparus aurata).

The aim of this study was to investigate the effects of different levels of substitution of fish oil by vegetable oils rich in oleic, linoleic and linolenic acids on gilthead seabream plasma and leukocyte fatty acid compositions and prostaglandin (PG) and leptin production. Juvenile seabream of 24 g initial body mass were fed four iso-energetic and iso-proteic experimental diets for 281 days. Fatty acid composition of plasma lipids was markedly affected by the inclusion of vegetable oils (VO). ARA (arachidonate), EPA (eicosapentaenoate) and DHA (docosahexaenoate) were preferentially incorporated into polar lipids of plasma, and DHGLA (di-homogammalinoleate) accumulated with increased vegetable oil inclusion. Dietary treatments resulted in alterations of DHGLA/ARA ratios, but not ARA/EPA. ARA-derived PGE(2) production in plasma was not affected by vegetable oils, in agreement with similar eicosanoid precursor ratio (ARA/EPA) in leukocytes total lipids and plasma phospholipids among fish fed with the different dietary treatments. Feeding vegetable oils leads to a decrease in plasma EPA which in turn reduced plasma PGE(3) concentration. Moreover, PGE(3) was the major prostaglandin produced in plasma of fish fed fish oil based diet. Such findings point out the importance of EPA as a precursor of prostaglandins in marine fish, at least for the correct function of the blood cells, and correlates well with the predominant role of this fatty acid in immune regulation in this species. A negative correlation was found between plasma PGE(2) and leptin plasma concentration, suggesting that circulating levels of leptin may act as a metabolic signal modulating PGE(2) release. The present study has shown that increased inclusion of vegetable oils in diet for gilthead seabream may profoundly affect the fatty acid composition of plasma and leukocytes, specially HUFA (highly unsaturated fatty acids), and consequently the production of PGE(3), which can be a major PG in plasma. Alteration in the amount and type of PG produced can be at least partially responsible for the changes in the immune system and health parameters of fish fed diets with high inclusion of VO.

Adaptation of lipid metabolism, tissue composition and flesh quality in gilthead sea bream (Sparus aurata) to the replacement of dietary fish oil by linseed and soyabean oils.

Linseed (LO) and soyabean (SO) oils were evaluated as fish-oil (FO) substitutes in the diets of marketable-sized gilthead sea bream (Sparus aurata). Practical diets were designed factorially with the lipid added as follows (%): FO 100, LO 60+FO 40, LO 80+FO 20, SO 60+FO 40, SO 80+FO 20. The effects of experimental diets on growth, fatty acids patterns in liver and muscle, flesh quality variables and activities of selected enzymes involved in lipid synthesis and catabolism were determined at the end of a 7-month trial. Fatty acid composition of liver and muscle generally reflected the fatty acid composition of the diets. The n-3 PUFA levels were significantly reduced by the inclusion of vegetable oils. This tendency was more pronounced for EPA than for docosahexaenoic acid. The n-3:n-6 fatty acid ratio reached the lowest values in fish fed the SO diets; this was associated with a higher liver lipid deposition. No differences were found in fillet texture and pH. However, under conditions of forced peroxidation, muscles from fish fed the SO diets had lower peroxidation levels. Vegetable oil substitution decreased lipogenesis in liver and this effect was greatest at the highest substitution level. In contrast, muscle beta-oxidation enzymes had increased activities with vegetable oil substitution. Thus, the lower hepatic lipogenesis was correlated with an increased lipid utilisation in muscle. It is concluded that growth and lipid metabolism were affected by experimental diets.

Glomerulonephritis and immunosuppression associated with dietary essential fatty acid deficiency in gilthead sea bream, Sparus aurata L., juveniles.

An experiment was conducted to determine the effect of an essential fatty acid (EFA) deficient diet on growth, immune status and renal morphology of juvenile gilthead sea bream, using two diets: a control diet containing 2% (DW) of n-3 high unsaturated fatty acid (n-3 HUFA) and a diet formulated to be deficient in EFA for this species and containing 0.5% DW of n-3 HUFA (diet NFA). After 9 weeks of feeding the EFA-deficient diet fish showed a reduction in growth compared with the control group (107.48 +/- 9.14 and 123.14 +/-11.87 g final weight respectively). Fish fed the NFA diet showed a reduction in the erythrocyte volume together with increased erythrocyte fragility, haemoglobin content and red blood cell count in comparison with fish fed the control diet. EFA deficiency also reduced cellular immunity in terms of neutrophil activity and the number of circulating lymphocytes. The serum alternative complement pathway was markedly reduced in fish fed the EFA deficient diet. Fish fed the control diet showed glomeruli with a well-defined Bowman's space and normal renal tubes. However, up to 88% of fish fed the EFA-deficient diet showed alterations in renal morphology affecting at least 50% of the glomeruli, which showed extreme dilation of capillaries and occlusion of Bowsman's capsule. Mesangial proliferation and diffuse thickening of the capillary walls, as well as renal tube degeneration, were also observed.

Low vitamin E in diet reduces stress resistance of gilthead seabream (Sparus aurata) juveniles.

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.

Hematological recovery in Sparus aurata after bleeding. A time course study.

To determine the hematological recovery after blood loss in gilthead sea bream juveniles, an experimental anemia was induced by caudal blood extraction. Seventy fish were used for experimental hemorrhage and another 35 were used as controls. Blood samples were taken after 0.5, 1, 2, 4, 8, 16 and 32 days in both control and anemic groups. After bleeding, besides a reduction in the direct hematic constants, a peak in the mean cell volume value, together with a decrease in mean cell hemoglobin concentration after bleeding, suggested erythrocyte swelling. The increase in MCH suggested the occurrence of hyperchromic erythrocytes. Recovery of RBC number started between 2 and 4 days after bleeding and seemed to be related to erythroblast release to circulation. Total recovery was completed after 8 days of bleeding. The recovery patterns for hematological parameters of sea bream are discussed in relation to applied work on this species.