Fatty acid-sensing mechanisms in the hypothalamus of European sea bass (Dicentrarchus labrax): The potential role of monounsaturated and polyunsaturated fatty acids.

This study aimed to evaluate the hypothalamus fatty acid (FA)-sensing mechanisms response to different FA in European sea bass. For that purpose, fish (body weight of 90 g) were intraperitoneally (IP) injected (time 0 h) with five long-chain unsaturated fatty acids, namely, docosahexaenoic acid (DHA; C22:5n3); eicosapentaenoic acid (EPA; C20:4n3); α-linolenic (ALA; C18:3n3); linoleic acid (LA; C18:2n6) and oleic acid (OA; C18:1n9) at a dose of 300 µg kg-1, or with 0.9% saline solution (control). Feed intake (FI) was recorded at 3, 6, and 24 h after the IP injection. One week later, fish were IP injected with the same FA, and the hypothalamus was collected 3 h after the IP injection for measurement of molecules related to FI regulation and FA-sensing mechanisms. Cumulative FI (g/kg/day) was not affected by treatments. However, compared to the control, FI increased with the OA treatment at 6 h after the IP injection. FI decreased with mealtime in the DHA and LA groups. Gene expression of orexigenic (npy/agrp) and anorexigenic (cart2/pomc1) neurons was not affected by the FA treatments. Attending the enzymes involved in the FA-sensing mechanisms activation, compared to the control carnitine palmitoyltransferase I (CPT1) and ATP citrate lyase (ACLY) activity were not affected by FA treatments. Contrarily the key enzymes of lipid metabolisms, malic enzyme and hydroxyacylCoA dehydrogenase was higher in fish that received the EPA and OA treatment, than fish treated to the control. Overall, the results of the present study indicate that gene expression of orexigenic and anorexigenic neurons was not affected at 3 h after IP injection with different FA. However, the activity of key enzymes of lipid metabolism was differently affected by circulating FA, indicating that FA-sensing mechanisms respond to different FA. Further studies are required involving different sampling times to further characterize the response of FA-sensing mechanisms to FA. These findings may be of relevance to the aquaculture industry in an era where alternative lipid sources are being increasingly used.

Hepatic, Muscle and Intestinal Oxidative Status and Plasmatic Parameters of Greater Amberjack (Seriola dumerili, Risso, 1810) Fed Diets with Fish Oil Replacement and Probiotic Addition.

The present study was conducted to investigate the effects of dietary fish oil replacement with a mixture of vegetable oils and probiotic supplementation on plasma biochemical parameters, oxidative stress, and antioxidant ability of Seriola dumerili. Specimens with an initial weight of 175 g were used. Four feeds were formulated with 0% (FO-100), 75% (FO-25), and 100% (FO-0 and FO-0+ with the addition of Lactobacillus probiotics) substitution of fish oil with a mixture of linseed, sunflower, and palm oils. After 109 days, no significant differences were observed in the activity of antioxidant enzymes in the liver, foregut, and hindgut, only glucose-6-phosphate dehydrogenase activity in the liver was higher in the fish fed the FO-100 diet than in those fed the FO-0 diet. No significant differences were observed in the total, reduced, and oxidized glutathione and the oxidative stress index in the liver. In addition, lipid peroxidation in the liver and red muscle values were higher in the fish fed the FO-100 diet than in the fish fed the FO-0+ diet, however, the foregut of the fish fed the FO-100 diet presented lower values than that of the fish fed the FO replacement diet, with and without probiotics. There were significant differences in cholesterol levels in the FO-100 group; they were significantly higher than those observed with the fish diets without fish oil. To sum up, fish oil can be replaced by up to 25% with vegetable oils in diets for Seriola dumerili juveniles, but total fish oil substitution is not feasible because it causes poor survival. The inclusion of probiotics in the FO-0+ diet had no effects on the parameters measured.

In vitro modulation of gilthead seabream (Sparus aurata L.) leukocytes by Bacillus spp. extracellular molecules upon bacterial challenge.

Stimulation of the fish immune system using immunostimulants is an environmentally friendly strategy to minimize bacterial outbreaks in aquaculture. Different biological and synthetic immunostimulants can enhance non-specific innate immune responses by directly activating immune cells. An example are Bacillus spp., known for their immunostimulatory effects, although the exact mechanisms by which Bacillus spp. offer protection against diseases remains to be elucidated. Furthermore, most studies have focused on Bacillus spp. cells, while the immunostimulant effect of their extracellular metabolome, known to harbour biologically important metabolites, including antimicrobial molecules, has been scarcely evaluated. Here, we evaluated the in vitro immune-modulatory properties of extracellular extracts of three Bacillus spp. strains (B. subtilis FI314, B. vezelensis FI436 and B. pumilus FI464), previously isolated from fish-guts and characterized for their in vitro and in vivo antimicrobial activity against a wide range of fish pathogens. Bacillus spp. extracellular extracts did not affect immune cells viability, but remarkably increased pathogens' phagocytosis when seabream head-kidney leukocytes were challenged with Vibrio anguillarum and Edwardsiella tarda. All extracts significantly increased the engulfment of bacterial pathogens 1 h post-infection. Cells stimulated with the extracellular extracts showed an up-regulation of the expression of immune-relevant genes associated with inflammation, including IL-1ß, IL-6, and COX-2. In cells challenged with E. tarda, FI314 extracellular extract significantly increased the expression of IL-1ß, IL-6, and COX-2, while FI436 and FI464 significantly increased IL-6 expression. The results of this study revealed that the extracellular molecules from Bacillus spp. fish isolates improved the in vitro response of gilthead seabream immune cells and are thus promising candidates to act as immunostimulants, helping fish fight diseases.

Feeding frequency and dietary protein/carbohydrate ratio affect feed intake and appetite regulation-related genes expression in gilthead seabream (Sparus aurata).

To evaluate the effects of feeding frequency (FF) and dietary protein/carbohydrate (P/CH) ratios on appetite regulation of gilthead seabream, two practical diets were formulated to include high protein and low carbohydrate (P50/CH10 diet) or low protein and high carbohydrate (P40/CH20 diet) content and each diet was fed to triplicate groups of fish until visual satiation each meal at a FF of 1, 2, or 3 meals per day. Feed intake and feed conversion ratio were higher in fish fed 2 or 3 meals than 1 meal per day and in fish fed the P40/CH20 than the P50/CH10 diet. The specific growth rate was only affected by FF, being higher in fish fed 2 or 3 meals per day than 1 meal per day. Expression of the cocaine-amphetamine-related transcript, corticotropin-releasing hormone, ghrelin receptor-a (ghsr-a), leptin, and neuropeptide y in the brain, cholecystokinin (cck) in the intestine, and leptin and ghrelin in the stomach was not affected by FF or dietary P/CH ratio. This is the first time that ghrelin cells were immune-located in the stomach of gilthead seabream. Fish fed 3 meals per day presented lower cck expression in the brain than those fed twice per day and higher hepatic ghsr-b expression than those fed once per day. Fish fed P40/CH20 diet presented higher hepatic leptin expression than those fed P50/CH10 diet. In conclusion, present results indicate that feeding a P40/CH20 diet at 3 meals a day seems to decrease the satiation feeling of gilthead seabream compared to fish fed higher P/CH ratio diets or fed 1 or 2 meals a day.

Maize distillers dried grains with solubles alter dietary digestibility and improve intestine health of pacu, Piaractus mesopotamicus juveniles.

Two trials were conducted to evaluate the effects of soyabean meal replacement by maize distillers dried grains with solubles (DDGS) in diets for pacu juveniles. Five diets were formulated with 0, 100, 200, 300 and 400 g of DDGS/kg diet replacing up to total dietary soyabean meal. In trial 1, the experimental diets were fed to five groups of fish to evaluate the apparent digestibility coefficients (ADC). In trial 2, four groups of fish were fed each experimental diet for 100 d to evaluate the effects of these diets on digestive enzyme activity, intestine oxidative stress and intestine morphology. The ADC of DM and energy was reduced with dietary DDGS inclusion, while the ADC of lipids was increased, and no differences were observed for the ADC of protein. Independent of dietary treatment, pH increased from anterior to the distal intestine with dietary DDGS inclusion. Digestive enzyme activities were higher on anterior than the distal intestine. Dietary DDGS decreased lipase, amylase, chymotrypsin and trypsin activities, while no differences were observed for total protease activity. Intestine glucose-6-phosphate dehydrogenase was reduced in fish fed the DDGS diets, while catalase activity increased. Lipid peroxidation was lower in fish fed DDGS diets than the control. Intestine histomorphology improved with dietary DDGS inclusion. Overall, the negative effects of soyabean meal could be decreased by dietary replacement with maize DDGS which may have a prebiotic effect, improving intestine health.

Bacillus spp. Inhibit Edwardsiella tarda Quorum-Sensing and Fish Infection.

The disruption of pathogen communication or quorum-sensing (QS) via quorum-quenching (QQ) molecules has been proposed as a promising strategy to fight bacterial infections. Bacillus spp. have recognizable biotechnology applications, namely as probiotic health-promoting agents or as a source of natural antimicrobial molecules, including QQ molecules. This study characterized the QQ potential of 200 Bacillus spp., isolated from the gut of different aquaculture fish species, to suppress fish pathogens QS. Approximately 12% of the tested Bacillus spp. fish isolates (FI). were able to interfere with synthetic QS molecules. Ten isolates were further selected as producers of extracellular QQ-molecules and their QQ capacity was evaluated against the QS of important aquaculture bacterial pathogens, namely Aeromonas spp., Vibrio spp., Photobacterium damselae, Edwardsiela tarda, and Shigella sonnei. The results revealed that A. veronii and E. tarda produce QS molecules that are detectable by the Chr. violaceum biosensor, and which were degraded when exposed to the extracellular extracts of three FI isolates. Moreover, the same isolates, identified as B. subtilis, B. vezelensis, and B. pumilus, significantly reduced the pathogenicity of E. tarda in zebrafish larvae, increasing its survival by 50%. Taken together, these results identified three Bacillus spp. capable of extracellularly quenching aquaculture pathogen communication, and thus become a promising source of bioactive molecules for use in the biocontrol of aquaculture bacterial diseases.

Functional Feeds to Tackle Meagre (Argyrosomus regius) Stress: Physiological Responses under Acute Stressful Handling Conditions.

Marine algae are recognised sources of bioactive compounds that have attracted great interest as nutritional supplements for aquaculture fish. Intensive rearing conditions often expose fish to husbandry-related stressors, rendering fish more susceptible to disease and reducing production yields. The present work evaluated the potential of two marine algae extracts (Fucus vesiculosus and Nannochloropsis gaditana) as nutritional supplements to mitigate stress effects in meagre (Argyrosomus regius) exposed to an acute handling stress (AS). A plant-based diet was used as a control, and three other diets were prepared, which were similar to the control diet but supplemented with 1% of each algal extract or a combination of the two extracts (0.5% each). The effects of supplemented diets on stress biomarkers, antioxidant enzyme activities, and immune response were analysed in fish exposed to AS after 4 weeks of feeding. Supplemented diets did not affect growth performance but the inclusion of F. vesiculosus promoted higher feed efficiency, as compared to the control group. Dietary algal extracts supplementation reduced plasma glucose levels, increased white blood cell counts, and reduced the expression of pro-inflammatory genes when compared with the control. N. gaditana supplementation led to a reduction in hepatic antioxidant enzyme activity and glutathione levels, while F. vesiculosus supplementation increased muscle glutathione reductase activity and reduced lipid peroxidation. These findings support the potential of algal extracts as nutraceuticals in aquafeeds to enhance the ability of fish to cope with husbandry-related stressful conditions and ultimately improve fish health and welfare.

Gilthead seabream (Sparus aurata) in vitro adipogenesis and its endocrine regulation by leptin, ghrelin, and insulin.

Leptin, ghrelin, and insulin influence lipid metabolism and thus can directly affect adipose tissue characteristics, modulating the organoleptic quality of aquaculture fish. The present study explored gilthead seabream (Sparus aurata) cultured preadipocytes development, and the regulation of adipogenesis by those three hormones. Preadipocytes presented a fibroblast-like phenotype during the proliferation phase that changed to round-shaped with an enlarged cytoplasm filled with lipid droplets after complete differentiation, confirming the characteristics of mature adipocytes. peroxisome proliferator-activated receptor-γ (pparγ) expression was higher at the beginning of the culture, while fatty acid synthase and 3-hydroxyacyl-CoA dehydrogenase gradually increased with cell maturation. The expression of lipoprotein lipase-like, lysosomal acid lipase (lipa), fatty acid translocase/cluster of differentiation-36 (cd36), and leptin receptor (lepr) were not affected during cell culture development; and undetectable expression levels were observed for leptin. Concerning regulation, leptin inhibited lipid accumulation significantly reducing pparγ and cd36 gene expression, both in early differentiating and mature adipocytes, while ghrelin decreased the expression of pparγ in the early differentiating phase but did not reduce intracellular lipid content significantly. Additional insulin past the onset of adipogenesis did not affect lipid accumulation either. In conclusion, at present culture conditions leptin has an anti-adipogenic function in differentiating preadipocytes of gilthead seabream and continues exerting this role in mature adipocytes, while ghrelin and insulin do not seem to influence adipogenesis progression. A better understanding of leptin, ghrelin, and insulin impact on the adipogenic process could help in the prevention of fat accumulation, improving aquaculture fish production and quality.

Taurine modulates hepatic oxidative status and gut inflammatory markers of European seabass (Dicentrarchus labrax) fed plant feedstuffs-based diets.

This study aimed to evaluate the effect of taurine (tau) supplementation to low fishmeal (FM) diets on growth performance, oxidative status, and immune response of European seabass juveniles. Four isoproteic (46% crude protein) and isolipidic (19% crude lipid) diets were formulated to contain either 25 or 12.5% FM and a mixture of plant feedstuffs, supplemented or not with 1% tau. Twelve groups of 20 fish (IBW = 9.4 g) were fed each diet for 9 weeks. Reduction of dietary FM from 25 to 12.5% impaired growth performance, feed efficiency, and protein efficiency ratio but had no effect on nitrogen retention (% N intake). Independently of FM level, dietary tau supplementation improved growth performance and nitrogen retention without affecting feed efficiency. Dietary FM level reduction increased liver G6PDH activity, but did not affect lipid peroxidation or activities of redox key enzymes. Contrarily, dietary tau supplementation decreased hepatic G6PDH and GPX activities and lipid peroxidation. Gene expression COX-2 was not affected either by FM or tau levels but TNF-α increased with the reduction of FM level but not with the tau level. Dietary tau supplementation decreased Casp3 and Casp9 expression regardless of dietary FM level. Overall, this study evidenced that dietary tau supplementation improved growth performance and antioxidant response and reduced intestine inflammatory and apoptosis processes.

Vegetable oil and carbohydrate-rich diets marginally affected intestine histomorphology, digestive enzymes activities, and gut microbiota of gilthead sea bream juveniles.

For an increased incorporation of plant ingredients in aquafeeds at the expense of fish meal (FM) and fish oil (FO), more knowledge is needed on the effects at the intestine level of dietary vegetable oils (VO) and carbohydrates (CH), and of possible interactions. For that purpose, in this study, the activities of digestive pancreatic enzymes (amylase, lipase, total alkaline proteases), gut microbiota, and histomorphology were assessed in gilthead sea bream (IBW 71.0 ± 1.5 g) fed four diets differing in lipid source (FO or a blend of VO) and carbohydrate content (0% or 20% gelatinized starch) for 81 days. No major changes in digestive enzyme activities were noticed in fish fed the experimental diets. Dietary VO, but not CH content, modified intestinal microbial profile, by increasing the similarity of bacterial communities. Especially when combined with CH, dietary VO promoted abnormal enterocyte architecture. Liver histology was also accessed, and an increased cytoplasmic vacuolization of hepatocytes was related with dietary CH inclusion, being only significantly different in fish fed FO-based diets. Overall, nutritional interactions between dietary lipid source and carbohydrate content were not observed on digestive enzyme activities and microbial profile. However, the intestine histological modifications observed in fish fed the VOCH+ diet suggest a negative interaction between dietary VO and CH. This requires a more in depth assessment in future studies as it can have negative consequences at a functional level.

Humoral and mucosal immune responses in meagre (Argyrosomus regius) juveniles fed diets with varying inclusion levels of carob seed germ meal.

Many studies have assessed the effects of incorporation of plant feedstuffs in fish diets on growth performance, whereas few studies have addressed the effects of fish meal replacement by plant protein sources on fish immune parameters. Thus, the aim of this study was to evaluate the effects on immune response of different inclusion levels of carob seed germ meal (CSGM) as partial replacement for fish meal in diets for meagre (Argyrosomus regius) juveniles. Fish were fed four experimental diets with increased CSGM inclusion levels [0% (control), 7.5% (CSGM7.5), 15% (CSGM15) and 22.5% (CSGM22.5)]. After 1, 2, and 8 weeks of feeding fish were sampled to determine haematological profile and several humoral parameters in plasma and intestine. Results showed that dietary inclusion of CSGM did not negatively affect the immune parameters of meagre. In addition, total numbers of red and white blood cells, as well as thrombocytes, lymphocytes, monocytes, and neutrophils counts were not affected by dietary treatments. All parameters evaluated in plasma were unaffected by dietary CSGM inclusion after 1 and 2 weeks of feeding, with only the haemolytic complement activity showing an increase in fish fed diets with CSGM after 1 week and in fish fed CSGM22.5 diet after 2 weeks. Regarding the innate immune parameters analysed in the intestine, it could be highlighted the increase in alkaline phosphatase and antiprotease activities in fish fed the diet with the higher inclusion of CSGM at 8 weeks. Overall, results suggest that high dietary CSGM inclusion do not compromise immune status or induce an inflammatory response in meagre juveniles.

The European seabass (Dicentrarchus labrax) innate immunity and gut health are modulated by dietary plant-protein inclusion and prebiotic supplementation.

Inclusion of prebiotics in aqua feeds, though a costly strategy, has increased as a means to improve growth. Still, its effects on health improvement are not fully disclosed. Regarding their immunestimulatory properties, research has focused on carbohydrates such as fructooligosaccharides and xylooligosaccharides demonstrating their modulatory effects on immune defences in higher vertebrates but few studies have been done on their impact on fish immunity. Replacing fish meal (FM) by plant protein (PP) sources is a current practice in the aquaculture business but their content in antinutrients is still a drawback in terms of gut well-functioning. This work intends to evaluate the short-term effect (7 or 15 days feeding the experimental diets) on juvenile European seabass (Dicentrarchus labrax) immune status of dietary i) replacement of FM by PP sources; ii) prebiotics supplementation. Six isoproteic (46%) and isolipidic (15%) diets were tested including a FM control diet (FMCTRL), a PP control diet (PPCTRL, 30 FM:70 PP) and four other diets based on either FM or PP to which short-chain fructooligosaccharides (scFOS) or xylooligosaccharides (XOS) were added at 1% (FMFOS, PPFOS, FMXOS, PPXOS). The replacement of FM by PP in the diets induced nitric oxide (NO) and lysozyme production, while immunoglobulins (Ig), monocytes percentage and gut interleukin 10 (IL10) gene expression were inhibited. Dietary scFOS supplementation inhibited total bactericidal activity and neutrophils relative percentage regardless protein source and increased plasma NO and thrombocytes percentage in fish fed FM-based diets, while monocytes percentage was increased in PPFOS-fed fish. XOS supplementation down-regulated immune gene expression in the gut while it partly enhanced systemic response. Inconsistency among results regarding FM replacement by PP-based ingredients exposes the need for further research considering both local and systemic responses. Distinct outcomes of prebiotic supplementation were highlighted reflecting sight-specific effects with no clear interaction with protein source.

Regulation of glucose and lipid metabolism by dietary carbohydrate levels and lipid sources in gilthead sea bream juveniles.

The long-term effects on growth performance, body composition, plasma metabolites, liver and intestine glucose and lipid metabolism were assessed in gilthead sea bream juveniles fed diets without carbohydrates (CH-) or carbohydrate-enriched (20 % gelatinised starch, CH+) combined with two lipid sources (fish oil; or vegetable oil (VO)). No differences in growth performance among treatments were observed. Carbohydrate intake was associated with increased hepatic transcripts of glucokinase but not of 6-phosphofructokinase. Expression of phosphoenolpyruvate carboxykinase was down-regulated by carbohydrate intake, whereas, unexpectedly, glucose 6-phosphatase was up-regulated. Lipogenic enzyme activities (glucose-6-phosphate dehydrogenase, malic enzyme, fatty acid synthase) and ∆6 fatty acyl desaturase (FADS2) transcripts were increased in liver of fish fed CH+ diets, supporting an enhanced potential for lipogenesis and long-chain PUFA (LC-PUFA) biosynthesis. Despite the lower hepatic cholesterol content in CH+ groups, no influence on the expression of genes related to cholesterol efflux (ATP-binding cassette G5) and biosynthesis (lanosterol 14 α-demethylase, cytochrome P450 51 cytochrome P450 51 (CYP51A1); 7-dehydrocholesterol reductase) was recorded at the hepatic level. At the intestinal level, however, induction of CYP51A1 transcripts by carbohydrate intake was recorded. Dietary VO led to decreased plasma phospholipid and cholesterol concentrations but not on the transcripts of proteins involved in phospholipid biosynthesis (glycerol-3-phosphate acyltransferase) and cholesterol metabolism at intestinal and hepatic levels. Hepatic and muscular fatty acid profiles reflected that of diets, despite the up-regulation of FADS2 transcripts. Overall, this study demonstrated that dietary carbohydrates mainly affected carbohydrate metabolism, lipogenesis and LC-PUFA biosynthesis, whereas effects of dietary lipid source were mostly related with tissue fatty acid composition, plasma phospholipid and cholesterol concentrations, and LC-PUFA biosynthesis regulation. Interactions between dietary macronutrients induced modifications in tissue lipid and glycogen content.

Effect of short chain fructooligosaccharides (scFOS) on immunological status and gut microbiota of gilthead sea bream (Sparus aurata) reared at two temperatures.

The effects of dietary short chain fructooligosaccharides (scFOS) incorporation on hematology, fish immune status, gut microbiota composition, digestive enzymes activities, and gut morphology, was evaluated in gilthead sea bream (Sparus aurata) juveniles reared at 18 °C and 25 °C. For that purpose, fish with 32 g were fed diets including 0, 0.1, 0.25 and 0.5% scFOS during 8 weeks. Overall, scFOS had only minor effects on gilthead sea bream immune status. Lymphocytes decreased in fish fed the 0.1% scFOS diet. Fish fed the 0.5% scFOS diet presented increased nitric oxide (NO) production, while total immunoglobulins (Ig) dropped in those fish, but only in the ones reared at 25 °C. Red blood cells, hemoglobin, bactericidal activity and NO were higher at 25 °C, whereas total white blood cells, circulating thrombocytes, monocytes and neutrophils were higher at 18 °C. In fish fed scFOS, lymphocytes were higher at 18 °C. Total Ig were also higher at 18 °C but only in fish fed 0.1% and 0.5% scFOS diets. No differences in gut bacterial profiles were detected by PCR-DGGE (polymerase chain reaction denaturing gradient gel electrophoresis) between dietary treatments. However, group's similarity was higher at 25 °C. Digestive enzymes activities were higher at 25 °C but were unaffected by prebiotics incorporation. Gut morphology was also unaffected by dietary prebiotic incorporation. Overall, gut microbiota composition, digestive enzymes activities and immunity parameters were affected by rearing temperature whereas dietary scFOS incorporation had only minor effects on these parameters. In conclusion, at the tested levels scFOS does not seem worthy of including it in gilthead sea bream juveniles diets.

Prebiotics effect on immune and hepatic oxidative status and gut morphology of white sea bream (Diplodus sargus).

The aim of this study was to evaluate the effects of short-chain fructooligosaccharides (scFOS), xylooligosaccharides (XOS) and galactooligosaccharides (GOS) on immune and hepatic oxidative status, and gut morphology of white sea bream juveniles. Four diets were formulated: a control diet with fish meal (FM) and plant feedstuffs (PF) (30FM:70PF) and three test diets similar to the control but supplemented with 1% of scFOS, XOS or GOS. Dietary prebiotic incorporation did not affect total blood cell counts, hematocrit, hemoglobin, red blood indices or differential white blood cell counts. Fish fed GOS had lower ACH50 and nitric oxide than fish fed control diet. XOS enhanced immune status through the increase in alternative complement pathway (ACH50), lysozyme and total immunoglobulin. The higher activity of glucose 6-phosphate dehydrogenase in fish fed FOS compared to the other dietary groups was the only related antioxidant enzyme affected by prebiotics in the liver. GOS ameliorated the precocious adverse effects of PF based diet on gut histomorphology, as denoted by the lower incidence of histological alterations in fish fed GOS for 15 days. In conclusion, XOS and GOS at 1% might have potential to be used as prebiotics in white sea bream juveniles.

Metabolic responses to dietary protein/carbohydrate ratios in zebra sea bream (Diplodus cervinus, Lowe, 1838) juveniles.

This study aims to evaluate the effect of diets with different protein to carbohydrate ratios (P:C) on the omnivorous zebra sea bream (Diplodus cervinus) juveniles growth performance, feed efficiency, N excretion and metabolic response of intermediary metabolism enzymes. Four isoenergetic and isolipidic diets were formulated to contain increasing protein levels (25, 35, 45 and 55%) at the expense of carbohydrates (43, 32, 21 and 9%): diets P25C43, P35C32, P45C21 and P55C9. Growth performance, feed efficiency (FE), N intake [(g kg(-1) average body weight (ABW) day(-1))], N retention (g kg(-1) ABW day(-1)) and energy retention (kJ kg(-1) ABW day(-1)) increased with the increase of P:C ratio. The best growth performance and FE were achieved with diet P45C21. Ammonia excretion (mg NH4­N kg(-1) ABW day(-1)) increased as dietary protein level increased. Alanine aminotransferase and glutamate dehydrogenase activities increased with the increase of dietary P:C ratio. The opposite was observed for malic enzyme activity. Aspartate aminotransferase, hexokinase, glucokinase, fructose-1, 6-bisphosphatase and fatty acid synthetase activities were unaffected by dietary treatments. Response of key amino acid catabolic enzymes and N excretion levels to dietary P:C ratio supports the metabolic adaptability of this species to dietary protein inclusion levels. Overall, zebra sea bream seems capable of better utilize dietary protein rather than dietary carbohydrates as energy source which may be an obstacle for using more economically diets and thus for reducing environmental N loads in semi-intensive aquaculture of this species.

Effects of fish oil replacement by vegetable oil blend on digestive enzymes and tissue histomorphology of European sea bass (Dicentrarchus labrax) juveniles.

The impact of replacing circa 70% fish oil (FO) by a vegetable oil (VO) blend (rapeseed, linseed, palm oils; 20:50:30) in diets for European sea bass juveniles (IBW 96 ± 0.8 g) was evaluated in terms of activities of digestive enzymes (amylase, lipase, alkaline phosphatase, trypsin and total alkaline proteases) in the anterior (AI) and posterior (PI) intestine and tissue morphology (pyloric caeca-PC, AI, PI, distal intestine-DI and liver). For that purpose, fish were fed the experimental diets for 36 days and then liver and intestine were sampled at 2, 6 and 24 h after the last meal. Alkaline protease characterization was also done in AI and PI at 6 h post-feeding. Dietary VO promoted higher alkaline phosphatase activity at 2 h post-feeding in the AI and at all sampling points in the PI. Total alkaline protease activity was higher at 6 h post-feeding in the PI of fish fed the FO diet. Identical number of bands was observed in zymograms of alkaline proteases of fish fed both diets. No alterations in the histomorphology of PC, AI, PI or DI were noticed in fish fed the VO diets, while in the liver a tendency towards increased hepatocyte vacuolization due to lipid accumulation was observed. Overall, and with the exception of a higher intestine alkaline phosphatase activity, 70% FO replacement by a VO blend in diets for European sea bass resulted in no distinctive alterations on the postprandial pattern of digestive enzyme activities and intestine histomorphology.

Gut morphology and hepatic oxidative status of European sea bass (Dicentrarchus labrax) juveniles fed plant feedstuffs or fishmeal-based diets supplemented with short-chain fructo-oligosaccharides and xylo-oligosaccharides.

The effects of short-chain fructo-oligosaccharides (scFOS) and xylo-oligosaccharides (XOS) on gut morphology and hepatic oxidative status were studied in European sea bass juveniles weighing 60 g. Fish were fed diets including fishmeal (FM diets) or plant feedstuffs (PF diets; 30 FM:70 PF) as main protein sources (control diets). Four other diets were formulated similar to the control diets but including 1 % scFOS or 1 % XOS. At the end of the trial, fish fed PF-based diets presented histomorphological alterations in the distal intestine, whereas only transient alterations were observed in the pyloric caeca. Comparatively to fish fed FM-based diets, fish fed PF diets had higher liver lipid peroxidation (LPO), superoxide dismutase (SOD) and catalase (CAT) activities, and lower glutathione peroxidase, glutathione reductase and glucose 6-phosphate dehydrogenase activities. In fish fed the PF diets, prebiotic supplementation decreased SOD activity and XOS supplementation further decreased CAT activity. In fish fed the FM diets, XOS supplementation promoted a reduction of all antioxidant enzyme activities. Overall, dietary XOS and scFOS supplementation had only minor effects on gut morphology or LPO levels. However, dietary XOS reduced antioxidant enzymatic activity in both PF and FM diets, which indicate a positive effect on reduction of hepatic reactive oxygen species production.

Dietary carbohydrate and lipid source affect cholesterol metabolism of European sea bass (Dicentrarchus labrax) juveniles.

Plant feedstuffs (PF) are rich in carbohydrates, which may interact with lipid metabolism. Thus, when considering dietary replacement of fishery by-products with PF, knowledge is needed on how dietary lipid source (LS) and carbohydrates affect lipid metabolism and other metabolic pathways. For that purpose, a 73-d growth trial was performed with European sea bass juveniles (IBW 74 g) fed four diets differing in LS (fish oil (FO) or a blend of vegetable oils (VO)) and carbohydrate content (0 % (CH-) or 20 % (CH+) gelatinised starch). At the end of the trial no differences among diets were observed on growth and feed utilisation. Protein efficiency ratio was, however, higher in the CH+ groups. Muscle and liver fatty acid profiles reflected the dietary LS. Dietary carbohydrate promoted higher plasma cholesterol and phospholipids (PL), whole-body and hepatic (mainly 16 : 0) lipids and increased muscular and hepatic glycogen. Except for PL, which were higher in the FO groups, no major alterations between FO and VO groups were observed on plasma metabolites (glucose, TAG, cholesterol, PL), liver and muscle glycogen, and lipid and cholesterol contents. Activities of glucose-6-phosphate dehydrogenase and malic enzyme - lipogenesis-related enzymes - increased with carbohydrate intake. Hepatic expression of genes involved in cholesterol metabolism was up-regulated with carbohydrate (HMGCR and CYP3A27) and VO (HMGCR and CYP51A1) intake. No dietary regulation of long-chain PUFA biosynthesis at the transcriptional level was observed. Overall, very few interactions between dietary carbohydrates and LS were observed. However, important insights on the direct relation between dietary carbohydrate and the cholesterol biosynthetic pathway in European sea bass were demonstrated.

Dietary carbohydrate and lipid sources affect differently the oxidative status of European sea bass (Dicentrarchus labrax) juveniles.

This study aimed to evaluate the effects of dietary lipid source and carbohydrate content on the oxidative status of European sea bass (Dicentrarchus labrax) juveniles. For that purpose, four diets were formulated with fish oil (FO) and vegetable oils (VO) as the lipid source and with 20 or 0 % gelatinised starch as the carbohydrate source, in a 2×2 factorial design. Liver and intestine antioxidant enzyme activities (catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPX), glutathione reductase (GR), glucose-6-phosphate dehydrogenase (G6PD)), hepatic and intestinal lipid peroxidation (LPO), as well as hepatic oxidative stress index (OSI), were measured in fish fed the experimental diets for 73 d (n 9 fish/diet). Carbohydrate-rich diets promoted a decrease in hepatic LPO and OSI, whereas the lipid source induced no changes. Inversely, dietary lipid source, but not dietary carbohydrate concentration, affected LPO in the intestine. Lower intestinal LPO was observed in VO groups. Enzymes responsive to dietary treatments were GR, G6PD and CAT in the liver and GR and GPX in the intestine. Dietary carbohydrate induced GR and G6PD activities and depressed CAT activity in the liver. GPX and GR activities were increased in the intestine of fish fed VO diets. Overall, effects of diet composition on oxidative status were tissue-related: the liver and intestine were strongly responsive to dietary carbohydrates and lipid sources, respectively. Furthermore, different metabolic routes were more active to deal with the oxidative stress in the two organs studied.