Dietary fatty acid supply can affect stress response in fish during early development. Although knowledge on the mechanisms involved in fatty acid regulation of stress tolerance is scarce, it has often been hypothesised that eicosanoid profiles can influence cortisol production. Genomic cortisol actions are mediated by cytosolic receptors which may respond to cellular fatty acid signalling. An experiment was designed to test the effects of feeding gilthead sea-bream larvae with four microdiets, containing graded arachidonic acid (ARA) levels (0·4, 0·8, 1·5 and 3·0 %), on the expression of genes involved in stress response (steroidogenic acute regulatory protein, glucocorticoid receptor and phosphoenolpyruvate carboxykinase), lipid and, particularly, eicosanoid metabolism (hormone-sensitive lipase, PPARα, phospholipase A2, cyclo-oxygenase-2 and 5-lipoxygenase), as determined by real-time quantitative PCR. Fish fatty acid phenotypes reflected dietary fatty acid profiles. Growth performance, survival after acute stress and similar whole-body basal cortisol levels suggested that sea-bream larvae could tolerate a wide range of dietary ARA levels. Transcription of all genes analysed was significantly reduced at dietary ARA levels above 0·4 %. Nonetheless, despite practical suppression of phospholipase A2 transcription, higher leukotriene B4 levels were detected in larvae fed 3·0 % ARA, whereas a similar trend was observed regarding PGE2 production. The present study demonstrates that adaptation to a wide range of dietary ARA levels in gilthead sea-bream larvae involves the modulation of the expression of genes related to eicosanoid synthesis, lipid metabolism and stress response. The roles of ARA, other polyunsaturates and eicosanoids as signals in this process are discussed.

Free radicals are continuously generated during an organism's lifetime. In order to understand the involvement in the oxidative status of fish, methionine and white tea were assayed as antioxidant supplements in diets for gilthead sea bream (Sparus aurata). For the purpose of this study, four isonitrogenous and isolipidic diets were formulated to contain 45 % of protein and 18 % lipid and 0·3 % methionine (Met diet), 2·9 % white tea dry leaves (Tea diet) and 2·9 % of white tea dry leaves+0·3 % methionine (Tea+Met diet). An unsupplemented diet was used as the control. Key enzymatic antioxidant defences, superoxide dismutase (SOD) isoenzyme profile, total, reduced and oxidised glutathione and oxidative damage markers were determined. The results showed that dietary methionine supplementation increased liver SOD activity, while white tea induced higher hepatic catalase activity. Dietary white tea induced a notable increase in Mn-SOD isoenzyme. This is the first study to provide evidence that dietary tea inclusion in fish feeding could be an important source of Mn with metabolic repercussions on antioxidant mechanisms.

In liver, through the reaction catalysed by alanine aminotransferase (ALT), alanine becomes an effective precursor for gluconeogenesis. In the present study amino-oxyacetate (AOA) was used to evaluate its effect on liver ALT activity of the carnivorous fish Sparus aurata. Moreover, the derived metabolic effects on metabolites and other key enzymes of glycolysis, gluconeogenesis and the pentose phosphate pathway were also studied. A dose-effect-dependent inhibition of AOA on hepatic cytosolic and mitochondrial ALT activity was observed in vitro. In vivo, AOA behaved as an inhibitor of hepatic cytosolic ALT activity. A long-term exposure to AOA increased pyruvate kinase activity in the liver irrespective of the composition of the diet supplied to fish. 1H NMR studies showed that inclusion of AOA to the diet decreased the hepatic levels of alanine, glutamate and glycogen. Moreover, 2H NMR analysis indicated a higher renewal rate for alanine in the liver of fish fed with a high-carbohydrate/low-protein diet, while AOA decreased alanine 2H-enrichment irrespective of the diet. The present study indicates that AOA-dependent inhibition of the cytosolic ALT activity could help to increase the use of dietary carbohydrate nutrients.

Age and growth of adult gilthead seabream, Sparus aurata were determined from 332 specimens (total N = 476), collected from purse-seiners, in the south-eastern Aegean Sea from November 2006 to January 2007. The fork length and body wet weight ranged from 26.5 to 51.5 cm (mean: 36.6 ± 0.18) and from 375 to 2600 g (mean: 1009.2 ± 14.16), respectively. Age groups were varied between 2 and 7. Growth parameters were estimated as L∞ = 64.97 ± 12.93 cm, K = 0.14 ± 0.07 year−1, t0 = −2.47 ± 1.09 year−1, and index of phi-prime was Φ′ = 2.772 ± 0.51. The length–weight relationship equation of all fish was calculated as W = 0.0515 × L2.737 (r2 = 0.95). The mean condition factor was estimated as 2.058. Mortalities (M, F and Z) and exploitation rate (E) of gilthead seabream from the Aegean Sea were 0.34 year−1, 0.77 year−1, 1.11 year−1and 0.69 year−1, respectively. The result of E indicated that the population was overexploited.

Live preys commonly used in fish larval rearing seem to be imbalanced in terms of amino acids. Manipulation of their amino acid composition is difficult, but the use of microencapsulated diets allows this manipulation. This study analysed the effect of amino acid supplementation, in order to compensate for dietary amino acid imbalances, on growth and survival of gilthead seabream (Sparus aurata) larvae. Larvae were reared until 32 days after hatching, in a closed recirculating water system (19 °C), using only live food (rotifers and Artemia). Thereafter, larvae were fed Artemia or one of the experimental microencapsulated diets: supplemented with indispensable amino acids (IAAsup diet), or supplemented with dispensable amino acids (DAAsup diet). Experiment lasted for 10 days. Dietary supplementation with indispensable amino acids resulted in A/E ratios [(each indispensable amino acid) × (total indispensable amino acids)−1 × 1000]more similar to the ones of larval seabream and in higher IAA:DAA ratios than in the DAAsup diet. Survival was similar in larvae fed the IAAsup diet (75%) or Artemia (87%), but was significantly lower in larvae fed the DAAsup diet (52%). Larvae from all treatments more than doubled their average dry weight during the experimental period. Final dry weight was similar in larvae fed both microcapsules, but these were lighter than larvae fed with Artemia. Relative growth rate (RGR) and total biomass production tended to be higher in larvae fed the IAAsup (RGR = 9% day−1) than the DAAsup diet (RGR = 7.5% day−1) and only in this last treatment these parameters were significantly lower than in larvae fed with Artemia. Therefore, dietary supplementation with indispensable amino acids resulted in a more balanced dietary amino acid profile, which significantly increased survival. Further studies introducing microdiets earlier in the development seem necessary in order to optimise growth.

To examine the role of glucose-6-phosphatase (G6Pase) in glucose homeostasis in the diabetes-like experimental model of carnivorous fish, we analysed postprandial variations and the effect of starvation, ration size and diet composition on the regulation of G6Pase expression at the enzyme activity and mRNA level in the liver of gilthead sea bream (Sparus aurata). G6Pase expression increased in long-term starved or energy-restricted fish. In contrast to data reported for other fish species, short-term regulation of G6Pase expression was found in regularly fed S. aurata. G6Pase mRNA levels were lowest between 4 and 15 h after food intake, whereas minimal enzyme activity was observed 10–15 h postprandially. Alterations of plasma glucose levels affect G6Pase in mammals. However, the carbohydrate content of the diet did not affect hepatic expression of G6Pase in S. aurata, suggesting that a different molecular mechanism is involved in the control of G6Pase expression in fish. Although G6Pase was unaffected, high-carbohydrate low-protein diets increased glucokinase (GK) expression and thus allowed a metabolic adaptation favouring glycolysis over gluconeogenesis. Interestingly, only the nutritional conditions that promoted variations in the blood glucose levels resulted in changes in the hepatic expression of G6Pase. These findings indicate a concerted regulation of G6Pase and GK expression and suggest that the direction and rate of the glucose–glucose-6-phosphate substrate cycle flux is finely regulated in the liver of S. aurata, challenging the role attributed to deficient regulation of G6Pase or GK expression in the low ability of carnivorous fish to metabolize glucose.

The effects of diet composition and ration size on the activities of key enzymes involved in intermediary metabolism were studied in the liver of gilthead sea bream (Sparus aurata). High-carbohydrate, low-protein diets stimulated 6-phosphofructo 1-kinase (EC 2.7.1.11), pyruvate kinase (EC 2.7.1.40), glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (EC 1.1.1.44) enzyme activities, while they decreased alanine aminotransferase (EC 2.6.1.2) activity. A high degree of correlation was found between food ration size and the activity of the enzymes 6-phosphofructo 1-kinase, pyruvate kinase, glucose-6-phosphate dehydrogenase (positive correlations) and fructose-1,6-bisphosphatase (EC 3.1.3.11) (negative correlation). These correlations matched well with the high correlation also found between ration size and growth rate in starved fish refed for 22 d. Limited feeding (5 g/kg body weight) for 22 d decreased the activities of the key enzymes for glycolysis and lipogenesis, and alanine aminotransferase activity. The findings presented here indicate a high level of metabolic adaptation to both diet type and ration size. In particular, adaptation of enzyme activities to the consumption of a diet with a high carbohydrate level suggests that a carnivorous fish like Sparus aurata can tolerate partial replacement of protein by carbohydrate in the commercial diets supplied in culture. The relationship between enzyme activities, ration size and fish growth indicates that the enzymes quickly respond to dietary manipulations of cultured fish.

The presence of protease inhibitors in different ingredients used in fish feeds may negatively affect their digestive utilisation. The effect of inhibitors present in several protein sources on the activity of digestive proteases of sea bream was assessed using ‘in vitro’ assays. Inhibition produced by extracts of plant proteins ranged from 25 to 50% of total activity, whereas that obtained using animal protein sources ranged from 1 to 20 %. The exception was ovoalbumin, which showed the highest measured value (62 %). A plot of the inhibition values obtained by changing the relative concentrations enzyme/inhibitor resulted in different curves for different protein sources. The use of sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) zymograms allowed visualisation of the aforementioned differences in inhibition. The importance of measuring protease inhibition in the preliminary evaluation of fish feed ingredients is discussed.

The common dentex (Dentex dentex) is a sparid fish which is considered a suitable candidate for Mediterranean aquaculture. A seven-weeks feeding trial was conducted over the summer period with common dentex, sea bream (Sparus aurata) and sea bass (Dicentrarchus labrax). All three species were fed to visual satiety with two practical diets with varying crude protein and crude fat levels (55 % protein, 9 % lipids; 46 % protein, 17 % lipids). The initial body weights were 8.2, 11.6 and 17.3 g for common dentex, sea bass and sea bream, respectively. In all cases, specific growth rates were not significantly affected by dietary treatment, but they varied among species (sea bass 1.7-1.8 %; sea bream 2.1-2.3 %; common dentex 3.1-3.2 %). When comparisons between fish species were made, we observed that the increase in growth rates was linked to a decrease of the whole body fat content, as a consequence of a greater utilisation of dietary lipids as energy substrates. Furthermore, in common dentex, the 17 % lipid diet was able to improve protein retention, but this diet effect was less significant in sea bream and sea bass. In contrast to sea bream, the 17 % lipid diet did not up-regulate plasma GH levels in common dentex, which provides additional evidence for a more efficient utilisation of dietary lipids.

A model to estimate the waste production from sea cage culture was established. Using known feed inputs of nitrogen, phosphorus and organic matter, the model quantifies waste discharge from seabream culture. Daily feed intake and growth in Sparus aurata fed a commercial diet with known composition were measured and found to be dependent on fish weight and water temperature. Digestibility of the commercial feed was measured using chromic oxide as a marker and collection of feces by stripping. The proximate composition of Sparus aurata at different sizes was determined and nitrogen and phosphorus content were on average 28.5 and 7.2 g·kg−1 body mass, respectively. Excretion of ammonia-nitrogen and inorganic phosphorus after metabolic processes was calculated as the difference.

On the basis of their respective metabolizable energy contents, supplemental marine oil was partially replaced with a maize starch/dextrin mixture in diets containing approximately 500 g crude protein per kg food thus lowering the total dietary lipid concentration from 141·6 to 100·9 g/kg. The basal diets were then supplemented with thiamin hydrochloride at 0·5, 5·0 and 10·0 mg/kg food to produce six test diets in all. After 182 days of feeding, the fish given the diets containing the higher lipid content with a thiamin supplement of 10·0 mg/kg had shown significantly better growth and food utilization (food conversion efficiency and apparent net protein utilization) than the fish presented with the remaining five diets. Thiamin supplements of 5 mg/kg or less resulted in poorer performance amongst the fish given the high lipid diets. The fish given all three of the low oil diets performed poorly by comparison with those given the high lipid, high thiamin diet and performance was comparable with that of the fish given the high lipid diets containing thiamin at 5 mg/kg or less. Proximate carcass composition in terms of moisture, protein and lipid was unaffected in response to diet. Glycogen accumulation in the liver was significantly elevated amongst the fish given the low lipid diet where the thiamin supplement was 5·0 mg/kg or more. The haematology of the fish was not significantly altered in response to diet although elevated haematocrit was associated with decreasing thiamin supplementation. It would appear that for the seabream, lipid and carbohydrate are not exchangeable on the basis of their metabolizable energy content. When given high lipid diets the thiamin requirement of the seabream is in excess of 5·0 mg/kg and increasing the thiamin supplement to 10 mg/kg in high carbohydrate diets made no impact on performance.

Semi-purified diets incorporating 495·4 g/kg crude protein and 72·0 g/kg oil, were used to identify the qualitative requirement of Spams aurata juveniles for thiamin, riboflavin, pyridoxine, niacin and pantothenic acid. Deficiency of these vitamins was associated with poor performance in terms of growth, food efficiency and apparent net protein utilization. All dietary vitamin deficiencies resulted in elevated moisture and a lowering of the lipid content of the carcass. In addition to increased mortality among the fish fed deficient diets, a small number of externally visible changes were observed including food refusal, anorexia and changes of skin colour. At the haematological level, all cases of vitamin deficiency were associated with significant reductions in haematocrit. In general, histological examination revealed that the pancreas of vitamin-deficient animals became atrophic with an accumulation of pigmented granules around this organ while additionally, the normal homogeneity of the liver parenchyma was lost.

Semi-purified diets were used to determine the nicotinic acid requirement of the gilthead seabream, Sparus aurata. Vitamin losses during food manufacture were minimal resulting in dietary levels which were close to the declared content. Dietary nicotinic acid contents of 52·21 mg/kg and below resulted in sub-optimum growth, reduced food efficiency and poor values for apparent net protein utilization. Diets containing less than 28·67 mg/kg gave a low proportion of carcass lipid concomitant with an elevation in the relative content of carcass moisture. Although gross deficiency symptoms were not observed, a reduced hepatosomatic index and a slightly lower plasma glucose concentration were detected amongst the fish given diets supplemented with nicotinic acid at the level of 25 mg/kg or less. Based upon the results of broken line analysis and data derived by modelling the weight gain of the fish, the nicotinic acid requirement of Sparus aurata fingerlings lies between 63 and 83 mg/kg of diet or 1·57 to 2·07 µg/kg body weight per day.

The interaction between essential dietary components and changes in tissue nutrient reserves, egg quality and egg composition, were studied from 60 d before and during the spawning of Sparus aurata broodstock. Fish were given isonitrogenous (550 g/kg dry weight) and isolipidic (100 g/kg dry weight) diets, based on protein and lipid extracts of squid meal. Diets differed in the levels of n−6 (10–30 mg/g dry weight) and n−3 (0–10 mg/g dry weight) essential fatty acids. The effects of these diets on biochemical and fatty acid composition of body tissues, and the subsequent effects on egg composition and egg viability were measured. Dietary essential fatty acids were mostly incorporated into the liver, ovaries, digestive tract and associated adipose tissues. The lipid composition of these tissues reached an equilibrium with dietary lipid composition within 15 d of feeding on any given diet. Muscle and gill cartilage tissues did not show any significant changes in their biochemical and fatty acid composition, even after 60 d feeding. Egg viability decreased significantly within 10 d of feeding the broodstock with a diet deficient in n−3 highly unsaturated fatty acids (n−3 HUFA). The levels of n−3 HUFA in both polar and neutral fractions of egg lipid were directly correlated with their levels in the broodstock diet. When the total amount of egg n−3 HUFA dropped below 17 mg/g dry weight, egg viability and larvae hatching rate decreased by 53% and 47 % respectively. These results suggest that the biochemical composition of organs involved in S. aurata reproduction are highly sensitive to the nutritional value of the diet, which affects egg and larval quality rapidly.