Histological alterations in the liver of sea bream, Sparus aurata L., caused by short- or long-term feeding with vegetable oils. Recovery of normal morphology after feeding fish oil as the sole lipid source

This study evaluated the effects of fish oil (FO) replacement by vegetable oils [soybean oil (SO), rapeseed oil (RO), linseed oil (LO)] and subsequent feeding with FO on the liver morphology of sea bream. A short-term trial (3 months) and long-term trial (6 months) were carried out feeding sea bream with the following experimental diets: FO100%; SO60% + FO40%; RO60% +FO40%; LO60% + FO40%; SO + RO +LO60% + FO40%. Finally, all groups from the long-term trial were fed with FO100% for 95 days (washout period). Liver samples were taken for histological and biochemical studies. In both the short- and long-term trials, livers of sea bream fed LO60% and SO + RO + LO60% showed a similar hepatic morphology to that observed in fish fed FO100%. In contrast, sea bream fed SO60% showed an intense steatosis, with foci of swollen hepatocytes containing numerous lipid vacuoles. After the washout period, a considerable reduction of the cytoplasmic vacuolation and the lipid vacuole accumulation were observed in the livers of fish fed the different experimental diets. The results of this study suggested that the type of non-essential fatty acid, characteristic of vegetable oils, induces the appearance of steatosis in the following order: linoleic acid > linolenic acid > oleic acid. However, the liver alterations found during the experimental periods with vegetable oils are reversible when the fish are re-fed with a balanced diet (FO100%), indicating the non-pathological character of these histological changes.     

Amino acid retention of gilthead sea bream (Sparus aurata,L.) fed with pea protein concentrate

A trial of 218 days of duration was carried out to assess the use of pea protein concentrate (PPC) as a substitute for fish meal in diets for juvenile gilthead sea bream (52 g average initial weight), using four diets (0, 16, 32 and 48) with PPC inclusion levels of 0, 162, 325 and 487 g kg^?1, respectively. At the end of the trial, the fish reached weights of 397, 385, 383 and 355 g for 0, 16, 32 and 48 diets, respectively; diet 48 gave the lowest specific growth rate (SGR, 0.88% per day) but 0%, 16% and 32% PPC did not present statistical differences. Feed conversion ratio (FCR), protein efficiency ratio (PER) and body composition were not affected by the diets. Digestible protein retention was significantly higher with the 0% diet, but no energy retention differences were observed. Retention rates of ingested and digestible amino acids were very similar; in general, the retention of essential amino acids decreased as dietary PPC increased, and lysine retention was the highest and phenylalanine retention was the lowest. From the results of this experiment, it may be concluded that fish meal can be replaced up to 32% by PPC in sea bream without negative effects on fish weight gain, FCR and PER.     

Influence of dietary levels of soybean meal on the performance and gut histology of gilthead sea bream (Sparus aurata L.) and European sea bass (Dicentrarchus labrax L.)

The present study was performed to determine the effect of soybean meal (SBM) on the performance and gut histology of gilthead sea bream and European sea bass. Three isonitrogenous and isolipidic extruded diets (crude protein, 470 g kg⁻¹ diet; crude fat, 200 g kg⁻¹ diet) were formulated containing 0 (0 SBM), 180 (180 SBM) and 300 (300 SBM) g kg⁻¹ diet SBM and tested on both species in two separate experiments. Fish at an initial average weight of around 18 g were randomly allocated to 800 L square tanks connected to a closed recirculating system. The trials lasted 80 days for sea bream and 89 days for sea bass. Fish were hand-fed to apparent satiation. Increasing the level of SBM had no significant effects on the specific growth rate, feed intake and feed conversion rate in both the species. In the sea bream distal intestine, lamina propria was moderately and diffusely expanded in some fish due to an increase in cell infiltration represented by mononuclear cells, this finding being more frequent in animals fed diet 300 SBM. No other morphological alterations in intestinal folds, enterocytes or other inflammation signs were noticed in the sea bream distal intestine of any group. No histological differences were found in sea bass in any experimental group.     

Preliminary evaluation of pea seed meal in diets for gilthead sea bream (Sparus aurata) juveniles

Juvenile gilthead sea bream with a mean initial body weight of 5 g were fed for 12 weeks with experimental diets containing 10% and 20% fishmeal protein (sole protein source in the control diet) replaced by processed pea seed meals. The processed pea seed meals were dehulled, defibred, extruded and microground pea seed meal (PSM1) or whole pea treated by infrared radiation and ground (PSM2). Apparent digestibility coefficients of the experimental diets were determined in a separate trial. At the end of the growth trial there were no significant differences in growth performance, feed utilization or whole-body composition among experimental groups. There were no differences in apparent protein digestibility among experimental groups (except for fish fed PSM1 at the lowest inclusion level). Both dry matter and energy digestibility of the diets, including PSM2 and with the highest inclusion level of PSM1, were significantly lower than those of the control diet. The results of this study suggest that pea seed meal may replace up to 20% fishmeal protein in diets for gilthead sea bream juveniles without affecting fish performance. Further studies should focus on technological treatments to increase utilization of pea seed meal carbohydrate, as both apparent dry matter and energy digestibility were affected by dietary inclusion level and by pea seed meal processing method.     

Toxicity of intraperitoneally injected ivermectin in sea bream, Sparus aurata

Ivermectin has been widely used as a treatment against sea lice infections of farmed fish (salmonids and non-salmonids) without any official license for use in aquaculture. This study presents data on the toxic side effects of ivermectin administered intraperitoneally in sea bream, Sparus aurata at single doses of 100, 200, 400 and 800 μg ivermectin kg⁻¹ fish. The toxicity of ivermectin was evaluated for a period of 35 days post treatment by determining changes in selected hematological parameters (hematocrit, hemoglobin, red blood cell count, leucocyte count, differential white blood cell count, plasma glucose, electrolytes, urea and alkaline phosphatase), pathological effects in the tissues and the organs of the fish using histological techniques and changes in the behavior of the fish. The results showed a decrease of the hematocrit value one hour after the injection for most of the doses along with an increase of the hemoglobin concentration, the number of the white blood cells, the lymphocytes and monocytes and plasma glucose. No mortality or any histopathological alteration at the tissues examined, were recorded. The fish, which were injected with the highest dose of ivermectin, experienced a loss of appetite, lethargy and darkening of their color during the first week post injection. All the studied parameters reached the normal level by the end of the experiment indicating a low and reversible toxicity of the drug. This revised version was published online in August 2006 with corrections to the Cover Date.     

A new tool for determining the optimum fish meal and vegetable meals in diets for maximizing the economic profitability of gilthead sea bream (Sparus aurata,L.) feeding

The economic profitability of fish meal replacement by soybean meal and pea‐rice concentrate mixture, was analysed considering the economic conversion ratio (ECR) and the economic profit index (EPI_st) as a proposal for evaluating the economic profitability in diets for sea bream. The optimal fish meal levels for minimizing the ECR were 33% (when soybean meal was included) and 14% (in the case of pea‐rice concentrate mixture) and for maximizing the EPI_st, optimal fish meal levels were 31% and 28.8%, respectively, considering the current market price of fish meal and vegetal ingredients. The EPI_st, seems to be a better index than the ECR to estimate diet profitability, because it considers weight increment, feed conversion ratio, diet cost and fish sale price. A sensibility analysis was conducted, considering variations in the price of fish meal and vegetal ingredients. Finally, several multiple regressions were developed for estimating the optimal level of fish meal with each vegetable ingredient and the expected value of the economic profit as a function of the market price of both fish meal and plant protein. When fish meal prices increase, the highest beneficial level of the inclusion fish meal and the highest economic profit would be achieved with pea‐rice mixture.     

Evaluation of corn gluten meal as a protein source in diets for gilthead sea bream (Sparus aurata L.) juveniles

A 12‐week growth trial was conducted to evaluate corn gluten meal as an alternative protein source to fish meal in diets for gilthead sea bream juveniles. The experimental diets were formulated to be isonitrogenous and isoenergetic and to have 20%, 40%, 60% and 80% of fish meal protein, the only protein source in the control diet, replaced by corn gluten meal. At the end of the growth trial only the group fed the diet with 80% corn gluten protein exhibited significantly reduced growth and feed efficiency compared with the fish meal‐based diet. This was most likely due to a dietary amino acid deficiency in that diet. A trend was noticed for feed efficiency to improve with the replacement of fish meal protein in the diets up to 60%. There were no significant differences among groups in protein and energy retention (as percentage of intake). At the end of the trial whole body water content of the experimental groups was significantly lower and the lipid content of groups including 60% and 80% corn gluten protein was significantly higher than that in the control. No other differences were observed in whole body composition among groups. Apparent digestibility coefficients (ADC) of the diets were evaluated in a separate trial. The ADC of dry matter of the experimental diets was significantly higher than in the control diet; there were no significant differences among diets in the ADC of energy and protein, except for the ADC of protein of diet with 80% corn gluten protein, which was significantly lower than the control. The results of this study indicate that corn gluten meal can replace up to 60% fish meal protein in diets for gilthead sea bream juveniles with no negative effects on fish performance.     

Effects of dietary orange peel on growth performance,antioxidant activity,intestinal microbiota and liver histology of Gilthead sea bream (Sparus aurata) larvae

This study investigated the effects of dietary orange peel (OP) on growth performance, feed utilization, antioxidant activity, intestinal microbiota and liver histology of Gilthead sea bream (Sparus aurata) larvae (0.32 ± 0.01 g/fish) (mean ± SD). Fish fed iso‐nitrogenous (480 g/kg protein) and iso‐energetic (23 MJ/kg) diets supplemented with OP at concentrations of 0, 1, 3 or 5 g/kg diet, for 60 days. Growth performance and feed utilization parameters were significantly improved by the elevation of dietary OP level, and the optimum level was 5 g/kg diet. The maximum activity of the total antioxidant capacity, superoxide dismutase, glutathione peroxidase, catalase, alkaline phosphatase enzyme and malondialdehyde in the liver was found at 5.5, 4.6, 3.4, 2.9, 3.7 and 3.8 g OP/kg diet, respectively. All tested bacteria (Staphylococcus spp., Vibrio spp. and Salmonela spp) and total bacterial count decreased significantly in the gut of fish fed high levels of OP (3.0 or 5.0 g/kg). No differences were found in the liver histo‐architecture among treatments after 60 days feeding on OP diets. In summary, dietary OP improved growth rate, antioxidant activity and intestinal microbiota of S. aurata larvae with an optimum range from 2.9 to 5.5 g/kg diet.     

Evaluation of immunological parameters in farmed gilthead sea bream, Sparus aurata L., before and during outbreaks of 'winter syndrome'

In recent years, 'winter syndrome' has caused severe economic losses in many marine farms in southern Europe. This study compared the activity of the immune system in healthy, asymptomatic and diseased gilthead sea bream. Serum protein and immunoglobulin content were evaluated as well as serum complement activity from these groups of fish. Differential leucocyte counts were also determined. Immunological assays were performed to determine intra- and extracellular 'respiratory burst' activity as non-specific immune response parameters. Diseased fish showed a significant reduction in haemolytic activity (90%), and in serum proteins and immunoglobulins in comparison with the other groups. A significant increase in the lymphocyte percentage (50%) and a decrease in the granulocyte percentage (over 70%) was found in asymptomatic fish. Respiratory burst activity was reduced in both the clinical and preclinical stages of the disease, compared with the controls. These observations confirm a severe immunodeficiency in diseased fish but also the presence of a cellular immune dysfunction in fish without clinical signs, before the onset of the disease.     

Effects of chronic confinement on physiological responses of juvenile gilthead sea bream, Sparus aurata L., to acute handling

Understanding how gilthead sea bream, Sparus aurata L., an important Mediterranean Sea species for aquaculture, respond physiologically to stressors commonly encountered in intensive rearing is important for effective production, as managing for stress is a major factor in maintaining healthy fish stocks. Our objective was to determine whether holding juvenile gilthead sea bream at a high density (HD), as a chronic stressor, would affect their physiological responses to a subsequent acute handling stressor. After acclimation at a low density (LD) of 6 kg m^?3 in 200‐L circular tanks containing 33–36 g L^?1 recirculating seawater at 19°C under a normal photoperiod, juvenile 37‐g gilthead sea bream were confined for 14 days at a HD of 26 kg m^?3 and then subjected to 30‐s aerial emersion in a dipnet. Plasma levels of cortisol, glucose, lactate, osmolality and chloride were determined in fish held in separate lots during LD (control) and HD confinement at 0, 1, 2, 7 and 14 days, and then after handling at 0, 1, 2, 4 and 8 h. Although plasma cortisol levels were similar in LD and HD fish groups after 14 d of confinement (15 and 23 ng mL^?1, respectively), the cortisol response in fish from the HD treatment at 1 and 2 h following acute handling (70 and 37 ng mL^?1, respectively) was only about half of that measured in the control group (139 and 102 ng mL^?1); plasma cortisol was similar in both groups by 4 and 8 h. In contrast, plasma glucose elevations in response to handling were higher at 4 and 8 h in the HD‐held fish (94 and 72 mg dL^?1, respectively) than in those from the LD treatment (59 and 51 mg dL^?1); glucose responses were similar in both groups at 1 and 2 h after handling and throughout confinement. Plasma lactate levels were higher in LD fish than in the HD group at the beginning of the experiment but were similar after 14 d confinement and responses to handling were similar (e.g. 33 and 35 mg dL^?1 at 1 h). Plasma osmolality showed increases during the first 2 h after acute handling but no differences were evident between the two density treatments at any time during confinement or posthandling. Plasma chloride levels did not change throughout the experiment. The reduced plasma cortisol response to acute handling likely resulted from negative feedback of mildly but chronically elevated circulating cortisol caused by the confinement stressor on the hypothalamic–pituitary–interrenal axis. While other post‐handling physiological changes also showed differences between treatment groups, the suppressed cortisol response in the HD‐held fish suggests a reduction in the gilthead sea bream's normal capacity to respond to an acute stressor.     

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.     

Biochemical differences in lateral muscle of wild and farmed gilthead sea bream (series Sparus aurata L.)

Red and white muscle from specimens of wild and farmed gilthead sea bream (Sparus aurata) were analyzed for histochemical ATPase activity, total protein content, fatty acids, trace element concentrations and myosin isoforms. The fibre type composition of muscle samples was confirmed histochemically by the ATPase reaction, which did not show any differences between the two groups of animals. Myosin ATPase activities, myosin and protein yields were significantly higher in white muscle than in the red muscle and for the red muscle the latter two parameters were higher in wild fish. Fatty acid profiles revealed differences between the two groups of animals, probably because of the fatty acid composition of the diets. Zinc, copper and iron concentrations were higher in red muscle than in white muscle; muscles from wild fish were significantly richer in trace elements. No separation of fast and slow heavy chains of myosin could be obtained on SDS-gel electrophoresis, but two dimensional electrophoresis revealed the presence of three light chains in white muscle (LC1F, LC2F, LC3F), and two main types in red muscle (LC1S, LC2S). Small, variable percentages of LC3F were found in the red muscle samples, especially in the wild fish. It is concluded that the different environmental conditions, experienced by wild and farmed fish, have significantly influenced the biochemical composition of their lateral muscle.     

Effect of diets containing a purified soybean trypsin inhibitor on growth performance,digestive proteases and intestinal histology in juvenile sea bream (Sparus aurata L.)

Juvenile sea bream were fed on diets containing 0.0, 2.0 or 4.0 g kg^?1 of a soybean trypsin inhibitor (SBTI) for 30 days. The growth performance, total protease activity and intestinal histology were studied after 0, 15 and 30 days of dietary treatment. No significant differences were found in the weight gain, specific growth rate (SGR) and feed conversion rate in fish fed on inhibitor‐supplemented diets when compared with those fed on an inhibitor‐free diet. Only the SGR at day 15 decreased significantly with protease inhibitor inclusion, although this effect was not observed at day 30. In relation to proteolytic activity at day 15, the total protease activity in the distal intestine decreased in fish fed on inhibitor‐supplemented diets. Zymograms of these extracts showed that the SBTI reduced the intensity of some proteolytic fractions in the distal intestine. A noticeable reduction in the protease activity of the intestinal content in fish fed on the highest level of soybean inhibitor (4.0 g kg^?1) was also observed. However, at day 30, the inhibition effect on these active bands was not detected, and the total protease activity was similar to that in fish fed on an inhibitor‐free diet. Histological examination revealed no perceptible differences in the intestinal structure between any of the diet groups. In addition, all fish were maintained under experimentation for 10 more days and fed on an inhibitor‐free diet to determine whether the possible effects caused by the protease inhibitor could be reverted. The administration of SBTI‐supplemented diets did not affect sea bream growth performance or intestine histology after 30 days, and only a decrease in the total alkaline protease activity was found at day 15.     

Intestinal bacterial flora of Mediterranean gilthead sea bream (Sparus aurata Linnaeus) as a novel source of natural surface active compounds

Microbial communities play different functions in the digestive tract of fish and represent a source of bioactive compounds. This study was aimed to detect the presence of biosurfactant (BS)‐producing bacteria in the intestine of gilthead sea bream (Sparus aurata L.) to select strains candidate for biotechnological applications in aquaculture. A total of 100 bacterial strains were isolated from the guts of three groups of fish and analysed by different qualitative screening tests and the tensiometric analysis. BSs were extracted, characterized by thin‐layer chromatography (TLC) and their antibacterial activity against bacterial pathogens of interest in aquaculture was determined. A total of 17 out of 100 strains, affiliated to the genera Pseudomonas (11 isolates), Acinetobacter (1 isolate), Sphingomonas (4 isolates) and Aeromonas (1 isolate), displayed a stable emulsion production with the E₂₄ index from 0% to 44%, a surface tension reduction from 8.3 to 30 mN/m and 11 of them exhibited antibacterial activity against fish pathogens. The TLC analysis indicated that the intestinal BSs consisted of compounds ascribed to the class of glycolipids–rhamnolipids. This is the first study reporting on the isolation and selection of BS‐producing bacteria from fish gut, a natural novel source of surface active compounds with potential biotechnological applications in aquaculture.     

Comparison of the swimming performance of farmed and wild gilthead sea bream, Sparus aurata

Farmed gilthead sea bream, Sparus aurata, frequently escape from the sea cages and interact with wild populations. The impact of these interactions on the wild populations will depend, in part, on differences in performance of the bream. This study compared the swimming performance of the wild and farmed fish in a current channel. The absolute critical swimming speed (U_crit) increased with increasing size while the relative U_crit decreased. Even at the same length there were noticeable performance differences between the individuals. The wild sea bream have significantly higher (P<0.05) absolute U_crit performance (0.86±0.01 m s⁻¹) than the farmed fish (0.79±0.01 m s⁻¹) and significantly higher (P<0.05) relative U_crit performance (4.52±0.05 BL s⁻¹) than the farmed fish (4.21±0.05 BL s⁻¹). The present study suggests that cultured sea bream may not have the ability to compete with wild sea bream in native seawaters.     

Florfenicol depletion in edible tissue of rainbow trout,Oncorhynchus mykiss (Walbaum), and sea bream,Sparus aurata L.

An increase in fish production has consequently brought an increase in infectious diseases in fish farms. The use of chemotherapic drugs is the most effective instrument against common bacterial agents. The number of registered drugs for use in aquaculture is limited and often veterinary practitioners resort to the off‐label use of chemotherapic agents authorized for different food‐producing animal species. Florfenicol is well known for its outstanding effect against various pathogenic bacteria affecting fish, and therefore, it may be a useful drug for off‐label use in aquaculture. The aim of this study was to evaluate the depletion of florfenicol and its major metabolite, florfenicol amine, from the edible tissue of two fish species, rainbow trout and sea bream, following treatment with medicated feed at a dosage of 10 mg kg^?1 of bw day^?1, for 10 consecutive days. At prefixed time points after the end of administration (0.25, 1, 2, 3, 4, 6, 7, 10, 14 and 21 days after treatment), edible tissues (muscle plus adherent skin) from 15 individuals in each group were collected and analysed by HPLC, to determine concentration of the drug in the tissue. On the basis of the obtained concentrations, withdrawal times of florfenicol in the two species were calculated. The results indicate that a drug withdrawal time of 500 °C‐day, as established by Directive 2004/28/EC, for off‐label drug use is more than satisfactory to guarantee the healthiness of fish products against the risk of drug residues.     

Effects of protein level of the diet on digestibility and growth of gilthead sea bream, Sparus auratus L.

A feeding trial with a duration of 12 weeks was conducted to determine the effects of various protein levels, in relation to diet digestibility and growth of the gilthead sea bream, Sparus aurata L. Four experimental fish meal/wheat meal based diets (A,B,C,D) containing 400, 450, 500 and 550 g kg^?1 protein, respectively, were tested. The increase of the fishmeal content of the diet led to an increment of dry matter digestibility. Apparent digestibility coefficients of protein and lipids were always very high (being near or even over 90%). Energy digestibility coefficients increased from diet A to diet D, which corresponds to a decrease in the wheat meal content of the diet. Voluntary feed intake increased with the decrease of protein content of the diet (from diet D to diet A). In the other sense, feed/gain ratio decreased regularly as protein percentage increased (from diet A to diet D). The most favourable feed/gain ratio, 1.07, was noted for the group receiving 55% protein (diet D). Fish on the lowest protein diet (Diet A) showed the highest protein efficiency ratio (PER) and the highest percentage retention of the digestible protein intake. Other than slight positive differences between fish fed diets with 500 and 550 g kg^?1 protein, no significant differences were observed for growth when dietary protein exceeded 450 g kg^?1. Beyond this level, no significant difference in final average individual weight was observed. Although it is generally considered that the dietary protein requirement for gilthead sea bream is 400 g kg^?1, our experiment demonstrates that to obtain high growth rates (>2.3% per day), a minimum of 450 g kg^?1 protein in the diet is necessary. However, the most favourable values for growth rates and feed/gain ratio are obtained with 550 g kg^?1 protein in diet, considering that no significant differences were observed for PER among diets B, C and D.     

Performance factors, body composition and digestion characteristics of gilthead sea bream (Sparus aurata) fed pelleted or extruded diets

The effects of steam pelleting or extrusion of a commercial feed on performance of 2 g (small) and 40 g (large) gilthead sea bream was studied. In addition the apparent digestibility of diets, gastric evacuation time (GET) and trypsin and amylase activities were measured in large fish. Fish size significantly increased protein and energy productive value, body protein and lipid, liver glycogen and liver lipid, however decreased daily growth index, feed intake and feed utilization, body moisture and hepatosomatic index. Extrusion processing decreased feed intake, more strongly for small fish, and increased daily growth index, feed utilization, protein and energy productive value, body protein and lipid. Digestibility of starch and energy increased with extrusion and GET of extruded feed was double that of steam-pelleted conditioned feed (i.e. 544 min versus 284 min). Digestive enzyme activities approached maximum values after 240 min for the pelleted diet, while those of the extruded continued to increase, at higher rates for amylase than trypsin. Pelleted diet indicated higher moisture for stomach digesta, while the moisture of the intestinal precipitate indicated a higher water-binding capacity for the extruded diet. The overall results indicate a better assimilation of the extruded diet, which could result from its prolonged gastric evacuation.