Pharmacokinetics of oxolinic acid in gilthead sea bream, Sparus aurata L.

This is the first study on the pharmacokinetic parameters of oxolinic acid (OA) in gilthead sea bream, Sparus aurata L. The kinetic profile of OA was studied after a single intravascular injection (20 mg kg⁻¹) in 100 g fish at 20 °C. The distribution half-life ( t _1/2α) and the elimination half-life ( t _1/2β) of the drug were found to be short (0.51 and 12.60 h, respectively). The drug penetration from the plasma to the tissues was adequate as the apparent volume of distribution of the drug at steady-state ( V _d(ss)) was found to be 2.11 L kg⁻¹. The mean residence time ( MRT ) of OA was short (14.25 h) and the total clearance rate ( Cl _T) of the drug was low (0.15 L kg⁻¹ h⁻¹). The bioavailability ( F %) of OA following oral administration (30 mg kg⁻¹) was also low (14%). Maximum values were observed for muscle at 0.5 h after injection, with levels declining as with subsequent sampling. At the first two time points (0.5 and 1 h) plasma levels of OA were higher than muscle, however, the reverse was evident for subsequent samples. Following oral administration, highest muscle levels were found at 16 h and, with the exception of the 24-h sampling, muscle OA concentrations were higher than plasma at all time points. The fast elimination of OA suggests short withdrawal times with reference to human consumption of treated fish.     

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.     

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.     

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.     

Consistency of stress response to repeated handling in the gilthead sea bream Sparus aurata Linnaeus, 1758

Gilthead sea bream Sparus aurata Linnaeus, 1758 were subjected to repeated stresses, namely confinement and handling once a month for 5 consecutive months. Plasma cortisol concentration was determined as a primary stress response, and plasma glucose and osmolality, serum agglutination and growth rate were analysed as indicators of the secondary and tertiary effects of stress. The results of the mean values after the five trials showed that most indicators followed a consistent pattern with time. Cortisol and growth rate were positively correlated, and cortisol and osmolality were negatively correlated after stress. Analysis of the individual responses throughout all 5 months showed that 6–20% of the fish displayed a consistently high or low response depending on the parameter analysed.     

Evaluation of micronized lupin seed meal as an alternative protein source in diets for gilthead sea bream Sparus aurata L. juveniles

A 12‐week growth trial was performed to evaluate the effect of lupin seed meal as a protein source in diets for gilthead sea bream (Sparus aurata) juveniles. Six experimental diets were formulated to be isonitrogenous and isoenergetic and to contain 10%, 20% and 30% of raw lupin (Lupinus angustifolius) seed meal protein or 20% and 30% lupin (L. angustifolius) seed meal processed by infrared radiation (micronized) in place of fish meal protein, the only protein source of the control diet. Fish accepted all diets well and no significant differences in feed utilization among groups were noticed during the trial. Final weight of fish fed the experimental diets was identical or higher than the control group. Final weight of fish fed diets including 20% micronized lupin protein was even significantly higher than that of fish fed the fish meal‐based control diet. Moreover, at the same dietary lupin seed meal protein inclusion levels, final weight of fish fed diets including micronized lupin was significantly higher than with raw lupin. A trend was also noticed for a decrease of final weight with the increase in lupin seed meal in the diets. At the end of the trial no significant differences in proximate whole‐body composition, hepatosomatic and visceral indices were observed among groups. It is concluded that lupin seed meal can replace up to 30% fish meal protein in diets for gilthead sea bream juveniles with no negative effects on growth performance. Furthermore, micronization of lupin seeds improves its dietary value for gilthead sea bream juveniles. At the same dietary lupin inclusion levels, diets including micronized lupin seeds promote significantly higher growth rates than raw lupin seeds.     

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.     

Incorporation and metabolism of 14C-labelled polyunsaturated fatty acids in juvenile gilthead sea bream Sparus aurata L. in vivo

The incorporation, and the capacity for desaturation and elongation in vivo, of intraperitoneally-injected, ¹⁴C-labelled n–3 and n–6 C₁₈ and C₂₀ PUFAs were investigated in juvenile gilthead sea bream, Sparus aurata. The results indicate that juvenile gilthead sea bream have only limited ability to convert CH PUFAs to C₂₀ and C₂₂ HUFAs in vivo. The data are consistent with the results from nutritional studies on larvae, postlarvae and fingerlings that have shown that gilthead sea bream require the provision of preformed eicosapentaenoic and docosahexaenoic acids in the diet. The impairment in the desaturase/elongase pathway was quantitatively and qualitatively similar to that found in turbot, Scophthalmus maximus, being at the level of the 5-desaturase. The low activity of 5-desaturase combined with the consistent finding that arachidonic acid is selectively retained in membrane phosphatidylinositol suggests that, in addition to eicosapentaenoic and docosahexaenoic acids, gilthead sea bream may also have a requirement for preformed arachidonic acid in the diet.Abbreviations AA 5,8,11,14-eicosapenaenoic acid (arachidonic acid, 20:4n–6) - CPL diradyl (diacyl + alkenylacyl + alkylacyl) glycerophosphocholine - DHA 4,7,10,13,16,19-docosahexaenoic acid (22:6n–3) - EPA 5,8,11,14,17-eicosapentaenoic acid (20:5n–3) - EPL diradyl (diacyl, alkenylacyl + alkylacyl) glycerophosphoethanolamine - HUFA highly unsaturated fatty acids ( C₂₀ and with 3 double bonds) - LA 9,12-octadecadienoic acid (linoleic acid, 18:2n–6) - LNA 9,12,15-octadecatrienoic acid (-linolenic acid, 18:3n–3) - PI phosphatidylinositol - PS phosphatidylserine - PUFA polyunsaturated fatty acid(s)     

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.     

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.     

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.     

Effect of broodstock diets on lipid classes and their fatty acid composition in eggs of gilthead sea bream (Sparus aurata L.)

Total lipid content, total lipid fatty acids, lipid classes and their fatty acid compositions were studied in eggs of gilthead sea bream from two different broodstocks. The two broodstock diets used had the same total lipid content, but differed with respect to their fatty acid levels. The lipid class composition of eggs from different origins was not significantly different. The predominant polar lipids were phosphatidylcholine, phosphatidylethanolamine and sphingomyelin, and the main neutral lipid classes were cholesterol esters and triacylglycerols. The fatty acid composition of total lipids and main lipid classes in the two groups of eggs showed marked differences, reflecting the influence of fatty acid levels in the broodstock diets.     

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.     

Seasonal variation in osmoregulatory and metabolic parameters in earthen pond-cultured gilthead sea bream Sparus auratus

Seasonal variations in osmoregulatory and metabolic parameters were assessed in juvenile gilthead sea bream ( Sparus auratus ) cultured in earthen ponds under a natural photoperiod and temperature. Specimens were sampled, and the plasma, gill, kidney and liver were collected during winter 2005 and 2006 (January), spring 2005 (April), summer 2005 (July) and autumn 2005 (October). Plasma osmoregulatory parameters showed higher values in summer, while metabolic parameters presented different patterns of variations. Gill Na⁺,K⁺-ATPase activity decreased significantly in winter, while gill metabolite levels showed different patterns of variations among seasons. The enzymatic activities tested did not present a clear pattern of variation [(glutamate dehydrogenase (EC 1.4.1.2) (GDH) and hexokinase (EC 2.7.1.11) (HK)] or significant differences along seasons [glucose 6-phosphate dehydrogenase (EC 1.1.1.49)]. Kidney Na⁺,K⁺-ATPase activity decreased during summer and autumn. Different patterns of variation were observed in kidney metabolite levels while all the enzymatic activities assessed [lactate dehydrogenase-oxidase (EC 1.1.1.27) (LDH-O), HK and GDH] presented the highest values during summer. In the liver, metabolite levels and enzymatic activities did not show significant variations or present clear patterns of variation along different seasons. These results indicated seasonal variations in the osmoregulatory and metabolic parameters of different organs (blood, gill, kidney and liver) in earthen pond-cultured gilthead sea bream ( S. auratus ), which could be mainly attributed to seasonal changes in temperature.     

Insect larvae as feed ingredient selectively increase arachidonic acid content in farmed gilthead sea bream (Sparus aurata L.)

This work is aimed at obtaining farmed fish designed to contain targeted PUFAs. To this end, an experiment was conducted with farmed specimens of gilthead sea bream (Sparus aurata L.), which were fed meal containing different percentages of common green bottle fly (Lucilia sericata Meigen) larvae, cultured on appropriate substrates, and then the fatty acid profiles in fish muscles were determined. Results indicate that arachidonic acid content in fish muscle is significantly increased when replacing different proportions of fishmeal by larvae from L. sericata, which contains arachidonic acid up to 10.6% of total fatty acids. Thus, larvae seem to be a suitable vector for introducing target PUFA in fish muscle and it could also contribute to reduce the use of wild fish stocks for fishmeal production.     

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.