Genetic variation in farmed populations of the gilthead sea bream Sparus aurata in Greece using microsatellite DNA markers

Genetic variation in seven reared stocks of gilthead sea bream Sparus aurata, originating from Greek commercial farms, was assessed using five polymorphic microsatellite markers and was compared with that of two natural populations from the Ionian and the Adriatic Seas. The total number of alleles per marker ranged from 11 to 19 alleles, and hatchery samples showed the same levels of observed heterozygosity with samples from the wild but substantially smaller allelic diversity and expected heterozygosity. The global genetic differentiation for the cultivated samples was significant as indicated by F_st analysis, which might indicate random genetic drift and inbreeding events operating in the hatcheries. On the contrary, no significant difference was found between the two wild populations. Population pairwise tests between farmed and wild stocks were also significant, with the exception of one hatchery sample, the Central Greece 1, which was not significantly different from the two wild samples perhaps due to its recent use in aquaculture from wild‐caught animals. The UPGMA tree topology grouped the wild samples together with the Central Greece 1 stock, and showed a clear division between wild and farmed sample sets for the six remaining hatchery samples. Knowledge of the genetic variation in S. aurata cultured populations compared with that in the wild ones is essential for setting up appropriate guidelines for the proper monitoring and management of the stocks either under traditional practices or for the implementation of selective breeding programmes.     

Transmission of lymphocystis disease virus to cultured gilthead seabream,Sparus aurata L., larvae

The transmission of lymphocystis disease virus (LCDV) to gilthead seabream, Sparus aurata L., larvae was investigated using fertilized eggs from a farm with previous reports of lymphocystis disease. LCDV genome was detected by PCR‐hybridization in blood samples from 17.5% of the asymptomatic gilthead seabream broodstock analysed. Using the same methodology, eggs spawned from these animals were LCDV positive, as well as larvae hatched from them. The presence of infective viral particles was confirmed by cytopathic effects development on SAF‐1 cells. Whole‐mount in situ hybridization (ISH) and immunohistochemistry (IHC) showed the presence of LCDV in the epidermis of larvae hatched from LCDV‐positive eggs. When fertilized eggs were disinfected with iodine, no viral DNA was detected either in eggs (analysed by PCR‐hybridization) or in larvae (PCR‐hybridization and ISH). These results suggest the vertical transmission of LCDV, the virus being transmitted on the egg surface. Larvae hatched from disinfected eggs remain LCDV negative during the endotrophic phase, as showed by PCR‐hybridization, ISH and IHC. After feeding on LCDV‐positive rotifers, viral antigens were observed in the digestive tract, which suggests that viral entry could be achieved via the alimentary canal, and that rotifers can act as a vector in LCDV transmission to gilthead seabream larvae.     

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.     

Early assessment of gilthead sea bream (Sparus aurata) spawning dynamics by mini‐broodstocks

The implementation of any sustainable breeding program requires a maximization of effective size (Ne) to maintain inbreeding rate at minimum levels. However, the control of Ne remains a major challenge for those species whose reproduction in captivity is based on a spawning strategy where there is no control over the established matings. The present study aims to assess the spawning dynamic of gilthead sea bream (Sparus aurata) at different dates from two mini‐broodstocks (4 ♀ and 4 ♂). Here we report the use of a simple protocol based on the genotyping of fertilized eggs from a reduced set of microsatellite loci which provides a quick evaluation of daily spawning in terms of family structuring (number of active breeders and number and family size). According to our results, the daily spawning dynamics is characterized by a fluctuation in the number of active breeders, reaching maximum values (until 100%) during peak production phase. However, the proportion of possible mating (families) in a single daily spawning never exceeded more than 81%. Unbalanced parental contributions were highly frequent, with extreme cases involving the assignment of 50%–90% of the offspring to a single family. Therefore, the use of mini‐broodstocks and the grouping of spawning harvested at different dates are proposed as strategies to minimize the inbreeding risk by providing a more optimal pattern of family structuring (increase in the number of families obtained and a more balanced parental contributions) that entails a more conservative Ne/N ratio.     

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.     

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.     

Effect of broodstock diets on total lipids and fatty acid composition of larvae of gilthead sea bream (Sparus aurata L.) during yolksac stage

Total lipid content and total lipid fatty acid compositions were studied in larvae at the yolksac stage of gilthead sea bream from two different broodstocks. The two broodstock diets had the same total lipid content but were different with respect to their fatty acid levels. There were differences in the fatty acid composition of total lipids in the two groups of larvae, reflecting the influence of fatty acid levels in the diets fed to the broodstock. The main fatty acids in both larvae lots during yolksac stage were 16:0, 18:1n-9 and 22:6n-3. The desaturation index Δx = [P + Σ (n − x) − Σ (n − x) diet]/P allowed the determination of the relative importance of the Δ9, Δ6, Δ5 and Δ4 desaturations during the egg and larval yolksac stages. The levels of total lipid fatty acids in eggs and larvae during yolksac period were almost constant and very low desaturation and elongation activities were apparent. A competitive inhibition was observed between the fatty acids of the n-6 series with respect to the ones of the n-3 series in those eggs and larvae from broodstock whose diet contained a high proportion of linoleic acid and its derivatives.     

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.     

A behavioural approach of dominance establishment in tank‐held sea bream (Sparus aurata L.) under different feeding conditions

Dominance is a strong behaviour exhibited by farmed species that very often impinges on fish growth and welfare. This study presents a behavioural approach of dominance, where colour pattern differentiation was tested as a signal of dominance presence in small sea bream population. The phenotype of dominance was first described in detail, referring to vertical dark stripes and splayed fins. Fish were kept in triplicate tanks for 21 days, during which they were exposed to three feeding conditions (well fed, limited fed and no‐fed) and continuous video recordings. Each tank was stocked with 15 individuals (~30.34 ± 1.70 g), and they were confined to the half volume of the tank via a removable net pen. Percentage of dominants per population was found up to 40%, while duration was calculated to 53.52 ± 7.44 s. Dominance behaviour was further quantified via colour pattern differentiation on sea bream body (CDA, contrast of dominance appearance). The results demonstrated that the body colour of sea bream is directly linked to species social hierarchies and such variations are visual signal of dominance rank inside the population. The fish feeding conditions had an influence on the dominance presence, but not on the dominance rank (as measured by the CDA) between fish groups. The described study provides state‐of‐the‐art knowledge on sea bream dominance. In relevance to aquaculture, dominance quantification would be a reliable tool to evaluate non‐equal food distribution in tank‐held populations, thus, avoid non‐regulate growth performance of fish before transfer to sea‐cage installations.     

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.     

Morphological and histological characterization of an ectopically mineralized structure in a gilthead sea bream Sparus aurata with opercular deformation

In this study, we describe an abnormal ectopically mineralized structure (EMS) that was found inside the skull of a juvenile Sparus aurata that also showed a bilateral opercular deformation. The overall phenotype and tissue composition were studied using micro‐CT scanning and histological analyses. The ectopic structure occupies a large volume of the brain cavity, partially extruding into the gill cavity. It shows a dense mineralization and an extracellular matrix‐rich phenotype, with variation in both the morphology and size of the cell lacunae, combined with an irregular fibre organization inside the matrix. This study is the first to report such an EMS in a juvenile teleost fish, where the tissue does not resemble any other connective tissue type described in bony fish so far. The tissue phenotype seems to rule out that the EMS corresponds to a tumorous cartilage. Yet, it is rather reminiscent of a highly mineralized structure found in cartilaginous fish, where it is suggested to be associated with damage repair.     

Effects of dietary mannan oligosaccharides in early weaning diets on growth,survival, fatty acid composition and gut morphology of gilthead sea bream (Sparus aurata,L.) larvae

Early weaning of marine fish larvae with dry diets delays gut maturation and reduces growth rates. In juvenile and adult forms of several marine fish species, inclusion of dietary mannan oligosaccharides (MOS) improves gut integrity and functionality, but the effects of MOS inclusion in gilthead sea bream (Sparus aurata, L.) larval diets have not been addressed yet. Thus, this study assesses the effects of dietary MOS inclusion on survival, growth performance, gut morphology, feed acceptance and quality of gilthead sea bream larvae. For that purpose, 16 days post‐hatched gilthead sea bream larvae were fed four graded levels of MOS (Biomos^®, Alltech, Nicholasville, KY, USA) in weaning diets as follows: 0 g kg^?1 MOS, 0.5 g kg^?1 MOS, 1.5 g kg^?1 MOS and 2 g kg^?1MOS. Dietary MOS did not affect feed acceptance in gilthead sea bream larvae (P > 0.05). MOS supplementation was correlated in a dose‐dependent way with higher larval survival (P = 0.026). After 15 days of feeding, dietary MOS increased whole larvae (P < 0.01) arachidonic acid, eicosapentaenoic acid and docosahexaenoic acid. Gilthead sea bream larvae fed 2 g kg^?1 MOS presented higher gut occupation with goblet cells after feeding compared with larvae fed the other dietary treatments. Overall, the results suggest that inclusion of MOS in early weaning diets for gilthead sea bream improves essential fatty acid utilization and may promote growth and final survival.     

Effect of dietary energy content on gilthead sea bream (Sparus aurata) feeding behaviour and nutritional use of the diet

We have studied how the diet energy level affects gilthead sea bream (Sparus aurata) daily pattern of demand‐feeding activity and nutritional use of the diet under summer‐ and winter‐like conditions. To that end, animals were kept in a closed circuit under controlled temperature and photoperiod, and fed one of two commercial diets with either high (H: 238 g kg^?1 fat) or low (L: 172 g kg^?1 fat) energy content. In summer conditions (26 °C; 12 : 12 L : D), both diets yielded similar growth rate (0.7 ± 0.0 and 0.7 ± 0.1) and thermal unit growth coefficient values (0.6 ± 0.0 and 0.6 ± 0.0), and although the daily rate of delivered feed was somewhat higher for fish under the high‐energy diet (16 ± 2 g kg^?1 of fish) the difference was not significant. In winter conditions (17 °C; 9 : 15 L : D), on the other hand, no differences were found for any of the parameters evaluated. Thus, the higher lipid content of the diet does not appear to have any protein sparing effect. The ‘summer’ demand‐feeding pattern displayed three daily peaks, which were quite apparent for fish under the low‐energy diet, but rather smoothed for those that ate the energy‐rich diet. This profile was replaced in winter conditions by a single peak around noon, and the animals also displayed a lower overall number of demands.     

Quality of reared gilthead sea bream (Sparus aurata) during ice storage,as affected by dietary fish oil substitution; an instrumental and sensory designation approach

The fillet fatty acids (FAs) and volatile compounds (VCs) of gilthead sea bream, fed either fish oil‐based diet (FO) or plant oils (PO), were studied in dorsal and ventral fillet parts over a 12‐day ice storage. Fillet FA reflected the FA composition of the respective diets. Monounsaturated FAs were reduced with storage, while no FA differentiations occurred between fillet parts. VCs varied between diet treatments, with food chain‐transported compounds (α‐pinene and dimethylsulphide) showing higher abundance in the FO group. VCs proposed as fish spoilage indicators (trimethylamine, pentanal, propanal, 3‐methylbutanal and 1‐penten‐3‐ol) increased with storage, while concentration of carbon disulphide associated with fresh fish aroma decreased during the same period. VCs varied between fillet parts, with ventral part exhibiting higher concentrations in fat‐soluble (terpenes, aromatic hydrocarbons) and spoilage‐associated VCs. Sensory analysis revealed no significant differences between diet groups with the exception of fattiness, which was found significantly higher in the plant oil group.     

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.     

Optimum krill phospholipids content in microdiets for gilthead seabream (Sparus aurata) larvae

The aim of the present study was to determine the optimum dietary levels of krill phospholipids (KPL) for sea bream (Sparus aurata) larvae, and its influence on larval development and digestive enzymes activity. Larvae were fed five formulated microdiets with five different levels of KPL. Complete replacement of live preys with the experimental microdiets for seabream larvae produced high survival and growth rates, particularly in fish fed the highest levels of KPL. In the present study, increase in dietary KPL up to 120 g kg^?1 (100 g kg^?1 total PL) significantly improved larval survival and growth, whereas further increase did not improve those parameters. An increase in alkaline phosphatase, trypsin and lipase activity with the elevation of KPL up to 120 g kg^?1 was also found denoting a better functioning of digestive system. Besides, there was a linear substrate stimulatory effect of dietary KPL on phospholipase A2 activity. Finally, increasing dietary KPL lead to better assimilation of n‐3 HUFA especially eicosapentaenoic acid, reflected in the higher content of these fatty acids in both neutral and polar lipids of the larvae. In summary, KPL were found to be an excellent source of lipids for seabream larvae. Optimum inclusion levels of this ingredient in microdiets to completely substitute live preys at this larval age were found to be 120 g kg^?1 KPL.     

Stock enhancement of gilthead sea bream (Sparus aurata, L.): assessment of season, fish size and place of release in SW Spanish coast

Gilthead sea bream, Sparus aurata L., is a highly level protein species in Mediterranean countries and can easily be produced in hatcheries. Despite the commercial interest in the species, alternatives to aquaculture such as ranching and stock enhancement, which have proven to be profitable for other species, have not been attempted for the gilthead sea bream. The aim of this paper is to evaluate the feasibility of these alternatives for the Atlantic coast of the southern Iberian Peninsula, where this species is highly valuable for professional and amateur fishermen. A total of 30 323 individuals marked with average weights of 15, 100, 316 and 854 g were released between 1993 and 1997 at six different locations. For each of these locations, the displacements, growths, the oldest recapture and the percentage of recapture were determined. The Bay of Cádiz turned out to be the most suitable location since the highest recapture (2.41%, S.E. 0.53) and specific growth rates (0.86, S.E. 0.09), as well as small displacements (8.16 km, S.E. 0.95), were found in this location. One hundred grams was the best size for enhancement as fish of 15 g showed very low recapture rates (0.12%, S.E. 0.08), while fish larger than 316 g made significant displacements (42.87 km, S.E. 4.73). Growth and displacement also point to summer versus spring, winter or fall as the best releasing period for stock enhancement of this species in the analysed area. These elements point to the feasibility of implementing a stocking program for gilthead sea bream. This must have the bay of Cádiz as the core place of interest, releasing 100-g fish in late summer.     

Cottonseed oil as a complementary lipid source in diets for gilthead seabream Sparus aurata juveniles

The efficacy of using cottonseed oil (CSO) as a fish oil (FO) substitute in gilthead seabream (Sparus aurata) juveniles feed was evaluated. Fish (BW_i 4.0 ± 2.9 g) were fed one of four isoproteic (~48% CP) and isolipidic (~18% L) diets for 9 weeks. Added oil was either FO (control diet, CTRL) or CSO, replacing 50% (CSO50 diet), 60% (CSO60 diet) and 70% (CSO70 diet) of dietary FO. Results indicated that FO replacement by CSO up to 60% level had no detrimental effects on growth or nutritive utilization and composition in fish muscles. Higher CSO intake (CSO70 diet, 56 g kg^?1) led to a 16% reduction in weight gain, 14% in feed utilization (FCR) and 57% in muscle n‐3 long‐chain polyunsaturated fatty acids (lc PUFA) as compared with CTRL and to abundant accumulation of lipid within the hepatocytes. Use of CSO altered fatty acid (FA) profiles of muscle and liver. Data suggested utilization of linoleic acid (LOA) by fish and retain of docosahexaenoic acid (DHA) in muscles. Therefore, limits of CSO inclusion as the main source of supplementary dietary lipid, with no negative effects on fish performance or nutritive composition and utilization in muscles, are: 40–48 g kg^?1 feed for gilthead seabream juveniles.     

Importance of the relative levels of dietary arachidonic acid and eicosapentaenoic acid for culture performance of gilthead seabream (Sparus aurata) larvae

The effect of different arachidonic acid (ARA) dietary contents at several dietary eicosapentaenoic acid (EPA) levels on the growth, survival and biochemical composition of gilthead seabream larvae was studied to better define the importance of this fatty acid as a function of EPA. Larvae of 18 days were fed one of the five isonitrogenous and isolipidic microdiets with three different EPA (0.3%, 2% and 4%) and ARA amounts (0.1%, 0.6% and 1.2%). Although a dietary increase in either ARA or EPA alone did not improve survival significantly, the increase in both fatty acids significantly enhanced growth and survival, suggesting an optimum dietary value of EPA:ARA close to 4:1.2. Dietary ARA was more efficiently incorporated into larval tissues than EPA. Increased dietary EPA or ARA contents reduced the incorporation of ARA or EPA into larval lipids, indicating their competition as substrates for different enzymes. The possible negative effect of further elevation of dietary ARA and its competition with EPA for phospholipids synthesis deserves further studies in marine fish larvae.