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.     

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.     

Application of in situ detection techniques to determine the systemic condition of lymphocystis disease virus infection in cultured gilt-head seabream, Sparus aurata L

Immunohistochemistry (IHC) and in situ hybridization (ISH) techniques have been used for the detection of lymphocystis disease virus (LCDV) in formalin-fixed, paraffin-embedded tissues from gilt-head seabream, Sparus aurata L. Diseased and recovered fish from the same population were analysed. IHC was performed with a polyclonal antibody against a 60-kDa viral protein. A specific digoxigenin-labelled probe, obtained by PCR amplification of a 270-bp fragment of the gene coding the LCDV major capsid protein, was used for ISH. LCDV was detected in skin dermis and gill lamellae, as well as in several internal organs such as the intestine, liver, spleen and kidney using both techniques. Fibroblasts, hepatocytes and macrophages seem to be target cells for virus replication. The presence of lymphocystis cells in the dermis of the skin and caudal fin, and necrotic changes in the epithelium of proximal renal tubules were the only histological alterations observed in fish showing signs of the disease.     

Improved performance of gilthead sea bream, Sparus aurata, larvae after ozone disinfection of the eggs

Ozone (O₃) dissolved in seawater (ODS) was evaluated, as an egg disinfectant, on the spawn of captive gilthead sea bream, Sparus aurata, brood stock. Four contact times (CT) were tested (0.6, 1.2, 2.4 and 4.8 mg min L⁻¹) where CT was calculated by multiplying the dissolved O₃ concentration (0.3 mg L⁻¹) by different exposure periods (2, 4, 8, 16 min). There was also a disinfected seawater treatment that contained no O₃ or derived compounds (CT 0) and an untreated seawater control. All ODS treatments reduced egg surface bacterial counts to zero, which was significantly (P<0.05) lower than the CT 0 and the control groups (194 and 1320 plate⁻¹ respectively). Nevertheless, the hatching rate was high in the control and the CT treatments 0, 0.6 and 1.2 (88.7%, 87.3%, 89.5% and 83.7% respectively) while eggs exposed to a CT 2.4 and 4.8 hatched poorly (36.5% and 20.4% respectively), which was likely due, at least in part, to larvae unable to break the egg chorion successfully. Swim‐bladder inflation was significantly higher in the ODS groups (>97%) compared with the control and CT 0 treatments (ca. 70%). The results suggest that a 2‐min exposure of eggs to 0.3 mg O₃ L⁻¹ of ODS (CT 0.6) would improve current protocols in marine larviculture.     

Towards an inert diet for first-feeding gilthead seabream Sparus aurata L. larvae

The development of an inert food to replace live prey during the early stages of marine fish larvae requires research in different fields and therefore a precise work strategy. Our research on this subject has been carried out in successive steps using the gilthead seabream Sparus aurata . The first step was the design of a food particle that would be well accepted and ingested by free-swimming marine larval fish during the first developmental stages. We chose microencapsulation by polymerization of the dietary protein as the most appropriate method for making the particles; different types of microcapsules were made using a basic diet containing only the major dietary components. In the second step, our aim was to keep the larvae alive in a routine rearing system in 300-L tanks, using exclusively this kind of food, long enough to detect any changes in growth, survival, or anatomical and histological status of the larvae, in order to verify whether the technological changes were positive. The third step focused on diet formulation and searching for clues to inefficient assimilation and growth. The use of ' in vitro ' digestibility techniques allowed us to detect the inhibitory effect of some diet ingredients on larval proteases and to determine more suitable sources of protein. We now have a microcapsule able to efficiently support growth and development of S. aurata larvae , at least during the first 2 weeks of life, although the larvae still need to feed on rotifers during the first 2–4 days of exogenous feeding. This microcapsule will make it possible to make advances in determining the specific nutritional requirements of larval fish.     

Study of the dose of thyme essential oil in feed to prolong the shelf life of gilthead seabream (Sparus aurata)

The effect of thyme essential oil (Thymus zygis, subspecies gracilis) on the quality and shelf life of gilthead seabream was studied, when added to the diet: a control diet and four experimental diets (T500, T1000, T1500, T2000) with 500, 1000, 1500 and 2000 mg kg^?1 of thyme essential oil, respectively. After 12 weeks of experimentation, the fish were stored on ice at 4 °C for 0, 7, 14 and 21 days. Physical–chemical, microbiological and sensory analyses were carried out at each sampling point to determine the degree of deterioration. A dose‐dependent effect was observed on the colour, TBARS and total volatile basic nitrogen during the storage. Microbiological counts were lower for Enterobacteriaceae and coliforms at high doses of the essential oil. The sensory analysis showed an effect on the quality index that was not dependent on the dose, which extended the shelf life of gilthead seabream from 17 to 18 days for all doses.     

Microsatellites markers to depict the reproductive and genetic patterns of farmed gilthead seabream (Sparus aurata): illustration by a case study on mass spawning

In the absence of breeding strategy, natural spawning constitutes the breeding ground for fish farmers to empirically manage their commercial broodstock. In this context, we used six microsatellite markers to characterize the genetic pattern of six commercial seabream broodfish tanks having a common history spanning four generations. The progeny of one tank single‐day mass‐spawning event, reared in two different environments, was used to estimate the genetic parameters for body weight. Limited genetic differentiation was observed among broodfish groups. A panel of nine loci allowed us to unambiguously assign 95.4% of the offspring (1692) and identify 37 parents (65% of the total broodfish). The limited effective population size (N_e = 15.3) was due to the elevated variance of parental contributions and to broodfish failing to contribute to the progeny. The fluctuation of the allele frequency highlighted the risks of genetic drift and reduction in the heterozygosity in the next generations. Heritability for body weight was moderate at commercial size (0.40 ± 0.10) and the high genetic correlation at later stages laid the groundwork for precocious selection criteria for growth. The discussion opens on the opportunity to use mass spawning for selective breeding.     

Plant protein and vegetable oil‐based diets modulate gilthead seabream (Sparus aurata) muscle biochemical status and proteolytic enzymes at an early postmortem stage

A 70 days feeding trial was carried out on juvenile gilthead seabream (Sparus aurata) (mean initial body weight: 362 ± 59 g) to evaluate whether the simultaneous replacement of fishmeal and fish oil by vegetable ingredients affected the muscle's proteolytic potential. Fish were fed either a fishmeal/fish oil diet, a fishmeal/vegetable oil diet, a plant protein/fish oil or a plant protein/vegetable oil diet. The use of dietary plant proteins resulted in lower sulphated glycosaminoglycans' content and lower glycogen phosphorylase activity in the muscle, while both plant proteins and vegetable oil increased muscle pH and reduced cathepsin B activity. Our results indicate that high replacement of fish meal and/or fish oil with plant ingredients has a significant impact on early postmortem metabolic processes and proteolytic potential of seabream muscle, especially when replacing both fish meal and fish oil.     

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.     

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.     

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.     

Total nitrogen,carbon and phosphorus digestibility in gilthead seabream (Sparus aurata) and European seabass (Dicentrarchus labrax) fed with conventional and organic commercial feeds: implications for particulate waste production

Organic fish farming is postulated as a more environmentally friendly activity than conventional activities. However, environmental sustainability as regards waste output has not been addressed. In this respect, the most important environmental problems associated with fish farming are those related with the discharge of particulate wastes. In this study, total nitrogen, carbon and phosphorus digestibility was estimated in a wide size range of gilthead seabream (Sparus aurata) and European seabass (Dicentrarchus labrax) fed a commercially available conventional diet and an organic feed, as a previous step to assessing particulate waste output. Neither fish size nor feed nutrient content was seen to influence nutrient digestibility in either species. The organic diet provided higher carbon but lower nitrogen and phosphorus digestibility than conventional feed in both species. Simulations under real ongrowing conditions resulted in lower values for nitrogen and higher values for carbon particulate wastes when conventional feed was supplied to both species. Particulate phosphorus wastes were 2.6‐ to 3.4‐fold higher for fish fed the organic diet. Restrictions concerning NO the raw materials used for organic diet formulation and differences in nutrient bioavailability between the diets seemed to be the underlying reason for the differences found.     

Tolerance response to water pH in larvae of two marine fish species, gilthead seabream, Sparus aurata (L.) and Senegal sole, Solea senegalensis (Kaup), during development

Gilthead seabream, Sparus aurata (L.) and Senegal sole, Solea senegalensis (Kaup) are two fish of primary importance in Mediterranean aquaculture. In the present study, the larvae of these species were exposed to different rearing‐water pH during 24 h to examine their tolerance. The 24‐h pHL50 values were calculated with low and high pH values at 7, 20 and 32 days after hatching (DAH) in S. senegalensis larvae, and 12, 20 and 52 DAH in S. aurata larvae. Low 24‐h pHL50 values ranged between 4.88 and 5.76, whereas high 24‐h pHL50 values ranged between 8.94 and 9.57 in S. senegalensis larvae. S. aurata larvae showed values of low 24‐h pHL50 that ranged between 4.82 and 5.55, whereas values of high 24‐h pHL50 ranged between 8.66 and 9.26. Both species showed similar tolerance response at all the tested ages. The high 24‐h pHL50 values found were close to pH values that eventually can be reached in the rearing tanks. The pH should be carefully controlled in rearing water during the first development stages of both species.     

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.     

Growth,tissue metabolites and digestive functionality in Sparus aurata juveniles fed different levels of macroalgae,Gracilaria cornea and Ulva rigida

The aim of this study was to evaluate the inclusion of two macroalgae as dietary ingredients on growth, body composition, intermediate metabolism, intestinal enzymes and gut histology of Sparus aurata juveniles. Six isocaloric and isonitrogenous diets incorporating 5%, 15% or 25% of Gracilaria cornea or Ulva rigida meal were tested against a control diet. Duplicate groups of fifteen fish (14 g) were fed the experimental diets for 70 days. Growth performance showed an inverse linear relationship with the inclusion level of Gracilaria. Feed conversion ratio increased significantly in fish fed 25% Gracilaria. Ulva presented a quadratic relationship with the lowest SGR at 15% of inclusion. Fish fed 25% Ulva exhibited significantly lower lipid content than those fed control diet. Overall, metabolites did not show differences with specimens fed control diet, except for plasmatic glucose and muscle lactate. Intestinal proteolytic activity was reduced with the macroalgae inclusion level, but all fish showed the same enzyme pattern. The histological study revealed no negative effect on liver and intestine structure by the macroalgae inclusion. The present work demonstrated that these ingredients can be used in practical diets for sea bream, but a more comprehensive research is needed to determine the optimum dietary level in a longer feeding trial.     

Effect of different culture conditions on the structural diversity of prokaryote communities in the sediment of earth ponds stocked with gilthead seabream Sparus aurata (Linnaeus, 1758)

In this study, we evaluate the use of polymerase chain reaction‐denaturing gradient gel electrophoresis (PCR‐DGGE) for monitoring the effect of different aquaculture practices on sediment prokaryote (Archaea and Bacteria) communities. The effect of initial fish (gilthead seabream Sparus aurata) stocking density on the structural diversity of prokaryote communities of earth ponds bottom sediments was evaluated using PCR‐DGGE after a 5 month grow‐out period. An identical approach was used to monitor the effect of supplying different fish feeds [commercial feed (CD) versus an ecofeed (ECO)]. One additional variable was the use of copper sulphate (CuSO₄) as an algicide in some of the experimental rearing tanks. The statistical analyses of prokaryote community profiles showed that the presence of fish in earth ponds significantly influenced the structure of sediment prokaryote communities, when compared with earth ponds without fish, independently of the stocking density. Our results also indicated that the structure of the prokaryote communities of earth ponds supplied with the ECO feed shared a strong similarity with that fed CD. Curiously, the use of CuSO₄ in ponds receiving the ECO feed promoted significant differences on the structural composition of the bacterial community, but not on the archaeal community. DGGE molecular fingerprints are suitable for fast evaluation of new management practices in food‐fish production on earth ponds by monitoring shifts on microbial communities in bottom sediments.     

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.