Debaryomyces hansenii L2‐enriched diet enhances the immunity status,gene expression and intestine functionality in gilthead seabream (Sparus aurata L.)

This study evaluates the effects of the dietary administration of the live yeast Debaryomyces hansenii strain L2 on the immune responses of gilthead seabream for 4 weeks. Cellular immune parameters were measured from serum and head‐kidney leucocytes respectively. The expression levels of immune‐associated genes were quantified by real‐time polymerase chain reaction. In addition, the profile of intestinal microbiota was studied by denaturing gradient gel electrophoresis. The results showed that seabream fed a diet containing D. hansenii had significantly increased cellular immune parameters. The yeast‐supplemented diet up‐regulated the expression of most seabream genes at week 2 and down‐regulated all of them at week 4, except in the head‐kidney. Finally, a reduction in the diversity of the intestinal microbiota was detected in those specimens receiving the yeast‐supplemented diet. These results support the idea that the live yeast D. hansenii strain L2 stimulates the immune system of gilthead seabream.     

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.     

Reproductive performance of gilthead seabream (Sparus aurata L., 1758) fed two combined levels of carotenoids from paprika oleoresin and essential fatty acids

Despite carotenoids and essential fatty acids seem to play important roles in fish reproduction, no studies have yet been conducted to determine the effect of dietary carotenoids on gilthead seabream broodstock performance and their relation as antioxidants with dietary n‐3 highly unsaturated fatty acid (HUFA) levels. In addition, the high cost of synthetic sources of carotenoids is leading to the search for new natural carotenoid sources such as paprika oleoresin. Four experimental diets containing two combined levels of carotenoids from paprika oleoresin (40 and 60 mg kg^?1) and n‐3 HUFA (25 and 40 g kg^?1) were respectively fed to triplicate groups of gilthead seabream (Sparus aurata) broodfish. Elevation of n‐3 HUFA dietary levels from 25 to 40 g kg^?1 significantly improved gilthead seabream broodstock performance in terms of egg viability, hatching rates and fecundity. Besides, it markedly increased egg contents in HUFAs which play important energetic and structural roles and improve embryo development. Both arachidonic acid and eicosapentaenoic acid (20:5n‐3) egg contents were more readily affected by dietary n‐3 HUFA than docosahexaenoic acid. HUFA levels did not caused any negative effect suggesting an optimized content of antioxidants in broodstock diets. Increase in dietary carotenoids from 40 to 60 mg kg^?1 increased carotenoid contents in eggs and significantly improved egg fertilization rates suggesting an important sperm cell’s protective role by reducing the risk of lipid peroxidation which is detrimental for sperm motility. The increased inclusion of dietary paprika oleoresin enhanced egg carotenoid deposition and improved fish reproductive performance, denoting the high nutritional value of this product as a source of carotenoids for broodstock of this species.     

Mono- and duoculture of juvenile sharpsnout seabream Diplodus puntazzo (Cetti) and gilthead seabream Sparus aurata L. in a recirculated water system

Although the growth of intensively reared sharpsnout seabream Diplodus puntazzo (DP) has been suggested to improve in duoculture with gilthead seabream Sparus aurata (SA), the stocking ratio for most effective duoculture rearing has not been investigated. For this purpose, juvenile D. puntazzo (1.68±0.011 g) and S. aurata (1.43±0.007 g) were reared in duplicated groups of 165 specimens each, in two monoculture (100% DP, 100% SA) and four duoculture combinations (80% DP–20% SA, 60% DP–40% SA, 40% DP–60% SA, 20% DP–80% SA) for 96 days under recirculated water system conditions. Both species exhibited the highest growth when stocked at 20% and the poorest growth when stocked at 80%. For each species, no major differences for coefficient of weight variation and carcass proximate composition were detected. As the percentage of stocked S. aurata increased, D. puntazzo aggressiveness and weight variability of whole population were reduced, while total biomass, food consumption and food conversion ratio were increased. The results obtained are discussed in relation to fish behaviour and social interactions. From the fish farming point of view, it is indicated that under the present experimental conditions, juvenile S. aurata should be reared in monoculture and D. puntazzo in duoculture 40% DP–60% SA.     

Effect of dietary vegetable oils on health and liver histology of gilthead seabream (Sparus aurata) growers

There is an increasing trend towards the replacement of fish oil (FO) by vegetable oils in feeds for farmed fish, particularly marine carnivores. Previous researches on partial or complete replacement of FO by alternate vegetable oils for young gilthead seabream (Sparus aurata) were successful. The aim of the present study was to investigate the effects of different dietary vegetable oils on seabream growers' health and liver structure. Two dietary vegetable oil blends (Mix 1 and Mix 2), namely sunflower oil, cottonseed oil and either linseed oil (LO) or soybean oil (BO), were tested as 60% FO substitutes versus the 100% FO control (CTR) diet. Three isonitrogenous (∼47% CP) and isolipidic (18.2% L) diets were fed, twice a day, 6 days a week to apparent satiation, to triplicate groups of seabream growers (mean IW, 130.85 g) in PVC tanks. The growth trial lasted for 20 weeks at an average ambient water temperature of 27.0±1.8°C. At the end of the experiment, fish attained market size (300–400 g) and their health status was evaluated by blood analyses, head kidney prints and liver histology. Haemoglobin and haematocrit contents, erythrocyte (RBC) and leucocyte (WBC) count remained significantly unaffected by any dietary treatments. Despite the apparent relatively higher thrombocyte counts for fish fed vegetable oil diets, especially Mix 2 as compared with those of CTR fish, variations were found to be insignificant (P>0.05). However, a slight deformation in erythrocyte shape was noticed in the blood films of fish fed the vegetable oil diets. Inclusion of either dietary vegetable oil blend had also elevated the number of both developing myelocytes and phagocytes in fish head kidney prints examined compared with those of CTR fish particularly for Mix 2 group. Liver from seabream fed vegetable oils showed slightly swelled hepatocytes due to accumulation of oil droplets in the cytoplasm. In the liver sections investigated ultra‐structurally, nuclei and cytoplasmic organelles were shifted towards the cell periphery. Hepatocytes of fish fed Mix 2 diet showed relative shrinkage and picnotic nuclei, compared with those of CTR fish, suggesting an effect on lipid transport and/or metabolism in fish. The comparative hepatic features between experimental fish groups were described. Accordingly, Mix 1 diet (with LO) was considered better than Mix 2 (with BO) in terms of influence on seabream growers' health status.     

A Novel Zero Discharge Intensive Seawater Recirculating System for the Culture of Marine Fish

Results are presented of a zero‐discharge marine recirculating system used for the culture of gilthead seabream Sparus aurata. Operation of the system without any discharge of water and sludge was enabled by recirculation of effluent water through two separate treatment loops, an aerobic trickling filter and a predominantly anoxic sedimentation basin, followed by a fluidized bed reactor. The fish basin was stocked for the first 6 mo with red tilapia Oreochromis niloticus × O. aureus at an initial density of 16 kg/m³. During this period salinity was raised from 0 to 20 parts per thousand. Then, gilthead seabream, stocked at an initial density of 21 kg/m³, replaced tilapia at day 167 and were cultured for an additional 225 d. Non steady‐state inorganic nitrogen transformations occurred as a result of these salinity changes. After day 210, the system operated at all times with those water quality parameters considered critical for successful operation of mariculture systems, within acceptable limits. Thus ammonia, nitrite, and nitrate concentrations did not exceed 1.0‐mg total ammonia‐N/ L, 0.5‐mg NO₂:‐N/L and 50‐mg NO₃‐N/L, respectively. Sulfide levels in the fish basin were below detection limits and oxygen > 6 mg/L after the oxygen generator was added at day 315. Ammonia, produced in the fish basin and to a lesser extent in the sedimentation basin, was converted to nitrate in the aerobic trickling filter. Nitrate removal took place in the sedimentation basin and to a lesser extent in the fluidized bed reactor. Sludge, remaining in the sedimentation basin at the end of the experimental period, accounted for 9.2% of the total feed dry matter addition to the system. The system was disease‐free for the entire year and fish at harvest were of good quality. Water consumption for production of 1 kg of tilapia was 93 L and 214 L for production of 1 kg of gilthead seabream. Additional growth performance data of gilthead seabream cultured in a similar but larger system are presented. During 164 d of operation of the latter system, maximum stocking densities reached 50 kgl M³ and fish biomass production was 27.7 kg/m³. Relatively poor fish survival and growth resulted from occasional technical failures of this pilot system.     

Eggs of gilthead seabream Sparus aurata L. as a potential enrichment product of Brachionus sp. in the larval rearing of gilthead seabream Sparus aurata L.

The gilthead seabream Sparus aurata L. is a sequential spawner whose reproductive cycle can be manipulated to obtain year-round egg and fry production. Hence, there is an overproduction of eggs in commercial hatcheries that is normally wasted. Two feeding experiments were conducted with the purpose of investigating the potential use of fresh and freeze-dried gilthead seabream eggs for the enrichment of live preys. The first experiment was also designed to study the possibility of using freeze-drying as a conservation process for eggs in excess. Larvae were reared for 20 days after hatching (DAH),on a diet of Brachionus sp. As experimental controls, the commercial enrichment emulsion SUPER HUFA was used in the first experiment, and the microalgae Nannochloropsis sp. in the second. This study demonstrated the potential of gilthead seabream eggs as an enrichment product to be used in the feeding of the first larval stages of Sparus aurata L. The data obtained emphasize the need to have an overall perspective of the nutritional requirements of the organisms in culture.     

Effect of different feeding strategies on gilthead sea bream (Sparus aurata) demand-feeding behaviour and nutritional utilization of the diet

We have studied the effects of time‐restricted food access and ration restriction on gilthead sea bream demand‐feeding behaviour and nutritional use of the diet (Sparus aurata), and also compared the nutritional efficiency of three different feeding systems: manual, automatic and modulated‐automatic. In the first trial, fish were allowed to feed from self‐feeders under three different conditions: ad libitum, ration restriction, and time‐restricted food access, and their demand‐feeding pattern, diet utilization and body composition were analysed. In the second trial, animals were fed by hand or using an automatic system, either fixed or modulated, and diet utilization and body composition were analysed as before. Restricting the amount of food modifies gilthead seabream self‐feeding behaviour, with fish increasing the number of demands provided these are rewarded with food. However, demand‐feeding activity does not increase if rewards are restricted to a certain time. Feeding gilthead sea bream by hand versus automatically, and distributing the daily food ration in two or three equal or unequal‐size daily meals, have no effect on the animals’ growth, nutritional use of the diet or body composition.     

Influence of lupin and rapeseed meals on the integrity of digestive tract and organs in gilthead seabream (Sparus aurata L.) and goldfish (Carassius auratus L.) juveniles

Two rearing trials were conducted to evaluate the effects of dietary lupin (LM) and rapeseed (RM) meals in gilthead seabream (Sparus aurata L.) and goldfish (Carassius auratus L.) juveniles. Each plant meal was incorporated at the rate of 200 g kg^?1 in two distinct diets, which were compared with a fishmeal‐based diet as control. After 1 month, the two plant diets did not influence the whole body growth, but the digestive systems were affected. The splenosomatic index was reduced with two plant meals in goldfish and with RM in seabream. The hepatosomatic index was only reduced in LM‐fed seabream. Cellular characteristics were also affected. The largest liver cells were observed in RM‐fed goldfish suggesting changes in metabolic function. The LM and RM diets stimulated in seabream, especially the reaction in haematopoietic tissues with the proliferation of melano‐macrophages centres, and a tendency for elongated villus height in the anterior intestine thus that possibly compensated for a reduction in digestive function. Such adaptive structural modifications and the absence of degenerative signs allowed concluding that the integrity of the digestive system was maintained in fish fed plant meals.     

Use of the probiotic Shewanella putrefaciens Pdp11 on the culture of Senegalese sole (Solea senegalensis, Kaup 1858) and gilthead seabream (Sparus aurata L.)

The application of probiotics on aquatic animals is increasing for a better fish welfare status as well as an environment-friendly activity which are actual demands of modern aquaculture industry. A bacterium from skin mucus of healthy gilthead seabream (Sparus aurata L.) has been isolated and identified as Shewanella putrefaciens Pdp11. Different studies have been done to know its application as probiotic in the Senegalese sole and gilthead seabream farming. This article reviews the studies carried out with this probiotic microorganism focusing on the current knowledge of its in vitro and in vivo mechanisms of action. The results suggested that the probiotic S. putrefaciens Pdp11, due to its beneficial effects, could be used in the aquaculture activity of both species.     

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.     

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.     

Effects of dietary dehydrated lemon peel on some biochemical markers related to general metabolism,welfare and stress in gilthead seabream (Sparus aurata L.)

Dehydrated lemon peel (DLP) at two levels (1.5% and 3%) was included for 30 days in the diet of gilthead seabream (Sparus aurata L.) and after 15 and 30 days the serum and skin mucus of fish were analysed. In serum, both experimental diets led to an initial decrease in glucose and lactate after which the levels became comparable to the control. Dietary DLP modulated the activity of transaminases, alanine aminotransferase and aspartate aminotransferase. The total antioxidant status showed a progressive increase in relation to levels of inclusion of DLP and time of administration (p < .05). In skin mucus, some biomolecular markers related to general stress, oxidative stress and apoptosis exhibited variations that suggest an adaptive response of fish to the new metabolic situation, resulting from the experimental diet. The inclusion of DLP in the diet seems to positively modulate the central metabolism and welfare of gilthead seabream.     

Interference of Listonella anguillarum with potential probiotic microorganisms isolated from farmed gilthead seabream (Sparus aurata, L.)

Four isolates obtained from gilthead seabream have been tested for their adhesion to the skin, gill and intestinal mucus of gilthead seabream, and for their ability to interfere with Listonella anguillarum, an important pathogen of farmed gilthead seabream. The ability to adhere to mucus was higher than 7% for all isolates. Three isolates showed an antagonistic effect against some of the pathogenic strains tested. They were assayed to interfere with the attachment of L. anguillarum to the mucus of gilthead seabream. Only two isolates significantly reduced the adhesion of L. anguillarum to all of the mucus assayed under exclusion, competition and displacement conditions. According to the criteria applied, the isolate Pdp11 was selected and its in vivo probiotic potential was assessed by oral administration followed by challenge with the pathogen L. anguillarum. For the feeding trial, a group of 50 gilthead seabreams received a commercial diet supplemented with lyophilized 10⁸ CFU g^?1 of this isolate for 15 days. An other group of similar characteristics received the non‐supplemented commercial diet. After the challenge, the mortality of the fish receiving the diet supplemented with the potential probiotic was significantly lower than that observed in the groups of fish receiving the non‐supplemented commercial diet.     

The effect of dietary exogenous digestive enzymes on ingestion,assimilation, growth and survival of gilthead seabream (Sparus aurata,Sparidae, Linnaeus) larvae

The success of microdiets commonly used in the cultivation of marine fish larvae is limited to serving as partial replacements for live food. This limited success is thought to be associated with a reduced digestive ability due to an incompletely developed digestive system. The enhanced growth obtained from live food has been partially attributed to the digestive enzyme activity of the food organism. The present study was designed to test the effect of an exogenous digestive enzyme incorporated. into a microdiet on the growth of Sparus aurata. Larval gilthead seabream, 20–32 days old, were fed ¹⁴C labelled microdiets containing a commercial pancreatic enzyme at different concentrations (0, 0.1 and 0.05g / 100 g dry diet). Rates of ingestion and assimilation were measured and their relationship to dry weight was determined. Our results show that the success of the microdiet as a food for larval gilthead seabream was limited by the larva's low ingestion rate which only approached its maintenance requirement. In addition, the presence of digestive enzyme in the microdiet enhanced its assimilability by 30%. Larval growth over ten days was 0, 100 and 200% on microdiet free of added enzymes, one with added enzymes and a live food regime, respectively. It is our opinion that successful development of microdiets for Sparus aurata must be based on diets improved both in digestibility and attraction to the larvae. Further studies are now underway to determine the nutritional requirements of gilthead seabream larvae using the experimental method developed in the present study. This research was carried out in partial fulfillment of the requirements for the M.Sc. degree.     

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.     

Effects of Muscari comosum Extract on Nonspecific Immune Parameters in Gilthead Seabream,Sparus aurata (L. 1758)

This study was conducted to determine the effects of Muscari comosum (L.) Mill. 1768, geophyte plant extract on some nonspecific immune parameters of gilthead seabream, Sparus aurata (L. 1758). For this purpose, the plant was collected and extracted in ethanol solvent. Three different fish groups were formed, and each group was duplicated (total 300 fish). The plant extract was applied to the fish by intraperitoneal injection in two different concentrations (0.5 mg/fish and 2 mg/fish). Following injections on the 1st, 7th, 14th, 21st, and 28th d, blood samples were collected from each group. Some of the nonspecific immune parameters in blood and serum samples such as leukocyte level, phagocytic activity (NBT (+) cells), lysozyme activity, and the percentage of white blood cells were determined. The results indicate that in line with the parameters observed, the group given 0.5 mg/fish was not as effective as the one given 2 mg/fish but more effective than the control group. The nonspecific immune parameters of blood samples of the gilthead seabream in the 2 mg/fish experimental group showed that maximum levels on measured parameters of blood were reached at the first week postinjection. The applied concentrations of plant extract did not inhibit growth performance in any of the groups.     

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.     

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.