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.     

Effects of Fish Meal Replacement by a Soybean Protein on Growth,Histology, Selected Immune and Oxidative Status Markers of Gilthead Sea Bream,Sparus aurata

The purpose of the trial was to study the impact of a 6‐mo dietary administration of soybean protein on growth, liver and intestine morphology, immune response, and oxidative stress in gilthead sea bream. The immune response was evaluated by performing immunological assays in blood, head kidney (HK), or serum (respiratory burst activity [RBA], myeloperoxidase content and bacteriolytic activity) and gene expression analysis of immune‐associated genes (MHCIIα [major histocompatibility complex IIα], β2m [β‐2‐microglobulin], CSF‐1R [colony‐stimulating factor‐1 receptor], NCCRP‐1 [nonspecific cytotoxic cell receptor protein 1], TGF‐β1 [transforming growth factor beta‐1], and HSP70 [heat‐shock protein 70]) in HK and intestine. Oxidative stress was evaluated by measuring the activity of liver enzymes associated with antioxidant system. The soybean protein was administrated in the diets at 20, 40, and 60% levels and its effects were evaluated compared with a fish meal diet. Growth and feed efficiency were affected negatively from 40% level. Increased RBA and expression levels of TGF‐β1 and β2m were found in HK only at 40% level. In intestine, at 60% level CSF‐1R expression was upregulated and some signs of inflammation were evident. In liver, at 60% level lipid accumulation in hepatocytes was observed and enzyme activity was increased. Dietary administration of soybean protein indicated changes from 40% in growth and immune response, and exerted an antioxidative effect at 60% level.     

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.     

Fish oil replacement by different microalgal products in microdiets for early weaning of gilthead sea bream (Sparus aurata,L.)

The aim of this study was to determine if algal products rich in DHA or ARA are able to completely replace fish oil in microdiets for marine fish larvae, gilthead seabream and if extra supplementation with EPA may further enhance larval performance. For that purpose, 20 day‐old gilthead seabream larvae of 5.97 ± 0.4 mm mean total length and 0.12 ± 0.001 mg mean dry body weight were fed with five microdiets tested by triplicate: a control diet based on sardine oil; a diet containing AquaGrow^® DHA (diet DHA) to completely substitute the sardine oil; a diet containing AquaGrow^® ARA (diet ARA); a diet containing both products, AquaGrow^® DHA and AquaGrow^® ARA to completely substitute the fish oil; and, a diet containing both products, AquaGrow^® DHA and AquaGrow^® ARA, together with an EPA source. Temperature, air and salinity activity tests were also performed to detect larval resistance to stress. At the end of the experiment, final survivals did not differ among groups. The microorganism produced DHA was able to completely replace fish oil in weaning diets for gilthead seabream without affecting survival, growth or stress resistance, whereas the inclusion of microorganism produced ARA did not improve larval performance. Moreover, addition of EPA to diets with total replacement of fish oil by microorganism produced DHA and ARA, significantly improved growth in terms of body weight and total length. The results of this study denoted the good nutritional value of microorganisms produced DHA as a replacement of fish oil in weaning diets for gilthead seabream, without a complementary addition of ARA. However, dietary supplementation of EPA seems to be necessary to further promote larval performance.     

Nano-delivery of trace minerals for marine fish larvae: influence on skeletal ossification,and the expression of genes involved in intestinal transport of minerals,osteoblast differentiation,and oxidative stress response

Currently, the larviculture of many marine fish species with small-sized larvae depends for a short time after hatching, on the supply of high-quality live zooplankton to ensure high survival and growth rates. During the last few decades, the research community has made great efforts to develop artificial diets, which can completely substitute live prey. However, studies aimed at determining optimal levels of minerals in marine larvae compound feeds and the potential of novel delivery vectors for mineral acquisition has only very recently begun. Recently, the agro-food industry has developed several nano-delivery systems, which could be used for animal feed, too. Delivery through nano-encapsulation of minerals and feed additives would protect the bioactive molecules during feed manufacturing and fish feeding and allow an efficient acquisition of active substances into biological system. The idea is that dietary minerals in the form of nanoparticles may enter cells more easily than their larger counterparts enter and thus speed up their assimilation in fish. Accordingly, we evaluated the efficacy of early weaning diets fortified with organic, inorganic, or nanoparticle forms of trace minerals (Se, Zn, and Mn) in gilthead seabream (Sparus aurata) larvae. We tested four experimental diets: a trace mineral-deficient control diet, and three diets supplemented with different forms of trace minerals. At the end of the feeding trial, larvae growth performance and ossification, and the level of expression of six target genes (SLC11A2β, dmt1, BMP2, OC, SOD, GPX), were evaluated. Our data demonstrated that weaning diets supplemented with Mn, Se, and Zn in amino acid-chelated (organic) or nanoparticle form were more effective than diets supplemented with inorganic form of minerals to promote bone mineralization, and prevent skeletal anomalies in seabream larvae. Furthermore, nanometals markedly improved larval stress resistance in comparison to inorganic minerals and upregulated mRNA copy number of OC gene. The expression of this gene was strongly correlated with mineralization degree, thus confirming its potency as a good marker of bone mineralization in gilthead seabream larvae.     

Improving the fitness of Holothuria arguinensis larvae through different microalgae diets

Development and design of adequate feeding protocols on sea cucumber larvae are necessary for improvements on larvae growth, survival and production of competent juveniles. This work assessed the Holothuria arguinensis larvae growth and survival, fed with three different single microalgae diets of Chaetoceros calcitrans, Isochrysis galbana and Tetraselmis chui, and two microalgae diets based on a combination of C. calcitrans and T. chuii (C:T; 1:1) and C. calcitrans, T. chuii and I. galbana (C:T:I; 1:1:1) during 2 months. Holothuria arguinensis larvae fed with T:C:I, showed higher survival and larger larval and stomach sizes than the ones fed with the other diets. Comparing the results obtained from the single diets, H. arguinensis larvae fed with I. galbana grew and survived better during the first 5 days. However, higher growth was obtained on the larvae fed with C. calcitrans from day 5 to day 12; since this day, larvae fed with T. chuii showed the largest size. These results could be related to the different microalgae size and morphology, in addition to the nutritional value of the different microalgae used. However, no competent larvae that metamorphosed into doliolaria were obtained during this experiment. The tanks used were transparent, which could be causing light stress to the larvae, since competent larvae were obtained in opaque production tanks in our aquaculture installations during the same time period. The combined diet of C. calcitrans, T. chuii and I. galbana (C:T:I) could improve the growth and larvae competency of H. arguinensis.     

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.     

Effect of Two Natural Carotenoid Sources in Diets for Gilthead Seabream,Sparus aurata,on Growth and Skin Coloration

To improve the unnatural fade-pigmented skin of cultivated gilthead seabream, Sparus aurata, (if shown) the present study was initiated. The effects of either red bell-pepper (Capsicum annum) meal or carrot (Daucus carota) meal as a natural dietary carotenoid source, on growth and skin coloration of gilthead seabream growers were investigated. A basal/control diet (D1/CTR) was firstly formulated to contain 48% crude protein and 14% lipids, with no added-carotenoids. With this basal diet, two other test diets were similarly prepared and supplemented each with about 40mg/Kg total carotenoids from either red-pepper meal (D2) or carrot meal (D3). In a feeding trial, fish (mean IW, 94.86±0.3g) were fed one of the three diets (D1, D2, D3), in triplicates for each treatment, for 6 weeks in light-blue background PVC tanks supplied with natural seawater flow. Total carotenoids content of skin was determined spectrophotometerically at initiation and end of the experiment. Neither growth nor feed utilization were significantly (P<0.05) affected by the red pepper-added diet (D2) as compared to CTR diet. However, the carrot fed fish recorded the lowest and significant (P<0.05) weight gain (g/fish) and specific growth rate (SGR, %/d) among dietary treatments. There were no considerable (P>0.05) differences in major nutrients composition between fish fed the experimental diets. Total carotenoids content was significantly (P<0.05) increased, in the skin-opercle area, of fish fed the red pepper diet (D2) as compared to initial fish and to either carrot fed fish or CTR fish. Results have suggested that gilthead seabream can effectively bio-absorb natural carotenoid pigments (mainly capsansin and capsorbin) in red-pepper but not in carrot (mainly β-and α-carotene).     

Characterization of β-haemolytic Group B Streptococcus agalactiae in cultured seabream, Sparus auratus L., and wild mullet, Liza klunzingeri (Day), in Kuwait

Streptococcus agalactiae was isolated from cultured gilthead seabream, Sparus auratus L., and diseased wild Klunzinger's mullet, Liza klunzingeri (Day), in Kuwait Bay, Arabian Gulf. Isolates were catalase negative, β‐haemolytic, Gram‐positive cocci and serogroup B. Experimental infectivity trials with mullet and seabream brain isolates in Nile tilapia, Oreochromis niloticus L., caused 100 and 90% mortality, respectively, within 7 days post‐inoculation indicating virulent S. agalactiae as the bacterial pathogen responsible for the epizootic in Kuwait Bay.     

Growth, survival, quality and digestive enzyme activities of larval shrimp fed microencapsulated, mixed and live diets

An artificial diet for shrimp larvae was microencapsulated with a polysaccharide blend [66% gum arabic, 17% mesquite gum and 17% maltodextrin 10 dextrose equivalent (DE)]. Microencapsulated diet (MD) was fed to mysis alone, as a co‐feed with the microalgae Chaetoceros cerastosporum and Tetraselmis chuii (mixed) and compared with a live diet (control) of C. cerastosporum, T. chuii and Artemia nauplii. No significant differences (P > 0.05) were detected in growth rates, development and quality indexes of larvae fed the three experimental diets. All diets supported survival percentages of >90%. Shrimp larvae fed MD and mixed diets had higher specific trypsin activity and soluble protein content than those fed live diet. Amylase activity decreased in larvae fed the mixed and MD apparently due to the carbohydrate composition of the diet. The results indicate that it is possible to substitute a live diet with a microencapsulated one (with a wall composition made up of a polysaccharide blend) in Litopenaeus vannamei mysis.     

Depletion of serum alternative complement pathway activity in gilthead seabream caused by α-tocopherol and n-3 HUFA dietary deficiencies

Studies on the alternative complement pathway (ACP) activity in fish have suggested the particular relevance of this pathway as a non-specific immune mechanism being its activation directly related with the availability of certain nutrients. In the present study ACP activity in gilthead seabream fed different dietary levels of a-tocopherol and n-3 HUFA deficiencies was studied under different stress conditions (overcrowding and repetitive chasing).A reduction in ACP activity was found when diets with deficiencies in -tocopherol and n-3 HUFA were assayed at any of the experimental conditions assayed. Levels of plasma cortisol were similar to those of fish after crowding stress subjected to dietary deficiencies at low stocking density, suggesting that dietary deficiencies produced a chronic elevation of cortisol. When fish were subjected to repetitive chasing, plasma cortisol from fish fed control diet showed a progressive increase whereas plasma cortisol levels from both dietary deficient groups showed a transitory peak 4 days after the beginning of the repetitive chasing. However, ACP activity levels of fish fed deficient diets remained lower than the control group and constant during chasing period. These results suggest that both a-tocopherol and n-3 HUFA nutritional deficiencies rather than stress are an important determinant of fish complement activity.     

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.     

Observations on the increase of wild gilthead seabream,Sparus aurata abundance,in the eastern Adriatic Sea: problems and opportunities

The recent increase of the local population of gilthead seabream, Sparus aurata, in three areas along the southeastern Adriatic Sea: Malostonski Bay (Croatia and Bosnia and Herzegovina), Neretva Estuary (Croatia) and Boka Kotorska Bay (Montenegro) and its adverse effects on shellfish culture by preying on Mediterranean mussels, Mytilus galloprovincialis, and the European flat oyster, Ostrea edulis, are studied. The results from the analysis of the existing information show that the main reason for the recent increase is the escapes from local fish farm which enrich the local population constantly with new gilthead sea bream. The existence of practically endless food in the area of the shellfish farms allows the concentration of the population in the region instead of its dispersion along the Adriatic coast. Moreover, ecological analysis indicates that the gilthead seabream is facing a very low competition from other local species which enhances its capacity to further populate the region. While the impact on the ecosystem is not yet known, the socio-economic impact of the increase of the gilthead seabream population is evident today. Many shellfish farms are closing today in the region since the damages may reach over 90 % of the production.     

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.     

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.     

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.     

Virulence factors and pathogenicity of Hafnia alvei for gilthead seabream, Sparus aurata L

Virulence factors (eae gene, haemolytic capacity, fimbriae, resistance to the bactericidal effect of serum, siderophore production) and pathogenicity for gilthead seabream, Sparus aurata L., were analysed for 23 Hafnia alvei strains. None of the strains used in LD50 studies were lethal for seabream at doses as high as >10(8) cfu mL(-1). In chronic challenge studies differences in severity of the inflammatory response were observed between strains. On the basis of correlation of the inflammatory response to different strains of H. alvei in seabream with those virulence factors studied, it was only possible to establish a positive correlation between pathogenicity and resistance to the bactericidal effect of fish serum. Gilthead seabream is thus a species with considerable resistance to experimental infection with H. alvei. The bacterium does, however, have the capacity to remain viable in seabream for up to 3 months, without any clinical signs. Hafnia alvei is a well-recognized human and animal pathogen. Thus, as the pathogen can coexist with aquaculture operations, cultured gilthead seabream could represent a risk to human health as a carrier in some circumstances.