Preliminary studies on the cryopreservation of gilthead seabream (Sparus aurata) embryos

Vitrification could provide a promising tool for the cryopreservation of fish embryos. In order to achieve successful cryopreservation, several parameters should be taken into account in the design of a vitrification protocol. In the present study some relevant factors were investigated (permeable and non-permeable cryoprotectant toxicity, toxicity of vitrificant solutions, adequate container for embryo loading and temperature of thawing) using two gilthead seabream embryonic development stages (tail bud and tail-bud-free). Permeabilized embryos were incubated in dimethyl sulfoxide (DMSO), methanol (MeOH), ethylene glycol (EG) and 1,2-propanediol (PROH) in concentrations ranging from 0.5 to 6 M for 10 and 30 min and in 5%, 10% and 15% polyvinyl pyrrolidone (PVP), 10%, 15% and 20% sucrose or 0.1%, 1% and 2% X1000® for 2 min. After treatment, embryos were washed and incubated in seawater until hatched. The toxicity of permeable cryoprotectants increased with concentration and exposure time. EG was best tolerated by the embryos. Exposure to non-permeable cryoprotectants did not affect the hatching rate except at F stage. Six vitrificant solutions (DMSO—V1, V2 and V3 and EG—V1, V2 and V3) were tested using a stepwise incorporation protocol. The DMSO-based solutions contained 5 M DMSO + 2 M MeOH + 1 M EG plus 5% PVP, 10% sucrose or 2% X1000® and the EG-based solutions contained 5 M EG + 2 M MeOH + 1 M DMSO plus 5% PVP or 10% sucrose. Before loading the embryos into 0.5 ml straws or 1 ml macrotubes, toxicity tests were effected with these solutions. Our results demonstrated that DMSO-based solutions were better tolerated by seabream embryos than EG-based solutions. After thawing (water bath, 0 or 25 °C), embryos were evaluated by stereoscopic microscopy and the percentage of embryos with intact morphology was registered. The highest percentage of embryos with intact morphology (28%) was observed in samples frozen in macrotubes and thawed at 25 °C. Several malformations associated with ice crystal formation inside the embryos were detected. None of these embryos achieved hatching. Our results suggest that the absence of a proper incorporation of cryoprotectants prior to vitrification is the main problem that must be overcome. This procedure should be optimized in order to avoid ice crystal formation inside embryo compartments.     

Tissue distribution and depletion of sarafloxacin hydrochloride after in feed administration in gilthead seabream (Sparus aurata L.)

Tissue distribution and depletion of sarafloxacin was studied in gilthead seabream, under experimental field conditions at 25 and 18 °C, after in-feed administration of sarafloxacin hydrochloride for 5 days (10 mg/kg body weight/day). Ten fish per sampling point were examined during and after treatment. Muscle plus skin (n=10), liver, kidney and vertebra (pooled) were collected and analyzed by HPLC. Sarafloxacin concentrations in these tissues increased during the medication period, and then decreased rapidly. The highest sarafloxacin concentrations were recorded in liver (335.2 and 49.8 μg/g at 25 and 18 °C, respectively). In muscle plus skin, sarafloxacin concentrations were 193.0 and 42.0 μg/kg at 25 and 18 °C, respectively. Sarafloxacin residues were eliminated rapidly; at 36 h post treatment, the levels in muscle plus skin were 10.2 and 8.5 μg/kg at 25 and 18 °C, respectively. Elimination half-lives (t_1/2) were 17.8 and 32.5 h at 25 and 18 °C, respectively. Withdrawal period for the Maximum Residue Limit (MRL) of 30 μg/kg sarafloxacin in muscle plus skin (95% tolerance limit) at 25 °C was 42.2 h. The slow elimination from vertebra, relative to other tissues, suggests that vertebra behaves as a reservoir in gilthead seabream.     

Optimum soybean lecithin contents in microdiets for gilthead seabream (Sparus aurata) larvae

The aim of the present study was to determine the optimum dietary levels of soybean lecithin (SBL) for seabream (Sparus aurata) larvae, and its influence on production performance and digestive enzymes activity. Larvae were fed five formulated microdiets with five levels of SBL. Complete replacement of live preys with the experimental microdiets for seabream larvae at 16 dph produced over 55% survival rates, particularly in fish fed with the highest levels of SBL. Moreover, increase in dietary SBL up to 80 g kg⁻¹ significantly improved larval growth, leading to high final total length and body weight. An increase in alkaline phosphatase activity with the elevation up to 80 g kg⁻¹ SBL was also found denoting a better maturation of the digestive system. Besides, there was a stimulatory effect of dietary SBL on PLA2 activity. Finally, increasing dietary SBL lead to better utilization of dietary highly unsaturated fatty acid, as it was reflected in their higher content in both neutral and polar lipid of the larvae. In summary, elevation of dietary SBL up to 80 g kg⁻¹ in microdiets for seabream significantly improved digestive enzymes activities, enterocyte maturation, utilization and deposition of dietary essential fatty acids and larval growth, as a consequence of a better digestion, absorption, transport and deposition of dietary nutrients.     

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.     

Association of a lordosis-scoliosis-kyphosis deformity in gilthead seabream (Sparus aurata) with family structure

Skeletal deformities constitute a major problem for aquaculture industry by decreasing the final value of the fish. An analysis of skeletal malformations in 11,640 fish was performed considering families and triplicates per family. Thirtynine different skeletal abnormalities were detected, such as lordosis, vertebral fusion, absence of one or both operculum, bent-jaw, etc. Moreover, a new unusual complex spinal column deformity consisting of a consecutive repetition of lordosis, scoliosis and kyphosis (LSK) from the head to the caudal fin was described. This syndrome was statistically associated with the family structure (Z_{2H–family,LSK}=3.49; p<0.05). The incidence of this deformity was 0.2% in the whole population, and 6.5% within the affected family (2H). The environmental and genetic causes are discussed.     

Characterization of digestive enzyme activity during larval development of gilthead seabream (Sparus aurata)

The evolution of the digestive enzyme equipment in seabream from hatching to 30 days old larvae was studied; there was a progressive increase in the activity of protease, amylase and acid and alkaline phosphatase from day 15 onwards. The use of specific inhibitors, and SDS-PAGE provided evidence to suggest that most of the proteases belonged to the serine group. A high -amylase activity was also denoted. Zymograms of larval extracts indicated that exogenous food has more a qualitative than a quantitative role in the secretion of digestive enzymes in this species.     

Predicting aquaculture waste from gilthead seabream (Sparus aurata) culture using a nutritional approach

A model to estimate the waste production from sea cage culture was established. Using known feed inputs of nitrogen, phosphorus and organic matter, the model quantifies waste discharge from seabream culture. Daily feed intake and growth in Sparus aurata fed a commercial diet with known composition were measured and found to be dependent on fish weight and water temperature. Digestibility of the commercial feed was measured using chromic oxide as a marker and collection of feces by stripping. The proximate composition of Sparus aurata at different sizes was determined and nitrogen and phosphorus content were on average 28.5 and 7.2 g·kg⁻¹ body mass, respectively. Excretion of ammonia-nitrogen and inorganic phosphorus after metabolic processes was calculated as the difference.     

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.     

Relation of gilthead seabream (Sparus aurata) seasonal reproductive activity to hematology,serum biochemistry,histopathology, and Brdt gene expression

Fish Physiology and Biochemistry - This study aimed to find the relation of Sparus aurata (gilthead seabream) reproductive activities to some blood parameters as complete blood count, liver...     

Nutritional stimuli of gilthead seabream (Sparus aurata) larvae by dietary fatty acids: effects on larval performance,gene expression and neurogenesis

The concept of nutritional programming raises the interesting possibility of directing specific metabolic pathways or functions in juvenile fish, for example, to improve the use of substitutes to fishmeal and oil, and hence to promote sustainability in aquaculture. The aim of the study was to determine effects of early nutritional stimuli of gilthead seabream larvae and check if nutritional programming of gilthead sea bream is possible between 16 days post hatching (dph) and 26 dph. A trial was conducted to determine the effects of early nutritional stimuli of gilthead seabream larvae. Five experimental microdiets (pellet size <250 μm) were formulated containing five different proportions of a marine lipid source rich in long‐chain polyunsaturated fatty acids (LC‐PUFA) and two vegetable lipid sources rich in linolenic and linoleic acids. The results of this study demonstrate that dietary n‐3 LC‐PUFA levels increased larval growth and survival affecting Δ6 desaturase gen (fads2) expression and retinal neurons density. However, the high mortalities obtained along on‐growing in fish fed low n‐3 LC‐PUFA at 16 dph constrained the feasibility of nutritional programming of gilthead seabream during this late developmental window and needs to be further investigated.     

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.     

Cloning of a second variant of somatolactin-encoding cDNA from the gilthead seabream Sparus aurata

cDNA clones which include coding sequences of the gilthead seabream (Sparus aurata) somatolactin (SL) have been isolated from a cDNA library prepared from seabream pituitary gland poly (A)⁺ RNA. Flounder – SL cDNA was used as a hybridization probe. The complete nucleotide (nt) sequence of the seabream somatolactin has been determined. The clone encodes a polypeptide of 231 amino acid (aa) residues including 24 amino acid residues of signal peptide. Northern blot hybridization detected one band of approximately 1.8 kb mRNA. By comparing the sequences of this SL cDNA to the one recently published, it is suggested that two variants of the SL exist in seabream. By comparing the sequences of the aa of SL to the deduced aa sequences, it is possible that even a third variant of SL exists in this species.     

Energy content and chemical composition of gilthead seabream, Sparus aurata L., eggs

Abstract. Gross energy, ash content and elemental composition of carbon, nitrogen and hydrogen in eggs of the gilthead seabream, Sparus aurata L., have been determined throughout several spawning seasons. Also, changes in the biochemical components during embryonic development have been analysed. Gross energy of seabream eggs was approximately 1 J per egg. Dry weight of individual eggs at fertilization varied from 39 to 50μg, the chorion accounting for about 20% of total dry weight. However, there were no differences in carbon, nitrogen, hydrogen and ash percentages. From fertilization to hatching eggs lost 7·2% of total energy (chorion not included). The possible relationship between egg energy content and recent emerged larvae is discussed.     

Energy and protein requirements for maintenance and growth in gilthead seabream (Sparus aurata L.)

Factorial determinations of energy and protein requirements in growing Sparus aurata were carried out at 23–24°C. The energy content in the whole fish was dependent on fish weight and ranged from 5 to 11 MJ kg⁻¹ body mass for 1–250 g fish, whereas the protein content remained constant at 179 g kg⁻¹.
During starvation the fish lost 42.5 kJ body weight (BW) (kg)^−0.83 day⁻¹ and 0.42 g protein BW (kg)^−0.70 day⁻¹. The maintenance requirement for energy was calculated to be 55.8 kJ BW (kg)^−0.83 day⁻¹ and for protein 0.86 g BW (kg)^−0.70 day⁻¹. Utilization of digestible energy and digestible crude protein below and at maintenance was determined as 0.72 and 0.51, respectively. Utilization of digestible energy and digestible crude protein for growth above maintenance was determined as 0.46 and 0.28, respectively.
These values allow estimation of requirements for growing Sparus aurata .     

The effect of spray‐dried porcine plasma on gilthead seabream (Sparus aurata) intestinal microbiota

The effect of spray‐dried porcine plasma (SDPP) on the intestinal histological organization and autochthonous microbiota composition was evaluated in Sparus aurata. Fish were fed a basal diet (51 g/kg protein, 17 g/kg fat, 20.6 MJ/kg gross energy) and a diet containing 3 g/kg SDPP for 95 days (initial body weight, BW = 9.5 ± 0.2g, mean ± SD). The inclusion of SDPP promoted growth (p < .05), being fish fed the SDPP diet 6.2% (BW = 88.2 ± 1.6 g) heavier than the control (BW = 82.7 ± 3.2 g). SDPP increased the density of intestinal goblet cells (p < .05), whereas no differences in villi height were found (p > .05) between both groups. Intestinal microbiota was dominated by Proteobacteria (>85%) and Firmicutes (5%–12%), whereas Bacteroidetes never represented more than 1.5%. γ‐Proteobacteria, and Bacilli and Clostridia were the predominant classes. The short administration of SDPP (20 days) resulted in changes in microbiota diversity and richness associated with an increase in the sequences of the genus Lactobacillus and to a decrease in the genus Vibrio, whereas these changes were reverted at 95 days. Intestinal goblet cell density was not correlated to microbiota diversity and richness changes rather than to the immunostimulatory effect of the SDPP.     

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.     

Regulation of growth, fatty acid composition and delta 6 desaturase expression by dietary lipids in gilthead seabream larvae (Sparus aurata)

The Δ6 and Δ5 desaturases and elongases show only very limited activity in marine fish, and little is known of the possibility of enhancing Δ6 desaturase gene expression in these fish. The use of plant oils in marine fish diets is limited by their lack of n−3 highly unsaturated fatty acids (HUFA) despite an abundant content of the 18C fatty acid precursor linoleic and α-linolenic acids. The objective of the present study was to determine the ability of larval gilthead seabream to utilize vegetable oils and assess the nutritional regulation of Δ6 desaturase gene expression. Seventeen-day-old gilthead seabream larvae were fed during a 17-day period with one of four different microdiets formulated with either sardine fish oil (FO), soybean, rapeseed or linseed oils, respectively, or a fifth diet containing defatted squid meal and linseed oil. Good larval survival and growth, both in terms of total length and body weight, were obtained by feeding the larvae either rapeseed, soybean or linseed oils. The presence of vegetable oils in the diet increased the levels of 20:2n−9 and 20:2n−6, 18:2n−9, 18:3n−6, 20:3n−6 and 20:4n−6, in larvae fed rapeseed and soybean oils in comparison to those fed FO. In addition, a sixfold increase in the relative expression of Δ6 desaturase-like gene was found in larvae fed rapeseed and soybean oils, denoting the nutritional regulation of desaturase activity through its gene expression in this fish species. However, feeding linseed oil did not increase the expression of the Δ6 desaturase gene to such a high extent.     

Vitamin C status and physiological response of the gilthead seabream, Sparus aurata L., to stressors associated with aquaculture

Gilthead seabream, Sparus aurata L., fed diets of equal vitamin C content were exposed to three different stressors–hyposalinity, shallow water and handling–and the effect of each on the internal homeostasis of the fish was evaluated. The individuals subjected to hyposaline stress exhibited no significant changes in plasma osmolality. Na⁺. Cl^- K⁺, glucose, protein and triglyceride concentrations. Hepatic glycogen content was significantly lower in the fish subjected to the lower salinity (12%o), while the red muscle glycogen was unaltered. Amongst the challenged individuals, tissue ascorbate content was not significantly altered. Shallow water stress significantly diminished the liver and red muscle glycogen content, and lowered the liver. kidney and spleen vitamin C concentrations in the stressed fish. No significant changes were recorded in the plasma glucose, protein and triglyceride concentrations. In response to handling stress, no significant differences were detected in any of the measured parameters, with the exception of plasma glucose levels and the splenic L-ascorbic acid and ascorbate-2-sulphate (vitamin C2) contents. The lack of existing standardization of both experimental designs and analytical methods made comparisons of the present data with the work of other authors difficult, thus highlighting the necessity of coordinated research in this field.