Seasonality of nuclear abnormalities in gilthead sea bream <Emphasis Type="Italic">Sparus aurata</Emphasis> (L.) erythrocytes

The frequencies of erythrocyte nuclear abnormalities, such as irregularly shaped (ISN), vacuolated nuclei (VN), binucleated cells (BN), including micronuclei (MN), were monitored in the peripheral blood of cultured gilthead sea bream (Sparus aurata L.) from the southern Adriatic Sea (Croatia) and assessed for a relation to seasonality. Fish were sampled in February, April, June, and September. The lowest frequencies of ISN and MN were measured in April, and the lowest frequencies of VN and BN were found in February. The highest values of ISN and BN were detected in September. The highest values of MN and the most frequent occurrence of VN occurred in June and April, respectively. The Spearman rank order correlation test revealed a significant positive correlation between the frequencies of ISN, BN and MN; VN did not correlate with any of the measured parameters. The ISN had significantly higher nuclear surface area values than normal nuclei (P < 0.001). Our results demonstrate that the measured parameters displayed a distinct seasonality. Consequently, we conclude that seasonal variations should be taken into account when nuclear abnormalities are used as biomarkers. This variation should be well understood in order to successfully separate true contamination effects from seasonal factors that have the potential to affect the frequency and occurrence of nuclear abnormalities.     

Olive oil by-product as a natural antioxidant in gilthead sea bream (<Emphasis Type="Italic">Sparus aurata</Emphasis>) nutrition

The aim of the study reported here was to evaluate the efficiency of a natural antioxidant substance in gilthead sea bream (Sparus aurata) feeds. An olive oil by-product, olive mill vegetation water (VW), contains polyphenols, which have a strong antioxidant activity. A 147-day growth trial was conducted (monofactorial balanced, 4 × 3) with diet as the experimental factor. Two diets [isonitrogenous (crude protein 40%) and isoenergetic (gross energy = 18MJ kg⁻¹ on a dry weight basis] were formulated with 1 and 5% of VW (VW1, VW5); the control diet did not contain VW. A total of 600 juvenile gilthead sea breams (mean body weight 114.1 ± 5.7 g) were utilised. Production parameters and somatic indexes were calculated at the end of the growth trial. Antioxidant activity in fish fillets was investigated using TBARS and DPPH assays. A number of haematological parameters and digestive enzyme patterns were measured in fish in the middle and at the end of the experiment. The TBARS values showed slight delays in the development of oxidation in the fillet of fish fed with VW. There was no statistical difference between fish fed with the experimental diet and the control group, except for maltase activity, which increased with increasing VW in the feed. We found that the use of VW in a gilthead sea bream diet did not have any detrimental effects on gilthead sea bream production and physiological parameters and slightly improved the conservation of the fish fillets.     

Oxygen consumption and feeding rates of gilthead sea bream (<Emphasis Type="Italic">Sparus aurata</Emphasis>) reveal lack of acclimation to cold

Low temperature has been implicated in inducing outbreaks of winter syndrome or winter disease in farmed gilthead sea bream (Sparus aurata). The responses of gilthead sea bream to reduced temperature followed by maintenance at low temperaturewere studied. In a first experiment, oxygen consumptionwas measured when water temperature was reduced from 18°C to 8 °C at either a rate of 1 °C· day-1 or as two sharp drops (from 18 °C to 12 °C, and from 12 °C to 8 °C). In a second experiment, the water temperature was reduced from 16 °C to 8 °C or 12 °C and then maintained for 20 days to study the fish acclimation to these temperatures. In both experiments, fish stopped feeding below 13 °C and did not resume feeding when maintained at low temperatures. The decrease in metabolic activity, expressed by the oxygen consumption rate, was directly related to the fall in water temperature: the Q10(18 °C-8 °C) values were between 2.2–2.5, independently of the descend rate in water temperature. However, we observed a more reduced metabolic rate when the water temperature was below 12 °C. Fish maintained at low temperatures showed only a partial recovery in oxygen consumption (15% at 8 °C and 20% at 12 °C) after 20 days. A higher metabolic rate together with a fasting-temperature condition meant that maintenance at 12 °C was more aggressive than at 8 °C, as revealed by the condition factor and energy needs. Data suggest that 12 .C could be a threshold temperature for the metabolic activity of gilthead sea bream. The relationship between low temperatures and their possible implication in the appearance of winter disease in gilthead sea bream is also discussed.     

Growth and feed utilization of gilthead sea bream (<Emphasis Type="Italic">Sparus aurata</Emphasis>, L.) fed to satiation and restrictively at increasing dietary energy levels

Three isoproteic (47% protein) diets were formulated to contain graded levels of crude fat (diet D16:16%, diet D24: 24% and diet D32: 32%). Each diet was fed to satiation in three and to 80% satiation in two replicate groups of gilthead sea bream (Sparus aurata), having an initial body weight of 72–74 g. The trial lasted 81 days. Groups fed to satiation showed higher final body weight (FBW; 238.8–252.3 g vs. 218.0–229.3 g) and daily growth index (DGI; 2.49–2.65%/day vs. 2.27–2.34%/day) than those fed to 80% satiation. Feed intake was significantly different both for feeding level and for diet composition. Fish fed to satiation had higher feed conversion rate (FCR) compared to the 80% satiation groups (1.33–1.44 vs. 1.13–1.17; P ≤ 0.001). Within satiation groups, FCR was significantly lower in fish fed D16 compared to fish fed D32 (1.33 vs. 1.44, P ≤ 0.05), whereas no statistical differences were found within the 80% satiation groups. The increase in dietary lipid level did not improve growth performance, feed efficiency and protein utilization but decreased gross lipid efficiency. Conversely, a reduction in ration from satiation to 80% satiation decreased DGI, thus improving FCR. Feed costs were influenced by dietary energy level and feeding ratio, the lowest energy diet at 80% satiation being the most profitable combination among the variables.     

A comparative study on the composition and importance of free amino acids in semen of gilthead sea bream, <Emphasis Type="Italic">Sparus aurata</Emphasis>, and perch, <Emphasis Type="Italic">Perca fluviatilis</Emphasis>

A comparative study was conducted on the free amino acid composition of gilthead sea bream, Sparus aurata, and perch, Perca fluviatilis. Also the effect of 21 free amino acids on sperm motility was investigated. Spermatozoa were incubated in species-specific motility-inhibiting saline solution containing the different amino acids for 48 h. Thereafter, the motility was activated and investigated using computer-assisted cell motility analysis. Twelve free amino acids, respectively, were detected in S. aurata and P. fluviatilis semen. Arginine, cysteine, glutamic acid, leucine, and methionine occurred in semen of both species. In S. aurata, arginine, glycine, hydroxyproline, lysine, and phenylalanine in concentrations of 1.25 and 2.50 mmol/l, methionine in a concentration of 2.5 mmol/l, and serine in a concentration of 1.25 mmol/l had a positive effect on the motility of spermatozoa. In P. fluviatilis, alanine, asparagine, cysteine, glycine, isoleucine, lysine, methionine, serine, threonine, and valine in concentrations of 2.50 mmol/l positively affected motility. From these data, it can be concluded that the amino acid composition and the effect on motility are species specific. Possible consequences for spermatozoa functionality are discussed.     

Partial characterization of pyloric-duodenal lipase of gilthead seabream (<Emphasis Type="Italic">Sparus aurata</Emphasis>)

In the present study, we report the isolation and characterization of seabream Sparus aurata pyloric caeca-duodenal lipase. Optimum activity was found at pH 8.5 and salinity of 50 mM NaCl. Lipase activity was sensitive to divalent ions, and extreme pH values (4, 5, and 12), being more stable at alkaline than acid pH. Optimum temperature was found at 50°C, but lipase was stable at temperatures below 40°C. Lipase has a bile salt sodium taurocholate requirement for increased activity. Gradient PAGE electrophoresis revealed the presence of four isoforms with apparent molecular masses of 34, 50, 68, and 84 KDa, respectively. Pyloric-duodenal lipase was able to hydrolyze emulsified alimentary oils. Results confirm the presence of true lipases in Sparus aurata digestive tract.     

Genetic assessment of three gilthead sea bream (<Emphasis Type="Italic">Sparus aurata</Emphasis> L.) populations along the Spanish coast and of three broodstocks managements

The gilthead sea bream (Sparus aurata L.) is one of the most important Sparid farmed in Europe, especially in the Mediterranean area. However, efficient breeding programs for this species are scarce and very little, and it is known concerning their population structure. The present study was mainly designed to genetically characterize, by microsatellite markers, three gilthead sea bream populations sampled along the Spanish coast (Cantabrian Sea, the Atlantic Ocean and Mediterranean Sea) and their progeny with the aim of studying its genetic variability and its genetic structure. Moreover, we evaluated different strategies of broodstocks management (breeders’ number, origin and sex combination) on parental contributions and on effective breeding numbers. That number of breeders in the stock was of great importance to the maximization of contribution since the larger broodstock resulted in larger proportion of parents contributing. Variation in dam and sire spawning incidence and in number of progeny produced per dam and per sire was translated into reduced effective breeding numbers and consequently into expected increased inbreeding rates. Our results highlight the high genetic variability of the studied sea bream populations, as well as the existence of three genetically differentiated populations along the Spanish coast. These findings should be relevant for the establishment of successful breeding programs in aquaculture of the gilthead sea bream.     

Influence of time,storage temperature and freeze/thaw cycles on the activity of digestive enzymes from gilthead sea bream (<Emphasis Type="Italic">Sparus aurata</Emphasis>)

In this study, we tested the effects of long-term storage (2 years) at ?20 °C and short-term storage (several hours) in ice and freeze/thaw cycles on the activities of pancreatic, gastric and intestinal (brush border and cytosolic) digestive enzymes in a teleost fish species. The results revealed a significant lose in activity of pancreatic (trypsin, chymotrypsin, total alkaline proteases and α-amylase) and intestinal cytosolic (leucine–alanine peptidase) enzymes between 140 and 270 days of storage at ?20 °C, whereas in contrast, the activity of all the assayed brush border enzymes remained constant during the first 2 years of storage at ?20 °C. During short-term storage conditions, the most stable enzymes assayed were those of the enterocytes of the brush border, which did not show any change in activity after being held for 5 h in ice. Five freezing and thawing cycles did not affect the activity of the intestinal brush border enzymes and the cytosolic ones, whereas the activity of trypsin, α-amylase and bile-salt-activated lipase was significantly affected by the number of freezing and thawing cycles. No changes in pepsin activity were found in samples exposed to 1 and 2 freezing and thawing cycles.     

Changes in muscle collagen content during <Emphasis Type="Italic">post mortem</Emphasis> storage of farmed sea bream (<Emphasis Type="Italic">Sparus aurata</Emphasis>): influence on textural properties

The changes in collagen content and its solubility in sea bream muscle were studied for variable storage times following the death of the fish, and these variables were related to the evolution of physical parameters important for consumer acceptance: firmness and water-holding capacity (WHC). The results show that the collagen content in muscle diminished slightly over storage time and that this variable was directly related to firmness but inversely related to the water-holding capacity. With regard to collagen solubility, a decline was detected in acid-soluble collagen (ASC) in the first few post mortem hours, perhaps related to the end of rigor mortis that occurs at these stages. Pepsin-soluble collagen (PSC) increased, while insoluble collagen (ISC) decreased from 96 h, coinciding with a loss of firmness. This softening can be explained as a result of specific collagenases acting on the insoluble fraction of the collagen.     

Influence of the dietary inclusion of <Emphasis Type="Italic">Gracilaria cornea</Emphasis> and <Emphasis Type="Italic">Ulva rigida</Emphasis> on the biodiversity of the intestinal microbiota of <Emphasis Type="Italic">Sparus aurata</Emphasis> juveniles

Partial substitutions of fish meal by 5, 15, or 25 % of Gracilaria cornea or Ulva rigida in experimental diets were evaluated to study their effects on biodiversity of intestinal microbiota composition in gilthead seabream (Sparus aurata) juveniles. The diets were offered to duplicate groups of 15 juvenile fish (14.0 ± 0.5 g) for 70 days, and at the end of the experiment the intestinal microbiota from four specimens of each treatment was analysed by denaturing gel gradient electrophoresis. Results showed that the substitution of fish meal by algae meal induced important modifications in the intestinal microbiota community, as a big reduction of the biodiversity when the highest percentage (25 %) of U. rigida was included. On the contrary, an increase on the number of species was detected when a 15 % of algae was included. Various Lactobacillus delbrueckii subspecies were selectively stimulated when G. cornea was included in the feed, and other bacterial species, such as those included in the Vibrio genus, were reduced.     

Origin of broodstock and effects on the deformities of gilthead sea bream (<Emphasis Type="Italic">Sparus aurata</Emphasis> L. 1758) in a Mediterranean commercial hatchery

The use of broodstock of different origin as a method to improve fry production performance and consequently to minimize deformities was examined at industrial scale in a commercial gilthead sea bream hatchery. The outcome of fry production from three different broodstock groups (BA: broodfish (Mediterranean) with multiannual hatchery presence, BB: selected offspring originating from the BA group, and BC: broodfish of Atlantic origin) was investigated in the same rearing conditions and feeding protocol. Performance factors assessed were the survival and weaning of the larvae; the mortality rates from the “weaning until the end of the hatchery stage” of the larvae/fry; the percentage of fry without swim bladder; the percentage of fry with skeletal deformities and the feed conversion ratio. In all factors, no statistical differences among the experimental groups were detected. However, due to early rejection of the deformed individuals, benefits are expected from the decrease of the supplied amount of food and the reduced labor cost.     

Vessel noise pollution as a human threat to fish: assessment of the stress response in gilthead sea bream (<Emphasis Type="Italic">Sparus aurata</Emphasis>, Linnaeus 1758)

This study examined the effects of boat noise pollution on the stress indices of gilthead sea bream (Sparus aurata, Linnaeus 1758). To assess the stress response in these fish, biometric values and plasma parameters such as ACTH, cortisol, glucose, lactate, haematocrit, Hsp70, total protein, cholesterol, triglycerides and osmolarity were analysed. After acclimatization of the animals, the experiment was carried out in a tank fitted with underwater speakers where the fish were exposed to sound treatments (in duplicate) consisting of: 10 days of no sound (control treatment; the animals were only exposed to the experimental tank’s background noise) and 10 days of noise derived from original recordings of motor boats, including recreational boats, hydrofoil, fishing boat and ferry boat (vessel noise treatment). The exposure to noise produced significant variations in almost all the plasma parameters assessed, but no differences were observed in weights and fork lengths. A PERMANOVA analysis highlighted significantly increased values (p < 0.05) of ACTH, cortisol, glucose, lactate, haematocrit, Hsp70, cholesterol, triglycerides and osmolarity in the fish exposed to vessel noise for 10 days. This study clearly highlights that anthropogenic noise negatively affects fish, and they are valuable targets for detailed investigations into the effects of this global pollutant. Finally, these experimental studies could represent part of the science that is able to improve the quality of the policies related to management plans for maritime spaces (Marine Strategy Framework Directive 56/2008 CE) that are aimed at stemming this pollutant phenomenon.     

Effects of additive iron on growth,tissue distribution,haematology and immunology of gilthead sea bream, <Emphasis Type=Italic>Sparus aurata</Emphasis>

The comparative effects of iron-supplemented levels on growth, tissue distribution, haematology and immunology of gilthead sea bream, Sparus aurata (2 g) were investigated, using four organic (50, 100, 200, 300 mg ORG/kg diet) and one inorganic iron source (200 INOR mg/kg diet). Fish were treated for 12 weeks with the experimental diets and maintained at a water temperature of 19–22°C. Growth (final weight and specific growth rate), tissue distribution (spleen, liver and muscle), haematological parameters (red blood cells, haematocrit, haemoglobin and mean corpuscular haemoglobin concentration) and non-specific immune indexes (respiratory burst activity and antibacterial activity of serum) were analysed. No significant differences were found in growth and iron tissue distribution among the tested groups. Red blood cell counting was statistically higher in fish given 50 ORG, 100 ORG, 200 ORG and 200 INOR feeds. However, haematocrit, haemoglobin and mean corpuscular haemoglobin concentration were not significantly affected by increasing dietary iron. Fish receiving the 100 ORG diet had the best performance with respect to the respiratory burst activity and significantly higher values for antibacterial activity of serum were obtained in fish fed with the 300 ORG diet. The present findings provided no clear evidence of the optimum iron concentration. However, there was adequate indication that iron supplementation enhanced the performance of gilthead sea bream, mainly from a haematological and immunological point of view.     

Using trace elements in otoliths to discriminate between wild and farmed European sea bass (<Emphasis Type="Italic">Dicentrarchus labrax</Emphasis> L.) and Gilthead sea bream (<Emphasis Type="Italic">Sparus aurata</Emphasis> L.)

Trace elements in otoliths of sea bass (Dicentrarchus labrax L.) and sea bream (Sparus aurata L.) from fish farms and coastal wild populations in the western Mediterranean Sea were analysed by inductively coupled plasma-mass spectrometry. Results showed that concentrations of Mg, K, and Mn differed significantly between wild and farmed sea bass, while concentrations of Mg, K, Mn, Fe, Zn, Sr, and Ba varied significantly between wild and farmed sea bream. Discriminate analysis and cross-validation classification showed that the trace element profile in otoliths can be used to separate farmed fish from wild stocks with high accuracy on both sea bass (individuals correctly classified: 90.7 %) and sea bream (individuals correctly classified: 96.6 %). Moreover, trace elements in otoliths resulted to be useful to discriminate among wild fish stocks within each species.     

Characterization and in vitro sensitivity of cholinesterases of gilthead seabream (<Emphasis Type="Italic">Sparus aurata</Emphasis>) to organophosphate pesticides

The characterization of cholinesterase activity in brain and muscle of gilthead seabream was carried out using four specific substrates and three selective inhibitors. In addition, K _m and V _max were calculated from the Michaelis–Menten equation for ASCh and BSCh substrates. Finally, the in vitro sensitivity of brain and muscle cholinesterases to three organophosphates (OPs) was also investigated by estimating inhibition kinetics. The results indicate that AChE is the enzyme present in the brain, whereas in muscle, a typical AChE form is present along with an atypical form of BChE. Very low ChE activity was found in plasma with all substrates used. The inhibitory potency of the studied OPs on brain and muscle AChEs based on bimolecular inhibition constants (k _i ) was: omethoate < dichlorvos < azinphosmethyl-oxon. Furthermore, muscle BChE was found to be several orders of magnitude (from 2 to 4) more sensitive than brain and muscle AChE inhibition by dichlorvos and omethoate.     

Molecular endocrine changes of Gh/Igf1 axis in gilthead sea bream (<Emphasis Type="Italic">Sparus aurata</Emphasis> L.) exposed to different environmental salinities during larvae to post-larvae stages

The influence of acclimation of the euryhaline gilthead sea bream (Sparus aurata) larvae/post-larvae to brackish water on growth, energetic contents, and mRNA levels of selected hormones and growth-regulating hypothalamic neurohormones was assessed. Specimens from 49 days post-hatching were acclimated during 28 days to two different environmental salinities: 38 and 20 psu (as brackish water). Both groups were then transferred to 38 psu and acclimated for an additional week. Early juveniles were sampled after 28 days of acclimation to both salinities and one week after transfer to 38 psu. Pituitary adenylate cyclase-activating peptide (adcyap1; pacap), somatostatin-I (sst1), growth hormone (gh1), insulin-like growth factor-I (igf1), and prolactin (prl) mRNA expression were all studied by QPCR. Post-larvae acclimated to 20 psu showed better growth performance and body energetic content than post-larvae maintained at 38 psu. prl, adcyap1, and igf1 mRNA expression levels increased in 20-psu-acclimated post-larvae but decreased upon transfer to 38 psu. GH1 expression did not show significant changes under both experimental conditions. Our results suggested an enhanced general performance for post-larvae in brackish water, supported by the actions of adcyap1, igf1, and prl.     

Physiological stress and innate immune response in gilthead sea bream (<Emphasis Type="Italic">Sparus aurata)</Emphasis> and sea bass (<Emphasis Type="Italic">Dicentrarchus labrax</Emphasis>) exposed to combination of trimethoprim and sulfamethoxazole (TMP-SMX)

The purpose of this study was to determine whether gilthead sea bream and sea bass treated with combination of trimethoprim and sulfamethoxazole (TMP-SMX) differed in terms of physiological and innate immune biomarkers. Fish were exposed to TMP-SMX at 40 ppm concentration for 1 h as a prophylactic usage. Plasma cortisol, glucose, electrolytes (Ca, P, Na, K, Cl, and Mg) as well as plasma lysozyme activity, C-reactive protein (CRP), and ceruloplasmin (Cp) were measured soon after treatment and following 24 and 48 h in normal sea water for recovery. Treatment with TMP-SMX in both gilthead sea bream and sea bass led to an increase in plasma cortisol and glucose. Fluctuations in some electrolytes were found after treatment and during recovery period, however, the ratios of monovalent ions in treated sea bream were similar to control. Hematocrit values as well as plasma lysozyme activity in gilthead sea bream and sea bass were not affected by the treatment. CRP in gilthead sea bream and Cp in sea bass responded to the treatment with decreased levels. Both gilthead sea bream and sea bass displayed a rapid physiological stress response and sensitivity to TMP-SMX exposure, which requires more than 48-h period for regaining homeostasis.     

Acute effects of benzo[a]pyrene on liver phase I and II enzymes,and DNA damage on sea bream <Emphasis Type="Italic">Sparus aurata</Emphasis>

In the present study biotransformation and detoxification responses to acute exposure to the polycyclic aromatic hydrocarbons benzo[a]pyrene (B[a]P) were investigated in the liver of Sparus aurata (sea bream). Sexually immature gilthead sea bream were treated by intraperitoneal injection of B[a]P (20 mg kg⁻¹) for 6, 12, 24, and 48 h. B[a]P accumulation was quantified in sea bream liver by mean of gas phase chromatography (GPC-MS) after the various exposure periods. The following biological responses were measured: (1) ethoxyresorufin-O-deethylase (EROD) activity, as a phase I biotransformation parameter; (2) liver glutathione S-transferase (GST) activity as a phase II conjugation enzyme. DNA damage was assessed over time using the single-cell gel electrophoresis comet assay. B[a]P bioaccumulation in the liver resulted in a biphasic curve with an increasing uptake up to 5.55 ± 0.67 μg g⁻¹ dry weight after only 6 h exposure and 4.67 ± 0.68 μg g⁻¹ dry weight after 48 h exposure. EROD activity showed a nonsymmetrical bell-shaped kinetic with a maximum at 24 h and lower but significant activities at 12 and 48 h with respect to control animals. Hepatic GST activities were only significant after 48 h exposure. Comet assay showed an increase in liver cells DNA damage with a maximum after 48 h exposure reaching up to 12.17 %DNA in the tail.     

Growth performance and metabolic utilization of diets with native and waxy maize starch by gilthead sea bream (Sparus aurata) juveniles

The effect of dietary starch source and level on growth performance, feed utilization, apparent digestibility coefficients and liver enzyme activities involved in intermediary metabolism of gilthead sea bream juveniles was studied. Five isonitrogenous (47% crude protein) and isolipidic (15% crude lipids) diets were formulated to contain 10% native (diet NS10) or waxy (diet WS10) maize starch; 20% native (diet NS20) or waxy (diet WS20) maize starch or no starch (control). Diets were adjusted with α-cellulose. Another diet was formulated without carbohydrates, and contained 70% crude protein and 15% crude lipids (diet HP). Each diet was fed to triplicate groups of 30 fish (initial weight: 20 g) for 12 weeks. The HP group was fed to near satiation and the other 5 groups were fed on a pair-feeding scheme according to the group that ingested less feed (control diet group). The reduction of dietary protein level from 70% to 47% by the incorporation of 20% starch did not significantly affect gilthead sea bream growth performance or feed efficiency. Compared to the control diet, neither the level nor the nature of starch had any measurable effect on growth performance and feed efficiency. Digestibility of starch was unaffected by source or dietary inclusion level. Diet had no effect on plasma glucose levels, but liver glycogen was higher in diet groups NS20, WS20 and HP. Dietary carbohydrates increased GK and G6PD enzyme activities and decreased ALAT and GDH enzyme activities while had only a minor effect on FBPase activity. The nature of dietary starch tested (native or waxy) had little influence on performance criteria.