Dietary lysine requirement of juvenile gilthead seabream Sparus aurata L.*

The dietary lysine requirement of juvenile gilthead seabream was determined by the growth response of duplicate groups of fish (3.5 g initial weight) fed on six isonitrogenous (427 g kg^?1) and isolipidic (135 g kg^?1) diets containing graded levels of crystalline l ‐lysine HCl, with dietary lysine content ranging from 36.3 to 79.7 g kg^?1 of protein. The final indispensable amino acid profile of the diets except for lysine was formulated so as to resemble that of wild seabream whole body. Except for the reduced growth performance of fish groups fed the lysine‐deficient diets no other deficiency signs were apparent. Survival observed throughout the feeding period of 6 weeks was excellent. Weight gain (in %), specific growth rate, feed efficiency and daily protein deposition (DPD) were significantly improved in response to the increasing levels of dietary lysine up to 52.7 g kg^?1 of protein and remained nearly constant thereafter. Whole‐body protein content followed a similar pattern as growth parameters in relation to dietary lysine level. Non‐linear regression analysis of DPD against dietary lysine level using the four‐parameter saturation kinetic model indicated a lysine requirement of 50.4 g kg^?1 of protein for this species to support growth.     

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

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

金头雕海水工厂化养殖技术

金头鲷（sparus aurata l．，teleostei）又称大西洋王鲷、洋鲷，属硬骨鱼纲，鲈形目，鲈亚目，鲷科，鲷属。该鱼通体为银灰色，背部颜色较深，两侧通常有六或七条深色垂直条纹。额头上有一条金色的条纹。主要分布于大西洋东海岸，不列颠群岛，直布罗陀海峡到佛得角以及金丝雀岛周围，同时地中海、新西兰也有分布，是世界闻名的优良品种。它以体态优美、色泽鲜艳、肉质细嫩、营养丰富而博得广大消费者的青睐，欧洲人称为“皇家鲷鱼”，在国际市场上深受欢迎，是一种名贵的经济鱼类。该鱼生长迅速，当年可达商品规格（≥500g），抗病力强，肉质鲜美细嫩，营养丰富。     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Development of a reference diet for use in indispensable amino acid requirement studies of gilthead seabream Sparus aurata L.

The development of a test diet supporting good fish growth to be used in gilthead seabream indispensable amino acid (IAA) requirement studies based on dose–response relationship was the objective of the present study. Four isonitrogenous diets, one serving as control (diet C) based on fish meal LT, and three others in which the fish meal component was reduced to 45% and the rest of the nitrogen was provided by mixtures of crystalline AA were formulated. Diet S resembled the whole body IAA profile of wild seabream. Diet D was made deficient in all IAA and a 10% excess of IAA compared with diet S was used in diet E. Triplicate groups of fish were fed the diets to satiation for 6 weeks. Growth parameters and nutrient utilization efficiencies among diets C and S were similar, while excessive supplementation of IAA negatively affected most of them, compared with the control. Whole body composition was differentiated in relation to the diet fed. No significant differences were observed among groups fed the control and diet S. As shown by the results, the presence of crystalline AA in diet S does not significantly affect growth, and could therefore serve as control diet in IAA requirement studies of gilthead seabream.     

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 .     

Ovarian contractility in reared gilthead seabream (Sparus aurata L.) in different phases of the reproductive cycle

Spontaneous ovarian tunica albuginea contractility was evaluated in gilthead seabream (Sparus aurata L.) at different phases of the reproductive cycle. Fourteen adult females were sampled from February to November 2012 in a commercial fish farm, and ovaries were removed and processed for histological and contractility analyses. Fish reproductive stages were evaluated on haematoxylin–eosin-stained ovary sections or by simple macroscopic observation of hydrated oocytes in spawning individuals. Tunica albuginea spontaneous contractility was recorded by using ovary wall strips mounted in an organ bath containing modified Ringer’s solution. Ovary macro- and microscopic analyses allowed the identification of three different reproductive conditions: vitellogenesis, spawning and regressing. The gilthead seabream tunica albuginea was capable to contract spontaneously, and significant differences were found in mean contraction amplitude among the three reproductive states, with the highest value recorded in individuals in regressing condition and the lowest in individuals at spawning stage. No differences in mean contractility frequency among the three different groups were found. Possible involvement of spontaneous contractility in facilitating developing follicle advancement towards the ovarian lumen within the ovary and in supporting recovery of regressing ovaries may be hypothesized. The low contractility observed during the final oocyte maturation and spawning phases does not seem to support a role of tunica albuginea during ovulation, which could conversely involve theca cell contraction. Alternatively, possible single instantaneous contractions of tunica albuginea muscle fibres, not detected in the present study, could occur during ovulation in response to neuro-hormonal stimulations; a role of abdominal wall musculature in ovary “squeezing” and consequent release of ovulated eggs cannot be excluded.     

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.     

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.     

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.     

The use of squid protein hydrolysate as a protein source in microdiets for gilthead seabream Sparus aurata larvae

In the present study, the use of predigested proteins as an ingredient of microdiets offered to gilthead seabream larvae was tested. The protein source (freeze-dried squid powder) was hydrolysed with protease (trypsin and pancreatin). Different levels of raw squid protein and hydrolysate (100% protein, 50% protein/50% hydrolysate, 100% hydrolysate) were added to the microdiets to produce a dietary protein level of 65%. For comparison, cofeeding of Artemia nauplii and microdiet as well as microdiet supplemented with pancreatin were also offered to the larvae. The final average dry weights of 32-day-old larvae were 1.65 ± 0.04 mg, 1.38 ± 0.06 mg and 1.13 ± 0.1 mg, respectively, for larvae cofed 0%, 50% and 100% hydrolysate microdiets and Artemia nauplii. Survival of larvae was not affected by protein source. The survival of larvae cofed Artemia nauplii and microdiet was significantly higher than that of larvae fed exclusively on microdiet (68% and 80%, respectively). These results suggest that the use of hydrolysate (at 50% greater) as a protein source in diets for seabream larvae is not to be recommended.     

Essential fatty acid requirements of larval gilthead sea bream, Sparus aurata (L.)

Abstract. Two experiments were carried out to study the effect of n -3 HUFA levels in rotifers on survival, growth, activity and fatty acid composition of gilthead bream, Sparus aurata (L.), larvae. From the third to the 15th day after hatching, gilthead bream larvae received one of the three kinds of rotifers containing different percentages of n –3 HUFA. Moisture, crude lipid, saponifiable matter and fatty acid composition of total lipids of rotifers and larvae were determined.
A good correlation was found between larval growth and n –3 HUFA levels in rotifers. Larval survival was also significantly improved by the elevation of the n –3 HUFA levels in rotifers. A high occurrence of hydrops was registered in larvae fed with EFA-deficient rotifers. The n –3 HUFA levels in the larvae were increased by the elevation of n –3 HUFA contents in rotifers. However, n –9 fatty acids in the larvae remained almost constant, regardless of the different 18:1 n –9 contents in rotifers. Therefore, the ratio of oleic acid to n –3 HUFA, known to be an indicator of the EFA deficiency in fish, was reduced by the elevation of the n –3 HUFA levels in rotifers.