Administration of Probiotic Strain to Improve Sea Bream Wellness during Development

Two bacterial strains Lactobacillus fructivorans (AS17B), isolated from adult sea bream (Sparus aurata) gut, and Lactobacillus plantarum (906), isolated from human faeces, were administered contemporaneously, during sea bream development using Brachionus plicatilis and/or Artemia salina and dry feed as vectors. The probiotic treatment influenced gut colonization: at 35 days post-hatching (p.h.) L. fructivorans was not present in the gut, but the treatment induced colonization by L. plantarum. At 66 days p.h., L. fructivorans was evident also in the control; moreover, when suitable environmental conditions appeared in the post-metamorphosis gastro-intestinal tract, competition between L. plantarum and L. fructivorans occurred. At 90 days p.h., L. plantarum was displaced by L. fructivorans that became significantly higher with respect to the control. In treated groups, probiotic administration significantly decreased larvae and fry mortality.     

Haematological, biochemical and immunological parameters as stress indicators in Dicentrarchus labrax and Sparus aurata farmed in off-shore cages

In 2000–2001, an investigation was performed in two Sicilian mariculture sites (Pachino and Castellammare) to monitor physiological and biochemical parameters in sea bream (Sparus aurata) and sea bass (Dicentrarchus labrax) specimens farmed in off-shore sea cages. Plasma cortisol and glucose levels, tissue lactate concentration, haematocrit and haemoglobin content, lysozyme, haemolytic and haemagglutinating activities were determined. During the experiment, an increase in tissue lactate and plasma cortisol levels and a reduction in haemolytic and haemagglutinating titers, were recorded. Changes occurring in these haematological, immunological and biochemical values suggest that these parameters can be useful indicators in assessing the condition of chronic stress induced by mariculture operations.     

A drop in ambient temperature results in a transient reduction of interrenal ACTH responsiveness in the gilthead sea bream (Sparus aurata, L.)

This study investigated the effects of a drop in water temperature (18 °C to 9 °C in 24 h) on the pituitary and interrenal hormones of the gilthead sea bream Sparus aurata. The in vitro sensitivity of the interrenal tissue to ACTH, plasma levels of cortisol, ACTH, -MSH, GH, glucose, lactate and ions were determined. In vitro ACTH, stimulated the release of cortisol from isolated interrenal glands from control gilthead sea bream in a concentration dependent fashion. However, the interrenal cells were less sensitive to ACTH as soon as 24 h following the onset of the temperature drop. At this time, plasma cortisol and ACTH levels were raised, and plasma GH concentrations were decreased, whereas no significant changes were found in plasma -MSH. After 96 h plasma ACTH levels had recovered whereas plasma cortisol levels were still higher than controls after 8 days of the beginning of the experiment. Interrenal sensitivity had recovered after 8 days. The results may help to clarify the relationship between the stress response and the aetiology of the winter syndrome in sea bream.     

Inhibition of HPI axis response to stress in gilthead sea bream (Sparus aurata) with physiological plasma levels of cortisol

This study investigates the effect of corticosteroid (cortisol) administration on the stress response of the gilthead sea bream Sparus aurata subjected to a 48 h confinement. The effect of (in-vitro and in-vivo ) cortisol administration on the in-vitro ACTH sensitivity of the interrenal tissue; the plasma levels and tissue concentration of cortisol; and the plasma levels of ACTH, -MSH, -endorphin and glucose were determined. Confinement caused a transient and concomitant increase in plasma cortisol and ACTH levels. However, in cortisol-fed fish the plasma ACTH levels were lower, indicating a suppresion of the ACTH release from the corticotropes by cortisol. In contrast to the activation of the corticotropes, the levels of plasma melanotrope derived peptides were not affected. In spite of the fact that interrenal cells of cortisol-fed gilthead sea bream released less cortisol than controls, the interrenal sensitivity to ACTH was not affected by in-vivo and in-vitro cortisol administration. This suggests that the interrenal sensitivity to ACTH in stressed (confinement) sea bream is probably not regulated by -MSH, N-ac--END, or by cortisol. Thus, in gilthead sea bream the interrenal sensitivity to ACTH could be regulated at the hypothalamus and/or pituitary and communicated via circulating ACTH levels.     

Induction of apoptosis in sea bream fibroblasts by Vibrio harveyi haemolysin and evidence for an anti‐apoptotic role of heat shock protein 70

In this study, we exposed black sea bream, Mylio macrocephalus (Basilewsky), fibroblast (BSF) and silver sea bream, Sparus sarba Forsskål, fibroblast (SSF) cell lines to a recombinant Vibrio harveyi haemolysin (VHH) and investigated mechanisms involved in apoptosis. A decrease in mitochondrial membrane potential, followed by an increase in caspase 3 activity, occurred within 2–8 h of VHH exposure, in both cell lines; however, VHH did not alter cellular levels of reactive oxygen species. As heat shock protein 70 (HSP70) is known to prevent the onset of apoptosis in certain mammalian cells, we aimed to test whether such a protective effect is operative in VHH‐exposed fibroblasts. The amounts of HSP70 were elevated in SSF and BSF via an acute heat shock or an acute heat shock followed by a 6 h recovery. It was found that the VHH‐mediated reduction in mitochondrial membrane potential was suppressed in cells that had a 6 h post‐heat shock recovery, and the protective effect of heat shock‐induced HSP70 was attenuated following treatment of cells with the HSP70 inhibitor, quercetin. This study demonstrates how haemolysin causes cell death via induction of apoptosis and provides evidence as to the role of HSP70 as an anti‐apoptotic factor.     

Effects of chronic confinement on physiological responses of juvenile gilthead sea bream, Sparus aurata L., to acute handling

Understanding how gilthead sea bream, Sparus aurata L., an important Mediterranean Sea species for aquaculture, respond physiologically to stressors commonly encountered in intensive rearing is important for effective production, as managing for stress is a major factor in maintaining healthy fish stocks. Our objective was to determine whether holding juvenile gilthead sea bream at a high density (HD), as a chronic stressor, would affect their physiological responses to a subsequent acute handling stressor. After acclimation at a low density (LD) of 6 kg m^?3 in 200‐L circular tanks containing 33–36 g L^?1 recirculating seawater at 19°C under a normal photoperiod, juvenile 37‐g gilthead sea bream were confined for 14 days at a HD of 26 kg m^?3 and then subjected to 30‐s aerial emersion in a dipnet. Plasma levels of cortisol, glucose, lactate, osmolality and chloride were determined in fish held in separate lots during LD (control) and HD confinement at 0, 1, 2, 7 and 14 days, and then after handling at 0, 1, 2, 4 and 8 h. Although plasma cortisol levels were similar in LD and HD fish groups after 14 d of confinement (15 and 23 ng mL^?1, respectively), the cortisol response in fish from the HD treatment at 1 and 2 h following acute handling (70 and 37 ng mL^?1, respectively) was only about half of that measured in the control group (139 and 102 ng mL^?1); plasma cortisol was similar in both groups by 4 and 8 h. In contrast, plasma glucose elevations in response to handling were higher at 4 and 8 h in the HD‐held fish (94 and 72 mg dL^?1, respectively) than in those from the LD treatment (59 and 51 mg dL^?1); glucose responses were similar in both groups at 1 and 2 h after handling and throughout confinement. Plasma lactate levels were higher in LD fish than in the HD group at the beginning of the experiment but were similar after 14 d confinement and responses to handling were similar (e.g. 33 and 35 mg dL^?1 at 1 h). Plasma osmolality showed increases during the first 2 h after acute handling but no differences were evident between the two density treatments at any time during confinement or posthandling. Plasma chloride levels did not change throughout the experiment. The reduced plasma cortisol response to acute handling likely resulted from negative feedback of mildly but chronically elevated circulating cortisol caused by the confinement stressor on the hypothalamic–pituitary–interrenal axis. While other post‐handling physiological changes also showed differences between treatment groups, the suppressed cortisol response in the HD‐held fish suggests a reduction in the gilthead sea bream's normal capacity to respond to an acute stressor.     

Growth hormone attenuates branchial HSP70 expression in silver sea bream

In this study the effects of growth hormone (GH) on silver sea bream branchial heat-shock protein 70 (HSP70) expression was investigated using in-vivo and in-vitro experiments. For in-vivo experiments, sea bream were administered recombinant bream GH or the GH secretagogue hexarelin. Pituitary levels of GH were unchanged in fish administered exogenous GH but decreased on hexarelin administration, in comparison with saline controls. Levels of HSP70 were measured using immunoanalysis and it was found that both GH and hexarelin administration caused a significant decrease in branchial HSP70 abundance. For in-vitro analysis, branchial filaments were exposed to a range of GH concentrations (1, 10, and 100 ng/ml) and it was found that HSP70 levels were significantly lowered in all cases. This study adds to the growing body of evidence surrounding the importance of hormones in regulating heat-shock protein expression in fish.     

Comparative study of antioxidant defence mechanisms in marine fish fed variable levels of oxidised oil and vitamin E

The aim of the study was to compare theantioxidant systems in juvenile marine fish ofcommercial importance to European aquaculture,namely turbot (Scophthalmus maximus),halibut (Hippoglossus hippoglossus) andgilthead sea bream (Sparus aurata). Thepresent dietary trial was specifically designedto investigate the antioxidant effects ofvitamin E under moderate oxidising conditions,including high dietary levels of highlyunsaturated fatty acids and the feeding ofoxidised oils. The objective was to induce astressful pro-oxidant status to enablecharacterisation of the biochemical responsesto peroxidative stress without causingunnecessary suffering to the experimentalanimals or high mortalities during the trials. Both sea bream and turbot showed excellentgrowth, whereas growth was poorer in halibut.Dietary oxidised oil significantly reducedgrowth in turbot and especially in halibut, butnot in sea bream. Vitamin E improved growth insea bream fed oxidised oil but not in turbot orhalibut. However, vitamin E supplementationappeared to improve survival in all threespecies. In sea bream and turbot, liverantioxidant defence enzyme activities weregenerally increased by feeding peroxidised oiland reduced by vitamin E. Conversely, inhalibut, the liver antioxidant defence enzymeactivities were not increased by feedingperoxidised oil and only superoxide dismutasewas reduced by feeding vitamin E. Consistentwith these data, feeding oxidised oil increasedlipid peroxidation products in halibut, butgenerally not in sea bream or turbot.Furthermore, lipid peroxidation products weregenerally reduced by dietary vitamin E in bothsea bream and turbot, but not in halibut. Therefore, halibut liver antioxidant defenceenzymes did not respond to dietary oxidised oilor vitamin E as occurred in turbot and,especially sea bream. This resulted inincreased levels of lipid peroxides in halibutcompared to turbot and sea bream in fish givendietary oxidised oil. In addition, supplementalvitamin E did not reduce lipid peroxides inhalibut as it did in turbot and sea bream. Theincreased peroxidation stress in halibut mayaccount for their poorer growth and survival incomparison to turbot and especially sea bream.Halibut were reared at a lower temperature,although relatively high for halibut, thaneither turbot or sea bream but they were alsoslightly younger/smaller fish and possibly,therefore, more developmentally immature, andeither or all of these factors may be importantin the lack of response of the liver enzymes inhalibut.     

Lactobacillus spp. bacteria as probiotics in gilthead sea bream (Sparus aurata, L.) larvae: Effects on growth performance and digestive enzyme activities

In this study, the influence of commercial probiotic, Lactobacillus spp., supplementation was investigated on growth parameters and digestive enzyme activities in gilthead sea bream, Sparus aurata, during larval development. All experiments were triplicated and designed in three different administrations of probiotic from 3 days after hatching (DAH) concurrently with starting of exogenous feeding. In the first group, probiotic was added to live food (rotifer and Artemia). In the second group, probiotic was supplemented directly to both live food and water. In the third group, probiotic was added directly to water. Also, no probiotic treatment was maintained in control group. Total bacterial counts among probiotic probiotic-supplemented groups were significantly different from total bacterial counts in controls in water and digestive tract of larvae (p < 0.05). The mean of total bacterial counts in control was approximately 4 × 10⁴-fold increased from the experimental groups in the sea water (p < 0.05). Besides, mean digestive enzyme activities of all probiotics treatment groups were significantly different (p < 0.05) with that of the control. Except probiotic water supplementation group, in all treatments, the specific activities of pancreatic and intestinal enzymes were significantly higher (p < 0.05) in larvae to which probiotic had been supplemented by live food and live food with water. Also, S. aurata larvae that had probiotic administered by live food with water demonstrated significant (p < 0.05) increases in both survival (13–105% higher) and specific growth rate (2–9% higher) as compared to controls. As a result, supplementation of probiotic to directly tank water could not significantly increase growth parameters and digestive enzyme activities and therefore, administration of probiotics by this method would not be effective in terms of husbandry parameters and nutritional condition.     

Physiological responses and HSP70 mRNA expression in GIFT tilapia juveniles,Oreochromis niloticus under short‐term crowding

Physiological responses and HSP70 mRNA expression to short‐term crowding were tested in juveniles genetically improved farmed tilapia (Oreochromis niloticus) (70.43 ± 4.43 g). Fish were kept at control group (5 g L^?1) and stress groups (low density 10 g L^?1, medium density 40 g L^?1 and high density 70 and 100 g L^?1) for 48 h. Each density was tested in triplicates. The effects of a short‐term exposure on the physiological responses of fish were determined before stress (0 h) and at 6, 12, 24 or 48 h post‐crowding. There was a significant increase in serum cortisol, total protein, lysozyme and aspartate aminotransferase (AST) activities in all stressed groups at 24 h post‐crowding (P < 0.05). Serum glucose and alanine aminotransferase (ALT) of 100 g L^?1 stressed group were lower than that of the control after 48 h of crowding stress (P < 0.05). The levels of serum triglyceride and cholesterol in crowding stress with high density were significantly decreased compared with the control group at 48 h. The mRNA expression data showed that hepatic HSP70 mRNA levels were markedly elevated at all stressed groups. HSP70 mRNA levels of 70 and 100 g L^?1 stressed groups decreased at 48 h compared with the 24 h post‐crowding. The protective ability of HSP70 was limited.     

Development of a black sea bream fibroblast cell line and its potential use as an <Emphasis Type="Italic">in vitro</Emphasis> model for stress protein studies

A fibroblast cell line (BSF) derived from a caudal fin explant of black sea bream (Mylio macrocephalus) was developed. The optimum fetal bovine serum (FBS) concentration for fibroblast cell line growth was found to be 15–20% v/v FBS and the optimum temperature range for growth was found to be 26–30 °C. The fibroblast cells displayed a diverse distribution in chromosome number with two modal chromosome numbers of 48 and 54. Upon acute heat shock (+8 °C) the cells displayed a 4.1 fold increase in hsp70 and this elevation was not prolonged as hsp70 returned to near basal levels following a 6 h recovery period. The effect of the hsp70 inducer L-azetidine- 2-carboxylic acid was tested and it was found that at a concentration of 10 mM this inducer caused a 2.3 fold increase in hsp70 levels. The sensitivity of the fibroblast cell line to heavy metal exposure was tested by treatment with Cu2+ and it was found that hsp70 was significantly elevated in the presence of micromolar concentrations of Cu2+. The data from this study demonstrates that the established black sea bream fibroblast cell line could serve as a useful in vitro model for stress protein studies.     

Effects of dietary mannan oligosaccharides in early weaning diets on growth,survival, fatty acid composition and gut morphology of gilthead sea bream (Sparus aurata,L.) larvae

Early weaning of marine fish larvae with dry diets delays gut maturation and reduces growth rates. In juvenile and adult forms of several marine fish species, inclusion of dietary mannan oligosaccharides (MOS) improves gut integrity and functionality, but the effects of MOS inclusion in gilthead sea bream (Sparus aurata, L.) larval diets have not been addressed yet. Thus, this study assesses the effects of dietary MOS inclusion on survival, growth performance, gut morphology, feed acceptance and quality of gilthead sea bream larvae. For that purpose, 16 days post‐hatched gilthead sea bream larvae were fed four graded levels of MOS (Biomos^®, Alltech, Nicholasville, KY, USA) in weaning diets as follows: 0 g kg^?1 MOS, 0.5 g kg^?1 MOS, 1.5 g kg^?1 MOS and 2 g kg^?1MOS. Dietary MOS did not affect feed acceptance in gilthead sea bream larvae (P > 0.05). MOS supplementation was correlated in a dose‐dependent way with higher larval survival (P = 0.026). After 15 days of feeding, dietary MOS increased whole larvae (P < 0.01) arachidonic acid, eicosapentaenoic acid and docosahexaenoic acid. Gilthead sea bream larvae fed 2 g kg^?1 MOS presented higher gut occupation with goblet cells after feeding compared with larvae fed the other dietary treatments. Overall, the results suggest that inclusion of MOS in early weaning diets for gilthead sea bream improves essential fatty acid utilization and may promote growth and final survival.     

Alternative matrices for cortisol measurement in fish

Plasma cortisol is the most commonly used indicator of stress in fish but, as the blood sampling procedure itself can be a source of stress, it would be helpful to measure cortisol using less invasive matrices. It is also necessary to find alternative matrices as stress indicators in dead fish in which blood sampling is impossible. In the present study, we investigated transport stress in three aquaculture species, European sea bass (Dicentrarchus labrax L.), common carp (Cyprinus carpio L.) and rainbow trout (Oncorhynchus mykiss Walbaum), by cortisol determination (radioimmunoassay) in plasma and other matrices (skin mucus, gut content, lateral muscle and caudal fin). Cortisol significantly increased after transport in all species and matrices, except in the sea bass gut content, where it remained unchanged. The three species responded to transport stress by producing different cortisol levels. In conclusion, the significant correlation found between plasma cortisol and most of the other matrices opens up the possibility of using them to evaluate stress in fish: mucus sampling is a less invasive method than blood sampling, and in addition to muscle and fin sampling, it can be used in postmortem fish.     

Intergeneric Hybridization in Sparidae

Early development of white sea bream, Diplodus sargus, ♀ X common dentex, Dentex dentex, ♂ hybrids was compared to those of the parental species. Percent fertilization and percent hatching in the hybrid was not significantly different from those of parental species. Mean hatching time of the hybrid was intermediate between those of the parental species and was significantly different from both parental species. Growth of white sea bream and hybrid fry form days 6 to 30 was not significantly different. All common dentex fry died during this period. Results from this study suggest that this hybrid may be a suitable candidate for fish farming.     

Quantitation of inert feed ingestion in larval silver sea bream (Sparus sarba) using auto-fluorescence of alginate-based microparticulate diets

The ingestion of an inert feed as a sole food source was investigated in larval silver sea bream (Sparus sarba) fed an alginate-based microparticulate diet. Using the auto-fluorescent properties of pigments associated with the alginate base, ingestion and gut content were investigated over a 6 h experimental period in fed and unfed larvae. By extracting and measuring chlorophyll a (Chl a) and phaeopigment content of feeding larval fish and relating this to standardized Chl a and phaeopigment content of the diet, relative to diet weight, it was determined that individual fed 7-day old larvae had a maximum gut content of 1.05±0.09 g diet while 14-day old fed fish had a maximum gut content of 3.17±0.90 g diet. On average, the gut content of 14-day old fish was 2.89 times greater than the gut content of 7-day old fish. The dry weight of larval sea bream increased from 43±4.2 g at day 7 to 134.3±20.4 g at day 14 indicating that growth of fish fed this inert feed was substantial. Gut pigment dynamics suggested that Chl a was degraded to phaeopigments by 7-day but not 14-day old larvae and the individual gut dietary content varied considerably in 14-day old fish. The maximum Chl a and phaeopigment content in larval sea bream was 0.4 ng ind^–1 and 0.55 ng ind^–1 for 7-day old fish and 1.54 ng ind^–1 and 2.81 ng ind^–1 for 14-day old fish respectively. The present method may potentially allow simple and direct assessment of larval fish feed ingestion in both an experimental and commercial setting.     

Effects on mortality and stress response in European sea bass, Dicentrarchus labrax (L.), fed mannan oligosaccharides (MOS) after Vibrio anguillarum exposure

The effects of dietary mannan oligosaccharides (MOS; 4 g kg^?1; Bio‐Mos, Alltech Inc, USA) in diets for European sea bass, Dicentrarchus labrax (L.), juveniles in relation to disease and stress resistance, combining intestinal infection with Vibrio anguillarum and stress challenge by confinement, were assessed in this study. After 8 weeks of MOS supplementation, fish were exposed to a pathogen challenge test against V. anguillarum by direct gut inoculation combined with a confinement stressor panel. Cumulative mortality of fish fed MOS caused by anally inoculated V. anguillarum decreased from 66% to 12.5% and from 54.1% to 25% in infected and infected + stressed fish, respectively, compared to fish fed control diet. Results for European sea bass revealed a positive effect of MOS dietary inclusion on disease resistance, in terms of cumulative mortality, against gut inoculated V. anguillarum, as well as reduced effects of stress on microbiota diversity. Both of these findings, together with the enhanced innate immune response and the higher gut mucus production and density of eosinophil granulocytes in gut mucosa obtained in previous studies after MOS supplementation ( Torrecillas et al. 2007, 2011a,b ) suggest that general reinforcement of the innate immune system, and particularly of the intestinal barrier efficiency, is the main defence mechanism of European sea bass fed MOS against pathogenic microorganisms.     

Effect of dietary medicinal herbs on lipid metabolism and stress recovery in red sea bream <Emphasis Type="Italic">Pagrus major</Emphasis>

The effect of dietary medicinal herbs on lipid metabolism and stress recovery was investigated in red sea bream Pagrus major. Fish (mean body weight 24.0 ± 0.2 g) were fed on test fish meal diets supplemented with either Massa Medicata (Mm), Crataegi Fructus (Cf), Artemisia capillaries (Ac), or Cnidium officinale (Co), or with a mixture of the four herbs (HM) for 12 weeks. A control group was fed a diet without herbs in the same manner. A high survival rate was observed in the herbal diet groups. The final mean body weight, feed efficiency, protein efficiency ratio, and apparent protein and lipid retention in the Ac, Co, and HM groups were higher than those in the control and Mm groups. Final carcass, hepatic lipid and triglyceride contents, and plasma triglyceride and nonesterified fatty acid levels were lower in the Ac, Co, and HM groups compared to those of control and Mm groups. However, final hepatic phospholipid, plasma phospholipid, and high density lipoprotein cholesterol levels were higher in the Ac, Co, and HM diets groups than in the control and Mm groups. The Cf, Co, Ac, and HM groups showed faster recovery time in the 2-phenoxyethanol anesthesia test and a higher recovery rate in the 10-min air exposure test than those of the control and Mm groups. Moreover, the Cf, Ac, Co, and HM diet groups had a significantly lower plasma cortisol level than the control and Mm diet group, but the glucose level in the herbal diet groups was higher than that in the control group after a 1-h air exposure. These results indicate that the addition of medicinal herbs to the fish diet improved lipid utilization and stress recovery in red sea bream.     

Stimulation of growth performance without causing stress response in young red sea bream, Pagrus major (Temminck & Schlegel), by photoperiod manipulation

Three photoperiods (12L:12D, 16L:8D and 24L:0D) were used to investigate the growth performance and stress response in red sea bream, Pagrus major (body weight 200–400 g). Fish were fed a commercial diet to apparent satiation, two times a day for 8 weeks. Fish exposed to a 24L:0D photoperiod showed a significantly higher weight gain (%) than those exposed to other photoperiods (P<0.05). The best specific growth rate and feed conversion efficiency were achieved at 24L:0D and 16L:8D, without significant differences among them. Although fish exposed to 16L:8D showed a significantly higher plasma level of cortisol than those exposed to other photoperiods in the third week, there was no major variation in the cortisol level among the treatments either at the sixth week or at the end of this study. There were no significant differences either in the haematocrit level or the plasma levels of glucose, total cholesterol and total protein among the treatments during this study. The results revealed that the growth performance of red sea bream reared from 200 to 400 g can be stimulated significantly using a continuous (24L:0D) photoperiod without any measurable significant stress response in fish.