The use of preserved copepods in sea bream small‐scale culture: biometric,biochemical and molecular implications

Considering the well‐known problems arising from the use of rotifers and Artemia as live prey in larval rearing in terms of fatty acid deficiencies, the aim of this study was to evaluate a partial or complete replacement of traditional live prey with preserved copepods during the larviculture of gilthead sea bream (Sparus aurata). Sea bream larvae were randomly divided into 4 experimental groups in triplicates: group A larvae (control) fed rotifers followed by Artemia nauplii; group B fed a combined diet (50%) of rotifers–Artemia and preserved copepods; group C fed rotifers followed by preserved copepods; and group D fed preserved copepods solely. Survival and biometric data were analysed together with major molecular biomarkers involved in growth, lipid metabolism and appetite. Moreover, fatty acid content of prey and larvae was also analysed. At the end of 40 days treatment, a stress test, on the remaining larvae, was performed to evaluate the effects of different diets on stress response. Data obtained evidenced a positive effect of cofeeding preserved copepods during sea bream larviculture. Higher survival and growth were achieved in group B (fed combined diet) larvae respect to control. In addition, preserved copepods cofeeding was able to positively modulate genes involved in fish growth, lipid metabolism, stress response and appetite regulation.     

Substrate-SDS-PAGE determination of protease activity through larval development in sea bream

Identification of alkaline proteases produced during larval stages of gilthead sea bream, Sparus aurata was achieved using SDS-PAGE and specific inhibitors. Such techniques were also applied to determine proteases existing in rotifers, Brachionus plicatilis, and Artemia nauplii, which are used as live food for these larvae, as well as proteases of adult fish. The results show a great prominence of trypsin-like proteases during the 4 weeks after hatching, but the number of enzyme species was reduced in adult fish. Alkaline proteases present in the rotifers and Artemia showed clear differences when compared with those of the larvae and were not detected in extracts obtained from fed larvae. The results obtained provide information about the role of exogenous enzymes in larval feeding of sea bream.     

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.     

Growth and Survival of Larval Red Sea Bream Pagrus major and Japanese Flounder Paralichthys olivaceus Fed Microbound Diets

To establish the practical use of microbound diets (MBD) for larval fish in mass seedling production, rearing experiments of larval red sea bream, Pagrus major , and Japanese flounder, Paralichthys olivaeeus , were conducted. A mixture of various protein sources was used, and dietary amino acid patterns were approximated to those of larval whole body protein. Two thousand red sea bream larvae and 1,000 Japanese flounder larvae, all 10 days old, were placed in 100 liter tanks with running sea water under ambient water temperature, which ranged from 18 to 20C. The particle size of MBD was 125 μm at the beginning of the experiment and adjusted as fish size increased thereafter. Employing MBD together with a small amount of live food could sustain the growth and survival of larval red sea bream and Japanese flounder. Thus, data from the present study indicates that substitution of artificial feeds for live foods is possible for larval fish production, although improvements in MBD diets may be necessary before they are adequate for large scale seedling production.     

Biomarkers of bone development and oxidative stress in gilthead sea bream larvae fed microdiets with several levels of polar lipids and α‐tocopherol

Although dietary marine phospholipids are able to improve culture performance of marine fish larvae in a further extend than soybean lecithin, both types of phospholipids (PL) markedly increase oxidative risk. The inclusion of a fat‐soluble antioxidant such as the vitamin E α‐tocopherol could allow a better control of oxidative stress. The objective of this study was to determine the combined effect of graded levels of α‐tocopherol with different levels and sources of krill phospholipids (KPL) and soybean lecithin (SBL) on growth, survival, resistance to stress, oxidative status, bone metabolism‐related genes expression and biochemical composition of sea bream larvae. Sea bream larvae were completely weaned at 16 dph and fed for 30 days seven microdiets with three different levels of PL (0, 40 and 80 g kg^?1 diet) and two of α‐tocopherol 1500 and 3000 mg kg^?1 diet. Sea bream larvae fed diets without PL supplementation showed the lowest survival, growth and stress resistance, whereas increase in PL, particularly KPL, markedly promoted larval survival and growth. However, feeding SBL markedly increased TBARs and GPX gene expression increasing the peroxidation risk in the larvae. Besides, KPL inclusion improved incorporation of n‐3 HUFA and, particularly, EPA into larval tissues, these fatty acids being positively correlated with the expression of BMP‐4, RUNX 2, ALP, OC and OP genes and to bone mineralization for a given larval size class. The increase in dietary α‐tocopherol tends to improve growth in relation to the n‐3 HUFA levels in the diet, denoting the protective role of this vitamin against oxidation. Indeed, dietary α‐tocopherol decreased the oxidative stress in the larvae as denoted by the reduction in larval TBARs contents and gene expression of SOD and CAT, but not GPX. Thus, increase in dietary α‐tocopherol effectively prevented the formation of free radicals from HUFA, particularly EPA, but did not affect the incidence of bone anomalies or the expression of genes related to osteogenetic processes.     

Dietary supplementation of mannan oligosaccharide on white sea bream (Diplodus sargus L.) larvae: effects on development,gut morphology and salinity tolerance

The influence of dietary mannan oligosaccharide (MOS) on the development, gut integrity and quality (in respect of stamina and survivability) of white sea bream Diplodus sargus L. larvae was investigated. White sea bream larvae were held under appropriate rearing conditions and fed Artemia, enriched by A1 DHA Selco^? with the addition or absence of MOS (Bio‐Mos®). The results indicated that larval growth performance and survivability were not affected by the MOS supplementation. Light microscopy revealed that MOS supplementation significantly improved the intestinal morphology by increasing the villi surface area by over 12%. Transmission electron microscopy revealed that MOS supplementation increased the microvilli length by 26% compared with the control. Salinity challenge experiments showed that MOS significantly increased larval stamina and survival in both 0 and 60 mg L^?1 salinity water by 13% and 22.9% respectively. These improvements in the larval quality at the early stages of fish development are important for the efficiency of intensive hatchery production.     

Studies on the effect of using the rotifer, Brachionus plicatilis, treated with different nutritional enrichment media and antibiotics on the growth and survival of blue-fin sea bream, Sparidentex hasta (Valenciennes), larvae

Studies were carried out to determine the effect of using the rotifer, Brachionus plicatilis (Muller) (S‐type), subjected to different treatments on the growth and survival of blue‐fin sea bream, Sparidentex hasta (Valenciennes), larvae. This was to illustrate the role of mixed algae added to the oil enrichments for the treatment of the rotifers to improve the sea bream larval survival. The highest sea bream larval survival (P < 0.05) was obtained while feeding the larvae with rotifers enriched in a mixture of algae plus half the recommended dose of Super Selco and DHA Protein Selco. No significant difference (P > 0.05) in the larval growth was observed between different treatments. However, larval survival was significantly high (P < 0.05) when rotifers were not treated with antibiotics. The results show that there is no need to use antibiotics to treat the rotifers before feeding the blue‐fin sea bream larvae, providing that the rinsing procedure for rotifers used in this study is followed.     

Supplementations of dl‐Methionine and Methionine Dipeptide in Diets are Effective for the Development and Growth of Larvae and Juvenile Red Sea Bream,Pagrus major

Growth trials for larvae and juvenile red sea bream, Pagrus major, were conducted to elucidate the efficacy of two molecular forms of methionine; dl ‐methionine (dl ‐Met) and methionine dipeptide (Met‐Met). For the larvae experiment, five experimental diets were formulated and fed to fish (42 mg) for 30 days. A diet which has 15% soy protein isolate served as the control diet. Similarly, test diets supplemented with dl ‐Met and Met‐Met at 0.5%, which were either precoated by zein or intact, were also formulated. For the juvenile experiment, five experimental diets were formulated wherein the control diet contained 25% soy protein isolate. Test diets were supplemented with dl ‐Met and Met‐Met at 0.75%, which were either coated by carboxymethycellulose or intact and fed to juveniles (0.75 g) for 56 days. The results of two feeding trials showed both dl ‐Met and Met‐Met can be equally utilized by red sea bream larvae and juveniles. Coating the amino acid significantly improved both fish larval and juvenile growth performance. The development of digestive protease activity of larvae was significantly influenced by coating the amino acid, but the type of methionine was not a factor in changing the protease activity of larvae.     

Modelling changes in the length–frequency distributions of fish larvae using field estimates of predator abundance and size distributions

The goal of this study is to determine if an individual-based size-dependent model can realistically simulate changes in the length–frequency distributions of several species of fish larvae collected in Conception Bay in 1993 and 1994, using field estimations of growth and predator abundance. We first model the length–frequency distribution of field samples with the best possible estimates of mean growth rate. Then, we add predation mortality given the characteristics of the predator community observed during our surveys, which was composed of macrozooplankton and adult capelin. The larval fish community is generally not affected by predation by macrozooplankton, as the average instantaneous mortality rate predicted by the model was 0.004 day^–1. Fish larvae appear to be more vulnerable to predation by the population of adult capelin. We estimate that an abundance of adult capelin ranging between 0.2 and 1.0 individuals per 1000 m^–3 may have a substantial impact on the larval fish community. The predictions of an individual-based model are directly related to the accuracy of estimates of the mean growth rates of the larval fish cohorts. We find that it is difficult to differentiate size-selective removal of individuals from random selection by analysing changes of the length–frequency distributions of the larval fish community.     

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.     

Potential of three new krill products for seabream larval production

The objective of this study was to evaluate three products derived from krill as sources of essential fatty acids, protein and, particularly, phospholipids in microdiets for larval gilthead seabream (Sparus aurata). Their effect on larval performance, biochemical composition and histological development was investigated. The addition of krill phospholipids, rich in highly unsaturated fatty acids, improved larval sea bream growth in terms of weight and length, enhanced hepatic utilization of dietary lipids and reduced the incidence of enterocyte injuries. These results confirm the higher nutritional value of marine phospholipids for the early development of marine fish larvae in comparison with soybean phospholipids.     

Comparison of iodine and glutaraldehyde as surface disinfectants for red porgy (Pagrus pagrus) and white sea bream (Diplodus sargus sargus) eggs

The efficacy of iodine and glutaraldehyde as fish egg surface disinfectants were assessed in red porgy (Pagrus pagrus) and white sea bream (Diplodus sargus sargus) eggs, two species of interest for Mediterranean aquaculture. Iodine was effective in reducing the bacterial load of the 1-day-old eggs when applied at 50 mg L⁻¹ for 5 min. The same concentration did not cause any significant change in hatching success or survival of the larvae for the first 5 days. Glutaraldehyde failed to reduce the bacterial load of the fish eggs at concentrations that were safe for the eggs (100 mg L⁻¹ for 5 min), as it had a significant effect in preventing hatching of the developed embryo. Disinfecting 0-day-old eggs with iodine resulted in a significant reduction of hatching percentage, while larval survival thereafter was unaffected. The results of the present study suggest that iodine may be an appropriate egg disinfectant for both red porgy and white sea bream.     

Effect of water temperature and light intensity on swim bladder inflation and growth of red sea bream <Emphasis Type="Italic">Pagrus major</Emphasis> larvae

This study aimed to examine the effect of different water temperatures and light intensities on swim bladder inflation (SBI) and growth of red sea bream Pagrus major larvae to improve rearing techniques for this species. Two sets of experiments were conducted: different rearing temperatures were used in experiment 1 (19, 21, 23, and 25 °C), and different light intensities in experiment 2 (250, 1000, 4000, and 16,000 lx). Water temperature did not affect SBI frequency, but SBI initiation was accelerated at higher temperature, i.e., it was initiated on 3 days post-hatching (dph) at 25 °C and on 6 dph at 19 °C, suggesting that the promotion period for SBI, which needs a surface skimmer to be  run, also accelerated with increasing temperature in red sea bream larviculture. A higher temperature also significantly promoted larval growth, although the notochord of larvae at SBI initiation was shorter at higher temperatures. Light intensity had no effect on either the initiation or the frequency of SBI. However, light intensity of 250 lx significantly reduced early larval growth compared to light intensities higher than 1000 lx. These results indicate that light at an intensity greater than 1000 lx at the water surface is suitable for the early larviculture of red sea bream.     

Preliminary Results on Rearing of Sparus Aurata × Pagrus Pagrus Hybrids. Performance Comparison With the Parental Species

Hybridization between gilthead sea bream, Sparus aurata, and red porgy, Pagrus pagrus, was undertaken and viable hybrids were produced by an artificial hormonal-induced cross. The hybrids, from the cross of female red porgy and male gilthead sea bream (Pp × Sa), survived through the yolk-sac larval stage. The hybrids produced from the cross of female gilthead sea bream and male red porgy (Sa × Pp) presented higher mortality in the larval stage (98.4%) compared to gilthead sea bream (80.0%) and red porgy (92.5%). Afterwards, the Sa × Pp hybrid was compared to the offspring of its parental species under the same rearing conditions (tanks of 500 l capacity, natural light, ambient temperature, self-feeding with commercial feed) in a 40–week experiment. In this comparative study, the Sa × Pp hybrid had intermediate mortality rate (6%), compared to gilthead sea bream (0%) and red porgy (21%). The growth profile of this hybrid resembled the slow growing phases of its parents (red porgy in warm period and gilthead sea bream in cold period). The feed conversion ratio between fish forms was not significantly different (1.54–1.84 in the Sa × Pp hybrid, 1.53–1.56 in the maternal species and 1.33–1.40 in the paternal one). The body weight variation in the Sa × Pp hybrid was quite high (31.7–37.9%), compared to that in gilthead sea bream (19.7–24.7%) and red porgy (18.0–31.3%). With respect to body coloration, the Sa × Pp hybrid was distinguishable and classified into groups of red porgy-like (55.1%), gilthead sea bream-like (40.6%), and intermediate forms (4.3%). The preliminary results of this study proved that the produced Sa × Pp hybrid had too low a performance for aquaculture use, but the interesting consequent hereditary potential could be a useful tool in fish hybridization practice.     

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.     

Mortality,growth and swim bladder stress syndrome of sea bass (Dicentrarchus labrax) larvae under varied environmental conditions

Responses to variations in light, temperature and salinity by sea bass larvae are described here. Massive mortality of larvae has been common in Mediterranean culture efforts. This mortality, associated with swim bladder malfunction, has been analysed regarding its association with individual stresses.Optimum temperatures for sea bass growth are clearly unsuited to larval rearing, while brackish sea water (25‰) just as clearly improves growth and survival. Light and nutritional regimes as well as water quality are other sources of environmental stress that may induce the swim bladder stress syndrome (SBSS) and reduce growth and survival. SBSS should not be confused with “gas bubble” disease and its most effective treatment depends on an understanding of larval environmental requirements.Larval growth was enhanced by both decreased salinity and increased temperature, but not by increased light. SBSS and mortality were avoided only when larvae were held at ambient temperature (12.5°C), natural light, and reduced salinity (25‰). Larvae dying with distended swim bladders also had other symptoms of environmental stress—spinal abnormalities, calculi in the urinary bladders, copious mucous and opaque (edemous) tissues. Elevations of temperature and light, as well as ambient sea water (36‰) are environmental stressors and induce SBSS.     

A closed water recirculation system for ecological studies in marine fish larvae: growth and survival of sea bass larvae fed with live prey

This paper describes the suitability of a closed recirculation system to study the development of fish larvae in a strictly controlled environment, where only feeding was varied (fed, starved, delayed and late fast treatments). The system served both as an incubator and hatchery. The time variation of physical and chemical parameters together with survival and growth of reared sea bass larvae (Dicentrarchus labrax) were studied over the first month of life. The recirculation design allowed for the maintenance of levels of ammonia, nitrite and nitrate below those cited as responsible for mortality or decreased feeding ability in other marine fish larvae. Almost no larval mortality occurred in the fed larvae from day 9 after hatching. The starved group of larvae showed a sharp decline in survival after 16 days of food deprivation. Larvae for which feeding was delayed until day 13 ceased dying 4 days after food was supplied. Fast feeding on days 24 and 25 had no effect on larval survival. Growth in length was similar during the first 2 weeks of larval life regardless of feeding treatment. Two days of late fast had no effect on larval growth. Growth patterns of fed larvae in this study were similar to those reported for larvae reared in flow through systems. We believe that survival and growth of the reared larvae was a direct function of diet, and that the type of rearing system did not adversely affect these parameters. The rearing design and the results obtained suggest that future nutritional studies of field-collected larvae will benefit from this kind of rearing experiment.     

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.