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.     

Effect of krill phospholipids versus soybean lecithin in microdiets for gilthead seabream (Sparus aurata) larvae on molecular markers of antioxidative metabolism and bone development

The objective of the present study was to compare the effectiveness of dietary marine phospholipids (MPL) obtained from krill and soybean lecithin (SBL) on the rearing performance and development of seabream (Sparus aurata) larvae. Larvae were fed from 16 to 44 day posthatching (dph) five formulated microdiets with three different levels (50, 70 and 90 g kg^–1) of phospholipids (PL) obtained either from an MPL or from a SBL source. Larvae‐fed MPL show a higher survival, stress resistance and growth than those‐fed SBL, regardless the dietary PL level. Overall, the increase in MPL up to 70 g kg^–1 total PL in diet was enough to improve larval gilthead seabream performance, whereas even the highest SBL inclusion level (90 g kg^–1 PL) was not able to provide a similar success in larval growth or survival. Inclusion of SBL markedly increased the peroxidation risk as denoted by the higher TBARs in larvae, as well as a higher expression of CAT, GPX and SOD genes. Moreover, SBL tends to produce larvae with a lower number of mineralized vertebrae and a lower expression of osteocalcin, osteopontin and BMP4 genes. Finally, increasing dietary MPL or SBL lead to a better assimilation of polyunsaturated fatty acids in the larvae, n‐3HUFA (especially 20:5n‐3) or n‐6 fatty acids (especially 18:2n‐6), respectively. In conclusion, MPL had a higher effectiveness in promoting survival, growth and skeletal mineralization of gilthead seabream larvae in comparison with SBL.     

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.     

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.     

Fish oil replacement by different microalgal products in microdiets for early weaning of gilthead sea bream (Sparus aurata,L.)

The aim of this study was to determine if algal products rich in DHA or ARA are able to completely replace fish oil in microdiets for marine fish larvae, gilthead seabream and if extra supplementation with EPA may further enhance larval performance. For that purpose, 20 day‐old gilthead seabream larvae of 5.97 ± 0.4 mm mean total length and 0.12 ± 0.001 mg mean dry body weight were fed with five microdiets tested by triplicate: a control diet based on sardine oil; a diet containing AquaGrow^® DHA (diet DHA) to completely substitute the sardine oil; a diet containing AquaGrow^® ARA (diet ARA); a diet containing both products, AquaGrow^® DHA and AquaGrow^® ARA to completely substitute the fish oil; and, a diet containing both products, AquaGrow^® DHA and AquaGrow^® ARA, together with an EPA source. Temperature, air and salinity activity tests were also performed to detect larval resistance to stress. At the end of the experiment, final survivals did not differ among groups. The microorganism produced DHA was able to completely replace fish oil in weaning diets for gilthead seabream without affecting survival, growth or stress resistance, whereas the inclusion of microorganism produced ARA did not improve larval performance. Moreover, addition of EPA to diets with total replacement of fish oil by microorganism produced DHA and ARA, significantly improved growth in terms of body weight and total length. The results of this study denoted the good nutritional value of microorganisms produced DHA as a replacement of fish oil in weaning diets for gilthead seabream, without a complementary addition of ARA. However, dietary supplementation of EPA seems to be necessary to further promote larval performance.     

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.     

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.     

The effect of dietary exogenous digestive enzymes on ingestion,assimilation, growth and survival of gilthead seabream (Sparus aurata,Sparidae, Linnaeus) larvae

The success of microdiets commonly used in the cultivation of marine fish larvae is limited to serving as partial replacements for live food. This limited success is thought to be associated with a reduced digestive ability due to an incompletely developed digestive system. The enhanced growth obtained from live food has been partially attributed to the digestive enzyme activity of the food organism. The present study was designed to test the effect of an exogenous digestive enzyme incorporated. into a microdiet on the growth of Sparus aurata. Larval gilthead seabream, 20–32 days old, were fed ¹⁴C labelled microdiets containing a commercial pancreatic enzyme at different concentrations (0, 0.1 and 0.05g / 100 g dry diet). Rates of ingestion and assimilation were measured and their relationship to dry weight was determined. Our results show that the success of the microdiet as a food for larval gilthead seabream was limited by the larva's low ingestion rate which only approached its maintenance requirement. In addition, the presence of digestive enzyme in the microdiet enhanced its assimilability by 30%. Larval growth over ten days was 0, 100 and 200% on microdiet free of added enzymes, one with added enzymes and a live food regime, respectively. It is our opinion that successful development of microdiets for Sparus aurata must be based on diets improved both in digestibility and attraction to the larvae. Further studies are now underway to determine the nutritional requirements of gilthead seabream larvae using the experimental method developed in the present study. This research was carried out in partial fulfillment of the requirements for the M.Sc. degree.     

The effect of dietary lecithin and lipase,as a function of age,on n-9 fatty acid incorporation in the tissue lipids of Sparus aurata larvae

The present study tested the effect of dietary lecithin and exogenous lipase on the incorporation of oleic acid in the tissue lipids of gilthead seabream larvae (Sparus aurata). Two of four microdiets were prepared by the addition of [¹⁴C]oleic acid as free fatty acid (FFA) to diets containing either 5% cuttlefish liver oil (CLO) or 5% soybean lecithin. Glycerol tri[1-¹⁴C]oleate was similarly incorporated in two other diets identical in lipid (4% cuttlefish liver oil, 1% soybean lecithin) and non-lipid composition but differed in that one contained a supplement of 0.05% porcine lipase. The effect of these diets was tested by following the incorporation of the label (dpm/mg larvae DBW) in the neutral and phospholipid fractions of seabream larvae at four different ages (21, 27, 32 and 45 days after hatching).A significant (p<0.05) effect of dietary lecithin on the incorporation of labelled FFA in both larval neutral and phospholipid fractions was demonstrated at all ages. This was particularly pronounced during early development (day 21) where fish fed the lecithin supplement incorporated 6.75 times more label than the diet containing [¹⁴C]oleic acid in CLO. The dietary lecithin enhancing effect diminished with age but was still significant at day 45 (2.17 times more label). In addition, the label was considerably higher in the phospholipid fraction compared to the neutral lipid, reflecting the high demand for membrane synthesis during rapid growth. Lecithin fed larvae demonstrated a higher consumption rate and efficiency of incorporation than fish consuming the cuttlefish liver oil diet, suggesting an emulsifying function for dietary phospholipid.In contrast, the supplementation with lipase showed a clear effect only in older fish where 45 day old larvae fed the lipase diet demonstrated a 3.42 times increase in radioactivity in their tissue lipids. This late lipase response may be the result of an insufficient level of dietary lecithin (M) and a short intestinal length being ineffective, in the early larval stages, in incorporating labelled free fatty acid from dietary glycerol tri[1-¹⁴C]oleate breakdown.     

Characterization and functional properties of digestive proteases in two sparids; gilthead seabream (Sparus aurata) and common dentex (Dentex dentex)

Digestive proteases present in two sparids, seabream (Sparus aurata) and common dentex (Dentex dentex), have been characterized using both biochemical and electrophoretic techniques. Although optimum pH and temperature for maximum activity of both acid and alkaline proteases were similar in the two species, important differences in total activity, as well as in thermal and pH stability were found. Specific inhibitors and SDS-PAGE zymograms were used to clarify such differences. Evidences support the existence of a more active and complex protease set in dentex. Results are discussed from the perspective of their application to the formulation of feeds for each species.     

Effect of dietary phospholipid level on the development of gilthead sea bream (Sparus aurata) larvae fed a compound diet

The aim of the study was to determine the influence of dietary phospholipid (PL) levels on survival and development of first feeding gilthead sea bream (Sparus aurata) larvae. Larvae were fed from day 4 to 23 posthatching with an isoproteic and isolipidic formulated diet with graded levels of PL from 90–150 g kg^?1 dry matter (DM). A dietary PL content of more than 90 g kg^?1 DM seems to be necessary for sustaining growth of first feeding sea bream larvae. The survival rates of larvae fed the formulated diets (31–40% at day 23) were similar to those generally observed in marine aquaculture hatcheries with live prey feeding sequence. However, this high survival rate was not associated with high growth and the larvae showed, at the end of the study, a high proportion of individuals with abnormal liver and calculi in the urinary bladder. It is concluded that although the diets used here cannot be used in total replacement of live preys, they constitute a solid starting point for further nutritional studies with first feeding gilthead sea bream larvae.     

Importance of the relative levels of dietary arachidonic acid and eicosapentaenoic acid for culture performance of gilthead seabream (Sparus aurata) larvae

The effect of different arachidonic acid (ARA) dietary contents at several dietary eicosapentaenoic acid (EPA) levels on the growth, survival and biochemical composition of gilthead seabream larvae was studied to better define the importance of this fatty acid as a function of EPA. Larvae of 18 days were fed one of the five isonitrogenous and isolipidic microdiets with three different EPA (0.3%, 2% and 4%) and ARA amounts (0.1%, 0.6% and 1.2%). Although a dietary increase in either ARA or EPA alone did not improve survival significantly, the increase in both fatty acids significantly enhanced growth and survival, suggesting an optimum dietary value of EPA:ARA close to 4:1.2. Dietary ARA was more efficiently incorporated into larval tissues than EPA. Increased dietary EPA or ARA contents reduced the incorporation of ARA or EPA into larval lipids, indicating their competition as substrates for different enzymes. The possible negative effect of further elevation of dietary ARA and its competition with EPA for phospholipids synthesis deserves further studies in marine fish larvae.     

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.     

Improved performance of gilthead sea bream, Sparus aurata, larvae after ozone disinfection of the eggs

Ozone (O₃) dissolved in seawater (ODS) was evaluated, as an egg disinfectant, on the spawn of captive gilthead sea bream, Sparus aurata, brood stock. Four contact times (CT) were tested (0.6, 1.2, 2.4 and 4.8 mg min L⁻¹) where CT was calculated by multiplying the dissolved O₃ concentration (0.3 mg L⁻¹) by different exposure periods (2, 4, 8, 16 min). There was also a disinfected seawater treatment that contained no O₃ or derived compounds (CT 0) and an untreated seawater control. All ODS treatments reduced egg surface bacterial counts to zero, which was significantly (P<0.05) lower than the CT 0 and the control groups (194 and 1320 plate⁻¹ respectively). Nevertheless, the hatching rate was high in the control and the CT treatments 0, 0.6 and 1.2 (88.7%, 87.3%, 89.5% and 83.7% respectively) while eggs exposed to a CT 2.4 and 4.8 hatched poorly (36.5% and 20.4% respectively), which was likely due, at least in part, to larvae unable to break the egg chorion successfully. Swim‐bladder inflation was significantly higher in the ODS groups (>97%) compared with the control and CT 0 treatments (ca. 70%). The results suggest that a 2‐min exposure of eggs to 0.3 mg O₃ L⁻¹ of ODS (CT 0.6) would improve current protocols in marine larviculture.     

Optimum dietary soybean meal level for maximizing growth and nutrient utilization of on-growing gilthead sea bream (Sparus aurata)

Six isonitrogenous [450 g kg⁻¹ crude protein (CP)] and isoenergetic diets (23 kJ g⁻¹) with six levels of defatted soybean meal inclusion (0, 132, 263, 395, 526 and 658 g kg⁻¹) in substitution of fish meal were evaluated in gilthead sea bream of 242 g initial weight for 134 days. Fish fed diets S0, S13, S26 and S39 had a similar live weight (422, 422, 438 and 422 g, respectively) but fish fed diets S53 and S66 obtained the lowest final weight (385 and 333g, respectively), and similar results were presented in specific growth rate (SGR). Fish fed diets S53 and S66 also obtained the highest feed conversion ratio (FCR). Quadratic multiple regression equations were developed for SGR and FCR which were closely related to dietary soybean level. The optimum dietary soybean levels were 205 g kg⁻¹ for maximum SGR and 10 g kg⁻¹ for minimum FCR. Sensorial differences were appreciated by judges between fish fed S0 and S39 soybean level, but after a re-feeding period of 28 days with diet S0, these differences disappeared.     

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

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

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

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