Dietary spermine supplementation induces intestinal maturation in sea bass (Dicentrarchus labrax) larvae

Sea bass (Dicentrarchus labrax) larvae were fed microparticulated compound diet containing 0 (FP0), 0.10 (FP10) and 0.33% (FP33) of a polyamine, spermine, from day 20 to day 38. LP group was fed live prey. This group exhibited the highest growth and survival. The addition of spermine did not lead to growth enhancement. A 33% survival improvement was obtained in FP33 group compared to FP0 group. The spermine addition affected the activity of pancreatic enzymes, trypsin, chymotrypsin and amylase, during larvae development. This non specific effect suggested that the action of spermine would be mediated by hormones. In the intestine, the FP33 group exhibited from day 31 higher activities of brush border membrane enzymes (leucine aminopeptidase and alkaline phosphatase) and lower level in a cytosolic enzyme (leucine-alanine peptidase) compared to FP10 and FP0 group. The diet containing the highest spermine level induced an enzymatic profile similar to that obtained in LP group and characteristic of a mature enterocyte. The initiation of enterocyte maturation at a proper development stage was associated to the survival improvement observed in FP33 group.     

Amylase and trypsin responses to intake of dietary carbohydrate and protein depend on the developmental stage in sea bass (Dicentrarchus labrax) larvae

Sea bass (Dicentrarchus labrax) larvae were fed from day 15 to day 35 with 4 isoenergetic formulated diets that varied in protein (30, 40, 50, 60%) and carbohydrate (37, 27, 17, 7%) content. The diets were designated as P30, P40, P50 and P60, respectively. Best growth and survival were noted for P50 larvae. Poor growth and survival were observed for P30 larvae. The specific activity of amylase increased in direct relation to the dietary carbohydrate level from day 18 onwards. This increase was the result of extensive amylase synthesis. Trypsin activity was positively correlated with dietary protein level only at day 35. This study shows that the mechanisms involved in amylase regulation are efficient in very young sea bass larvae, while those related to trypsin appear later in ontogeny and thus suggests an age-dependent regulation of enzyme synthesis.     

Effect of the molecular form of dietary nitrogen supply in sea bass larvae: Response of pancreatic enzymes and intestinal peptidases

Sea bass (Dicentrarchus labrax) larvae were weaned at day 25 with microparticulated diets in which 10% of the nitrogen supply had different molecular forms: amino acid mixture (SLAA), casein hydrolysate (SLH) or fish meal (SLP). The control group (LP) was fed live prey. No difference was observed in larval growth between the weaned groups, but the survival was significantly higher in the SLH group. Trypsin secretion was stimulated in the SLAA group, whereas the SLH diet reduced the secretion from the exocrine pancreas. The activity of the leucine-alanine peptidase, located in the cytosol of enterocytes, remained high in all weaned groups. However, the activity of the peptidases of the brush border membrane increased during the development phase in the control group. These results suggest that weaning with a classic compound diet delays enterocyte differentiation by maintaining the larval features of digestion. A compound diet containing protein hydrolysate can attenuate the delay of intestinal maturation.     

Development and response to a diet change of some digestive enzymes in sea bass (Dicentrarchus labrax) larvae

Variations in some enzyme activities during larval development of sea bass fed live prey were investigated from hatching to day 40. Fluctuations in the enzyme specific activities (except for trypsin) occurred in three phases: initially a sharp increase until day 12, followed by a plateau and subsequently a decrease around day 23. Then activities remained constant until day 40. Trypsin activity kept rising until day 23, then fell. Enzymatic adaptation to a change in diet was studied by feeding larvae with microparticulate diet from day 25. Adaptation to dietary change was observed for amylase, alkaline phosphatase and leucine aminopeptidase, assayed in whole larvae. In larvae fed microparticulate dry diet, the activities of these three enzymes tended to be higher than in those fed natural prey. Although poor growth was observed in larvae fed microparticles, the brush border enzyme activities purified from whole body homogenate, were not impaired.     

Saponins and phytosterols in diets for European sea bass (Dicentrarchus labrax) juveniles: effects on growth,intestinal morphology and physiology

This study aimed to elucidate the effects of two soy antinutrients, saponins and phytosterols, on growth, intestinal health and function of European sea bass juveniles. Seven fishmeal‐based diets were formulated: a control diet without antinutrients and six experimental diets containing low or high levels of soyasaponins (SapL and SapH, respectively), low or high levels of phytosterols (PhytL and PhytH, respectively) and low or high levels of soyasaponins + phytosterols (SapPhytL and SapPhytH, respectively). A feeding trial was conducted for 59 days after which blood was collected for plasmatic cholesterol quantification and intestinal samples were collected for histology, digestive enzymes activity and gene expression analysis. Histology was also performed at day 15. PhytL resulted in high distal intestine (DI) relative weight and decreased plasma cholesterol, while PhytH caused inflammatory changes in the DI. SapH depressed maltase and alkaline phosphatase activity in DI and the histological evaluation indicated some inflammatory changes. The SapPhytH resulted in decreased maltase activity in the DI and indications of inflammatory changes that were supported by the results on gene expression profiles. The antinutrients tested did not compromise growth, however, caused some gastrointestinal disturbance that may affect fish in the long term.     

Peptide molecular weight distribution of soluble protein fraction affects growth performance and quality in European sea bass (Dicentrarchus labrax) larvae

Dicentrarchus labrax were fed from 10 to 40 days posthatching with six microdiets differing in the inclusion level (60 g kg^‐1 and 120 g kg^‐1) and type of protein hydrolysate (PH; yeast, YPH; pig blood, PBPH; pig red blood cells, PRBCPH). A microdiet containing 120 g kg^‐1 fish PH (FPH) was used as a control. PH differed in their amino acid (AA) profile and molecular weight distribution and therefore the tested microdiets too. The estimated content in FAA and di‐ and tripeptides in the FPH microdiet was 2 g kg^‐1 and 44 g kg^‐1, respectively. FAA estimated levels in YPH and PBPH microdiets were 26 g kg^‐1 and 53 g kg^‐1, whereas levels of di‐ and tripeptides were 30 g kg^‐1 and 60 g kg^‐1, respectively. The estimated levels of FAA in PRBCPH microdiets were 8 g kg^‐1 and 17 g kg^‐1, whereas estimated levels of di‐ and tripeptides were 11 g kg^‐1 and 22 g kg^‐1, respectively. Results revealed that FPH may be replaced by alternative PH from yeast and pig blood products, as fish fed those diets performed, in terms of growth, survival, digestive function and incidence of skeletal deformities, as well as those fed the FPH microdiet. Using YPH, PBPH and PRBCPH, the inclusion level of PH in microdiets might be reduced to a half with respect to current practices using FPH.     

Effect of a dietary phospholipid supplementation on growth and fatty acid composition of European sea bass (Dicentrarchus labrax L.) and turbot (Scophthalmus maximus L.) juveniles from weaning onwards

Two 40-day feeding trials using extruded diets were conducted to assess the effect of a dietary phospholipid (PL) supplementation on growth, survival and fatty acid composition of European sea bass (Dicentrarchus labrax) and turbot (Scophthalmus maximus) from weaning onwards. Two dietary treatments (FO and PL) were tested; both had an identical extruded basis (92.5% total diet weight) coated with a different lipid fraction (7.5% total diet weight). Diet PL contained 2% egg yolk PL (69% pure). In diet FO the PL was replaced by hydrogenated coconut oil. The isolipidic diets contained an equal amount of fish oil ethyl esters providing 1.6% (% diet dry weight) of n-3 highly unsaturated fatty acids (HUFA). A diet water stability test showed no effect of the PL supplementation on the leaching of the dietary fatty acids. In both fish species weight, but not survival, significantly increased as a result of PL supplementation. Weaning onto the experimental diets resulted in similar changes in the relative percent levels of fatty acids in both species. In general, the percentage of saturated fatty acids levelled off after a rapid increase, while monoenes increased after an initial decrease. Total n-3 polyunsaturated fatty acids (PUFA) decreased and total n-6 PUFA remained almost constant. The major effect of the dietary PL on fish fatty acid composition was a 50% increase in n-6 and n-3 HUFAs compared to the PL-free FO diet. The rise in n-6 HUFA may have reflected the higher moiety in the dietary PL. On the other hand this was not the case for the n-3 HUFA since they represented only low levels in the PL fraction (0.1%) compared to that provided by the ethyl esters (1.6%) suggesting a more efficient incorporation of the PL n-3 HUFA than of the ethyl ester n-3 HUFA. A second hypothesis is that the dietary PL may have favored the incorporation of the dietary ethyl ester n-3 HUFA.     

Effects of different dietary oils in sea bass (Dicentrarchus labrax) nutrition

Five diets having the same proximate composition but containingdifferent types of supplemental oils, singly or in combination, were used forgrowing sea bass from 95 g to about 200 g in smallseacages. The oils tested were olive oil, soybean oil and fish oil. The dietsformulated contained EPA and DHA levels ranging from 0.88 to 1.35% of the diet.Growth parameters and fish body composition were not significantly affected bythe type of oil used. The same was generally apparent for liver andhematological characteristics. The content of phospholipids in EPA and DHA washighest in the livers of fish fed diets supplemented with fish oil. A positivecorrelation was found between dietary and liver n-6 PUFA. Histological sectionsindicated extended pathological symptoms (intensive liver degeneration andhemorrhages, changes in the gill structure) in the fish receiving dietssupplemented only with plant oils. These symptoms existed but to a smallerfrequency and degree in livers of fish fed diets supplemented with plant andfish oil, while were not apparent in those fed the fish oil diet.     

Proteolysis by juvenile sea bass (Dicentrarchus labrax) gastrointestinal enzymes as a method for the evaluation of feed proteins

Protein digestibility by gastrointestinal homogenates of Dicentrarchus labrax, was investigated in 5 feeds in relation to their amino acids composition and their ability to sustain growth of D. labrax post larval juveniles. Gastric proteolysis was found to contribute <25% of the total proteolytic capacity. Correlation was found between intestinal proteolytic capacity and the feed protein content of three pairs of amino acids. The sum of basic amino acids, arginine + lysine in the feed protein exhibited a positive correlation with susceptibility to proteolysis. The sum of the acid amino acids glycine and proline showed a negative correlation.Comparison of the intestinal proteolytic enzymatic activities in 3 growth stages of this species revealed similar profiles. Post larval and large juveniles had similar proteolytic capacities when compared on the basis of equal activity on an artificial substrate of Trypsin. Specific growth rates of post larval fish fed on these 5 feeds correlated positively with their protein digestibility as measured with large juvenile's intestinal homogenates. The results suggest that in this species, the efficiency of feeds in sustaining growth at the post larval stage can be estimated by measuring the feed protein digestibility by the proteolytic system of larger sized juveniles.     

The effects of dietary marine protein hydrolysates on the development of sea bass larvae,Dicentrarchus labrax,and associated microbiota

Protein hydrolysate is an essential component of dry starter diets for fish larvae, as promoting healthy development. Peptides are also suitable substrates for many intestinal microbes. Five experimental diets were compared to a control diet (CONT) supplemented with a commercial fish protein hydrolysate. Each diet contained one marine protein hydrolysate, which differed by the proportion of di‐ and tri‐peptides, and by raw materials. Two diets (HYD4 and HYD5) stimulated larval growth compared with CONT. Two other diets (HYD1 and HYD2) yielded inferior growth. HYD1 was detrimental for survival, which was associated with an up‐regulation of genes involved in inflammation and antioxidative responses, while the gene coding for osteocalcin was down‐regulated in this group. The relative activity of enzymes in the brush border membrane of enterocytes was significantly stimulated with diets HYD3 and HYD4. Gut microbiota were influenced by the diet, but the bacterial community profiles observed with HYD3 and HYD4 were the only ones that were not significantly dissimilar. Groups HYD1 and HYD2 showed the most dissimilar microbiota. It was concluded that the nature of the marine protein hydrolysate is important for larval development and health, but a high proportion of small peptides is not a sufficient criterion to assess dietary value.     

Improved feed utilization,intestinal mucus production and immune parameters in sea bass (Dicentrarchus labrax) fed mannan oligosaccharides (MOS)

In the present study, the effects of dose dietary administration of mannan oligosaccharides (MOS) (Bio‐Mos; Alltech Inc.) on growth, digestibility, liver morphology, lipid and carbohydrate metabolism enzymes, organoleptic properties, immune parameters and gastrointestinal tract mucus production were assessed. MOS dietary inclusion significantly reduced lipogenic enzymes activity in liver and resulted in a lower lipid vacuolization and more regular‐shaped morphology of hepatocytes around the sinusoidal spaces, which could be associated to a better energy and feed utilization because of the improvement of feed utilization and the reduction of feed intake found in the current experiment. Moreover, MOS inclusion enhanced phagocytic activity of head kidney leukocytes in fish fed 4 and 6 g kg^?1 MOS diets at days 30, 45 and 60. Gut quantitative morphological analyses showed a marked dose‐dependent enhancement in the number of cells secreting acid mucins by unit of area which could be related to the previous improvement in resistance to bacterial infection. No effects on sensorial parameters and biochemical composition of flesh were detected.     

Marine raw material choice,quality and weaning performance of Ballan wrasse (Labrus bergylta) larvae

Previous results show that weaning success of Ballan wrasse larvae greatly depends on the quality of the dietary marine raw materials. In the present study, six moist or agglomerated experimental weaning diets containing different combinations of high‐quality marine raw materials, being fish meal (FM), cod muscle meal (CMM), shrimp meal (SM) and krill hydrolysate (KH), were tested in a 2‐month weaning trial with Ballan wrasse larvae of 34.5 mg initial body weight. Larvae performance was good in all dietary treatments except those fed diet D1 containing only FM. The Ballan wrasse larvae fed weaning diets D4 and D5 containing FM and either SM or KH, respectively, had the highest final body weight (0.7 g) but also the highest mortality (50%). Best weaning survival (77%) was obtained using the dry agglomerated diet D3 containing CMM and SM. During the first month, fish survival correlated positively with dietary free amino acid and soluble protein levels and negatively with the combined levels of dietary lipid oxidation metabolites and ethoxyquin. During the second month, mortality rates were lower in all treatments. Fish larvae final body weight correlated negatively with total dietary fatty acids and positively with dietary cholesterol, phosphorous and DHA/EPA ratio.     

Growth,development and survival of European sea bass (Dicentrarchus labrax) larvae cultured under different light spectra and intensities

We assessed the effects of light intensity and spectrum on the growth, development and survival of Dicentrarchus labrax larvae from 30 to 96 days after hatching. Twelve lighting regimes were applied using 0.3, 1 and 2 W/m² full spectrum white, blue, red and green light. By day 66 of exposure, growth performance was significantly better in larvae reared under green or red light compared with white or blue light (p < 0.05), while it was significantly better at 2.0 W/m² compared with 1.0 or 0.3 W/m² (p < 0.05). Larvae reared under white or blue light had significantly more deformities (p < 0.05) than did larvae reared under light of other spectra, while larvae reared under 0.3 W/m² light intensity had a significantly higher proportion of jaw malformations than reared under 1.0 and 2.0 W/m² (p ? 0.05). Dicentrarchus labrax larvae subjected to green light showed significantly lower survival compared with the other light spectra (p < 0.05). Moreover, light significantly affected brain aanat1a, aanatt1b, mellc, mt1, mt2, gh, tsh and crf mRNA expression. Therefore, the red and green light groups (or under 2.0 W/m²) could improve the growth performance and enhance secretion of melatonin, gh and tsh, and reduce the proportion of jaw malformations.     

Changes in plasma glucagon and insulin associated with fasting in sea bass (Dicentrarchus labrax)

Juvenile sea bass (Dicentrarchus labrax) were fasted for 22 days and changes in plasma insulin, glucagon and glucose levels, as well as glycogen and protein content in liver and muscle were analyzed. Glucagon increased initially on the 4th day of fasting. The glucagon/insulin ratio (G/I) increased from a value of 0.11±0.02 (24h of fasting) to 0.21±0.05 (4th day of fasting). Thereafter, both glucagon and insulin levels decreased and remained at low concentrations until the 22nd day of fasting. Plasma glucose levels fell at the beginning of fasting, stabilized between the 4th and 8th day, and gradually declined during the rest of the experiment. There was a body weight loss of 15% and a significant decrease in both the hepatosomatic index and mesenteric fat. The decrease in the percentage of muscle proteins was not significant, while liver glycogen content showed a sharp decline.     

Evaluation of weaning performance of California halibut (Paralichthys californicus) larvae using growth,survival and digestive proteolytic activity

This study evaluated weaning success of California halibut, Paralichthys californicus, larvae onto a microdiet at various stages of development utilizing growth, survival and digestive enzyme activity. Weaning onto a microdiet was evaluated at 16, 26, 36 and 46 days posthatch (dph). Alkaline and acid proteases and leucine aminopeptidase activities were measured after weaning and compared between the weaned treatment and Artemia‐fed controls. Survival was significantly lower in the microdiet‐fed treatments compared to the control groups. Growth was significantly reduced in all weaning treatments compared to the control, except for the 46 dph group. No differences in enzyme activities were detected between treatment and diet at 16 and 26 dph; however, activities were higher for the microdiet‐fed larvae at 36 and 46 dph. This study demonstrates that California halibut larvae possess a differentiated and effective digestive system early in development and can be weaned with relative success (>40% survival) before completion of the metamorphosis (i.e., 36 dph). The lack of weaning success at an early date cannot be entirely because of the absence of a functional stomach but could be related to, among other factors, the low‐microdiet ingestion rates observed and higher leaching of smaller microdiets.     

Characterization of mevalonate metabolism in the sea bass (Dicentrarchus labrax L) liver

The activities of mevalonate kinase, mevalonate 5-phosphate kinase and mevalonate 5-pyrophosphate decarboxylase, were examined in sea bass (Dicentrarchus labrax L) liver. The activities of the three enzymes were studiedin vitro in relation to the influence of protein content, time of incubation, pH, temperature, mevalonate, ATP and Mg⁺⁺ concentration. Protein content in the assay medium affected the three enzymes differently. Mevalonate kinase, mevalonate 5-phosphate kinase, and mevalonate 5-pyrophosphate decarboxylase activities were linear up to 0.2, 0.4 and 0.8 mg protein, respectively. With respect to the time course studies, the enzymes also behaved differently. Mevalonate kinase activity increased over forty minutes, reaching a plateau thereafter, while mevalonate 5-phosphate kinase and decarboxylase increased over the entire assay period. All the three enzymes showed a maximum in activity at pH 7.5. The effect of reaction temperature showed that phosphorylation increased to maximum around 35°C for mevalonate kinase and 30°C for mevalonate 5-phosphate kinase while decarboxylation rates remained constant well until 30°C temperature decreasing afterwards. The enzymes behaved differently as a function of mevalonate concentration. Mevalonate 5-phosphate formed was maximal when the initial mevalonate concentration was 272 M, whereas mevalonate 5-pyrophosphate and CO₂ were formed maximally at mevalonate concentrations of 136 M and 68M, respectively. Optimal ATP concentration in the medium was 3 mM for decarboxylase and 6 mM for kinases, and Mg⁺⁺ requirements varied from 4 mM for decarboxylase to 6 mM for kinases.     

Effect of vitamin A on the skeletal morphogenesis of European sea bass,Dicentrarchus labrax (Linnaeus, 1758)

The purpose of this study was to examine the effects of vitamin A levels in the larval diet of Dicentrarchus labrax, (a) on the shape of juvenile skeleton and (b) on the development of pelvic fins. To exclude the side‐effects of skeletal abnormalities on skeleton shape, morphometric analysis was performed on normal individuals only. Geometric morphometric analysis revealed a significant effect of larval nutrition on the shape of juvenile skeleton (Wilk's λ=0.0188, P<0.001), 55 days after the application of the different nutritional regimes. Between‐groups shape differences mainly concerned the position of anterior dorsal pterygiophore, which was associated with a significant (P<0.05) increase of the number of dorsal lepidotrichia and hard spines at low levels of vitamin A (3–9 × 10³ IU kg^?1 dry food). At the lowest vitamin A levels (3 × 10³ IU kg^?1 dry food), the development of pelvic fins was abnormal, with severe bilateral (44±10%, mean±SD) or unilateral (22±9%) lack of the fin lepidotrichia, hard spines and their supporting elements. In the 82–94% of the unilaterally deformed individuals, deformation concerned the left pelvic fin. Concerning the shape of pelvic fins in the normally developed individuals, results demonstrated a significant effect of nutritional conditions on the size and position of all the elements of the pelvic fin area (basipterygium, lower metacleithrum, cleithrum, scapula) (Wilk's λ=0.3087, P<0.001). Our results demonstrate a significant nutritionally driven phenotypic plasticity in D. labrax, with dietary levels of vitamin A determining the skeleton shape and the left–right symmetry of pelvic fins.     

Effect of dietary protein and lipid level on metabolic utilization of diets by european sea bass (Dicentrarchus labrax) juveniles

Two trials were performed with sea bass juveniles to study the effect of dietary protein (trial I) and lipid (trial II) levels on the metabolic utilization of diets at 25 °C. The effect of water temperature (18 and 25 °C) on metabolism was also tested in trial I. For that purpose, oxygen consumption and ammonia excretion were measured both in fed and in 9-days starved fish. In trial I, diets were formulated to be isoenergetic (GE: 19.4 kJ g^–1) and to have 36, 42, 48 and 56% protein; in trial II, diets were formulated to be isonitrogenous (48% protein) and to have 12, 18, 24 and 30% lipid. In trial I, feed intake (g kg^–1 day^–1), and daily ammonia excretion and oxygen consumption significantly increased with water temperature. However, when expressed relatively to intake there was no significant effect of temperature on ammonia excretion (% N intake) or heat production (% GE intake). Heat increment of feeding (% GE intake) was neither affected by diet composition nor by water temperature. The relative contribution of protein catabolism to total energy expenditure significantly increased with dietary protein level, but was not affected by water temperature. In trial II, both daily ammonia excretion and oxygen consumption were inversely correlated to dietary lipid levels. Nitrogen excretion, heat production, heat increment of feeding, non-fecal losses (% intake) and the relative contribution of protein to total energy expenditure were also inversely related to dietary lipid levels. Results of this study indicate that the main effect of water temperature was to modify feed intake, not the metabolic utilization of diets. Indeed, expressed relatively to nitrogen or energy intakes, both nitrogen and energy budgets were not significantly affected by water temperature. A decrease of dietary protein to energy ratio, by modifying either dietary protein or lipid levels, spared protein utilization for metabolism, and this effect was essentially due to a decrease of non-fecal nitrogen excretion and of the heat increment of feeding.