Dietary protein hydrolysate and trypsin inhibitor effects on digestive capacities and performances during early-stages of spotted wolffish: suggested mechanisms.

Growth rate is dependent upon adequate provision of amino acids especially in newly-hatched fish which experience very high growth rate. The replacement of a fraction of protein content by partially hydrolyzed (pre-digested) proteins was carried out and the digestive capacities and performances of larval/juvenile spotted wolffish (Anarhichas minor) were measured. The goal of this study was to verify whether the scope for growth is principally dictated by the proteolytic capacity of the digestive system by examining the effect of protein hydrolysates (PH) and trypsin inhibitor dietary inclusion on protein digestion/assimilation capacities, growth and survival. Four experimental diets were examined: C (control) I (supplemented with 750 mg/kg soybean trypsin inhibitor (SBTI)) H (supplemented with 20% PH) and HI (supplemented with 20% PH and 750 mg/kg SBTI). Protein hydrolysate supplementation gave significantly higher body mass than control at day 15 post-hatching. Unexpectedly, at day 30 and 60, fish administered diet HI (containing trypsin inhibitor) were heavier than the other groups. Suggested mechanisms are presented and discussed. The main conclusions of this study are that wolffish larval stage lasts roughly 15 days and that juvenile growth is linked to proteolytic capacity, but also very likely to absorption capacity of peptides and amino acids.

Regulation of FADS2 expression and activity in European sea bass (Dicentrarchus labrax, L.) fed a vegetable diet.

Supplies of marine fish oils are limited, and continued growth in aquaculture production dictates that lipid substitutes in fish diets must be used without compromising fish health and product quality. In this study, the total substitution of a fish meal and fish oil by a blend of vegetable meals (corn, soybean, wheat and lupin) and linseed oil in the diet of European sea bass (Dicentrachus labrax) was investigated. Two groups of European sea bass were fed with fish diet (FD) or vegetable diet (VD) for 9months. VD, totally deprived of eicosapentaenoate (EPA; 20:5n-3) and docosahexaenoate (DHA; 22:6n-3), revealed a nutritional deficiency and affected growth performance. Whilst VD induced a significant increase in fatty acid desaturase 2 (FADS2) and sterol binding regulatory element-binding protein 1 (SREBP-1) mRNA levels, the desaturation rate of [1-(14)C]18:3n-3 into [1-(14)C]18:4n-3, analysed in microsomal preparations using HPLC method, did not show an upregulation of FADS2 activities in liver and intestine of fish fed VD. Moreover Western-blot analysis did not revealed any significant difference of FADS2 protein amount between the two dietary groups. These data demonstrate that sea bass exhibits a desaturase (FADS2) activity whatever their diet, but a post-transcriptional regulation of fads2 RNA prevents an increase of enzyme in fish fed a HUFA-free diet. This led to a lower fish growth and poor muscle HUFA content.

Gene expression patterns during the larval development of European sea bass (dicentrarchus labrax) by microarray analysis.

During the larval period, marine teleosts undergo very fast growth and dramatic changes in morphology, metabolism, and behavior to accomplish their metamorphosis into juvenile fish. Regulation of gene expression is widely thought to be a key mechanism underlying the management of the biological processes required for harmonious development over this phase of life. To provide an overall analysis of gene expression in the whole body during sea bass larval development, we monitored the expression of 6,626 distinct genes at 10 different points in time between 7 and 43 days post-hatching (dph) by using heterologous hybridization of a rainbow trout cDNA microarray. The differentially expressed genes (n = 485) could be grouped into two categories: genes that were generally up-expressed early, between 7 and 23 dph, and genes up-expressed between 25 and 43 dph. Interestingly, among the genes regulated during the larval period, those related to organogenesis, energy pathways, biosynthesis, and digestion were over-represented compared with total set of analyzed genes. We discuss the quantitative regulation of whole-body contents of these specific transcripts with regard to the ontogenesis and maturation of essential functions that take place over larval development. Our study is the first utilization of a transcriptomic approach in sea bass and reveals dynamic changes in gene expression patterns in relation to marine finfish larval development.

Dietary vitamin mix levels influence the ossification process in European sea bass (Dicentrarchus labrax) larvae.

The influence of dietary vitamins on growth, survival, and morphogenesis was evaluated until day 38 of posthatching life in European sea bass larvae (Dicentrarchus labrax). A standard vitamin mix (VM), at double the concentration of the U.S. National Research Council's recommendations, was incorporated into larval feeds at 0.5%, 1.5%, 2.5%, 4.0%, and 8.0% to give treatments VM 0.5, VM 1.5, VM 2.5, VM 4.0, and VM 8.0, respectively. The group fed the VM 0.5 diet all died before day 30. At day 38, the larvae group fed VM 1.5 had 33% survival, while the other groups, with higher vitamin levels, showed at least 50% survival. The higher the percentage VM in the diet, the lower the percentage of column deformities. High dietary vitamin levels positively influenced the formation of mineralized bone in larvae: the higher the dietary vitamin level, the higher the ossification status. In the larvae group fed at the highest vitamin levels, we observed a temporal sequence of coordinated growth factor expression, in which the expression of bone morphometric protein (BMP-4) preceded the expression of IGF-1, which stimulated the maturation of osteoblasts (revealed by high osteocalcin expression levels). In groups fed lower proportions of vitamins, elevated proliferator peroxisome-activated receptors (PPAR-gamma) expression coincided with low BMP-4 expression. Our results suggest that high levels of PPAR-gamma transcripts in larvae-fed diets with a low VM content converted some osteoblasts into adipocytes during the first two weeks of life. This loss of osteoblasts is likely to have caused skeletal deformities.

Effects of different dietary levels of fish protein hydrolysates on growth, digestive enzymes, gut microbiota, and resistance to Vibrio anguillarum in European sea bass (Dicentrarchus labrax) larvae.

Two fish protein hydrolysates (FPH) were incorporated into four diets prepared for start-feeding sea bass larvae, at two different levels (10% and 19% of total ingredients): a commercial FPH, CPSP, in which the molecular mass of the main fraction of soluble peptides (51%) was between 500-2500 Da, and an experimental FPH obtained by acidic silage of sardine offal, SH, with a main portion of soluble peptides (54%) ranging from 200 to 500 Da. The diet with 10% of the commercial FPH gave the best results in terms of growth, survival and intestinal development, as evaluated by the early activity of digestive enzymes in the brush border membrane (alkaline phosphatase and aminopeptidase N). This was related to the low level of Vibrio spp. counted in the larvae of group C10. The high dose of FPH, especially in the experimental preparation rich in short peptides, seemed to favour the dominance of Vibrio sp. TYH3, which behaved opportunistically. The effect of the experimental FPH was ambiguous, since early larvae challenged with Vibrio anguillarum were more resistant to the pathogen, especially at high FPH dose (group S19). This might be due either to direct antagonism between V. anguillarum and Vibrio sp. TYH3, or to the stimulation of the immune response in the larvae. These results indicate that different molecular weight fractions and concentrations of feed-soluble peptides may affect the growth performance and immunological status of sea bass larvae. Consequently, a low dose of commercial FPH seems advisable, both for larval development and for the bacterial environment, although further research is required to determine and characterize peptide fractions that may have a beneficial effect on growth and immune response, and to determine their optimal inclusion levels in diets for sea bass larvae.

Reduced lipid intake leads to changes in digestive enzymes in the intestine but has minor effects on key enzymes of hepatic intermediary metabolism in rainbow trout ( Oncorhynchus mykiss).

For sustainable aquaculture, the removal of marine resource ingredients in fish diets is an important objective. While most studies focus on the replacement of fish oil by vegetable oil, little is known on the nutritional effects of presence (which corresponds to the control diet) or absence of dietary fish oil. We studied fatty acid composition of brush-border membranes and digestive enzyme activities of the intestine and measured the expression and activities of several enzymes involved in the hepatic intermediary metabolism of rainbow trout (Oncorhynchus mykiss) fed for 7 weeks with or without fish oil. The diets were pair-fed to ensure that fish fed either diet had comparable carbohydrate and protein intakes. Absence of fish oil significantly reduced growth rate, protein efficiency and plasma lipid components. Activities of intestinal digestive enzymes were significantly decreased in the anterior intestine in fish fed without fish oil. In liver, dietary fish oil removal did not affect the transcript levels or activities of the main enzymes involved in lipogenesis (fatty acid synthase) and fatty acid ß-oxidation (3-hydroxyacyl-CoA dehydrogenase), glycolysis or amino acid oxidation. It lowered the expression of the genes coding for gluconeogenic enzymes (glucose-6-phosphatase and phosphoenolpyruvate carboxykinase), but their enzyme activities were not affected. The activities, but not gene expression of lipogenic enzymes, involved in NADPH and malonyl-CoA formation were also modified after fish oil removal as reflected by higher activities of isocitrate dehydrogenase/glucose-6-phosphate dehydrogenase and acetyl-CoA carboxylase enzymes. Overall, our results indicate that the intestinal digestive capacity was strongly modified by dietary fish oil removal, while hepatic intermediary metabolism was only marginally affected, in fed rainbow trout.

Dietary phospholipids are more efficient than neutral lipids for long-chain polyunsaturated fatty acid supply in European sea bass Dicentrarchus labrax larval development.

We evaluated the effects of dietary lipid class (phospholipid vs. neutral lipid) and level of n-3 long-chain PUFA (LC-PUFA) on the growth, digestive enzymatic activity, and histological organization of the intestine and liver in European sea bass larvae. Fish were fed from the onset of exogenous feeding at 7 to 37 d post-hatch with five isoproteic and isolipidic compound diets with different levels of EPA and DHA. Diet names indicated the percentage of EPA and DHA contained in the phospholipids (PL) and neutral lipids (NL), as follows: PL5, PL3, PL1, NL1, and NL3. Histological observations showed different patterns of lipid absorption and accumulation in the intestinal mucosa depending on the level and nature of the dietary lipid fraction. Fish fed high levels of neutral lipids (11%, NL3 diet: 2.6% of EPA + DHA in the NL fraction) showed large intracellular and intercellular lipid deposits in the anterior intestine, but no such lipid accumulation was detected when larvae were fed with low and moderate levels of EPA and DHA in the phospholipid and neutral lipid fractions of the diet (PL and NL1 diets). PL were preferentially absorbed in the postvalvular intestine, and the accumulation of marine PL was inversely correlated to their dietary level. The postvalvular intestinal mucosa and liver showed signs of steatosis; large lipid vacuoles were observed in this region of the intestine and in the liver and were inversely correlated with the level of dietary neutral lipids. The best results in terms of growth, survival, and development (maturation of the digestive system and histological organization of the liver and intestinal mucosa) were obtained in the group fed with 2.3% of EPA and DHA in the PL fraction of the diet (PL3 diet), revealing that European sea bass larvae use the LC-PUFA contained in the PL fraction more efficiently than those from the NL fraction of the diet.

Dietary TAG source and level affect performance and lipase expression in larval sea bass (Dicentrarchus labrax).

The influence of dietary TAG source (fish oil, triolein, and coconut oil) and level (7.5 and 15% of the diet) on growth, lipase activity, and mRNA level was studied in sea bass larvae, from mouth opening until day 24 and from day 37 to 52. Fish oil and triolein induced better growth in both experiments, this being significant at a higher dietary level. Coconut oil significantly decreased growth at the higher level, possibly as the result of an excessive supply of medium-chain TAG. Growth was not related to lipase specific activity, suggesting a production in excess to dietary needs. Body lipid content was positively related to dietary lipid level and was affected by lipid quality. In addition, larval FA composition generally reflected that of the diet. The source of dietary lipid, but not the quantity, was shown to affect lipase activity significantly. Coconut oil diets induced the highest lipase activity, whereas the effect of fish oil was age dependent-it was similar to coconut oil at day 24 but induced the lowest lipase activity in 52-d-old larvae. The differential lipase response was probably caused by differences in the FA composition of the diet, related to the specificity of lipase toward FA differing in chain length and degree of saturation. No significant differences were found in lipase/glyceraldehyde-3-phosphate dehydrogenase mRNA, which suggests the existence of a posttranscriptional regulation mechanism.

Farmed and wild fish in the prevention of cardiovascular diseases: assessing possible differences in lipid nutritional values.

AIM: The consumption of fish and fish-derived products is recommended as a means of preventing cardiovascular and other diseases, and has considerably increased over recent decades. However, as the world's wild fish stocks are limited, consumers are now being proposed farmed fish as an alternative. The aim of this study was to compare the fat composition of farmed and wild fish in order to estimate whether the expected health effects of the former (especially in relation to cardiovascular diseases) are potentially the same as those of the latter. DATA SUMMARY: The data presented in this paper were collected from the recently published literature. The lipid composition of farmed fish is more constant and less affected by seasonal variations than that of wild fish because, as it is largely dependent on the fatty acid composition of their feed, it can be customised by adjusting dietary intakes. Vegetable food is increasingly replacing fishmeal in fish feeds, and may induce a relative decrease in n-3 polyunsaturated fatty acids (PUFAs), expressed as a percentage of total fatty acids. However, as farmed fish generally have higher total lipid levels than wild fish, 100 g of farmed fish fillet can provide a higher amount of n-3 PUFAs (especially EPA and DHA) than 100 g of wild fish. Furthermore, quite high levels of (alpha-tocopherol in farmed fish can theoretically provide better EPA and DHA protection against peroxidation. Sensory analyses by trained consumer panels have not revealed any significant differences between wild and farmed fish. Moreover, fresh fish storage conditions (including the time from slaughtering to consumer sales) are more easily verifiable in the case of farmed fish, in which the content of potentially toxic heavy metals (a major health concern in certain areas) is also theoretically more easily controlled. CONCLUSIONS: Provided that they are raised under appropriate conditions, the nutritional content of farmed fish is at least as beneficial as that of wild fish (particularly in terms of the prevention of cardiovascular diseases), and they also have the advantages of freshness and apparent non-toxicity.

Ontogeny of the gastrointestinal tract of marine fish larvae.

Marine fish larvae undergo major morphological and cellular changes during the first month of life. The ontogeny of the gastrointestinal tract combines these two aspects of the larval development and is very interesting in that the timing of functional changes appears genetically hard-wired. The goal of this paper is to give an overview of the gastrointestinal development process in marine fish larvae, with particular attention to three species: sea bass; red drum; and sole, since the description of gut maturation in fish larvae was initiated during the last decade with these species. During the early stages, marine fish larvae exhibit particular digestive features. Concerning the exocrine pancreas, amylase expression decreases with age from the third week post-hatching in sea bass and red drum (approximately 400 degree days), whereas expression of other enzymes (trypsin, lipase, phospholipase A2...) increases until the end of the larva period. Moreover, secretory function of the exocrine pancreas progressively develops and becomes efficient after the third week of life. Concerning the intestine, enzymes of the enterocyte cytosol (in particular peptidase) have higher activity in young larvae than in older. Approximately in the fourth week of post-hatching development in sea bass, red drum and sole larvae, the cytosolic activities dramatically decline concurrently with a sharp increase in membranous enzyme activities of the brush border, such as alkaline phosphatase, aminopeptidase N, maltase. This process characterises the normal maturation of enterocytes in developing fish larvae and also in other vertebrates' species. The establishment of an efficient brush border membrane digestion represents the adult mode of digestion of enterocytes. This paper also describes the role of diet on the development of the gastrointestinal tract. Indeed, the maturational process of digestive enzyme can be enhanced, stopped, or delayed depending on the composition of the diet.

High dietary lipid levels enhance digestive tract maturation and improve dicentrarchus labrax larval development.

This study was designed to determine the nutritional lipid requirement of seabass larvae and to understand the effects of dietary fat concentration on their digestive tract maturation. Seabass (Dicentrarchus labrax) larvae were fed, from d 15 to 38 of life, one of five isonitrogenous compound diets with different lipid levels, ranging from 10 to 30 g/100 g. The higher the lipid level, the greater the growth and survival of the larvae (P < 0.05). The lipolytic enzymes assayed, lipase and phospholipase A2, were stimulated by the increase in their respective dietary substrates, triglycerides and phospholipids, in 38-d-old larvae (P < 0.05). Nevertheless, a plateau in the activity of these two lipolytic enzymes was observed from 20% dietary lipids onwards. The similar mRNA levels of phospholipase A2 in the three groups fed the highest lipid levels suggested that the maximal synthesis level of lipolytic enzyme was reached at 20% dietary fat. Pancreatic secretion of trypsin and amylase were positively affected by the dietary lipid level; a possible involvement of a cholecystokinin-releasing factor is discussed. Diets containing >20% lipids led to the increase in activities of brush border membrane enzymes to the detriment of a cytosolic enzyme in enterocytes, leucine-alanine (Leu-Ala) peptidase. This enzymatic change reveals the earlier maturation of enterocytes in larva groups fed high lipid levels.

Partial substitution of di- and tripeptides for native proteins in sea bass diet improves Dicentrarchus labrax larval development.

To determine whether incorporation of peptides into diets can improve larval development, sea bass (Dicentrarchus labrax) larvae were fed for 21 d one of three isonitrogenous, isoenergetic semipurified diets in which enzymatic hydrolysate (75% di- and tripeptides) of fish meal proteins was substituted for 0, 20 or 40% of native fish meal proteins. Growth and survival were significantly greater (P < 0.05) in larvae fed peptide diets compared to those fed only native protein, with the best performance exhibited by those fed the 20% level of peptides. Chymotrypsin activity was much higher in groups fed peptide diets compared to that fed all native protein (P < 0.001), indicating a greater proteolytic capacity of the pancreas. At the intestinal level, activities of the brush border enzymes, aminopeptidase, maltase and gamma-glutamyl transpeptidase, increased with age while the cytosolic enzyme, leu-ala peptidase, decreased with age (P < 0.001). These changes in enzymatic activities correspond to the normal development of intestinal digestion. This development occurred earlier in the group fed 20% peptide-substituted diet than in the two other groups. The better larval performances observed in groups fed diets containing peptides can be related to the enhanced proteolytic capacity of the pancreas and the earlier development of intestinal digestion.

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.

Maturation of the pancreatic and intestinal digestive functions in sea bass (Dicentrarchus labrax): effect of weaning with different protein sources.

The maturation of the digestive functions in sea bass (Dicentrarchus labrax) larvae was evaluated by the enzymatic profile of pancreas and intestine brush border membranes. Sea bass larvae were weaned at day 25 with three simplified diets different by their protein nature: 100% fish meal (FP), 100% casein mixture (CP) and 50% fish meal-50% casein mixture (CFP). The casein mixture contained 35% of hydrolysate. The control group was fed live preys. The specific activity of amylase decreased with age irrespectively of the diets whereas the specific activity of trypsin was enhanced. The casein mixture reduced pancreatic secretion in amylase and trypsin. The CFP group differed from the other groups fed on compound diets, exhibiting as soon as day 32 high activities of brush border enzymes, similar to controls. This sharp increase between day 25 and 32 appeared to be crucial for larval survival. The addition of a protein hydrolysate in a weaning diet seems to facilitate this maturation process.

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.