Chantal Cahu

Substitution of live food by formulated diets in marine fish larvae

Until recently, it was considered impossible to feed newly hatched marine fish species with a compound diet. Substituting a compound diet for live prey was performed several weeks after hatching, depending on the species. Compound diets were well ingested at the early stage but larvae died with a gut full of food, suggesting that larvae were unable to digest the compound diet. The hypothesis was that younger larvae have insufficient digestive enzymes to thrive on compound diets, and that exogenous enzymes, provided from live prey, are necessary for early stages. The organogenesis of marine fish larvae is not completely achieved at hatching and histological studies have revealed that the anatomy of the digestive tract undergoes developmental changes over some weeks. Nevertheless, biochemical studies over 20 years have shown that most of the digestive enzymes are present in young larvae. Recent studies have provided better understanding of digestion mechanisms in larvae and have led to proposed dietary compositions meeting larvae nutritional requirements. Pancreatic digestive enzymes are detected before mouth opening. Their synthesis is not induced by diet ingestion, but secretory mechanisms in the pancreas, and so enzymatic action, become efficient chronologically after those of synthesis. Inadequate diets can delay the onset of secretion mechanisms. The ratio of secreted enzymes to total enzymes indicates the nutritional value of the diet ingested by the larvae. At the intestinal level, cytosolic enzymes, which are peptidases, exhibit high activity in the early stages, suggesting a high capacity in larvae to digest protein hydrolysate. Indeed, larvae growth and survival is improved by the incorporation of a moderate concentration of peptide or hydrolysate in the diet. Peptidase activity abruptly decreases around day 25 in sea bass, concurrent with an increase in enzymes of the brush border membranes. This corresponds to a normal maturation process of enterocytes. Compound diets can slightly delay the onset of this maturation process, and inadequate diet can prevent it, leading to near death of the larvae. A proper onset of the maturation process has been associated with high larvae survival. The early developmental stage larvae exhibit high hydrolytic capacity, related to their weight. Enzyme activity pattern is age-dependent, but can be modulated by diet composition. Thus, larvae have the ability to digest and thrive on compound diet, if this diet is well adapted. Larvae have different specificities in digestion and nutritional requirements when compared to juveniles. Taking these specificities into consideration, recent research has led to the formulation of a compound diet that was well adapted for larvae from mouth opening, and could totally replace live prey.

Nutritional components affecting skeletal development in fish larvae

Marine fish larvae undergo major functional and morphological changes during the developmental stages and several factors can interfere with the normal development of larvae and affect fry quality. Skeletal malformations, such as spinal malformation—scoliosis, lordosis, coiled vertebral column-, missing or additional fin rays, bending opercle or jaw malformations, are frequently observed in hatchery-reared larvae. This paper reviews the effects of some nutritional components on skeletal development in larvae of a number of fish species. In the dietary lipid fraction, for instance, it was proven that the phospholipid concentration affected the spinal malformation rate in sea bass fed a compound diet from mouth opening onwards. Phosphatidylinositol, in particular, seems to prevent skeletal deformities. Highly unsaturated fatty acids, and particularly DHA enrichment in live prey, induce a decrease of opercular deformities in milkfish. It is known that highly unsaturated fatty acids have profound effects on gene expression, leading to changes in metabolism, growth and cell differentiation, and these effects are worth investigating in developing fish. The nature of the dietary protein fraction also affects the quality of fish larvae development. It appears that dietary incorporation of 20 amino acid peptides or di- and tripeptides leads to a reduction of spinal malformations in sea bass. Among vitamins, the teratogenic effect of retinoic acid is now well documented in vertebrates. High dietary retinoic acid levels result in higher incidence of bone deformities, such as vertebral curvature, central fusion and compression of vertebra in Japanese flounder larvae. The teratogenic effect of retinoic acid observed in embryonic and postembryonic stages was explained by a depression of shh expression. As for vitamin C, opercular abnormalities in milkfish larvae, associated with distortion of gill filament cartilages, were reduced by 50% when feeding larvae with ascorbic acid enriched rotifers and Artemia, compared to control fish.

Development of digestive enzymes in larvae of Solea senegalensis, Kaup 1858

Abstract The activities of some digestive enzymes were studied in sole larvae fed live prey from first feeding until the first month of life. Whole larvae body homogenates were used for enzymatic determination in larvae younger than 21 days after hatching (DAH). Older larvae were dissected in order to obtain the pancreatic and intestinal segment. Brush border membranes of enterocytes were purified from intestinal segment. From 2 DAH to 18 DAH, activities of pancreatic and intestinal enzymes exhibited a pattern characteristic of developing animals: an increase during the first 10 days post-hatching, followed by a decrease. From 21 DAH to 27 DAH, the strong increase in alkaline phosphatase activity reflected the development of the brush border membranes of enterocytes, which occurred concurrently with a decrease in a cytosolic enzyme, leucine–alanine peptidase. These opposite patterns indicate a maturation of enterocytes and the acquisition of an adult mode of digestion.

Early weaning of sea bass (Dicentrarchus labrax) larvae with a compound diet: Effect on digestive enzymes

Abstract Sea bass (Dicentrarchus labrax) larvae were weaned with a microparticulated diet at various times after hatching: 10, 15, 20 and 25 days; a control group was fed live prey (Artemia salina). The earlier the weaning, the lower the larval growth obtained. The amylase activity in the pancreatic segment increased swiftly after the weaning in all groups. This increase was the result of an extensive synthesis induced by the starch content (12%) of the compound diet. The enhancement of specific activities of intestinal peptidases, leucine aminopeptidase and γ-glutamyl transpeptidase after weaning was the result of compensatory adaptation, as was described in the case of malnutrition. The depressed activities of alkaline phosphatase, in the brush border membrane fraction, indicated malnutrition in weaned groups. Weaning before day 20 stops or delays larval development, particularly maturation of some digestive processes in larvae, such as the onset of pancreas secretory functions.

Effect of dietary phospholipid level and phospholipid:neutral lipid value on the development of sea bass ( Dicentrarchus labrax ) larvae fed a compound diet

The aim of the study was to determine the influence of dietary phospholipid concentration on survival and development in sea bass (Dicentrarchus labrax) larvae. Larvae were fed from day 9 to day 40 post-hatch with an isoproteic and isolipidic formulated diet with graded phospholipid levels from 27 to 116 g/kg DM and different phospholipid:neutral lipid values. The best growth (32 mg at the end of the experiment) survival (73 %) and larval quality (only 2 % of malformed larvae) were obtained in the larvae fed the diet containing 116 g phospholipid/kg DM (P<0.05). These results were related to the amount of phosphatidylcholine and phosphatidylinositol included in this diet (35 and 16 g/kg respectively). Amylase, alkaline phosphatase and aminopeptidase N activities revealed a proper maturation of the digestive tract in the two groups fed the highest phospholipid levels. Regulation of lipase and phospholipase A2 by the relative amount of their substrate in the diet occurred mainly at the transcriptional level. The response of pancreatic lipase to dietary neutral lipid was not linear. As in mammals 200 g triacylglycerol/kg diet seems to represent a threshold level above which the response of pancreatic lipase is maximal. The response of phospholipase A2 to dietary phospholipid content was gradual and showed a great modulation range in expression. Sea bass larvae have more efficient capacity to utilize dietary phospholipid than neutral lipids. For the first time a compound diet sustaining good growth, survival and skeletal development has been formulated and can be used in total replacement of live prey in the feeding sequence of marine fish larvae.

Effect of live yeast incorporation in compound diet on digestive enzyme activity in sea bass (Dicentrarchus labrax) larvae

Yeasts produce polyamines, and some strains have a strong adhesion potential to intestinal mucus, an important condition for probiotic efficiency. The aim of this study was to explore an in situ production of polyamines by Debaryomyces hansenii HF1 (DH), a yeast strain isolated from fish gut, in comparison with Saccharomyces cerevisiae X2180 (SC) (Goteborg University Collection). The production of polyamines by DH was three times higher than that of SC. The main polyamines were spermine and spermidine, produced at a similar level. Both strains adhered to the gut of sea bass larvae. When the yeasts were introduced into a compound diet, the colonization was effective in the larvae (104 CFU g−1 on a body weight basis). The DH diet led to an increase in amylase secretion in 27-day-old larvae in comparison with the control diet. The secretion of amylase and trypsin was lower in the SC diet, and some delay in trypsin secretion was still observed in this group at day 42. At day 27, the activity of brush border membrane enzymes was stimulated by the DH diet, and delayed by the SC diet, in comparison with the control diet. The survival of the larvae was also increased in the DH diet, but the growth rate was lower than that in the control group. This may be due to the introduction of live yeast into the diet, which needs to be optimised.

Protein hydrolysate vs. fish meal in compound diets for 10-day old sea bass Dicentrarchus labrax larvae

Abstract Four groups of Dicentrarchus labrax larvae were fed from day 10 post-hatching to day 41 with four isoenergetic and isonitrogenous (60%) compound diets differing only by the incorporation level of a commercial fish protein hydrolysate, CPSP G. In diet H0, the protein fraction was fish meal. In diets H19, H38 and H58, 25, 50 and 75% of the fish meal was replaced by CPSP, respectively. The four compound diets sustained larval growth throughout the experiment. The highest survival was obtained in the group fed H19 (47±5.0%). Final weights of larvae fed H0 and H19 diets (5.5±2.61 and 5.7±2.77 mg, respectively) were significantly higher than those of larvae fed diets H38 and H58 (3.3±1.03 and 2.6±0.70 mg, respectively). At day 41, the highest trypsin secretion levels were obtained in groups fed diets H0 and H19, suggesting a proper maturation of pancreatic digestive function. The incorporation of 19 and 38% hydrolysate in diets induced a high level of two membranous enzymes of intestine, alkaline phosphatase (AP) and aminopeptidase N (lap) as early as day 20. The cytosolic enzyme leucine–alanine peptidase (leu–ala) was also assayed. The ratios of AP/leu–ala and lap/leu–ala revealed that the development of the intestine was more advanced in the H19 fed group than in the others. The proper onset of intestinal digestive function was associated with good larvae survival. The experiment showed that the incorporation of a moderate dietary level of fish protein hydrolysate facilitates the onset of the adult mode of digestion in developing fish.

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.

Dietary regulation of activities and mRNA levels of trypsin and amylase in sea bass (Dicentrarchus labrax) larvae

Specific activities and mRNA levels of trypsin and amylase were studied in sea bass larvae. From day 20 to day 40, Dicentrarchus labrax were fed two rations of one day old Artemia: satiation (LP) and one-eighth of the satiation ration (LP/8) or two isoenergetic compound diets that varied in protein (30 and 60%) and carbohydrate (37 and 7%) content (FP30 and FP60 respectively). Trypsin mRNA levels and specific activities were mainly influenced by the nature of dietary protein and the Artemia ration. By using fish meal as protein source, dietary protein concentration did not affect either mRNA level nor specific activity of trypsin. These results suggested that the trypsin synthesis was not affected at a transcriptional level by the protein ration, i.e., Artemia ration. Decrease in amylase mRNA observed from day 29 in the four dietary groups suggested that this decrease in amylase expression is genetically programmed during sea bass larvae development. Nevertheless, the composition and the quantity of the diet influenced the amylase specific activities revealing primarily translational regulation of amylase. This study shows for the first time that the molecular mechanisms which control the dietary adaptation of trypsin and amylase are independently regulated, age-dependent and influenced by the composition and the quantity of the diet.

Algal addition in sea bass (Dicentrarchus labrax) larvae rearing: effect on digestive enzymes

Abstract Two groups of Dicentrarchus labrax were reared from day 5 to day 20 post-hatching with live prey in running seawater with or without algae. The concentration of algae Isochryisis galbana was maintained between 20 000 to 40 000 cells ml −1 . On day 20, each group was divided into two subgroups, one still receiving live prey, the other fed a compound diet until day 32. On day 32, larvae fed live prey in the presence of algae exhibited an 18% higher survival than larvae reared without algae; in group fed compound diet, the algae supply induced a 26% survival enhancement. At day 16, it was observed a 40% weight improvement in the group reared in the presence of algae compared to the group reared without algae. At day 32, a slight but significant positive effect of algae on larvae weight was still noted in the group fed compound diet. From day 8 to day 16, the presence of algae in the rearing water resulted in an increase in trypsin activity whereas amylase and chymotrypsin were not affected. At day 26, alkaline phosphatase and maltase assayed in purified brush border membranes of intestine were significantly higher in larvae reared in seawater with algae than in those reared in clear water. This result suggests that the presence of algae facilitates the onset of hydrolytic functions of cell membranes. The significant improvement in survival of larvae reared in the presence of algae was related to the early development of brush border membranes. It is concluded that the algae acts by triggering digestive enzyme production, at both the pancreatic and intestinal level.