Evaluation of the Inclusion of the Green Seaweed Ulva ohnoi as an Ingredient in Feeds for Gilthead Sea Bream (Sparus aurata) and European Sea Bass (Dicentrarchus labrax).

This study evaluated the use of Ulva ohnoi as an ingredient in feeds for aquaculture in three different experiments. Experiment 1 was oriented to confirm the negative effect of U. ohnoi on fish digestion. Experiment 2 assessed the effect on growth, feed efficiency, and immune status of juvenile sea bass (Dicentrarchus labrax) fed on diets including U. ohnoi, previously treated or not with carbohydrases used to partially hydrolyze indigestible polysaccharides. Experiment 3 was aimed to evaluate the potential protective effect of U. ohnoi on the oxidative status of sea bream (Sparus aurata) challenged by the consumption of a feed formulated with the oil fraction completely oxidized. Results show a negligible effect of U. ohnoi meal on protein digestion when included in feeds at levels of 10% or less. Moreover, results of growth and feed use evidenced the possibility of using up to 5% inclusion of algal meal in feeds without adverse effects on the zootechnical parameters, while the enzyme pretreatment was ineffective to improve its nutritional use. Finally, the inclusion of U. onhoi in feeds determined both an immunostimulatory effect, evidenced by an increase in skin mucus lysozyme in the two mentioned fish species, and a positive influence on the oxidative metabolism of seabream when fed on a diet including rancid oil.

Seasonal changes in kinetic parameters of trypsin in gastric and agastric fish.

The objective of the present study was to assess if trypsin, a key enzyme involved in protein digestion, presents some kind of functional adaptations to seasonal changes in water temperature in freshwater fish. In order to test this hypothesis, individuals of two fish species Carassius gibelio (agastric) and Perca fluviatilis (gastric) were sampled in the basin of Chany Lake (Siberia, Russia) at two different seasons (spring and summer). Apparent kinetic parameters (Km and Vmax) were determined for both species and seasons at the actual pH values in fish guts, and at actual temperatures. Results showed a significant effect of both the species and sampling season on the apparent kinetic parameters of trypsin. In the case of Prussian carp, Km and Vmax were lower for each assayed temperature (for 5 and 15 °C the differences were significant) for fish sampled in summer when compared to those sampled in spring. In contrast, values of Km in perch tended to be lower in spring at 5 and 25 °C but these differences were not significant, while Vmax showed a significant decrease in summer samples. This suggests a sort of functional adaptation of the same trypsin enzymes to seasonal changes, oriented to maximize protein digestion under variable conditions.

Daily rhythms of intestinal cholecystokinin and pancreatic proteases activity in Senegalese sole juveniles with diurnal and nocturnal feeding.

The influence of diurnal and nocturnal feeding on daily rhythms of gut levels of cholecystokinin (CCK) and the activity of two key pancreatic proteases, trypsin and chymotrypsin, were examined in juveniles of Senegalese sole (Solea senegalensis), a species with nocturnal habits. Four feeding protocols were performed: P1) One morning meal; P2) Six meals during the light period; P3) Six meals during the dark period; and P4) 12 meals during 24 h. Daily activity patterns of both proteases were remarkably similar and showed a high correlation in all the experimental protocols. In P1, daily patterns of CCK and digestive enzymes showed a single maximum. In P2, CCK levels exhibited two peaks. Digestive enzymes activities showed slightly delayed peaks compared to CCK, although their daily fluctuations were not significant. In P3, intestinal CCK concentration exhibited two peaks at the end of light and dark periods, but only the second one was significant. The first maximum level of chymotrypsin activity occurred 4 h after the first CCK peak, while the second one coincided with the second CCK peak. Fluctuations of trypsin activity were not significant. In P4, CCK concentration showed three small peaks. Digestive enzymes daily fluctuations were not significant, although they showed an inverted trend with respect to CCK. The daily pattern of the gut CCK content in our study is in agreement with the anorexigenic function of this hormone. Our results support the existence of a negative feedback regulatory loop between CCK and pancreatic proteolytic enzymes in Senegalese sole juveniles.

Ontogeny of Expression and Activity of Digestive Enzymes and Establishment of gh/igf1 Axis in the Omnivorous Fish Chelon labrosus.

Thick-lipped grey mullet (Chelon labrosus) is a candidate for sustainable aquaculture due to its omnivorous/detritivorous feeding habit. This work aimed to evaluate its digestive and growth potentials from larval to early juvenile stages. To attain these objectives the activity of key digestive enzymes was measured from three until 90 days post hatch (dph). Expression of genes involved in digestion of proteins (try2, ctr, pga2, and atp4a), carbohydrates (amy2a), and lipids (cel and pla2g1b), together with two somatotropic factors (gh and igf1) were also quantified. No chymotrypsin or pepsin activities were detected. While specific activity of trypsin and lipase were high during the first 30 dph and declined afterward, amylase activity was low until 57 dph and increased significantly beyond that point. Expression of try2, ctr, amy2a, and cel increased continuously along development, and showed a peak at the end of metamorphosis. Expression of pla2g1b, pga2 and atp4a increased until the middle of metamorphosis and decreased afterwars. Most of these trends contrast the usual patterns in carnivorous species and highlight the transition from larvae, with high protein requirements, to post-larvae/juvenile stages, with omnivorous/detritivorous feeding preferences. Somatotropic genes, gh and igf1, showed approximately inverse expression patterns, suggesting the establishment of the Gh/Igf1 axis from 50 dph.

Modelling digestive hydrolysis of nutrients in fish using factorial designs and desirability function.

Models simulating the in vitro digestive hydrolysis of nutrients by different animal species are frequently used to obtain a better understanding of factors affecting this process. Optimization algorithm of a model may be used to prospect the more favourable combination of selected factors resulting in the higher performance. This study was conducted to determine the combination of factors (pH, enzyme:substrate ratio, and reaction time) leading to highest bioavailability of proteins and carbohydrates in the gilthead seabream gastrointestinal tract. Besides, a novel multi-objective algorithm, desirability function, was introduced for optimization of the digestive hydrolysis of nutrients within the simulated gut of the species, using models based on the Response Surface Methodology. Design of experiment was defined based on the physiology and culture conditions of the species, and in vitro assays were performed in a two-phase (stomach ad intestine) digestion process, using the species-specific enzyme extract. According to results, intestinal phase of digestion makes the major contribution to the total protein hydrolysis, being the efficiency of the process directly correlated to all the three studied factors. In contrast, the efficiency of carbohydrate hydrolysis was directly correlated to the amount of substrate and inversely to the pH, while reaction time did not exert a significant effect. The physiological range of the factors studied in the assays favoured the hydrolysis of proteins over carbohydrates, a similar scenario to that observed in the live fish. Results from the mathematical models and their simultaneous optimization obtained from this work may have practical applications in design of feeds for this species.

Acidic digestion in a teleost: postprandial and circadian pattern of gastric pH, pepsin activity, and pepsinogen and proton pump mRNAs expression.

Two different modes for regulation of stomach acid secretion have been described in vertebrates. Some species exhibit a continuous acid secretion maintaining a low gastric pH during fasting. Others, as some teleosts, maintain a neutral gastric pH during fasting while the hydrochloric acid is released only after the ingestion of a meal. Those different patterns seem to be closely related to specific feeding habits. However, our recent observations suggest that this acidification pattern could be modified by changes in daily feeding frequency and time schedule. The aim of this study was to advance in understanding the regulation mechanisms of stomach digestion and pattern of acid secretion in teleost fish. We have examined the postprandial pattern of gastric pH, pepsin activity, and mRNA expression for pepsinogen and proton pump in white seabream juveniles maintained under a light/dark 12/12 hours cycle and receiving only one morning meal. The pepsin activity was analyzed according to the standard protocol buffering at pH 2 and using the actual pH measured in the stomach. The results show how the enzyme precursor is permanently available while the hydrochloric acid, which activates the zymogen fraction, is secreted just after the ingestion of food. Results also reveal that analytical protocol at pH 2 notably overestimates true pepsin activity in fish stomach. The expression of the mRNA encoding pepsinogen and proton pump exhibited almost parallel patterns, with notable increases during the darkness period and sharp decreases just before the morning meal. These results indicate that white seabream uses the resting hours for recovering the mRNA stock that will be quickly used during the feeding process. Our data clearly shows that both daily illumination pattern and feeding time are involved at different level in the regulation of the secretion of digestive juices.

Gut pH as a limiting factor for digestive proteolysis in cultured juveniles of the gilthead sea bream (Sparus aurata).

After the development of the gastric function in juvenile fish, dietary proteins enter a two-phase digestive process comprising an acidic gastric phase followed by an alkaline intestinal phase. However, the main gastric protease, pepsin, is strictly dependent on the existence of a low-enough environmental pH. In 20-g gilthead sea bream, Sparus aurata, the mean minimal gastric pH is close to 4.5, while the mean pH in the duodenal portion of the intestine was nearly fixed at 6.5. The mean maximal gastric content of HCl was approximately 20 microEq for a low-buffering diet. Gastric proteases were more severely affected than intestinal proteases when assayed at actual sub-optimal pH values, 4.5 and 6.5, respectively. When the gastric proteases of juvenile fish were pre-incubated with a citric acid buffer at pH 6.0, the activity at pH 4.5 was very low, whereas when they were pre-incubated with the same buffer at pH 3.0, the activity at pH 4.5 was significantly increased; this fact suggests a deficient activation of zymogens during the gastric digestion and points to a potential approach to improve protein digestion in juvenile gilthead sea bream.

New members of the brachyurins family in lobster include a trypsin-like enzyme with amino acid substitutions in the substrate-binding pocket.

Crustacean serine proteases (Brachyurins, EC 3.4.21.32) exhibit a wide variety of primary specificities and no member of this family has been reported for spiny lobsters. The aim of this work was to study the diversity of trypsins in the digestive gland of Panulirus argus. Several trypsin-like proteases were cloned and the results suggest that at least three gene families encode trypsins in the lobster. Three-dimensional comparative models of each trypsin anticipated differences in the interaction of these enzymes with proteinaceous substrates and inhibitors. Most of the studied enzymes were typical trypsins, but one could not be allocated to any of the brachyurins groups due to amino acid substitutions found in the vicinity of the active site. Among other changes in this form of the enzyme, conserved Gly216 and Gly226 (chymotrypsin numbering) are substituted by Leu and Pro, respectively, while retaining all other key residues for trypsin specificity. These substitutions may impair the access of bulky residues to the S1 site while they make the pocket more hydrophobic. The physiological role of this form of the enzyme could be relevant as it was found to be highly expressed in lobster. Further studies on the specificity and structure of this variant must be performed to locate it within the brachyurins family. It is suggested that specificity within this family of enzymes is broader than is currently believed.

Development of a novel casein-protamine based microparticles for early feeding of fish larvae: in vitro evaluation.

OBJECTIVE: The objective of this study was to develop novel type of protein walled microparticles suitable for using in early feeding of fish larvae. METHODS: The microparticles were made of casein and protamine through complex coacervation and did not require further cross-linking or use of environmentally problematic reagents. The methodology was oriented to generate microparticles with an appropriate size range for easy recognition and ingestion by fish larvae (50-200 microm), adequate floating properties in saline, sufficient stability in terms of protein leakage and appropriate digestibility by the gut enzymes of fish larvae. RESULTS: Desired particle size and stability against protein leakages over 8 h were successfully achieved by optimizing the coacervation process conditions. The floating properties under static conditions were considered appropriate as a main particle fraction remained in suspension during at least 10 min. Very importantly, an enzyme extract from larval gut readily digested the particles. The digestibility of the casein-protamine particles was similar to that measured for Artemia nauplii and for two previously developed casein-based microparticles produced by interfacial polymerization and ionic gelation; the latter microparticle type had previously achieved good results of digestibility in early feeding of marine fish larvae. CONCLUSION: The in vitro evaluation of the newly developed casein-protamine microparticles revealed promising characteristics as artificial larval feed. Thus, these particles merit further development with respect to entrapping nutrients and testing them in larval cultures for their nutritional value.

Effects of high-molecular-mass substrates on protein migration during sodium dodecyl sulfatepolyacrylamide gel electrophoresis.

Substrate-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) has become a popular procedure for the separation and identification of active fractions present in enzyme mixtures due to its relative simplicity. Procedures including high-molecular-mass substrates within the gel, such as starch for identification of amylase activity, and protein substrates, including gelatin, casein, and collagen, for revealing protease activity, have been described. SDS-PAGE separation under denaturing conditions is dependent on the molecular mass of the proteins and on the effective pore size of the gels, the last factor being affected by the inclusion of high-molecular-mass substrates into the polyacrylamide matrix. In order to quantify the effect of the addition of increasing concentrations of such substrates on protein migration, starch, gelatin, and casein were included in gels in which polyacrylamide concentration was kept constant. High-molecular-mass substrates decreased migration of proteins ranging from 6.5 to 205 kDa, although the migration pattern, and thereby the accuracy of the assignation of relative molecular masses to proteins separated on those gels, was practically unaffected. The substitution of glycine, as the carrying ion, by Tricine in denaturing electrophoresis buffer systems resulted in an improvement of the migration of proteins in substrate-containing gels. Results suggested that zymograms including substrates remain a valuable procedure for the separation and the relative molecular mass assignation of active enzyme fractions.