Effect of diets containing a purified soybean trypsin inhibitor on growth performance, digestive proteases and intestinal histology in juvenile sea bream (Sparus aurata L.)

Juvenile sea bream were fed on diets containing 0.0, 2.0 or 4.0 g/kg of a soybean trypsin inhibitor (SBTI) for 30 days. The growth performance, total protease activity and intestinal histology were studied after 0,15 and 30 days of dietary treatment. No signi¢cant di¡erences were found in the weight gain, speci¢c growth rate (SGR) and feed conversion rate in fish fed on inhibitor-supplemented diets when compared with those fed on an inhibitor-free diet. Only the SGR at day 15 decreased signi¢cantly with protease inhibitor inclusion, although this effect was not observed at day 30. In relation to proteolytic activity at day 15, the total protease activity in the distal intestine decreased in ¢sh fed on inhibitor-supplemented diets. Zymograms of these extracts showed that the SBTI reduced the intensity of some proteolytic fractions in the distal intestine. A noticeable reduction in the protease activity of the intestinal content in fish fed onthe highest level of soybean inhibitor (4.0 g/kg) was also observed. However, at day 30, the inhibition e¡ect on these active bands was not detected, and the total protease activity was similar to that in ¢sh fed on an inhibitor-free diet. Histological examination revealed no perceptible differences in the intestinal structure between any of the diet groups. In addition, all ¢shweremaintained under experimentation for 10 more days and fed on an inhibitor-free diet to determine whether the possible effects caused by the protease inhibitor could be reverted.The administration of SBTI-supplemented diets did not affect sea bream growth performance or intestine histology after 30 days, and only a decrease in the total alkaline protease activity was found at day 15

Sequence analysis and tissue expression pattern of Sparus aurata chymotrypsinogens and trypsinogen

Two apparently full-length cDNA clones encoding chymotrypsinogens I and II (CHTRI, 1022 bp; CHTRII, 909 bp) and one eDNA clone encoding trypsinogen II (TRPII, 848 bp) were isolated from a cDNA library prepared from gilthead sea bream (Sparus aurata) liver. The deduced amino acid sequences of the isolated cDNAs contain highly conserved residues essential for serine protease catalytic activity and conformational maintenance. The deduced amino acid sequences of CHTRI and CHTRII are 261 as and 277 as long, respectively, and share only 61% identity. Sea bream CHTRII appears to be the longest of all known teleostean chymotrypsinogen forms and contains a high number of methionine residues. Compared with CHTRI, CHTRII is more hydrophobic and has a lower isoelectric point. On the other hand, deduced amino acid sequence of TRPII is 241 as long and has a signal peptide of thirteen amino acid residues and an activation peptide of seven amino acids long. In contrast to CHTRI and CHTRII, TRPII has a low isoelectric point (4.95), which makes it anionic at neutral pH. Northern blot analysis revealed that liver is the major transcription site for all zymogens. As expected, all zymogen transcripts were detected in parts of the digestive tract (stomach, pyloric cacca, anterior and posterior intestine) and pyloric caeca presented the most intense expression. In all tissues and amongst all zymogens, TRPII constitutive expression was the highest. (c) 2007 Elsevier Inc. All rights reserved

The response of digestive proteases to abrupt salinity decrease in the euryhaline sparid Sparus aurata L

The response of the digestive proteases to abrupt salinity change was studied in juvenile gilthead sea bream (Sparus aurata) for 15 days after transfer from 33 parts per thousand to 21 parts per thousand. Salinity decrease affected significantly neither the activity of total acid proteases in stomach, nor the activities of total alkaline proteases and major serine proteases - trypsin and chymotrypsin - in the alkaline part of the intestine. The activity of the major proteases was significantly different between the alkaline segments of the intestine, with the posterior intestine presenting the highest activities followed by the pyloric caeca. This distribution pattern remained unaffected by salinity decrease. Notably, salinity change led to significant alterations in elastase and carboxypeptidase activity. The changes were more prominent in the upper part of the intestine (pyloric caeca and anterior intestine) than in the posterior intestine. In pyloric caeca significant alteration of carboxypeptidase A and B activities was observed, elastase changes were confined to anterior intestine together with alterations in carboxypeptidase B activity, while in posterior intestine the changes were restricted to carboxypeptidase A activity. The results are discussed in relation to the osmoregulatory action of the intestinal segments and dietary protein digestion. (C) 2007 Elsevier Inc. All rights reserved