Fig. 1. Trypsin activity in the intestinal contents of Trial I and Trial 2 samples. Trypsin activity in the intestinal contents of 5 different intestinal segments of Atlantic salmon fed either fishmeal (FM) or soybean meal (SBM). A and B represent the trypsin activities of Trial I and Trial II respectively. The segments are: the proximal half of the pyloric intestine (PI1), the distal half of the pyloric intestine (PI2), midintestine (MI), proximal half of the distal intestine (DI2) and the distal half of the distal intestine (DI2). Columns indicate mean trypsin activity of the FM and SBM diet respectively. Vertical lines indicate 95% confidence intervals resulting from the statistical comparison between diets with respect to mean trypsin activity in identical intestinal segment. ^a,bDifferent lowercase letters denote significant differences between the same intestinal segment (P < 0.05). ^A,B,C,DDifferent uppercase letters denote significant differences between the intestinal segments of the FM diet (P < 0.05). ^I,II,IIIDifferent roman letters denote significant differences between the intestinal segments of the SBM diet (P < 0.05).

Fig. 2. Expression of trypsin mRNA in the pancreatic tissue of Trial I samples. Expression of trypsin mRNA normalized to 18S rRNA in pancreatic tissue of Atlantic salmon fed either fishmeal (FM) or soybean meal (SBM). A representative template from a FM fed fish was chosen as the calibrator. Reverse transcriptase (RT) was performed in duplicates for each individual sample and template from each RT reaction was run in duplicates in quantitative PCR. An average normalized-calibrated ratio for each individual was calculated. Columns indicate the mean normalized calibrated ratio for all individuals in the two feeding groups respectively. Bars indicate 95% confidence intervals.

Fig. 3. Multiple tissue expression analysis. Reverse transcriptase PCR (RT PCR) performed on reverse transcribed RNA extracted from 18 different tissues in a control fish. Equal amounts of RNA were used in the PCRs. A) RT PCR using primers amplifying trypsin I, II and III. B) RT PCR using primers amplifying trypsin I and II. C) RT PCR using primers amplifying trypsin III. D) RT PCR using primers amplifying beta-actin. A, B, C and D) Es, esophagus; St, stomach; Pa, pancreas; Pi, pyloric tissue; Mi, midintestine; Di, distal intestine; Li, liver; Hk, head kidney; Ki, kidney; He, heart; Sp, spleen; Th, thymus; Br, brain; Ey, eye; Gi, gills; Go, gonads; Mu, muscle; Sk, skin; Neg, negative control.

Fig. 4. Inhibition of trypsin-like activity with soybean trypsin inhibitors. Detection of trypsin-like activity and Coomasie staining of intestinal wall and brain homogenates. A: Fluorescent in-gel assay using the substrate Boc–Gln–Ala–Arg–AMC copolymerized in the SDS-polyacrylamide gel. B: Protein staining of tissue homogenates using Page Blue^™ Protein Staining Solution after SDS-polyacrylamide gel electrophoresis. Each tissue (midintestine, distal intestine and brain) represents homogenates from the identical sample. (Sample loading is identical (1–8) in A and B). Lanes 1, 3, 5 and 7 represent SDS-polyacrylamide gel electrophoresis of 25 ng bovine trypsin, 10 μg midintestine homogenate, 15 μg distal intestine homogenate and 50 μg brain homogenate respectively. Lanes 2, 4, 6 and 8: identical samples of bovine trypsin, midintestine, distal intestine and brain treated with 1.25 μg soybean trypsin inhibitor (+). Apparent molecular size is indicated by kiloDalton (kDa).

Fig. 5. Trypsin-like activity in tissues of the intestine and brain. Detection of trypsin-like activity and total protein staining of intestinal wall and brain homogenates of fishmeal (FM) and soybean meal (SBM) fed fish. A, C and E: Fluorescent in-gel assay using the substrate Boc–Gln–Ala–Arg–AMC copolymerized in the SDS–polyacrylamide gel on tissue homogenates from midintestine (MI)(A), distal intestine (DI)(C) and brain (E). B and D: Silver staining of tissue homogenates from MI and DI respectively after SDS-polyacrylamide gel electrophoresis. F: Protein staining of tissue homogenates from brain using Page Blue^™ Protein Staining Solution after SDS-polyacrylamide gel electrophoresis. Lanes 1–4 represent tissue homogenates of 4 different individuals fed FM. Lanes 4–8 represent tissue homogenates of 4 different individuals fed SBM. Identical samples of the same tissue are represented by the same lane number. All samples from the same tissue are loaded in equal amounts (0.5 μg for MI and DI and 50 μg for brain). Apparent molecular size is indicated by kiloDalton (kDa).

Fig. 6. Histological sections of the distal intestine of Trial II samples. Sections showing representative details of simple mucosal folds of the distal intestine. (A) Fish fed fishmeal (FM): regular, vacuolated enterocytes (asterisk) and basally located nuclei (arrowhead) are seen. A sparse lamina propria (lp), intraepithelial lymphocytes (arrow) and a few goblet cells (g) are also seen. (B) Fish fed soybean meal (SBM): loss of nuclei regularity (arrowhead) and loss of vacuolization of the enterocytes are seen. The lamina propria (lp) is heavily infiltrated with a mixed population of inflammatory cells. (H and E. Bars = 500 μm).

Formulation, proximate analysis and trypsin inhibitor activity (TIA) of the experimental diets

Oligonucleotide primers used in PCR applications, annealing temperature and product size