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

Abstract

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.

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1
Comparative Biochemistry and Physiology -
Part A: Molecular & Integrative Physiology
April 2011, Volume 158, Issue 4, Pages 525-530
http://dx.doi.org/10.1016/j.cbpa.2010.12.017
Copyright © 2011 Elsevier Inc. All rights reserved.
Archimer
http://archimer.ifremer.fr
Dietary protein hydrolysate and trypsin inhibitor effects on digestive
capacities and performances during early-stages of spotted wolffish:
Suggested mechanisms
A. Savoiea , N.R. Le Françoisa, b, *, S.G. Lamarrea, c, P.U. Bliera , L. Beaulieua, d and C. Cahue
a Département de biologie, Université du Québec à Rimouski, Rimouski, QC, Canada G5L 3A1
b Biodôme de Montréal, 4777, Ave Pierre-De Coubertin, Montréal, QC H1V 1B3
c Memorial University of Newfoundland, St-John, NL, A1C 5S7
d Institute of Nutraceuticals and Functional Food (INAF), Université Laval, Québec, Québec, G1V 0A6, Canada
e Ifremer, Nutrition, Aquaculture & Genomic Research Unit, F-29280 Plouzane, France
*: Corresponding author : N.R. Le François, Tel.: + 1 514 868 3072.,
email address : NLe_Francois@ville.montreal.qc.ca , Nathalie_Le-Francois@uqar.qc.ca
Abstract:
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.
Keywords : Spotted wolffish; Anarhichas minor; Early-stages; Trypsin inhibitor; Protein hydrolysate;
Digestive capacity; SBTI

Keywords:... 35
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Introduction
Along with the dynamic and complex processes of organ differentiation and morphogenesis,
the newly-hatched fish experience the highest growth rate that they will ever achieve in their
entire lifetime (Gisbert and Williot, 2002; Sala et al. 2005). Growth rate is mainly a function
of protein synthesis and deposition and is the net income of behavioural and physiological
processes that begins with food intake (Lamarre et al. 2010). Accordingly, and especially
during the early stages, rapid growth is reliant upon adequate provision of amino acids (aa). It
has been suggested that protein digestion and amino acids assimilation could set a limit on
growth rate in juvenile fish (Blier et al. 1997). Not surprisingly, trypsin activity, a key-
enzyme of protein digestion, is correlated to growth rate and survival in various fish species
(Sharma and Chakrabarti, 1999; Lemieux et al., 1999) including wolffishes (Lamarre et al.,
2004; 2007). Growth performances are thus clearly limited by the efficiency of the digestive
system to provide amino acids for protein synthesis and energy production.
Protein is usually the main component of fish feed and fish rely on proteases for their
digestion. Many studies have shown that the replacement of a fraction of protein content by
partially hydrolyzed (pre-digested) proteins could enhance performances of larval fish. If
digestive capacity is a limiting factor at first-feeding, offering more digestible dietary proteins
should improve both growth and survival, since proteins are absorbed mainly as smaller
peptides or single amino acids (Silk et al., 1985; Rust, 1995; Ronnestad et al., 2001;
Kotzamanis et al. 2007).
Positive effects of dietary protein hydrolysate supplementation on growth and survival have
only been reported on larval stages of fish when the digestive system is actively developping.
Medium level of inclusion of protein hydrolysate enhanced survival and/or growth in larval
stages of seabass (Dicentrarchus labrax, Zambonino Infante et al. 1997; Cahu et al. 1999),
common carp (Cyprinus carpio, Carvalho et al., 1997) and barramundi (Lates calcarifer,
Nankervis and Southgate, 2009). On the other hand, no or negative effects of PH have been
reported on juvenile fish such as rainbow trout (Oncorhynchus mykiss, Stone et al., 1989) and
juvenile turbot (Scophthalmus maximus, Oliva-Teles et al., 1999). Most marine fish larvae
hatch with a rudimentary digestive system and undergo a metamorphosis prior to the onset of
exogenous feeding. Newly-hatched larvae generally display low activity of digestive enzymes

(Kolkovski, 2001) and many species do not even present a functional stomach (Govoni et al.,
1986). On the other hand, spotted wolfish (Anarhichas minor, Olafsen) is a particular case
because it displays external morphology and internal organs that are not typical of larval fish
(Falk-Petersen and Hansen, 2001). This species hatches well developed at a relatively large
size (20-24 mm), displays a fully functional digestive system, fairly high trypsin activity
(Desrosiers et al., 2008; Savoie et al., 2008) and readily accept exogenous food (Falk-Petersen
& Hansen 2001; Lamarre et al. 2004; 2007). Accordingly, the distinction betwen larval and
juvenile stages is rather arbitrary in fish (Paine and Balon 1984; Flegler-Balon 1989, Copp
and Kovac, 1996) and wolffish hatch with very few remaining larval characteristics (Falk-
Petersen and Hansen, 2001). In this experiment, fish up to 15 days post-hatching (DPH) were
considered as newly-hatched fish and after 30 DPH as true juveniles. This bottow-dwelling
marine fish has been identified as a threatened species in Canadian coastal waters (Kulka et
al., 2007), a species of concern in the USA (AWBRT, 2009) and is the object of directed
research and development efforts that include aquaculture interests as well as population
conservation concerns (Le François et al. 2002; 2010). Hence, a better understanding of the
early-life processes governing survival and growth of spotted wolffish is warranted.
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Trypsin (TRYP) and chymotrypsin (CHY) are proteolytic enzymes secreted by the pancreas
and liberated in the digestive tract as inactive precursors, respectively trypsinogen and
chymotrypsinogen, activated once in the digestive tract. They are both endopeptidases that
break peptide bonds of specific non-terminal amino acids within the protein. Aspartate amino
transferase (AST) is indicative of the capacity to oxidize amino acids for energy production or
of transamination for protein synthesis.
The goal of this study was to verify whether the scope for growth is dictated by the proteolytic
capacity by examining the effect of protein hydrolysates (PH) and trypsin inhibitor dietary
inclusion on protein digestion/assimilation capacities, growth and survival. The inhibitor used
in this study was soybean trypsin inhibitor (SBTI), which inhibits trypsin and to a lesser
extent, chymotrypsin. We suggest that the capacity to digest proteins sets a limit to the
expression of growth potential in first-feeding wolfish. The first prediction that we formulate
to test this hypothesis is that growth will be enhanced by the presence of PH in the feed but
hindered by the addition of a trypsin inhibitor. The second prediction is that the decrease in
growth rate induced by the inhibitor of trypsin will be counterbalanced by the inclusion of
PH.

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Material and method
Experimental animals and rearing conditions
The study was carried out at the facilities of the Centre Aquacole Marin de Grande-Rivière,
Québec, Canada. Fertilized spotted wolffish eggs originating from captive female wild
broodstock were incubated as described in Savoie et al. (2006). Hatching began on February
28th 2007 and entire hatching was stimulated on March 13th by gentle mixing of the eggs.
Day 0 of the experiment was considered to be March 14th. Newly hatched fish (mean weight
0.103 ±0.01g and mean length 24.5 ±1.4 mm) were randomly placed in each of the 12 low-
level (2 cm) rearing units containing 1.5 litres with a water supply of 1 litre/minute and
individual aeration (n=50 per unit). Feeding of all experimental groups was initiated on
March 14th. Mean salinity during the sixty days of the experiment was of 31.3 ± 0.1, oxygen
saturation was always above 85% (mean 86.8 ± 2.2%) and a 12/12-h light/dark cycle was
adopted. Once a day, mortality was recorded, dead fish were removed and rearing units
carefully cleaned.
Nutrition
The different treatments (diets) were randomly assigned to twelve rearing units (4 diets in
triplicate). Fish were fed by hand each hour from 8am to 5pm for the entire experimental
period. As suggested by Savoie et al. (2006), no live prey was distributed to avoid possible
interference with negative or positive effects of experimental diets on survival rates. The
composition of the four diets and processing details (previously described in Savoie et al.
2006) are provided in Table 1. The compound diets were formulated and processed at
Ifremer, Centre de Brest (France). It was formulated in order to be isonitrogenous. The four
experimental diets contained exactly the same ingredients except for hydrolysates and SBTI
content. In the diets “C” and “I”, the protein fraction was fish meal. In the diets “H” and
“HI”, the fish meal was replaced by 20% of PH. Two pellet sizes were used: 400-800 and
800-1200 μm according to the fish length and according to the feeding tables adopted in
Savoie et al. (2006).
Sampling
Twenty-five fish were sampled at day 0 and four fish per tank were sampled at day 15, 30 and
60 (total: 205 fish). Fish were fasted for 18 hours before sampling. Individual weight (g) and
length (cm) were noted and the fish were quickly frozen at –80°C until analysis. A

supplementary 11 fish were weighed (g) and measured (mm) at each sampling date in order to
evaluate growth and calculate productivity of the different experimental groups. Productivity
(total biomass produced/tank) was calculated as follow: mean final fish weight X remaining
individuals in a tank (Lopes et al., 2001).
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Table 1. Composition of the experimental diets for spotted wolffish
Ingredients1 (in %) C I H HI
Fish meal
HPC 90
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-
74
-
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20
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20
Precooked Potatoe starch
Cod liver oil
Soy lecithin
Vitamin Mixture2
Mineral Mixture3
Betaine
SBTI
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3
5
8
4
1
5
3
5
8
4
1
750 mg/kg
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3
5
8
4
1
5
3
5
8
4
1
750 mg/kg
1Dietary ingredients were commercially obtained. Fish meal, and cod liver oil were from
La Lorientaise (Lorient, France). The soy lecithin was from Ets Louis François (St Maur
des Fossés, France). The potato precooked starch (Nutralys) was from Roquette (Lille,
France). HPC 90 was from Ocean NutraSciences (Matane, Québec, Canada), SBTI
(Trypsin soybean inhibitor) was from Sigma (T-9128).
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2Per kg of vitamin mix: retinyl acetate 1 g; cholecalciferol 2.5 mg; all-rac- α-tocopherol
acetate 10 g; menadione 1 g; thiamin 1 g; riboflavine 0.4 g; D- calcium pantothenate 2 g;
pyridoxine HCl 0.3 g; cyanocobalamin 1 g; niacin 1 g; choline chloride 200 g; ascorbic
acid 20 g; folic acid 0.1 g; biotine 1 g; meso-inositol 30 g.
3Per kg of mineral mix: KCl 90 g; KI 40 mg; CaHPO4.2H2O 500 g; NaCl 40 g;
CuSO4.5H2O 3 g; ZnSO4.7H2O 4 g; CoSO4.7H2O 20 mg; FeSO4.7H2O 20 g; MnSO4.H2O 3
g; CaCO3 215 g; MgSO4.7H2O 124 g; NaF 1 g.

Enzymatic assays 156
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Whole individuals were thawed on ice and homogenised in 9 volumes of Tris-HCl buffer
(100mM, pH 7.5) using a Ultra Turrax T25 (IKA Labortechnik) electrical homogeniser for
three 10-s periods. Between each period, samples were kept on ice for 1 min. Homogenate
was centrifuged at 7000 g for 1 minute prior to the enzymatic assays. The different enzyme
activities were measured using a Lambda 11 UV/VIS spectrophotometer equipped with a
thermostated cell holder (Perkin Helmer inc.). Conditions for enzyme assays were as follows:
Aspartate aminotransferase (AST, E.C. 2.6.1.1): Aspartate (22mM), phosphate buffer
(100mM) pH 7.4 (Schwartz, 1971). Trypsin (TRY, E.C. 3.4.21.4): Benzoyl-L-arginine-p-
nitroanilide (2.3 mM), Tris-HCl 0.2 M and CaCl2 0.04 M buffer, pH 8.0 (Erlanger et al., 1961
Desrosiers et al. 2008, Lemieux and Blier, 2007). Chymotrypsin (CHY, E.C. 3.4.21.1):
Succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (2 mM), Tris-HCl 0.1 M and CaCl2 0.01 M buffer,
pH 7.8 (Delmar et al., 1979). Total protein content of muscle was determined using the
bicinchoninic acid method (Smith et al. 1985). Enzyme assays were conducted at 15 °C and
protein analyses were conducted at room temperature (≈23 °C). All enzymatic assays were
run in duplicate and protein assays in triplicate.
Molecular weight distribution of the protein hydrolysate
Size exclusion chromatography (SEC). Molecular weight distribution of the HPC90 protein
hydrolysate (Ocean NutraScience Inc., Matane (QC), Canada) was determined by gel
permeation chromatography (GPC) on a Superdex Peptide HR 10/300 GL column (GE
Healthcare, Baie-d’Urfé, QC, Canada) with an exclusion limit of 1x105 Da (13 μm, 10 x 300-
310 mm) using a high-pressure liquid chromatography system (HPLC Waters, Mississauga,
ON, Canada)(Beaulieu et al., 2009). The system was equipped with a Waters 996 Photo
Diode Array detector, a Waters 600 solvent delivery pump and a Waters 717 plus
autosampler. The mobile phase (isocratic) consisted of 50 mM sodium phosphate buffer
containing 150 mM of NaCl at pH 7.0. In accordance with instructions from the supplier, the
column was calibrated using peptides of known molecular weight (GE Healthcare, Baie-
d’Urfé, QC, Canada) as reference samples. Ribonuclease A (1mg/ml), aprotinin (1mg/ml),
vitamin B12 (0.1mg/ml) and cytidine 5’-monophosphate (Sigma, Oakville, ON, Canada)
(0.1mg/ml) were mixed together for the calibration of the Superdex Peptide HR 10/300 GL
column. This yielded a near linear correlation between the retention time and the log of the
molecular mass of peptides in the range of 367 to 13700 Da. Millenium32 (version 3.2)

software was used to analyse the chromatographic data. The protein hydrolysate sample (50
μl, 1 mg/ml) prepared in 50 mM sodium phosphate,150 mM NaCl, pH 7.0 was eluted at a
flow rate of 0.5 ml/min and monitored at an absorbance of 280 nm.
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Statistical analyses
To detect the potential effect of diet on survival rate, productivity and final weight, an
analysis of variance was performed using the general linear model procedure in Systat 10.2
computer software. Survival rates were arcsin transformed. When a significant difference
was detected, a Fisher LSD post hoc test was used. To detect possible interaction between the
effect of diet and weight on enzymatic activities, analysis of covariance (factors: diet, weight
and diet × weight) was performed using the general linear model procedure. When no effect
of weight on enzymatic activity was detected within a group, an analysis of variance with diet
as factor was performed. When a significant interaction was detected between diet and
weight, a linear regression of weight and enzymatic activity was performed for each diet.
Enzymatic activities were log transformed prior to analysis. Treatments were considered
significantly different when P<0.1.
Results
Inhibitory capacity of the formulated diets containing SBTI
In order to confirm the effectiveness of SBTI when processed in the experimental feed, gross
inhibitory activity was initially evaluated and compared for diets C, H, HI and I. Pellets of
the four different diets were homogenized with 9 volumes of Tris-HCl as described above.
Trypsin activity was measured according to Erlanger et al. (1961). The concentration of
trypsin added to the mixture was 100 U/ml. Assay was repeated 8 times for each diet. The
trypsin activity was 1.58 ± 0.27, 1.62 ± 0.18, 0.55 ± 0.09 and 0.51 ± 0.12 U for diet C, H, HI
and I respectively. There was no significant difference between C and H diet nor between HI
and I but in diets containing SBTI, trypsin activities were reduced by three-fold and
significantly lower than in the two other diets. As a result, the inhibitory activity of SBTI in
the diets HI and I was confirmed and judged effective.
Analysis of the shrimp protein hydrolysate HPC 90
The result of the molecular weight analysis (SEC) is presented in Fig. 1. Our estimation
revealed that the experimental hydrolysate was composed of 87% of peptides of around 1900
Da and 13 % of peptides of less than 303 Da. This suggest that the protein hydrolysate was

mostly composed of oligopeptides of 10-20 amino acids residues and contained very few free
amino acids
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Survival, productivity and weight
At the end of the trial, there was a significant effect of diet on survival and productivity
(Figure 1). Survival was 67.3 ± 3.5, 82.7 ± 5.7, 84.7 ± 5.9, 44.7 ± 10.9 % and productivity
was 38.1 ± 1.0, 46.2 ± 3.7, 58.8 ± 2.8 and 24.5 ± 5.5 g of fish/tank for group C, H, HI and I
respectively. Diet HI enhanced both productivity and survival compared to the control group
(p=0.02 and 0.085 respectively) while diet enhancements of productivity and survival failed
to reach significance (p=0.155 and 0.132 respectively). Productivity and survival were
significantly lower in group fed diet I compared to diet HI (p=0.001 and 0.006 respectively)
and diet I was different from control diet for productivity only (p=0.029). It is noteworthy
that a great variability between replicates, especially in the group I likely precluded
differences between groups to be significant.
Fish mass according to diet and time are presented in Fig. 2. At day 15, fish mass was higher
in both groups receiving diets containing hydrolysates (H and HI, p<0.000 and p=0.002
respectively) and lower for the group I (p=0.021) compared to control diet. At day 30 and 60,
group H had the same mass as the control but group HI was heavier (p=0.001 and 0.008 for
day 30 and 60 respectively). Group I (SBTI without PH) had a lower body mass of all groups
after 15 and 30 days but at day 60 achieved a mean mass equivalent to group C and H (Fig.3).
Enzymatic activities
Mean enzymatic activities at day 0 of the experiment were 7.932 ± 0.325, 0.084 ± 0.008 and
1.208 ± 0.034 U/g fish for AST, CHY and TRY respectively (U per mg protein was calculated
revealed the same trends but data are not presented). At day 15, there were some differences
between groups but none were significantly different: AST activity was lower when inhibitor
was present in the diet (groups HI and I), CHY was higher in group HI and TRY was lower in
group I. At day 30, CHY and TRY activities were significantly higher in group HI than in
group I. At day 60, only CHY activities were different between groups, CHY activity was
higher in group HI than control and group I (Fig. 4).

Discussion 256
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It was anticipated that PH would to some extent improve growth rate and survival and that
SBTI, would reduce protein digestion capacities through direct trypsin inhibition, and
significantly impair those same performance traits. Indeed, diet I (containing SBTI) caused
more mortality and slower growth and fish administered diet H had a higher body mass at day
15 (see Fig. 3). However, diet H did not led to better performances after 30 and 60 days
suggesting sufficient maturation of the digestive system after 15 days to fulfill the digestive
requirements in order to maintain a fair growth rate. This is in accordance with many studies
which found that hydrolysates are beneficial to fish larvae (Szlaminska et al., 1993; Cahu and
Zambonino-Infante, 1995; Carvalho et al., 1997; Zambonino Infante et al., 1997) but do not
affect juvenile growth (Cahu and Zambonino-Infante, 2001). According to those results,
newly-hatched wolffish up to 15 DPH would benefit from a diet supplemented with PH but
not thereafter, when they could be considered as ‘’true juveniles’’.
In a previous study on the same species, Savoie et al. (2006) obtained some indications that
protein hydrolysates could be beneficial to the young spotted wolfish but failed to obtain
significant results. The proposed explanation for the absence of a clear benefit following PH
dietary incorporation in this previous experiment, was linked to the insufficient degree of
hydrolysis of the commercial protein hydrolysates used (> 6 500 Da: Asta-Pep™). In
comparison, HPC90™ used in the present study presents a higher degree of hydrolysis (1900
Da).
The deleterious effect of the added SBTI was clearly demonstrated: mass and TRY activity
were lower at day 15 and 30 in group I for which mean survival was the lowest (45%) of all
experimental group. Fish in this group were probably in very bad condition by having a
restricted access to appropriate amount of amino acids, normally obtained via trypsin
breakdown of whole proteins. In this group, digestive capacities were hampered by the
presence of SBTI and contrarily to group HI, access to amino acids was not insured by the
supply of pre-digested proteins.
We foresaw some level of compensatory effect of PH when administered in combination with
a trypsin inhibitor in the diet which would in the best of cases, display growth rates equivalent
(or similar) to the control group. Unexpectedly, a highly significant positive impact of the
combination of trypsin inhibitor and protein hydrolysate (HI) on growth performance was

observed after 60 days: survival and mass of group HI were around 25% higher and 20%
higher than that of the control group. Adding only protein hydrolysate to the diet (group H)
enhanced survival almost as much as group HI but did not enhance final fish mass. Even if it
is well know that PH is not beneficial to juveniles, we were expecting the group H to perform
better than HI since their digestive capacities were not hampered by an inhibitor. Different
processes that might help to explain those results are discussed below.
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Sveier et al. (2001) working on Atlantic salmon achieved best growth rates when both
protease inhibitor and protein hydrolysate were added to the diet. They suggested that the
changes in digestion and absorption patterns caused by the inhibitor might result in an
extended digestion time and better protein utilization for growth.
Pre-digested proteins are more readily absorbed than intact proteins and result in a high
postprandial peak of amino acids in the plasma (Espe et al., 1993, 1999). Carvalho et
al.(2004) suggested that dietary excess of di- and tri-peptides was linked to reduced early-
stage performance in common carp (Cyprinus carpio) either due to saturation of the peptide
transport mechanisms and/or to the rapid hydrolysis of low-molecular weight peptides, that
produced an excessive amino acid load, that in turn saturated the amino acid intestinal
transport mechanisms.
In adult fish and in mammals, cholecystokinin (CCK) plays a major role in the pancreatic
enzyme secretion (Singer, 1993). This gastro-intestinal hormone is secreted in response to the
presence of nutrients in the lumen (Liddle, 1997) and stimulates trypsin and chymotrypsin
secretion (Einarsson et al., 1997). On the other hand, trypsin acts with a feedback signal by
degrading the CCK releasing factor and terminating CCK secretion (Herzig, 1998; Liddle,
2000; Cahu et al., 2004). In fact, food (proteins) act as a substrate for trypsin, bind it and
prevent degradation of CCK releasing factor. In this particular case, trypsin was inactivated
by SBTI and did not give the signal to stop pancreatic secretion. Growth could then be
enhanced via an overcompensation of digestive enzyme secretion. This would imply that
other digestive enzymes than trypsin would set digestive capacity and growth at these young
stages (Rungruangsak-Torrissen, 2006).
Chymotrypsin activity was always higher in group HI (significant at day 30 and 60) but in
group I, values were similar to control group. Fish of group I were likely in bad condition and

overcompensation of chymotrypsin secretion was not expressed in this group. In the case of
TRY this overcompensation seemed to allow a steady state of the activity level despite the
presence of the inhibitor. These results suggest that the improved performances of the groups
provided with trypsin inhibitor and protein hydrolysate partly depend on overcompensation
which is induced only when supplemented with small peptides. In a recent study Lilleeng et
al. (2007) reported an upregulation of trypsin-like activity in the distal intestine wall. This
increased activity may contribute to higher trypsin activity in the intestinal content and partly
explain improved growth preformance when PH is supplemented in presence of trypsin
inhibitor. This hypothesis could be also validated via the quantification of trypsin and
chymotrypsin messenger RNA because enzyme activities do not necessarily equal their
synthesis rate. In the case of trypsin and chymotrypsin, these are secreted in the form of
inactive zymogens: respectively trypsinogen and chymotrypsinogen. These have to be
activated by enterokinase or by their own active form. Consequently, if the enzymes are
produced but readily inactivated by SBTI, their normal “self-activation” could be impaired
resulting in a large quantity of zymogens but no measurable activity.
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The PH used in our experiment was processed by enzymatic hydrolysis with endopeptidase
(Seanergy inc. technical information on HPC90™). Endogenous exoproteases (which non-
specifically cuts terminal amino acids from the peptide chain) may therefore play a more
important role in the final digestion of PH than trypsin which is an endopeptidase. SBTI may
extend the life of these exoproteases in the gut by blocking their digestion by trypsin thus
increasing their net activity. Completion of the final steps of peptide digestion could then be
facilitated and scope for growth of fish enhanced (Ross, N. Pers. Comm..). This hypothesis
could be tested by estimating synthesis and expression of this exopeptidase under the same
conditions used in the present study.
Acknowledgement
The authors would like to express their gratitude to Tony Grenier and Dany Ouellet for their
technical assistance throughout the experiment. We are grateful to Dr. Neil Ross (CNRC-
PARI, Halifax, NS, Canada) for his constructive comments on an earlier version of the
manuscript. The financial support of MAPAQ and Université du Québec à Rimouski to NLF
is also acknowledged.

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Figure captions 513
Figure 1 Molecular weight distribution of the HPC90 shrimp protein hydrolysate (Ocean 514
NutraScience Inc., Matane (QC), Canada) determined by gel permeation chromatography 515
516
Figure 2 Survival (%) and productivity (g/tank, mean weight * survival) of spotted wolfish at 517
the end of the experiment (60 DPH). 518
519
Figure 3 Mean weight of spotted wolfish according to diet (C, H, HI, I) and days post-520
hatching 521
522
Figure 4 Enzymatic activities of aspartate aminotransferase (AST), chymotrypsin (CHY) and 523
trypsin (TRY) of entire larvae of spotted wolffish according to diet (C, H, HI, I) and days 524
post-hatching 525

Arbitrary absorbance units
25
510 15 20 30
35 40 45
55
50 60 65 70
1914
303184
12
Time (minutes)
0.00
0.02
0.04
0.06
0.08

0
10
20
30
40
50
60
70
80
90
100
C H HI I
Diet
Survival - Productivity
Productivity
Survival
a
a
ab
ab
b
bc
c

0
0.2
0.4
0.6
0.8
1
1.2
1.4
015 30 60
Time (DPH)
Weight (g)
C
H
HI
I
a
a
a
a
a
b
b
b
bc
c
c

0
2
4
6
8
10
AST (U/g fish)
C H HI I
0
0.1
0.2
0.3
0.4
0.5
0.6
CHY (U/g fish)
0
0.5
1
1.5
2
015 30 60
Time (DPH)
TRY (U/g fish)
a
ab b
c
ab
ab
ab
ab
a
b
b
c
a