Growth and protein utilization in Atlantic salmon(Salmo salar L.) given a protease inhibitor in the diet

H. SVEIER1, B.O. KVAMME2 & A.J. RAAE2

1EWOS Innovation AS, Dirdal, Norway; 2Institute of Molecular Biology, Hùyteknologisenteret, Bergen, Norway

Abstract

In a series of experiments the e�ects of dietary protease

inhibitor inclusions on growth and amino acid absorption

rate were investigated in Atlantic salmon (Salmo salar L.).

An optimal inclusion of inhibitor was found by conducting

dose±response studies with speci®c growth rate as the main

response variable. No negative e�ects on feed conversion

ratio or nitrogen digestibility were observed at this level. In

protein sources studies the addition of potato protease

inhibitor to a ®shmeal based diet increased speci®c growth

rate by 14%. When a proportion of the ®shmeal was replaced

with hydrolysed protein (®sh silage) the addition of the

inhibitor resulted in a 31% increase in speci®c growth rate.

Absorption of amino acids from the gastrointestinal tract

into the blood was investigated in two experiments using14C-labelled algal (Synechoccus leopoliensis) protein. The

absorption pattern of 14C-labelled amino acids was altered by

adding potato protease inhibitor when the algal protein was

supplied in intact form, but not when the algal protein was

hydrolysed. The absorption of amino acids from a hydro-

lysed protein was signi®cantly faster than from intact

protein. The enzymatic activity of pepsin in the stomach

and of trypsin, chymotrypsin, carboxypeptidases A and B in

the di�erent segments of the intestine changed signi®cantly

with increasing inclusion of potato protease inhibitors in the

diet.

KEY WORDSKEY WORDS: Atlantic salmon, digestive proteases, gastro-

intestinal tract, growth, hydrolysed protein, protease

inhibitors

Received 8 November 1999, accepted 23 February 2001

Correspondence: H. Sveier, EWOS Innovation AS, N-4335 Dirdal,

Norway.

e-mail: harald.sveier@ewos.com

Introduction

E�cient protein utilization is a prerequisite for a high growth

rate and low feed conversion ratio in ®sh. In the wild Atlantic

salmon (Salmo salar L.) feed on prey containing primarily

native body protein while manufactured diets for salmonids

consist of dried, ®nely ground, denatured protein. Feed

particle sizes have been shown to in¯uence gastric emptying

(Jobling 1987; Sveier et al. 1999) without a�ecting nitrogen

and fat digestibility (Sveier et al. 1999). Thus, the absorption

of dietary amino acids and small peptides from the intestine

may be faster feeding a formulated feed (small particles)

compared with natural prey (large particles), which may lead

to less e�cient protein accretion. Dos Santos et al. (1993)

reported higher protein retention in cod (Gadus morhua L.)

fed coarsely chopped herring (Clupea harengus L.) than in

those fed minced herring, but Sveier et al. (1999) did not ®nd

such an e�ect when feeding Atlantic salmon coarsely or ®nely

ground ®shmeal. Another factor a�ecting amino acid

absorption is the amount and activity of proteolytic enzymes

in the gastrointestinal tract. Protease inhibitors in the diets

may in¯uence both the activity and amount of proteolytic

enzymes and therefore overall growth rate and feed utiliza-

tion. Krogdahl et al. (1994) and Olli et al. (1994) examined

the e�ects of dietary soyabean protease inhibitor inclusion on

rainbow trout (Oncorhynchus mykiss) and Atlantic salmon.

In general they found negative e�ects on growth and nutrient

digestibility when protease inhibitors were added. Soyabean

protease inhibitor reduces the activity of the endopeptidases

trypsin and/or chymotrypsin (Liener 1979; Birk 1989),

whereas protease inhibitors from potato inhibit trypsin,

chymotrypsin and carboxypeptidase A and B activities (Ryan

1974; Pearce et al. 1982; Aksnes 1989). Potato protease

inhibitors may therefore be used to modulate the gastro-

intestinal proteolytic activity of both endo- and exo-peptid-

ases. The mechanism of action of protease inhibitors from

potato is thought to be a transient and reversible blocking of

the active site of the proteases, resulting in a time-dependent

255

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Ó 2001Blackwell Science Ltd

reduction of the proteolytic process (Laskowski & Kato

1980).

The aim of the present study was to investigate the

in¯uence of a protease inhibitor isolate prepared from potato

on the proteolytic activity in the gastrointestinal tract of

Atlantic salmon. Further, the aim was to examine the speci®c

growth rate, feed utilization and the absorption pattern of

amino acids and small peptides using dietary intact or partly

hydrolysed protein when potato protease inhibitor isolate

was added to the diet.

Materials and methods

Three separate studies were carried out at the EWOS

Innovation Research Station in Dirdal, in the south-west

of Norway, to examine the e�ect of dietary protease

inhibitor addition on gastrointestinal peptidase activity

and growth performance of Atlantic salmon. In the ®rst

study the e�ect of di�erent levels of dietary inhibitor on

growth was examined. In the second study two experi-

ments were carried out to investigate the e�ect of inhibitor

inclusion in diets containing intact or hydrolysed protein.

In the third study two experiments were carried out to

examine the e�ect of inhibitor inclusion on the absorption

of 14C-labelled amino acids derived from hydrolysed or

intact algal protein.

Atlantic salmon derived from NLA (Norsk Lakseavl,

Kyrksñterùra, Norway) broodstock were exposed to con-

tinuous light from smolti®cation until the end of the

experiment. Water temperature and salinity were recorded

daily, while oxygen saturation in the water outlet was

recorded and adjusted weekly to ensure a minimum level of

7 mg L)1. The ®sh were acclimatized to the experimental

conditions for 4 weeks prior to the experiments.

In all experiments ®sh were fed to excess three daily meals

using automatic belt feeders (Hùlland Teknologi, Sandnes,

Norway). The ®rst meal was fed between 20.00 and 20.30 h

(20% of expected daily ration), the second from 02.00 to

02.30 h (20% of expected daily ration) and the third from

06.30 to 07.30 h (60% of expected daily ration).

The protease inhibitor preparation used was produced

from potato juice by Norsk Potetindustri, Gjùvik, Norway.

The chymotrypsin inhibitor activity of the inhibitor prepar-

ation was determined to be 1.22 ´ 105 inhibiting unit per

gram of protein or 6.49 ´ 104 chymotrypsin BTEE unit per

gram of inhibitor preparation as described in `inhibitor assay'

later. One inhibitor unit is de®ned as the amount of inhibitor

that inhibits one standard chymotrypsin BTEE unit 50% per

min at 23 °C.

Study one: dose^response experiments

Experimental conditions The study was carried out in two

parts (Exp. 1a and 1b) consisting of a preliminary study and

a main experiment. In Exp. 1a, a dose±response design with

®ve dietary protease inhibitor levels and one experimental

unit per level was used. Groups of 15 ®sh averaging

342 � 7.7 g (mean � 1 SD, n � 5) were distributed into

180 L circular tanks. In Exp. 1b a dose±response design with

six dietary inhibitor levels and two replicates per level was

used. Groups of 35 ®sh averaging 117 � 13 g (mean �

1 SD, n � 420) were assigned to tanks of 0.5 m3 volume

(1 ´ 1 ´ 0.5 m). Seawater of 28.8 � 0.2& at 9.6 � 0.5 °Cand 29.0 � 0.3& at 7.8 � 0.4 °C was used in Exp. 1a and

1b, respectively.

Diet composition and feeding In Exp. 1a and 1b the diets were

produced by coating uncoated commercial extruded pellets

(4 mm Nostra, NorAqua AS, Stavanger, Norway) with

yttrium oxide (Y2O3) and protease inhibitors. The protease

inhibitor isolate was added to the diet in concentrations of

0, 1, 5, 10 and 20 g kg)1 feed and 0, 0.1, 0.2, 0.5, 1.0 and 5.0 g

in Exp. 1a and 1b, respectively. Yttrium oxide was added to

the diets as an inert indicator to measure apparent nitrogen

digestibility. Protease inhibitor isolate and Y2O3 were gently

mixed with some of the oil and coated on the pellets using a

vacuum coater. The remaining oil was then coated under

vacuum to give a feed with 50% protein and 28% oil.

In Exp. 1a, uneaten pellets were collected using a box with

a mesh bottom placed under the water outlet and amount of

feed eaten was quanti®ed daily. In Exp. 1b uneaten feed was

collected on a moving mesh belt that removed the pellets

from the outlet water and transferred them to a collection

box (Excess Fishfeed collector, Hùlland Teknologi, Sandnes,

Norway) (Sveier et al. 1997). Daily inspection of the amount

of uneaten feed ensured adequate overfeeding of ®sh.

Fish sampling and sample treatment Prior to the start of Exp.

1a feed was withheld for 4 days before the ®sh were counted

and weighed in bulk. At the end of a 21-day feeding period,

six ®sh per tank were killed by a blow to the head and the

gastrointestinal tract removed and immediately frozen. The

frozen gastrointestinal tract was divided into four parts;

stomach, pyloric caeca, mid gut and hindgut, and the

contents were removed for measurement of pepsin (stomach

only), trypsin, chymotrypsin and carboxypeptidase A and B

activity. Faeces from the remaining ®sh were removed by

stripping (Austreng 1978). All ®sh were individually weighed

before sampling. Prior to Exp. 1b, feed was withheld for

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5 days and 10 ®sh were randomly sampled for initial whole

body analysis of fat and protein. The number of ®sh in each

tank was adjusted to 15 and the ®sh were weighed

(118 � 2.6 g, n � 12). At the end of an 84-day feeding

period, faeces were removed from all ®sh by stripping. Fish

were deprived of food for 4 days and then weighed indi-

vidually. For ®nal whole body analysis of fat and protein ®ve

®sh per tank were used. Another 10 ®sh per tank were

sampled for measurement of condition factor, dressing out

percentage (DOP) (see below for de®nition) and fat content

in the cutlet (pooled samples) according to the Norwegian

quality cut (NQC, NS 9401 1994).

Study two: protein sources experiments

Experimental conditions The experiment was carried out in

two parts (Exp. 2a and 2b). Groups of 16 ®sh (285 � 51 g,

n � 192 and 297 � 46 g, n � 192 part 2a and 2b, respect-

ively) were distributed to each of the 12 tanks of 0.5 m3

volume (1 ´ 1 ´ 0.5 m, six replicates per treatment in each

part). The experiment was run in seawater of 31 � 0.9& at

8.1 � 0.2 °C.

Diet composition, feeding and ®sh sampling In Exp. 2a,

®shmeal based diets either with or without protease inhibitor

were used. The diets used in Exp. 2b were similar to those of

Exp. 2a but with approximate 10% of the ®shmeal replaced

by hydrolysed ®sh protein (FPCÒ, Rieber Co., Bergen,

Norway). Semi-moist diets were produced according to

Table 1 using AlgibindÒ (Algca, Oslo, Norway) as a binder.

The hydrolysed ®sh protein, which replaced some of the

®shmeal, was added to give a calculated trinitrobenzo

sulphonic acid (TNBS) value of 1.2 a-amino-LL-leu equiva-

lents (Espe et al. 1999). Based on the results from Exp. 1a

and 1b, the protease inhibitor inclusion level in study two

was ®xed at 1 g kg)1. Collection of uneaten feed from the

outlet, using the same equipment as in Exp. 1b, ensured

su�cient overfeeding of the ®sh. In Exp. 2a and 2b feed was

withheld for 4 days at the start and end of the 45-day feeding

period before individually measuring weight and length of

®sh. Diet formulations are presented in Table 1.

Study three: uptake of 14C-labelled amino acids

Experimental conditions The experiment was carried out in

two parts (Exp. 3a and 3b), using two dietary treatments and

six replicates per treatment in each part. Groups of 16 ®sh

(328 � 57 g, n � 192 and 697 � 108 g, n � 192 in part 3a

and 3b, respectively) were distributed to each of 12 square

tanks of 0.5 m3 volume (1 ´ 1 ´ 0.5 m). Water temperature

and salinity were 8.2 � 0.2 °C and 31 � 0.9&, respectively.

Diet composition and feeding Fish were fed the same

experimental diets used in Exp. 2 for at least 4 weeks before

they were given a single meal of a 14C-labelled diet. The14C-source used was 14C-labelled algal protein from

Table 1 Dietary ingredients and chemi-

cal calculations of diets used in study

two (protein source) and study three

(14C-absorption)

Exp. 2a Exp. 2b Exp. 3a Exp. 3b

Ingredients (g kg)1)Fish meal (Norse LT-94Ò) 364 323 364 323Capelin oil (Norsamoil) 190 196 190 196Suprex maize 149 142 149 142Binder (Algibind, Algea) 35 35 35 35Shrimpmeal 50 50 50 50Premix (vitamin, mineral, betain,

pigment)17 7 7 7

Indicator (Y2O3) 0.1 0.1 0.1 0.1Protease inhibitor þ1 þ1 þ1 þ1Hydrolysed protein (FPC, Rieber & Co) 0 123 0 123Intact 14C-labelled algal protein2 +Hydrolysed 14C-labelled algal protein2 +Water 205 124 205 124

Chemical composition (gram DM)(kg feed))1

Protein 408 365 408 365Fat 247 272 247 247Rest 345 363 345 363

1 V|tamins andminerals according to or higher than recommended by NRC (1993).2 When 14C-labelled algal protein was included in the diets no correction in the other ingredientswas performed because of the small inclusion volume. Norse LT-94Ò; Norsildmel, Fyllingsdalen, Norway.

Growth and protein utilization in Atlantic salmon using protease inhibitors

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Synechoccus leopoliensis (Sigma Chemical Co., St Louis,

MO, USA) and was included in the diets to give a calculated

intake of 2 lCi (100 g ®sh))1 based on expected feed intake.

In Exp. 3a the 14C-labelled algal protein was hydrolysed by

mixing 1 mL 14C-labelled algal protein solution with 5 g

homogenized salmon muscle and 2.2% formic acid. The

protein was allowed to hydrolyse for 4 days at room

temperature with continuous stirring (Espe & Lied 1999).

In Exp. 3b intact 14C-labelled algal protein was used.

Chromic oxide (Cr2O3) at 1% level was included in the diets

with 14C-labelled algal protein to allow feed intake to be

measured. Diet formulations are presented in Table 1.

Fish sampling and sample treatment Fish were sampled at 0, 2,

4, 6, 9 and 12 h after feeding the meal with the experimental

diets containing 14C-labelled algal protein and Cr2O3. Feed

was withheld for 18 h before the meal to ensure high feed

intake. At each time interval, two ®sh from two tanks were

sampled, to give two replicates per sampling time and two

samplings per tank. A blow on the head killed the ®sh and a

blood sample was then collected from the caudal vein using

heparin vacumtainers. Plasma obtained by centrifugation at

3000 g for 10 min was stored at ±20 °C until analysis of14C-activity. Each ®sh was weighed, and the gastrointestinal

tract removed, weighed and stored at ±20 °C until analysis of

Cr2O3 content. After the 12 h sample the remaining ®sh were

fed the experimental diets without Cr2O3 and 14C-labelled

algal protein. At 72 h postfeeding the 14C-labelled meal, ®ve

®sh per tank were taken for analyses of radioactivity in the

fat and protein fractions in pooled samples of the whole

body.

Chemical analyses

The diets were analysed in duplicate for dry matter, protein,

fat and ash. Protein (n ´ 6.25) was determined colorimeter-

ically after micro-Kjeldahl digestion according to Crooke &

Simpson (1971). Fat was determined gravimetrically in feed,

whole ®sh and cutlet (NQC) after extraction with ethyl

acetate; dry matter after drying at 105 °C for 24 h and ash

after combustion at 550 °C for 16 h. Protein in ®sh and

faeces was analysed using a nitrogen gas analyzer (Perkin

Elmer Series II Nitrogen analyzer 2410, Perkin Elmer,

Welleley, MA, USA) according to the manufacturers proce-

dure.

Yttrium oxide in feed and faeces was analysed using ICP-

MS (Perkin Elmer/SciX Elan 5000). Chromic oxide was

analysed by atomic absorption spectrophotometry according

to Lied et al. (1982). The Institute of Nutrition, Directorate

of Fisheries, Bergen, Norway, performed both element

analyses. All analyses of samples from ®sh were performed

on pooled samples from each tank.

Inhibitor assay

One inhibitor unit of the potato protease preparation was

de®ned as the amount that inhibited one bovine a-chymo-

trypsin BTEE (N-benzoyl-LL-tyrosine esthyl ester) unit 50%

per min at 23 °C. A 10-mg mL)1 inhibitor stock solution was

made by dissolving the dried potato inhibitor preparation in

a bu�er (50 mMM Tris±HCl, 100 mMM NaCl) at pH 8.0. To

determine the inhibitor activity in the inhibitor preparation,

varying concentrations of inhibitor were mixed with a ®xed

number of units of a-chymotrypsin. After incubation for

20 min at 23 °C, the mixtures were added to 0.1 mMM Suc-

Ala-Ala-Pro-Phe-pNa (Sigma Chemical Company, St Louis,

MO, USA) in the bu�er, and inhibitor activity measured

spectrophotometrically.

For determination of protease activity in the digestive

tract, extracts were prepared by mixing 0.2 g of the contents

of the di�erent segments with 1 mL 50 mMM Tris±HCl,

100 mMM NaCl, pH 8.0 and vortex mixing to homogeneity.

The solution was cleared by centrifugation and the super-

natant (digestive enzyme extract ± DEE) transferred to fresh

tubes. Protein concentrations in DEE samples were measured

using the Bio-Rad DC protein assay for microtiter plates

using bovine serum albumin (BSA) as the standard protein.

The DEE samples were then assayed for pepsin (stomach

only), chymotrypsin, trypsin and carboxypeptidase A and B

activity using the procedures described below. All enzyme

substrates were supplied by Sigma Chemical Company.

Chymotrypsin activity was measured spectrophotometri-

cally at A410 in 5 lL DEE. One unit is de®ned as the amount

of chymotrypsin that hydrolyses 1 lmol of Suc-Ala-Ala-Pro-

Phe-pNA per min at 23 °C (DelMar et al. 1979).

Trypsin activity was measured spectrophotometrically by

adding 5 lL DEE to 1 mL 0.5 mMM BAEE (benzoyl-arginyl

ethyl-ester), dissolved in 50 mMM Tris±HCl, 100 mMM NaCl,

pH 8.0 at 23 °C and recording DA253 min±1. The number of

trypsin units in DEE was determined by comparing with a

trypsin standard solution.

Carboxypeptidase A activity was measured spectrophoto-

metrically by adding 25 lL DEE to 1 mL 1 mMM Hippuryl-

Phe (Wol� et al. 1962; Folk 1963), dissolved in 25 mMM

Tris±HCl, 500 mMM NaCl, pH 7.5 at 23 °C and recording

DA254 min±1. The activity was correlated to Hippuryl-Phe

units by comparison with carboxypeptidase A standard

solution.

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Carboxypeptidase B activity was measured spectrophoto-

metrically by adding 25 lL DEE to 1 mL 1 mMM Hippuryl-

Arg (Wol� et al. 1962; Folk 1963), dissolved in 25 mMM

Tris±HCl, 100 mMM NaCl, pH 7.65 at 23 °C and recording

DA254 min±1. The activity was correlated to Hippuryl-Arg

units by comparison to carboxypeptidase B standard solu-

tion.

Pepsin activity in the stomach was measured essentially

after the method described by Kassel & Meitner (1970).

Haemoglobin (2% w/v) was dissolved in 60 mMM HCl and

®ltered. The activity was measured spectrophotometrically

by incubating 10 lL DEE in 1 mL 2% Hb-solution for

60 min at 37 °C. After incubation, ice cold 20% TCA was

added to a ®nal concentration of 3.1% (w/v) and stored for

5 min on ice. After centrifugation for 15 min at 12 500 r.p.m.,

´13975 g the absorbance was measured at 280 nm. All

calculations of enzymatic activity were normalized using the

protein concentration in the DEE sample.

Isotope activity in plasma was determined by scintillation

counting according to Berge et al. (1994). Samples of the

experimental diets with 14C-labelled algal protein were

solubilized overnight in Soluene-350 before counting. For

measurement of isotope activity in whole body fat and fat-

free fraction, homogenized whole body samples of 1 g were

treated with 5 mL chloroform:methanol 2:1 for 1 h and then

centrifuged. After that, 2.5 mL supernatant was solubilized

in Soluene-350 and the radioactivity determined. The

precipitate was washed three times with chloroform:metha-

nol (2:1). After the last washing, the supernatant was

removed, and the precipitate was allowed to air-dry over-

night at room temperature before solubilizing in Soluene-350

and the radioactivity determined.

The degree of hydrolysis of the 14C-labelled algal protein

was checked by separating the proteins on a 12.5% SDS±

PAGE PHAST-gel (Pharmacia Biotech, Uppsala, Sweden),

and thereafter a FLA2000 PhosphoImager (Fuij®lm) was used

for measuring the radioactivity in the di�erent protein bands.

Calculation and statistics

Speci®c growth rate (SGR) was calculated as:

SGR � 100 �lnW2 ÿ lnW1�tÿ1

where W1 and W2 are the initial and ®nal weight, respect-

ively, and t is the number of days in the feeding period.

Feed intake (FI) was calculated as:

FI� �feed givenÿ feed collected�� �ratio of collection efficiency�ÿ1 �Helland et al: 1996�:

For calibration of the collecting e�ciency of uneaten feed,

the procedure described by Helland et al. (1996) was used.

Feed conversion ratio (FCR) was calculated as:

FCR � �FI��B2 � Bdead ÿ B1�ÿ1

where B1 and B2 are the biomass at the start and end,

respectively, and Bdead is the biomass of dead ®sh.

Dressing out percentage (DOP) was calculated as:

DOP � �1ÿ �BWguttedBWÿ1ungutted��100

where BWgutted and BWungutted are the weights of gutted and

ungutted unbled ®sh, respectively.

Condition factor was calculated as:

CF � W Lÿ3 100

where W is the weight in gram and L the length of the ®sh

in cm.

Productive protein value (PPV) was calculated as:

PPV � �P2 � Pdead ÿ P1�Pÿ1in

where P1 and P2 are estimates of protein content of the

biomass at the start and end of the experiment and Pdead is

the estimated protein content ((P1 + P2) 2±1) of the dead/

sample ®sh. Pin is the protein intake.

Apparent nutrient digestibility (AD) as:

AD � 100)100 ((Ifeed Nfaeces) (Ifaeces Nfeed)±1)

where Ifeed and Ifaeces are the concentrations of indigestible

marker in the feed and faeces. Nfeed and Nfaeces are the

nutrient concentrations in feed and faeces, respectively.

Figure 1 The e�ect of increasing inclusion of a protease inhibitor

mixture (PI) on nitrogen digestibility (%). The inhibitor activity of

the protease preparation was estimated to 6.49 ´ 104 chymotrypsin

BTEE (N-benzoyl-LL-tyrosine esthyl ester) units per gram.

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Amount of 14C-labelled algal protein eaten was calculated

as:

�Crorgan 14C activityfeed�Crÿ1feed

where Crorgan and Crfeed are the chromic dioxide concentra-

tion in the total gastrointestinal tract and feed, respectively,

and 14C activityfeed is the isotopic activity in the feed.

The concentration of 14C-labelled amino acids in the

plasma was calculated relative to amount eaten 14C-labelled

amino acids as:

�14C activityplasma14C activityÿ1eaten�100:

The inhibitor activity of the crude potato inhibitor was

calculated as:

IA � aes aÿ1e

where aes and ae is the measured activity in the enzyme-

inhibitor and enzyme solutions, respectively.

The data from Exp. 1 (dose±response study, Table 3,

Fig. 1) and Exp. 3a (amino acid uptake, Fig. 4) were

analysed by regression choosing the best ®tting model. The

enzyme activity data from Exp. 1a are presented as

mean � 1 SD (Fig. 2). Data from Exp. 2 and 3 (Table 4,

Fig. 3) were tested using an analysis of variance (one-way

ANOVAANOVA) using tank mean values. Where signi®cant

(P < 0.05) di�erences were found, a Tukey HSD multiple

range test was used to rank the treatments. The software

Figure 2 The activity of pepsin, trypsin, chymotrypsin, carboxypeptidase A and carboxypeptidase B in the di�erent segments of the

gastrointestinal tract measured as units (g water soluble protein)±1. Data is presented as average � SEM. Di�erent letters indicates signi®cant

di�erences within an organ. The inhibitor activity of the potato protease preparation was estimated to 6.49 ´ 104 chymotrypsin BTEE

(N-benzoyl-LL-tyrosine esthyl ester) units per gram.

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Statgraphics, version 3.1 (Statistical Graphics Corporation,

Manugistics, Inc., MD, USA) was used.

Results

Study one: dose^response experiments

Speci®c growth rate and feed conversion rate data from

Exp. 1a and 1b are presented in Table 2. Statistical analyses

of relative SGR values achieved in both experiments setting

the control diet � 100% gave a signi®cant correlation

between inhibitor level and SGR (y � ±0.9828x2 +

4.2936x + 96.825, r2 � 0.970). Based on this correlation an

optimum inhibitor inclusion level of about 0.5 g kg±1 was

estimated. Higher inclusion levels gave a marked reduction in

speci®c growth rate. In the same way there was a signi®cant

correlation between FCR and dietary inhibitor level

(y � 2.1853x + 93.253, r2 � 0.4721). At the lowest inclusion

levels of protease inhibitor there was a tendency for

decreased FCR compared with the control group and there

were only small changes in FCR until the dietary inclusion of

protease inhibitors reaches 5 g kg±1. Protein utilization and

body trait data from Exp. 1b are listed in Table 3. There was

no e�ect of protease inhibitor level on any of the measured

parameters, except for nitrogen digestibility which showed a

decreasing tendency when protease inhibitor inclusion excee-

ded 5 g kg±1 (Fig. 1). Fat digestibility was not in¯uenced by

protease inhibitor inclusion (Table 3).

The enzymatic activities in the di�erent segments of the

gastrointestinal tract from Exp. 1a are shown in Fig. 2(a±e).

Pepsin, chymotrypsin, carboxypeptidase A and B all showed

a signi®cantly reduced activity at high inclusion levels of

protease inhibitors. Trypsin showed a signi®cant reduction in

the pyloric caeca region even at relatively moderate inclusion

levels.

Study two: protein source experiments

The results from the experiments are shown in Fig. 3 setting

the SGR of the control groups � 100%. In both experiments

speci®c growth rate was higher when the potato protease

inhibitor preparation was added to the diet. The growth

improvement was almost signi®cant (P � 0.058) when only

®shmeal protein was used as dietary protein source.

When approximately 10% of the ®shmeal was replaced by

Figure 3 The relative speci®c growth rate (%) achieved with a ®sh-

meal based diet or a diet where a part of the ®shmeal was replaced

with hydrolysed protein. Both diets were tested with or without

inclusion of protease inhibitor. Di�erent letters indicate signi®cant

e�ect of inhibitor inclusion.

Table 2 Speci®c growth rate (SGR) and feed conversion rate (FCR)

achieved in Exp. 1a and 1b

Inhibitor

Specific growthrate (% day)1)

Feed conversion rate

added (g kg)1) Exp.1a Exp.1b Exp.1a Exp.1b

0 0.85 1.17 þ 0.07 1.13 0.82 þ 0.030.1 1.21 þ 0.03 0.79 þ 0.020.2 1.12 þ 0.04 0.84 þ 0.000.5 1.16 þ 0.01 0.83 þ 0.011.0 0.80 1.16 þ 0.05 1.08 0.86 þ 0.035.0 0.76 1.16 þ 0.02 1.15 0.84 þ 0.04

10.0 0.64 1.2620.0 0.59 1.31

Values as mean (1a) andmean þ 1SD (n = 2) (1b).

Table 3 Productive protein value (PPV),

apparent nitrogen (N-digapp) and fat

(Fat-digapp) digestion, fat in cutlet

(NQC) and condition factor (C-factor)

achieved in Exp. 1b

Inhibitor added(g kg)1) PPV N-digapp (%) Fat-digapp (%) NQC (%) C-factor

0 0.398 þ 0.008 84.8 þ 1.2 93.9 þ 0.3 8.47 þ 0.15 1.24 þ 0.040.1 0.422 þ 0.05 86.2 þ 2.1 94.6 þ 1.1 8.57 þ 0.15 1.29 þ 0.040.2 0.398 þ 0.007 86.4 þ 1.1 95.0 þ 0.3 8.24 þ 0.06 1.26 þ 0.020.5 0.383 þ 0.017 85.7 þ 1.3 93.4 þ 2.5 8.66 þ 0.06 1.24 þ 0.011.0 0.365 þ 0.013 85.4 þ 0.5 93.6 þ 0.3 8.40 þ 0.42 1.24 þ 0.035.0 0.385 þ 0.017 86.6 þ 1.2 94.7 þ 1.0 8.36 þ 0.23 1.26 þ 0.01

Values as mean þ 1SD (n = 2).

Growth and protein utilization in Atlantic salmon using protease inhibitors

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261

hydrolysed protein, growth rate was signi®cantly increased.

Fish length was signi®cantly increased when protease inhib-

itors were added to the diet without hydrolysed protein

inclusion (Table 4). There was no e�ect of protease inhibitors

in diets with or without hydrolysed protein inclusion on

condition factor (Table 4).

Study three: uptake of 14C-labelled amino acids

The relative concentrations of 14C-labelled amino acids

in plasma at di�erent times post feeding are shown in

Fig. 4(a,b). There was a tendency for a higher absorption

rate of 14C-labelled amino acids derived from intact protein

when protease inhibitor was added, compared to when

protease inhibitor was not added (Fig. 4a). There was no

e�ect of protease inhibitors when added to feeds containing

partly hydrolysed 14C-labelled protein.

The relative deposition of 14C from dietary protein into

whole body fat and protein was not a�ected by protease

inhibitor (Table 4), but there was a signi®cant e�ect of using

hydrolysed protein in the feed. The use of hydrolysed protein

resulted in higher distribution ratio into the whole body

protein and consequentially a lower distribution in the whole

body fat.

Discussion

The time needed to digest and absorb ®nely ground dietary

particles of denatured protein is shorter than that of larger

particles as found in natural diets (Jobling 1987; Sveier et al.

1999). Altering the activity of gastrointestinal proteolytic

enzymes by including protease inhibitors in the diet may

change the digestion and absorption rate of dietary protein.

In the present study, a protease inhibitor preparation with

multiprotease inhibiting activity isolated from potato (Aksnes

1989) was used. In a parallel study, we have demonstrated

that the inhibitor reversibly inhibits chymotrypsin in caecal

extracts from Atlantic salmon (data not shown) that indicate

that the enzyme activity may be changed and not totally

blocked by using this protease inhibitor. The dose±response

study showed a dose dependant e�ect of adding a moderate

level of protease inhibitor to feed on speci®c growth rate

without a�ecting feed conversion ratio or nitrogen digesti-

bility. Higher levels of protease inhibitors (>5 g kg±1) in the

diet resulted in a clear negative e�ect on growth rate, feed

conversion ratio and nitrogen digestibility. Olli et al. (1994)

made the same observation associated with moderate inclu-

Table 4 Body traits (Exp. 2a and 2b) and relative distribution of 14C

in whole body protein and fat fraction (Exp. 3a and 3b) using diets

with or without silage combined with or without proteinase inhibitor

addition

Without silage With silage

Condition factor^Inhibitor 1.28 þ 0.013 1.04 þ 0.009+Inhibitor 1.27 þ 0.011 1.03 þ 0.013

Length increase (cm)^Inhibitor 2.3 þ 0.11a 2.0 þ 0.11+Inhibitor 2.8 þ 0.15b 2.3 þ 0.13

Relative distribution of 14C inwhole body fat fraction (%)^Inhibitor 20.8 þ 1.1B 14.6 þ 0.6A

+Inhibitor 20.1 þ 0.7B 15.3 þ 0.7A

Relative distribution of 14C inwhole body protein fraction (%)^Inhibitor 79.2 þ 1.1B 85.4 þ 0.6A

+Inhibitor 79.9 þ 0.7B 84.7 þ 0.7A

Different capital letters indicate significant effect of silage inclusion,while small letters indicate significant effect of proteinase inhibitorinclusion.Values as average þ SEM (n = 6).

Figure 4 The relative uptake in the plasma of 14C labelled amino acids from intact (a) or hydrolysed (b) 14C labelled algal protein without (Ð)

or with (- - - -) protease inhibitor inclusion.

H. Sveier et al.

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262

sions of soyabean trypsin inhibitor in Atlantic salmon diets.

Krogdahl et al. (1994) examining rainbow trout (Oncorhyn-

chus mykiss) also observed a reduction in nitrogen digesti-

bility. Neither author found an e�ect of inhibitor use on fat

digestion, which is in agreement with the present study.

The use of approximately 10% of partly hydrolysed ®sh

protein (silage) in the diet has been found to improve growth

rate in Atlantic salmon (Espe et al. 1999). This has been

explained by a di�erent uptake rate of amino acids and

peptides derived from hydrolysed protein as compared with

intact protein (Cowey & Sargent 1979; Cowey & Walton

1988). This could conceivably lead to a better overall protein

utilization caused by an extended ¯ow of dietary amino acids

from the gastrointestinal tract into the site of protein synthesis

in cells (Boirie et al. 1997). By adding protease inhibitors to a

diet where some silage is included changes in the digestion and

absorption pattern might result in an improved dietary

protein utilization for protein synthesis and growth. The

present study showed an almost signi®cantly higher growth

rate when a protease inhibitor preparation from potato was

added to ®shmeal-based diets. By replacing some of the

®shmeal with hydrolysed ®sh protein the growth performance

using protease inhibitors was further increased. This supports

the hypothesis that an extended digestion and absorption time

of the dietary protein may be advantageous to ®sh.

In this study, the absorption pattern of 14C-labelled amino

acids was not in¯uenced by the dietary inclusion of protease

inhibitors when the 14C-labelled algal was hydrolysed. This

may indicates that the digestion and absorption capacity of

the gastrointestinal tract has little in¯uence on the absorption

pattern of hydrolysed protein. There was, however, a tendency

for faster uptake of 14C-labelled amino acids from intact14C-labelled algal protein when inhibitors were added. This

may be caused by a more rapid enzymatic degradation of the

protein in the intestines; a degradation which is dependent on

the amount of active digestive protease. At a dietary inclusion

of 1 g kg±1 protease inhibitor preparation the enzymatic

activity in the di�erent compartments of the gastrointestinal

tract was not signi®cantly in¯uenced. The amount of absorbed14C-labelled amino acids and small peptides was signi®cantly

higher at any sampling time when the protein was hydrolysed

compared with intact protein (Fig. 4). This demonstrates that

using partly hydrolysed protein in the diets alters the

absorption pattern of dietary protein, a ®nding in agreement

with the results of Espe et al. (1999).

The enzyme activities in the di�erent segments of the

gastrointestinal tract were in general reduced with increasing

inclusions of protease inhibitors in the feed. An inhibition of

trypsin, chymotrypsin and carboxypeptidase A and B by

potato inhibitor has previously been reported (Ryan 1974;

Pearce et al. 1982; Aksnes 1989). At moderate inhibitor

inclusions, chymotrypsin activity in the pyloric caecae and

mid gut region showed an increasing tendency before it was

markedly reduced at higher inclusion levels. Trypsin activity

on the other hand, showed a clear reduction in the pyloric

caecae even at moderate inclusion levels. This is not in

agreement with Olli et al. (1994) who found increased trypsin

secretion when including a moderate amount of soyabean

trypsin inhibitor in diets for Atlantic salmon. Krogdahl et al.

(1994) found a similar result as Olli et al. (1994) using

rainbow trout. Carboxypeptidase A and B show the same

tendencies as chymotrypsin. In this study, enzymatic activity

was measured at one time. Considering the reversible binding

of the inhibitor to the enzyme one has to assume that

enzymatic activity in the gastrointestinal tract will change

over time. It is, therefore, not possible to discuss the overall

activity of the di�erent protease enzymes in the gastrointes-

tinal tract based on the results from this study.

The enzyme activity results together with the response

curve for growth and nitrogen digestion indicates that an

increased enzymatic secretion compensates for a moderate

dietary inclusion of proteolytic enzyme inhibitors. This

increased secretion in combination with the reversible bind-

ing of the inhibitors to the enzymes results in an increase in

the amount of active digestive proteases in the intestines.

In a series of experiments we have demonstrated a positive

e�ect on growth in Atlantic salmon when small amounts of a

protease inhibitor are included in the diets. This did not have

negative e�ects on feed conversion ratio or nitrogen digest-

ibility. Moreover, the positive e�ect of protease inhibitors

was further enhanced when some of the intact protein was

replaced with hydrolysed protein. The absorption pattern of14C-labelled amino acids was a�ected when using intact

protein but no measurable di�erences when using hydrolysed

protein.

Acknowledgements

Marianne Kaland Gjesdal, Leif Pedersen, Henny Dirdal,

Anne Brit Fjermedal and AÊ sveig Tillung at EWOS Innova-

tion Research Station are thanked for taking care of the

experimental ®sh in a conscientious way. The skilled analytic

assistance of Edel Erdal and Anita Birkenes at the Institute

of Nutrition and Einar Odland at the Institute of Molecular

Biology is highly appreciated. Thanks to Oddvar Garathun-

Tjeldstù for helping with the Phast-gel analysis, and Einar

Lied at the Institute of nutrition for valuable discussion of

the experimental set up.

Growth and protein utilization in Atlantic salmon using protease inhibitors

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263

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