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Comparative Biochemistry and Physio

Enzyme activities of intestinal triacylglycerol and phosphatidylcholine

biosynthesis in Atlantic salmon (Salmo salar L.)

Anthony Oxleya,b,T, Bente E. Torstensenb, Arild C. Rustanc, Rolf E. Olsena

aInstitute of Marine Research, Matre Aquaculture Research Station, N-5984 Matredal, NorwaybNational Institute of Nutrition and Seafood Research (NIFES), P.O. Box 2029 Nordnes, 5817 Bergen, Norway

cDepartment of Pharmacology, School of Pharmacy, University of Oslo, P.O. Box 1068 Blindern, 0316 Oslo, Norway

Received 2 November 2004; received in revised form 25 January 2005; accepted 25 January 2005

Abstract

The substitution of fish oil with plant-derived oil in diets for carnivorous fish, such as Atlantic salmon, has previously revealed the

potentially deleterious supranuclear accumulation of lipid droplets in intestinal cells (enterocytes) which may compromise gut integrity, and

consequently, fish health. This suggests that unfamiliar dietary lipid sources may have a significant impact on intestinal lipid metabolism,

however, the mode of lipid resynthesis is largely unknown in teleost fish intestine. The present study aimed at characterising three key

lipogenic enzymes involved in the biosynthesis of triacylglycerol (TAG) and phosphatidylcholine (PC) in Atlantic salmon enterocytes:

monoacylglycerol acyltransferase (MGAT), diacylglycerol acyltransferase (DGAT), and diacylglycerol cholinephosphotransferase (CPT).

Furthermore, to investigate the dietary effect of plant oils on these enzymes, two experimental groups of fish were fed a diet with either

capelin (fish oil) or vegetable oil (rapeseed oil:palm oil:linseed oil, 55:30:15 w/w) as the lipid source. The monoacylglycerol (MAG) pathway

was highly active in the intestinal mucosa of Atlantic salmon as demonstrated by MGAT activity (7 nmol [1-14C]palmitoyl-CoA incorporated

min�1 mg protein�1) and DGAT activity (4 nmol [1-14C]palmitoyl-CoA incorporated min�1 mg protein�1), with MGAT appearing to also

provide adequate production of sn-1,2-diacylglycerol for potential utilisation in PC synthesis via CPT activity (0.4 nmol CDP-[14C]choline

incorporated min�1 mg protein�1). Both DGAT and CPT specific activity values were comparable to reported mammalian equivalents,

although MGAT activity was lower. Nevertheless, MGAT appeared not to be the rate-limiting step in salmon intestinal TAG synthesis. The

homology between piscine and mammalian enzymes was established by similar stimulation and inhibition profiles by a variety of tested

cofactors and isomeric substrates. The low dietary n-3/n-6 PUFA ratio presented in the vegetable oil diet did not significantly affect the

activities of MGAT, DGAT, or CPT under optimised assay conditions, or in vivo intestinal mucosa lipid class composition, when compared to

a standard fish oil diet.

D 2005 Elsevier Inc. All rights reserved.

Keywords: Atlantic salmon; Intestine; Microsomes; Monoacylglycerol pathway; Lipid metabolism; Dietary oil; Lipid composition

1. Introduction

The demand for feed in the salmonid aquaculture

industry has increased over recent years in parallel with

increases in total fish production (Sargent and Tacon, 1999;

Watanabe, 2002). Up to the present, fish feeds have relied

1096-4959/$ - see front matter D 2005 Elsevier Inc. All rights reserved.

doi:10.1016/j.cbpc.2005.01.012

T Corresponding author. National Institute of Nutrition and Seafood

Research (NIFES), P.O. Box 2029 Nordnes, 5817 Bergen, Norway.

Tel.: +47 55905139; fax: +47 55905299.

E-mail address: anthony.oxley@nifes.no (A. Oxley).

mainly on fish meal and oil as protein and lipid sources.

However, traditional marine resources are exploited to the

highest possible level (FAO, 1998) and further sustainable

growth in the carnivorous fish aquaculture industry will

depend on new feed sources becoming available. The most

viable alternatives at present are vegetable protein and oil

sources. Although data suggests that these can be included

into salmonid diets at a high inclusion level, there is still

some concern that these may, in certain circumstances,

compromise fish health and welfare. For example, it has

been shown that the inclusion of plant-derived oils in diets

logy, Part B 141 (2005) 77–87

A. Oxley et al. / Comparative Biochemistry and Physiology, Part B 141 (2005) 77–8778

for species such as Artic charr (Olsen et al., 1998, 1999,

2000), rainbow trout (Caballero et al., 2002; Olsen et al.,

2003) and gilthead seabream (Caballero et al., 2003) may

result in supranuclear accumulation of lipid droplets in

enterocytes, possibly resulting in tissue damage and

compromised gut integrity. This suggests a significant

impact on intestinal lipid metabolism (Olsen et al., 2000;

Caballero et al., 2002), for instance, the biosynthesis of

triacylglycerol (TAG) which is the principal intestinal

synthetic lipid class product and subsequently, along with

phosphatidylcholine (PC), the main constituents required for

the assembly of chylomicrons and VLDL lipoproteins

before exportation from the enterocyte (Chapman, 1980;

Green and Glickman, 1981; Babin and Vernier, 1989). It has

been suggested that the accumulation is caused by an

imbalance in the reacylation of digested lipid into TAG

compared to PC synthesis. Previous studies have shown that

the clearance of enterocytic lipid droplets is dependent on

dietary PC, and also, saturated fatty acids such as palmitic

acid (Olsen et al., 1999, 2000, 2003). Despite these

observations, the enzymes involved in the biosynthetic

pathways of TAG and PC have received little attention in

the intestine of fish.

The biosynthesis of TAG is well known in the intestine

of mammals where the monoacylglycerol (MAG) pathway

utilises absorbed sn-2-monoacylglycerol (2-MAG) tem-

plates for TAG re-esterification and predominates over de

novo TAG synthesis via the glycerol-3-phosphate (G-3-P)

pathway (Johnston, 1977). In the MAG pathway, absorbed

2-MAG is acylated, in the presence of fatty acyl-CoA, at

either the sn-1 or sn-3 position by monoacylglycerol

acyltransferase (MGAT) to yield sn-1,2-diacylglycerol

(1,2-DAG) and, to a lesser extent, sn-2,3-diacylglycerol

(2,3-DAG) (Manganaro and Kuksis, 1985; Lehner et al.,

1993; Lehner and Kuksis, 1996). The resulting 1,2(2,3)-

DAG undergoes subsequent fatty acyl-CoA-dependent

esterification at the sn-3 or sn-1 position to TAG by

diacylglycerol acyltransferase (DGAT). In contrast, G-3-P is

formed from glucose metabolism, or glycerol via glycerol

kinase, and can result in TAG synthesis through a more

energy consuming series of enzyme-catalysed reactions

(Johnston, 1977). However, these pathways converge at

the synthesis of 1,2-DAG where a branch-point, not only

into TAG production occurs, but also into phosphatidyle-

thanolamine (PE) through ethanolaminephosphotransferase,

or PC through cholinephosphotransferase (CPT) (Bell and

Coleman, 1983; Coleman and Lee, 2004).

In fish, current opinion speculates that significant

intestinal TAG biosynthesis may also occur via the MAG

pathway (Henderson and Tocher, 1987; Tocher, 2003)

relating to products of TAG digestion and recent evidence

of a specific pancreatic lipase–colipase system (reviewed by

Olsen and Ringo, 1997). Furthermore, it was concluded that

the mechanism of lipid absorption and lipoprotein formation

in fish intestine does not differ fundamentally to that in

mammals (Sire et al., 1981). To date, the possible

importance of the MAG pathway in fish intestinal TAG

synthesis has been overlooked, and as yet, to the authors’

knowledge, has not been verified in fish. The present study

therefore aims to increase current knowledge on the

lipogenic processes in the intestinal mucosa of fish, with

special emphasis on TAG and PC biosynthesis, by studying

the existence and activities of three key lipogenic enzymes

in Atlantic salmon enterocytes: MGAT, DGAT, and CPT.

Furthermore, to compare the characteristics of these

enzymes in salmon to the mammalian intestinal isoforms

with reference to selected activators and inhibitors. In

addition, to assess the impact and possible interaction

between dietary lipid sources and enzyme activities asso-

ciated with altered enterocyte lipid metabolism.

2. Materials and methods

2.1. Fish and feeding

The dietary feeding trial began in April 2002 at Lerang

Research Station (Nutreco ARC, Stavanger, Norway) where

approximately 2000 Atlantic salmon (Salmo salar L.)

juveniles, having an average weight of 0.160 g (F0.052

g), were randomly distributed equally between six 1 m3

freshwater tanks. Fish were fed the respective diets until

satiation under a 24 h lighting regime and at a mean

temperature of 12.6 8C. During February 2003, the fresh-

water in the tanks was replaced with seawater to coincide

with smoltification. In May 2003, 600 salmon from each

tank were transferred to individual 25 m3 seawater net pens.

The two diets were fed throughout the whole period of April

2002 to August 2003 at the point of sampling, increasing

pellet size with increasing fish weight accordingly.

Throughout the period of May 2003 to August 2003, fish

were subjected to natural light conditions with mean

temperatures ranging from 7.9 to 16.6 8C. Tanks/net penswere assigned randomly with respect to dietary treatment,

with the average values of fish measurements from each

tank treated as one replicate.

The Atlantic salmon used in optimisation experiments

had an approximate weight of 2 kg and were kept in

seawater net pens at Matre Aquaculture Research Station

(Institute of Marine Research, Matredal, Norway). These

fish were fed a standard commercial salmon diet and were

unfed 24 h prior to sampling.

2.2. Diets

Diets were produced by Nutreco ARC (Stavanger,

Norway) based on a typical salmonid feed formula, the

only difference between the two diets being the lipid source:

the fish oil diet containing 100% capelin oil, and the

vegetable oil diet containing rapeseed oil:palm oil:linseed

oil (55:30:15, w/w). The vegetable oil diet was formulated

to obtain a saturated, monounsaturated, and polyunsaturated

A. Oxley et al. / Comparative Biochemistry and Physiology, Part B 141 (2005) 77–87 79

fatty acid profile as similar to the fish oil diet as possible.

Dietary lipids were extracted and identified as described in

detail previously (Torstensen et al., 2004).

2.3. Sampling

Sampling was carried out after 17 months of feeding in

August 2003 by dissecting out the whole intestinal tract of

10 fish from each of the six tanks. Upon sampling, 10 fish

were collected randomly from each net pen and dispatched

humanely after prior sedation with methomidate (1-(1-

phenylethyl)-1 H-imidazole-5-carboxylic acid methyl ester;

7 g L�1). The intestinal tract was excised, removed of

surrounding mesenteric fat using a metal spatula, and the

lumen rinsed through with ice-cold STE buffer (0.25 M

sucrose, 10 mM Tris—pH 7.4, 1 mM EDTA) using a 10 mL

syringe. The intestine was cut open longitudinally and the

mucosa scraped, with the aid of a glass microscope slide,

from the fore and midgut. The mucosa was then homoge-

nised in 4 vol. (w/v) STE buffer, containing 1 mg mL�1

trypsin inhibitor and 5 Ag mL�1 aprotinin, using 10 up-and-

down strokes of an Ultra TurraxR T25 homogeniser (IKA

Works NC, USA) on a medium setting. The homogenates

were subsequently rapidly frozen by immersion in liquid

nitrogen, transported on dry ice, and stored at �80 8C.

2.4. Preparation of microsomes

Intestinal mucosa sampling and microsome preparation

was adapted from Balk et al. (1985) and utilised a rapid two-

step centrifugation scheme. Prior to centrifugation, homo-

genates were rapidly thawed according to Deutscher (1990)

and, due to high viscosity, diluted to 10% (w/v) in ice-cold

STE buffer before centrifuging at 25,000 �gav for 15 min to

remove unbroken cells and both nuclear and mitochondrial

fractions. The supernatant was carefully aspirated off, taking

care not to disturb the pellet or floating fat layer, and

centrifuged at 200,000 �gav on a Beckman Optimak XL-

100 K ultracentrifuge (Beckman Instruments CA, USA) for

150 min at 4 8C. The resultant microsomal pellet was

resuspended in a small volume of STE buffer and stored at

�80 8C. Storage under these conditions has previously

shown not to adversely affect microsomal enzyme activity

(Balk et al., 1985; Pearce et al., 1996). Before use,

microsomes were sonicated twice with a Vibra-Cell 50 T

sonicator (Sonics and Materials CT, USA) set at half setting,

in an ice bath, for two 10 s periods to ensure a uniform

microsomal vesicle distribution.

The yield and purity of the microsome preparation was

assessed by measuring subcellular marker enzyme activity

in the 25,000 �gav pellet (P1), 200,000 �gav pellet (P2),

and 200,000 �gav supernatant (S) compared to the original

homogenate. The marker enzymes assayed for were:

NADPH-cytochrome c reductase for microsomes, succinate

dehydrogenase for mitochondria, acid phosphatase for

lysosomes, and catalase for peroxisomes. Enzymes were

measured according to Graham (1993) under optimal

temperature and pH conditions for the respective enzymes,

using appropriate blanks, and were within linear protein

concentrations and time periods. Relative specific activity

(RSA) was calculated as % enzyme activity recovered in

fraction/% amount of protein recovered in fraction.

2.5. Enzyme assays

An attempt to partially characterise monoacylglycerol

acyltransferase (MGAT, EC 2.3.1.22), diacylglycerol acyl-

transferase (DGAT, EC 2.3.1.20) and diacylglycerol chol-

inephosphotransferase (CPT, EC 2.7.8.2) was made in

reference to known activators and inhibitors of the

mammalian intestinal isoforms of the enzymes. Pilot

studies were also undertaken to quantify the initial rates

of reaction with respect to time and microsomal protein

concentrations. These reactions were all carried out at an

approximately physiological temperature and pH for

Atlantic salmon of 15 8C and pH 7.8 to simulate in vivo

enzyme activities, and also, to compare different enzyme

activities under natural conditions. Microsome preparations

from the intestinal mucosa of each fish were assayed

individually.

2.6. Monoacylglycerol acyltransferase assay

The determination of MGAT activity was adapted from

the method of Coleman and Haynes (1986) based on

optimisation experiments for Atlantic salmon as aforemen-

tioned. The final MGAT reaction mixture contained 150

mM Tris (pH 7.8), 1 mg mL�1 BSA, 25 AM [1-14C]pal-

mitoyl-CoA (specific activity = 50 ACi Amol�1), and 50 AgPC:PS (1:1, w/w). A 100 AL aliquot of this mixture was

taken in triplicate to determine precise specific activity of

the [1-14C]palmitoyl-CoA with respect to dpm. The PC:PS

mixture was dispersed in 10 mM Tris (pH 7.8) by

sonication before addition to the reaction mixture. The

reaction was carried out in a shaking water bath where the

reaction mixture was allowed to equilibrate to 15 8C for 10

min. The reaction was started by the sequential addition of

5 Ag of microsomal protein and 25 AL of 1.0 mM sn-2-

monooleoylglycerol (2-MAG) dispersed in ice-cold ace-

tone followed by brief vortexing. The final reaction

volume was 500 AL with an acetone concentration of

5% which did not significantly inhibit MGAT or DGAT

activity (results not shown). The reaction was terminated

after 10 min of incubation by the addition of 10 mL ice-

cold chloroform:methanol (2:1, v/v) followed by rapid

vortexing.

The lipid products were extracted by the method of Folch

et al. (1957): 2 mL of 0.88% aqueous KCl was added and

shaken with the 10 mL of chloroform:methanol (2:1, v/v)

stop solution before centrifugation at 800 �g for 10 min.

The upper phase was aspirated off and discarded while the

remaining lower phase was evaporated under a stream of

A. Oxley et al. / Comparative Biochemistry and Physiology, Part B 141 (2005) 77–8780

nitrogen. The lipid residue was resuspended in 50 AL of

hexane and applied as a 2 cm streak on silica gel 60 plastic-

backed TLC plates (Merck, Darmstadt, Germany) along

with 1 AL of a MAG: DAG: FFA: TAG: FAME (1:1:1:1:1,

w/w) mixed lipid standard (50 mg mL�1), solubilised in

hexane, applied as a spot at the centre of the streak. Lipid

classes were separated using a hexane:diethyl ether:acetic

acid (65:35:1, v/v) solvent system and visualised by

exposure to iodine vapour. The DAG and TAG bands were

then cut into 20 mL plastic scintillation vials and 10 mL of

Ultima Gold scintillation fluid was added to each vial before

determining radioactivity in a Tri-Carb 1900TR liquid

scintillation counter (Packard Instrument Company, Mer-

iden, CT, USA). Total MGAT activity was calculated by the

sum of dpm recovered in DAG and half the dpm recovered

in TAG (Grigor and Bell, 1982). Greater than 92% of lipid

soluble products were recovered in DAG and TAG.

2.7. Diacylglycerol acyltransferase assay

The standard DGAT assay was modified from Coleman

and Bell (1976) due to optimisation experiments previously

described in Section 2.5. The reaction mixture contained

150 mM Tris buffer (pH 7.8) and 25 AM [1-14C]palmitoyl-

CoA (specific activity = 50 ACi Amol�1). The reaction was

again started by the addition of approximately 5 Ag of

microsomal protein and 25 AL of 1.25 mM sn-1,2-

dioleoylglycerol (1,2-DAG) dispersed in ice-cold acetone

and was performed in a final volume of 500 AL at 15 8C for

10 min. The reaction was terminated, lipids extracted and

identified as described above. DGAT activity was quantified

by the dpm recovered in TAG.

2.8. Diacylglycerol cholinephosphotransferase assay

CPT activity was determined using the reaction con-

ditions of Coleman and Bell (1977) except the final reaction

volume was 500 AL and the pH and temperature were

adjusted to 7.8 and 15 8C, respectively for the same reasons

stated above in Section 2.5. The final reaction mixture

contained 150 mM Tris buffer (pH 7.8), 100 AM CDP-

[14C]choline (specific activity = 10 ACi Amol�1), 8 mM

MgCl2, and 5 mM EGTA. The reaction was started by

addition of 50 Ag of microsomal protein and 25 AL of 2 mM

sn-1,2-dioleoylglycerol dispersed in ice-cold ethanol (5%

final volume) as 5% acetone has previously been shown to

inhibit CPT activity (Coleman and Bell, 1977). The reaction

was stopped after 15 min by addition of chloroform:metha-

nol (2:1, v/v) and the lipid extracted by adding 2 mL of

0.88% aqueous KCl as described above. The lower phase

was then rinsed twice with 1.5 mL of methanol:water (1:1,

v/v) before evaporation under a stream of nitrogen. The lipid

residue was resuspended in 100 AL of chloroform:methanol

(2:1) and transferred to a scintillation vial for radioactivity

counting as described above. More than 96% of the lipid

soluble radioactivity was recovered as PC when analysed

using a double development TLC solvent system of methyl

acetate: propan-2-ol: chloroform: methanol: 0.25% aqueous

KCl (25:25:25:10:9, v/v) and hexane: diethyl ether: acetic

acid (80:20:2, v/v) outlined by Henderson and Tocher

(1992).

2.9. Lipid class and fatty acid determination of mucosa

Sampled intestinal mucosa was homogenised in 4 vol.

STE buffer containing protease and lipase inhibitor as

described above. Homogenates were stored at �80 8C. Totallipid was extracted from 0.5 mL of homogenates following

the method of Folch et al. (1957) with the dried residue

resuspended in approximately 0.5 mL of chloroform.

For fatty acid analysis, the samples were saponified and

methylated using 12% BF3 in methanol. Methyl esters were

separated using a Trace Gas Chromatograph 2000 (Thermo-

Quest CE Instruments, Milan, Italy) (bcold on columnQinjection, 60 8C for 1 min25 8C/min, 160 8C for 28 min25 8C/

min, 190 8C for 17 min25 8C/min, 220 8C for 10 min),

equipped with a 50 m CP-sil 88 (Chromopack, Middelburg,

The Netherlands) fused silica capillary column (id: 0.32

mm). Fatty acids were identified by retention time using

standard mixtures of methyl esters (Nu-Chek Prep, Elyian,

MN, USA). All samples were integrated using Totalchrom

software (version 6.2, Perkin Elmer, Boston, MA, USA)

connected to the GLC.

High performance thin-layer chromatography (HPTLC)

was used to separate and quantify lipid classes. HPTLC

plates were pre-run in hexane:diethyl ether (1:1, v/v) and

activated at 110 8C for 30 min. Ten micrograms of total

lipid was applied to the plate and developed to 5.5 cm in a

methyl acetate:chloroform:methanol:0.25% aqueous KCl

(25:25:25:10:9, v/v) solvent system (Olsen and Henderson,

1989). After drying, plates were developed fully in

hexane:diethyl ether:acetic acid (80:20:2, v/v). Lipid classes

were visualised by spraying the plate with 3% copper

acetate (w/v) in 8% phosphoric acid (v/v) and charring at

160 8C for 15 min. Lipid classes were quantified by

scanning densitometry using a CAMAG TLC Scanner 3

(CAMAG, Muttenz, Switzerland) and calculated with an

integrator (WinCATS-Planar Chromatography Manager,

version 1.2.0, CAMAG). Further, quantitative determination

of lipid classes was achieved by utilising established

standard equations for each lipid class within a linear area,

in addition to including a standard mixture of all lipid

classes on each HPTLC plate to correct for between plate

variations.

2.10. Protein determination

Protein concentration of microsome preparations was

determined by the method of Smith et al. (1985) using a

bicinchoninic acid (BCA) assay kit. Individual samples

were assayed in triplicate and read on a microplate reader at

562 nm using bovine serum albumin (BSA) as a standard.

Table 1

Proximate composition of experimental diets, and fatty acid and lipid class

composition of intestinal mucosa from fish oil (FO)- and vegetable oil

(VO)-fed fish

FO diet VO diet FO mucosa VO mucosa

Proximate composition (% d.w. of feed)

Protein 45.0 45.4 – –

Lipid 27.9 28.1 – –

Ash 8.9 8.7 – –

Moisture 6.1 7.0 – –

Fatty acid composition (% w.w. of total fatty acids)

14:0 6.6 1.0 3.5 F0.7 0.8 F0.1

16:0 13.7 15.9 16.1 F0.8 15.3 F0.6

18:0 2.5 3.4 6.0 F0.8 6.3 F0.5

16:1n-7 4.1 0.8 3.0 F0.7 0.6 F0.1

18:1n-7 2.1 2.2 2.5 F0.2 1.8 F0.0

18:1n-9 11.5 42.7 10.3 F1.4 26.6 F3.0

20:1n-9 9.1 1.3 7.7 F1.7 2.1 F0.3

22:1n-11 15.2 0.7 5.8 F1.7 0.6 F0.3

18:2n-6 2.6 14.6 2.6 F0.4 10.3 F0.1

18:3n-3 1.1 11.4 0.5 F0.1 4.5 F0.5

18:4n-3 2.5 0.2 0.7 F0.2 0.5 F0.1

20:5n-3 6.6 1.2 5.7 F1.3 3.7 F1.3

22:5n-3 1.0 0.2 1.5 F0.1 1.0 F0.2

22:6n-3 10.1 2.1 23.2 F3.7 18.4 F1.6Psaturates 24.6 21.9 26.8 F1.1 23.0 F1.1

Pmonoenes 45.4 47.9 32.9 F6.2 32.5 F3.9

Pn-6 PUFA 3.5 14.7 4.9 F0.3 14.7 F0.5Pn-3 PUFA 22.5 15.2 32.2 F4.7 28.6 F2.5

P

A. Oxley et al. / Comparative Biochemistry and Physiology, Part B 141 (2005) 77–87 81

2.11. Materials

[1-14C]palmitoyl-CoA (55 mCi mmol�1) and CDP-

[14C]choline (55 mCi mmol�1) were purchased from

American Radiolabeled Chemicals (St. Louis, MO, USA).

sn-1- and sn-2-monooleoylglycerol, sn-1,2- and sn-1,3-

dioleoylglycerol, phosphatidylcholine (from beef brain),

phosphatidylserine (from egg lecithin), essentially fatty

acid-free bovine serum albumin, cytochrome c (from bovine

heart), and NADPH were obtained from Sigma-Aldrich (St.

Louis, MO, USA). Ultima Gold scintillation cocktail was

from Packard BioScience (Groningen, The Netherlands).

Plastic-backed silica gel 60 TLC plates and all organic

solvents, of analytical grade, were obtained from Merck

(Darmstadt, Germany). The BCA protein assay kit was

purchased from Pierce (Rockford, IL, USA).

2.12. Statistical analysis

A one-way analysis of variance (ANOVA) test was

performed on MGAT, DGAT and CPT activity values to

discern levels of significance with regard to dietary treatment

using SPSS software (SPSS, Chicago, IL). Although micro-

somes from each fish were assayed individually, the mean

respective enzyme activities from each tank were considered

as n=1 to counter pseudo replication.

PUFA 26.0 29.9 37.3 F4.7 43.3 F2.9

n-3/n-6 ratio 6.4 1.0 6.6 F1.2 1.9 F0.1

Lipid class composition (% w.w. of total lipid)

PC 4.1 4.2 17.1 F1.7 16.7 F2.3

PE 1.8 1.9 8.9 F1.0 9.7 F0.7

MAG 2.8 3.3 – – – –

C 7.2 8.9 14.1 F1.1 13.1 F0.3

FFA 11.5 7.8 5.2 F1.1 7.0 F1.8

TAG 63.9 65.8 34.9 F7.6 35.1 F1.8

SE 5.8 5.3 – – – –

Abbreviations: PUFA, polyunsaturated fatty acid; FA, fatty acid; PC,

phosphatidylcholine; PE, phosphatidylethanolamine; C, cholesterol; FFA,

free fatty acid; TAG, triacylglycerol; SE, sterol ester.

The mean total extracted lipid from intestinal mucosa for FO- and VO-fed

fish was 57.6 and 58.1 mg g�1 mucosa (w.w.), respectively. Values are

means of triplicate measurementsFSD.

3. Results

3.1. Diets and fish growth

The experimental vegetable oil diet was formulated to

match the fish oil diet as closely as possible with respect to

total saturated, monounsaturated and polyunsaturated fatty

acid composition (Table 1). Both diets had a lipid content of

28% containing similar proportions of saturated and mono-

unsaturated fatty acids. However, the fish oil diet contained

considerably more of the n-3 highly unsaturated fatty acids

(HUFA), such as 20:5n-3, 22:5n-3 and 22:6n-3, with the

vegetable oil diet containing more n-6 PUFA, resulting in a n-

3/n-6 fatty acid ratio of 6.4 and 1.0 for respective diets. The

majority of n-3 PUFA in the vegetable oil diet was 18:3n-3.

There were no significant differences between the fish oil and

vegetable oil diets with respect to proximate composition,

total saturates, monoenes and PUFA composition, and TAG

and PL classes. No significant differences in specific growth

rate (SGR) or feed conversion rate (FCR) were observed

during the whole feeding trial. The final average weight of the

fish at the point of sampling was 960F48 g in the 100% fish

oil group and 915F43 g in the 100% vegetable oil group.

3.2. Intestinal microsome preparation

A relatively high force and time period was required to

sediment microsomes from intestinal mucosal homogenates

during the final ultracentrifugation step. Attempts to isolate

microsomes from intestinal mucosa using a more conven-

tional centrifugal force of 106,000 �gav for 60 min only

resulted in the recovery of 5% total protein and 5%

NADPH-cytochrome c reductase activity from original

homogenates (data not shown). Increasing the force from

106,000 to 200,000 �gav for 150 min yielded 20% of

protein from the original homogenate along with approx-

imately 50% of total NADPH-cytochrome c reductase

activity giving a relative specific activity (RSA) of 2.4

(Fig. 1). Utilising a force of 25,000 �gav (15 min) for the

initial centrifugation step ensured that most of the mito-

chondria and peroxisomes were sedimented in the P1

fraction, and did not contaminate the P2 fraction, resulting

in a relatively pure microsomal preparation. Acid phospha-

Fig. 1. Distribution of marker enzymes for lysosomes (acid phosphatase), peroxisomes (catalase), mitochondria (succinate dehydrogenase), and microsomes

(NADPH-cytochrome c reductase) in the 25,000 �g pellet (P1) and 200,000 �g pellet (P2) plus supernatant (S). Results are expressed as bDe Duve plotsQ (DeDuve et al., 1955) with bars representing the mean of 3 separate subcellular fractionation preparations.

A. Oxley et al. / Comparative Biochemistry and Physiology, Part B 141 (2005) 77–8782

tase activity had a more even distribution between fractions.

The supernatant contained the greatest proportion of protein

(ca. 50%) and also accounted for 26% of the NADPH-

cytochrome c reductase activity. Freezing the microsomal

preparation at �80 8C for 2 months did not affect enzyme

activity. The preparation was also stable for up to 5 h at 4 8Cfollowing thawing, however, rapid loss of enzyme activity

was observed over 1 h when incubated at 25 8C decreasing

to 60% of starting activity (data not shown).

3.3. Monoacylglycerol acyltransferase (MGAT) activity

dependences

MGAT displayed a high incorporation rate of [1-14C]pal-

mitoyl-CoA which was linear during the first 15 min of

incubation with 10 Ag of microsomal protein (Fig. 2) with

an average specific activity of 7 nmol [1-14C]palmitoyl-CoA

min�1 mg protein�1. Conversely, the reaction was linear up

to 10 Ag of microsomal protein with a 10 min reaction time.

The MGAT reaction was dependent on the presence of BSA

and was inhibited by high concentrations of MgCl2 (8 mM),

especially in the absence of BSA (Table 2). With respect to

divalent cations at a concentration of 2.5 mM, the reaction

was inhibited by Mn2+NCa2+NMg2+, with all divalent ions

tested having an inhibitory effect. Addition of 50 Ag of an

equimolar mixture of PC and PS markedly stimulated

MGAT activity by 188%. Endogenous synthesis of DAG

and TAG proceeded at 24% the rate when the 2-MAG

substrate was omitted. The utilisation of a 2-MAG substrate

was greater than for sn-1(3)-monoacylglycerol (1(3)-MAG)

proceeding at 7.0 and 5.5 nmol [1-14C]palmitoyl-CoA

min�1 mg protein�1, respectively (Fig. 3). However, the

DAG:TAG ratio increased substantially to 13 when 1(3)-

MAG was used compared to the 2-MAG where the ratio

was 2.8.

3.4. Diacylglycerol acyltransferase (DGAT) activity

dependences

Linearity of [1-14C]palmitoyl-CoA incorporation into

TAG was observed up to 30 min for DGAT activity with

respect to optimal reaction conditions and 10 Ag of

microsomal protein (Fig. 2). However, the reaction was

more influenced by increasing microsome protein concen-

tration, plateauing out at 15 Ag after 10 min of incubation.

The mean specific activity within the linear region of

reaction for DGAT was approximately 4 nmol [1-14C]pal-

mitoyl-CoA min�1 mg protein�1.

The initial reaction conditions selected proved to be the

most suitable for optimal DGAT activity (Table 3). DGAT

did not have a requirement for BSA, with MgCl2 being a

potent inhibitor at a concentration of 8 mM. However, when

BSA and MgCl2 were presented simultaneously, a rather

paradoxical non-additive inhibition of DGAT activity

seemed to occur; in fact DGAT activity returned to 73%

of the optimum. In accordance with MGAT, MnCl2 was a

potent inhibitor at 2.5 mM, while in contrast to MGAT,

CaCl2 seemed not to have a marked effect on DGAT activity

at the same concentration. Endogenous DGAT activity

proceeded at 8.6% of the exogenous rate when the DAG

substrate was omitted. DGAT was highly specific towards

the 1,2-DAG substrate isomer with little incorporation of

Table 2

MGAT reaction dependences

Relative activities (%)

Initial systema 100.0

�1 mg/mL BSA 72.1

�1 mg/mL BSA,+8.0 mM MgCl2 21.3

+2.5 mM MgCl2 90.6

+8.0 mM MgCl2 60.2

+2.5 mM MnCl2 26.9

+2.5 mM CaCl2 51.9

+50 Ag PC:PS (1:1, w/w) 188.5

�MAG 24.2

�Microsomes 0.0

a The initial system contained 150 mM Tris (pH 7.8), 1 mg mL�1 BSA,

25 AM [1-14C]palmitoyl-CoA. The amount of microsomal protein and

reaction conditions employed are as described in the Materials and Methods

section. The mean specific activity from 3 individual microsome

preparations was 7 nmol [1-14C]palmitoyl-CoA incorporated min�1 mg

protein�1 (corrected for endogenously synthesized acylglycerols).

A. Oxley et al. / Comparative Biochemistry and Physiology, Part B 141 (2005) 77–87 83

radioactivity into TAG when the 1,3-DAG isomer was

utilised (Fig. 3). There was, unusually, a production of

radioactive DAG, the rate of which, proceeded at similar

rates irrespective of isomeric substrate.

3.5. Diacylglycerol cholinephosphotransferase (CPT)

activity dependences

The initial rate of reaction for CPT was linear up to

around 20 min when 50 Ag of microsomal protein was

employed under optimal reaction conditions (Fig. 2). The

sluggishness of the reaction meant that PC formation was

linear up to a protein amount of 70 Ag for an incubation

period of 15 min. BSA was not important for the reaction

system, while an absence of EGTA reduced activity by 30%

(Table 4). CPTwas the most heavily affected out of the three

enzymes by the inclusion of 8 mM MgCl2, reducing activity

to just 3%. CPT was also the enzyme most inhibited by 2.5

mM CaCl2 and also the most resistant to 2.5 mM MnCl2resulting in a relative activity of 76%. The presence of 25

AM palmitoyl-CoA did not have a marked effect on CPT

activity. Endogenous PC formation was considerable at 57%

when the 1,2-DAG substrate was omitted.

3.6. Effect of dietary lipids on intestinal mucosa lipid

composition and enzyme activities

Differences between fish oil- and vegetable oil-fed fish

were not significant regarding MGAT, DGAT and CPT for

the dietary vegetable oil-fed fish (Table 5). MGAT displayed

Fig. 2. Progress of the MGAT (A), DGAT (B), and CPT (C) reactions,

under optimal conditions described in the Materials and Methods section,

with respect to time and protein. Both MGAT and DGAT reactions were

carried out with 10 Ag of microsomal protein for time determinations and a

10 min incubation period for protein determinations, with 50 Ag and 15 min

utilised for CPT time and protein determinations respectively. Data points

represent meansFSD for 3 individual microsome preparations, and are

corrected for endogenously synthesized lipid products.

Fig. 3. Substrate specificity of the monoacylglycerol acyltransferase

(MGAT) and diacylglycerol acyltransferase (DGAT) reactions using 1-

monoolein (1-MAG) and 2-monoolein (2-MAG) for MGAT and 1,2-diolein

and 1,3-diolein (1,3-DAG) for DGAT. Results are expressed as the total

mean incorporation of [14C]palmitoyl-CoA into diacylglycerol (DAG) and

triacylglycerol (TAG)FSD for 3 microsome preparations, and are corrected

for endogenously synthesized lipid products. Reactions were carried out as

described in the text.

Table 4

CPT reaction dependences

Relative activities (%)

Initial systema 100.0

�8 mM MgCl2 3.3

�5 mM EGTA 69.6

+1 mg/mL BSA 87.0

+2.5 mM MnCl2 76.1

+2.5 mM CaCl2 6.0

+25 AM palmitoyl-CoA 88.6

�DAG 57.1

�Microsomes 0.0

a The initial system, amount of microsomal protein and reaction

conditions employed are as described in the Materials and Methods section.

The mean specific activity from 3 individual microsome preparations was

0.4 nmol CDP-[14C]choline incorporated min�1 mg protein�1 (corrected for

endogenously synthesized phosphatidylcholine).

A. Oxley et al. / Comparative Biochemistry and Physiology, Part B 141 (2005) 77–8784

the highest activity of the three enzymes measured at a mean

of 6.23 and 6.89 nmol [1-14C]palmitoyl-CoA incorporated

min�1 mg protein�1 for fish oil- and vegetable oil-fed fish

respectively. DGAT exhibited approximately 3.5 times lower

activity than MGAT, with mean activities of 1.12 and 1.53

nmol [1-14C]palmitoyl-CoA incorporated min�1 mg

protein�1 for respective dietary treatments. CPT activity

was substantially lower than MGAT or DGAT activity with

average specific activities of 0.10 and 0.18 CDP-[14C]chol-

ine incorporated min�1 mg protein�1 for individual dietary

exposure. The DGAT and CPT activities were lower in

assays for the dietary fish compared to fish used in

optimisation experiments which could be attributable to

partial isomerisation of the 1,2-DAG, dissolved as an

acetone stock solution, to the 2,3-DAG or 1,3-DAG isoform.

Table 3

DGAT reaction dependences

Relative activities (%)

Initial systema 100.0

+1 mg/mL BSA 52.9

+8.0 mM MgCl2 34.4

+1 mg/mL BSA,+ 8.0 mM MgCl2 73.3

+2.5 mM MgCl2 60.0

+2.5 mM MnCl2 14.5

+2.5 mM CaCl2 85.6

�DAG 8.6

�Microsomes 0.0

a The initial system, amount of microsomal protein and reaction

conditions employed are as described in the Materials and Methods section.

The mean specific activity from 3 individual microsome preparations was 4

nmol [1-14C]palmitoyl-CoA incorporated min�1 mg protein�1 (corrected

for endogenously synthesized triacylglycerol).

The lipid class composition of the intestinal mucosa was

between 34.9% and 35.1% TAG and 17.1% and 16.7% PC

for the fish oil and vegetable oil diets, respectively (Table 1).

These differences were not significant, as were none of the

other listed lipid classes listed. The total saturated, mono-

unsaturated, and polyunsaturated composition of the intes-

tinal mucosa (Table 1) did not differ significantly with

dietary treatment. Also, the n-3/n-6 PUFA ratio was similar

to that represented in diets, although the 22:6n-3 proportion

in mucosa substantially increased in vegetable oil-fed fish

corresponding with a decrease in 18:3n-3. The absolute

values of total lipid extracted from mucosa, along with

relatively low proportions of TAG, are consistent with

successful lipid clearance from enterocytes.

4. Discussion

Monoacylglycerol acyltransferase (MGAT), diacylgly-

cerol acyltransferase (DGAT) and diacylglycerol choline-

phosphotransferase (CPT) are intrinsic membrane-bound

enzymes associated with the endoplasmic reticulum (Cole-

man, 1992; Lehner and Kuksis, 1996; McMaster and Bell,

1997). Therefore, microsomes were isolated, via subcellular

fractionation, and used to assay these enzymes for determi-

nation of activities. The aim of subcellular fractionation

from Atlantic salmon intestinal mucosa was to prepare a

microsomal fraction that was high in yield and purity,

without significant loss of enzyme activity associated with

the preparation and storage of microsomes.

Table 5

Influence of dietary vegetable oil (VO) on intestinal MGAT, DGAT and

CPT microsome activities compared to dietary fish oil (FO)

MGAT DGAT CPT

FO (n=3) 6.23F1.06 1.12F0.37 0.10F0.03

VO (n=3) 6.89F1.65 1.53F1.15 0.18F0.06

Values represent nanomoles of [1-14C]palmitoyl-CoA or CDP-[14C]choline

incorporated min�1 mg protein�1 as described in the text FSD, and are

corrected for endogenously synthesized lipid.

A. Oxley et al. / Comparative Biochemistry and Physiology, Part B 141 (2005) 77–87 85

The centrifugal force required to sediment 50% of

microsomes was 200,000 �gav for a period of 150 min

which is higher than that stated for mammalian intestinal

mucosa (Hubscher et al., 1965). In this study, increasing the

centrifugal force and period from 106,000 �gav for 60 min

to 200,000 �gav for 150 min increased the microsome yield

10-fold, resulting in a relative specific activity (RSA) of 2.4.

A microsomal yield of 50% and purity of 2.4 (RSA) is

consistent with previous fractionations in fish intestinal

mucosa (Balk et al., 1985) and other fish tissues (Statham et

al., 1977; Andersson, 1992). However, significant NADPH-

cytochrome c reductase activity remained in the post

200,000 �gav supernatant which has previously been

attributed to solubilisation of active enzyme fragments due

to endogenous proteases (Balk et al., 1985). This is

confirmed by the use of trypsin inhibitor in a previous

study (Pesonen and Andersson, 1987) to prevent solubilisa-

tion of microsomal enzymes into the cytosol.

Little cross-contamination occurred in the microsomal

fraction with respect to mitochondria and peroxisomes due

to the majority of these organelles being sedimented in the

initial 25,000 �gav (15 min) centrifugation step. However,

there was an even distribution of acid phosphatase activity

across all fractions reiterating the labile nature of lysosomes

during fractional centrifugation (Hinton and Mullock,

1997). Due to the relative purity of the microsomal

preparation, there was no significant loss in enzyme activity

due to protein degradation when stored at �80 8C for 2

months and when kept at 4 8C for 8 h following thawing.

However, appreciable loss occurred in enzyme activity over

an hour when incubated at 25 8C. Nevertheless, assays forMGAT, DGAT and CPT were conducted at near physio-

logical temperatures for Atlantic salmon of 15 8C and for 15

min or less. Moreover, previous studies regarding, at least,

DGAT stability have reported that microsomal preparations

from rat adipocytes remained viable at 23 8C for 15 min

(Coleman and Bell, 1976).

The monoacylglycerol (MAG) pathway is the predom-

inant triacylglycerol (TAG) synthetic pathway in mammals,

following prior digestion of TAG to sn-2-monoacylglycerol

(2-MAG) by specific pancreatic lipase, and subsequent

absorption into the enterocyte. The MAG pathway was

clearly active in Atlantic salmon intestinal mucosa as

evidenced by the high activities of both MGAT and DGAT.

DGAT specific activity was remarkably comparable (4 nmol

[1-14C]palmitoyl-CoA min�1 mg protein�1) to mammalian

DGAT (1.2–5.5 nmol [1-14C]palmitoyl-CoA min�1 mg

protein�1) (Mansbach, 1973; Coleman and Bell, 1976;

Lehner and Kuksis, 1996). However, the specific activity of

MGAT was much lower (7 nmol [1-14C]palmitoyl-CoA

min�1 mg protein�1) than that generally described for

mammalian intestinal microsomes (65–181 nmol

[1-14C]palmitoyl-CoA min�1 mg protein�1) (Grigor and

Bell, 1982; Coleman and Haynes, 1986). The low MGAT

specific activity could imply the greater contribution of the

glycerol-3-phosphate (G-3-P) pathway to provide diacyl-

glycerol (DAG) for TAG synthesis in fish. This is consistent

with the view that the digestion products from teleost fish

are both 2-MAG and glycerol relating to the specific and

non-specific nature of intestinal lipases (Tocher, 2003).

Mammalian isoforms of the MGAT, DGAT and CPT

enzymes have been previously extensively characterised

with respect to substrate specificity, specific activity and

various stimulatory/inhibitory cofactors. To elucidate the

presence of an analogous MAG pathway in Atlantic salmon

intestinal mucosa, microsomal preparations were subjected

to a series of optimisation experiments to draw comparisons

with mammalian MGAT and DGAT.

In the current study, MGAT exhibited similar inhibition

characteristics with Mg2+ and Mn2+, and stimulation

characteristics with BSA and PC: PS, compared to mamma-

lian MGAT (Bierbach, 1983; Coleman and Haynes, 1986).

MGAT also displayed a broad specificity towards isomeric

MAG substrates with the production of DAG proceeding at

75% the rate with 1(3)-MAG than 2-MAG. This broad

utilisation by intestinal MGAT has been described in

mammals where the enzyme is capable of acylating both

the sn-1 and sn-3 positions of sn-2-MAG to yield sn-1,2- or

sn-2,3-DAG, and the sn-3 position of sn-1-MAG yielding

sn-1,3-DAG (sn-3-MAG is not utilised at all) (Lehner et al.,

1993). However, neither MGAT nor DGATcan acylate at the

sn-2 position which explains the observed low further

incorporation of DAG into TAG with the 1(3)-MAG

substrate. The residual TAG formation is probably due to

intermediate isomerisation of sn-1(3)-MAG to sn-2-MAG,

or sn-1,3-DAG to sn-1,2-or sn-2,3-DAG. The rapid accu-

mulation of DAG, and higher specific activity of MGAT

utilising the preferred 2-MAG substrate, compared to TAG

indicates that DGAT, and not MGAT, is the rate-limiting step

in the MAG pathway in Atlantic salmon.

Regarding DGAT, Ca2+ and Mn2+ inhibition was in

accordance with mammalian DGAT, although BSA inhib-

ition was not (Coleman and Bell, 1976). Analysis of all lipid

class products revealed a substantial proportion (ca. 15%) of

radioactivity in DAG. This phenomenon has been described

previously in rat intestinal microsomes which was attributed

to MGAT activity synthesising endogenous MAG (Chautan

et al., 1991). It has also been shown that enterocytes contain

appreciable amounts of pancreatic lipase (Tsujita et al.,

1996) which may act on the exogenous 1,2-DAG substrate

providing 2-MAG for reacylation.

The production of sn-1,2-diacylglycerol (1,2-DAG) via

the MAG pathway, or G-3-P pathway, provides a template

for TAG, PE, or PC synthesis. Cholinephosphotransferase

(CPT) provides a synthetic route for PC at this branch-point,

where appreciable contribution from 2-MAG can occur

(Lehner and Kuksis, 1992). Therefore, there maybe some

competition between DGAT and CPT for 1,2-DAG as TAG

and PC are the respective main intestinal synthetic lipid

products for lipoprotein export from the enterocyte. CPT

activity in intestinal microsomes from Atlantic salmon

commenced at approximately 10% the rate (0.4 nmol

A. Oxley et al. / Comparative Biochemistry and Physiology, Part B 141 (2005) 77–8786

CDP-[14C]choline incorporated min�1 mg protein�1) of

DGAT. Compared to mammalian CPT, values between 0.9

and 8.7 nmol CDP-[14C]choline incorporated min�1 mg

protein�1 have been reported (Gurr et al., 1965; Coleman

and Bell, 1977; Cornell, 1992). Interestingly, Holub et al.

(1975) described a specific activity of approximately 0.2

nmol CDP-[14C]choline incorporated min�1 mg protein�1

for CPT in hepatic microsomes from rainbow trout when

assayed at 15 8C. Therefore, the value of CPT specific

activity obtained from this study correlates well with CPT

from another salmonid tissue.

The homology between mammalian and piscine CPT

activity has been demonstrated previously in fish liver and

brain (Holub et al., 1975; Hazel, 1990) but not intestine. The

absolute requirement of CPT for Mg2+ demonstrated in this

study, along with Ca2+ inhibition and EGTA stimulation,

agree with the mammalian enzyme. CPT also appeared to be

able to utilise a high proportion of endogenous DAG (57%)

when the exogenous 1,2-DAG was omitted. This effect has

been demonstrated in microsomes derived from rainbow

trout liver at 15 8C, although endogenous activity decreased

at higher incubation temperature (Holub et al., 1975). Also,

significant endogenous synthesis of PC from CPT activity is

described in microsomes from mammalian tissues (Kanoh

and Ohno, 1981; Cornell, 1992).

Previous studies where plant-derived oils have been fed

to carnivorous fish have revealed potentially deleterious

morphological effects on enterocytes which could compro-

mise gut integrity (Olsen et al., 1998, 1999, 2000, 2003;

Caballero et al., 2002, 2003). Therefore, enzymes synthe-

sising TAG and PC, the two principal lipid components for

lipoprotein export, were investigated with regard to eluci-

dating the relationship between dietary lipid source and the

activities of MGAT, DGAT and CPT.

There was no significant effect on MGAT, DGAT or CPT

activity in the intestinal mucosa of Atlantic salmon between

fish fed diets supplemented with vegetable oil of fish oil.

Further, the lipid class composition of the intestinal mucosa

was not affected by dietary treatment. This could be

explained by the close similarity in total saturated, mono-

unsaturated, and polyunsaturated fatty acid and lipid class

composition provided by both diets. Although the vegetable

oil diet had an inferior n-3/n-6 fatty acid ratio compared to

the fish oil diet, both diets contained similar levels of

saturated fatty acids (such as 16:0) that are required for PC

synthesis. Mammalian CPT shows maximal activity with

1,2-DAG containing a saturated fatty acid and unsaturated

fatty acid at the sn-1 and sn-2 positions respectively

(Morimoto and Kanoh, 1978). Therefore, it maybe the

availability of saturated fatty acids for acylation at the sn-1

position that dictates the synthesis of PC for lipoprotein

assembly and clearance of TAG. It is concluded that the

formulated vegetable oil diet in this study does not

significantly affect the biosynthesis of TAG, via the MAG

pathway, or PC, via 1,2-DAG intermediates, in the intestinal

mucosa of Atlantic salmon.

Acknowledgements

This work was carried out with financial support from

the Norwegian Research Council bKrill as feed source

for fishQ (146871/120) and the Commission of the Eu-

ropean Communities, Quality of Life and Management of

Living Resources programme, project Q5RT-2000-31656

bGastrointestinal Functions and Food Intake Regulation in

Salmonids: Impact of Dietary Vegetable LipidsQ (GUTIN-TEGRITY). This work does not represent the opinion of the

European Community, which is thus not responsible for any

use of the data presented. The fish feeding experiment was

part of the RAFOA project Q5RS-2000-30058: bResearchingAlternatives to Fish Oil in AquacultureQ. The authors are

indebted to Prof. Livar Frbyland for his enthusiastic and

skilled support during initiation of the project.

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