Aquaculture 285 (2008) 193–200

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Aquaculture
j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / a q u a - o n l i n e

Novel production of Atlantic salmon (Salmo salar) protein based on combined

replacement of fish meal and fish oil with plant meal and vegetable oil blends
B.E. Torstensen a,⁎, M. Espe a, M. Sanden a, I. Stubhaug a, R. Waagbø a, G.-I. Hemre a, R. Fontanillas c,
U. Nordgarden b, E.M. Hevrøy a, P. Olsvik a, M.H.G. Berntssen a
a
National Institute of Nutrition and Seafood Research, NIFES, Bergen, Norway
b
Marine Research Institute, Matredal, Norway
c
Skretting Aquaculture Research Centre (ARC), P.O. Box 48, 4001, Stavanger, Norway

a r t i c l e i n f o a b s t r a c t

Article history: The aim of the present study was to combine maximum replacement of fish meal and fish oil with plant
Received 3 July 2008 ingredients in feed for Atlantic salmon, in order to gain a sound and sustainable net fish protein production. The
Received in revised form 19 August 2008 design implied that all known nutrient requirements should be met. Atlantic salmon smolts with an initial
Accepted 21 August 2008
weight of 0.3 kg were fed in triplicate either a fully marine control diet or one of three plant based diets through
the seawater production phase for 12 months, until final weight of approximately 4 kg. In a maximum plant
Keywords:
Vegetable oil
based diet, 80% of the fish meal was replaced with a mixture of plant protein ingredients and krill meal, while
Plant protein 70% the fish oil was replaced with a mixture of vegetable oils. Two intermediate replacement diets contained
Fish meal either one half of this fish meal replacement level and maximum fish oil replacement, or one half replacement
Fish oil level of fish oil and maximum fish meal replacement. Fish performance was assessed by measuring mortality,
Growth feed intake, growth, nutrient digestibility and nutrient utilisation. Specific growth rate was significantly lower
Nutrient utilisation in the combined high replacement group compared to the other experimental groups, both for the first 3-
Nutrient digestibility month period (12%) and for the complete 12 months (9%) of feeding. The final fish weights were 17% lower in
the combined high replacement group and 9% lower in the high plant protein and intermediate vegetable oil
group, compared to the marine control and the intermediate plant protein group. Significantly reduced feed
intake during the first period and slightly reduced digestibility of 16:0 and starch were identified as possible
causes for growth depression, since minor differences in protein or lipid digestibility, feed conversion ratio, and
protein and lipid retention were observed.
The maximum fish meal and fish oil replacement represented a net production of fish protein, with 2 kg salmon
protein produced per kg fish meal protein fed. This being four-fold more efficient usage of fish meal in the 80%
plant protein diets compared to the 100% fish raw material diet.
© 2008 Elsevier B.V. All rights reserved.

1. Introduction
Farmed Atlantic salmon have traditionally been fed diets containing
large amounts of fish oil and fish meal. The steady increase in production
volume in aquaculture of 8–10% a year (Tacon, 2004; Tacon et al., 2006),
has resulted in increasing use of alternative proteins and oils in aqua
feeds. Sustainable fish farming includes the use of diets formulated using
economical, suitable and ethical acceptable feed ingredients. The use of
Abbreviations: FM, fish meal; FO, fish oil; VO, vegetable oil; PP, plant protein; IAA,
indispensable amino acids; PPV, protein productive value; LPV, lipid productive value;
CF, condition factor; FCR, feed conversion ratio; SGR, specific growth rate; PER, protein
efficiency ratio; LER, lipid efficiency ratio; EPV, energy productive value; ADC, apparent
digestibility coefficient.
⁎ Corresponding author. P.O. Box 2029, Strandgt 229, 5817 Bergen, Norway. Tel.: +47
55905200; fax: +47 55905299.
E-mail address: bente.torstensen@nifes.no (B.E. Torstensen).
fish meal and oil, representing 3–5 kg fresh fish per kg farmed fish have
been criticised both for exploitation of the global fishery resources and
the misuse of high quality marine protein and lipid sources that could be
directly used for human consumption. In this respect, vegetable oils (VO)
have been proposed as sustainable alternatives to fish oil (Torstensen
et al., 2005). VOs are however devoid of n − 3 PUFAs (EPA, DPA and DHA)
while the levels of 18:2n − 6 and monoene fatty acids are usually high,
resulting in low dietary n − 3/n − 6 ratios. Likewise, sustainable alter-
natives to fish meal have included various plant protein sources, such as
vegetable meals with crude protein content of 20 to 50 % (Hertrampf and
Piedad-Pascual, 2000). However, the indispensable amino acid (IAA)
profile in plant proteins differs from fish meal. Sensible blending of
different protein sources are necessary to balance the IAA composition,
while low levels of certain crystalline AAs may have to be added to fulfil
AA requirements (NRC, 1993; Halver and Hardy, 2002). When including
plant protein sources, the carbohydrate fraction as well as anti nutrients

0044-8486/$ – see front matter © 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.aquaculture.2008.08.025
194 B.E. Torstensen et al. / Aquaculture 285 (2008) 193–200

may alter digestion and nutrient utilisation (Francis et al., 2001;
Krogdahl et al., 2005).
Several studies have reported no growth reduction when the lipid
from fish oils were completely replaced by selected single or mixed VOs
in diets for Atlantic salmon, leaving the fish meal lipid (80–100 g kg− 1) as
the only source of n − 3 PUFA (Waagbø et al., 1991; Torstensen et al.,
2000, 2004, 2005; Bell et al., 2001, 2002). In fact, both growth and
protein utilisation were significantly improved in Atlantic salmon when
fed VO diets at low (6 °C) water temperatures (Torstensen et al., 2005).
Apparent digestibility of protein, lipid and fatty acids were highly
influenced by environmental temperature, with less saturated and more
unsaturated fatty acids having a positive effect on digestibility at lower
water temperatures (Torstensen et al., 2000; Caballero et al., 2002;
Bendiksen et al., 2003; Menoyo et al., 2003; Ng et al., 2004).
A large body of literature presents studies on alternative protein
sources to fish meal in feeds for salmonids, both by using blends of
plant proteins with or without amino acid supplementation, as well as
total replacement of fish meal (Gomes et al., 1995; Kaushik et al., 1995;
de Francesco et al., 2004; Espe et al., 2006). Increased replacement of
fish meal by plant proteins in diets for salmonids has resulted in
reduced growth performance, however this was believed to be caused
by reduced feed intake and impact on gut integrity, while apparent
protein and amino acid digestibilities were unaffected (Kaushik et al.,
2004; Espe et al., 2006). In these studies lipid retention increased in
rainbow trout (Kaushik et al., 2004) but decreased in Atlantic salmon
(Espe et al., 2006) at high plant protein inclusions.
The current experiment was designed to maximise the replacement
of fish meal and oil with plant ingredients in feeds for Atlantic salmon
growers, based on recent knowledge gained from research focused on
protein (e.g. EU-project PEPPA Q5RS-CT-2000-30068; Kaushik et al.,
2004; Espe et al., 2006) and lipid replacements (e.g. EU-project RAFOA
Q5RS-2000-30058; Bell et al., 2002, 2003a,b; Torstensen et al., 2004,
2005). The present focus was to combine maximum replacement of fish
meal protein and fish oil with plant ingredients in diets fed throughout
the major production phase in sea, in order to gain a sound and
sustainable net production of fish for human consumption. Feed intake,
digestibility, nutrient retention and growth were assessed in four
samplings during the 12 month experimental production period from
smolt to slaughter size. This paper is the first in a series of publications
from the current IP-EU project “AQUAMAX” (016249-2) that evaluates
the consequence of the combined replacement of fish meal and fish oil.
Further communications will focus on metabolic aspects, fish health and
welfare, as well as fillet quality.
2. Material and methods
2.1. Feeding trial
The feeding trial was carried out at the Institute of Marine Research,
Matre (Matredal, Norway; 60°52′N, 05°35′E) during the period of June
22nd 2006–June 15th 2007. The Atlantic salmon (Salmo salar) were
obtained from AkvaGen A/S (Tingvoll, Norway). In June 2006 approxi-
mately 6000 smolts with a mean weight of 355± 92 g were distributed
equally into 12–10 m3 indoor fibreglass tanks containing 7 m3 seawater,
with a continuous flow-through (~52 L min− 1) of seawater (salinity
34.9 g L− 1) from a deepwater inlet (80 m deep, Matrefjorden).
Temperature was kept constant at 8.9 ± 0.1 °C, with continuously
recording and automatic regulation. Oxygen was also automatically
recorded in the outlet water and was never less than 80% saturation. The
fish were acclimated to the experimental conditions for 2.5 weeks
before being fed the experimental diets on the 22nd of June. Fish in 3
randomised tanks were fed 1 of the 4 different extruded diets; 1) a diet
with maximum inclusion of fish meal (FM) and fish oil (FO) (FMFO), 2) a
diet with an estimated safe maximum replacement of both fish meal and
fish oil with plant meal (80% PP) and vegetable oil (70% VO) (80PP70VO),
3) a diet with one half the maximum replacement with plant meal and
maximum replacement with vegetable oil (40PP70VO), and 4) a diet
with maximum replacement with plant meal and one half the
maximum replacement of vegetable oil (80PP35VO). Diets were
produced by Skretting ARC (Stavanger, Norway). The four experimental
diets were fed throughout an entire seawater production phase, with all
diets changing in pellet size and lipid content after a three month
feeding period (Table 1). Diets with pellet size 4 mm were fed from June
to 20th September 2006, and from 20th September to June 2007 the fish

Table 1
Feed composition (g kg− 1), proximate composition (g 100 g− 1, w.w.), and energy (kJ g− 1) of two pellet sizes of the four experimental diets

Pellet size 4 mm 6 mm
FMFO 80PP35VO 40PP70VO 80PP70VO FMFO 80PP35VO 40PP70VO 80PP70VO
Ingredient and proximate composition
Wheat (Statkorn, Norway) 127 110 104 111 157 123 127 124
Wheat gluten (Cerestar Scandinavia AS, Denmark) – 150 96 150 – 150 90 150
Corn gluten (Cargill, USA) – 150 150 150 – 150 150 150
Soybean meal extracted (Denofa, Norway) – 130 50 130 – 110 14 110
Krill meal (Aker Seafoods Antartic ASA ,Norway) – 50 25 50 – 50 25 50
LT South American (Consortio, Peru) 620 120 300 120 560 120 300 120
Linseed oil (Elbe Fetthandel GmbH, Germany) – 17 30 30 – 18 36 36
Palm oil (Denofa, Norway) – 33 60 60 – 32 61 61
Rapeseed oil (Emmelev AS, Denmark) – 61 110 110 – 56 110 110
Fish oil Nordic (Nordsildmel, Norway) 250 160 65 70 280 188 84 86
L-lysine (Ajinomoto Europe S.A.S., France) – 13 6 13 – – – –
Histidine-HCl (Kyowa Hakko Kogyo Co., Ltd, Japan) – 1 – 1 – – – –
DL-methionine (Adisseo, France) – 1 – 1 – – – –
Vitamins and mineralsa 3 4 4 4 3 3 3 3

Proximate composition (g kg− 1)

Crude fat 286 281 286 281 343 318 339 328
Crude protein 431 442 433 442 422 424 412 426
Ash 108 58.2 72.4 58.4 67.2 54.4 65 54.5
Starch 85.7 97.4 97.4 101 91.2 88.4 80.7 86.6
Dry matter 928 939 932 943 923 925 924 932
Rest of dry matter 17.3 60.4 43.2 60.6 0.4 40.2 27.3 36.9
Energy (kJ g− 1) 238 249 245 248 251 254 254 253

FMFO: 100% fish meal and 100% fish oil, 80PP35VO: 80% plant protein and 35% vegetable oil blend; 40PP70VO: 40% plant protein and 70% vegetable oil blend, 80PP70VO: 80% plant
protein and 70% vegetable oil blend.
a
Vitamin and mineral supplementation is estimated to cover requirements according NRC (1993).
 B.E. Torstensen et al. / Aquaculture 285 (2008) 193–200
were fed 6 mm diets. The feed batches of all four l diets used from
November 2006 until February 2007 included yttrium oxide (1 g kg− 1) in
order to assess digestibility midway in the experiment (Dec 2006).
Capelin oil (Fish oil Nordic, Nordsildmel, Norway) was used as the main
source for long chain highly unsaturated n −3 polyunsaturated fatty acids
(HUFA). A mixture of rapeseed, palm, and linseed oil (55/30/15, v/v/v) was
used as replacement for fish oil (Table 1). The mixture was selected to
obtain a lipid profile of saturated, monounsaturated and n−3 PUFA as
similar as possible to capelin oil (Torstensen et al., 2005).
As replacement for fish meal, a mixture of corn gluten, wheat
gluten, and soy concentrate, and krill meal were used (Table 1). A
minor (b50 g kg− 1) inclusion of krill meal (Aker Seafoods Antartic
ASA ,Norway) was added to the replacement diets, with the aim to
improve the palatability and thus feed intake (Gaber, 2005; Olsen
et al., 2006). All diets were formulated to meet nutrient
requirements of fish according to NRC (1993) recommendations.
After 0, 3, 5, 8, and 12 months fish were weighed, and the amount
of feed fed was adjusted in accordance with biomass. Fish were
reared under a natural light regime until October 2006 when a
10 h light: 14 h dark regime was maintained throughout the
winter until March 2007, after which the fish returned to the
natural light regime. From June 2006 until February 2007, the feed
consumption per tank was recorded daily. Fish were fed in excess
twice a day with automatic feeders for 0.5 h, followed by feed
collection 0.5 h after each feeding.
Samples of each experimental diet and the oil and meal ingredients
used in the feeds for each feed production batch were stored at −20 °C.
Fish sampled for whole body proximate composition were unfed
2 days prior to sampling. Fish from each tank were anesthetised with
benzocain (7 g L− 1) and killed by a blow to the head. Individual
weighing of at least 30% of the biomass per tank and sampling of fish
for whole body analysis (6 pooled fish per tank) were performed in
June 2006 (Start), September 2006 (T = 3 months), November 2006
(T = 5 months), February 2007 (T = 8 months), and June 2007
(T = 12 months). During the December sampling faeces samples were
collected by stripping of 30 fish per tank for digestibility analyses.
2.2. Chemical analysis
All chemical analyses were run in duplicate. Nitrogen was determined
after total combustion using a Nitrogen-Analyser (Perkin Elmer, 2410 Ser.
II, Norwalk, CT, USA), and crude protein content calculated assuming that
proteins contain 16% N. Dietary and faecal lipid content was determined
gravimetrically as the sum of free and bound fat. Free or loosely bound fat
was extracted with petroleum ether and dried at 103 ±1 °C. The samples
were thereafter hydrolysed with HCl in a Tecator Soxtec Hydrolysing unit
to release the bound fat, which was extracted with petroleum ether and
dried at 103 ± 1 °C. Dry weight and ash content was determined
gravimetrically after freeze-drying the samples and dried to constant
weight in an oven at 550 °C, respectively. Gross energy was analysed by
the use of an adiabatic bomb calorimeter (IKA Laborteknik, Sweden).
Yttrium was determined in feed and faeces by ICP-MS (Agilent 7500,
Japan). Dietary and faecal amino acid composition was determined after
being hydrolysed in 6N HCl at 110 °C for 22 h and pre-derivatisation with
phenylisothiocyanate (PITCH®) as described by Cohen and Strydom
(1988). Dietary tryptophan was determined after basic hydrolyses in Ba
(OH)2 for 20 h at 110 °C using HPLC (Supelcosil LC-18 column) as
previously described (Liaset et al., 2003). The cysteine content in feed was
performed by the Norwegian Institute of Fisheries and Aquaculture
Research (Bergen, Norway). Starch in homogenized samples of feed and
faeces was determined as previously described by Hemre et al. (1989).
2.3. Lipid extraction and fatty acid analysis
Total lipid was extracted from diets and faeces by homogenization
in chloroform/methanol (2:1, v/v) with 19:0 methyl ester as internal
195
standard, basically according to Folch et al. (1957). Fatty acid methyl
esters (FAME) were prepared from total lipid by boron trifluoride
following saponification, essentially as described previously (Lie and
Lambertsen, 1991; Torstensen et al., 2004). A Thermo Finnegan Trace
2000 GC equipped with a fused silica capillary column was used (CP-
sil 88; 50 m × 0.32 mm id.; Chrompak Ltd.) with temperature
programming of 60 °C for 1 min, 160 °C for 28 min, 190 °C for
17 min, and finally 220 °C for 10 min with all intervening temperature
ramps being at 25 °C min− 1. Individual methyl esters were identified
by comparison to known standards and by reference to published data
(Ackman, 1980). Data were collected and processed using the
Totalchrom software (ver. 6.2, Perkin Elmer).
2.4. Calculations
Fultons condition factor (CF) was calculated as:
 
CF ¼ BWðgÞ FL−3 ðcmÞ  100
where BW = body weight, FL = fork length.
Feed conversion ratio (FCR) was calculated from the amount of diet
consumed (kg dry matter) and the total biomass (kg) gained:
FCR ¼ ðkg diet consumedÞ ðkg final biomass−kg initial biomass
þ kg sampled fish þ mortalitiesÞ−1
Specific growth rate (SGR) was calculated as % daily growth increase


SGR ¼ ln BW2 − ln BW1  days of experiment−1  100
Where BW1 and BW2 represent the initial and final body weights in
g, respectively.
Protein efficiency ratio (PER) was calculated as weight gain (kg) for
each kg protein consumed
 
PER ¼ ðBW2 −BW1 Þ protein fed
−1
Where BW1 and BW2 represent the initial and final body weights in
g, respectively.
Lipid efficiency ratio (LER) was calculated as weight gain (kg) for
each kg lipid consumed
 
LER ¼ ðBW2 −BW1 Þ lipid fed
−1
Protein productive value (PPV) was calculated as retained protein
(kg) in whole fish of consumed protein (kg)
 
PPV ¼ ðfinal protein content−initial protein contentÞ protein consumed
−1
Likewise the energy productive value (EPV) was calculated
 
EPV ¼ ðfinal energy content−initial energy contentÞ energy consumed
−1
 
LPV ¼ ðfinal lipid content−initial lipid contentÞ lipid consumed
−1
Apparent digestibility (ADC) was calculated as the ratio between
the inert marker, yttrium and the nutrient within diet and faeces
calculated based on wet weight.


ADC ¼ ð1− feed marker content  faecal nutrient content
ðfeed nutrient content  faecal marker contentÞ−1 Þ 100
Fish meal protein utilisation (FM PPV) from June 2006 to February
2007:
FM PPV ¼ ððfinal protein content in whole fish
− initial protein content in whole fishÞ
protein consumed from fishÞ
196 B.E. Torstensen et al. / Aquaculture 285 (2008) 193–200

Table 2
Amino acid composition (g/16gN) in the experimental diets at two pellet sizes

Pellet size 4 mm 6 mm
Composition FMFO 80PP35VO 40PP70VO 80PP70VO FMFO 80PP35VO 40PP70VO 80PP70VO
AAs
Ala 5.7 4.8 5.4 4.7 5.4 4.9 5.7 4.8
Arg⁎ 5.4 4.7 4.9 4.7 5.6 4.9 5.1 4.8
Asp 8.2 6.9 7.3 6.8 8.3 6.8 7.2 6.9
Glu 12.6 21.7 19.2 21.3 12.5 21.4 18.3 21.4
Gly 5.5 3.7 4.4 3.6 5.4 3.6 4.4 3.7
His⁎ 2.1 1.5 1.8 1.7 3.4 1.5 2.2 1.7
OH-pro 0.7 0.2 0.4 0.2 1.0 0.2 0.4 0.2
Ile⁎ 3.8 3.6 4.0 3.5 3.6 4.0 3.8 3.7
Leu⁎ 7.4 9.1 8.9 9.0 6.9 9.1 9.4 9.1
Lys⁎ 7.2 5.9 6.2 6.0 6.8 5.3 5.7 5.4
Met⁎ 2.9 2.2 2.3 2.4 2.6 2.6 3.2 2.5
Phe⁎ 4.0 4.8 4.7 4.7 3.7 5.0 4.8 4.9
Pro 4.1 7.4 6.7 7.3 4.2 7.9 7.0 7.7
Ser 3.8 4.7 4.4 4.7 3.9 4.5 4.4 4.6
Tau 0.9 0.2 0.4 0.2 0.9 0.2 0.5 0.3
Thr⁎ 3.7 3.0 3.3 3.0 3.9 3.3 3.7 3.3
Tyr 3.2 3.7 3.7 3.7 3.1 3.9 4.0 3.9
Val⁎ 4.6 4.1 4.4 4.0 4.5 4.4 4.7 4.3
Trp⁎ 1.1 0.9 0.9 0.9 1.1 0.9 0.9 0.9
Cys 1.0 1.4 1.4 1.4 1.1 1.4 1.4 1.6
IAA/DAA 0.92 0.73 0.78 0.74 0.92 0.75 0.82 0.74
Non AA-N 12.1 5.5 5.3 6.2 12.1 4.2 3.2 4.3

Abbreviations of dietary groups are given in Table 1.

Amino acids (AAs) followed by an asterisk are considered indispensable for Atlantic salmon. Non AA-N is the percentage not being accounted for as AAs analysed.

Calculations were based on the following protein content of the 2.5. Statistics
different protein sources: fish meal, 700 g kg− 1; wheat gluten, 500 g kg− 1;
corn gluten, 650 g kg− 1; soy protein concentrate, 500 g kg− 1; krill meal, All statistics were performed using the program Statistica (Statsoft
650 g kg− 1. Inc., Tulsa, USA). To account for the variance among experimental

Table 3
Fatty acid composition (area %, w.w.) of the experimental diets at two different pellet sizes

4 mm diets 6 mm diets
FMFO 80PP35VO 40PP70VO 80PP70VO FMFO 80PP35VO 40PP70VO 80PP70VO
14:0 4.5 2.8 1.8 1.7 6.4 5.0 2.6 2.8
15:0 0.5 0.3 0.2 0.2 0.6 0.4 0.2 0.2
16:0 14.0 15.1 16.7 16.5 15.2 15.0 16.3 16.1
17:0 0.7 0.4 0.2 0.2 0.4 0.3 0.2 0.2
18:0 2.0 2.1 2.6 2.5 2.5 2.4 2.8 2.6
20:0 0.2 0.3 0.4 0.4 0.2 0.3 0.4 0.4
22:0 0.0 0.1 0.2 0.2 0.0 0.1 0.2 0.4
Sum saturated 21.7 21.3 22.1 21.8 25.4 23.5 22.8 22.8
16:1n − 7 5.3 3.1 1.8 1.8 4.7 4.1 1.9 2.1
16:1n − 9 0.4 0.2 0.1 0.1 0.4 0.3 0.2 0.2
18:1n − 7 3.2 2.8 2.5 2.5 2.0 2.2 2.4 2.3
18:1n − 9 11.0 24.5 33.6 34.9 9.5 17.6 30.0 28.9
18:1n − 11 0.5 0.3 0.0 0.1 0.2 0.2 0.0 0.2
20:1n − 7 0.4 0.3 0.1 0.1 0.3 0.2 0.1 0.1
20:1n − 9 9.7 6.6 3.3 3.5 6.7 5.3 3.0 3.3
20:1n − 11 1.6 1.1 0.5 0.5 0.5 0.4 0.2 0.2
22:1n − 9 1.9 1.3 0.7 0.8 0.9 1.1 1.3 1.3
22:1n − 11 11.8 7.8 3.3 3.6 10.4 7.7 3.5 4.1
24:1n − 9 1.1 0.6 0.3 0.4 1.1 0.8 0.5 0.5
Sum monoenes 47.0 48.7 46.3 48.4 36.7 39.8 43.3 43.3
18:2n − 6 1.6 8.9 13.7 13.4 2.3 7.5 12.8 12.7
20:2n − 6 0.3 0.2 0.0 0.0 0.3 0.2 0.1 0.1
20:3n − 6 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
20:4n − 6 0.6 0.3 0.2 0.1 0.9 0.5 0.3 0.3
Sum n − 6 2.5 9.3 13.9 13.5 3.4 8.2 13.3 13.1
16:4n − 3 0.4 0.1 0.2 0.0 0.7 0.5 0.3 0.2
18:3n − 3 0.8 5.7 9.1 9.7 1.4 5.3 9.4 9.3
18:4n − 3 2.2 1.3 0.7 0.6 3.5 2.6 1.2 1.3
20:3n − 3 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
20:4n − 3 0.6 0.4 0.2 0.2 0.7 0.5 0.2 0.3
20:5n − 3 8.6 4.8 2.9 2.5 9.8 7.5 3.5 3.9
22:5n − 3 1.0 0.5 0.3 0.3 1.2 0.8 0.4 0.4
22:6n − 3 11.4 6.1 3.8 3.0 13.0 8.5 5.0 4.7
Sum n − 3 25.2 18.8 17.1 16.3 30.5 26.0 19.9 20.3
n − 3/n − 6 9.9 2.0 1.2 1.2 8.9 3.2 1.5 1.6

Abbreviations of dietary groups are given in Table 1.

 B.E. Torstensen et al. / Aquaculture 285 (2008) 193–200 197

tanks within a dietary treatment as well as variance among Table 4

individual fish within an experimental tank, differences in Weight (g), length (cm) and condition factor (CF; g cm− 3) and mean tank specific growth
rate (SGR, %, n = 3) in Atlantic salmon fed on four different replacement diets for
individual weight and length, and conditions factor were assessed 12 months (mean ± SD)
using two-way analysis of variance (nested ANOVA; (Zar, 1984)
with the (random) experimental tanks nested in dietary exposure FMFO 80PP35VO 40PP70VO 80PP70VO ANOVA

group. Significant difference among dietary treatments for whole Weight

June 2006 353 ± 48 353 ± 39 351 ± 51 354 ± 39 P = 0.93
tank parameters such as FCR, SGR, PER, LER, PLV, and PPV was
September 786 ± 111a 774 ± 140ab 779 ± 185a 716 ± 127b P b 0.05
assessed by one-way analysis of variance (ANOVA) (Zar, 1984). November 1381 ± 256ab 1381 ± 251ab 1387 ± 253a 1311 ± 230b P b 0.05
Where the null hypothesis (H0: no difference between treatments February 2294 ± 414a 2227 ± 380ab 2426 ± 384a 2118 ± 328b P b 0.05
or within treatment at different time intervals) was rejected, June 2007 3943 ± 835a 3590 ± 766b 3967 ± 882a 3280 ± 736b P b 0.05
significant differences were tested using Tukey's HSD test (P b 0.01
Length
and P b 0.05; Sokal and Rohlf, 1981). A Kolmogorov–Smirnov test June 2006 32.9 ± 1.92 32.1 ± 2.45 32.1 ± 2.12 32.9 ± 2.26 P = 0.98
was used to assess normality of distribution of each treatment September 40.6 ± 2.55ab 40.4 ± 2.84ab 41.2 ± 2.97a 40.1 ± 2.87b P b 0.05
(Zar, 1984). All data were found to be normally distributed. November 48.2 ± 3.18 48.2 ± 2.93 47.7 ± 3.07 47.4 ± 2.89 P = 0.08
Dependent variables were checked for homogeneity of variance February 56.9 ± 2.13a 56.2 ± 3.15ab 57.0 ± 3.38a 54.9 ± 2.75b P b 0.05
June 2007 67.4 ± 4.15a 66.3 ± 4.18b 67.7 ± 4.47a 64.1 ± 4.16c P b 0.05
by the Levene test and transformed whenever necessary (Zar,
1984). CF
June 2006 0.99 ± 0.13 1.06 ± 0.12 1.06 ± 0.14 0.99 ± 0.11 P = 0.98
3. Results September 1.11 ± 0.12 1.17 ± 0.16 1.18 ± 0.29 1.12 ± 0.17 P = 0.12
November 1.22 ± 0.09a 1.23 ± 0.09a 1.26 ± 0.11b 1.22 ± 0.08a P b 0.05
February 1.27 ± 0.11ab 1.25 ± 0.10a 1.31 ± 0.13b 1.27 ± 0.09ab P b 0.05
After three months of feeding, pellet sizes were adjusted according
June 2007 1.28 ± 0.11b 1.21 ± 0.11a 1.29 ± 0.11b 1.23 ± 0.11a P b 0.05
to fish size from 4 to 6 mm pellets. Lipid level increased as pellet size
increased, resulting in slightly increased lipid to protein ratio, while SGR
the ratio of plant ingredients relative to marine ingredients was kept June–September 1.00 ± 0.03a 0.99 ± 0.03a 0.99 ± 0.02a 0.88 ± 0.02b P b 0.05
September– 1.04 ± 0.07 1.02 ± 0.10 1.02 ± 0.03 1.06 ± 0.06 P = 0.89
constant. The dietary amino acids differed as a consequence of plant
November
protein substituting for fish meal (i.e. 80PP, Table 2) which reflected November– 0.60 ± 0.03 0.60 ± 0.03 0.61 ± 0.04 0.55 ± 0.05 P = 0.05
the amino acid composition of the different protein ingredients used. February
The ratio of IAA (indispensable amino acids) to DAA (dispensable February–June 0.44 ± 0.05 0.37 ± 0.05 0.42 ± 0.06 0.35 ± 0.02 P = 0.74
amino acids) was lower in the replacement diets than in the FMFO diet Total period 0.94 ± 0.02a 0.90 ± 0.01a 0.94 ± 0.02a 0.86 ± 0.01b P b 0.05

(Table 2). Among the IAA, dietary histidine was 14–29% lower in the
plant diets as compared to the FMFO in the 4 mm pellets and from 35
to 56% lower in the 6 mm pellets. Further, methionine in the 4 mm
diets was reduced by 17–24% compared to the FMFO diet. Due to the
ingredients used, cysteine was higher in the plant protein diets.
Taurine was 56–78% lower in the 4 mm plant protein diets and from
44 to 78% lower in the 6 mm plant protein diets than in the FMFO diet.
All diets had an equal level of starch of around 90 g kg− 1. The rest of the
dry matter (probably fiber) was neglectable in the FMFO diet, close to
40 g kg− 1 in both 80PP diets and close to 27 g kg− 1 in the 40PP70VO
diet.
The VO blend was formulated to mimic FO in total saturated,
monounsaturated and polyunsaturated fatty acid content but with
no HUFA and this was largely achieved. Replacement of FO with the
VO blend resulted in increased percentages of 18:3n − 3, 18:2n − 6 and
18:1n − 9, with concomitant decreased proportions of n − 3 HUFA and
long chain monoenoic fatty acids like 20:1 and 22:1. These
differences were quantitatively greater in the diets with the higher
level of FO replacement, in diets 40PP70VO and 80PP70VO. In
contrast to protein replacement, replacement of FO with the VO
blend had similar effects in the 4 and 6 mm diets except for the total
level of monoenoic fatty acids which were similar in the 4 mm diets
but higher in the 6 mm plant protein diets mainly due to 18:1n − 9
(Table 3).
Mortality was negligible (b1%) in all diet groups during the
experimental period. Atlantic salmon fed the maximum plant
protein and vegetable oil (80PP70VO) diets had significantly lower
weight and length compared to fish fed the control diet (FMFO)
from first sampling (September 2006) and onwards. Fish fed the
diet with maximum inclusion of plant protein, but only one half of
the maximum inclusion of vegetable oil (80PP35VO), showed no
significant difference in weight compared to the marine control
treatment until the end of the experiment (Table 4). Final weight,
length and condition factor in both maximum plant protein
inclusion groups 80PP35VO and 80PP70VO were significantly
lower compared to fish fed the FMFO diet and the diet with
moderate plant protein inclusion 40PP70VO (Table 4). Likewise,
Abbreviations of dietary groups are given in Table 1.
Different letters denote statistically significant differences revealed by ANOVA.
Due to the cross-over design feed intake data could not be recorded during the period
February to June 2007.
specific growth rate (SGR) was significantly lower in the first
growth period (June–September 2006) and over the entire experi-
mental period (June 2006–June 2007) for fish fed the maximum
replacement level diet 80PP70VO compared to the other dietary
groups. In the second growth period (September–November), SGR
in this group tended to increase compared to the other groups
followed by a decrease again in the third (November–February) and
fourth (February–June) growth period, but none of these changes
were significant (Table 4). A significantly (P b 0.05) lower feed
intake (g feed fish− 1 day− 1) was observed during the first three
months of the experiment in the maximum replacement group
(80PP70VO) compared to the all marine control (FMFO) (Fig. 1).
During the following two periods when feed intake was recorded,
no difference in feed intake was observed. No differences were
observed in feed conversion ratio, or protein and lipid efficiency
ratios or protein or lipid productive values, among the dietary
treatments during in three periods from June to February (Table 5).
Some trends, however, were observed. The feed conversion ratio
(FCR) in the 80PP70VO group increased during all feeding periods
and showed the highest values in the third feeding period.
Furthermore, protein efficiency ratio (PER) and protein productive
value (PPV) were lowest in the 80PP70VO group compared to the
other dietary groups from September 2006 to February 2007, but
not during the first feeding period from June to September.
Similarly, lipid retention assessed by lipid efficiency ratio (LER)
and lipid productive value (LPV) were lower in both groups fed
maximum inclusion of vegetable oil (70% VO) compared to the
groups with one half (35%) VO during the second and third feeding
periods but not during the initial feeding period (Table 5).
Calculated for the experimental period when feed collection was
done, from June 2006 to February 2007, neither PPV nor LPV were
198 B.E. Torstensen et al. / Aquaculture 285 (2008) 193–200

Table 6
Digestibility of protein, fat, starch, energy, dry matter, indispensable amino acids (IAA)
and fatty acids in Atlantic salmon fed on different combined replacement diets (mean ±
SD, n = 3) from June to December '06

FMFO 80PP35PO 40PP70PO 80PP70PO

Lipid 95.0 ± 0.0 96.2 ± 0.5 95.1 ± 0.4 95.1 ± 1.1
Protein 97.7 ± 0.3b 98.5 ± 0.1a 98.4 ± 0.0a 98.7 ± 0.1a
Starch 68.9 ± 2.7a 62.9 ± 3.3b 69.4 ± 0.5a 61.7 ± 4.8b
Dry matter 72.0 ± 3.6 69.4 ± 4.0 77.7 ± 1.0 71.9 ± 5.5
Energy 85.0 ± 2.0b 83.9 ± 0.2b 87.5 ± 0.1a 84.9 ± 1.4b

IAA
His 83.5 ± 1.6ab 82.4 ± 1.3b 86.3 ± 0.1a 85.9 ± 0.9a
Arg 92.9 ± 0.9b 94.4 ± 0.1a 94.5 ± 0.1a 94.9 ± 0.5a
Thr 86.9 ± 1.4 89.2 ± 1.7 89.4 ± 0.4 88.8 ± 0.9
Val 89.2 ± 1.8b 91.3 ± 0.3ab 91.6 ± 0.7ab 92.2 ± 1.0a
Met 88.1 ± 2.3b 91.4 ± 0.4b 92.1 ± 1.1a 91.9 ± 0.9a
Ile 90.0 ± 1.3b 91.0 ± 0.3ab 91.9 ± 0.4ab 92.1 ± 0.7a
Fig. 1. Feed intake (g feed fish− 1day− 1) from June 2006 to February 2007 divided into
Leu 90.9 ± 1.4b 93.6 ± 0.2a 93.5 ± 0.2a 94.1 ± 0.8a
three experimental periods. FMFO: 100% fish meal and 100% fish oil, 80PP35VO: 80%
Phe 89.2 ± 1.6b 93.0 ± 0.3a 92.8 ± 0.4a 93.9 ± 0.6a
plant protein and 35% vegetable oil blend; 40PP70VO: 40% plant protein and 70%
Lys 91.9 ± 1.0 91.6 ± 0.4 92.7 ± 0.4 92.1 ± 1.0
vegetable oil blend, 80PP70VO: 80% plant protein and 70% vegetable oil blend. Data are
Trp n.a. n.a. n.a. n.a.
presented as mean ± std (n = 3). Different letters denote significant differences among
dietary treatments revealed by 1-way ANOVA.
Fatty acids
14:0 92.4 ± 0.9 92.4 ± 4.3 92.2 ± 2.3 88.9 ± 6.2
16:0 88.0 ± 1.4a 86.0 ± 0.6a 83.8 ± 3.1a 74.1 ± 9.7b
18:0 84.7 ± 1.9 87.7 ± 0.8 87.6 ± 2.4 82.5 ± 5.9
significantly different among the dietary treatments. However, a 20:0 79.1 ± 1.8 89.3 ± 1.6 88.1 ± 2.5 83.6 ± 4.9
trend toward poorer LPV in the 80PP70VO group was observed Sum saturated FA 88.7 ± 1.3 87.6 ± 1.1 85.4 ± 2.9 77.5 ± 8.6
(Table 5). 18:1n − 9 96.7 ± 1.3 98.1 ± 0.3 98.2 ± 0.2 97.1 ± 1.2
Digestibility of protein, starch and energy was highest in the 18:1n − 7 96.6 ± 0.8 97.6 ± 0.1 97.9 ± 0.3 97.0 ± 1.1
20:1n − 11 97.4 ± 0.4 98.0 ± 0.1 98.4 ± 1.3 98.0 ± 1.8
40PP70VO group (Table 6). Starch digestibility was significantly lower
20:1n − 9 95.9 ± 0.6b 97.8 ± 0.2a 97.3 ± 0.4a 96.4 ± 1.2a
in the two maximum plant protein replacement groups compared to 20:1n − 7 92.7 ± 0.6b 96.2 ± 0.2b 95.3 ± 0.2b 98.2 ± 3.1a
the marine control and moderate plant protein replacement groups. 22:1n − 11 95.1 ± 0.7b 97.5 ± 0.3a 96.6 ± 0.6a 95.9 ± 1.3a
Focusing on the individual IAA, the digestibility was generally 22:1n − 9 92.6 ± 1.4 96.6 ± 0.3 96.5 ± 0.9 96.3 ± 1.1
significantly lower in the FMFO dietary group compared to the Sum monenes 95.9 ± 0.8b 97.8 ± 0.2a 97.9 ± 0.3a 96.9 ± 1.2b
18:2n − 6 96.3 ± 0.6b 97.4 ± 0.3b 98.5 ± 0.3a 97.6 ± 1.1b
replacement groups (Table 6). Of the fatty acid digestibilities, no 20:2n − 6 96.9 ± 1.0 95.8 ± 0.2 96.4 ± 3.7 94.4 ± 5.9
significant difference was found for most of the fatty acids (Table 6), 20:4n − 6 97.8 ± 0.7 97.8 ± 1.0 96.9 ± 0.1 98.1 ± 3.3
Total n − 6 FA 96.7 ± 0.6 97.4 ± 0.3 98.4 ± 0.2 97.6 ± 1.2
18:3n − 3 97.7 ± 0.7b 98.6 ± 0.2b 99.3 ± 0.2a 98.9 ± 0.6b
18:4n − 3 98.8 ± 0.5 99.4 ± 0.0 99.3 ± 0.3 98.9 ± 0.6
Table 5 20:5n − 3 98.7 ± 0.6 98.9 ± 0.1 98.8 ± 0.3 98.3 ± 1.0
Mean feed conversion ratio (FCR), protein efficiency ration (PER), protein productive 22:5n − 3 97.9 ± 0.7 97.9 ± 0.1 97.6 ± 0.1 96.8 ± 2.5
value (PPV), lipid efficiency ration (LER), and lipid productive value (LPV) (mean ± SD, 22:6n − 3 98.0 ± 0.6 98.3 ± 0.0 97.8 ± 0.5 97.6 ± 1.1
n = 3) Total n − 3 FA 98.3 ± 0.6 98.7 ± 0.1 98.8 ± 0.3 98.5 ± 0.9
Total FA 94.8 ± 0.6 95.6 ± 0.2 95.3 ± 0.8 92.9 ± 2.8
FMFO 80PP35VO 40PP70VO 80PP70VO ANOVA
Abbreviations of dietary groups are given in Table 1. Different letters denote statistically
FCR
significant differences revealed by 1-way ANOVA. Na = not analysed.
June–September 0.82 ± 0.08 0.80 ± 0.04 0.78 ± 0.02 0.80 ± 0.02 P = 0.46
September–November 0.77 ± 0.07 0.79 ± 0.14 0.78 ± 0.02 0.85 ± 0.03 P = 0.32
November–February 0.82 ± 0.04 0.79 ± 0.09 0.83 ± 0.14 0.91 ± 0.02 P = 0.13 while 16:0 was significantly less digestible in the 80PP70VO group.
The fatty acids in the 40PP70VO diet were generally significantly more
PER
digestible than fatty acids in the FMFO diet.
June–September 2.80 ± 0.15 2.84 ± 0.15 2.97 ± 0.39 2.83 ± 0.08 P = 0.76
September–November 3.03 ± 0.26 2.95 ± 0.52 2.88 ± 0.06 2.82 ± 0.10 P = 0.85
November–February 2.81 ± 0.21 2.83 ± 0.16 2.81 ± 0.11 2.53 ± 0.10 P = 0.12

PPV
June–September 0.47 ± 0.01 0.50 ± 0.14 0.50 ± 0.01 0.51 ± 0.04 P = 0.81
September–November 0.56 ± 0.04 0.55 ± 0.13 0.57 ± 0.03 0.53 ± 0.02 P = 0.90
November–February 0.53 ± 0.02 0.57 ± 0.05 0.53 ± 0.11 0.46 ± 0.03 P = 0.32
Total period June–Feb 0.50 ± 0.01 0.53 ± 0.06 0.53 ± 0.05 0.50 ± 0.03 P = 0.72

LER
June–September 4.10 ± 0.35 4.36 ± 0.24 4.20 ± 0.14 4.15 ± 0.13 P = 0.20
September–November 4.22 ± 0.36 4.35 ± 0.73 3.78 ± 0.07 3.69 ± 0.13 P = 0.25
November–February 3.98 ± 0.29 4.21 ± 0.78 3.42 ± 0.54 3.39 ± 0.22 P = 0.28

LPV
June–September 0.67 ± 0.06 0.70 ± 0.04 0.73 ± 0.06 0.66 ± 0.05 P = 0.37
September–November 0.79 ± 0.09 0.76 ± 0.15 0.67 ± 0.08 0.66 ± 0.05 P = 0.37
Fig. 2. Fish meal protein productive value (FM PPV) for the period from June 2006 to
November–February 0.72 ± 0.03 0.75 ± 0.12 0.64 ± 0.13 0.63 ± 0.09 P = 0.31
February 2007. FMFO: 100% fish meal and 100% fish oil, 80PP35VO: 80% plant protein
Total period June–Feb 0.65 ± 0.04 0.68 ± 0.08 0.60 ± 0.07 0.58 ± 0.06 P = 0.29
and 35% vegetable oil blend; 40PP70VO: 40% plant protein and 70% vegetable oil blend,
These values are not given for the total feeding period since all feeds changed 80PP70VO: 80% plant protein and 70% vegetable oil blend. Calculations were made
composition by higher inclusion of fat in the larger pellets sizes. From February onwards based on the protein levels of each ingredient used. Data are presented as mean ± std
a cross-over design was applied during which no feed collection per experimental diet (n = 3). Different letters denote significant differences among dietary treatments
could be recorded. Abbreviations of dietary groups are given in Table 1. revealed by 1-way ANOVA.
 B.E. Torstensen et al. / Aquaculture 285 (2008) 193–200
Fish meal protein utilisation (FM PPV) for the period from June
2006 to February 2007 is presented in Fig. 2. By calculating how much
Atlantic salmon protein was produced per kg fish meal protein fed in
the four dietary groups, the high plant protein groups, 80PP70VO and
80PP35VO, produced four-fold more salmon protein per kg fish meal
protein consumed than the marine control group (Fig. 2).
4. Discussion
Dietary fish meal and fish oil were replaced partially and at
maximum levels without compromising known nutrient require-
ments (NRC, 1993). By replacing 80% of the fish meal with plant
protein in combination with either 35 or 70% vegetable oil inclusion,
the utilisation of fish meal protein was four-fold more efficient, with
2 kg of Atlantic salmon protein being produced from 1 kg of fish meal
protein consumed.
The corn gluten, wheat gluten and soy meal were selected based on
earlier documentation showing their support of adequate growth in fish
(de Francesco et al., 2004; Kaushik et al., 2004; Espe et al., 2006).
Interestingly, the 80PP70VO group had significantly lower growth and
feed intake during the first feeding period, whereas this was not
observed in any of the other dietary groups. If the reduced feed intake
was predominantly due to reduced palatability due to low fish meal
inclusion levels, then the 80PP35VO should have the same reduction in
feed intake and growth. As this was not the case, a combined effect of
high replacement of both the lipid and protein fractions seems obvious.
In the final period, however, from February to June 2007, both the
80PP35VO and 80PP70VO groups had reduced growth and significantly
lower final weight (9 and 17% reduction, respectively), indicating plant
protein as the major factor affecting growth. The 40PP70VO diet group,
however, showed no negative effects on growth, nutrient utilisation or
apparent digestibilities, being in line with previous fish oil replacement
studies (Bell et al., 2001, 2002; Rosenlund et al., 2001; Torstensen et al.,
2004, 2005). Increased growth during the second feeding period for the
80PP70VO group can be regarded as compensatory, and concomitant
with no reduced feed intake which indicates that the Atlantic salmon
needed a longer adaptation time before fully accepting the high plant
protein diet. Earlier studies suggest that salmon are able to compensate
growth after periods with restricted feed intake (Johansen et al., 2001).
However, the reduced growth that appeared at different times during
the experiment for the two high plant protein diets with respective 35
and 70% plant oil, clearly points to an interaction between dietary
protein and lipid replacement levels. While at the 70% VO level, the
reduced weight was related to lower feed intake during the first period,
at the 35% VO level, the growth reduction during the final experimental
period remains unclear since feed intake was not recorded during this
final feeding period. The outcome on feed intake and growth during the
first 3 months, as compared to the following experimental 12 months
period, demonstrate the importance of running long term feeding trials
when high inclusion levels of alternative ingredients are being
evaluated.
Since our diets were not identical to fish meal protein in IAA
composition but met the NRC (1993) recommendations, the results
appear to confirm the need for updating the amino acid requirement
data according to growth rate and life stage (Espe et al., 2006, 2007).
NRC (1993) recommendations are mostly based on performance in
juvenile fish, and for freshwater stages of salmonids. High fish meal
replacement levels as used in the current experiment may result in
IAA intakes that are borderline of requirements when diets are high in
plant protein. Hence, reduced feed intake and daily IAA intake below
the requirements for the growth potential of salmon of this size may
explain the reduced growth performance observed in the 80PP70VO
group.
Changes in palatability and physical texture of the pellets for the
80PP70VO diet may be the reasons for the reduced feed intake during
the initial experimental period, commonly reported in experiments
199
with total (de Francesco et al., 2004; Espe et al., 2006) or partial
replacement of fish meal (Gomes et al., 1995; Kaushik et al., 2004). A
minor inclusion of krill meal was added to all replacement diets to
increase acceptability of the plant protein and oil diets (Olsen et al.,
2006).
No significant difference was found in nutrient utilisation when
Atlantic salmon were fed increasing inclusions of VO and PP, but a
trend toward lower PPV in the 80PP70VO group compared to the other
three diets during the third feeding period (November'06 to
February'07), further confirms the combined effect of replacing both
the protein and lipid simultaneously at maximum levels. However,
PPV calculated for the first experimental period (June'06 to Febru-
ary'07) showed no difference between the maximum replacement
group and control. This is in agreement with earlier studies on Atlantic
salmon, where high inclusion of plant protein did not affect protein
utilisation (PPV and PER) as long as the diet amino acid composition
was balanced to mimic a fish meal based diet and the feed intake was
close to that of the control (Espe et al., 2006, 2007, 2008). Limitations
in dietary lysine reduced protein utilisation (Rodehutscord et al., 1997;
Espe et al., 2007), while limitations in methionine seemed not to affect
either protein or energy utilisation (Espe et al., 2008). The 40PP70VO
group generally had somewhat higher nutrient digestibilities but this
was not reflected in improved PPV or growth compared to the FMFO
group. This may, in part, be explained by the stable temperature at 9 °C
used in the current experiment. Environmental factors, such as low
water temperature (2–6 °C), has been found to affect protein
utilisation and protein efficiency ratio when Atlantic salmon were
fed VO based diets (Bendiksen et al., 2003; Torstensen et al., 2005).
The VO blend used in the current experiment was identical to the one
used in Torstensen et al. (2005) and the resulting fatty acid
composition of the experimental diets (40PP70VO and 80PP70VO)
were similar. The significantly increased PPV and growth previously
reported for 100% VO fed Atlantic salmon (Torstensen et al., 2005) may
be due to increased digestibility of dietary lipid and protein in the VO
diet at lower water temperatures, with a clear protein sparing effect of
the VO blend used (Bendiksen et al., 2003). Furthermore, Atlantic
salmon fed rapeseed oil as a replacement for fish oil at higher water
temperatures (11.6 °C) significantly increased fatty acid digestibility
(Karalazos, 2007). However in these diets the level of saturated fatty
acids decreased from 40 % in the FO diet to 22 % in the rapeseed oil
diet. In the current experiment, dietary saturated fatty acid levels were
similar in the fish oil and VO diets. High levels of dietary saturated
fatty acids have been shown to dramatically reduce digestibility of
fatty acids and protein at 8 °C (Torstensen et al., 2000) but not at
higher water temperatures of 11 °C (Bell et al., 2002). Thus, both the
relatively lower levels of saturated fatty acids in the fish oil based diet
and the slightly higher water temperatures resulted in no additional
positive effect of VO mix inclusion on fatty acid digestibility in the
present experiment, and thus no change in nutrient utilisation.
Starch digestibility studies have identified a correlation between
the presence of indigestible matter and reduced digestibility of starch
and lipids (Krogdahl et al., 2005). The significantly higher starch
digestibility in the FMFO and 40PP70VO groups, and the low levels of
dietary indigestible remains in the respective diets, support this
earlier observation. Normally dietary protein or lipid do not affect
starch digestibility (Krogdahl et al., 2005). All dietary groups in the
present study showed starch digestibility in the upper ranges reported
for Atlantic salmon (Hemre et al., 1995), which adds to the potential to
include plant ingredients in diets for Atlantic salmon.
In conclusion, the results demonstrate a sound production of
Atlantic salmon protein with 2 kg of fish protein produced per 1 kg of
fish meal protein consumed. However, by replacing 80% of fish meal
with plant proteins and 70% of the fish oil replaced with a VO blend, a
reduction in growth of approximately 9% was observed during the
production phase in seawater. The growth retardation was related to
reduced feed intake during the early feeding stages, due to an
200 B.E. Torstensen et al. / Aquaculture 285 (2008) 193–200
interaction effect of high plant protein and VO replacement. Possible
metabolic interactions between dietary lipid and protein will be
addressed in future papers.
Acknowledgements
The study was funded by the IP-EU project “AQUAMAX” (016249-2).
We would like to thank Arnor Gullanger (IMR, Matre Aquaculture
Research Station) for excellent fish husbandry, and the technical
assistance of Jacob Wessels (NIFES) is greatly appreciated. Technical
staffs at NIFES are thanked for excellent assistance with the chemical
analysis.
References
Ackman, R.G., 1980. Fish Lipids. Fishing News Books, Farnham, UK, pp. 87–103.
Bell, J.G., McEvoy, J., Tocher, D.R., McGhee, F., Campbell, P.J., Sargent, J.R., 2001.
Replacement of fish oil with rapeseed oil in diets of Atlantic salmon (Salmo salar)
affects tissue lipid compositions and hepatocyte fatty acid metabolism. J. Nutr. 131,
1535–1543.
Bell, J.G., Henderson, R.J., Tocher, D.R., McGhee, F., Dick, J.R., Porter, A., Smullen, R.P.,
Sargent, J.R., 2002. Substituting fish oil with crude palm oil in the diet of Atlantic
salmon (Salmo salar) affects muscle fatty acid composition and hepatic fatty acid
metabolism. J. Nutr. 132, 222–230.
Bell, J.G., McGhee, F., Campbell, P.J., Sargent, J.R., 2003a. Rapeseed oil as an alternative to
marine fish oil in diets of post-smolt Atlantic salmon (Salmo salar): changes in flesh
fatty acid composition and effectiveness of subsequent fish oil “wash out”.
Aquaculture 218, 515–528.
Bell, J.G., Tocher, D., Henderson, R.J., Dick, J.R., Crampton, V.O., 2003b. Altered fatty acid
compositions in Atlantic salmon (Salmo salar) fed diets containing linseed and
rapeseed oils can be partially restored by a subsequent fish oil finishing diet. J. Nutr.
133, 2793–2801.
Bendiksen, E.Å., Berg, O.K., Jobling, M., Arnesen, A.M., Måsøval, K., 2003. Digestibility,
growth and nutrient utilisation of Atlantic salmon (Salmo salar L.) in relation to
temperature, feed fat content and oil source. Aquaculture 224, 283–299.
Caballero, M.J., Obach, A., Rosenlund, G., Montero, D., Gisvold, M., Izquierdo, M., 2002.
Impact of different dietary lipid sources on growth, lipid digestibility, tissue fatty
acid composition and histology of rainbow trout, Onchorhynchus mykiss. Aqua-
culture 214, 253–271.
Cohen, S.A., Strydom, D.J., 1988. Amino-acid analysis utilizing phenylisothiocyanate
derivatives. Anal. Biochem. 174, 1–16.
de Francesco, M., Parisi, G., Medale, F., Kaushik, S.J., Poli, B.M., 2004. Effect of long term
feeding with a plant protein mixture based diet on growth and body/fillet quality
traits of large rainbow trout (Oncorhynchus mykiss). Aquaculture 263, 413–429.
Espe, M., Lemme, A., Petri, A., El-Mowafi, A., 2006. Can Atlantic salmon grow on diets
devoid of fish meal? Aquaculture 255, 255–262.
Espe, M., Lemme, A., Petri, A., El-Mowafi, A., 2007. Assessment of lysine requirement for
maximal protein accretion in Atlantic salmon using plant protein diets. Aquaculture
263, 168–178.
Espe, M., Hevrøy, E.H., Liaset, B., Lemme, A., El-Mowafi, A., 2008. Methionine intake
affect hepatic sulphur metabolism in Atlantic salmon, Salmo salar. Aquaculture 274,
132–141.
Folch, J., Lees, M., Sloane-Stanley, G.H., 1957. A simple method for the isolation and
purification of total lipids from animal tissues. J. Biol. Chem. 226, 497–509.
Francis, G., Makkar, H.P.S., Becker, K., 2001. Antinutritional factors present in plant-
derived alternate fish feed ingredients and their effects in fish. Aquaculture 199,
197–227.
Gaber, M.M.A., 2005. The effect of different levels of krill meal supplementation of
soybean-based diets on feed intake, digestibility, and chemical composition of
juvenile Nile tilapia Oreochromis niloticus, L. J. World Aquac. Soc. 36, 346–353.
Gomes, E.F., Rema, P., Kaushik, S.J., 1995. Replacement of fish meal by plant proteins in
the diet of rainbow trout (Oncorhynchus mykiss): digestibility and growth
performance. Aquaculture 130, 177–186.
Halver, J.E., Hardy, R.W. (Eds.), 2002. Fish Nutrition. Elsevier Science, San Diego, CA.
824 pp.
Hemre, G.-I., Lie, Ø., Lied, E., Lambertsen, G., 1989. Starch as an energy source in feed for
cod (Gadus morhua): digestibility and retention. Aquaculture 80, 261–270.
Hemre, G.-I., Sandnes, K., Lie, Ø., Torrisen, O., Waagbø, R., 1995. Carbohydrate nutrition
in Atlantic salmon. Growth and feed utilization. Aquac. Res. 26, 149–154.
Hertrampf, J.W., Piedad-Pascual, F., 2000. Handbook on Ingredients for Aquaculture
Feeds. Kluwer Academic Publishers, Dordrecht, The Netherlands. 573 pp.
Johansen, S.S., Ekli, M., Stangnes, B., Jobling, M., 2001. Weight gain and lipid deposition
in Atlantic salmon, Salmo salar, during compensatory growth: evidence for
lipostatic regulation? Aquac. Res. 32, 963–974.
Kaushik, S.J., Cravedi, J.P., Lalles, J.P., Sumpter, J., Fauconneau, B., Laroche, M., 1995.
Partial or total replacement of fish meal by soybean protein on growth, protein
utilization, potential estrogenic or antigenic effects, cholesterolemia and flesh
quality in rainbow trout (Oncorhynchus mykiss). Aquaculture 133, 257–274.
Kaushik, S.J., Covès, D., Dutto, G., Blanc, D., 2004. Almost total replacement of fish meal
by plant protein sources in the diet of a marine teleost, the European sea bass
(Dicentrarchus labrax). Aquaculture 230, 391–404.
Karalazos, V., 2007. Sustainable alternatives to fish meal and fish oil in fish nutrition:
effects on growth, tissue fatty acid composition and lipid metabolism. PhD thesis,
University of Stirling, UK., pp. 96–134.
Krogdahl, Å., Hemre, G.-I., Mommsen, T.P., 2005. Carbohydrates in fish nutrition:
digestion and absorption in postlarval stages. Aquac. Nutr. 11, 103–122.
Liaset, B., Julshamn, K., Espe, M., 2003. Chemical composition and theoretical nutritional
evaluation of the produced fractions from enzymatic hydrolysis of salmon frames
with Protamex™. Process Biochem. 38, 1747–1759.
Lie, Ø., Lambertsen, G., 1991. Fatty acid composition of glycerophospholipids in seven
tissues of cod (Gadus morhua), determined by combined high-performance liquid
chromatography and gas chromatography. J. Chromatogr. 565, 119–129.
Menoyo, D., Lopez-Bote, C.J., Bautista, J.M., Obach, A., 2003. Growth, digestibility and
fatty acid utilization in large Atlantic salmon (Salmo salar) fed varying levels of n − 3
and saturated fatty acids. Aquaculture 225, 295–307.
Ng, W.-K., Sigholt, T., Bell, J.G., 2004. The influence of environmental temperature on the
apparent nutrient and fatty acid digestibility in Atlantic salmon (Salmo salar L.) fed
finishing diets containing different blends of fish oil, rapeseed oil and palm oil.
Aquac. Res. 35, 1228–1237.
NRC (National Research Council), 1993. Nutrient Requirements of Fish. National
Academy Press, Washington DC.
Olsen, R.E., Suontama, J., Langmyhr, E., Mundheim, H., Ringø, E., Melle, W., Malde, M.K.,
Hemre, G.-I., 2006. The replacement of fish meal with Antarctic krill, Euphausia
superba in diets for Atlantic salmon, Salmo salar. Aquac. Nutr. 12, 280–290.
Rodehutscord, M., Becker, A., Pack, M., Pfeffer, E., 1997. Response of rainbow trout
(Oncorhynchus mykiss) to supplements of individual essential amino acids in a
semipurified diet, including an estimate of the maintenance requirement for
essential amino acids. J. Nutr. 127, 1166–1175.
Rosenlund, G., Obach, A., Sandberg, M.G., Standal, H., Tveit, K., 2001. Effect of alternative
lipid sources on long-term growth performance and quality of Atlantic salmon
(Salmo salar L.). Aquac. Res. 32, 323–328.
Sokal, R.R., Rohlf, F.J., 1981. Biometry. W.H. Freeman & Co., New York, NY, pp. 56–76.
Tacon, A.G.J., 2004. Use of fish meal and fish oil in aquaculture: a global perspective.
Aquat. Res. Cult. Dev. 1, 3–14.
Tacon, A.G.J., Hasan, M.R., Subasinghe, R.P., 2006. Use of fishery resources as feed inputs
for aquaculture development: trends and policy implications. FAO Fish. Circ. 1018
99 pp.
Torstensen, B.E., Lie, Ø., Frøyland, L., 2000. Lipid metabolism and tissue composition in
Atlantic salmon (Salmo salar L.) — effects of capelin oil, palm oil, and oleic acid-
enriched sunflower oil as dietary lipid sources. Lipids 35, 653–664.
Torstensen, B.E., Frøyland, L., Ørnsrud, R., Lie, Ø., 2004. Tailoring of a cardioprotective
fillet fatty acid composition of Atlantic salmon (Salmo salar) fed vegetable oils. Food
Chem. 87, 567–580.
Torstensen, B.E., Bell, J.G., Rosenlund, G., Henderson, R.J., Graff, I.E., Tocher, D.R., Lie, Ø.,
Sargent, J.R., 2005. Tailoring of Atlantic salmon (Salmo salar L.) flesh lipid
composition and sensory quality by replacing fish oil with a vegetable oil blend.
J. Agric. Food Chem. 53, 10166–10178.
Waagbø, R., Sandnes, K., Sandvin, A., Lie, Ø., 1991. Feeding three levels of n − 3
polyunsaturated fatty acids at two levels of vitamin E to Atlantic salmon (Salmo
salar). Growth and chemical composition. Fisk. Dir. Skr., Ser. Ernaering 4, 51–63.
Zar, J.H., 1984. Biostatistical Analysis. Prentice-Hall, Englewood Cliffs, New York, NY,
pp. 253–260.