www.elsevier.nlrlocateraqua-online
Long-term protein and lipid growth of Atlantic
ž
/
salmon Salmo salar fed diets with partial
replacement of fish meal by soy protein products at
medium or high lipid level
Stale Refstie
a, Trond Storebakken
a,), Grete Baeverfjord
a,
˚
Andries J. Roem
b,1a
( )
AKVAFORSK Institute of Aquaculture Research AS , N-6600 Sunndalsøra, Norway
b
Nutreco Aquaculture Research Centre, N-4000 StaÕanger, Norway
Received 10 April 2000; received in revised form 5 July 2000; accepted 6 July 2000
Abstract
This study investigated long-term effects on nutrient digestibility and protein and lipid growth of soy protein and lipid levels used in commercial grower diets for Atlantic salmon. Two series of
Ž .
extruded diets were formulated to contain either 45% protein and 32% lipid medium fat or 40%
Ž .
protein and 39% lipid high fat . Each series consisted of six diets in which a soy protein source
Ž . Ž . Ž .
partially replaced low-temperature LT dried fish meal FM : FM only control , soy protein
Ž . Ž
concentrate SPC, 30% of crude protein, CP ; SPC added 0.2% DL-methionine 30% of CP,
. Ž . Ž . Ž
SPCqmet ; dehulled, defatted soybean meal HP-SBM, 20% of CP ; full-fat FF SBM 10% of
. Ž .
CP , and; defatted SBM 10% of CP . Each diet was fed to triplicate groups of 0.56-kg salmon kept in sea pens. The experiment lasted 235 days, during which the salmon reached 2.5 to 2.8 kg. Growth was slower in the FM-control and HP-SBM treatments than in the other treatments. The HP-SBM diet induced enteritis in the distal intestine of the salmon and, at day 215, the apparent digestibilities of nitrogen, lipid and energy were lower in the HP-SBM treatments than in all other treatments. Except a slightly reduced muscle protein concentration in the HP-SBM treatment, no effects of dietary soy were detected on final body composition. Salmon fed the high-fat diets
)Corresponding author. Tel.:q47-71-69-5314; fax:q47-71-69-5301.
Ž .
E-mail address: [email protected] T. Storebakken .
1
Present address: Trouw France, Le Pont de Pierre, 02140 Fontaine les Vervins, France. 0044-8486r01r$ - see front matterq2001 Elsevier Science B.V. All rights reserved.
Ž .
( )
S. Refstie et al.rAquaculture 193 2001 91–106
92
reached on average 122 g higher final weight than those fed the medium-fat diets; however, 91 g of this was in the form of lipid, and corresponded with enlarged deposits of visible fat.q2001 Elsevier Science B.V. All rights reserved.
Keywords: Feedstuff; Fish meal–Soy protein concentrate–Soybean meal; Dietary lipid level; Growth–Digesti-bility–Fat deposition–Soybean meal-induced enteritis-Atlantic salmon Salmo salar
1. Introduction
The development of feeds for salmonid fish has focused on protein economy. There have been strong efforts to define and develop cost-effective protein sources that can, at
Ž .
least partly, substitute for expensive high-quality fish meals FMs in least-cost feed
Ž .
formulations Hardy, 1994 . Soy protein products are among the most interesting alternatives due to high protein content, reasonable price, and steady supply of soybeans
ŽStorebakken et al., 2000 . The dietary protein has also gradually been replaced by lipid. Ž
to reduce the catabolic loss of ingested protein Austreng, 1976; Hillestad and Johnsen,
.
1994; Einen and Roem, 1997; Hemre and Sandnes, 1999 . Consequently, current commercial salmon grower diets contain 34% to 47% protein and 28% to 40% lipid.
Ž . Ž .
Full-fat and defatted solvent extracted soybean meals SBMs typically contain 35%
Ž . Ž .
to 40% and 45% to 50% crude protein CP , respectively Lusas and Riaz, 1995 ; however, such meals also contain several compounds that may interfere with the
Ž . Ž .
digestive process in fish Rackis, 1974 . As reviewed by Storebakken et al. 2000 ,
Ž .
toasting steam-cooking of the meals inactivates proteinase inhibitors and agglutinating lectins. Further extraction with ethanol andror acidic water produce soy protein
Ž . Ž .
concentrates SPCs , which typically contain 65–70% CP Lusas and Riaz, 1995 . This additional extraction removes water-soluble carbohydrates, soybean isoflavones,
anti-Ž .
genic storage proteins, and factor s inducing distal enteritis, but not phytic acid
Žreviewed by Storebakken et al., 2000 . No adverse effects were reported when.
Ž .
including 20% SBM in Low Temperature LT dried FM-based diets for Atlantic salmon
ŽOlli et al., 1995 and rainbow trout Kaushik et al., 1995 . SPC may replace 50% of the. Ž .
dietary LT-FM without adverse effects on growth and nutrient retention of rainbow trout
ŽMedale et al., 1998; Mambrini et al., 1999 . Although soy protein has a well-balanced
´
.Ž .
amino acid profile for fish, it is low in methionine Storebakken et al., 2000 .
When increasing the dietary lipid level, a corresponding increase in lipid deposition is
Ž
observed in Atlantic salmon Grisdale-Helland and Helland, 1997; Einen and Skrede,
.
1998; Helland and Grisdale-Helland, 1998; Hemre and Sandnes, 1999 . This lipid is
Ž
stored as subcutaneous fat, in muscle, and as visceral fat Henderson and Tocher, 1987;
.
Aursand et al., 1994 . Muscle fibres and bundles of muscle fibres are surrounded by
Ž .
adipocytes in Atlantic salmon Zhou et al., 1996 , and there is a strong inverse
Ž .
relationship between percent water and percent lipid in the muscle Shearer, 1994 . Thus, moderately increased lipid content does not result in more visible fat except in the
Ž .
viscera Hillestad and Johnsen, 1994; Hillestad et al., 1998 ; however, the belly-flap, the subcutaneous fat layer, and the myosepta between muscle segments are major sites for
Ž . Ž .
Ž .
mainly in the form of triacylglycerides Zhou et al., 1995 . Hence, obesity in salmon is
Ž .
characterised by enlargement of adipocyte-rich tissues Bjerkeng et al., 1997 . Bjerkeng
Ž .
et al. 1997 reported flesh-quality and sensory attributes of selected groups of slaugh-tered fish from the present study.
Ž .
The objectives of the present investigation were to study 1 the effects on growth and body composition of long-term use of various soy protein products in diets during
Ž .
the saltwater grow-out phase of Atlantic salmon; 2 how the lipid level of current
Ž .
commercial salmon diets influence protein and lipid growth during this phase, and 3 the interactional effects between soy protein product and lipid level in the diet. The levels of soy and lipid were based on the literature reviewed above.
2. Materials and methods
2.1. Diets
Ž .
Twelve diets were manufactured by Skretting Stavanger, Norway , using extrusion
Ž .
technology Table 1 . Two series of six diets were formulated to contain 45% CP and
Ž . Ž .
32% lipid medium fat or 40% CP and 39% lipid high fat , respectively. Within each series, diets were formulated with six different combinations of LT-FM and soy protein
Ž . Ž .
meals: 1 100% of CP from LT-FM; 2 LT-FM and 30% of CP from soy-protein
Ž . Ž . y1
concentrate SPC ; 3 LT-FM and 30% of CP from SPCq added 2 g kg DL
-Ž . Ž .
methionine SPCqmet ; 4 LT-FM and 20% of CP from defatted, dehulled
high-pro-Ž . Ž . Ž . Ž .
tein SBM HP-SBM ; 5 LT-FM and 10% of CP from full-fat SBM FF-SBM ; 6
Ž .
LT-FM and 10% of CP from defatted SBM with hulls; SBM, 10% CP . The diets were formulated to contain similar amounts of available phosphorus. Each diet was pelleted into feed particle sizes of 6 and 9 mm. The 6-mm pellets contained 100 mg yttrium
Ž . y1
oxide Y O ; Sigma, St. Louis, MO, USA kg2 3 dry mix as an inert marker to permit
Ž .
apparent digestibility measurements Austreng et al., 2000 . The diets were sealed in plastic bags and stored dark aty208C until used.
2.2. Fish, rearing conditions, and sampling
The experiment was carried out at Nutreco ARC’s Lerang Research Station,
Jørpe-Ž .
land, Norway. Atlantic salmon Salmo salar; 564"2 g, mean"S.E.M. were fed the experimental diets for a period of 235 days, starting September 20.
Ž
Before the experiment started the fish were fed commercial FM-based diets
Skret-.
ting . As juveniles the salmon were exposed to continuous light until the previous July, then to ambient day until the previous December, and finally to continuous light until transfer to saltwater, at an average weight of approximately 60 g, in April. Before being transfer to saltwater the fish were vaccinated against Vibrio anguillarum, V. salmonicida
Ž
and Aeromonas salmonicida using a Atriple vaccineB Biojec, 1900, Biomed, Bergen,
.
()
Formulation and composition of the diets
Lipid level Medium fat High fat
Protein combination FM-control SPC SPCqmet HP-SBM FF-SBM SBM FM-control SPC SPCqmet HP-SBM FF-SBM SBM
Ingredients, grkg
a
Fish meal 600.0 423.0 423.0 491.0 556.4 547.6 501.0 360.0 360.0 409.0 464.2 456.5
b
Capelin oil 231.0 249.0 249.0 240.0 218.0 237.0 322.2 337.2 337.2 332.2 312.0 327.0
Wheat 127.9 89.9 87.9 46.9 97.0 80.4 125.7 95.7 93.7 56.4 101.4 87.5
f
DL-methionine 2.0 2.0
g
Constant 41.1 41.1 41.1 41.1 41.1 41.1 41.1 41.1 41.1 41.1 41.1 41.1
( )
Composition 6-mm particle sizer9-mm particle size y1
Dry matter, g kg 961r973 966r977 968r973 966r974 954r967 960r964 976r968 976r973 989r974 980r965 980r959 979r951 y1
In DM, kg
Crude protein, g 492r480 475r464 476r476 474r467 480r466 485r451 415r407 405r393 400r393 410r398 409r400 409r405 Lipid, g 317r333 333r338 330r329 318r334 317r327 321r350 397r402 391r417 383r413 394r401 384r412 391r406
Ash, g 79r79 73r70 70r73 73r79 75r81 78r77 75r75 70r70 68r69 71r73 77r75 79r73
Gross energy, MJ 25.0r25.5 24.8r25.4 25.3r25.6 25.0r25.7 23.9r25.0 24.4r25.2 25.4r26.5 25.5r26.4 25.8r26.4 25.6r26.5 25.4r26.3 25.5r26.3
a
Norse LT-94, Norsildmel, Bergen, Norway.
b
Soycomill, ADM, Rotterdam, Holland.
c
HP meal, Farcital Trouw Produkten, Gent, Belgium.
d
gConstant ingredients: 40 g vitamin and mineral premix and digestible binder proprietary composition, Skretting, Stavanger, Norway kgŽ y1 feed: Vitamin A, 4000
IU; vitamin D , 1500 IU;3 a-tocopherol, 0.15 g; thiamin, 0.01 g; riboflavin, 0.015 g; pyridoxine, 0.01 g; panthotenate, 0.045 g, niacin, 0.06 g; folate, 3.0 mg; vitamin
.
B , 0.02 mg; biotin, 0.15 mg; vitamin K , 0.05 g; choline, 5.5 mg; inositol, 0.08 g; Mn, 18.4 mg; Zn, 78 mg ; 0.7 g Carophyll Pink, 8% astaxanthin formulation12 3
One week before initiating the experiment 270 salmon were allocated into each of 36
3 Ž . Ž
125-m 5=5=5 m net-pens that were equipped with automatic feeders Sterner
.
Maxi, Sterner, Leksand, Sweden . The pens were blocked according to water currents and thus inner vs. outer location in the farm, and each diet was fed to three pens in a randomised block design. The fish were fed according to apparent appetite, aiming at 10% overfeeding. Each meal lasted 1.5 to 2 h, aiming for 80% automatic feeding and 20% by hand. The salmon were fed twice a day during fall and winter and three times a day during spring and summer. The meal frequency was changed when the sun set at
Ž .
0700 h. Five of the high-fat diets had a high proportion 2% to 40% of floating pellets, and feeding of these diets was increased accordingly. Care was taken when hand feeding, and the pens were equipped with a shallow, fine-meshed fence to avoid drifting of floating pellets into other pens. Water temperature, salinity, and oxygen were
Ž .
measured daily at 0, 2, 4 Fig. 1 , and 8 m.
Ž . Ž . Ž y1.
Fig. 1. Experimental conditions: Daylight hours civil twilight and temperature 8C , salinity g l , and
Ž y1.
( )
S. Refstie et al.rAquaculture 193 2001 91–106
96
The fish were weighed individually at the start of the experiment and at days 99 and 235. Before each weighing, feed was withdrawn for two days. The fish were stripped to
Ž .
collect faeces at days 45 and 215, as described by Austreng 1978 . The feed particle size was increased from 6- to 9-mm at day 60, and sufficient amounts of the Y O2 3
fortified 6-mm diets were stored aty208C until fed for 14 days before the last stripping. Before starting the experiment, three samples of four fish each were collected. The fish were euthanised by cutting the gill arches, and bled for a minimum of five minutes.
Ž
Whole fish, carcass, and viscera were weighed, and samples of carcass including
. Ž .
kidney and viscera including liver and gonads from four pooled fish were frozen for analysis of body composition. At day 235, this procedure was repeated, sampling four fish per pen. At day 228, four fed fish per pen were euthanised, weighed, and filleted for evaluations of myosepta-stripe index. Four additional fed fish per pen from the low-fat FM, SPC, HP-SBM, and FF-SBM treatments were euthanised and sampled for intestinal histology. At day 235 the carcasses were scanned by computerised X-ray tomography to estimate area of visible fat on transversal carcass sections before pooling of the samples.
2.3. Assessments ofÕisible body fat
Ž .
The assessments of visible body fat are described in detail by Bjerkeng et al. 1997 . Myosepta-stripe index was measured on the fillets in the abdominal cavity between the pectoral and abdominal fins, related to the width of the pale white area on both sides of
Ž . Ž .
the myosepta S and the width of the coloured part of the myotomes T . It was defined
Ž .
by the following ratio: 100=Sr SqT , where S and T were determined as mean
width of five different stripes. Computerised X-ray tomography was conducted with a
Ž .
whole body scanner Somatom 2 CT, Siemens, Munich, Germany equipped with 512
Ž .
detectors, as described by Rye 1991 . The fish were scanned anterior to the dorsal fin.
Ž .
The data were analysed by the AutoCAT software Jopson et al., 1995 , dividing the
Ž
transversal carcass sections into visible fat deposits, lean muscle, and bone Rye et al.,
.
1995 . The area of visible fat was estimated as percentage computer-estimated fat deposits of the total scan area.
2.4. Calculations
Ž .
Daylight hours Fig. 1 were estimated as civil twilight, beginning in the morning and ending in the evening when the centre of the sun was geometrically 68 below the horizon. The data were obtained from the Astronomical Applications Department of the U.S. Naval Observatory, Washington, DC. Thermal-unit growth coefficient was
calcu-Ž . Ž .1r3 Ž .y1
lated according to Cho 1992 as: TGCs W yW = ÝD8 , where W and W
1 0 0 1
Ž .
are the respective initial and final weights pen means , and ÝD8 is the thermal sum Žfeeding days=average temperature measured at 4 m . Apparent feed conversion ratio. ŽFCR was calculated as F. =Gy1, where F is the dry weight of fed feed and G is the
wŽ . Ž .x
Žpooled samples of four fish per pen , respectively, and the subscripts denote initial 0. Ž . Ž .
and final 1 .
2.5. Analyses of diets, faeces, and body composition
Ž . Ž
The diets and faeces were analysed for dry matter 1058C, 14–16 h , ash 5508C, 16
. Ž Ž .
h , yttrium inductivity coupled plasma ICP mass spectroscopy, as described by Refstie
. Ž .
et al., 1997 , nitrogen Kjeltec Autoanalyser, Tecator, Hoganas, Sweden , and fat
¨
¨
Žpreextraction with diethylether and hydrolysis with 4 M HCl prior to diethyletherŽ ..
extraction in a Soxtec HT-6 apparatus Tecator by Nutreco ARC laboratory. Gross
Ž .
energy Parr 1271 Bomb calorimeter, Parr, Moline, IL, USA in diets and faeces was
Ž .
analysed at AKVAFORSK. Carcass and viscera were dried 1058C, 46–50 h , and
Ž . Ž
analysed for nitrogen Kjeltec Autoanalyser and fat dichloromethane extraction in the
.
Soxtec HT-6 apparatus by Nutreco ARC laboratory. Due to high lipid content, the viscera were mixed with 500 g kgy1 celite to assure proper drying.
2.6. Histological eÕaluation of distal intestine
Samples for histological evaluation of the distal intestine were immersed in phosphate
Ž .
buffered formalin 4%, pH 7.2 . The samples were subsequently dehydrated and embedded in paraffin according to standard histological procedures. Hematoxylin and Eosin-stained sections were made, and examined under a light microscope. The sections
Ž .
were evaluated according to the following criteria: 1 widening and shortening of the
Ž .
mucosal foldings; 2 loss of the supranuclear vacuolisation in the absorptive cells in the
Ž .
intestinal epithelium; 3 widening of the central stroma within the mucosal foldings,
Ž .
with increased amounts of connective tissue; and 4 infiltration of a mixed leukocyte population in the lamina propria and submucosa. These are the characteristics of a
Ž
condition previously described as SBM-induced enteritis in Atlantic salmon
Baever-.
fjord and Krogdahl, 1996; Ingh et al., 1991, 1996 . In order to be classified as affected, individuals need to meet all of these four criteria.
2.7. Statistical analyses
The results were analysed by the General Linear Model procedure in the SAS
Ž .
computer software SAS, 1985 . Mean results per pen were subjected to two-way
Ž .
analysis of variance ANOVA with interaction according to the randomised block-de-sign, with dietary protein combination and fat level as the independent variables. When not significant, block was excluded from the model. The results from the ANOVA are presented as the proportion of total variation explained by each of the factors and their interaction, calculated as the marginal contribution of the mean square of the parameter
Žtype I sum of squares as a percentage of the corrected total sum of squares. Significant.
differences within the protein combination=fat level treatments were indicated by least-squares means comparison. The level of significance was chosen at PF0.05, and
Ž .
( )
S. Refstie et al.rAquaculture 193 2001 91–106
98
3. Results
Ž .
The lipid content was slightly but systematically higher 5.0"3.6%, mean"S.D. in the 9-mm than in the 6-mm diets. In consequence, the CP content was 2.4"1.9% lower, whereas the gross energy concentration was 3.1"1.0% higher in the 9-mm diets. Growth rates, measured as TGC, were twice as high during the first 99 days than
Ž .
from days 99 to 235 Table 2 . There were significant effects of block on weight gain, TGC, and protein gain. No block effects were observed for the other variables. Hence, the systematic variation caused by pen location in the farm did not alter growth sufficiently to affect the body composition of the salmon. Overfeeding was confirmed by
Ž .
high apparent FCR, which reached 1.25"0.1 mean"S.D. during the first 99 days and 2.21"0.99 during days 99 to 235.
3.1. Dietary protein combination
No significant effects of dietary protein combination were observed on weight gain or TGC during the first 99 days. The treatments fed the FM-control diets and the diets with 20% of CP from HP-SBM grew significantly slower than the other treatments from days
Ž .
99 to 235, resulting in significantly lower final weights at day 235 Table 2 . Fish in the FM-control and HP-SBM treatments gained significantly less protein than the fish in the
Table 2
Ž . Ž .
Mean "S.E.M., ns3 weights, growth rates TGC and chemical partition of the total gain
Ž . Ž .
Weights g TGC X 1000 Accretion g
Initial Day 99 Day 272 0–99 days 99–272 days Protein Fat Protein combination
Two-way ANOVA: proportion type I SS of totalÕariation % explained by main effects and interaction
) ) ) ) ) ) ) ) ) )
Protein combination 2.4 17.6 34.6 21.2 37.9 43.9 14.9
) ) ) ) ) ) )
Dietary lipid level 3.3 4.0 10.4 7.1 10.1 1.5 56.5
) ) ) ) ) ) ) ) ) ) )
Block 1.6 28.2 26.4 17.0 17.6 23.9
Protein x Lipid level 13.2 9.7 6.7 10.5 12.0 8.0 4.0
Different superscript letters denote significant differences among protein combinations or between fat levels
other treatments, whereas the lipid gain was significantly lower only in the HP-SBM treatment.
Ž
At day 45, the faecal dry-matter content was lowest in the HP-SBM treatment Table
.
3 . When compared with the FM-control treatment, it was also significantly reduced in treatments fed the diets with 30% of CP from SPC, but not in treatments fed the diets with 10% of CP from FF-SBM or SBM. The digestibility of nitrogen was lowest in the SBM treatment, intermediate in the HP-SBM and FF-SBM treatments, and highest in the FM-control and SPC treatments. The digestibility of lipid was highest in the FM control and HP-SBM treatments, intermediate in the FF-SBM treatment and the SPC treatment
Ž .
supplemented with DL-methionine SPCqmet , and lowest in the SBM and
unsupple-mented SPC treatments. The digestibility of energy was higher in the FM-control and HP-SBM than in the other treatments.
At day 215, the HP-SBM treatment had markedly reduced faecal DM content and
Ž .
lower digestibilities of nitrogen, lipid, and energy, than all other treatments Table 3 . The faecal DM content was only slightly higher in the FM-control treatment than in the SPC, SPCqmet, FF-SBM and SBM treatments. The digestibilities of nitrogen and lipid were higher in the SPC and SPCqmet treatments than in the FM-control treatment.
The final CP content in carcass was slightly lower in fish from the HP-SBM
Ž .
treatment than in fish from the FM-control and SPCqmet treatments Table 4 . Other effects of dietary protein combination on final body composition were not observed.
No morphological changes were observed in the posterior intestinal tissue of fish fed the medium-fat FM-control, SPC, and FF-SBM diets; however, soybean-induced inflam-matory conditions were identified in the distal intestine of 10 out of the 12 examined fish fed medium-fat HP-SBM diet.
3.2. Dietary lipid leÕel
No significant effects of dietary lipid were observed on weight gain or TGC during the first 99 days. The fish in the high-fat treatment grew significantly faster than those in the medium-fat treatment from days 99 to 235, resulting in a 6% higher final weight at
Ž .
day 235 Table 2 . The total protein, or lean gain was not significantly affected by dietary lipid level; however, the fish in the high-fat treatment gained 24% more fat than those in the medium-fat treatment.
Ž .
The faecal dry-matter content was not affected by dietary lipid level Table 3 . At day 45, the digestibilities of nitrogen, lipid, and energy were slightly but significantly higher in the medium-fat than in the high-fat treatment. At day 215, the digestibility of lipid no longer differed between the treatments, whereas the digestibilities of nitrogen and energy were slightly higher in the high-fat treatment.
The final dress-out percentage of the fish was lower in the high-fat than in the
Ž
medium-fat treatment, with a corresponding 26% higher content of visceral fat Table
.
()
Mean "S.E.M., ns3 faecal dry matter DM content % and apparent digestibility of nutrients and energy
Lipid level Day 45 Day 215
Ž . Ž .
Protein combination Faecal DM Apparent digestibility % of Faecal DM Apparent digestibility % of
Nitrogen Lipid Energy Nitrogen Lipid Energy
Medium-fat diets
Two-way ANOVA: proportion type I SS of totalÕariation % explained by main effects and interaction
) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) )
Protein combination 63.6 53.7 28.9 43.2 83.6 58.3 75.3 40.5
) ) ) ) ) ) ) ) ) ) )
Dietary lipid level 1.5 6.3 34.9 13.8 0.2 4.4 0.5 13.2
) ) ) ) ) ) ) ) ) ) ) )
Protein x Lipid level 7.0 23.7 18.4 30.1 5.8 17.5 9.3 20.7
Ž .
()
S.
Refstie
et
al.
r
Aquaculture
193
2001
91
–
106
101
Ž .
Mean "S.E.M., ns3 final dress-out percentage, visceral fat content, chemical composition of the fish, area of visible fat in transversal sections in front of the
Ž . Ž .
dorsal fin %, estimated by computerised X-ray tomography , and myosepta-stripe index of fillets % of total width
Ž . Ž .
Dress-out Visceral fat, Chemical composition % of Visible fat deposits % y1
percentage g kg fish Carcass Viscera Area of fat tissue Myosepta stripe index
Protein Lipid Protein Lipid
Protein combination
a
FM control 92.2"0.2 31.5"2.2 18.4"0.1 16.6"0.5 9.6"0.3 42.2"2.0 6.3"0.3 23.4"1.0
a,b
SPC 92.4"0.1 29.4"1.3 18.2"0.1 16.9"0.4 9.9"0.3 39.5"1.2 6.4"0.3 23.5"1.2
a
SPCqmet 92.2"0.2 28.3"1.9 18.4"0.2 16.7"0.7 10.2"0.3 38.6"1.5 6.5"0.2 22.6"0.7
b
HP-SBM 92.3"0.3 26.3"2.1 18.0"0.2 16.5"0.7 10.6"0.3 36.8"2.0 6.2"0.3 24.0"1.3
a,b
FF-SBM 92.4"0.5 29.4"2.1 18.2"0.1 16.7"0.6 9.7"0.3 40.3"1.6 6.4"0.2 25.1"1.3
a,b
SBM 92.2"0.4 29.6"2.0 18.2"0.2 16.8"0.5 9.8"0.4 39.5"1.8 6.6"0.4 22.8"1.0
Dietary lipid leÕel
a b a b a b b b
Medium fat 92.8"0.2 25.8"0.8 18.4"0.1 15.6"0.2 10.4"0.1 37.1"0.9 5.9"0.1 22.4"0.6
b a b a b a a a
High fat 91.9"0.1 32.4"0.8 18.0"0.1 17.8"0.2 9.5"0.2 41.9"0.8 6.9"0.1 24.7"0.5
( ) ( )
Two-way ANOVA: Proportion type I SS of totalÕariation % explained by main effects and interaction
)
Protein combination 9.4 11.4 19.3 1.1 21.2 15.6 2.6 10.1
) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) )
Dietary lipid level 41.4 50.3 35.5 74.8 29.5 32.0 50.2 19.6
Protein x Lipid level 8.0 5.6 10.9 3.4 3.0 8.1 7.6 8.7
Ž .
Different superscript letters denote significant differences among protein combinations or between fat-levels PF0.05 . )
PF0.05.
) )PF0.01.
) ) )
( )
S. Refstie et al.rAquaculture 193 2001 91–106
102
3.3. Interactions between dietary protein combination and lipid leÕel
No interactions between dietary protein combination and lipid level for growth or final body composition were observed. At day 45, there was no interaction for faecal DM content; however, at day 215, the faecal DM content was highest in the SBM group within the medium-fat treatment, whereas it was highest in the FM-control group within
Ž .
the high-fat treatment Table 3 . There were several interactions for the estimates of
Ž .
apparent digestibility Table 3 . At day 45, the digestibilities of nitrogen, lipid, and energy were highest in the FM-control group within the medium-fat treatment, whereas they were highest in HP-SBM group within the high-fat treatment. At day 215, the digestibility of nitrogen in the FM-control group ranked among the lowest estimates within the medium-fat treatment, whereas it ranked among the highest estimates within the high-fat treatment. At this sampling, there was no interaction for digestibility of lipid, while the digestibility of energy was highest in the SPCqmet group within the medium-fat treatment, but highest in the FM-control group within the high-fat treatment.
4. Discussion
There were two major findings resulting from this experiment: First, the condition commonly known as SBM-induced distal enteritis did not affect long-term growth in Atlantic salmon, although it was paralleled by diarrhoea, digestive disturbances, and a slight reduction in the concentration of muscle protein. Second, the use of high-fat diets
Ž40% protein and 39% lipid resulted in similar protein gain but 24% higher lipid gain.
Ž .
than with medium-fat diets 45% protein and 32% lipid . This difference was largely in the form of visible adipose tissue, hence apparently expressing obesity.
Previous growth rates obtained with Atlantic salmon of comparable size and raised at
Ž . Ž .
similar temperatures Austreng et al., 1987 correspond to TGC values X 1000 of 2.5
Ž .
to 2.7 Einen and Mørkøre, 1997 . In comparison, the present TGCs were approximately 50% higher during days 1 to 99, but 20% lower during days 99 to 235.
SPC may supply up to 50% of the protein in LT-FM-based diets for rainbow trout
Ž
without adverse effects on growth Stickney et al., 1996; Medale et al., 1998; Mambrini
´
.et al., 1999 . Supplementation with methionine is not necessary at this level of SPC
ŽMambrini et al., 1999 . Both rainbow trout Kaushik et al., 1995 and Atlantic salmon. Ž . ŽOlli et al., 1995 tolerate 20% inclusion of SBM in LT-FM-based diets. Hence, the.
absence of adverse effects resulting from the present dietary use of soy products is in keeping with previous results; however, the faster growth with the SPC, FF-SBM, and SBM diets than with the LT-FM-control diets was unexpected. It may have resulted from slower and more even absorption of amino acids when mixing FM and soy in the diets. In rainbow trout, the availability of amino acids is high from both FM and SBM
ŽYamamoto et al., 1998a , but the postprandial absorption of amino acids is slower from.
Ž .
SBM Yamamoto et al., 1998b . Absorbed amino acids are rapidly removed from the
Ž .
portal blood by the liver Murai et al., 1987 , and excessive levels andror unbalanced
Ž
.
1984; Kaczanowski and Beamish, 1996 and, hence, poorer utilisation of dietary protein for deposition and growth.
The reduced absorption of lipid with the HP-SBM diet at day 215 was in keeping
Ž
with previous experiments with Atlantic salmon Refstie et al., 1998, 1999, 2000;
. Ž .
Storebakken et al., 1998 . Storebakken et al. 2000 suggested that this, at least in part, is an effect of soybean soluble nonstarch polysaccharides impairing diffusion, convec-tive transport, andror micelle formation within the gastrointestinal contents of fish.
The normal intestinal tissue of Atlantic salmon fed SPC and the inflammatory conditions in the distal intestine of salmon fed HP-SBM confirmed previous studies
ŽIngh et al., 1991, 1996; Baeverfjord and Krogdahl, 1996; Refstie et al., 2000 . The.
inflammation, known as SBM-induced enteritis, is also induced when feeding velasse
Ž .
extracted from the soy during SCP manufacturing Ingh et al., 1996 . SBM-induced
Ž
enteritis results in total absence of absorptive vacuoles in the distal intestine
Baever-.
fjord and Krogdahl, 1996 , and dietary velasse impair the lipid absorption by Atlantic
Ž .
salmon Olli and Krogdahl, 1995 . Thus, although the major site for lipid absorption in
Ž
salmonid fish is the proximal small intestine with associated pyloric caeca Buddington
.
et al., 1997; Krogdahl et al., 1999 , the enteritis of fish fed HP-SBM may have worsened
Ž .
the reducing effect of this SBM on lipid absorption. The soybean factor s inducing enteritis in the distal intestine of Atlantic salmon are still not identified. The absence of enteritis in salmon fed FF-SBM suggests that the condition does not develop at less than
Ž .
10% SBM in the diet; however, it may also indicate that the enteritis-inducing factor s are less potent in full-fat than in defatted SBMs.
Ž . Ž .
The optimal dietary ratio of digestible protein DP to digestible energy DE for
y1 Ž .
Atlantic salmon growing from 1 to 3 kg is 19 g MJ Einen and Roem, 1997 . The present salmon grew on average from 0.5 to 2.7 kg, while the DP DEy1 ratio was 20.7 g
MJy1 with the medium-fat diets and 16.8 g MJy1 with the high-fat diets. Salmon grown
on high-fat diets gained 122 g more weight than those grown on medium-fat diets; however, 91 g of this was lipid, which was related to more visceral fat, lower dress-out percentage, larger visible fat deposits on transversal carcass sections, and wider myosepta stripes. As reviewed in the Introduction, these are main sites for adipocytes in Atlantic salmon. Hence, the slightly higher weight gain, in response to high dietary lipid level, may have expressed obesity.
The differences among apparent digestibility estimates at days 45 and 215 may have
Ž .
been influenced by different fish size; however, the water temperature at 4 m also dropped from 9.58C at day 45 to 6.68C at day 215. As the present diets were formulated with different protein ingredients and lipid levels, this may explain the deviations among nutrient digestibility estimates obtained at days 45 and 215. In salmonids, the
digestibil-Ž
ity of macronutrients is reduced at low water temperature Watanabe et al., 1996; Olsen
. Ž
and Ringø, 1998 . Different proteins are digested at different rates Dabrowski and
.
Dabrowska, 1981; Yamamoto et al., 1998b , and digestive proteases markedly lose
Ž .
activity at low temperature Kitamikado and Tachino, 1960b; Torrissen, 1984 . This may explain why water temperature affects the digestibility of nitrogen from different protein
Ž .
sources differently Watanabe et al., 1996 . The lipase activity remains high at low
Ž .
temperature Kitamikado and Tachino, 1960a , but the digestibility of saturated fatty
Ž .
( )
S. Refstie et al.rAquaculture 193 2001 91–106
104
Ž .
is rich in saturated and monounsaturated fatty acids Austreng et al., 1979 . Thus, the fluidity of the dietary oil may have differed at days 45 and 215, with effects on the absorption of lipid as well as other nutrients. The high lipid digestibility with the HP-SBM diet at day 45 suggests that the negative effect of dietary SBM on lipid absorption is less at high water temperature.
To conclude, moderate replacement of LT-FM by commercial soy protein products was fully acceptable in grower diets for Atlantic salmon. High-fat diets resulted in a slightly higher weight gain than medium-fat diets, mainly in the form of visible fat deposits.
Acknowledgements
The authors want to acknowledge the skilful technical assistance of A. Moen, A.L. Løland, and G. Baardsen at Nutreco ARC. Financial support for this experiment was provided by a grant AProtein Economy in Fish Feed by Using SoyaB funded by The
Ž .
Research Council of Norway NTNF grant a 29843 , Trouw Aquaculture, Skretting, and Denofa. Stale Refstie was supported by grant
˚
a 111572r122 — AEvaluation of Feed Ingredients in FishB, provided by The Research Council of Norway.References
Aursand, M., Bleivik, B., Rainuzzo, J.R., Jørgensen, L., Mohr, V., 1994. Lipid distribution and composition of
Ž .
commercially farmed Atlantic salmon Salmo salar . J. Sci. Food Agric. 64, 239–248.
Austreng, E., 1976. Fat and protein in diets for salmonid fishes: I. Fat content in dry diets for salmon parr
ŽSalmo salar, L. . Sci. Rep. Agric. Univ. Norw. 55, 16 pp. in Norwegian with English abstract .. Ž .
Austreng, E., 1978. Digestibility determination in fish using chromic oxide marking and analysis of contents from different segments of the gastrointestinal tract. Aquaculture 3, 265–272.
˚
Austreng, E., Skrede, A., Eldegard, A., 1979. Effect of dietary fat source on the digestibility of fat and fatty acids in rainbow trout and mink. Acta Agric. Scand. 29, 119–126.
˚
Austreng, E., Storebakken, T., Asgard, T., 1987. Growth rate estimates for cultured Atlantic salmon and˚
rainbow trout. Aquaculture 60, 157–160.
Austreng, E., Storebakken, T., Thomassen, M.S., Refstie, S., Thomassen, Y., 2000. Evaluation of selected trivalent metal oxides as inert markers used to estimate apparent digestibility in salmonids. Aquaculture 188, 65–78.
˚
Baeverfjord, G., Krogdahl, A., 1996. Development and regression of soybean meal induced enteritis in Atlantic salmon, Salmo salar L., distal intestine: a comparison with the intestines of fasted fish. J. Fish Dis. 19, 375–387.
Bjerkeng, B., Refstie, S., Fjalestad, K.T., Storebakken, T., Rødbotten, M., Roem, A.J., 1997. Quality
Ž .
parameters of the flesh of Atlantic salmon Salmo salar as affected by dietary fat content and full-fat soybean meal as a partial substitute for fish meal in the diet. Aquaculture 157, 297–309.
˚
Buddington, R.K., Krogdahl, A., Bakke-McKellep, A.M., 1997. The intestines of carnivorous fish: structure and functions and the relation with diet. Acta Physiol. Scand. 161, 67–80, Suppl. 638.
Cho, C.Y., 1992. Feeding systems for rainbow trout and other salmonids with reference to current estimates of energy and protein requirements. Aquaculture 100, 107–123.
Ž .
Ž
Einen, O., Mørkøre, T., 1997. Foringslære for akvakulturLandbruksforlaget, Oslo, Norway, 235 pp. Inˆ .
Norwegian .
Einen, O., Roem, A.J., 1997. Dietary proteinrenergy ratios for Atlantic salmon in relation to fish size: growth, feed utilization and slaughter quality. Aquacult. Nutr. 3, 115–126.
Einen, O., Skrede, G., 1998. Quality characteristics in raw and smoked fillets of Atlantic salmon, Salmo salar, fed high-energy diets. Aquacult. Nutr. 4, 99–108.
Grisdale-Helland, B., Helland, S.J., 1997. Replacement of protein by fat and carbohydrate in diets for Atlantic
Ž .
salmon Salmo salar at the end of the freshwater stage. Aquaculture 152, 167–180.
Ž .
Hardy, R.W., 1994. Current issues in salmonid nutrition. In: Lim, C., Sessa, D.J. Eds. , Nutrition and Utilization Technology in Aquaculture. AOAC Press, Champaign, USA, pp. 26–35.
Helland, S.J., Grisdale-Helland, B., 1998. The influence of replacing fish meal in the diet with fish oil on
Ž .
growth, feed utilization and body composition of Atlantic salmon Salmo salar during the smoltification period. Aquaculture 162, 1–10.
Hemre, G.-I., Sandnes, K., 1999. Effect of dietary lipid on the muscle composition in Atlantic salmon Salmo salar. Aquacult. Nutr. 5, 9–16.
Henderson, R.J., Tocher, D.R., 1987. The lipid composition and biochemistry of freshwater fish. Prog. Lipid Res. 26, 281–347.
Hillestad, M., Johnsen, F., 1994. High-energyrlow-protein diets for Atlantic salmon: effects on growth, nutrient retention and slaughter quality. Aquaculture 124, 109–116.
˚
Hillestad, M., Johnsen, F., Austreng, E., Asgard, T., 1998. Long-term effects of dietary fat level and feeding˚
rate on growth, feed utilization and carcass quality of Atlantic salmon. Aquacult. Nutr. 4, 89–97.
˚
Ingh, T.S.G.A.M.v.d., Krogdahl, A., Olli, J.J., Hendriks, H.G.C.J.M., Koninkx, J.G.J.F., 1991. Effects of
Ž .
soybean-containing diets on the proximal and distal intestine in Atlantic salmon Salmo salar : a morphological study. Aquaculture 94, 297–305.
˚
Ingh, T.S.G.A.M.v.d., Olli, J.J., Krogdahl, A., 1996. Alcohol-soluble components in soybeans cause morpho-logical changes in the distal intestine of Atlantic salmon, Salmo salar L. J. Fish Dis. 19, 47–53. Jopson, N.B., Kolstad, K., Sehested, K., Vangen, O., 1995. Computed tomography as an accurate and cost
effective alternative to carcass dissection. Proc. Aust. Assoc. Anim. Breed. Genet. 11, 635–638. Kaczanowski, T.C., Beamish, F.W.H., 1996. Dietary essential amino acids and heat increment in rainbow trout
ŽOncorhynchus mykiss . Fish Physiol. Biochem. 15, 105–120..
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.
Kitamikado, M., Tachino, S., 1960a. Studies of the digestive enzymes of rainbow trout: II. Protease. Bull. Jpn. Soc. Sci. Fish. 26, 691–694.
Kitamikado, M., Tachino, S., 1960b. Studies of the digestive enzymes of rainbow trout: III. Esterases. Bull. Jpn. Soc. Sci. Fish. 26, 685–689.
˚
Krogdahl, A., Nordrum, S., Sørensen, M., Brudeseth, L., Røsjø, C., 1999. Effects of diet composition on apparent nutrient absorption along the intestinal tract and of subsequent fasting on mucosal disaccharidase activities and plasma nutrient concentration in Atlantic salmon Salmo salar L. Aquacult. Nutr. 5, 121–133.
Lusas, E.W., Riaz, M.N., 1995. Soy protein products: processing and use. J. Nutr. 125, 573S–580S. Mambrini, M., Roem, A.J., Cravedi, J.P., Lalles, J.P., Kaushik, S.J., 1999. Effects of replacing fish meal with` `
soy protein concentrate and ofDL-methionine supplementation in high-energy, extruded diets on the growth and nutrient utilization of rainbow trout, Oncorhynchus mykiss. J. Anim. Sci. 77, 2990–2999.
Medale, F., Boujard, T., Vallee, F., Blanc, D., Mambrini, M., Roem, A.J., Kaushik, S.J., 1998. Voluntary feed´ ´
Ž .
intake, nitrogen and phosphorus losses in rainbow trout Oncorhynchus mykiss fed increasing dietary levels of soy protein concentrate. Aquat. Living Resour. 11, 239–246.
Murai, T., Ogata, H., Hirasawa, Y., Akiyama, T., Nose, T., 1987. Portal absorption and hepatic uptake of amino acids in rainbow trout force-fed complete diets containing casein or crystalline amino acids. Nippon Suisan Gakkaishi 53, 1847–1857.
˚
( )
S. Refstie et al.rAquaculture 193 2001 91–106
106
˚
Olli, J.J., Krogdahl, A., Vabenø, A., 1995. Dehulled solvent-extracted soybean meal as a protein source in˚
diets for Atlantic salmon, Salmo salar L. Aquacult. Res. 26, 167–174.
Olsen, R.E., Ringø, E., 1998. The influence of temperature on the apparent nutrient and fatty acid digestibility of Arctic charr, SalÕelinus alpinus L. Aquacult. Res. 29, 695–701.
Rackis, J.J., 1974. Biological and physiological factors in soybeans. JAOAC 51, 161A–174A.
Refstie, S., Helland, S., Storebakken, T., 1997. Adaptation to soybean meal in diets for rainbow trout, Oncorhynchus mykiss. Aquaculture 153, 263–272.
Ž
Refstie, S., Storebakken, T., Roem, A.J., 1998. Feed consumption and conversion in Atlantic salmon Salmo
.
salar fed diets with fish meal, extracted soybean meal or soybean meal with reduced content of
oligosaccharides, trypsin inhibitors, lectins and soya antigens. Aquaculture 162, 301–312.
Refstie, S., Svihus, B., Shearer, K.D., Storebakken, T., 1999. Non-starch polysaccharides in soybean meals and effects on the absorption of nutrients in farmed Atlantic salmon and broiler chickens. Anim. Feed Sci. Technol. 79, 331–345.
Refstie, S., Korsøen, Ø.J., Storebakken, T., Baeverfjord, G., Lein, I., Roem, A.J., 2000. Differing nutritional
Ž . Ž
responses to dietary soybean meal in rainbow trout Oncorhynchus mykiss and Atlantic salmon Salmo
.
salar . Aquaculture 190, 49–63.
Rye, M., 1991. Prediction of carcass composition in Atlantic salmon by computerized tomography. Aquacul-ture 99, 35–48.
Rye, M., Baeverfjord, G., Refstie, T., 1995. Metodeutvikling for bestemming av fettfordeling hos laks.
Ž .
AKVAFORSK-report No. 10r95, ivq17 pp. In Norwegian . SAS, 1985. SASrSTATw
Guide for personal computers, Version 6 Ed. Cary, NC: SAS Institute Inc., 378 pp. Shearer, K.D., 1994. Factors affecting the proximate composition of cultured fishes with emphasis on
salmonids. Aquaculture 119, 63–88.
Stickney, R.R., Hardy, R.W., Koch, K., Harrold, R., Seawright, D., Massee, K., 1996. The effect of substituting selected oilseed protein concentrates for fish meal in rainbow trout Oncorhynchus mykiss diets. J. World Aquacult. Soc. 27, 57–63.
Storebakken, T., Kvien, I.S., Shearer, K.D., Grisdale-Helland, B., Helland, S.J., Berge, G.M., 1998. The apparent digestibility of diets containing fish meal, soybean meal or bacterial meal fed to Atlantic salmon
ŽSalmo salar : evaluation of different faecal collection methods. Aquaculture 169, 195–210..
Storebakken, T., Refstie, S., Ruyter, B., 2000. Soy products as fat and protein sources in fish feeds for
Ž .
intensive aquaculture. In: Drackley, J.K. Ed. , Soy in Animal Nutrition. Fed. Anim. Sci. Soc., Savoy, IL, USA, pp. 127–170.
Ž .
Torrissen, K.R., 1984. Characterization of proteases in the digestive tract of Atlantic salmon Salmo salar in
Ž .
comparison with rainbow trout Salmo gairdneri . Comp. Biochem. Physiol. 77B, 669–674.
Walton, M.J., Cowey, C.B., Adron, J.W., 1984. The effect of dietary lysine levels on growth and metabolism
Ž .
of rainbow trout Salmo gairdnerii . Br. J. Nutr. 52, 115–122.
Watanabe, T., Takeuchi, T., Satoh, S., Kiron, V., 1996. Digestible crude protein contents in various feedstuffs determined with four freshwater species. Fish. Sci. 62, 278–282.
Yamamoto, T., Akimoto, A., Kishi, S., Unuma, T., Akiyama, T., 1998a. Apparent and true availabilities of amino acids from several protein sources for fingerling rainbow trout, common carp and red sea bream. Fish. Sci. 64, 448–458.
Yamamoto, T., Unuma, T., Akiyama, T., 1998b. Postprandial changes in plasma free amino acid concentra-tions of rainbow trout fed diets containing different protein sources. Fish. Sci. 64, 474–481.
Ž
Zhou, S., Ackman, R.G., Morrison, C., 1995. Storage of lipids in the myosepta of Atlantic salmon Salmo
.
salar . Fish Physiol. Biochem. 14, 171–178.
Zhou, S., Ackman, R.G., Morrison, C., 1996. Adipocytes and lipid distribution in the muscle tissue of Atlantic
Ž .
