Directory UMM :Data Elmu:jurnal:A:Aquaculture:Vol183.Issue3-4.Mar2000:

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www.elsevier.nlrlocateraqua-online

Influence of dietary soy and phytase levels on

performance and body composition of large

ž

/

rainbow trout Oncorhynchus mykiss and algal

availability of phosphorus load

Jouni Vielma

a,)

, Timo Makinen

¨

, Petri Ekholm

, Juha Koskela

Finnish Game and Fisheries Research Institute, Laukaa Fisheries Research and Aquaculture, Valkola FIN-41360, Finland

Finnish Game and Fisheries Research Institute, P.O. Box 6, Helsinki FIN-00271, Finland

Finnish EnÕironment Institute, P.O. Box 140, Helsinki FIN-00251, Finland Accepted 23 August 1999

Abstract

Ž .

A feeding trial was designed to evaluate the influence of partial replacement of fish meal FM protein for soy-derived protein in large rainbow trout fed practical, high-energy diets with and

without supplemental phytase. A 2=2 factorial arrangement with two soy levels 0% and 69.4%

. Ž y1_.

of the dietary protein from soybeans and two phytase levels 0 and 1200 U kg was used. Soy

Ž . Ž .

protein was derived from soy protein concentrate SPC and soybean meal SBM with a

SPC:SBM protein ratio of 4:1. Diets were formulated to contain 36% and 28% crude protein and

Ž .

fat, respectively, and supplemented with lysine and methionine, but not with phosphorus P . Consequently, the dietary P contents were 10.5 and 6.9 g P kgy1 _{for diets without and with}

soy-derived proteins, respectively. Three replicate groups of fish per treatment were hand-fed once daily to apparent satiety for 24 weeks. Fish grew from 0.25 kg to an average of 2.02 kg, with soy-fed fish being significantly larger at the end of the trial. Phytase had no influence on weight gain of fish. Dietary treatments did not affect feed efficiency, which averaged 0.90. Percent bone

ash was statistically significantly lower in fish fed soy diets than in fish fed FM diets means 56.4

and 57.7 , but weight performance and whole body composition analyses did not confirm the modest P deficiency in fish fed soy without supplemental phytase. Phytase supplementation did not significantly increase bone ash of fish fed soy diets. P load significantly decreased from 8.5 to 4.6 g P kgy1 _{weight gain due to the partial replacement of FM protein for soy protein. Algal}

Ž .

availability of P was lower with soy-based diet than with FM-based diet 23% vs. 35% , and lower

)_{Corresponding author. Tel.:}_q_{358-2057-51510; fax:}_q_{358-2057-51519; e-mail: [email protected]}

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( ) J. Vielma et al.rAquaculture 183 2000 349–362

350

Ž .

in fecal matter of fish fed soy-based diets than in fish fed FM-based diets 9% vs. 27% . Results from this study show that a significant part of FM can be replaced by soy proteins for low-pollution diets, without compromising weight gain or feed efficiency in large rainbow trout fed practical diets.q_{2000 Elsevier Science B.V. All rights reserved.}

Keywords: Fish meal; Soybean; Phosphorus; Phytase; Algal availability; Rainbow trout

1. Introduction Ž .

Fish meal FM supplies the largest part of dietary protein for salmonid culture. Alternative protein sources have been studied intensively for at least two reasons. Firstly, long-term scenarios indicate an increasing demand for the world fisheries catch. In salmonid cage farming, feed costs represent around 50% of the operating costs

ŽTveteras and Bjørndal, 1998 , of which the largest single cost derives from protein

˚

feedstuffs. Higher global demand for fish landings may increase the price of FM, which

was recently demonstrated during the decline in catches due to the latest El Nino.

˜

Ž .

Secondly, the need to formulate diets which minimize phosphorus P excretion of fish and consequent eutrofication of waters requires replacement of FM with low-P protein

Ž .

sources Lall, 1991 . Suitability of soybean products to partially replace FM has been

assessed for cost-effective, sustainable and low-P fish feed formulations e.g., Murai,

1992; National Research Council, 1993 . Soybean products vary in their nutrient and

Ž .

antinutritional factor ANF contents and distinct fish species and size-related

differ-Ž

ences in nutrient requirements and tolerance to dietary ANF exist National Research

Council, 1993 . Therefore, careful review of available data on the nutritional value of soybean products to a given husbandry condition is necessary.

Processing of soybeans into various meals, concentrates and isolates has a major

Ž .

influence on their nutritional properties Wolf and Cowan, 1971 . Growth and feed

Ž .

utilization responses of rainbow trout to replacing FM for soybean meals SBM and

Ž .

soybean protein concentrates SPC have been reported in more than 30 scientific papers. SPC has successfully partially replaced FM in studies by Olli and Krogdahl

Ž1994 , Pfeffer and Henrichfreise 1994 , Kaushik et al. 1995 and Medale et al. 1998 ,. Ž . Ž .

´

Ž .

whereas weight gain reduction was reported by Rumsey et al. 1993; 1994 , Stickney et

Ž . Ž .

al. 1996 and Kim et al. 1998 . In contrast to SPC, SBM has more frequently been

assessed as an inferior protein source to FM e.g., Pfeffer and Beckmann-Toussaint,

1991; Davies and Morris, 1997 . However, SBM replaced 25%, 30% and 40% of the

Ž . Ž .

FM protein according to Dabrowski et al. 1989 , Oliva-Teles et al. 1994 and Sanz et

Ž .

al. 1994 , respectively, with no decrease in weight gain of fish. That growth perfor-mance of SPC-fed fish has been better than SBM-fed fish may be attributed to a decrease in the content of several ANF, including oligo-, di- and polysaccharides

Ž_{Liener, 1994 .}. Ž .

Murai et al. 1989 suggested that fish weight affects utilization of soy flour by fingerling rainbow trout. Most of the experiments have been conducted in juvenile fish with no trials conducted in fish weighing more than 300 g. Rainbow trout farmed in Chile, Denmark, Norway and Finland are typically grown to 1–4 kg in both land-based

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and sea-cage operations, and feed costs per fish are largest in the final stages of the production cycle. Therefore, more information on alternative protein feedstuffs for large rainbow trout are clearly needed.

Several ANF present in soybeans can be partially removed by proper heat treatment

Ž .

and extraction procedures Liener, 1994 . However, phytate, a cyclic inositol compound containing six phosphate groups, is relatively heat-stable and cannot be effectively removed without enzymatic reactions. Phytate-bound P is not available to monogastric

Ž .

animals including fish National Research Council, 1993 . Furthermore, phytate may

Ž .

interfere with the availability of other minerals Liener, 1994 and can bind trypsin and

Ž .

decrease protein availability in fish Singh and Krikorian, 1982; Spinelli et al., 1983 . Supplemental phytase effectively releases phosphate groups of phytate in rainbow trout

ŽCain and Garling, 1995; Rodehutscord and Pfeffer, 1995 , but the benefits of its.

supplementation in practical diets, containing high levels of soybean products, has not been reported. In most studies on nutritional properties of SPC and SBM, supplemental phosphate has been provided to meet or exceed the requirement of rainbow trout. Therefore, the influence of marginal P supply in trout fed soy-based diets has not been

reported. Sub-optimum P supply may induce poor bone mineralization Vielma and Lall,

. Ž .

1998 , increase carcass fat deposition Eya and Lovell, 1997 and decrease resistance to

Ž .

disease Eya and Lovell, 1998 .

In this paper, we report effects of partial replacement of FM for SPC and SBM

Ž_{SPC:SBM protein ratio 4:1 on weight gain, feed efficiency, body composition and}.

nutrient load of large rainbow trout. An attempt was also made to study the algal availability of dietary and fecal P. The experiment was conducted in adult rainbow trout fed practical, high-energy diets without supplemental phosphate.

2. Materials and methods

2.1. Diet preparation, experimental system, fish and feeding

Ž .

Two experimental diets FM and SM were formulated to contain 36% protein and

28% fat. For SM diet, FM was substituted with the mixture of SPC Soycomil, Loders

. Ž .

Croklaan, Denmark and SBM defatted, with hulls, Raisio Group, Finland . Protein ratio in the SPC:SBM mixture was 4:1. Levels of fish oil and wheat meal were adjusted to achieve isonitrogenous diets. Lysine and methionine were supplemented to meet the

y1 _Ž

requirements of 27.7 and 8.0 g kg , respectively, in large rainbow trout Rodehutscord

et al., 1995, 1997 . The experimental diets were extruded to 5 and 7 mm pellets with a full-scale Clextral extruder at Raisio Group. After the extrusion and fat application,

Ž w_.

liquid phytase BASF Natuphos was sprayed onto the pellets in 1% water solution to

y1 _Ž _.

supply 1000 U of phytase kg diet. Phytase levels will be referred to as 0 no phytase

Ž .

and 1 supplemental phytase in the paper. According to analyses, 0-diet had no phytase activity whereas 1-diet had phytase activity of 1200 U of phytase kgy1 _{diet. The diets} were stored at room temperature until feeding. Proximate composition of dietary ingredients, formulation and chemical composition of the diets and share of protein from the feed ingredients are presented in Tables 1–3.

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( ) J. Vielma et al.rAquaculture 183 2000 349–362

352 Table 1

Ž y1_.

Proximate composition g kg of the main protein ingredients

Water Protein Crude fat Ash Fibre N-free extracts Phosphorus

Fish meal 81 692 111 104 0 12 22.0

SPC 68 627 14 64 40 187 8.0

SBM 108 434 19 53 73 313 6.4

Calculated by difference.

Steam-dried capelin meal, Island.

Soycomill, Loders Croklaan, Denmark.

Defatted, with hulls, Raisio Group, Finland.

The trial was conducted at the experimental cage farm of the Finnish Game and

Ž .

Fisheries Research Institute in June–November 1998 144 days . At the beginning, 1800

Ž . Ž

rainbow trout Oncorhynchus mykiss were individually weighed initial weight 252

. 3 _Ž

g"30 g SEM and randomly distributed in twelve 48 m net cages 150 fish per cage,

three cages per treatment . Water temperature at 2 m depth gradually increased from 10.48C to peak at 19.08C in August, and thereafter gradually decreased to 9.28C by the

end of the experiment, averaging 12.48C. Oxygen saturation stayed above 70% during

Table 2

Formulation and proximate composition of the diets Diet

FM SM

( )

Ingredients g kg

Fish meal 489.0 133.0

SPC – 315.0

SBM – 121.0

Wheat meal 221.4 102.8

Mineral–vitamin premix 39.0 39.0

L-LysinePHCl 2.6 7.2

DL-Methionine – 2.0

Fish oil 248.0 280.0

( )

Proximate composition g kg

Moisture 44 33

Crude protein 364 351

Crude fat 266 285

Ash 64 51

Crude fiber 13 27

Ž .

Gross energy MJ kg 23.6 24.2

( )

Amino acid composition g kg

Lysine 28.8 28.2

Methionine 10.8 8.4

Mineral composition

Ž .

P g kg 10.5 6.9

Ž .

Phytate P percentage of total P 9.3 38.0

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Table 3

Ž .

Share of protein % from the feed ingredients

FM SM

Ž .

Fish meal % 91.4 25.2

Ž .

SPC % – 55.6

Ž .

SBM % – 13.8

Ž .

Wheat meal % 7.8 3.5

Ž .

Amino acids % 0.8 1.9

Total 100.0 100.0

the trial. The fish were carefully hand-fed to apparent satiety without undue feed wastage once a day, 7 days a week. Pellet size was changed from 5 to 7 mm at the 6th week of the trial. Feed consumption and mortalities were recorded daily.

2.2. Sampling and analyses of tissues

At the end of the experiment, food was withheld for 5 days before individual weighing. After weighing, 20 individuals from each cage were randomly selected and

Ž .

gutted to determine dressing percentage gills intact, kidney removed . Left operculum was removed for bone ash analyses and 10 g muscle sample was dissected for muscle fat analysis. The muscle sample was obtained by cutting 2 cm slice behind the dorsal fish,

and by dissecting approximately 8 cm3 piece of dorsal muscle with no skin or bones.

Thereafter, intestines and carcasses of the 20 individuals were pooled and minced for one whole body sample per cage for proximate analyses. At the beginning of the trial, a pooled sample of 20 fish was taken for whole body proximate analyses.

Operculum samples were dipped in boiling distilled water for 30 s, and skin and

connective tissues were removed. Bones were dried for 24 h at 608C, and thereafter

ashed at 5508C for 16 h. Muscle, whole body and diet samples were freeze-dried for

Ž . Ž

crude protein proteins6.25=Kjeldahl nitrogen , fat extraction in petroleum

ether:di-. Ž .

ethylether, 1:1 and ash 5508C for 16 h analysis. Diets were also analyzed for fibre

ŽAOAC, 1995 , gross energy adiabatic bomb calorimetry , and phytic acid Harland and. Ž . Ž .

Oberleas, 1986 . P concentration of diets and whole bodies was measured after acid

Ž . Ž .

digestion AOAC, 1995 according to Taussky and Shorr 1953 . Sample moisture

content was measured by drying 24 h at 1058C. All tissue analyses was performed on

duplicates on pooled samples, i.e., on three samples per diet. Analyses of amino acids

were performed according to the European standard method 98r64rEU with a Alpha

Ž .

Plus LKB amino acid analysator at the laboratory of the Plant Production Inspection

Ž .

Centre Vantaa, Finland .

2.3. Determination of algal-aÕailable P

On the 13th week of the trial, 30 fish per cage from treatments FM0 and SM0 were

Ž .

anesthetized and feces stripped as described by Austreng 1978 to obtain samples for the determination of algal-available P. Equal amounts of fecal matter were taken from each replicate cage, mixed and pooled into one fecal sample per treatment. Fecal and corresponding diet samples were placed on ice, and immediately taken for the assay to

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354

Fig. 1. Two-chambered vessel used in the dual culture assays.

the laboratory. Algal-available P was determined by incubating fecal samples and

Ž .

pulverized diets for 3 weeks with P-starved algae Selenastrum capricornutum in

Ž . Ž

two-chambered vessel Fig. 1 in dual culture algal assays DePinto et al., 1981;

modified by Ekholm, 1994 . Fecal samples and pulverized diets were suspended into a

Ž .

P-free algal nutrient medium 5% Z8 of Kotai, 1972 . Except for P, the conditions in the

Ž .

assays 20"18C, 4200"200 lx, pH 8 were optimal for the growth of the algae. P was determined from the fecal and diet samples and algae by ammonium molybdate method with K S O digestion. Algal-available P was assumed to equal the cumulative amount₂ ₂ ₈ of P taken up by the algae during the 3 weeks. The assays were performed in duplicate.

2.4. Statistical analyses

Data were subjected to 2=2 factorial analysis of variance with a probability of

P-0.05 considered significant. Before analysis, homogeneity of error variances was

tested with Levene’s test. Mean values for three replicate cages were used for statistical analyses with the exception of final weight and dressing percentage data, which were

analyzed with nested model using individual measurements within a cage Ruohonen,

1998 . Statistical analyses were conducted with Systat 6.0 for Windows.

3. Results

Fish grew from 0.25 kg to an average of 2.02 kg during the trial. Specific growth rate

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Table 4

Performance of large rainbow trout fed practical-type diets containing two protein sources FM or SPC:SBM

mixture supplemented with and without phytase

a b

Variables Final weight SGR Mortality FE

Žkg. Ž% day . Ž .% Main protein Phytase

ŽU kg .

FM0 Fish meal 0 1.95 1.42 2.7 0.91

FM1 Fish meal 1000 2.00 1.44 3.1 0.89

SM0 SPC:SBM 0 2.06 1.46 1.3 0.89

SM1 SPC:SBM 1000 2.08 1.47 1.8 0.90

Pooled SEM 0.01 0.01 1.3 0.01

Main protein 0.009 0.006 0.340 0.927

Phytase 0.202 0.130 0.744 0.727

Main protein=phytase 0.549 0.584 1.000 0.291

a _Ž _.

SGR, specific growth rates100=lnfinal weightylninitial weightrdays.

Gain per feed.

supplying 69% of the protein from soybean ingredients grew significantly faster and

Ž .

weighed significantly more than fish fed FM-based diet Table 4 . Supplemental phytase did not affect weight gain of fish. Mortality and feed efficiency averaged 2.2% and 0.90%, respectively, among the treatments with no significant effect caused by the protein source, phytase or their interaction.

Replacing FM with soy meals significantly decreased operculum bone ash concentra-tion. In fish fed soy diet, percent bone ash were 56.0% and 56.7% in fish fed without

Ž .

and with phytase, and the overall effect of phytase was not significant Ps0.102 . That phytase acted differently in FM-based diet was suggested by the P-value of 0.060 of the interaction term. Whole body P contents were similar among the treatments and

y1 _Ž _.

averaged 3.35 mg g Table 5 .

Whole body composition, and dressing percentage and muscle fat contents are presented in Table 6. The dietary treatments did not have a significant influence on

Table 5

Bone ash and whole body P concentration of large rainbow trout fed practical-type diets containing two

Ž .

protein sources FM or SPC:SBM mixture supplemented with and without phytase

Variables Bone ash Body P

Ž .% Žmg g .

Main protein Phytase

ŽU kg .

FM0 Fish meal 0 57.7 3.43

FM1 Fish meal 1000 57.7 3.37

SM0 SPC:SBM 0 56.0 3.28

SM1 SPC:SBM 1000 56.7 3.31

Pooled SEM 0.2 0.08

Main protein -0.001 0.241

Phytase 0.102 0.914

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Table 6

Whole body composition, dressing percentage and muscle fat levels of large rainbow trout fed practical-type diets containing two protein sources FM or SPC:SBM

mixture supplemented with and without phytase

Variables Proximate composition of whole body Dressing Muscle fat

Ž% whole weight. percentage %Ž . Ž% whole weight.

Main protein Phytase Water Protein Fat Ash

ŽU kg .

FM0 FM 0 59.2 16.5 21.6 1.74 16.1 8.7

FM1 FM 1000 58.5 16.8 22.2 1.71 16.2 8.6

SM0 SPC:SBM 0 59.3 16.7 21.7 1.66 15.4 8.3

SM1 SPC:SBM 1000 59.1 16.5 21.8 1.70 15.7 9.2

Pooled SEM 0.4 0.2 0.5 0.04 0.3 0.4

Main protein 0.415 0.836 0.737 0.330 0.016 0.795

Phytase 0.354 0.707 0.390 0.966 0.436 0.361

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Table 7

Protein efficiency ratio, protein retention and nitrogen and phosphorus load of large rainbow trout fed

Ž .

practical-type diets containing two protein sources FM or SPC:SBM mixture supplemented with and without phytase

a_Ž y1 _.

Variables Protein utilization Nutrient load g kg gain

b c

Main protein Phytase PER Retention Nitrogen Phosphorus

ŽU kg . Ž .%

FM0 Fish meal 0 2.49 40.9 37.9 8.34

FM1 Fish meal 1000 2.45 41.1 38.5 8.61

SM0 SPC:SBM 0 2.55 42.4 36.2 4.62

SM1 SPC:SBM 1000 2.57 42.3 36.0 4.51

Pooled SEM 0.03 0.8 0.9 0.18

Main protein 0.026 0.128 0.042 -0.001

Phytase 0.732 0.965 0.847 0.847

Main protein=phytase 0.306 0.838 0.664 0.664

a _Ž _{w x} _{w x}_. _w _x

Nutrient loadsnutrient fed gynutrient deposited g rbiomass gain kg .

b _w _x _w _x

PERsbiomass gain kgrprotein fed kg .

c _{w x} _{w x}

Protein retentions100=protein deposited grprotein fed g .

proximate composition of fish with water, protein, fat and ash concentration averaging at 59.0%, 16.6%, 21.8% and 1.70%, respectively. Fish fed the FM diets had significantly

Ž .

higher dressing percentage than fish fed with SM diets means 16.2 vs. 15.6 , whereas dressing percentages were similar in fish fed with or without phytase. Muscle fat content averaged at 8.7%, with no significant differences induced by the main effects or their interaction.

Ž .

Fig. 2. Algal available P percentage of the total P in the diets and fecal matter of treatments FM0 and SM0.

Ž .

Values are means"SEM of two replicates per treatment see text for details . Two-way ANOVA gave the following P values: Diets vs. Fecal Matter, Ps0.012; FM0 vs. SM0, Ps0.003; Interaction, Ps0.279.

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Protein efficiency ratio was higher and nitrogen load was lower in groups fed with

Ž .

SM than in groups fed with FM Table 7 . Protein retention averaged at 41.7% of intake with no significant differences among the treatments. Supplemental phytase did not affect protein utilization by the fish. P load was significantly lower in soy-fed groups

Ž .

than in FM-fed groups means 8.48 vs. 4.57 . Supplemental phytase had no effect on P load values. Algal availability of P was significantly higher in the diets than in the fecal

Ž . Ž

matter 35% vs. 23% , and lower in fecal matter of SM than FM groups 9% vs. 27%;

Fig. 2 .

4. Discussion

Decreasing the share of FM protein from 91% to 25% significantly improved the growth of large rainbow trout. Previous papers have reported unaltered or inferior growth rate by replacement of FM for soy products, while the present experiment is the first to show that growth of trout can be increased by partially replacing FM for

Ž .

commercially available soy products. Olli and Krogdahl 1994 , Pfeffer and

Henrich-Ž . Ž .

freise 1994 and Kaushik et al. 1995 have reported no growth reduction by FM

Ž . Ž .

replacement for SPC. Pfeffer and Henrichfreise 1994 and Kaushik et al. 1995 totally replaced FM for SPC and supplied the SPC diet with methionine and inorganic

Ž .

phosphate in 60–80 g trout, whereas Olli and Krogdahl 1994 replaced up to 56% of the dietary FM protein in 70 g trout. In the latter paper, methionine was supplied but composition of mineral premix was not presented. On the contrary, despite phosphate, lysine, methionine and threonine supplements, 75% replacement level decreased the

Ž . Ž .

growth rate of 12 g trout in Stickney et al. 1996 . In Rumsey et al. 1994 , 100% replacement of FM drastically decreased growth of 3 g trout fed diets with phosphate

Ž .

but without amino acid supplements, whereas in Medale et al. 1998 , increasing the

´

protein ratio between SPC and FM from 3:1 to 4:0 decreased the weight gain of 97 g trout fed closed formula diets. The soy diets in the present trial were supplied to meet

the requirement for lysine and methionine in large trout Rodehutscord et al., 1995,

1997 . In addition to differences in dietary amino acid and mineral concentrations, levels

Ž .

of ANF in soy products Rumsey et al., 1993 and quality of FM may affect how SPC and SBM compare to FM.

Supplemental phytase at 1200 U kgy1did not improve growth of fish. Several recent papers show enhanced amino acid availability by supplemental phytase in land animals

Ž_{e.g., Sebastian et al., 1997; Martin et al., 1998 . The negative effect of phytates on}. Ž

protein utilization by fish has been shown Singh and Krikorian, 1982; Spinelli et al.,

1983 , but in feeding experiments with practical-type diets, plant protein utilization has

Ž .

been reported to increase Storebakken et al., 1998; Vielma et al., 1998 , remain

Ž . Ž .

unchanged Lanari et al., 1998 or even decrease Teskeredzic et al., 1995 by phytase

Ž .

supplementation of diets or feedstuffs. In Storebakken et al. 1998 , soy protein and in

Ž .

Teskeredzic et al. 1995 , rapeseed protein concentrate were pre-treated with phytase,

Ž . Ž .

whereas in Lanari et al. 1998 and Vielma et al. 1998 , phytase were sprayed onto dry

pellets at 1000 U kgy1_{. According to the only dose–response assay we are aware of,}

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y1 _Ž _{. Ž} _.

U kg in channel catfish Ictalurus punctatus Jackson et al., 1996 . Differences in

protein quality are better assessed by feeding a range of dietary protein levels in a

Ž .

slope–ratio design March et al., 1985 , and the absence of positive response in the present study may indicate that the use of supplemental phytase is not economically feasible at soy and protein levels used.

Ž .

Bone ash is a sensitive criteria for available P supply Vielma and Lall, 1998 , which was also shown in the present trial. The slight decrease in operculum ash content in fish fed high soy levels without supplemental phytase shows that dietary P content of 7 g

kgy1 _{was marginally deficient for maximal bone mineralization under the present}

experimental conditions. Requirement of large trout for dietary P has not been deter-mined, but trout weighing 50–200 g require 0.25 g available P per megajoule available

Ž . Ž .

energy Rodehutscord, 1996 . Based on the data by Vielma and Lall 1998 , the decrease of 1.7% U in bone ash, as found in the present study, corresponds to about 0.6 g deficiency in available P supply per kilogram diet. Whole body ash or P was not significantly lower in fish fed soy-based diets, which also suggests that P deficiency was

not severe. P deficiency may increase fat deposition in tissues Eya and Lovell, 1997;

Skonberg et al., 1997 , but no such effect was evident, thus further indicating only a marginal P deficiency of fish. Decreased bone ash without changes in growth response is consistent with several P requirement studies which show that to some extent, fish can

adapt to sub-optimum P supply by homeostatic regulation of phosphate balance Vielma,

1998 .

Supplemental phytase tended to increase bone ash of soy-fed fish, thus indirectly indicating successful gastrointestinal hydrolysis of phytate in the soy diet. Enhanced phytate P availability by supplemental phytase has been reported in rainbow trout by

Ž . Ž . Ž .

Cain and Garling 1995 , Rodehutscord and Pfeffer 1995 , Lanari et al. 1998 ,

Ž . Ž .

Storebakken et al. 1998 and Vielma et al. 1998 .

Due to the similar feed efficiencies and whole body P contents, P load decreased by the decrease in dietary P content in fish fed soy-based diets. To further decrease dietary P content of low-pollution diets, P availability of feed ingredients should be increased to avoid P deficiency syndromes. As demonstrated by the factorial model of Shearer

Ž1995 , better feed efficiency would increase the requirement for dietary P. Therefore,.

changes in factors influencing feed efficiency, e.g., dietary available energy content or fish size, should also be accompanied by changes in dietary P concentration or availability.

Algal availability of P was lower for fecal matter than in the diets. This would agree with the fact that hydrolyzed phosphates are gradually absorbed from the intestine

Ž_{Nakamura, 1985 , and therefore, the percentage of poorly available P complexes is}.

expected to rise in the food chyme during the food passage. Accordingly, Petterson

Ž1986 reported that the share of organic P is lower and HCl-soluble P is higher in the. Ž .

fecal matter than in the diets, and Vielma and Lall 1997 found that the percentage of reactive phosphate in the food chyme is higher in the proximal than in the distal intestine. Algal availabilities of P both in soy diets and in fecal matter of soy-fed fish

Ž .

were lower than those of FM group, thus suggesting that similarly to fish Lall, 1991 , P present in phytic acid is poorly available to algae. Low algal availability of P present in fecals of soy-fed fish may also indicate that under hypophosphatemic threat, fish utilized

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360

available P effectively, thus increasing the share of poorly algal available P in the fecal matter. It is of future interest to investigate the possibilities to reduce algal availability of fecal P by a feed formulation.

5. Conclusion

Positive effects on growth performances and nutrient load were achieved by partial replacement of FM for soy products in practical-type diets for large rainbow trout. Supplemental phytase did not enhance growth rate of fish nor decrease nutrient load of the production. Our result would suggest that algal availability of P present in fish diets and fecals is relatively low and that algal availability and therefore eutrophying impact of fish farming could be manipulated by a diet formulation. These results suggest that significant economical and ecological benefits could be achieved by a combination of protein feedstuffs in diets for large rainbow trout.

Acknowledgements

The authors thank Ms. Leena Kytomaa, Ms. Soili Nikonen, Mr. Olli Norrdahl and

¨

Mr. Tero Nieminen for technical help. The help of Raisio Group in diet manufacturing, and BASF in supplying phytase and in phytic acid and phytase analyses is gratefully acknowledged. The study was funded by the Finnish Game and Fisheries Research

Ž .

Institute, European Union PESCA initiative and the Ministry of Agriculture and

Forestry of Finland.

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Dabrowski, K., Poczyczynski, P., Kock, G., Berger, B., 1989. Effect of partially or totally replacing fish meal¨ protein by soybean meal protein on growth, food utilization and proteolytic enzyme activities in rainbow

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Ž .

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lower Great Lakes tributaries. J. Great Lakes Res. 7, 311–325.

Ekholm, P., 1994. Bioavailability of phosphorus in agriculturally loaded rivers in southern Finland. Hydrobi-ologia 287, 179–194.

Eya, J.C., Lovell, R.T., 1997. Available phosphorus requirements of food-size channel catfish Ictalurus

punctatus fed practical diets in ponds. Aquaculture 154, 283–291.

Eya, J.C., Lovell, R.T., 1998. Effects of dietary phosphorus on resistance of channel catfish to Edwardsiella

ictaluri challenge. J. Aquat. Anim. Health 10, 28–34.

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(13)

Jackson, L.S., Li, M.H., Robinson, E.H., 1996. Use of microbial phytase in channel catfish Ictalurus

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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.

Kim, J.D., Kaushik, S.J., Breque, J., 1998. Nitrogen and phosphorus utilisation in rainbow trout

Oncor-.

hynchus mykiss fed diets with or without fish meal. Aquat. Living Resour. 11, 261–264.

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Ž .

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Ž .

treatment of plant protein diets for rainbow trout Oncorhynchus mykiss . Aquaculture 161, 345–356. Liener, I.E., 1994. Implications of antinutritional components in soybean foods. Crit. Rev. Food Sci. Nutr. 34,

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Ž .

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intake, nitrogen and phosphorus losses in rainbow trout Oncorhynchus mykiss fed increasing dietary levels of soy protein concentrate. Aquat. Living Resour. 11, 239–246.

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Ž .

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Ž .

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Ž .

rainbow trout Salmo gairdneri, R. . Arch. Anim. Nutr. 41, 223–228.

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ŽOncorhynchus mykiss . Arch. Anim. Nutr. 45, 371–377..

Ž .

Rodehutscord, M., 1996. Response of rainbow trout Oncorhynchus mykiss growing from 50 to 200 g to supplements of dibasic sodium phosphate in a semipurified diet. J. Nutr. 126, 324–331.

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Ž .

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Ž .

Rodehutscord, M., Jacobs, S., Pack, M., Pfeffer, E., 1995. Response of rainbow trout Oncorhynchus mykiss growing from 50 to 150 g to supplements ofDL-methionine in a semipurified diet containing low or high levels of cystine. J. Nutr. 125, 964–969.

Ž .

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Ž .

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(14)

( ) J. Vielma et al.rAquaculture 183 2000 349–362

362

response, non-specific defense mechanisms, growth, and protein utilization in rainbow trout. Vet. Im-munol. Immunopathol. 41, 323–339.

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Ž .

as partial substitutes for fish meal in rainbow trout Oncorhynchus mykiss diets: protein and energy utilization. Aquaculture 128, 287–300.

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Skonberg, D.I., Yogev, L., Hardy, R.W., Dong, F.M., 1997. Metabolic response to dietary phosphorus intake

Ž .

in rainbow trout Oncorhynchus mykiss . Aquaculture 157, 11–24.

Spinelli, J., Houle, C.R., Wekell, J.C., 1983. The effect of phytates on the growth of rainbow trout Salmo

gairdneri fed purified diets containing varying quantities of calcium and magnesium. Aquaculture 30,

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Ž .

Vielma, J., 1998. Utilization of dietary phosphorus in rainbow trout Oncorhynchus mykiss and Atlantic

Ž .

salmon Salmo salar . Nat. Environ. Sci., Vol. 79. Kuopio University Publications C. University of

Kuopio, Finland, 42 pp.

Vielma, J., Lall, S.P., 1997. Dietary formic acid enhances apparent digestibility of minerals in rainbow trout,

Ž .

Oncorhynchus mykiss Walbaum . Aquacult. Nutr. 3, 265–268.

Vielma, J., Lall, S.P., 1998. The control of phosphorus homeostasis of Atlantic salmon, Salmo salar L., in fresh water. Fish Physiol. Biochem. 18, 83–93.

Vielma, J., Lall, S.P., Koskela, J., Schoner, F.-J., Mattila, P., 1998. Effects of dietary phytase and¨ cholecalciferol on phosphorus bioavailability in rainbow trout, Oncorhynchus mykiss, W. Aquaculture 163, 307–321.

(1)

Table 7

Protein efficiency ratio, protein retention and nitrogen and phosphorus load of large rainbow trout fed

Ž .

practical-type diets containing two protein sources FM or SPC:SBM mixture supplemented with and without phytase

a_Ž y1 _.

Variables Protein utilization Nutrient load g kg gain

b c

Main protein Phytase PER Retention Nitrogen Phosphorus

ŽU kg . Ž .%

FM0 Fish meal 0 2.49 40.9 37.9 8.34

FM1 Fish meal 1000 2.45 41.1 38.5 8.61

SM0 SPC:SBM 0 2.55 42.4 36.2 4.62

SM1 SPC:SBM 1000 2.57 42.3 36.0 4.51

Pooled SEM 0.03 0.8 0.9 0.18

Main protein 0.026 0.128 0.042 -0.001

Phytase 0.732 0.965 0.847 0.847

Main protein=phytase 0.306 0.838 0.664 0.664

a _Ž _{w x} _{w x.} _w _x

Nutrient loadsnutrient fed gynutrient deposited g rbiomass gain kg .

b _w _x _w _x

PERsbiomass gain kgrprotein fed kg .

c _{w x} _{w x}

Protein retentions100=protein deposited grprotein fed g .

proximate composition of fish with water, protein, fat and ash concentration averaging at

59.0%, 16.6%, 21.8% and 1.70%, respectively. Fish fed the FM diets had significantly

Ž

.

higher dressing percentage than fish fed with SM diets means 16.2 vs. 15.6 , whereas

dressing percentages were similar in fish fed with or without phytase. Muscle fat content

averaged at 8.7%, with no significant differences induced by the main effects or their

interaction.

Ž .

Fig. 2. Algal available P percentage of the total P in the diets and fecal matter of treatments FM0 and SM0.

Ž .

Values are means"SEM of two replicates per treatment see text for details . Two-way ANOVA gave the following P values: Diets vs. Fecal Matter, Ps0.012; FM0 vs. SM0, Ps0.003; Interaction, Ps0.279.

(2)

Protein efficiency ratio was higher and nitrogen load was lower in groups fed with

Ž

.

SM than in groups fed with FM Table 7 . Protein retention averaged at 41.7% of intake

with no significant differences among the treatments. Supplemental phytase did not

affect protein utilization by the fish. P load was significantly lower in soy-fed groups

Ž

.

than in FM-fed groups means 8.48 vs. 4.57 . Supplemental phytase had no effect on P

load values. Algal availability of P was significantly higher in the diets than in the fecal

Ž

.

Ž

matter 35% vs. 23% , and lower in fecal matter of SM than FM groups 9% vs. 27%;

.

Fig. 2 .

4. Discussion

Decreasing the share of FM protein from 91% to 25% significantly improved the

growth of large rainbow trout. Previous papers have reported unaltered or inferior

growth rate by replacement of FM for soy products, while the present experiment is the

first to show that growth of trout can be increased by partially replacing FM for

Ž

.

commercially available soy products. Olli and Krogdahl 1994 , Pfeffer and

Henrich-Ž

.

Ž

.

freise 1994 and Kaushik et al. 1995 have reported no growth reduction by FM

Ž

.

Ž

.

replacement for SPC. Pfeffer and Henrichfreise 1994 and Kaushik et al. 1995 totally

replaced FM for SPC and supplied the SPC diet with methionine and inorganic

Ž

.

phosphate in 60–80 g trout, whereas Olli and Krogdahl 1994 replaced up to 56% of

the dietary FM protein in 70 g trout. In the latter paper, methionine was supplied but

composition of mineral premix was not presented. On the contrary, despite phosphate,

lysine, methionine and threonine supplements, 75% replacement level decreased the

Ž

.

Ž

.

growth rate of 12 g trout in Stickney et al. 1996 . In Rumsey et al. 1994 , 100%

replacement of FM drastically decreased growth of 3 g trout fed diets with phosphate

Ž

.

but without amino acid supplements, whereas in Medale et al. 1998 , increasing the

´

protein ratio between SPC and FM from 3:1 to 4:0 decreased the weight gain of 97 g

trout fed closed formula diets. The soy diets in the present trial were supplied to meet

Ž

the requirement for lysine and methionine in large trout Rodehutscord et al., 1995,

.

1997 . In addition to differences in dietary amino acid and mineral concentrations, levels

Ž

.

of ANF in soy products Rumsey et al., 1993 and quality of FM may affect how SPC

and SBM compare to FM.

Supplemental phytase at 1200 U kg

did not improve growth of fish. Several recent

papers show enhanced amino acid availability by supplemental phytase in land animals

Ž

_{e.g., Sebastian et al., 1997; Martin et al., 1998 . The negative effect of phytates on}

.

Ž

protein utilization by fish has been shown Singh and Krikorian, 1982; Spinelli et al.,

.

1983 , but in feeding experiments with practical-type diets, plant protein utilization has

Ž

.

been reported to increase Storebakken et al., 1998; Vielma et al., 1998 , remain

Ž

.

Ž

.

unchanged Lanari et al., 1998 or even decrease Teskeredzic et al., 1995 by phytase

Ž

.

supplementation of diets or feedstuffs. In Storebakken et al. 1998 , soy protein and in

Ž

.

Teskeredzic et al. 1995 , rapeseed protein concentrate were pre-treated with phytase,

Ž

.

Ž

.

whereas in Lanari et al. 1998 and Vielma et al. 1998 , phytase were sprayed onto dry

pellets at 1000 U kg

_{. According to the only dose–response assay we are aware of,}

(3)

_Ž

_{. Ž}

_.

U kg

in channel catfish Ictalurus punctatus

Jackson et al., 1996 . Differences in

protein quality are better assessed by feeding a range of dietary protein levels in a

Ž

.

slope–ratio design March et al., 1985 , and the absence of positive response in the

present study may indicate that the use of supplemental phytase is not economically

feasible at soy and protein levels used.

Ž

.

Bone ash is a sensitive criteria for available P supply Vielma and Lall, 1998 , which

was also shown in the present trial. The slight decrease in operculum ash content in fish

fed high soy levels without supplemental phytase shows that dietary P content of 7 g

kg

_{was marginally deficient for maximal bone mineralization under the present}

experimental conditions. Requirement of large trout for dietary P has not been

deter-mined, but trout weighing 50–200 g require 0.25 g available P per megajoule available

Ž

.

Ž

.

energy Rodehutscord, 1996 . Based on the data by Vielma and Lall 1998 , the decrease

of 1.7% U in bone ash, as found in the present study, corresponds to about 0.6 g

deficiency in available P supply per kilogram diet. Whole body ash or P was not

significantly lower in fish fed soy-based diets, which also suggests that P deficiency was

Ž

not severe. P deficiency may increase fat deposition in tissues Eya and Lovell, 1997;

.

Skonberg et al., 1997 , but no such effect was evident, thus further indicating only a

marginal P deficiency of fish. Decreased bone ash without changes in growth response is

consistent with several P requirement studies which show that to some extent, fish can

Ž

adapt to sub-optimum P supply by homeostatic regulation of phosphate balance Vielma,

.

1998 .

Supplemental phytase tended to increase bone ash of soy-fed fish, thus indirectly

indicating successful gastrointestinal hydrolysis of phytate in the soy diet. Enhanced

phytate P availability by supplemental phytase has been reported in rainbow trout by

Ž

.

Ž

.

Ž

.

Cain and Garling

1995 , Rodehutscord and Pfeffer

1995 , Lanari et al.

1998 ,

Ž

.

Ž

.

Storebakken et al. 1998 and Vielma et al. 1998 .

Due to the similar feed efficiencies and whole body P contents, P load decreased by

the decrease in dietary P content in fish fed soy-based diets. To further decrease dietary

P content of low-pollution diets, P availability of feed ingredients should be increased to

avoid P deficiency syndromes. As demonstrated by the factorial model of Shearer

Ž

1995 , better feed efficiency would increase the requirement for dietary P. Therefore,

.

changes in factors influencing feed efficiency, e.g., dietary available energy content or

fish size, should also be accompanied by changes in dietary P concentration or

availability.

Algal availability of P was lower for fecal matter than in the diets. This would agree

with the fact that hydrolyzed phosphates are gradually absorbed from the intestine

Ž

_{Nakamura, 1985 , and therefore, the percentage of poorly available P complexes is}

.

expected to rise in the food chyme during the food passage. Accordingly, Petterson

Ž

1986 reported that the share of organic P is lower and HCl-soluble P is higher in the

.

Ž

.

fecal matter than in the diets, and Vielma and Lall 1997 found that the percentage of

reactive phosphate in the food chyme is higher in the proximal than in the distal

intestine. Algal availabilities of P both in soy diets and in fecal matter of soy-fed fish

Ž

.

were lower than those of FM group, thus suggesting that similarly to fish Lall, 1991 , P

present in phytic acid is poorly available to algae. Low algal availability of P present in

fecals of soy-fed fish may also indicate that under hypophosphatemic threat, fish utilized

(4)

available P effectively, thus increasing the share of poorly algal available P in the fecal

matter. It is of future interest to investigate the possibilities to reduce algal availability of

fecal P by a feed formulation.

5. Conclusion

Positive effects on growth performances and nutrient load were achieved by partial

replacement of FM for soy products in practical-type diets for large rainbow trout.

Supplemental phytase did not enhance growth rate of fish nor decrease nutrient load of

the production. Our result would suggest that algal availability of P present in fish diets

and fecals is relatively low and that algal availability and therefore eutrophying impact

of fish farming could be manipulated by a diet formulation. These results suggest that

significant economical and ecological benefits could be achieved by a combination of

protein feedstuffs in diets for large rainbow trout.

Acknowledgements

The authors thank Ms. Leena Kytomaa, Ms. Soili Nikonen, Mr. Olli Norrdahl and

¨

Mr. Tero Nieminen for technical help. The help of Raisio Group in diet manufacturing,

and BASF in supplying phytase and in phytic acid and phytase analyses is gratefully

acknowledged. The study was funded by the Finnish Game and Fisheries Research

Ž

.

Institute, European Union

PESCA initiative and the Ministry of Agriculture and

Forestry of Finland.

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rainbow trout, Oncorhynchus mykiss Walbaum , fed soya-based diets. Aquacult. Res. 28, 65–74. DePinto, J.V., Young, T.C., Martin, S.C., 1981. Algal-available phosphorus in suspended sediments from

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punctatus fed practical diets in ponds. Aquaculture 154, 283–291.

Eya, J.C., Lovell, R.T., 1998. Effects of dietary phosphorus on resistance of channel catfish to Edwardsiella ictaluri challenge. J. Aquat. Anim. Health 10, 28–34.

Harland, B.F., Oberleas, D., 1986. Anion-exchange method for determination of phytate in foods: collabora-tive study. J. Assoc. Off. Anal. Chem. 69, 667–670.

(5)

Jackson, L.S., Li, M.H., Robinson, E.H., 1996. Use of microbial phytase in channel catfish Ictalurus punctatus diets to improve utilization of phytate phosphorus. J. World Aquacult. Soc. 27, 309–313. 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.

Kim, J.D., Kaushik, S.J., Breque, J., 1998. Nitrogen and phosphorus utilisation in rainbow trout

Oncor-.

hynchus mykiss fed diets with or without fish meal. Aquat. Living Resour. 11, 261–264.

Ž .

31–67.

March, B.E., MacMillan, C., Ming, F.W., 1985. Techniques for evaluation of dietary protein quality for the

Ž .

rainbow trout Salmo gairdneri . Aquaculture 47, 275–292.

Medale, F., Boujard, T., Vallee, F., Blanc, D., Mambrini, M., Roem, A., Kaushik, S., 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., 1992. Protein nutrition of rainbow trout. Aquaculture 100, 191–207.

Murai, T., Ogata, H., Villaneda, A., Watanabe, T., 1989. Utilization of soy flour by fingerling rainbow trout having different body size. Nippon Suisan Gakkaishi 55, 1067–1073.

Nakamura, Y., 1985. Sodium-dependent absorption of inorganic phosphate by the carp intestine. Comp. Biochem. Physiol. 80A, 437–439.

Ž .

National Research Council NRC , 1993. Nutrient Requirements of Fish. National Academic Press, Washing-ton, DC, 114 pp.

Olli, J.J., Krogdahl, A., 1994. Nutritive value of four soybean products as protein sources in diets for rainbow trout reared in fresh water. Acta Agric. Scand., Sect. A, Anim. Sci. 44, 185–192.

Petterson, K., 1986. Betydelsen av fiskfoders fosforsammansattning for fosforlackage till vatten fran foderspill¨ ¨ ¨ ¨ ˚

Ž .

och fekalier. Limnologiska institut Uppsala, LIU B:18, 34 pp. in Swedish .

Pfeffer, E., Beckmann-Toussaint, J., 1991. Hydrothermically treated soy beans as source of dietary protein for

Ž .

rainbow trout Salmo gairdneri, R. . Arch. Anim. Nutr. 41, 223–228.

Pfeffer, E., Henrichfreise, B., 1994. Evaluation of potential sources of protein in diets for rainbow trout

ŽOncorhynchus mykiss . Arch. Anim. Nutr. 45, 371–377..

Ž .

Rodehutscord, M., 1996. Response of rainbow trout Oncorhynchus mykiss growing from 50 to 200 g to supplements of dibasic sodium phosphate in a semipurified diet. J. Nutr. 126, 324–331.

Rodehutscord, M., Pfeffer, E., 1995. Effects of supplemental microbial phytase on phosphorus digestibility and

Ž .

utilization in rainbow trout Oncorhynchus mykiss . Water Sci. Technol. 31, 143–147.

Ž .

Rumsey, G.L., Hughes, S.G., Winfree, R.A., 1993. Chemical and nutritional evaluation of soya protein

Ž .

preparations as primary nitrogen sources for rainbow trout Oncorhynchus mykiss . Anim. Feed Sci. Technol. 40, 135–151.

(6)

response, non-specific defense mechanisms, growth, and protein utilization in rainbow trout. Vet. Im-munol. Immunopathol. 41, 323–339.

Ruohonen, K., 1998. Individual measurements and nested designs in aquaculture experiments: a simulation study. Aquaculture 165, 149–157.

Sanz, A., Morales, A.E., de la Higuera, M., Cardenate, G., 1994. Sunflower meal compared with soybean meal

Ž .

as partial substitutes for fish meal in rainbow trout Oncorhynchus mykiss diets: protein and energy utilization. Aquaculture 128, 287–300.

Shearer, K.D., 1995. The use of factorial modeling to determine the dietary requirements for essential elements in fishes. Aquaculture 133, 57–72.

Singh, M., Krikorian, A.D., 1982. Inhibition of trypsin activity in vitro by phytate. J. Agric. Food Chem. 30, 799–800.

Skonberg, D.I., Yogev, L., Hardy, R.W., Dong, F.M., 1997. Metabolic response to dietary phosphorus intake

Ž .

in rainbow trout Oncorhynchus mykiss . Aquaculture 157, 11–24.

Spinelli, J., Houle, C.R., Wekell, J.C., 1983. The effect of phytates on the growth of rainbow trout Salmo

gairdneri fed purified diets containing varying quantities of calcium and magnesium. Aquaculture 30, 71–83.

Taussky, H.H., Shorr, E., 1953. A microcolorimetric method for the determination of inorganic phosphorus. J. Biol. Chem. 202, 675–685.

Ž .

concentrate as sources of dietary protein for juvenile rainbow trout Oncorhynchus mykiss . Aquaculture 131, 261–277.

Tveteras, R., Bjørndal, T., 1998. Production, competition and markets: the evolution of the salmon aquaculture˚

industry. SNF Working paper No. 33. Foundation for Research in Economics and Business Administration, Bergen, Norway, 22 pp.

Ž .

Vielma, J., 1998. Utilization of dietary phosphorus in rainbow trout Oncorhynchus mykiss and Atlantic

Ž .

salmon Salmo salar . Nat. Environ. Sci., Vol. 79. Kuopio University Publications C. University of Kuopio, Finland, 42 pp.

Vielma, J., Lall, S.P., 1997. Dietary formic acid enhances apparent digestibility of minerals in rainbow trout,

Ž .

Oncorhynchus mykiss Walbaum . Aquacult. Nutr. 3, 265–268.

Vielma, J., Lall, S.P., 1998. The control of phosphorus homeostasis of Atlantic salmon, Salmo salar L., in fresh water. Fish Physiol. Biochem. 18, 83–93.

Vielma, J., Lall, S.P., Koskela, J., Schoner, F.-J., Mattila, P., 1998. Effects of dietary phytase and¨

cholecalciferol on phosphorus bioavailability in rainbow trout, Oncorhynchus mykiss, W. Aquaculture 163, 307–321.