Influence of Dietary Phytic Acid on Growth, Feed Intake, and Nutrient Utilization in Juvenile Japanese Flounder, Paralichthys olivaceus

JOURNAL OF THEWORLD AQUACULTURE SOCIETY

Vol. 41, No. 5October, 2010

Influence of Dietary Phytic Acid on Growth, Feed Intake, andNutrient Utilization in Juvenile Japanese Flounder, Paralichthys

olivaceus

Asda Laining, Rex F. Traifalgar, Moe Thu, Connie Fay Komilus, andMd. Abdul Kader

Science of Marine Resources, The United Graduate School of Agricultural Science, KagoshimaUniversity, 1-21-24 Korimoto, Kagoshima 890-8580, Japan

Shunsuke Koshio, Manabu Ishikawa1, and Saichiro Yokoyama

Laboratory of Aquatic Animal Nutrition, Faculty of Fisheries, Kagoshima University, 4-50-20Shimoarata, Kagoshima 890-0056, Japan

AbstractA feeding trial was conducted to determine the effect of phytic acid (IP6) on growth, feed intake,

nutrient digestibility, body composition, nutrient retention, and plasma mineral contents in juvenileJapanese flounder, Paralichthys olivaceus. Five test diets containing different levels of IP6 (0, 5.1,10.4, 13.5, and 20.6 g IP6/kg diet) were fed to juveniles (average body weight = 4.58 g). After a 50-dfeeding trial, there were no negative effects on growth and feed intake in fish fed diets supplementedwith IP6 up to 10.4 g IP6/kg. However, weight gain and feed intake of fish fed diets containing 13.5and 20.6 g IP6 were significantly lower than those of control group. Total phosphorus (P) contentsof fish were not significantly different among fish groups fed the diets containing up to 10.4 g IP6.Plasma inorganic P and magnesium (Mg) contents significantly lowered with increased dietary IP6.Dietary IP6 significantly reduced zinc level in the fish vertebra. The significantly lower contents ofvertebral Ca and Mg were found in fish fed diets containing the highest level of IP6 (20.6 g/kg).This study demonstrated that dietary IP6 with more than 13 mg/kg negatively affected the growthperformances, body composition, and nutrient utilization in juvenile Japanese flounder.

The use of plant feedstuffs and their deriva-tives in aquafeeds is limited because of theirunbalanced amino acid composition and thepresence of a wide variety of antinutritionalcompounds in particular phytic acid (Franciset al. 2001). Phytic acid (IP6), also knownas myoinositol hexaphosphate, is the majorphosphorus (P) storage in plant such as seeds,legumes, and cereal grains. Approximately 70%of the total P in plant feedstuff is boundas IP6 (Eeckhout and De Paepe 1994). Con-centration of IP6 in plant-based ingredientsvaries depending on the product types (Higgset al. 1995). Because of the high density ofnegatively charged phosphate groups, IP6 ishighly reactive and easily binds with di- and

1 Corresponding author.

trivalent cation and even larger molecules likeprotein, starch (Selle et al. 2000), and lipid(Leeson 1993). A major concern on IP6 isthat it cannot be digested by fish and hasseveral negative effects on growth, feed con-version (Usmani and Jafri 2002; Portz andLiebert 2003), and macronutrient digestibility(Sajjadi and Carter 2004a; Debnath et al. 2005).Results obtained from in vitro studies showedthat IP6–protein complexes are poorly solubleand hence decrease the availability of protein(Ravindran et al. 1995). However, results fromfeeding trials are still variable and inconsistent(Denstadli et al. 2006). It has also been foundthat IP6 reduces the digestibility of lipid in vitro(Knuckles 1988) and in vivo in the rat (Nymanand Bjorck 1989). Moreover, IP6 may affect thedigestibility of starch because IP6 and starch are

© Copyright by the World Aquaculture Society 2010

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INFLUENCE OF DIETARY PHYTIC ACID IN JUVENILE JAPANESE FLOUNDER 747

structurally capable of combining via phosphatelinkages (Thompson 1986). At present, resultsconcerning the effects of IP6 on both lipid andstarch digestibility in fish species are very rarelyavailable.

Effect of IP6 in decreasing the availabil-ity of several minerals in particular P, mag-nesium (Mg), calcium (Ca), and zinc (Zn) hasbeen widely accepted. Even though many ofthese studies have mainly observed the indi-rect effect of IP6 by evaluating the effect ofphytase supplementation in diet (Storebakkenet al. 1998; Forster et al. 1999; Vielma et al.2004), several studies have investigated thedirect effect of IP6 by adding graded levels ofsynthetic IP6 inclusion in diet and had focusedon freshwater fish including rainbow trout,Oncorhynchus mykiss (Spinelli et al. 1983);Chinook salmon, Oncorhynchus tshawytscha(Richardson et al. 1985); common carp, Cypri-nus carpio (Hossain and Jauncey 1993); tilapia,Oreochromis niloticus x O.aureus (Riche andGarling 2004); and Atlantic salmon, Salmosalar (Denstadli et al. 2006). So far, there isno available information on the effects of IP6for marine species including Japanese flounder.

The objectives of this study were to deter-mine the effect of different levels of dietary IP6on growth, feed intake, nutrient digestibility,nutrient retention, vertebral, and plasma mineralcontents in juvenile Japanese flounder.

Materials and Methods

Test Diets

Basal diet composition (Table 1) was for-mulated to satisfy nutritional requirement ofJapanese flounder using fishmeal, krill meal,and casein as protein sources. Phosphorus-free mineral mixture was applied for thetrial according to Uyan et al. (2007). Sodiummonophosphate was used as a P source tomeet the P requirement level of Japanese floun-der. Five different levels (0, 7.5, 15, 20, and30 g/kg) of synthetic IP6 (72.5% purity; Sigma-Aldrich, St. Louis, MO, USA) were supple-mented to a basal diet to obtain expectedcontents of IP6. Diet without IP6 was used as apositive control. All dry ingredients were mixed

Table 1. Basal diet composition.

Ingredient g/kg

Brown fishmeal 320Casein (vitamin free) 170Krill meal 150Dextrin-hydrate 40α-Starch 40Pollack liver oil 80n-3 HUFAa 10Activated gluten 50Vitamin mixtureb 30APMc 1P-free mineral mixtured 30Sodium monophosphatee 40Phytic acidf 0Attractantg 5α-Celluloseh 34Total 1000

aPowash A, Oriental Yeast Co., Ltd, Tokyo, Japan.bVitamin mixture (g/kg diet): β-carotene, 0.192; vitamin

D3, 0.019; menadione, 0.0917; α-tocopherol acetate, 0.77;thiamin nitrate, 0.115; riboflavin, 0.385; pyridoxine-HCl,0.092; cyanocobalamin, 0.00018; d-biotin, 0.0115; inositol,7.698; nicotinic acid, 1.539; Ca-pantothenate, 0.5391; folicacid, 0.0288; choline chloride, 15.738; ρ-aminobenzoicacid, 0.7665; cellulose, 2.849.

cAscorbic acid monophosphate-Mg.dPhosphorus-free mineral mixture (g/kg diet): KCl,

1.392; MgSO4.5H2O, 3.8; Fe citrate, 0.82; Ca lactate, 9.07;Al(OH)3, 0.0052; ZnSO4.7H2O, 0.099; CuSO4, 0.0028;MnSO4.5H2O, 0.022; K(IO3)2, 0.0045; CoSO4.7H2O,0.028; cellulose, 14.75.

eWako Pure Chemical Industries, Ltd, Osaka, Japan.fSodium phytate, P8810; Sigma-Aldrich, St. Louis, MO,

USA (72.5% purity).gAttractant (g/kg diet): betaine 2.0; taurine 3.0.hPhytic acid was added at the expense of α-cellulose.

using commercial mixer, and then lipid sourceswere added and mixed well. Distilled waterwas added to the diets and mixed again. Dietswere pelletized using meat chopper and thendried in the oven at 50 C until moisture con-tent becomes 12–15%. Test diets were storedat −20 C until use and during the trial.

Feeding Trial

Juvenile Japanese flounders were obtainedfrom a commercial hatchery (Matsumoto SuisanCo., Miyazaki, Japan) and maintained withcommercial feed (Higashimaru, Kagoshima,Japan) for 1 wk. Fish (average initial weightof 4.58 g) were distributed into 15 tanks of

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100 L capacity at a density of 12 fish/tank withtriplicates. Each tank was supplied with fil-tered seawater with flow-through system at aflow rate of 1.5 L/min. During the feeding trial,water temperature, salinity, and dissolved oxy-gen ranged from 24 to 29 C, 31–33 ppt, and4.4–4.8 mg/L, respectively. Each diet was fedto fish at nearly satiation level twice a day (0800and 1600). Fish were weighted every 10 d toadjust the ration size, and a feeding trial wasconducted for 50 d.

Whole Body, Blood, and Vertebral Sampling

At the beginning of a feeding trial, 15 fishwere randomly taken, freeze-dried, and crushedusing blender for whole body sample. At theend of a feeding trial, the samples of threefish from each tank representing the meanbody weight were taken for the analysis andwere stored at −20 C until analysis. At thesame time, blood samples were drawn fromthe caudal vein with heparinized (1000 IU/ml)syringe (1 ml, needle size 25Gx1; Terumo Co.,Tokyo, Japan) from three fish in each tank.Blood samples from three fish were pooledinto 1.5 mL microtube to make one sample pertank. Plasma was separated by centrifugation(1000 x g, 10 min at 4 C) and stored at −20 Cuntil analysis.

From the same fish used for plasma analysis,vertebral samples were obtained by heating thefish in microwave for 3 min, and connectivetissues were removed and finally washed withdistilled water. All samples from each tankwere also pooled for the analysis, and thosewere defatted by chloroform–methanol (2:1)extraction according to Folch et al. (1957).Defatted vertebrae were dried and pulverizedwith mortar and pestle, and stored at −20 Cfor total P analysis.

Digestibility Assessment

A digestibility trial was carried out after a50-d feeding trial using remaining live fishin each dietary treatment. Fish from the sametreatments were pooled and redistributed ran-domly into duplicate tanks with density ofsix fish per tank (ABW = 43.2 g). Chromium

oxide (Cr2O3) was added to test diets as an inertmarker at a level of 0.5%. Test fish were fed thediets containing Cr2O3 under the same condi-tion of a feeding trial. Fecal collection was car-ried out after 5 d adaptation of chromium dietsfrom first feeding. After each feeding, all tankswere cleaned up to remove uneaten diet andfecal residues. Feces were collected by siphon-ing at a 3-h interval between morning andafternoon feeds. Collected feces were gentlyrinsed with distilled water to remove contam-inating seawater, frozen after every sampling,and then freeze-dried immediately. Becausefeces of Japanese flounder were well intact,very rapidly settled to the bottom of the tank,and did not easily break out in rearing water,nutrient and marker losses from the feces wereminimized under the experimental condition.Obtained feces were kept at −20 C for protein,lipid, total P, and chromium analysis. Fecescollection continued for 2 wk until sufficientamounts of feces were obtained for analysis.

Chemical Analysis

Proximate analysis including moisture, crudeprotein, and ash for test diets, whole body,and feces were carried out according to AOAC(1995). IP6 contents of diets were determinedspectrophotometrically based on Haugh andLantzsch (1983). Total lipids were analyzedbased on Bligh and Dyer (1959). Analyses oftotal P were carried out based on the methodof Lowry and Lopez (1946). Concentrationsof Cr2O3 in test diets and feces were deter-mined according to Furukawa and Tsukahara(1966). Analyses of plasma inorganic P andMg were carried out by using blood ana-lyzer (Spotchem™ EZ SP 4430, Arkray Inc.,Kyoto, Japan).

Calculation and Statistical Analysis

Nitrogen and P retention (R) were calculatedaccording to the following formula: R = 100 ×(FBW × Nf) − (IBW × Ni )/(feed intake × Ndiet), where IBW and FBW are initial andfinal body weights, respectively, and N isthe concentration of nutrient in question infish and diet. Subscripts i and f represent

INFLUENCE OF DIETARY PHYTIC ACID IN JUVENILE JAPANESE FLOUNDER 749

initial and final sampling days. Apparentnutrient digestibility was calculated by thefollowing equation: ADC (%) = 100 × [1 −(Ni /Nf ) × (Cf /Ci )] where Ni and Nf are theconcentrations (%, dry matter) of nutrients inthe diet and feces, respectively, and Ci and Cf

are the concentrations (% dry matter) of themarker in the diet and feces, respectively.

Data were statistically analyzed by one-waynalysis of variance (P < 0.05), followed byDuncan multiple range test to identify signif-icant differences among treatments.

Results

Chemical Composition of Test Diets

Analytical values of protein and lipid wererelatively close to those of calculated ones indiet formulation. Detected levels of dietary IP6were 0, 5.1, 10.4, 13.5, and 20.6 g/kg diet. Ashand total P increased with increasing level ofIP6 supplementation (Table 2).

Growth Performances

Results of growth performances after a 50-dfeeding trial are presented in Table 3. Fish fed adiet without IP6 supplementation (positive con-trol group) had the highest specific growth rate(SGR, 4.64 %/d) among groups. However, 5.1or 10.4 g/kg IP6 supplementation groups didnot show any significant differences comparedwith positive control group. In this study, thehighest level of dietary IP6 (20.6 g/kg) had thelowest weight gain and SGR, but did not signif-icantly differ from the group fed 13.5 g IP6/kgdiet. With the exception of few dead fish foundin groups fed 13.5 and 20.6 g IP6/kg at Day 30,

Table 2. Chemical composition (% dry basis) of testdiets.

Dietary IP6 (g/kg)

Composition 0 5.1 10.4 13.5 20.6

Moisture 9.8 10.7 9.8 10.2 9.4Crude protein 49.7 49.2 49.6 49.3 49.2Total lipid 9.8 9.8 9.4 9.7 9.8Ash 10.1 10.7 11.3 11.5 11.9Phytic acid 0.0 0.51 1.04 1.35 2.06Total phosphorus 1.87 2.02 2.33 2.57 2.74

there were no dead fish observed during a 50-dfeeding trial.

At the end of feeding trial, the lowest cumu-lative feed intake was observed in fish fed dietwith highest level of IP6 (20.6 g/kg). Dietaryinclusion levels of IP6 significantly reduced thefeed intake, which was highest in control group,but did not differ between 5.1 and 10.4 g IP6/kggroups. The feed intake of fish fed diets withhigher IP6 of 13.5 and 20.6 g/kg had signifi-cantly lower level than that in positive controlgroup. There were no significant differences infeed conversion ratio (FCR) among groups.

Nutrient Digestibility

Results of nutrient digestibility are presentedin Table 4. Protein and lipid digestibility sig-nificantly decreased with increasing level ofdietary IP6. High inclusion level of dietary IP6(20.6 g/kg diet) showed the lowest digestibil-ities of both protein and lipid. A decreasingtrend toward high supplementation levels of IP6in diets was indicated although there were nosignificant differences in P digestibility amonggroups.

Whole-Body Composition and NutrientRetention

Dietary IP6 did not influence dry matter andprotein content of whole fish body (Table 5).Lipid and P contents slightly decreased withincreasing level of dietary IP6. Fish fed dietsupplemented with 20.6 g IP6/kg showed thelowest P content but did not differ from thatfed a diet with 13.5 g/kg. Dietary IP6 resultedin an increase in ash content.

Inclusion of IP6 up to 10.4 g/kg did notaffect nitrogen retention, but eventually loweredthe values with increased IP6 supplementation(Table 5). P retention significantly decreasedwith increasing level of dietary IP6. Fish feddiet with the higher IP6 (more than 13.5 g/kg)indicated significantly lower P retention thanother groups.

Mineral Content in Plasma and Vertebrae

In the blood analysis (Table 6), dietary IP6was a significant factor that lowered the level

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Table 3. Growth performances of juvenile Japanese flounder fed diets with different levels of IP61.

Dietary IP6 (g/kg)

Parameter 0.0 5.1 10.4 13.5 20.6 Pooled S.E.M

Initial weight (g) 4.59 ± 0.01 4.59 ± 0.01 4.56 ± 0.03 4.58 ± 0.03 4.58 ± 0.01 0.01Final weight (g) 46.7 ± 2.3 45.6 ± 1.1 42.5 ± 1.1 41.5 ± 2.2 39.8 ± 2.2 1.02Weight gain (%)2 579.0 ± 10c 563.0 ± 47bc 544.8 ± 36abc 499.7 ± 110ab 479.6 ± 112a 11.93Specific growth rate (%/d)3 4.64 ± 0.10c 4.59 ± 0.05bc 4.54 ± 0.04abc 4.46 ± 0.11ab 4.39 ± 0.11a 0.05Survival rate (%) 100 ± 0.0a 100 ± 0.0a 100 ± 0.0a 96.7 ± 5.8a 96.7 ± 5.8a 1.33Feed intake (g/fish) 36.7 ± 0.9c 35.9 ± 0.4bc 35.3 ± 0.3bc 33.7 ± 1.6ab 33.0 ± 2.2a 0.62FCR (g/g)4 0.92 ± 0.04a 0.93 ± 0.03a 0.99 ± 0.01a 0.9 ± 0.07a 1.0 ± 0.08a 0.03

1Values are expressed as means of three replicates. Values with different superscripts are significantly different(P < 0.05).

2Weight gain (%) = (final body weight – initial body weight)/initial body weight × 100.3Specific growth rate (%/d) = (ln final body weight − ln initial body weight)/time (50 d) × 100.4Feed conversion ratio = feed intake (g)/weight gain (g).

Table 4. Apparent digestibility coefficient (%) of dry matter, protein, lipid, and phosphorus of juvenile Japaneseflounder fed different levels of IP61.

Dietary

Parameter 0.0 5.1 10.4 13.5 20.6 Pooled S.E.M

Dry matter 81.9 ± 0.1 82.0 ± 0.2 81.7 ± 0.1 81.0 ± 0.8 80.2 ± 0.3 0.19Crude protein 95.0 ± 0.1d 94.9 ± 0.2d 94.3 ± 0.0c 93.9 ± 0.2b 93.0 ± 0.1a 0.08Lipid 92.2 ± 0.5c 92.4 ± 0.4c 91.6 ± 0.3b 90.6 ± 1.1b 88.6 ± 0.0a 0.34Phosphorus 52.1 ± 0.5 51.7 ± 1.9 50.9 ± 0.7 50.1 ± 2.4 49.1 ± 0.0 0.79

1Values are expressed as means of three replicates. Values with different superscripts are significantly different(P < 0.05).

of plasma inorganic P and Mg. P level ofthe fish fed the highest dietary IP6 (20.6 g/kg)was significantly lower than those of fish fedthe diet containing 5.1 g and the control diet.A similar trend was also observed in plasmaMg. Level of plasma Mg was highest inpositive control group (1.97 mg/dL) and furtherdecreased with increasing levels of dietary IP6.However, only fish fed diet with the highest

level of IP6 showed significantly lower plasmaMg (1.53 mg/dL) than that fed the control dietwithout IP6 supplement.

P contents in vertebrae ranged from 17.8to 18.7% among treatments, and there was nosignificant difference among groups (Table 6).Only the highest level of dietary IP6 (20.6 g/kg)demonstrated significantly lower Ca and Mglevels in vertebrae than those of other groups.

Table 5. Chemical composition (% dry matter) of whole body and nutrient retention of Japanese flounder fed differentlevels of IP61.

Dietary IP6 (g/kg)

Parameter 0.0 5.1 10.4 13.5 20.6 Pooled S.E.M

Dry matter 27.4 ± 1.5 27.1 ± 0.1 26.8 ± 0.2 26.9 ± 0.4 26.9 ± 0.4 0.31Crude protein 61.1 ± 0.6 61.5 ± 0.5 61.8 ± 0.0 62.1 ± 0.4 62.0 ± 0.3 0.26Lipid 14.2 ± 0.2b 14.2 ± 0.1b 14.0 ± 0.2b 13.8 ± 0.1b 13.0 ± 0.0a 0.10Ash 13.1 ± 0.0a 13.2 ± 0.1a 13.4 ± 0.1b 13.4 ± 0.0b 13.7 ± 0.1c 0.05Total phosphorus 2.20 ± 0.01b 2.22 ± 0.03b 2.23 ± 0.03b 2.11 ± 0.02a 2.09 ± 0.0a 0.02Nitrogen retention 33.7 ± 0.5a 31.1 ± 0.3a 30.1 ± 1.2ab 29.8 ± 1.4b 30.1 ± 1.6b 0.71Phosphorus retention 32.9 ± 0.4d 28.0 ± 0.1c 23.7 ± 1.3b 19.7 ± 1.0a 18.5 ± 0.7a 0.47

1Values are expressed as means of three replicates. Values with different superscripts are significantly different(P < 0.05).

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Table 6. Plasma inorganic P and Mg and vertebral minerals in juvenile Japanese flounder fed different levels of IP61.

Dietary IP6 (g/kg)

Parameter 0.0 5.1 10.4 13.5 20.6 Pooled S.E.M

Plasma in P (mg/dL) 11.37 ± 0.78b 11.23 ± 1.24b 10.5 ± 0.52ab 10.37 ± 1.10ab 9.33 ± 1.01a 0.54Plasma Mg (mg/dL) 1.97 ± 0.35b 1.87 ± 0.21ab 1.67 ± 0.06ab 1.6 ± 0.17ab 1.53 ± 0.06a 0.10Vertebral mineralsP (g/100 g) 17.78 ± 0.13 17.96 ± 0.28 18.04 ± 0.50 18.23 ± 0.26 18.67 ± 0.01 0.34Ca (g/100 g) 26.04 ± 0.31b 25.95 ± 0.06b 26.00 ± 0.06b 24.70 ± 0.81b 23.08 ± 0.76a 0.45Mg (g/100 g) 3.02 ± 0.08b 2.96 ± 0.16b 3.03 ± 0.08b 2.91 ± 0.07b 2.25 ± 0.09a 0.07Zn (mg/kg) 114.89 ± 7.06d 99.75 ± 0.07c 94.86 ± 7.13bc 89.96 ± 0.06abc 79.96 ± 7.01a 3.02

1Values are expressed as means of three replicates. Values with different superscripts are significantly different(P < 0.05).

Inclusion of dietary IP6 significantly decreasedZn levels in vertebrae. Those of fish fed alldiets containing IP6 were significantly lowerthan that of fish fed the control diet.

Discussion

In this study, the growth of floundersdeclined by feeding the diets containing IP6after 30 d of feeding period. Even thoughthere was a tendency for lower growth towardincreased level of IP6, there was no differencein growth among fish with a control group,5.1 g, and 10.4 g/kg supplemented groups. Onthe other hand, higher supplementation level ofdietary IP6 more than 13.5 g/kg significantlylowered the weight gain of flounders. A sim-ilar trend was also observed in feed intake.The effects were more clearly shown at Day40. Difference in growth among treatments wasthought to be because of the difference in feedintake. Recent study demonstrated similar pat-terns of growth and feed intake in Atlanticsalmon fed different levels of dietary IP6 (Den-stadli et al. 2006). They reported that Atlanticsalmon could accept dietary IP6 at a range of4.7–10.0 g/kg for the normal growth and feedintake. Sajjadi and Carter (2004a) also observedno effect on feed intake in the same species afterfeeding a diet with 8 g IP6/kg. In the case ofChinook salmon, high inclusion of synthetic IP6at 25.8 g/kg dramatically depressed the growthrate because of the lowered feed and pro-tein conversion ratios (Richardson et al. 1985).However, the pure synthetic IP6 (5 and 10 g/kgdiet) resulted in lower growth performances

on common carp (Hossain and Jauncey 1991).Because the results of this study on growthperformance and feed intake agree with the pre-vious studies, it seemed that inclusion of IP6more than 10 g/kg would not be recommendedin aquafeeds. Responses on feed intake seem tovary by fish species; however, it is still unclearwhether IP6 has effects in reducing appetiteor changes the physiological properties of fish.Several studies found that feed intake was stim-ulated by dietary phytase that catabolize phyticacids, inducing the growth stimulation (Jacksonet al. 1996; Portz and Liebert 2003). However,the effects of phytase on feed intake and FCRare still contradictory as reported by Sajjadi andCarter (2004a) that Atlantic salmon fed dietaryphytase did not significantly improve the feedintake.

A tendency toward a decrease of P digestibil-ity by increasing IP6 level was observedin this study without the statistical confir-mation. We might be able to predict thatlong-term feeding duration would show moreclear effect of dietary IP6 on P digestibil-ity. Even though the effect of IP6 on pro-tein digestibility was marginal, a high inclusionlevel of IP6 (20.6 g/kg) significantly decreaseddigestibility of protein from 95% in controlto 93%. Sajjadi and Carter (2004a) reportedthat Atlantic salmon fed the diet with 8 gIP6/kg showed lower protein digestibility com-pared with that without IP6 supplementation.Similarly, Spinelli et al. (1983) observed thatinclusion of 5 g IP6/kg decreased the proteindigestibility in rainbow trout. Another studyon the same species also found that phytate

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complexes with protein reduced the availabil-ity of protein and amino acids, suggesting closerelationship between IP6 and protein digestibil-ity (Sugiura et al. 2001). In contrast, Denstadliet al. (2006) found that a high inclusion levelof IP6 did not decrease the protein digestibil-ity of Atlantic salmon. In addition, Riche andGarling (2004) reported that inclusion level ofIP6 at 26 g/kg diet did not significantly affectthe protein efficiency ratio in tilapia. It seemspossible that the protein sources in diets maycontribute to the different effects of IP6 on pro-tein digestibility in fish as assumed by Denstadliet al. (2006). In the latter study and the studyby Riche and Garling (2004), fishmeal was usedas a major protein source, while in the stud-ies by Sajjadi and Carter (2004b) and Spinelliet al. (1983), casein was used as a major pro-tein source. Compared with the present study,even though fishmeal was the major proteinsource, 17% of casein was also used as a pro-tein source which possibly contributed to theresults. Knuckles et al. (1989) found that IP6is capable of producing a small but signifi-cant decrease in the in vitro pepsin digestion ofcasein and bovine serum albumin. Decreaseddigestion correlated with increased phospho-rylation of myoinositol which might relate tocapacity of casein to bind to IP6.

Lower lipid digestibility observed in fishfed highest dietary IP6 (20.6 g/kg) supportedthe in vitro study by Knuckles (1988), whostated that IP6 could bind ternary complexbetween lipase–mineral–IP6. Denstadli et al.(2006) found that high inclusion level of IP6had marginally but significantly reduced lipiddigestibility in Atlantic salmon. At present, onlyone in vivo study reported about the relationshipbetween IP6 and lipid digestibility in fish. Inthis study, decreased lipid digestibility corre-lated with lipid content in the whole body, inparticular, the group fed high IP6. Inclusion of20.6 g IP6/kg significantly lowered body lipidcompared with the other four groups. This isin agreement with Sajjadi and Carter (2004a)who found lower lipid body concentration(11.8%) in fish fed 10 g IP6/kg diet comparedwith control group (12.6%). Furthermore, theydetermined energy digestibility instead of lipid

digestibility and found that energy digestibilityof fish fed 10 g IP6/kg was lower comparedwith a diet without IP6, although there was nosignificant difference among groups. Richard-son et al. (1985) and Usmani and Jafri (2002)observed a similar response in Chinook salmonand Cirrhinus mrigala that fish fed more than5 g IP6/kg diet had lower body lipid content.Possible mechanisms underlying the decreasedlipid or energy digestibility in fish is stillunclear. In the case of poultry, there is evi-dence of IP6 to interact with lipid and proteinso-called lipophytins as discussed by Cosgrove(1966). These complexes may be involvedin the formation of insoluble metallic soapsin the gut of lumen of poultry, which arethe major constraints on utilization of energyderived from lipid, particularly saturated fats(Leeson 1993). Alternatively, ternary bindingof lipase–mineral–IP6 might reduce the enzy-matic capacity of lipase to breakdown the lipid(Knuckles 1988). Starch digestibility was notdetermined. However, as reported by Denstadliet al. (2006), starch digestibility of fish fed thediet without IP6 was not significantly differ-ent from that of fish fed IP6. This result doesnot support the study by Cawley and Mitchell(1968) who demonstrated that IP6 has a potentinhibitory on amylase activity contributing todecrease starch digestibility.

P retention decreased gradually from 33%(a control group) to 18% (20.6 g/kg group).This indicates low P availability in IP6, andthe results agreed with other studies in Atlanticsalmon (Storebakken et al. 1998; Denstadliet al. 2006) and rainbow trout (Vielma et al.2004). The trend of P retention correlated withthe P digestibility, even though there was nosignificant difference in P digestibility amonggroups.

Plasma Mg and inorganic P tended todecrease by increasing level of dietary IP6. Inthis study, only fish fed a diet with the high-est IP6 (20.6 g/kg) showed significantly lowerplasma Mg and inorganic P compared withthose of fish fed a diet without IP6. Eventhough there was no clear trend in P digestibil-ity among groups, decreasing level of plasmaP indicated that the fish could not absorb P in

INFLUENCE OF DIETARY PHYTIC ACID IN JUVENILE JAPANESE FLOUNDER 753

IP6. Atlantic salmon fed the diet containing 8 gIP6/kg had significantly lower P digestibilitycompared with that of fish fed a diet without IP6supplement (Sajjadi and Carter 2004b). Vielmaet al. (2002) observed lower P digestibility andplasma P in rainbow trout when fed the dietcontaining undephytinized soy products. Fur-thermore, Denstadli et al. (2006) found a pro-gressively decreased Mg and Zn digestibility inAtlantic salmon. In this study, digestibility ofboth trace minerals was not determined. How-ever, the data on plasma Mg level observedin this study suggest that availability of tracemineral was reduced by IP6 intake as widelyreported by other studies on poultry (Ravin-dran et al. 2000), Chinook salmon (Richard-son et al. 1985), Atlantic salmon (Sajjadi andCarter 2004b; Denstadli et al. 2006; Hellandet al. 2006), and rainbow trout (Shearer andAsgard 1990).

Vertebral P levels were not affected by inclu-sion of IP6 in diets, which may indicate thatsupplementation of dietary inorganic P was suf-ficient for normal bone mineralization. A sim-ilar response was observed in Atlantic salmonfed the diets with different IP6 levels, in whichthe levels of dietary P ranged from 16.2 to22.5 g/kg (Helland et al. 2006). It has been wellrecognized that P requirement for the maximumbone ash and bone strength is higher than thatfor the growth in terrestrial animals (McDowell2003) and also in fish (Rodehutscord 1996;Bureau and Cho 1999). Ogino and Takeda(1976) reported that the dietary requirementof available P for the maximum growth ofjuvenile carp, Cyprinus carpio, was 0.6–0.7%and that for the maximum bone mineralizationwas higher (1.5%) than that for the optimumgrowth. Skonberg et al. (1997) also reportedthat ash, P, and Ca levels in whole body andskin (with scales) were highly responsive todietary P levels (0.23–1.16% P), but the growthand feed efficiency were unaffected by dietaryP. Furthermore, Sugiura et al. (2004) in thereview of the pathological P deficiency in fishstated that estimated dietary requirement of Pbased on growth rate may not be accurate andis likely to underestimate the true requirement,unless the study period is very long. This

study indicated that high dietary IP6 loweredCa and Mg absorption because the contents ofthose minerals in vertebrae were lowered. Onthe other hand, there were no effects of thedietary IP6 on vertebral Ca and Mg contentsin Atlantic salmon (Helland et al. 2006). More-over, Zn concentration significantly decreasedby increasing the level of dietary IP6 in thisstudy. This agrees with the study of Atlanticsalmon, in which fish fed the diet with a highlevel of IP6 had lower vertebral concentrationof Zn compared with fish fed a diet withoutIP6. Further studies should be required to clar-ify the metabolic relationship between IP6 andminerals in fish.

In this study, no mineral deficiency signswere observed. However, in the study by Hel-land et al. (2006), hyper dense (HD) vertebraewere observed as a result of poor mineraliza-tion. The number of fish with HD vertebraeincreased from initially 16% to 45–60%, witha tendency of more fish with HD vertebrae withincreasing dietary IP6 content. Only few studieshave reported the effect of IP6 on pathologi-cal signs in fish including incidence of cataractindicating mineral-chelating action of IP6 and atoxic effect on the epithelial layer of the pyloriccaecae in Chinook salmon (Richardson et al.1985), also hypertrophy and vacuolization ofthe cytoplasm of the intestinal epithelium incommon carp (Hossain and Jauncey 1991).

The polyanionic IP6 molecule has a substan-tial capacity to chelate divalent cations includ-ing Ca, Mg, and Zn to form mineral–IP6complexes. It is generally believed that fish, likeother monogastric animals, do not or partiallydigest the P bound to IP6 (also called phytate-P)and the mineral–IP6 complexes because oflack of phytase, an enzyme that is capableto hydrolyze the mineral from inositol ring(Ellestad et al. 2002). As reviewed by Franciset al. (2001), even though the tolerated levelof IP6 differed according to fish species, itseems to be advisable to maintain the level ofIP6 below 5 g/kg in fish diet. In this study,if P retention and vertebral Zn are taken intoaccount, it appeared that Japanese floundercould only tolerate the lowest levels of dietaryIP6, 5.1 g/kg diet. In the case of Atlantic

754 LAINING ET AL.

salmon as discussed by Denstadli et al. (2006),the maximum tolerance level of IP6 regard-less of the normal feed intake was 4.7 g/kg. Ifthe diet was supplemented with 20% soy pro-tein concentrate, which contains 12.4–21.7 gIP6/kg (Ravindran et al. 1995), then the dietwould approximately contain IP6 in a range of2.5–4.3 g/kg. Replacement of a similar portionof soy protein concentrate with canola proteinconcentrate containing 53–75 g IP6/kg (Higgset al. 1995) would lead the diet to contain IP6in a range of 10.6–15 g/kg.

This study clearly demonstrated that phyticacids have several negative effects on the per-formances of marine species as observed injuvenile Japanese flounders, which have to becarefully taken into account when supplement-ing plant feedstuffs in aquafeeds.

Acknowledgments

We thank the Ministry of Education, Cul-ture, Sport, Science, and Technology (Monbuk-agakusho), Japan, for supporting this researchwork.

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