biotropia book final.indd 110 EFFECT OF PHYTASE SUPPLEMENTATION IN SOYBEAN MEAL BASED DIET ON NUTRIENT DIGESTIBILITY AND GROWTH PERFORMANCE OF GREEN CATFISH (Hemibagrus nemurus) YULISMAN, DEDI JUSADI* and ING MOKOGINTA Department of Aquaculture, Faculty of Fisheries and Marine Sciences, Bogor Agricultural University, Kampus IPB Darmaga, Bogor, Indonesia ABSTRACT Th is experiment was conducted to evaluate the eff ect of phytase supplemented to the diet on phosphorus (P) digestibility and growth performance of the green catfi sh Hemibagrus nemurus. Five kinds of experimental diets were used in this experiment, namely diets A, B, C, D and E. Diet A, as a control, was supplemented with inorganic P, without phytase supplementation. Diet B, C, D and E were supplemented with 0, 20, 40 and 60 mg phytase/100 g soybean meal (SBM), respectively, without inorganic P supplementation. Fifteen fi sh with initial body weight of 6.9 + 0.2 g were stocked into each 60-l aquarium. Fish were fed on the diets for 60 days. Results indicated that P digestibility increased from 64.5% to 87.0% as phytase supplement increased from 0 mg in diet B to 60 mg phytase/100 g SBM in diet E. P digestibility in diet E was higher than that in diet A (77.6%). Th e daily growth rate and feed conversion ratio followed similar trend. P, Ca and Zn concentration in the whole body and bone of fi sh fed diet E were higher than the fi sh fed diet B, C and D, but were insignifi cant compared to the fi sh fed diet A. Nitrogen (N) and P loading by fi sh fed diet E were, respectively, 76% and 20% lower than those in fi sh fed the control diet. It is concluded that the inclusion of 60 mg phytase/100 g SBM in the diet of the green catfi sh could replace the utilization of inorganic P increase the digestibility of the diet thereby resulting in increased growth rate and reduced excretion of P and N into the waters. Key words: Hemibagrus nemurus, phytase, soybean meal, phosphorus. INTRODUCTION Th e expansion of global aquaculture production increases the demand for aquaculture feeds. For carnivore species as green catfi sh Hemibagrus nemurus, protein can form up to 60% of the diet and fi shmeal is the main source of dietary protein. Fishmeal is one of the most expensive and demanded ingredients and has become the main and most critical ingredient in aquafeed production. Th e increasing cost and demand of fi shmeal has encouraged feed manufacturers to search for cheaper alternative protein source as plant protein. Soybean meal (SBM) is a common plant protein source used as a substitute for *Corresponding author: dedijusadi@yahoo.com BIOTROPIA VOL. 15 NO. 2, 2008 : 110 - 118 111 fi shmeal in the fi sh diet. It could replace 75% of fi shmeal as a protein source in the diet of the green catfi sh (Pebriyadi 2004). A major obstacle in using SBM is their high phytic acid content. Phytic acid is a substance that contains unavailable phosphorus (P) to monogastric animals, including fi sh, because these animals lack phytase, which can hydrolyze phytic acid (Baruah et al. 2004). By using phytase supplement to the diet, the P digestibility can be improved. Li et al. (2004) reported that 250 units of phytase per kg diet could eff ectively replace di-calcium phosphate supplement in the diet of channel catfi sh without aff ecting growth, feed effi ciency or bone phosphorus deposit. Masumoto et al. (2001) showed that 0.2 g of phytase supplement to the diet containing 67% SBM increased the growth rate and feed effi ciency of the Japanese fl ounder Paralicthys olivaceus. Th erefore, using phytase supplement in the fi sh diet could increase the bioavailability of phosphorus of SBM. Hence, replacing the inorganic P of the diet, resulting in lower fecal P loss. Th is experiment was conducted to evaluate the eff ect of phytase supplement to the diet on P digestibility and growth performance of the green catfi sh Hemibagrus nemurus. MATERIALS AND METHODS Experimental diets Five experimental diets were used in this experiment, namely diets A, B, C, D and E. Diet A, as a control, was supplemented with inorganic P (NaH2PO4.H2O, KH2PO4, Ca2(PO4)3) only, without phytase supplement. Diet B, C, D and E were supplemented with 0, 20, 40 or 60 mg phytase (Natuphos® 5000)/100 g SBM, respectively, without inorganic P supplement (Table 1). Th e proximate composition of experimental diets is shown in Table 2. Table 1. Ingredients composition of experimental diet (g/kg diet) Ingredients Treatments A B C D E Fish meal1 130 130 130 130 130 Soybean meal1 640 640 640 640 640 Fish oil 40 40 40 40 40 Soybean oil 44 44 44 44 44 Tapioca1 57 57 47,87 47,74 47,62 Vitamin mix 15 15 15 15 15 Choline chloride 5 5 5 5 5 L-Methionine 5 5 5 5 5 Taurin 6 6 6 6 6 Mineral mix2 58 0 0 0 0 P-free mineral mix3 0 58 58 58 58 Phytase 0 0 0.13 0.26 0.38 Citric acid 0 0 9 9 9 P content (%): Total P 1.46 0.55 0.55 0.56 0.58 Water-soluble P 0.59 0.19 0.45 0.48 0.53 Phytase for diet of green catfi sh (Hemibagrus nemurus) – Yulisman et al. 112 Ingredients Treatments A B C D E Water-soluble P/total P ratio 40.29 34.87 82.79 86.06 91.13 1 Crude protein (dry weight) concentration of: fish meal 70.39%, SBM 42.75%, tapioca meal 0.91%. 2 The mineral mix had the following composition (g/kg dry diet): NaCl 0.5; MgSO4.7H2O 7.5; NaH2PO4H2O 12.5; KH2PO4 16; Ca2(PO4)3 14.49; Fe-citric 1.25; filler 3.60 and trace element mix (0.5 g) had the following composition: ZnSO4.7H2O 17.65; MnSO4 8.1; CuSO4.5H2O 1.55; CoCl.6H2O 0.05; KIO3 0.15; and filler 30.5 (Takeuchi 1988). 3 P-free mineral mix had the following composition (g/kg dry diet): NaCl 0.5; MgSO4.7H2O 7.5; KCl 17.53; Fe-citric 1.25; CaCl2.2H2O 13.34; filler 30.5 and trace element mix (0.5 g) had the following composition: ZnSO4.7H2O 17.365; MnSO4 8.1; CuSO4.5H2O 1.55; CoCl.6H2O 0.05; KIO3 0.15; and filler 30.5. Table 2. Proximate composition of experimental diets (% wet weight) Proximate Composition Treatments A B C D E Crude Protein 31.19 29.49 29.30 30.11 29.19 Crude Lipid 10.58 10.62 10.18 10.30 9.92 Ash 11.09 8.74 8.92 9.12 8.79 Water 14.01 18.98 20.38 17.04 20.23 Fiber 4.20 3.21 2.62 2.92 2.24 NFE1 28.93 28.96 28.60 30.51 30.87 Total Energy2 (kcal/100g) 392.73 383.71 377.03 390.53 387.38 Energy/Protein (kcal/g protein) 12.59 13.01 12.87 12.97 13.27 1 Nitrogen Free Extract. 2 Total Energy (GE) was calculated based on equivalent values of: protein 5.6 Kcal/g, lipid 9.4 Kcal/g, and NFE 4.1 Kcal/g (Takeuchi, 1988). For the pretreatment of SBM with phytase, 640 g of SBM was mixed with 2240 ml water, and then phytase was added to the SBM-water mixed at 20, 40, 60 mg/100g, respectively, in the diets C, D and E. Th e pH was adjusted to 5.5 with citric acid. Th e mixtures were incubated at 37 °C for 2 h (Matsumoto et al. 2001). Th en, the mixtures were mixed with other ingredients and formed into granules. Th e pellets were stored at -20 °C until use. Feeding trial Green catfi sh juveniles were obtained from the Main Center for Freshwater Aquaculture Development, Sukabumi, West Java, Indonesia. Upon arrival at the Laboratory of Fish Nutrition, of the Bogor Agricultural University, the fi sh were acclimated to experimental conditions for 10 days and fi sh readily adjusted to the experimental diets. Th ereafter, fi fteen juveniles with an initial body weight of 6.9 + 0.2 g were stocked into each 60-l aquarium. Each aquarium was part of a closed recirculation system. During Table 1. Continued BIOTROPIA VOL. 15 NO. 2, 2008 113 rearing period, each aquarium was supplied with continuous aeration and water was allowed to fl ow through at the rate of 200-300 ml/min. Every day, impurities in the water of each aquarium were removed and 50% of the water was renewed to maintain water quality. From each aquarium, water was fl own to the physical and biological fi lters, then in the conditioning tank. Th ereafter, the water was drained back to each aquarium by using pump. Dissolved oxygen contents were 4.18-5.13 mg/l, water temperatures 28 to 30 ºC, and pH 6.30 to 6.35. Each experimental diet was fed to the fi sh in three aquaria. Th e diets were randomly assigned to groups of fi sh in the aquarium. Th e fi sh were fed on the diets to satiation three times a day at 08.00, 13.00 and 18.00 hrs for 60 days. At the end of the feeding trial, the fi sh of each aquarium were bulk weighed from each aquarium. Growth of the fi sh (as measured by the percentage of daily growth rate), feed conversion ratio (FCR), protein retention (PR) were calculated as described previously (Huisman 1976; Steff ens 1989; Takeuchi 1988). After the fi nal weighing, fi ve fi sh were randomly sampled from each aquarium for body proximate and mineral compositions analysis, and three fi sh were randomly sampled from each aquarium for analyses of bone mineral (P, Ca and Zn) composition. Th e proximate composition and mineral analyses were carried out according to the methods described by Takeuchi (1988). Digestibility trial Th e eff ect of phytase supplementation to the diet on the P digestibility was determined by the indirect method with 0.5% of chromic oxide (Cr2O3) as an inert reference substance. Th e green catfi sh with the same size as that for the feeding trial were used in this digestibility trial. Twelve fi sh were stocked into each 60-l aquarium. Triplicate groups of fi sh were fed on each experimental diet to satiation three times a day for 7 days prior to collection of feces. Feces were collected after 2 h of feeding by siphoning into a plastic sieve. After each collection, the samples from each aquarium were pooled, frozen at -20 ºC and stored for subsequent analyses. Feces collections were conducted for 21 days. Th ereafter, the samples of feces were analyzed for chromium (Cr), P and protein content according to the methods described by Takeuchi (1988). Apparent digestibility of total nutrient, protein, and P were calculated as follows: % digestibility = 1 – (% Cr in feed/ % Cr in feces) x (% ingredient in feces/ % ingredient in feed) X 100 P and Nitrogen (N) losses through feces were calculated as follows: P in feces (g) = undigestible P (%) x P consumption during culture period N in feces (g) = (undigestible N (%) x N consumption during culture period)/6.25 Statistical methods Th e data were statistically analyzed for diff erences among the means by the one- way analysis of variance. Th e Duncan’s test was used to compare treatment means using the statistical software SPSS 12 for windows. Diff erences were considered signifi cant at P<0.05. Phytase for diet of green catfi sh (Hemibagrus nemurus) – Yulisman et al. 114 RESULTS AND DISCUSSION Results indicated that P and protein digestibility increased from 64.5% to 87.0%, and 87.1% to 90.5%, respectively, as phytase supplement increased from 0 mg in diet B to 60 mg phytase/100 g SBM in diet E. P digestibility of diet E was higher than that of diet A (77.6%) (Table 3). P digestibility was correlated with the water-soluble P. Th e water-soluble P content in control diet was 0.59 %. As the phytase supplement increased, water-soluble P increases from 0.19 % in diet B to 0.53 % in diet E (Table 1). It could be observed that the lowest P loss through feces was found for diet E, and the lowest nitrogen (N) loss through feces for diet D. Diet E gave the second lowest N loss through feces. Level of N and P loading by fi sh fed diet E were 76% and 20%, respectively, lower than those in the fi sh fed control diet (A). Table 3. Phosphorus (P) and protein digestibility of experimental diets Parameters Experiment A B C D E P digestibility (%) 77.6 64.5 70.4 73.3 87.0 P consumption (g) 10.4+0.1d 3.2+0.1a 3.2+0.1a 3..4+0.1b 4.0+0.0c P digested (g) 8.1+0.1e 2.0+0.0a 2.3+0.0b 2.5+0.1c 3.5+0.1d P output via feces (g) 2.3+0.0e 1.1+0.0d 1.0+0.0c 0.9+0.0b 0.5+0.0a Protein digestibilty (%) 89.1 87.1 85.1 89.7 90.5 Protein consumption (g) 257.4+2.8d 207.6+3.5a 215.6+3.5b 218.2+4.1b 251.8+1.0c Nitrogen (N) digested (g) 36.7+0.4c 28.9+0.5a 29.4+0.5a 31.3+0.6b 36.5+0.2c N output via feces (g) 4.5+0.1d 4.3+0.1c 5.1+0.1e 3.6+0.1a 3.8+0.0b *) Values within the same row with different superscript letters are significantly different, P<0.05. Concentrations of P, Ca and Zn in the whole body and bone of the fi sh fed diet E were higher than those for the groups of fi sh fed on the diets supplemented with 0-40 mg phytase/100 g SBM, but were not signifi cantly diff erent from those of the fi sh fed diet A (Table 4). Table 4. P, Ca and Zn content in green catfish (% dry weight) fed experimental diets Parameters Experiment A B C D E Whole body P 3.18 + 0.28b 2.08 + 0.27a 2.34 + 0.39a 2.97 + 0.19b 3.20 + 0.12b Ca 3.26 + 0.06c 1.92 + 0.30a 2.12 + 0.07a 2.90 + 0.20b 3.30 + 0.09c Zn 0.012 + 0.001b 0.009 + 0.001a 0.012 + 0.000b 0.012 + 0.001b 0.013 + 0.002b BIOTROPIA VOL. 15 NO. 2, 2008 115 Parameters Experiment A B C D E Bone P 21.41+ 1.12c 13.44 + 0.26 a 16.65 + 0.77b 16.99 + 1.24b 21.46 + 0.43c Ca 33.33 + 0.11d 21.44 + 1.15a 24.42 + 0.76b 26.04 + 1.15c 33.60 + 1.66d Zn 0.032 + 0.002b 0.021 + 0.002a 0.023 + 0.002a 0.024 + 0.003a 0.032 + 0.001b *) Values within the same row with different superscript letters are significantly different, P<0.05. Th e mean body weight gain of the fi sh fed diet E was higher than that of the fi sh fed with the other diets. Th e daily growth rate and feed conversion ration (FCR) followed similar trend, while the fi sh fed diet B had the lowest daily growth rate and FCR (Table 5). Th e protein retention had the similar trend with the protein digestibility, which increased from 36.8% from 0 mg phytase/100 g SBM in the diet B to 44.4% from 60 mg phytase/100 g SBM in the diet E. Table 5. Initial body weight (Wo), final body weight (Wt), protein retention (PR), daily growth rate (DGR), feed consumption (FC), feed conversion ratio (FCR) and survival rate (SR) of green catfish Parameters Treatments A B C D E Wo (g) 7.0+0.01 7.0+0.02 6.9+0.12 6.8+0.09 6.8+0.05 Wt (g) 43.8+0.8 31.9+1.1 35.2+ 0.9 40.2+0.4 47.7+0.4 DGR (%) 3.1+0.0d 2.6+0.1a 2.8+0.1b 3.0+0.0c 3.3+0.0e PR (%) 39.5+0.2b 36.8+0.4a 40.1+1.4b 41.6+1.4b 44.4+1.9c FC (g) 709.7+7.6e 570.4+9.7a 585.9+9.5b 601.1+11.2c 688.1+2.7d FCR 1.3+0.0 c 1.5+0.1e 1.4+0.0 d 1,2+0.0 b 1.1+0.0 a SR (%) 100+0.0 100 + 0.0 100+0.0 100+0.0 100+0.0 *) Values within a row with different superscript letters are significantly different, P<0.05. Increasing levels of phytase level from 0 mg to 60 mg/100g of SBM resulted in increasing levels of water-soluble P, thereby increasing water-soluble P/total P ratios in the diets from 34.87% to 91.13%. Diet E produced the highest P and protein digestibility, followed by diet A (control diet) (Table 3). It means that phytase released P from the insositol ring of phytate to be water-soluble P (Baruah et al. 2004). Th e water-soluble P was digested in the intestine of fi sh, and improved the absorption and utilization of P. Phytase also released some protein and amino acids from the phytic acid, hence the absorption and utilization of protein also increased. Th e same results were found for other fi sh species, such as the Atlantic salmon Salmo salar (Sajjadi and Carter 2004), striped bass Morone saxatilis (Papatryphon and Shoares 2001), rainbow trout Onchorhynchus mykiss (Cheng and Hardy 2004), Korean rockfi sh Sebastes schlegeli (Yoo et al. 2005), and tilapia Oreochromis niloticus (Liebert and Portz 2005). On the other hand, the fi sh fed on the diet B had the lowest P digestibility. It appears that the concentration of phytase in diet B could not enough release all P from phytic acid in the SBM. Table 4. Continued Phytase for diet of green catfi sh (Hemibagrus nemurus) – Yulisman et al. 116 Table 4 shows that not only P availability in the diet could be improved by phytase supplementation, but also the other minerals as Ca and Zn which could be available to the fi sh. Accumulation of Ca, P and Zn in the body and bone of fi sh also increased as the phytase levels of the diets increased from 0 to 60 mg/100 g SBM. Other experiments also showed that phytase supplemented diet increased Ca, P and Mn contents of the African catfi sh Clarias gariepinus (Nwanna et al. 2005), striped bass Morone saxatilis (Hughes and Soares 1998), and Atlantic salmon Salmo salar (Sajjadi and Carter 2004). It is known that phytic acid is the major P storage compound in plant seeds, including SBM. Phytic acid is also a strong chelator of important minerals such as Ca, Mg, K, Fe, Cu, Zn and forms poorly soluble complexes. Apart from minerals, phytic acid also forms complexes with protein and amino acids. Th e digestibility of these complexes by fi sh are very limited due to the lack of intestinal phytase (Pointillart et al.1987). Th e supplementation of phytase in this experiment could release those minerals and protein from phytic acid, resulted in increasing their (minerals and protein) digestibility for fi sh (Table 1 & 3), hence the absorption and utilization of minerals and protein also increased (Table 4 & 5). On the other hand, minerals also play a role in many processes of protein, lipid and carbohydrate metabolisms. Increasing the availability of some minerals in the diet by phytase supplementation also appears to improve the protein synthesis, which can be seen from the protein retention data (Table5). Th e highest protein retention was found in the groups of fi sh fed on diet E, while the fi sh fed on diet B had the lowest protein retention. Th e increased protein retention also improved the protein deposit in the body, hence increasing the daily growth rate (Table 5). In this experiment, the fi sh fed on diet E had the highest protein retention and daily growth rate. Th is indicates that phytase could increase the nutrient (protein) digestibility leading to higher protein retention, resulting in the increase of the growth rate and reduce feed conversion ratio. Th e same conclusions were also indicated by other authors that the phytase supplementation to SBM improved the weight and growth rate of juvenile Korean rockfi sh Sebates schlegeli (Yoo et al. 2005), tilapia Oreochromis niloticus (Liebert and Ports 2005; Furuya et al. 2001), rainbow trout Oncorhynchus mykiss (Vandenberg et al. 2003), and Pangasius pangasius (Debnath et al. 2005). Nwanna et al. (2005) also found that supplementation of phytase to a diet containing SBM gave better feed conversion than that for diet without phytase supplementation for the African catfi sh Clarias gariepinus, and the striped bass Morone saxatilis (Hughes and Soares 1998). As earlier explained some of the diet consumed by fi sh was undigested, and will be excreted through feces. Increasing phytase concentration in the diet from 0 mg to 60 mg/100g SBM reduced the levels of P and N excretion through feces. Th is indicated that phytase was able to release nutrient (protein) and minerals of phytate, resulting in the improvement of fi sh growth and also reduced P and N excretion through feces. Hughes and Soares (1998) concluded that phytase in a diet containing high level of plant protein could reduce the P excretion of striped bass Morone saxatilis, seabass Dicentrarchus labrax (Teles et al. 1998), Japanese fl ounder Paralichthys olivaceus (Masumoto et al. 2001), rainbow trout Oncorhynchus mykiss (Vielma et al. 2002), Atlantic salmon Salmo salar (Sajjadi and Carter 2004), tilapia Oreochromis niloticus (Furuya et al. 2001; Liebert and Portz 2005), and the African catfi sh Clarias gariepinus (Van Weerd et al. 1999; Nwanna et al. 2005), thereby reducing the loading of P waste into the environment. BIOTROPIA VOL. 15 NO. 2, 2008 117 CONCLUSIONS Th e supplement of 60 mg phytase/100 g SBM in the diet improved P digestibility and the growth of the green catfi sh Hemibagrus nemurus, hence reducing the loading of P and N waste to the environment. REFERENCES Baruah K., N.P. Sahu, A.K. Pal, and D. Debnath . 2004. Dietary phytase: an ideal approach for a cost eff ective and low-polluting aquafeed. NAGA, Worldfi sh Center Quarterly 27 (3) 3 & 4 Jul-Dec 2004. p. 15-19. Cheng ZJ, and R.W. Hardy . 2004. Eff ect of microbial phytase supplementation in corn distiller’s dried grain with solubles on nutrient digestibility and growth performance of rainbow trout, Oncorhynchus mykiss. Journal of Applied Aquaculture. 15 (3/4): 83-100. Debnath D., A.K. Pal, N.P. Sahu, K.K. Jain, S. Yengkokpam and S.C. Mukherjee. 2005. Eff ect of dietary microbial phytase supplementation on growth and nutrient digestibility of Pangasius pangasius fi ngerlings. Aquaculture Research (36) 2: 180 – 187. Furuya W.M., G.S. Gonclaves, V.R.B. Furuya , C. and Hayashi. 2001. Phytase as feeding for Nile Tilapia (Oreochromis niloticus). Performance and digestibility. Rev. Bras. Zootec. 30: 924 – 929. Hughes K.P. and J.H. Soares Jr. 1998. Effi cacy of phytase on phosphorus utilization in practical diets fed to striped bass Morone saxatilis. Aquaculture Nutrition, 4: 133 – 140. Huisman E.A. 1976. Food conversion effi ciencies at maintenance and production levels for carp (Cyprinus carpio Linn) and rainbow trout (Salmo gairdneri Ric.). Aquaculture, 9 (2): 259-273. Liebert F. and L. Portz . 2005. Nutrient utilization of Nile tilapia (Oreochromis niloticus) fed plant based low phosphorus diets supplemented with graded levels of diff erent sources of microbial phytase. Aquaculture, 248: 111 – 119. Li M.H., B.B. Manning and E.H. Robinson. 2004. Summary of phytase studies for channel catfi sh. Mississippi Agricultural and Forestry Experiment Station (23) 23 No. 13: 1-5. Masumoto T., B. Tamura and S. Shimeno. 2001. Eff ects of phytase on bioavailabilty of phosphorus in soybean meal-based diets for japanese fl ounder (Paralichthys olivaceus). Fisheries Science, 67:1075-1080. Nwanna L.C., O.A. Fagbenro and A.O. Adeyo. 2005. Eff ect of diff erent treatments of dietary soybean meal and phytase on the growth and mineral deposition in African catfi sh Clarias gariepinus. Journal of Animal and Veterinary Advances. 4: 980 – 987. Papatryphon E., and J.H. Soares Jr. 2001. Th e eff ect of phytase on apparent digestibility of four practical plant feedstuff s fed to striped bass, Morone saxatilis. Journal Aquaculture Nutrition. 161-167. Pebriyadi, B. 2004. Supplementation of methionine and tryptophan in the high soybean meal diets of juvenile green catfi sh (Mystus nemurus). Th esis (in Indonesian). Graduate School, Bogor Agricultural University. 53 p. Pointillart A., A. Fourdin and N. Fontaine. 1987. Importance of cereal phytase activity for phytate phosphorus utilization by growing pigs fed diets containing triticale or corn. Journal of Nutrition 29: 907- 912. Sajjadi M. and C.G. Carter. 2004. Dietary phytase supplementation and the utilization of phosphorus by Atlantic salmon (Salmo salar L.) fed a canola-meal-based diet. Aquaculture 240: 417 – 431. Takeuchi T. 1988. Laboratory work-chemical evaluation of dietary nutrients. p. 179-233, In Watanabe (Ed) Fish nutrition and mariculture. Kanagawa International Fisheries Training. Japan International Cooperation Agency (JICA), Japan. Phytase for diet of green catfi sh (Hemibagrus nemurus) – Yulisman et al. 118 Teles A.O., J.P. Pereira, A. Gouveia and E. Gomes. 1998. Utilization of diets supplemented with microbial phytase by seabass (Dicentrarchus labrax) juvenils. Aquaculture Living Resources, 11 (4): 255- 259. Vandenberg G.W., V. Dallaire, S.L. Scott and J. De la Noue . 2003. Encapsulation of microbial phytase : eff ects on phosphorus bioavailability in rainbow trout (Oncorhynchus mykiss). Aquaculture Nutrition- Contributed papers. http://www.aquacultureassociation.ca/ac03/abstracts/nutrition.htm Van Weerd J.H., K.H.A. Khalaf, F.J. Aartsen and P.A.T. Tjissen. 1999. Balance trials with African catfi sh Clarias gariepinus fed phytase-treated soybean meal based diets. Aquaculture Nutrition, 5:135- 142. Vielma J., K. Ruohonen and M. Peisker . 2002. Dephytinization of two soy proteins increases phosphorus and protein utilization by rainbow trout, Oncorhynchus mykiss. Aquaculture. 204: 145 – 156. Watanabe T. 1988. Fish nutrition and mariculture. JICA Textbook. Th e general aquaculture course. Department of Aquatic Biosciences. Tokyo University of Fisheries. 233 p. Yoo G.Y., X. Wang, S. Choi , K. Han, J.C. Kang and S.C. Bai SC. 2005. Dietary microbial phytase increased the phosphorus digestibility in juvenile Korean rockfi sh Sebastes schlegeli fed diets containing soybean meal. Aquaculture 243. 315 – 322. BIOTROPIA VOL. 15 NO. 2, 2008