Effects of the Dietary Microbial Phytase Supplementation on Bioavailability of Phosphorus in Juvenile Olive Flounder Paralichthys olivaceus Fed Soybean Meal based Diets

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  • ABSTRACT

    An 8 weeks feeding trial was carried out to evaluate the effects of dietary microbial phytase (P) supplementation on nutrient digestibility, and body composition in juvenile olive flounder Paralichthys olivaceus fed soybean meal-based diets. Seven experimental diets were formulated to be isonitrogenous and isocaloric to contain 50.0% crude protein (CP) and 16.7 kJ of available energy/g with or without dietary phytase supplementation. White fish meal (FM) provided 92.4% of the total protein in the basal diet (S0), in the other 6 diets, 30% or 40% FM protein was replaced by soybean meal: 70% FM + 30% soybean meal (S30); 70% FM + 30% SM + 1000 U phytase/kg diet (S30P1000); 70% FM + 30% SM + 2000 U phytase/kg diet (S30P2000); 60% FM + 40% SM (S40); 60% FM + 40% SM + 1000 U phytase/kg diet (S40P1000); and 60% FM + 40% SM + 2000 U phytase/kg diet (S40P2000). After two weeks of the conditioning period, triplicate groups of 25 fish initially averaging 6.15 ± 0.04 g (mean ± S.D.) were randomly distributed into the aquarium and were fed one of the experimental diets for 8 weeks. After feeding trial, supplementation of phytase significantly improved the apparent digestibility coefficients of phosphorus in flounder diets (P<0.05) containing 30% and 40% soybean meal regardless the levels. However, phytase had no significant influence on growth performance and whole body composition of fish. Based on the experimental results, we conclude that dietary supplementation of phytase could improve the apparent digestibility coefficient of phosphorus in olive flounder.


  • KEYWORD

    Paralichthys olivaceus , Phytase , Posphorus , Digestibility , Olive flounder

  • Introduction

    Production of finfish culture increased gradually these years in Korea. Aquaculture of olive flounder Paralichthys olivaceus in Korea started in the 1970s and commercial production became extensive in the 1980s with development of seed production and rearing techniques. Olive flounder is currently the most important marine aquaculture species in Korea and its aquaculture production has been stable during last 10 years ranging from 40,000-50,000 metric tons (Ministry of Oceans and Fisheries, 2013). However, feed costs constitute a large part of the total expenses of an aquaculture facility, because of the use of the expensive fish meal with a large dietary proportion. Many studies have been conducted to seek less expensive protein source alternatives to fish meal, due to the expensive cost and availability issue of this ingredient (Lim et al., 2004; Jang et al., 2005; Barrows et al., 2007). Soybean meal is one of the plant sources that can be used as an alternative protein source. It has a high level of available protein with well-balanced amino acid profile, low price and wide availability (Choi et al 2004; Lim et al., 2011). One of the major problems limiting the use of soybean meal in fish feed is the presence of phytate. Phytate is the primary form of phosphorus (P) in soybean, account for approximately two-thirds of the total P bound as phytate P (Cheng and Hardy, 2002; Cao et al., 2008). Phytate is an indigestible P form, which is practically not available for fish because of lack of an intestinal phytase for efficient hydrolysis of phytate during digestion (NRC, 1993). Consequently, the available P in plant diets may not meet the requirements of fish, and inorganic P is usually supplemented to the diets. On the other hand, undigested phytate P excreted via faeces and contributes to environmental pollution. Excessive excretion of phosphorus in water can stimulate the growth of phytoplankton, thus reducing dissolved oxygen and causing water pollution. Many studies have demonstrated that addition of phytase to fish feeds can improve the utilization of phytate P and decrease the total P load to the environment for several species (Chong et al., 2003; Tantikitti et al., 2005; Kikuch and Furuta, 2009).

    The present study investigated the effects of dietary microbial phytase supplementation on the nutrient digestibility, growth performance and body composition in the juvenile olive flounder fed soybean meal-based diets.

    Materials and Methods

      >  Experimental diets

    Composition of the experimental diets is shown in Table 1. Seven experimental diets were formulated to be isonitrogenous and isocaloric to contain 50% crude protein (CP) and 16.7 kJ of available energy/g with or without dietary phytase supplementation (Natuphos-5000TM, BASF, NJ, USA). Estimated available energy of the experimental diets was calculated as 16.7, 16.7 and 37.7 kJ/g for protein, carbohydrate and lipid, respectively (NRC, 1993). White fish meal (FM) provided 89.1% of the total protein in the basal diet (S0), in the other 6 diets 30% or 40% FM protein was replaced by soybean meal as: 70% FM+30% soybean meal (S30); 70% FM+30% SM+1000 U phytase/kg diet (S30P1000); 70% FM+30% SM+2000 U phytase/kg diet (S30P2000); 60% FM+40% SM (S40); 60% FM+40% SM+1000 U phytase/kg diet (S40P1000); and 60% FM+40% SM + 2000 U phytase/kg diet (S40P2000). All the ingredients were mixed completely and then pelleted by using a 2 mm diameter module. After processing, all the diets were packed into small bags and kept at -80˚C until use.

      >  Fish and feeding trial

    Juvenile olive flounder were transferred from Pukyong Aquaculture Company, Tongyoung, Korea to the Department of Fisheries Biology, Pukyong National University, Korea. Before the start of the experiment, all the fish were reared in a 3000-L round tank and were fed a basal diet (S0) for 2 weeks to acclimated with the experimental diet and conditions. The feeding trial was conducted by using a flow-through system with 60-L aquaria receiving filtered sea water at a rate of 1 L/min. Supplemental aeration was provided to maintain dissolved oxygen near saturation. Water temperature was maintained at 15 ± 1˚C. Fish averaging 6.15 ± 0.04 g (mean ± SD) were distributed randomly to each aquarium as a group of 25 fish and were fed one of seven experimental diets in triplicate groups at a rate of 3% of wet body weight for 8 weeks. Total fish weight in each aquarium was measured every 2 weeks, and the amount of diet fed to fish was adjusted accordingly.

      >  Sample collection and analysis

    At the end of the feeding trial, all fish were weighed and counted to calculate percent weight gain (WG), feed efficiency ratio (FER), specific growth rate (SGR), protein efficiency ratio (PER), hepatosomatic index (HSI), condition factor (CF), and survival. Blood samples were obtained from the caudal vein by using a heparinized syringe. Hematocrit (PCV) was determined from five individual fish per aquarium by the microhematocrit method (Brown, 1980) and hemoglobin (Hb) was measured in the same five fish by the cyanmethemoglobin procedure using Drabkin’s reagent. A hemoglobin standard prepared from human blood (Sigma Chemical, St. Louis, MO) was used. Five fish per aquarium were used for proximate analysis. Crude protein, moisture and ash of whole-body samples were analyzed by AOAC methods (1995). Crude lipid was determined by using an ether extraction procedure (Soxtec System 1046, Foss, Sweden) after freeze-drying samples for 12 h. Fecal collections and digestibility determination were conducted according to the method described by Lim et al. (2004).

      >  Statistical analysis

    All data were subjected to one way ANOVA using SPSS for Windows (release 14.0). When a significant treatment effect was observed, an Honestly Significant Difference (HSD) test was used to compare means. Treatment effects were considered significant at P<0.05.

    Results and Discussion

    Apparent digestibility coefficients (ADCs) of phosphorus, dry matter and crude protein of diets containing different levels of soybean meal and phytase fed to juvenile olive flounder for 8 weeks are summarized in Table 2. ADCs of phosphorus in diets S30P1000, S30P2000, S40P1000 and S40P2000 were significantly higher than those in diets S0, S30 and S40 (P<0.05). The supplementation of phytase significantly improved the ADC of phosphorus in experimental diets (P<0.05) containing 30% or 40% soybean meal regardless of the level in olive flounder. Fish can only utilize approximately 1/3 of the phosphorus contained in soybean meal. Because about 60% phosphorus in soybean meal is in the form of phytate, fish cannot digest (Sajjadi and Carter, 2004). Based on the present experiment, supplementation of phytase can improve the ADCs of phosphorus in soybean meal based diets, and then it is possible to improve the phosphorus retention of diets and reduce the phosphorus discharge into water that was considered as one of the main pollution elements in water environment. Many studies found that addition of phytase had positive effects on ADC of phosphorus in red sea bream (Biswas et al., 2007), rockfish (Yoo et al., 2005) and rainbow trout (Sugiura et al., 2001; Wang et al., 2008) fed soybean meal based diets. There was no significant effect of phytase supplementation on ADCs of dry matter and protein in the present study.

    Growth performance, hepatosomatic index (HSI) and survival rate of juvenile olive flounder fed seven experimental diets for 8 weeks were shown in Table 3. There was no significant difference in weight gain (WG), specific growth rate (SGR), feed efficiency ratio (FE) and protein efficiency ratio (PER) of fish fed S0, S30, S30P1000 and S30P2000. However, WG, SGR, FE and PER of 40% soybean meal groups were significantly lower than those of fish fed S0 and 30% soybean meal. The reduced growth of 40% soybean meal groups could be because of the effect of difference in amino acid profiles and amount of antinutritional factors in the diets. Soybean contains numerous antinutritional factors like proteinase inhibitors and Suragglutinating lectins, as reported by many researchers (Refstie et al., 2001; Sun et al., 2007). In this study, supplementation of 1000 or 2000 U phytase did not improve WG. Similar results were observed in Korean rockfish, red sea bream and parrot fish (Yoo et al., 2005; Biswas et al., 2007; Lim and Lee, 2009). However, growth improvement was also observed in salmon, rainbow trout and Nile tilapia fed the diets with phytase pretreated ingredients (Cain and Garling 1995; Vielma et al., 2002; Cao et al., 2008). According to Cao et al., (2008), pretreatment of plant ingredients using microbial phytase significantly increased nutrient digestibility and growth. To improve the nutritive value of plant products for fish, ingredients are being modified by chemical, mechanical and biological methods (Cao et al., 2007). While differences in dietary formulations could be one of the reasons contributing to differences in feeding response of fish to phytase, species differences is another possible factor (Venou et al., 2006). Therefore, further studies should be conducted to compare the economic benefits among using chemical, mechanical and biological methods to the plant protein sources. There was no significant difference in HSI, condition factor (CF), hematocrit (PCV), hemoglobin (Hb) and survival rate among all the dietary treatments. Survival rate of fish ranged from 94.6 to 100 with flounder fed S40P2000 showed the lowest value of survival.

    Whole-body proximate compositions were shown in Table 4. In this study, whole body composition resulted to be not affected by soybean meal and phytase level. This result is in agreement with several studies on other species (Kim et al., 2009). Whole body protein slightly increased with increasing soybean meal in diets regardless the supplementation levels of phytase. There was a negative correlation to whole body lipid and ash content a slight decrease was observed when soybean meal inclusion increased. Similar results were also reported by Lin et al. (2012) and Bonaldo et al. (2008). Based on the experimental results, we conclude that supplementation of phytase could significantly increase available P content in feed, indicating that the indigestible phytate P successfully converted to available P by phytase.

  • 1. Cunniff P. 1995 Official Methods of Analysis of the Association of Official Analytical Chemists google
  • 2. Barrows FT, Gaylord TG, Stone DA, Smith CE 2007 histology and plasma amino acid concentration of rainbow trout (Oncorhychus mykiss Walbaum) [Aquacul Res] Vol.38 P.1747-1758 google
  • 3. Biswas AK, Kaku H, Ji SC, Seoka M, Takii K 2007 Use of soybean meal and phytase for partial replacement of fish meal in the diet of red sea bream, Pagrus major [Aquaculture] Vol.267 P.284-291 google
  • 4. Bonaldo A, Roem JA, Fagioli P, Pecchini A, Cipollini I, Gatt PP 2008 Influence of dietary levels of soybean meal on the performance and gut histology of gilthead seabream (Sparus aurata L.) and European seabass (Dicentrarchus labrax L.) [Aquacul Res] Vol.39 P.970-978 google
  • 5. Brown B.A. 1980 Routine hematology procedures.; Hematology: Principles and Procedures P.71-112 google
  • 6. Cain KD, Garling DL 1995 Pretreatment of soybean meal with phytase for salmonid diets to reduce phosphorus concentrations in hatchery effluents [Prog Fish-Cult] Vol.57 P.114-119 google
  • 7. Cao L, Yang Y, Wang WM, Yakupitiyage A, Yuan DR, Diana JS 2008 Effects of pretreatment with microbial phytase on phosphorous utilization and growth performance of Nile tilapia (Oreochromis niloticus) [Aquacult Nutr] Vol.14 P.99-109 google
  • 8. Cao L, Yang Y, Wang WM, Yang CT, Yang Y, Diana JS, Yakupitiyage A, Luo Z, Li DP 2007 Application of microbial phytase in fish feed [Enzyme Microb Technol] Vol.40 P.497-507 google
  • 9. Cheng ZJ, Hardy RW 2002 Effect of microbial phytase on apparent nutrient digestibility of barley, canola meal, wheat and wheat middlings, measured in vivo using rainbow trout (Oncorhynchus mykiss) [Aquacult Nutr] Vol.8 P.271-277 google
  • 10. Choi SM, Wang XJ, Park GJ, Lim SR, Kim KW, Bai SC, Shin IS 2004 Dietary dehulled soybean meal as a replacement for fish meal in fingerling and growing olive flounder Paralichthys olivaceus (Temminck et Schlegel) [Aquac Res] Vol.35 P.410-418 google
  • 11. Chong A, Hashim R, Ali A 2003 Assessment of soybean meal in diets for discus (Symphysodon aequifasciata Heckel) farming through a fishmeal replacement study [Aquacult Res] Vol.34 P.913-922 google
  • 12. Jang HS, Kim KD, Lee SM 2005 Effect of various commercial fish meals as dietary protein sources on growth and body composition of juvenile flounder, Paralichthys olivaceus [J Aquacult] Vol.18 P.267-271 google
  • 13. Kikuchi K, Furuta T 2009 Use of defatted soybean meal and blue mussel meat as substitute for fish meal in the diet of tiger puffer, Takifugu rubripes [J World Aquacult Soc] Vol.40 P.472-482 google
  • 14. Kim KW, Wang XJ, Bai SC 2001 Reevaluation of the optimum dietary protein level for the maximum growth of juvenile Korean rockfish, Sebastes schlegeli (Hilgendorf) [Aquacult Res] Vol.32 P.119-125 google
  • 15. Kim KW, Wang XJ, Bai SC 2002 Optimum dietary protein level for maximum growth of juvenile olive flounder Paralichthys olivaceus (Temminck et Schlegel) [Aquacult Res] Vol.33 P.673-679 google
  • 16. Kim YC, Bae SS, Lee JH, Park GH, Lee JY, Bai SC 2009 Dietary squid liver powder (SLP) with dehulled soybean meal (DHSM) as a fish meal (FM) subsititute for olive flounder Paralichthys olivaceus [Kor J Fish Aquat Sci] Vol.42 P.243-249 google
  • 17. Lim SJ, Lee KJ 2009 Partial replacement of fish meal by cottonseed meal and soybean meal with iron and phytase supplementation for parrot fish Oplegnathus fasciatus [Aquaculture] Vol.290 P.283-289 google
  • 18. Lim SR, Choi SM, Wang XJ, Kim KW, Shin IS, Min TS, Bai SC 2004 Effects of dehulled soybean meal as a fish meal replacer in diets for fingerling and growing Korean rockfish Sebastes schlegeli [Aquaculture] Vol.231 P.457-468 google
  • 19. Lim SJ, Kim SS, Ko GY, Song JW, Oh DH, Kim JD, Kim JU, Lee KJ 2011 Fish meal replacement by soybean meal in diets for tiger puffer, Takifugu rubfipes [Aquaculture] Vol.313 P.156-170 google
  • 20. Lin H, Chen X, Chen S, Zhuojia L, Huang Z, Niu J, Wu K, Lu X 2013 Replacement of fish meal with fermented soybean meal in practical diets for pompano Trachinotus ovatus [Aquacult Res] Vol.44 P.151-156 google
  • 21. 2013 Statistical Yearbook of Maritime Affairs & Fisheries google
  • 22. 1993 Nutritional requirements of fish google
  • 23. Peres H, Lim C, Klesius PH 2003 Nutritional value of heat treated soybean meal for channel catfish (Ictalurus punctatus) [Aquaculture] Vol.225 P.67-82 google
  • 24. Refstie ST, Storebakken G, Roem AJ 2001 Long-term protein and lipid growth of Atlantic salmon (Salmo salar) fed diets with partial replacement of fish meal by soy protein products at medium or high lipid level [Aquaculture] Vol.193 P.91-106 google
  • 25. Sajjadi M, Carter CG 2004 Dietary phytase supplementation and the utilization of phosphours by Atlantic salmon (Salmo Salar L.) fed a canola-meal-based diet [Aquaculture] Vol.240 P.417-431 google
  • 26. Sugiura SH, Gabaudan J, Dong FM, Hardy RW 2001 Dietary microbial phytase supplementation and the utilization of phosphorus, trace minerals and protein by rainbow trout [Oncorhynchus mykiss (Walbaum)] fed soybean meal-based diets [Aquacult Res] Vol.32 P.581-592 google
  • 27. Sun MH, Kim YC, Okorie OE, Devnath S, Yoo GW, Lee SH, Jo YK, Bae SC 2007 Use of fermented fisheries by-products and soybean curd residues mixture as a fish meal replacer in diets of juvenile olive flounder, Paralichthys olivaceus [J World Aquacul Soc] Vol.38 P.543-549 google
  • 28. Tantikitti C, Sangpong W, Chiavareesajja S 2005 Effects of defatted soybean protein levels on growth performance and nitrogen and phosphorus excretion in Acian seabass (Lates calcarifer) [Aquaculture] Vol.248 P.41-50 google
  • 29. Venou B, Alexis MN, Fountoulaki E, Haralabous J 2006 Effects of extrusion and inclusion level of soybean meal on diet digestibility, performance and nutrient utilization of gilthead sea bream (Sparus aurata) [Aquaculture] Vol.261 P.343-356 google
  • 30. Vielma J, Ruohonen K, Peisker M. 2002 Dephytinization of two soy proteins increases P and protein utilization by rainbow trout (Oncorhynchus mykiss) [Aquaculture] Vol.204 P.145-156 google
  • 31. Vielma J, Ruohonen K, Gabaudan J, Vogel K 2004 Top-spraying soybean meal-based diets with phytase improves protein and mineral digestibilities but not lysine utilization in rainbow trout [Oncorhynchus mykiss (Walbaum)] [Aquacult Res] Vol.35 P.955-964 google
  • 32. Wang F, Yang YH, Han ZZ, Dong HW, Yang CH, Zou ZY 2009 Effects of phytase pretreatment of soybean meal and phytase-sprayed in diets on growth, apparent digestibility coefficient and nutrient excretion of rainbow trout (Oncorhynchus mykiss Walbaum) [Aquacult Int] Vol.17 P.143-157 google
  • 33. Yoo GY, Wang XJ, Choi SM, Han K, Kang JC, Bai SC 2005 Dietary microbial phytase increased the P digestibility in juvenile Korean rockfish (Sebastes schlegeli) fed diets containing soybean meal [Aquaculture] Vol.243 P.315-322 google
  • [Table 1.] Composition and proximate analysis of experimental diets for juvenile olive flounder Paralichthys olivaceus
    Composition and proximate analysis of experimental diets for juvenile olive flounder Paralichthys olivaceus
  • [Table 2.] Apparent digestibility coefficients of phosphorus (ADPh), dry matter (ADM) and crude protein (ADPr) of the diets containing different levels of soybean meal and phytase in juvenile olive flounder Paralichthys olivaceus1
    Apparent digestibility coefficients of phosphorus (ADPh), dry matter (ADM) and crude protein (ADPr) of the diets containing different levels of soybean meal and phytase in juvenile olive flounder Paralichthys olivaceus1
  • [Table 3.] Growth performance, hepatosomatic index (HSI), and survival rate of juvenile olive flounder Paralichthys olivaceus fed seven experimental diets for 8 weeks1
    Growth performance, hepatosomatic index (HSI), and survival rate of juvenile olive flounder Paralichthys olivaceus fed seven experimental diets for 8 weeks1
  • [Table 4.] Whole body proximate composition of juvenile olive flounder Paralichthys olivaceus fed experimental diets for 8 weeks (% of as-is basis)1
    Whole body proximate composition of juvenile olive flounder Paralichthys olivaceus fed experimental diets for 8 weeks (% of as-is basis)1