Korean rockfish Sebastes schlegeli fed diets
Aquaculture 243 (2005) 315 – 322
Dietary microbial phytase increased the phosphorus digestibility in juvenile Korean rockfish Sebastes schlegeli fed diets containing soybean meal
Gwang-Yeol Yooa, Xiaojie Wanga, Semin Choia, Kyungmin Hana,
Ju-Chan Kangb, Sungchul C. Baia,*
aDepartment of Aquaculture/Feeds and Foods Nutrition Research Center, Pukyong National University, 599-1 Daeyeon-dong, Nam-Gu,
Busan 608-737, South Korea
bDepartment of Aquatic Life Medicine, Pukyong National University, 599-1 Daeyeon-dong, Nam-Gu, Busan 608-737, South Korea
Received 1 July 2004; received in revised form 22 October 2004; accepted 25 October 2004
This study was conducted to evaluate the effects of dietary microbial phytase supplementation on nutrient digestibility, growth performance and body composition in juvenile Korean rockfish fed soybean meal-based diets.
Nine experimental diets were formulated to be isonitrogenous and isocaloric to contain 48.6% crude protein (CP) and 15.9 kJ of available energy/g with or without dietary phytase (Natuphos-5000k, BASF, NJ, USA) supplementation. White fish meal (FM) provided 89.1% of the total protein in the basal diet (S0), in the other eight diets, 30 or 40% FM protein was replaced by soybean meal: 70% FM+30% soybean meal (S30); 70% FM+30% 1000 U phytase pretreated SM (S30PP1000); 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% 1000 U Ptre SM (S40PP1000); 60% FM+40% SM+1000 U phytase/kg diet (S40P1000); and 60%
FM+40% SM+2000 U phytase/kg diet (S40P2000). After 2 weeks of the adaptation, triplicate groups of 20 fish initially averaging 7.25F0.04 g (meanFS.D.) were randomly distributed into the aquarium and were fed one of the experimental diets for 8 weeks.
By the end of the 8-week feeding trial, supplementation of phytase significantly improved the apparent digestibility coefficient (ADC) of phosphorus in rockfish diets ( Pb0.05) containing 30% and 40% soybean meal regardless of the level and method of phytase supplementation. Supplementation of phytase significantly increased the ADC of dry matter in diets containing 30% soybean meal ( Pb0.05). However, phytase had no influence on growth performance and whole body composition of fish. The pretreatment of soybean meal with 1000 U phytase improved weight gain (WG) when 30% fish meal
* Corresponding author. Tel.: +82 51 620 6137; fax: +82 51 628 6873.
E-mail address: firstname.lastname@example.org (S.C. Bai).
0044-8486/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2004.10.025
protein was replaced by soybean meal. Based on the experimental results, we conclude that supplementation of phytase can improve the apparent digestibility coefficient of phosphorus in Korean rockfish.
D 2004 Elsevier B.V. All rights reserved.
Keywords: Phytase; Phosphorus; Digestibility; Korean rockfish
Recently, aquaculture has become one of the fastest growing food-producing industries in the world including Korea. Production of Korean rock- fish and flounder, considered as the main finfish culture species in Korea, reached 23,711 and 34,533 tons in 2003, respectively (Statistical Year Book of Ministry of Maritime Affairs and Fisheries, 2003). One of the most important protein sources in aquatic fish feeds, fish meal, might fluctuate periodically by over 20% in El Nin˜o years (Hardy, 1995; Cheng and
Hardy, 2002). Fish meal is also more expensive than plant protein such as soybean meal that are exten- sively used as protein sources in aquatic fish feeds (Choi et al., 2004; Lim et al., 2004). Although fish meal has a good amino acid profile, fish meal contains relatively high levels of phosphorus and other minerals and the availability of phosphorus is relatively low for many fish species including trout and salmon (NRC, 1993; Sugiura et al., 1998a). Phosphorus is an important constituent of nucleic acids and cell membranes, a major constituent of the structural components of skeletal tissues, and is directly involved in all energy-producing cellular reactions (NRC, 1993). Therefore, phosphorus is an essential nutrient for growth (Baeverfjord et al.,
1997) and reproduction of fish (Hardy and Shearer, 1985). Meanwhile, phosphorus is a critical pollutant in the aquatic environment; excessive excretion of phosphorus into water can stimulate the growth of algae and phytoplankton, thus reducing dissolved oxygen and causing water pollution (Miller et al.,
1974; Boyd, 1990; Sugiura et al., 1999). Soybean meal is considered a promising alternative protein source because of its ready supply, high protein content and low phosphorus content relative to fish meal (Hardy, 1995). However, about two-third of phosphorus in soybean meal is present as phytate (inositol hexaphosphate) which is not efficiently
1993). Previous researches indicated that heat treat- ment may reduce phytate in plant feed ingredients (Hafez et al., 1989; Satoh et al., 1998) and the use of phytase is also effective (Cain and Garling, 1995;
Therefore, the present study investigated the effects of dietary microbial phytase supplementation on the nutrient digestibility, growth performance and body composition in juvenile Korean rockfish fed diets containing soybean meal.
- Materials and methods
- Experimental diets
Composition of the experimental diets is shown in Table 1 and chemical analysis of the experimental diets is shown in Table 2. Nine experimental diets were formulated to be isonitrogenous and isocaloric to contain 48.6% crude protein (CP) and 15.9 kJ of available energy/g with or without dietary phytase supplementation (Natuphos-5000k, BASF, NJ, USA). Estimated available energy of the experimental diets was calculated using 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 eight diets 30% or 40% FM protein was replaced by soybean meal as: 70% FM+30% soybean meal (S30); 70% FM+30% 1000 U phytase pretreated SM (S30PP1000); 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% 1000 U Ptre SM (S40PP1000); 60% FM+40%
SM+1000 U phytase/kg diet (S40P1000); and 60% FM+40% SM+2000 U phytase/kg diet (S40P2000). Phytase pretreated soybean meal was prepared according to Nelson et al. (1968). One hundred milliliters of distilled water containing phytase (1000
Composition (% as fed) of experimental diets for Korean rockfish
White fish meal2
Corn gluten meal2
1 White fish meal (FM) provided 89.1% of the protein in the basal diet (S0), in the other eight diets 30% or 40% FM was replaced by soybean meal (SM) as: 70% FM+30% soybean meal (S30); 70% FM+30% 1000 U phytase pretreated SM (S30PP1000); 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% 1000 U phytase pretreated SM (S40PP1000); 60% FM+40% SM+1000 U phytase/kg diet (S40P1000); 60% FM+40% SM+2000 U phytase/kg diet (S40P2000).
2 Fisheries Co-operative of Korea Feeds, Uiryeong County, Uiryeong Town, Korea.
3 American Soybean Association/Korea.
4 United States Biochemical, Cleveland, OH 44122, USA.
5 0.5% l-lysined HCl (0.4% lysine).
6 Ilshin Chemical, Yangsan, Kyungnam 626-110, Korea.
7 Natuphos 5000k, BASF, NJ, USA.
- U) was added to 277.3 and 369.7 g soybean meal, which was the 30% and 40% replacement levels of fish meal in experimental diets, respectively, and Then 100 ml of 1.2 N HCl was added, mixed, and the resulting mixture incubated at 50–55 8C for 24
- All the ingredients were mixed completely and then pelleted by a laboratory pellet machine without heating using a 2-mm diameter module (Baokyong Commercial, Busan, Korea). After processing, all the diets were packed into small bags and kept at 80 8C until used.
- Fish and feeding trial
Korean rockfish (Sebastes schlegeli) were trans- ferred from Manseung Aquatic, Geoje, Korea to the Institute of Fisheries Science, 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 adjust to the experimental diet. The feeding trial was conducted by using a flow-through system with 60-l aquaria receiv- ing filtered seawater at a rate of 1 l/min. Supplemental
Chemical analysis of experimental diets
Proximate analysis (dry matter basis)
Total phosphorus (g/kg diet)
Analyzed phytase activity (U/kg diet)
1 Refer to Table 1, footnote #1.
aeration was provided to maintain dissolved oxygen near saturation. Water temperature was maintained at 19F1 8C. Fish averaging 7.25F0.04 g (meanFS.D.) were distributed randomly to each aquarium as a group of 20 fish and were fed one of nine 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 deter- mined from five individual fish per aquarium by the microhematocrit method (Brown, 1980) and hemoglo- bin (Hb) was measured in the same five fish by the cyanmethemoglobin procedure using Drabkin’s reagent. A hemoglobin standard prepared from human blood (Sigma, St. Louis, MO) was used. Six fish randomly selected per aquarium were used for prox- imate analysis. Crude protein, moisture and ash of whole-body samples were analyzed by AOAC meth- ods (1995). Crude lipid was determined by using an ether extraction procedure (Soxtec System 1046, Foss, Hoganas, Sweden) after freeze-drying samples for 12
- Fecal collections and digestibility determination were conducted according to the method described by
USA). When a significant treatment effect was observed, a Least Significant Difference (LSD) test was used to compare means. Treatment effects were considered significant at Pb0.05.
- Results and discussion
Apparent digestibility coefficients (ADCs) of phos- phorus, dry matter and crude protein (CP) of diets containing different levels of soybean meal and phytase fed to juvenile Korean rockfish for 8 weeks are summarized in Table 3. ADC of phosphorus in diets S30PP1000, S30P1000, S30P2000, S40PP1000, S40P1000 and
S30P2000 were significantly higher than those in diets
S0, S30 and S40 ( Pb0.05). The supplementation of phytase significantly improved the ADC of phospho- rus in experimental diets ( Pb0.05) containing 30% or 40% soybean meal regardless of the level and method of phytase supplementation in rockfish. The positive effect of phytase supplementation on ADC of phos- phorus has also been observed in salmonids (Cain and
Sugiura et al., 2001) fed soybean meal based diets. Although no significant effect of pretreatment of soybean meal with phytase was observed in the present experiment, the ADC of phosphorus value of diets S30PP1000 and S40PP1000 showed the highest values
Apparent digestibility coefficients of phosphorus, dry matter and crude protein of the diets containing different levels of soybean meal and phytase in juvenile Korean rockfish1
Lim et al. (2004). The apparent digestibility coeffi- cients of dry matter, crude protein and phosphorus of
Diets2 Phosphorus (%)
Dry matter (%)
Crude protein (%)
nine diets were determined by the chromic oxide S0 62.4b 65.9a 90.9a
(Cr O ) method (Hanley, 1987). Cr O
S30 57.6b 61.4bc 90.1a
2 3 2 3
92.0a 64.5ab 91.5a
were determined by flame atomic absorption spectro-
photometers following combustion of the sample in a
2.4. Statistical analysis
All data were subjected to one-way ANOVA using
S30P2000 87.0a 62.8ab 90.2a
S40 61.8b 57.1d 86.7bc S40PP1000 92.8a 57.5cd 87.9b S40P1000 84.0a 56.8d 87.8b
S40P2000 89.5a 55.2d 85.8c Pooled S.E.M.3 2.82 0.93 0.47
1 Values are means of triplicate groups, values in the same column not sharing a common superscript are significantly different ( Pb0.05).
2 Refer to the Table 1, footnote #1.
Statistix 3.1 (Analytical Software, St. Paul, MN,
Pooled standard error of mean: S.D./ n.
among the dietary treatments as 92% or 92.8%, respectively. Sugiura et al. (2001) observed that in rainbow trout fed diet containing 50% soybean meal with 4.21 g/kg diet total phosphorus, the ADC of phosphorus in diet containing pretreated soybean meal (200 U phytase/kg dry soybean meal) reached 93%. However, when the total phosphorus level increased to
14.7 g/kg diet, the ADC of phosphorus in diet containing 1000 U phytase/kg diet was only 62%. In the present experiment, there was no significant difference in ADC of phosphorus in diets containing different total phosphorus contents in 30% and 40% soybean diets. The total phosphorus contents of the experimental diets ranged 21.3–15.1 g/kg diet. Although the dietary phosphorus requirement in Korean rockfish has not been well determined, we estimated that the available phosphorus content in the experimental diets might surpass the dietary phospho- rus requirement in Korean rockfish. Therefore, the effect of dietary phytase supplementation could not be detected well. Because about 60% phosphorus in soybean meal is in the form of phytate (Sajjadi and
Carter, 2004), which fish cannot digest; fish can only utilize approximately 1/3 of the phosphorus contained in soybean meal. Based on the present experiment together with the other results concerning the supple- mentation of phytase in fish diets (Forster et al., 1999;
Sugiura et al., 2001; Cheng and Hardy, 2002), supplementation of phytase can improve the ADC of phosphorus in soybean meal or canola meal-based diets, 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. Supplemen- tation of phytase somewhat increased the ADC of dry matter of diets containing 30% soybean meal. There was no significant difference in ADC of dry matter among diets S0, S30PP1000, S30P1000 and S30P2000, and also among diets S30, S30PP1000, S30P1000 and S30P2000, however, S0 diet has a significantly higher ADC of dry matter than did S30 diet ( Pb0.05). The supplementa- tion of phytase in diets containing 40% soybean meal did not improve the ADC of dry matter, S0 diet had a significantly higher ADC of dry matter than did diets S40, S40PP1000, S40P1000 and S40P2000 ( Pb0.05), and
there was no significant difference in ADC of dry matter among diets S40, S40PP1000, S40P1000 and S40P2000. There was no significant effect of phytase
supplementation on ADC of protein in the present study.
55 8C, respectively; the rate of enzyme hydrolysis activity was much faster at 37 than at 15 8C. Therefore, the rearing temperature of 19F1 8C in the present experiment might have reduced the efficacy of supplemental phytase. However, the pretreatment of soybean meal with phytase might be a feasible alternative if low temperature limits the efficiency of supplemental phytase as observed in the present experiment and other previous studies (Stone et al., 1984; Cain and Garling, 1995; Sugiura et al.,
2001). Forster et al. (1999) also reported that the efficacy of dietary phytase to dephosphorylate phytic acid may be enhanced at higher water temperatures than low water temperatures. Rodehutscord and Pfeffer (1995) noted that at 10 8C water temperature, supplementation of 1000 U/kg diet phytase into phytate-containing feeds improved phytate phospho- rus availability, without a concomitant beneficial effect on growth or feed efficiency of rainbow trout. By contrast, these researchers observed that dietary phosphorus availability as well as the growth and feed efficiency of trout were improved by phytase supple- mentation when the fish were held at 15 8C. In carp reared at 23 8C (Scha¨fer et al., 1995) and channel catfish (Jackson et al., 1996) reared at 29–31 8C, supplementation with as little as 500 U/kg diet phytase significantly improved growth, feed effi- ciency and phytate phosphorus availability. The results from the present experiment indicated that supplementation of 1000 U/kg diet phytase could improve the ADC of phosphorus, but did not improve the growth and feed efficiency ratio of Korean rockfish. However, pretreating the 30% soybean meal diet with 1000 U/kg diet phytase significantly improved phosphorus digestibility and somewhat improved growth of Korean rockfish. Such effect was not observed in 40% soybean meal diet.
Growth performance, hepatosomatic index (HSI) and survival of juvenile Korean rockfish fed nine experimental diets for 8 weeks were shown in Table 4. After 8 weeks of feeding trial, weight gain (WG) of rockfish indicated that the replacement of fish meal protein by 30% or 40% soybean meal significantly decreased the WG ( Pb0.05), and supplementation of
Growth performance, hepatosomatic index (HSI), and survival of juvenile Korean rockfish fed nine experimental diets for 8 weeks1
Survival (%) 100 96.7 98.3 96.7 95.0 100 98.3 96.7 86.7 1.03
1 Values are means of triplicate groups, values in the same row not sharing a common superscript are significantly different ( Pb0.05).
2 Refer to Table 1, footnote #1.
Pooled standard error of mean: S.D./ n.
Weight gain: [(final wt.-initial wt.)/initial wt.]×100.
Specific growth rate: [(ln final wt.-ln initial wt.)/days]×100. Feed efficiency ratio: (wet wt. gain/dry feed intake)×100.
7 Protein efficiency ratio: wet wt. gain/protein intake.
Hepatosomatic index: (liver wt./body wt.)×100.<