Fish Physiology and Biochemistry 25: 181–194, 2001. © 2002 Kluwer Academic Publishers. Printed in the Netherlands.
181
Apparent digestibility of crude protein and apparent availability of individual amino acids in tilapia (Oreochromis niloticus) fed phytase pretreated soybean meal diets M. Riche 1,3 , N.L. Trottier 2 , P.K. Ku2 and D.L. Garling 1,∗ 1 Department of Fisheries and Wildlife,
and 2 Department of Animal Science, Michigan State University, East Lansaddress: United States Department of Agriculture, Agricultural Research Service, ing, MI 48824, USA; Harbor Branch Oceanographic Institution, 5600 Hwy U.S. 1 North, Fort Pierce, FL 34946, USA; ∗ Author for correspondence (
[email protected]) 3 Present
Accepted 23 July 2002
Key words: crystalline amino acids, fish, lysine, methionine, phytates, phytic acid
Abstract Soybeans contain phytates, the anionic forms of 1,2,3,5/4,6-hexakis (dihydrogen phosphate) myoinositol, with the potential to reduce amino acid (AA) availability. Tilapia lack the intestinal enzyme phytase to hydrolyze phytates. Oreochromis niloticus (approximately 68 g) were fed diets containing either phytase pretreated or untreated soybean meal (SBM) incorporated at 0, 25, 50, 75, or 100% of the crude protein (CP) in a 33% CP diet to determine whether phytates reduce CP digestibility and AA availability. There were no differences in apparent CP digestibility. Dietary and available methionine (Met), and available lysine (Lys), decreased with increasing incorporation of phytase pretreated SBM. Reduced availability of Met and Lys from the phytase pretreated diets was likely due to removal of phytates. Phytates may reduce the effect of other antinutritional factors, protect amino acids from degradation, or decrease leaching of water soluble components.
Introduction Most tilapia culture in temperate regions is conducted in recirculating systems to maintain suitable water temperatures. However, recirculating systems are prone to accumulation of nitrogenous wastes and solids. The difficulty and expense with nitrogen (N) and solids removal makes reducing wastes entering the system desirable. Increasing N retention is an obvious starting point for reducing N inputs into the system. In fish fed high quality fish meal (FM), approximately 10% of ingested N is excreted in the feces, and as much as 66% excreted as ammonia (Cho 1993). In diets with similar protein and energy levels, both fecal N and ammonia excretion are typically higher with plant protein substituted diets. However, to increase sustainability FM products are currently being replaced with less expensive plant proteins. Some of the most commonly utilized plant proteins are soybean products. However, reports of growth and
efficiency in tilapia fed soybean products are conflicting (Davis and Stickney 1978; Jackson et al. 1982; Viola and Arieli 1983; Shiau et al. 1987, 1989, 1990; Davies et al. 1989; De Silva and Gunasekera 1989; El-Dahhar and El-Shazly 1993). Poor performance is generally in direct relationship to level of soybean incorporation (Shiau et al. 1990). Soybeans have anti-nutritional factors (ANF) that reduce their biological value (Rackis 1974; Liener 1994). Phytic acid (1,2,3,5/4,6-hexakis (dihydrogen phosphate) myoinositol) is an ANF existing as a salt of mono- and divalent cations in legumes and cereals. Phytates, the anionic forms of phytic acid, bind divalent cations making them unavailable (Maga 1982). Reduced mineral bioavailability was demonstrated in rainbow trout (Spinelli et al. 1983; Cain and Garling 1995; Riche and Brown 1996), channel catfish (Satoh et al. 1989), carp (Hossain and Jauncey 1993), and
182 tilapia (McClain and Gatlin 1988), fed diets containing phytate. Phytates also form complexes with proteins reducing availability of amino acids (Cheryan 1980; Reddy et al. 1989). Both binary complexes (phytate-protein), and ternary complexes (phytate-mineral-protein) occur (Cheryan 1980; Reddy et al. 1989). In acidic environments, such as the tilapia stomach (pH 1.0–2.0), half of the phosphate moieties are negatively charged. This environment favors the binding of soluble proteins at ε-amino groups on lysine (Lys), imidazole groups on histidine (His), and guanidyl groups on arginine (Arg). In alkaline environments, such as the tilapia intestine (pH 8.5–8.8) the ternary complexes are favored. Both complexes are resistant to proteolytic digestion (Singh and Krikorian 1982; Satterlee and Abdul-Kadir 1983; Grabner and Hofer 1985; Vaintraub and Bulmaga 1991; Caldwell 1992). Trypsin inhibition is dependent on phytate concentration, temperature, Ca2+ concentration, and contact time (Singh and Krikorian 1982). Explanations for inhibition are decreased activation of trypsinogen, increased autolysis, and competitive sequestration of Ca2+ ions (Singh and Krikorian 1982; Vaintraub and Bulmaga 1991; Caldwell 1992). Phytate inhibition of pepsin activity occurs in a linear fashion (Knuckles et al. 1985). Maximal inhibition occurs near pH 2.0 (Camus and Laporte 1976; Vaintraub and Bulmaga 1991), suggesting the potential for decreased pepsin activity in tilapia. Inhibition is also correlated to degree of phytate hydrolysis. Tilapia lack the intestinal enzyme phytase [EC 3.1.3.8] that hydrolyzes phytate. However, exogenous phytase has been used successfully to hydrolyze phytate, thus increasing nutrient digestibility (Cain and Garling 1995; Mroz et al. 1994; Riche and Brown 1996). Phytase pretreated diets fed to Rainbow trout resulted in increased growth, which was attributed to improved protein utilization (Cain and Garling 1995). Swine fed diets containing phytase resulted in increased total tract digestibility of crude protein (CP), and all amino acids (AA) except cystine (Cys) and proline (Pro) (Mroz et al. 1994). Ileal digestibilty of methionine (Met) and Arg were also increased (Mroz et al. 1994). Additionally, daily urinary N excretion was reduced 20–25% suggesting an improved AA balance. Trypsin-like enzymes from tilapia behave similarly to porcine trypsin (El-Shemy and Levin 1997) suggesting the potential for inhibition by phytate. We hypothesized hydrolysis of phytate in soybean meal (SBM) would increase digestibility of CP and
individual AA. The purpose of this study was to determine if phytate decreases CP and individual AA digestibility in the tilapia Oreochromis niloticus. Materials and methods Experimental protocol O. niloticus spawned at Michigan State University were held in flow-through well water heated to 27±1.0 ◦ C until stocking. Fish held during this period were fed a standard commercial trout diet. The experimental system was a 4,700 l recirculating system. Temperature was maintained at 28 ◦ C, and flow rate at 2–3 l min−1 . Fish were maintained on a 16 h light:8 h dark photoperiod. Dissolved oxygen was monitored three times daily. Ammonia, nitrite, and nitrate were measured three times weekly. All water quality parameters were within acceptable limits for tilapia (Cherivinski 1982; Daud et al. 1988; Papoutsoglou and Tziah 1996). Two replicates, for each experimental diet, were run in each of two blocks. Fifteen fish each, from a mixed sex population, were randomly stocked into one of eighteen 125 l tanks. Each tank was defined as an experimental unit, and randomly assigned a dietary treatment. Mean weight at stocking was 69.9±1.04 g (n = 270), and 66.9±0.63 g (n = 270), for block one and two, respectively. Fish were fed a commercial trout diet during a 4 d acclimation period. Following acclimation, experimental diets were fed 10 d for dietary adaptation. Fish were offered 2.4% of their body weight d−1 on a dry matter basis, divided between three equal meals (9:00, 13:00, and 18:00). On day 15 following stocking, fecal collection began 2 h after the 13:00 feeding. Fish were euthanized in 500 mg l−1 MS-222 (tricaine methanesulfonate). Incisions were made along the mid-ventral line. The exposed GI tract was clamped 10 cm from the vent and the posterior section excised. Feces within the excised section was collected and pooled by experimental unit. Pooled fecal samples were stored at −20 ◦ C for subsequent analysis. Feed samples and pooled fecal samples were freeze dried (Virtis 25 SRC, Gardiner, New York) for AA, N and hydrolysis resistant organic matter (HROM) analysis. Apparent digestibility coefficients (ADC) and apparent availability coefficients (AAC) were calculated in the manner of Maynard and Loosli (1969):
183
(ADC) or (AAC) =
% HROM in feed % Nutrient in feces 100 − (100) × % HROM in feces % Nutrient in feed
Diet preparation Solvent extracted SBM was obtained from Zeeland Farm Services (Zeeland, Michigan), and herring meal from Zeigler Brothers, Inc. (Gardners, Pennsylvania). The SBM and herring meal were ground to pass an 850 µm sieve. Dextrin (type II from corn), L-methionine, and L-ascorbic acid were obtained from Sigma Chemical Co. (St. Louis, Missouri). Choline chloride, Carboxymethyl cellulose, and α-cellulose were obtained from ICN Biochemicals (Cleveland, Ohio). Alkali refined, bleached, and pressed menhaden oil stabilized with 200 ppm Coviox was supplied by Zapata Protein, Inc. (Reedville, Virginia). Feed grade soybean oil was purchased from a local retailer. Soybean meal was substituted into a basal FM diet for an isonitrogenous mixture of FM and cellulose, to provide 0, 25, 50, 75, or 100% of the CP (Table1). Crystalline L-methionine was added to diets containing SBM substituted at 50, 75, and 100% of the CP to meet the Met and total sulfur amino acid (TSAA) requirements (Santiago and Lovell 1988). Addition of supplemental Met was at the expense of cellulose. All diets were formulated to contain 33% CP on a dry matter basis, and a protein:digestible energy (DE) of 25.0 g MJ−1 based on predicted DE values for O. niloticus (Anderson et al. 1991; NRC 1993). All diets were supplemented with a complete mineral (Table 1) and vitamin premix (Hoffman-La Roche, Inc., Nutley, NJ). Microbial phytase [EC 3.1.3.8] (BASF, 5,000 IU g−1 ) was activated by hydration in a 0.1 M citrate solution (pH 5.0) at room temperature. The enzyme solution (5,000 IU l−1 ) was mixed slowly on a magnetic stirrer for 15 min. The buffered phytase solution was added to the SBM 1:1 (w/v) and mixed thoroughly for 1 h at room temperature. The resultant mash was covered and incubated for 6 h at 50 ◦ C. Following incubation, the SBM was dried in a forced air convection oven at 60 ◦ C. The re-dried SBM was reground to pass an 850 µm sieve. The SBM used in diets without phytase pretreatment was mixed 1:1 (w/v) with a 0.1 M citrate buffer (pH 5.0). The sham
treated SBM was mixed, incubated, dried, and reground in the same manner as the phytase pretreated SBM. Dry ingredients were mixed in an 18 kg liquidsolids V-mixer (Patterson-Kelly, Inc., East Stroudsberg, Pennsylvania) for a minimum of 12 h. Water and lipids were added under continuous mixing in a Univex mixer (Univex Corp., Salem, New Hampshire). The moist diet was cold extruded using a 2 mm diameter die. Pelleted diets were dried in a forced air oven at 60 ◦ C for 12 h. Dried diets were stored at −20 ◦ C until fed. Sample analysis Freeze-dried feed and fecal samples were analyzed for HROM with slight modifications to the method described by Buddington (1980). A standard curve was prepared using α-cellulose (ICN Biochemicals, Cleveland, Ohio). Approximately 50 mg fecal samples, or 100 mg feed samples were placed in 15.0 ml of 80% C2 H4 O2 and 1.5 ml HNO3 and gently boiled for 20 min. Samples were filtered through1 µm pore glass filter paper (Whatman GF/B) under vacuum, and sequentially washed with 6 ml hot ethanol, 6 ml hot benzene, 6 ml petroleum ether, and 6 ml ethanol. Filtered samples were dried at 105 ◦ C for 12 h. Dried samples were weighed and ashed at 525 ◦ C for 16 h before re-weighing. Hydrolysis resistant organic matter was calculated as amount of material lost on ignition expressed as a percentage of the original sample weight. Approximately 15 mg freeze dried fecal samples were analyzed for N with a N-analyzer (Leco FP-2000, Leco Corp., St. Joseph, Michigan) following manufacturers specifications. Feed N was determined by the Kjeldahl method (AOAC 1990). Crude protein was estimated as N × 6.25. Analyses were performed in triplicate on pooled samples. Feed and feces were analyzed for AA, except tryptophan. Samples were freeze-dried, pulverized, and hydrolyzed with 6 N HCl at 110 ◦ C for 24 h. Hydrolysate AA were derivatized with phenylisothiocyanate (PITC) before analysis (Waters Manual 1989). Derivatized AA were determined on a C-18 reverse phase HPLC column using a Waters HPLC separation system (Waters Chromatography Division, Millipore Corp., Milford, Massachusetts). Phytate was determined colorimetrically (Latta and Eskin 1980). Samples were prepared as previously described (Riche and Brown 1996). Absorbance was
184 Table 1. Composition of experimental diets.
Ingredient
International feed #
Controla
Percent soybean meal substitution 25 50 75 100
Herring meal Solvent extracted SBM L-Methionine Dextrin Mineral premixb Vitamin premixc Carboxymethyl cellulose Ascorbic acid Choline chloride α-Cellulose Menhaden oil Soybean oil Total
5-02-000 5-04-612
463.7 – – 125.0 60.0 3.0 20.0 1.0 0.8 226.5 75.0 25.0 1000.0
347.8 166.7 – 125.0 60.0 3.0 20.0 1.0 0.8 175.7 75.0 25.0 1000.0
4-08-023
7-08-049 4-07-983
231.9 333.4 0.5 125.0 60.0 3.0 20.0 1.0 0.8 124.4 75.0 25.0 1000.0
115.9 500.0 1.5 125.0 60.0 3.0 20.0 1.0 0.8 72.8 75.0 25.0 1000.0
– 666.7 2.5 125.0 60.0 3.0 20.0 1.0 0.8 21.0 75.0 25.0 1000.0
a Values are g kg−1 of the dry diet. b Mineral premix provided the following as mg kg−1 dry diet: Ca, 6,182; PO , 6,780; Mg, 167; Zn, 41; Fe, 4 34; Mn, 39; Cu, 22; Al, 6.7; F, 9.8; I, 4.5; SeO3 , 2.8; Co, 2.6; MoO4 , 1.5. c (Hoffman-La Roche, Inc., Nutley, NJ) supplied the following kg−1 dry diet: vitamin A, 10,582 IU; vitamin
D3 , 2,381 IU; vitamin E, 132, IU; menadione, 2 mg; folic acid, 5.3 mg; riboflavin , 17.2 mg; pantothenic acid, 42.3 mg; niacin, 105.8 mg; choline-Cl, 529.1 mg; thiamin, 11.9 mg; pyridoxine, 13.2 mg; biotin, 0.165 mg; cyanocobalamine, 0.0044 mg.
read at λ = 500 nm (DU 640 UV/VIS Spectrophotometer, Beckman Instruments, Fullerton, California). Residual trypsin inhibitor activity (TIA) was determined using a modification of American Association of Cereal Chemists Method 71-10 (AACC 1983). A 1.0-g sample of SBM was extracted with 50 ml 0.01 N NaOH for 3 h. Samples were transferred to 50 ml scintillation tubes and centrifuged at 4000 rpm for 10 min. Aliquots of 0, 0.6, 1.0, 1.4, and 1.8 ml of extract were brought to 2.0 ml with deionized distilled water, mixed with 2.0 ml trypsin solution (4 mg Type I-from bovine pancreas, (Sigma Chemical Co., St. Louis, Missouri) in 200 ml 0.001 M HCl), and warmed to 37 ◦ C. Following addition of 5.0 ml BAPNA (N∀-benzoyl-DLarginine-p-nitroanilide hydrochloride, Sigma Chemical Co., St. Louis, Missouri) solution (40 mg BAPNA dissolved in 1.0 ml DMSO and diluted to 100 ml with 0.05 M tris buffer (pH 8.2)) the solution was incubated at 37 ◦ C for exactly 10 min before stopping the reaction with 1.0 ml 30% C2 H4 O2 . The solution was twice filtered through Whatman # 2 filter paper and absorbance read at λ = 410 nm (DU 640 UV/VIS Spectrophotometer, Beckman Instruments, Fullerton, California). Activity was expressed as trypsin inhibitor units (increase of 0.01 absorbance units at 410 nm per 10.0 ml reaction volume) and converted to TIA
expressed as TI mg g−1 sample (Hamerstrand et al. 1981). Statistical analysis A randomized complete block design was employed. The fixed classification effects were SBM treatment (phytase pretreated and untreated) and levels of SBM substitution (0, 25, 50, 75, and 100% of the CP). The basal diet (0% SBM substitution) was used as the control group for statistical comparisons. Insufficient material for analytical analysis in some samples necessitated testing means by the least-squares estimates of marginal means (lsmeans) method. All experimental diets were tested against the control using Dunnett’s t-test. Orthogonal contrasts were run between phytase pretreated and untreated groups at each level of substitution (Cody and Smith 1997). All analyses were performed using SAS/STAT statistical software. Significant differences were reported at the 0.05 level for all analyses.
Results Trypsin inhibitor activity in the solvent extracted SBM was 2.3 mg g −1 SBM. The calculated residual TI in
185 the diets was 0.04, 0.08, 0.12, and 0.15% of the dry diet for diets incorporating SBM at 25, 50, 75, and 100% of the CP, respectively. Phytate concentrations in the untreated SBM diets increased in a linear fashion with increasing levels of SBM substitution, whereas phytate was not detected in the phytase pretreated SBM diets (Table 2). Dietary HROM decreased with increasing levels of SBM substitution (Table 2) due to lower levels of α-cellulose incorporation (Table 1). The apparent crude protein digestibility (ACPD) of the FM control diet was 81.7%. The ACPD of the experimental diets ranged from 78.9–84.6% (Table 3). No significant differences were detected in ACPD. The mean apparent availability of individual AA are summarized in Table 4. The means for the phytase pretreated SBM incorporated at 100% of the CP are based on only two values. The standardized residuals of two replicates tested as outliers (Montgomery 1991) and were excluded from the calculation. Relative to the other diets, the apparent availability of Ala was significantly lower in the phytase pretreated diet incorporating SBM at 100% CP. Additionally, apparent availability of Lys in this same diet was lower than that of the FM control. Apparent availability of Ser in the control diet was lower than in the diet containing untreated SBM at 75% of the CP. The only difference in apparent AA availability detected by orthogonal contrasts was between the two diets containing SBM at 100% of the CP. At the 100% level of substitution the apparent availability of Ala, Asp, Arg, Gly , His, Lys, Met, Thr, and Val were significantly lower (P < 0.05) in the group receiving the phytase pretreated diet. Available Met decreased relative to dietary Met with increasing substitution of phytase pretreated SBM (Figure 1A). In contrast, dietary and available Met in the untreated SBM diets paralleled each other (Figure 1B). Dietary and available Lys followed a similar trend to Met (Figure 2). However, whereas Met availability began to decrease relative to dietary Met at 100% substitution, the Lys availability began to decrease relative to dietary Lys when the level of substitution surpassed 25% of the CP.
Discussion Phytates reduce digestion and availability of amino acids for uptake across the gastrointestinal tract
(Cheryan 1980; Reddy et al. 1989). Decreased CP digestibility of diets supplemented with salts of phytic acid leads to depressed growth in Chinook salmon, rainbow trout, and carp (Spinelli et al. 1983; Richardson et al. 1985; Hossain and Jauncey 1993). Conversely, feeding rainbow trout plant based diets pretreated with phytase result in superior growth relative to fish fed untreated diets (Cain and Garling 1995). Moreover, performance and N retention increase in terrestrial animals fed diets containing phytase (Atwal et al. 1980; Satterlee and Abdul-Kadir 1983; Mroz et al. 1994; Sebastian et al. 1997; Martin et al. 1998). In this experiment, no differences were detected in ACPD among the SBM diets. Similar findings were observed in hybrid striped bass fed phytase supplemented diets (Papatryphon et al. 1999). In other digestibility studies, tilapia fed diets with SBM substitution resulted in similar (Shiau et al. 1989), or slightly higher ACPD (Shiau et al. 1987; Shiau et al. 1990). Sufficiently high levels of residual TIA reduce growth and protein digestibility in tilapia (Wee and Shu 1989; Shiau et al. 1990). The two diets incorporating SBM at 100% of the CP were calculated to contain 0.15% TI kg−1 of the dry diet, which is similar to the levels reported to decrease growth in tilapia (Wee and Shu 1989). However, an affect on apparent protein digestibility, apparent net protein utilization, or protein efficiency ratio relative to a fish meal control diet could not be demonstrated until the residual TIA reached 0.38% TI kg−1 of the dry diet. Similarly we were unable to detect differences in apparent CP digestibility even at the highest rates of SBM inclusion. The biological value of a protein is estimated from retention following digestion and catabolism of AA. Relative to mammals, protein synthesis in fish is more dependent upon dietary AA than AA from protein turnover (Fauconneau 1985). However, fish fed an excess of AA, or an imbalanced mixture of AA, catabolize the unutilizable AA thereby increasing NH3 /NH+ 4 excretion (Walton 1985; Cai et al. 1996). Therefore, increasing protein retention is critical in recirculating systems. Soybean meal has a favorable indispensable amino acid (IAA) profile for replacing FM in formulated diets for tilapia (Tacon and Jackson 1985). However, at high rates of incorporation the diets become deficient in one or more IAA. The first two limiting IAA for O. niloticus are Met and Lys. Soybean meal diets have been supplemented with IAA to compensate for this deficiency. Reports of success with this approach vary (Rumsey and Ketola 1975; Walton et al.
186 Table 2. Dietary phytic acid and hydrolysis resistant organic matter in the experimental diets SBMa substitution (% dry diet)
SBM incorporation (% dry diet)
Phytic acidb (% dry diet)
HROMc
Phytase treated SBM
0 25 50 75 100
0 16.7 33.3 50.0 66.7
ND ND ND ND ND
25.81 20.35 15.04 11.47 5.01
Untreated SBM
0 25 50 75 100
0 16.7 33.3 50.0 66.7
ND 0.20 0.39 0.58 0.77
25.81 21.53 16.68 14.79 5.26
(% crude protein)
a Soybean meal. b ND indicates none detected. c Hydrolysis resistant organic matter.
Table 3. Meana apparent crude protein digestibility for Oreochromis niloticus fed diets containing graded levels of phytase pretreated or untreated soybean meal Level of SBM substitution (% CP)
Apparent crude protein digestibility Phytase pretreated SBM Untreated SBM
0 25 50 75 100
81.7 (3.68) 78.9 (3.28) 79.1 (3.16) 84.4 (2.63) 84.6 (0.21)
81.7 (3.68) 80.9 (3.30) 82.3 (1.56) 82.1 (7.29) 82.5 (4.17)
a Parenthetical values represent standard error of the mean
1982; Viola et al. 1981; Shiau et al. 1989; El-Sayed 1989; El-Dahhar and El-Shazly 1993; Davis et al. 1995; Ng et al. 1996; Coyle et al. 2000). A frequently used method for increasing dietary IAA is supplementation with L-crystalline forms. Suitability of crystalline AA supplementation has been demonstrated in hybrid striped bass (Griffin et al. 1992), red drum (Brown et al. 1988), rainbow trout (Davies and Morris 1997), Atlantic salmon (Olli et al. 1995), carp (Nose et al. 1974), Tilapia zilli (El-Sayed 1989), and channel catfish (Wilson et al. 1977). Diets were formulated to contain sufficient IAA to meet the known requirements of O. niloticus. Crystalline L-methionine supplementation was used to meet the Met requirement. Despite Met supplementation, analysis indicated the diets contained sufficient levels of IAA, except Met in the two diets containing SBM as 75% of the CP, and Met in the diet containing untreated SBM at 50% of the CP. However, Cys can
contribute up to 50% of the Met requirement in tilapia (Jackson and Capper 1982). Therefore emphasis is placed on meeting the TSAA requirement. Assuming 20% of the dietary Cys was used for this purpose, the diets met the putative TSAA requirement. Amino acid availability coefficients for the untreated SBM diets were similar to values reported by Sadiku and Jauncey (1995) for O. niloticus fed graded levels of SBM in a SBM:poultry meat meal blend diet (Table 5). However, Met availabilities were lower in the present study. Incorporating sufficient IAA in the diet does not ensure bioavailability to the animal. Thus, apparent availability coefficients were applied to dietary IAA concentrations to determine available IAA (Table 6). Available TSAA and Thr were below reported dietary requirements in all diets. A deficiency of available Thr with high levels of SBM incorporation was also observed in rainbow trout (Davies and Morris 1997).
187
Figure 1. Dietary and available methionine in diets containing graded levels of phytase pretreated soybean meal (A), or untreated soybean meal (B) as a percent of dietary crude protein.
Available Met decreased relative to dietary Met in the diet containing phytase pretreated SBM (Figure 1); however, this phenomenon was not observed in the diets containing untreated SBM. In the diets containing untreated SBM dietary and available Met paralleled each other (Figure 1), which was similar to observations made in rainbow trout fed increasing levels of SBM (Dabrowski et al. 1989). This suggests an unknown physical or chemical property associated with the phytase pretreated SBM, or the phytate hydrolysis process was responsible for the observed decrease in Met availability in fish fed diets substituting phytase pretreated SBM at 75% and 100% of the CP.
Crystalline L-methionine was incorporated in the diets containing 50% or more of the CP as SBM. The crystalline Met appeared to be utilized by tilapia fed the diets containing untreated SBM (Figure 3). As percent of dietary Met from the intact protein decreased with increasing levels of SBM substitution, available Met as a percent of the dietary requirement improved slightly, suggesting crystalline Met was utilized. Conversely, Met supplementation in the diets containing phytase pretreated SBM appeared to be unutilized (Figure 3). However, this could not be verified. Applying the AAC to the Met contributed by the intact protein and to the Met contributed by the purified sup-
188
Figure 2. Dietary and available lysine in diets containing graded levels of phytase pretreated soybean meal (A), or untreated soybean meal (B) as a percent of dietary crude protein.
plement indicates they may have been utilized equally (Table 7). There is evidence crystalline AA are readily leached from diets (Wilson 1989). Tilapia prefer smaller sized feeds than other species of comparable size (NRC 1993). A smaller pellet increases the surface to volume ratio thereby increasing the potential for leaching. It cannot be determined if available Met was solely from the intact protein or partitioned between the two sources. This is an important area for future investigation. Caution should be exercised in interpreting the results of Met and TSAA availability. The feed and
fecal values of Cys and Met may be underestimated. Cysteine and Met can be unstable under the conditions used to hydrolyze the samples and are therefore typically converted to more stable derivatives such as cysteic acid and methionine sulfone prior to hydrolysis (Waters 1989). Insufficient fecal material precluded this step. However, measured dietary Met was similar to values predicted from the formulation suggesting loss upon hydrolysis was minimal. As with Met, Lys availability decreased relative to dietary Lys in fish fed the phytase pretreated SBM. A dietary interaction between Lys and Arg is well documented in some animals (Wilson 1989). In fish, as with
189 Table 4. Mean∗ apparent amino acid availability coefficients in Oreochromis niloticus fed graded levels of untreated or phytase pretreated soybean meal substituted into a control diet. Values with different superscripts across a row are significantly different (P < 0.05) AA∗∗
Arg His Ile Leu Lys Met Phe Thr Val Ala Asp Cys Glu Gly Pro Ser Tyr
Control diet 80.9a (2.10) 77.5a (2.49) 74.8a (2.00) 78.2a (1.58) 85.1a (1.49) 79.7a (1.57) 72.7a (2.37) 75.3a (2.12) 76.6a (2.51) 80.6a (1.94) 76.6 (2.51) 78.8 (4.83) 84.9 (2.43) 77.8 (2.88) 76.2 (3.30) 71.9b (4.29) 65.4 (5.05)
Phytase pretreated SBM (% CP) 25 50 75 100
Untreated SBM (%CP) 25 50 75
100
82.0a (2.77) 77.7a (2.73) 73.1a (3.43) 77.0a (2.64) 82.8a (2.61) 77.0a (2.77) 73.5a (3.49) 76.2a (3.27) 73.1a (3.33) 77.3a (2.89) 73.1 (3.33) 77.0 (4.54) 83.0 (2.46) 77.4 (2.76) 76.5 (3.70) 76.3a (3.35) 62.2 (6.46)
80.9a (2.10) 77.5a (2.49) 74.8a (2.00) 78.2a (1.58) 85.1a (1.49) 79.7a (1.57) 72.7a (2.37) 80.3a (1.49) 78.0a (3.98) 80.8a (2.84) 78.0 (3.99) 80.9 (5.09) 84.6 (2.57) 78.2 (3.46) 79.3 (3.69) 78.4a (2.86) 72.3 (3.98)
89.0a (5.11) 84.6a (7.55) 82.2a (8.72) 83.2a (8.36) 89.2a (4.79) 84.7a (7.23) 82.5a (8.44) 79.1a (6.57) 74.0a (8.84) 73.2a (9.13) 74.0 (8.84) 78.7 (7.91) 86.1 (5.39) 76.5 (7.97) 77.2 (10.56) 82.4a (5.39) 65.9 (8.84)
82.4a (1.86) 78.6a (2.35) 74.7a (3.19) 77.9a (2.46) 81.8a (2.09) 78.5a (2.23) 75.0a (3.20) 75.5a (2.01) 74.3a (3.35) 77.0a (2.99) 74.3 (3.35) 77.7 (3.87) 83.9 (2.60) 77.1 (3.01) 76.5 (3.23) 78.7a (2.61) 65.6 (3.34)
85.2a (2.13) 79.2a (4.01) 76.9a (3.72) 78.0a (3.90) 79.3a (3.71) 78.2a (3.73) 78.7a (3.12) 75.2a (6.26) 74.2a (4.32) 75.4a (4.24) 74.2 (4.32) 79.3 (2.60) 85.1 (2.61) 77.7 (3.92) 76.7 (4.14) 79.6a (4.75) 69.4 (4.32)
71.0a (17.95) 64.1a (19.93) 57.4a (23.53) 60.3a (22.35) 60.6b (23.28) 56.8a (25.07) 63.1a (20.51) 56.3a (25.19) 50.0a (27.66) 48.1b (29.6) 50.0 (27.66) 64.0 (21.65) 73.7 (20.25) 53.7 (26.45) 59.9 (22.05) 66.0a (26.45) 45.6 (27.66)
84.3a (3.04) 82.3a (3.29) 77.2a (3.72) 80.1a (2.92) 86.7a (2.59) 80.0a (4.06) 77.1a (3.79) 78.7a (1.91) 75.7a (2.73) 77.6a (2.46) 75.7 (2.73) 77.2 (2.15) 84.8 (1.62) 75.8 (2.19) 77.2 (2.01) 78.3a (1.38) 72.3 (2.73)
84.5a (1.09) 80.7a (1.28) 76.3a (2.84) 78.7a (2.67) 85.8a (1.40) 77.2a (2.15) 76.9a (2.60) 83.0a (8.90) 82.2a (8.29) 82.3a (8.39) 82.2 (8.29) 85.1 (6.27) 89.2 (5.16) 83.6 (7.51) 84.3 (7.07) 85.8a (6.57) 75.9 (8.29)
∗ Calculated on four replicates of pooled samples (n = 15), except the diet containing phytase pretreated
SBM at 100% CP which was calculated on two replicates. Parenthetical values represent standard error of the mean.
∗∗ Amino acid.
mammals, Arg and Lys compete for the same intestinal transporters (Ash 1985). Although an antagonistic relationship has yet to be demonstrated in fish (NRC 1993), there is insufficient evidence to rule out the possibility high levels of Arg did not exacerbate the low availability of Lys observed. However higher available Arg in the untreated SBM diets would suggest against this possibility.
The feeding behavior of tilapia likely contributes to reduced availability of AA from the phytase pretreated diets. Tilapia are equipped with palatine teeth and pharyngeal gill rakers allowing them to grind feed and filter material before swallowing (Jauncey and Ross 1982; Bowen 1982). This process creates small particles with high surface to volume ratio. Additionally, tilapia repeatedly draw in and expel their food before
190 Table 5. Comparison of apparent amino acid availability coefficients for Oreochromis niloticus fed graded levels of soybean meal substituted into a fish meal based dieta , or a poultry meat based dietb SBM 25% crude protein Fish meal Poultry meat
SBM 50% crude protein Fish meal Poultry meat
SBM 75% crude protein Fish meal Poultry meat
Indispensable AA ARG HIS ILE LEU LYS MET PHE THR VAL
84.3 82.3 77.2 80.1 86.7 80.0 77.1 80.3 78.0
76.2 74.6 72.9 77.1 77.9 89.4 74.6 71.5 72.3
84.5 80.7 76.3 78.7 85.8 77.2 76.9 78.7 75.7
83.1 82.7 80.0 79.9 84.1 91.5 79.9 77.5 76.7
89.0 84.6 82.2 83.2 89.2 84.7 82.5 83.0 82.2
82.9 84.6 81.0 83.3 85.7 93.2 82.1 80.6 76.7
Dispensable AA ALA ASP CYS GLU GLY PRO SER TYR
80.8 78.0 80.9 84.6 78.2 79.3 8.4 72.3
76.7 73.2 83.4 78.6 80.0 78.8 72.6 74.4
77.6 75.7 77.2 84.8 75.8 77.2 78.3 72.3
77.1 80.1 82.2 82.8 82.1 83.2 79.2 77.8
82.3 82.0 85.1 89.2 83.6 84.3 85.8 75.9
78.2 74.1 85.1 83.8 79.0 84.3 84.1 80.3
a Values from this study. b Values from Sadiku and Jauncey (1995).
Figure 3. Available methionine (% dietary requirement) as a function of L-crystalline methionine substitution. Curves were fitted by quadratic regression.
consumption (Hanley 1987; personal observations). These processes increase the potential for leaching of soluble components. Phytates decrease solubility of nutrients (Reddy et al. 1989) and its removal may increase solubility of essential nutrients.
Phytates may also serve in a protective capacity. Phytates bind to the globular proteins glycinin, and βconglycinin (Reddy et al. 1989), both of which are ANFs in fish (Kaushik et al. 1995; Rumsey et al. 1995). Phytates also may protect some of the more
191 Table 6. Dietary requirement and available indispensable amino acids for Oreochromis niloticus fed graded levels of untreated or phytase pretreated soybean meal as a percent of dietary crude protein
IAA
Requirementa
Control
Arg His Ile Leu Lys Met TSAA Phe Thr Val
1.18 0.48 0.87 0.95 1.43 0.75 0.90 1.05 1.05 0.78
1.55 0.49 1.00 1.91 1.67 0.67 0.81 0.87 0.85 1.24
IAA
Requirement
Arg His Ile Leu Lys Met TSAA Phe Thr Val
1.18 0.48 0.87 0.95 1.43 0.75 0.90 1.05 1.05 0.78
Untreated SBM (% Dietary CP) 25 50 75 100 1.89 0.60 1.15 2.14 1.92 0.70 0.87 1.10 0.96 1.34
1.87 0.58 1.09 2.02 1.70 0.55 0.70 1.11 0.89 1.21
2.21 0.66 1.24 2.21 1.71 0.61 0.80 1.30 1.00 1.36
2.30 0.69 1.22 2.16 1.67 0.61 0.81 1.37 0.93 1.26
Phytase treated SBM (% Dietary CP) 25 50 75 100 1.83 0.57 1.12 2.14 1.76 0.66 0.81 1.08 0.92 1.29
1.86 0.60 1.16 2.15 1.55 0.61 0.77 1.13 0.88 1.30
2.03 0.62 1.19 2.11 1.47 0.55 0.73 1.29 0.89 1.25
1.81 0.56 0.98 1.73 1.14 0.42 0.58 1.15 0.66 0.89
a Requirements from NRC (1993).
readily damaged AA, such as Lys, from degradation during processing or pelleting. The behavioral response of O. niloticus to the diet substituting phytase pretreated SBM at 100% of the CP may be indicative of a poor quality diet. The fish displayed avoidance behavior generally attributed to poor palatability; however, terrestrial animals behave similarly to diets with AA deficiencies or poorly balanced amino acids (Gietzen 1993). Moreover, the poor acceptance of the diet containing phytase pretreated SBM at 100% of the CP resulted in little feces being collected from this treatment, with some individuals containing no useable feces. As a result of the small fecal sample size the endogenous contribution may have been over-represented relative to the other treatments. Dabrowski et al. (1989) reported increased endogenous excretions of Met, Leu, and Thr with increasing levels of SBM. A measure of true AA availability would help elucidate these endogenous contributions.
We conclude hydrolysis of phytates, with the enzyme phytase, in the SBM incorporated in the experimental diets decreased the apparent availability of one or more of the IAA, but not the overall ACPD. Further research is required to conclusively determine whether phytate is beneficial to amino acid availability, or the absence of phytate decreases AA availability in diets incorporating high concentrations of SBM.
Acknowledgements The authors respectfully acknowledge the contributions of David I. Haley, Jon Amberg, Steve Hart, Betsy Haley, and Corissa Riche. We also thank Zeeland Farm Services (Zeeland, Michigan); Zeigler Brothers, Inc. (Gardners, Pennsylvania); Zapata Protein, Inc. (Reedville, Virginia), and BASF Corporation’s Chemical Division (Parsippany, New Jersey); for their generous contributions of materials. Partial funding
0.67 0.66 0.61 0.55 0.42
for this project was provided by the Michigan Soybean Promotion Committee. Additional support was provided by the North Central Regional Aquaculture Center under grant 96-38500-2631 from the U.S. Department of Agriculture, and by the Michigan State University Agricultural Experiment Station. The U.S. Government and the North Central Regional Aquaculture Center are authorized to produce and distribute reprints for governmental purposes notwithstanding any copyright notations appearing hereon.
0.77 0.65 0.56 0.45 0.26
0 0 0.03 0.13 0.17
0.77 0.65 0.59 0.58 0.43
References
a Methionine. b Met values for Herring meal and SBM from NRC (1993). c Apparent availability coefficient.
79.7 77.0 78.5 78.2 56.8 0.96 0.84 0.71 0.58 0.45 0 25 50 75 100
0 0 0.04 0.17 0.30
Measured available Met (g kg−1 dry diet) Estimated available Met (g kg−1 dry diet) Available Met supplement (g kg−1 dry diet) Available Met intact protein (g kg−1 dry diet) AACc (%) Met supplement (g kg−1 dry diet) Meta,b intact protein (g kg−1 dry diet) SBM (% CP)
Table 7. Dietary methionine supplied by intact protein and L-crystalline methionine, apparent availability coefficients, estimated availability of methionine from intact protein and supplemental methionine, and measured available methionine in the experimental diets
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