Encapsulation of microbial phytase: Effects on phosphorus bioavailability in rainbow trout (Oncorhynchus mykiss)

Animal Feed Science and Technology 169 (2011) 230–243

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Encapsulation of microbial phytase: Effects on phosphorus bioavailability in rainbow trout (Oncorhynchus mykiss) G.W. Vandenberg ∗ , S.L. Scott 1 , P.K. Sarker, V. Dallaire, J. de la Noüe Groupe de recherche en recyclage biologique et aquiculture, Département des sciences animales, Université Laval, Québec, QC, G1V 0A6, Canada

a r t i c l e

i n f o

Article history: Received 22 November 2010 Received in revised form 4 July 2011 Accepted 7 July 2011

Keywords: Fish Phytase Encapsulation Phosphorus Mineral Digestibility

a b s t r a c t This study was undertaken to investigate the effects of adding microbial phytase to plant protein-based diets for rainbow trout (Oncorhynchus mykiss). A plant protein-based basal diet was formulated to be isonitrogenous, isolipidic and isoenergetic to a nutrient-dense, fish meal-based control diet (Starter salmonid diet; Ontario Ministry of Natural Resources (MNR), University of Guelph). The basal plant protein-based diet was supplemented with 3000 FTU microbial phytase kg−1 included either in a free form or encapsulated in chitosanalginate microcapsules. A second control group was fed the basal plant protein-based diet supplemented with monosodium phosphate to NRC requirements. Apparent digestibility coefficients (ADC) were determined for dry matter, energy, protein, ash, P, Ca, Mg, Mn, Cu, Zn and Fe. The above diets were fed to triplicate tanks of rainbow trout for 56 days; growth and feed efficiency were monitored at 2-week intervals. At the start of the study and at 28-day intervals, fish were sampled to determine composition of the whole carcass and tissues and blood concentration of inorganic P. Proteolytic enzyme activity in intestine was measured on days 0, 4 and 28. Feeding the fishmeal-based control diet increased (P<0.001) ADC for a number of macro- and micro-nutrients, growth rate, feed efficiency, and tissue ash and P concentrations compared with the plant protein-based diet; however, supplementation of the plant protein-based diet with microbial phytase improved (P<0.001) the ADC for energy, protein (P<0.05), ash, P and a number of macro- and micro-nutrients. Fish growth, feed efficiency, concentrations of tissue ash and P, and retention of P (P<0.001) and N (P<0.05) were also increased by supplementation of the plant protein-based diet with microbial phytase; values for these parameters approached those of the fish meal control diet in the final 28 days of the experiment. In the same period, inclusion of microbial phytase resulted in lower (P<0.05) total, solid and dissolved P excretion versus all other treatment groups. There was no effect (P>0.05) of phytase supplementation on proteolytic enzyme activity. Encapsulation of microbial phytase tended (P<0.10) to diminish its ability to liberate P. The data suggest that microbial phytase can effectively increase the apparent digestibility and bioavailability of a range of nutrients from plant protein-based diets for rainbow trout, resulting in increased growth performance and tissue mineralization. Encapsulation of phytase reduces this effect, likely due to hindered interaction between the enzyme and dietary phytate-P. © 2011 Published by Elsevier B.V.

Abbreviations: P, phosphorus; Pi, inorganic P; FTU, phytase unit; N, nitrogen; DM, dry matter; CP, crude protein; FE, feed efficiency; GE, gross energy; NFE, nitrogen free extract; ADC, apparent digestibility coefficients; TGC, thermal growth coefficient; MNR, Ontario Ministry of Natural Resources. ∗ Corresponding author at: Département des sciences animales, Université Laval, Québec (QC), 2425, rue de l’Agriculture, Québec, QC, G1V 0A6, Canada. Tel.: +1 418 656 2131x6541; fax: +1 418 656 3766. E-mail address: [email protected] (G.W. Vandenberg). 1 Present address: Brandon Research Centre, Agriculture and Agri-Food Canada, P.O. Box 1000A, Brandon, Manitoba, R7A 5Y3, Canada. 0377-8401/$ – see front matter © 2011 Published by Elsevier B.V. doi:10.1016/j.anifeedsci.2011.07.001

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1. Introduction Mounting restrictions related to phosphorus (P) discharge from intensive fish production have limited expansion of the aquaculture industry in a number of countries. Denmark was the first country to place restrictions on fish production and establish legislation on feed quality to reduce pollution from aquaculture production (Iversen, 1995). Since then, a number of regions in the United States of America and Canada have instituted limitations on effluent P concentration, which in turn requires significantly reduced P excretion if industry growth is to be sustained. Phosphorus discharge from fish culture facilities arises from uneaten feed and from feces, the latter comprising particulate and dissolved forms. Several technologies aimed at reducing nutrient discharge from aquaculture production have been evaluated, including reduced feeding levels (Summerfelt et al., 1995), capture and removal of uneaten feed and feces (Bergheim and Cripps, 1998), or treatment of effluent to remove P using biological (Dumas et al., 1998; Hussenot et al., 1998) or chemical (Drizo et al., 1997) processes. Many of these technologies suffer from high initial capital investment, difficulty in controlling critical operational parameters, and lack of application to cage culture operations (Cripps, 1994). Given that the ultimate source of P waste is the feed, nutritional strategies to reduce P excretion at the source are among the most efficient methods to affect P output from aquaculture production. Nutritional strategies to reduce waste output from aquaculture include: fine-tuning of feed formulae, processing/refining of ingredients and use of additives (organic acids, vitamin D and phytase, etc.) (Bureau and Hua, 2010). Several study demonstrated that high P fish meal can be partially or fully replaced with plant protein-based ingredients using a variety of oilseed, legume and processing byproduct sources (Adelizi et al., 1998; Riche and Brown, 1999; Carter, 2000). Unfortunately, these plant protein sources may contain antinutritional factors (Tacon, 1997), specifically phytate, which represents 50–90% of the P found in plant protein sources (Graf, 1986). Phytate-bound nutrients are largely unavailable to monogastrics, who lack sufficient endogenous phytase activity to liberate phytate-P (Pointillart et al., 1984). Furthermore, the phytate molecule, being negatively charged, forms complexes with a variety of divalent cations (Davies and Nightingale, 1975) and proteins (Knuckles et al., 1985), leading to reduced availability of a number of essential nutrients. Addition of microbial phytase significantly increases P digestibility in monogastrics; Jongbloed et al. (1992) reported that the addition microbial phytase increased P digestibility in a practical swine diet, which subsequently resulted in a 40% increase in overall P retention (Mroz et al., 1994). The effect of phytase on P availability has been evaluated in a number of fish species, either following pre-treatment of ingredients with phytase (Cain and Garling, 1995; Ramseyer et al., 1999) or direct addition of phytase to non-salmonid (Yoo et al., 2005; Sarker et al., 2006; Nwanna et al., 2008; Sarker and Hosokawa, 2009; Laining et al., 2011) and salmonid (Vielma et al., 2000; Cheng et al., 2003; Sajjadi and Carter, 2004; Denstadli et al., 2007; Dalsgaard et al., 2009; Wang et al., 2009; Carter and Sajjadi, 2011) diets. Phytase is increasingly considered as the important additive for eco-friendly aquafeed formulation as it can enhance the bio-availability of phytate-bound P and nitrogen and thus reduce P discharged into the aquatic environment (Rodehutscord and Pfeffer, 1995; Storebakken et al., 2001; Cao et al., 2007). However, microbial phytase is thermally instable, resulting in loss of activity at processing temperatures of >80 ◦ C (Simons et al., 1990); occurrence of temperatures over 87 ◦ C during steam pelleting of feed results in a reduction of the phytase activity by more than half. Phytase thermal instability is particularly problematic in commercial finfish diets, given that a large proportion of compound diets are currently extruded; leading to, extreme temperatures (110–150 ◦ C), pressure and shear forces during processing (Autin, 1997). Approaches used to improve the thermal stability of various enzymes include microencapsulation and immobilization in polymers (HeichalSegal et al., 1995; Ortega et al., 1998); thus, microencapsulation of phytase may improve its thermal stability during feed processing. The objectives of the current studies were to investigate the effect of feeding a plant protein-based diet supplemented with either mineral P, free microbial phytase, or encapsulated microbial phytase on nutrient digestibility and mineral bioavailability, growth performance and proteolytic enzyme activity in rainbow trout, compared with a practical fish meal-based starter diet.

2. Materials and methods Sodium alginate was purchased from the BDH Company (Montreal, QC, Canada). This low molecular weight alginate is isolated from the stips of the brown algae Laminaria hyperborea. Low-viscosity chitosan with a 93% degree of deacetylation was obtained from Pronova Biopolymers (Washington, OR, USA). Low-P fish meal was obtained from International Seafoods of Alaska Inc. (Kodiak, AK, USA). Other feed ingredients were obtained from a commercial feed mill (Martin Feed Mills, Elmira, ON, Canada). Phytase (NatuphosTM ), vitamin and mineral supplements (Ontario Ministry of Natural Resources Formulation VIT-9608 and MIN-9504, respectively) were supplied by BASF Corporation (Georgetown, ON, Canada). Sipernat 50TM [source of acid insoluble ash (AIA)] was obtained from Degussa-Hüls Canada Inc. (Brampton, ON, Canada). Finstimm as feeding stimulant was provided by Finnsugar Bioproducts (Helsinki, Finland). Synthetic enzyme substrates, plasma inorganic P (Pi ) diagnostic kits (# 360-UV) and all other reagent grade chemicals were purchased from Sigma–Aldrich Canada Ltd. (Oakville, ON, Canada). Soluble protein assays were performed using a modified Coomassie protein assay (Pierce Inc., New York, NY, USA) in 96-well ELISA plates.

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Table 1 Experimental diet formulation (as fed basis) and chemical composition (dry matter basis). Ingredient

Diets Plant protein-based experimental diet

Animal protein-based MNR control diet

Formulation (g/kg) Fish meal (CP, 672.5) Maize gluten meal (CP, 604.3) Blood meal (CP, 820.0) Whey protein (CP, 120.0) Poultry by-product meal (CP, 640.0) Wheat middlings (CP, 156.6) Canola filter fines (CP, 380.0) Soybean meal (CP, 480.0) Pea protein (CP, 490.0) Brewers yeast (CP, 450.0) Fish oil Vitamin premixa Mineral premixb Sipernat 50c Finstimm dl-Methionine l-Lysine

150.0 380.0 70.0 0.0 0.0 50.0 50.0 50.0 50.0 0.0 160.0 10.0 5.0 10.0 10.0 0.0 5.0

300.0 240.0 92.5 80.0 50.0 0.0 0.0 0.0 0.0 40.0 160.0 10.0 5.0 10.0 10.0 1.5 1.0

Composition (g/kg)d Crude protein (N × 6.25) Gross energy (MJ/kg DM) Crude lipid Ash Calcium Magnesium Copper (mg/kg) Iron (mg/kg, ×101 ) Manganese (mg/kg) Zinc (mg/kg) Lysin (calculated, g/kg)e Methionine (calculated, g/kg) Cystein (calculated, g/kg)

525.6 24.7 196.5 53.4 3.8 1.1 236 29 236 967 21.5 8.8 6.3

532.7 24.8 195.9 65.3 8.6 0.89 216 24.4 216 903 29.8 11.2 5.9

a Based on Ontario Ministry of Natural Resources (MNR) formulation #VIT-9608. Provides (per kg diet as-fed): vitamin A (as retinyl acetate): 3750 IU; vitamin D3 (as cholecalciferol): 3000 IU; vitamin E (as dl-␣-tocopheryl acetate): 75 mg; vitamin K (as menadione Na-bisulfate): 1.5 mg; vitamin B12 (as cyanocobalamin): 0.03 mg; ascorbic acid (as ascorbyl polyphosphate): 75 mg; d-biotin: 0.21 mg; choline (as chloride): 1500 mg; folic acid: 1.5 mg; niacin (nicotinic acid): 15 mg; pantothenic acid: 30 mg; pyridoxine: 7.5 mg; riboflavin: 9 mg; thiamin: 1.5 mg. b Based on Ontario Ministry of Natural Resources (MNR) formulation #MIN-9504. Provides (per kg diet as-fed): sodium chloride (1199.25 mg Na, 1875.75 mg Cl): 3075 mg; ferrous sulfate (FeSO4 ·7H2 O, 13 mg Fe): 65 mg; copper sulfate (CuSO4 ·5H2 O, 7.5 mg Cu): 30 mg; manganese sulfate (MnSO4 , 32.4 mg Mn): 90 mg; potassium iodide (2.4 mg K, 7.6 mg I): 10 mg; zinc sulfate (ZnSO4 ·7H2 O, 60 mg Zn): 150 mg. c Sipernat 50: source of acid insoluble ash comprised of 985 g/kg SiO2 with an average particle size of 50 ␮m. d Analyzed according to AOAC (1990) methods. e Amino acids were calculated using Feed Formulation Template, University of Guelph, Canada.

2.1. Alginate microcapsule formation Phytase-loaded alginate microcapsules were produced as previously described (Vandenberg and de la Noüe, 2001a,b). Alginate was dissolved in water at 2% (w/v) and liquid phytase, NatuphosTM 5000 L (5000 FTU/g, where 1 FTU liberates 1 ␮mol Pi /min from 5.1 × 10−3 M sodium phytate at 37 ◦ C and pH 5.5) was added at a loading rate of 0.1% (w/w). Chitosan was dissolved in 2% acetic acid (w/v) to a final concentration of 0.25% (w/v) by gentle warming followed by filtration to remove any undissolved particles. The acidic solution of chitosan was mixed with 4 M NaOH, followed by addition of 1.5% (w/v) calcium chloride. Batches of approximately 500 mL of alginate/phytase were extruded drop-wise through a series of 8–21 gauge needles equipped with coaxial air jets into 1000 mL of the chitosan/calcium chloride solution and allowed to react for 45 min prior to rinsing in dilute (0.1 N) HCl and acetone. Microcapsules were dried overnight in a forced-air oven at 25 ◦ C and the final dry mass carefully recorded. Production of phytase-loaded alginate/chitosan microcapsules in this manner produced dried microcapsules with a phytase activity of 300 FTU phytase/g microcapsules. Microcapsules were produced one day prior to their incorporation into experimental diets, and thus were not exposed to storage-related losses of phytase activity. 2.2. Diet preparation The control diet containing animal-based protein sources was formulated according to the Ontario Ministry of Natural Resources (MNR) salmonid starter diet (Table 1). An isonitrogenous, isolipidic experimental diet was formulated by replacing

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fifty percent of the fish meal in the control diet with a variety of plant-based protein sources. For both types of diets, the levels of available methionine and lysine were estimated using protein availability values; if required to ensure an adequate amino acid balance, diets were supplemented with crystalline forms of methionine and lysine. Vitamin and mineral supplements were added to recommended levels (NRC, 1993). The basal plant protein-based diet was used to create the following four treatment diets: 1) N-0: no supplemental phytase added; 2) N-3000: 3000 FTU unencapsulated phytase/kg dry matter (DM); 3) E-3000: 3000 FTU encapsulated phytase/kg DM; and 4) P-Min: Addition of sufficient NaH2 PO4 (15 g/kg diet) in the expense of wheat middling to bring available P levels to NRC (1993) recommendations. Phytase dose (3000 FTU) was chosen based on our preliminary study that showed superior performance (Vandenberg et al., 2011). To prepare experimental diets, prior to heat conditioning and pelleting, micro-ingredients, encapsulated or unencapsulated phytase were first mixed and then slowly added to the macro-ingredients to ensure a homogenous mixture. Fifty percent of the indicated lipid was added to the mass and thoroughly incorporated. The mash was steam-conditioned and pelleted using a laboratory mill (California Pellet Mill, Crawfordsville, IN, USA) equipped with the appropriately sized die, the temperature immediately following pelleting was 78–83 ◦ C. The resulting pellets were dried in a forced-air oven at room temperature (25 ◦ C) for 24 h, sieved, and the remaining lipid was sprayed onto the pellets with regular mixing. The resulting pellets were stored in airtight containers at 5 ◦ C until fed. 2.3. Experimental rearing system Experiments were carried out at the Laboratoire régional des sciences aquatiques (LARSA; Université Laval, Québec, QC). Using the diets outlined in Table 1, digestibility study and a 56-day feeding study were carried out. All experimental tanks were supplied with water from a recirculation system as described previously (Vandenberg and de la Noüe, 2001a,b). Water temperature and dissolved oxygen were continuously monitored by a computer and were automatically maintained at 15.0 ± 0.5 ◦ C and 10.3 ± 0.5 mg/L, respectively. 2.4. Fish, feeding and experimental design All experimental animals were kept in accordance with the guidelines of the Canadian Council on Animal Care (CCAC, 2005) and the Comité de protection des animaux, Université Laval. For the digestibility study, rainbow trout (initial mass 45.5 ± 2.2 g) were obtained from a certified disease-free hatchery (Pisciculture des Alleghanys, St. Philemon, QC, Canada). Fish (n = 150) were randomly distributed into six 60-L cylindroconical tanks and fed a commercial diet (Martin Feed Mills, Elmira, ON, Canada) for 7 days prior to the start of the experiment. Experimental tanks were divided into groups of tanks, with effluent water being directed to a series of automatic fecal collection devices. Each dietary treatment was randomly assigned to 3 tanks (total 15 tanks) and was fed for 7 days to acclimate fish to the new diet prior to a 5-day fecal collection period. Diets were fed to apparent satiation once daily (08:00 h), following the recovery of collected feces. All tanks and system piping were thoroughly cleaned immediately following feeding to minimize subsequent contamination of feces with uneaten feed. For all treatment groups, daily fecal collections were pooled and stored at −80 ◦ C until required for further processing. For the feeding study, rainbow trout (n = 640; initial mass 33.19 ± 1.46 g) were randomly distributed among fifteen 80-L cylindroconical tanks and fed a commercial diet for 7 days prior to the start of the experiment. Dietary treatments were randomly assigned to the tanks. Fish were hand-fed to apparent satiation twice daily and tanks were then thoroughly brushed and purged immediately after feeding to assure optimal water quality. Feed consumption was monitored weekly; at the start of the experiment and at 14-day intervals. At the start of the experiment, 40 fish in total were randomly euthanized using a lethal dose of tricaine methanesulfonate (MS-222; 300 mg/L), and on days 28 and 56, ten fish/tank were removed and similarly euthanized. Along with those ten fish, remaining total fish in the tanks were also individually weighed and fork length measured. Whole blood was withdrawn into heparinized syringes, placed on ice and centrifuged (13,000 × g, 10 min) within 1 h of collection; plasma was analyzed immediately for concentration of Pi . Following blood collection, half of the fish removed from each tank were individually frozen for determination of proximate carcass composition [DM, crude protein (CP), ash, P]. The scales were removed from the lateral portion of one flank of the remaining fish removed from each tank. In addition, on days 0 and 4 and at the end of week 4, the distal portion of the small intestine (including the pancreatic tissue), was dissected from these same fish; it was frozen in liquid nitrogen (N), and stored at −80 ◦ C until required for analysis of proteolytic enzyme activity. 2.5. Sample preparation Frozen fecal samples were freeze-dried, ground using a 1-mm screen, and then stored at −30 ◦ C until required for analysis. Individual carcass samples were autoclaved for 15 min. For fish carcasses from which the scales of one flank had been removed, the entire skin was removed from the opposite flank, weighed and retained for mineral analysis. The spinal column from these same carcasses was removed, washed to remove the surrounding tissue, and retained for mineral analysis. The remaining carcasses were homogenized in a food processor, freeze dried, weighed, reground, and stored at −30 ◦ C until required for analysis. Scales removed from each carcass were resuspended twice in saline followed by a brief centrifugation,

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and washed in deionized water. Vertebrae were oven dried (110 ◦ C for 24 h), and ground and lipid was extracted in a chloroform:methanol (1:1, v/v) mixture. 2.6. Analytical methods Using standard methods (AOAC, 1990), tissues, whole carcasses, feed and feces were analyzed for DM (vacuum oven at 65 ◦ C for 18 h) and ash (incineration at 550 ◦ C for 18 h). Crude protein (N × 6.25) was quantified in feed, feces and whole carcasses using the semi-automatic Kjedahl (Model 16210, A/S N, Føss Electric, Hillerød, Denmark) method (AOAC method 7.B01-7.B04). Crude fat (CF) in the feed was analyzed via ethyl ether extraction (Soxtec System HT12, Føss Tecator AB; Hoganas, Sweden) and in the feces following acid hydrolysis (Lynch et al., 1963). Gross energy (GE) was quantified using an adiabatic bomb calorimeter (Parr Instrument Co., Moline, IL, USA) and N-free (NFE) extract was calculated by difference. Phosphorus was determined in all samples following digestion of ash in 10% (w/v) nitric acid using a Technicon AutoAnalyzer® apparatus (Technicon Corporation, TarryTown, NY, USA) employing the vanadate/molybdate method of analysis (Varley, 1966). For feed and fecal samples, other minerals (Ca, Mg, Mn, Cu, Zn, Fe) were analyzed following digestion of ash in 2 N hydrochloric acid using an atomic absorption spectrophotometer employing standard methods (AOAC, 1990). Phytase activity and phytate were measured according to standard methods (Radecki, 1999; Radecki and Chen, 1999). Acid insoluble ash was measured according to Atkinson et al. (1984), with particular care taken to thoroughly rinse the filtrate with boiling, de-mineralized water. Apparent digestibility coefficients (ADC) were calculated as described by Cho and Slinger (1979). 2.7. Enzyme assays Thawed intestinal sections were blended on ice using a high-speed tissue homogenizer in 5 volumes of cold 50 mM tris–HC1 buffer containing 10 mM CaC12 and 50 mM KC1 at pH 8.0. The homogenates were centrifuged (25,000 × g for 30 min at 4 ◦ C) and the supernatant collected and placed on ice until required for enzyme analysis. Specific enzyme activity was measured at 25 ◦ C using a temperature-controlled recording spectrophotometer. Enzyme reactions were allowed to continue for 2 min and enzyme activity was calculated within the linear portion of the curve. Protein determinations of the enzyme extracts were performed using Lowry’s method, modified by Hartree using Bovine Serum Albumin as standard (Hartree, 1972). The reaction mixture for carboxypeptidase and chymotrypsin assays contained 2.0 mL of 1.0 mM substrate, 50 mM tris–HC1 buffer, and 1.0 mM NaCl. Carboxypeptidase A (peptidyl-l-amino acid hydrolase, EC 3.4.17.1) activity was measured using N-(2-furanacryloyl)-l-phenylalanyl-l-phenylalanyl (FAPP) as the substrate according to the method of Riordin and Holmquist (1984). The reaction mixture was combined with 20 ␮L of enzyme extract and the change in absorbance was recorded at 350 nm. Carboxypeptidase B (protaminase; peptidyl-l-lysine[l-arginine] hydrolase, EC 3.4.17.2) activity was measured with furylacryloyl-l-alanyl-l-lysine (FAAL) as the substrate (Skidgel and Erdos, 1984). The reaction mixture was combined with 200 ␮L of enzyme extract and the change in absorbance was recorded at 336 nm. Chymotrypsin (EC 3.4.21.1) activity was measured with glutaryl-l-phenylalanine-p-nitroanalide (GPNA) as the substrate (Erlanger et al., 1966). The reaction mixture was combined with 400 ␮L of enzyme extract and the change in absorbance was recorded at 410 nm. Trypsin (EC 3.4.21.4) activity was measured with benzoyl-dl-arginyl-p-nitroanalide (BAPNA) as the substrate as described by Fritz et al. (1974). The reaction mixture contained 2.0 mL of 0.8 mM substrate, 100 mM triethanolamine buffer, and 10 mM CaCl2 . The reaction mixture was combined with 200 ␮L of enzyme extract and the change in absorbance was recorded at 405 nm. Enzyme specific activity within each treatment was expressed as mM substrate hydrolyzed/min/mg protein in the extract. 2.8. Calculations and statistical analyses Dissolved P output was predicted using the biological method of Cho et al. (1994), using the P ADC values from the digestibility experiment and the P retention calculations from the final 28 days of the feeding experiment. For the digestibility experiment, all data presented are mean values of duplicate analyses from three replicate tanks. Apparent digestibility values were subjected to analysis of variance using treatment, tank replicate and their interactions in the statistical model (least square means) (SAS, version 1, 2004). Means were compared using the Duncan’s multiple range post-hoc test and were considered significant at P<0.05. For the feeding study, growth and performance data were analyzed in a completely randomized design, with three replicate tanks/treatments. Tissue and enzyme samples were analyzed in a split-plot design using treatment as main plot values and values from individual fish as within plot values. 3. Results Using a variety of plant protein sources, 50% of the fish meal in the control diet (MNR) was replaced, resulting in similar levels of CP, CF and GE (Table 1), but differing greatly in phytate-P and thus available P levels (Table 2). The basal plant proteinbased diet (N-0) had low levels of endogenous phytase activity (123.4 ± 5 FTU/kg; mean ± SEM; as-fed basis), but the MNR control diet did not have any detectible phytase activity. Diets incorporating non-encapsulated microbial phytase (N-3000) had a mean phytase activity of 2638 ± 55 FTU/kg prior to steam processing, and 1352 ± 106 FTU/kg following heating and

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Table 2 Total phosphorus, phytate phosphorus (g/kg dry matter) and phytate phosphorus/total phosphorus (as proportion of 1) of the plant protein-based macroingredients used in the dietary formulation. Ingredient

Total Pa

Phytate-Pb

Phytate-P/total P

Soybean meal Maize gluten meal Pea protein Wheat middlings Canola filter fines Plant protein-based diet OMNR control diet

8.1 2.1 8.9 10.6 10.9 5.4 6.6

4.6 1.1 5.7 9.3 7.0 2.1 0.2

0.56 0.52 0.64 0.87 0.64 0.38 0.03

Values based on 3 duplicate samples. a Analyzed according to AOAC (1990) methods. b Analyzed according to Radecki (1999).

pelleting. Diets incorporating encapsulated phytase (E-3000) had a mean phytase activity of 273 ± 12 FTU/kg prior to steam processing, and 265 ± 7 FTU/kg following heating. As expected, the digestibility of macro- and micro-nutrients of the MNR control diet was higher (P<0.001) than other diets (Table 3). Similarly, a number of macro-nutrients in the N-0 diet were highly digestible in terms of DM, CP and GE; however, the ADC of ash, P, Ca, Mg and Mn were lower (P<0.001) compared with the MNR control. Adding non-encapsulated phytase (N-3000) increased (P<0.001) the availability of GE, CP (P<0.05), ash, and all macro- and micro-minerals (except copper) measured in the digestibility experiment. Adding encapsulated phytase (E-3000) improved ADC of ash, P, Ca and Mg, although the extent of the increase was less than that of the non-encapsulated phytase. Fig. 1 shows the effect of dietary regime on fish mass over the 8-week study. Fish receiving the MNR control diet grew significantly (P<0.05) faster than those fed plant protein-based diets (N-0, N-3000, E-3000, and P-Min), irrespective of phytase or mineral P supplementation. When growth rate is expressed as thermal growth coefficient (TGC; Table 4), fish fed the MNR control diet consistently showed higher (P<0.05) growth rates. Fish fed the plant protein-based diets showed a clear adaptation period during the initial two weeks of the study; there was no clear effect of phytase administration on TGC during the initial four weeks of the study. As the experiment progressed through weeks 6–8, the TGC was lower (P<0.05) for fish fed the plant protein-based diets without microbial phytase (N-0 and P-Min) compared with that for fish fed the diets containing either encapsulated or non-encapsulated phytase. The effects of phytase addition on feed efficiency (FE) (Table 4) are similar to those observed for TGC; there were no treatment-related differences were observed from the final 2 weeks until the end of the experiment. Although not directly measured, it was observed that feed wastage was prevalent in the plant protein-based diets during the first week of the study, irrespective of treatment, and in the N-0 diet group over the final four weeks of the experiment. There was no consistent effect of phytase supplementation on activity of any of the proteolytic enzymes on either day 4 or 28 of the study. Of interest, however, is the time-related increase in enzyme activity in all groups, as well as the lower activity of all four enzymes in fish fed the MNR control diet for 28 days (Table 5). This effect was not observed following short-term feeding. The influence of dietary treatment on blood Pi concentration on days 28 and 56 is shown in Fig. 2. At both sampling times, the fish fed the MNR control diet and the P-Min diet had the highest concentrations of plasma Pi. Fish receiving N-0 and E-3000 had the lowest concentration of Pi , with the N-3000 group having intermediate levels of plasma Pi . The ash and P content of a variety of tissues, including whole carcass (Table 6), scales (Table 7), skin (scales and skin; Table 8) and defatted vertebrae (Table 9), were evaluated as response criteria for P availability in different diets. In general, Table 3 Influence of dietary treatments on apparent digestibility coefficients (ADC) of nutrients fed to rainbow trout. Item

Dry matter Energy Protein Ash Phosphorus Calcium Magnesium Iron Zinc Manganese Copper

Treatmenta N-0b

N-3000

E-3000

P-min

MNR

0.758z 0.833z 0.911y 0.161z 0.363z −0.743z 0.528z 0.358y 0.738xy 0.384z 0.735

0.795y 0.862y 0.927x 0.277y 0.612x −0.263x 0.772w 0.500x 0.835w 0.610x 0.822

0.780y 0.831z 0.902z 0.240y 0.553y −0.155w 0.732x 0.272z 0.631z 0.332z 0.775

0.759z 0.851y 0.916y 0.306x 0.626x −0.650y 0.624y 0.503x 0.788x 0.582x 0.817

0.822x 0.884x 0.917y 0.397w 0.715w 0.364v 0.763w 0.274z 0.700y 0.508y 0.762

Pooled SEM

P-value

0.006 0.004 0.002 0.015 0.012 0.035 0.018 0.022 0.016 0.023 0.010

<0.001 <0.001 0.005 <0.001 <0.001 <0.001 <0.001 <0.001 0.004 <0.001 0.190

a Treatment groups are as follows: N-0: no supplemental phytase; N-3000: 3000 FTU unencapsulated phytase; E-3000: 3000 FTU encapsulated phytase; P-Min: NaH2 PO4 to NRC recommendations; MNR: fish meal-based control diet. b Within individual nutrients, mean values having different superscripts letters are significantly different (P<0.05). Least square means; n = 3.

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100

90

Fish Mass (g×fish-1)

80

70

60

50

40

30

0

2

4

6

8

Period (week) N-0

N-3000

E-3000

P-Min

MNR

Fig. 1. Average fish mass (mean ± SEM) over the 56 day feeding experiment. Each point represents the mean of n = 3 tanks/treatment.

these tissues responded in a similar manner to dietary treatment, with fish fed the N-0 diet showing a lower amount of ash and P, and those fed the P-Min and MNR control diets showing the highest amount of ash and P. The N-3000 and E-3000 diets resulted in intermediate levels of ash and P, with the levels in fish fed the encapsulated form of phytase (E-3000) being lower than those in fish fed the free form (N-3000). The exceptions to these observations were the percentages ash and P in skin, where treatment-related differences were of lower magnitude, or statistical differences were masked by increased variation. The effects of dietary treatments on P and N retention are shown in Figs. 3 and 4, respectively. Although the fish meal-based diet led to high P and N retention throughout the study, consistent treatment-related differences were not clear for the first 28 days of the experiment for either variable. During the final four weeks of the experiment, however, the addition of microbial phytase improved both P (P<0.001) and N retention (P<0.05). In this study, all forms of P waste expressed as a function of live fish mass were lowest for the plant protein-based diet supplemented with unencapsulated phytase (Table 10).

Table 4 Thermal growth coefficient (TGC) and feed efficiency (FE) of rainbow trout fed different dietary regimes. Treatmenta

Item

b

TGCc Day

FEd Day

Pooled SEM

N-0

N-3000

E-3000

P-min

MNR

0–14 14–28 28–42 42–56

0.082y 0.134y 0.112 0.110z

0.063y 0.137y 0.131 0.162x

0.076y 0.120y 0.125 0.148x

0.088y 0.130y 0.118 0.133y

0.141x 0.171x 0.141 0.182w

0.007 0.009 0.003 0.007

0–14 14–28 28–42 42–56

0.81y 0.93 0.84 0.75

0.74y 0.88 0.96 1.25

0.72y 0.87 0.86 0.95

0.79y 0.92 0.90 0.88

1.25x 1.07 0.88 1.19

0.04 0.05 0.05 0.09

P-value

0.001 0.050 0.142 0.008 <0.001 0.130 0.807 0.133

a Treatment groups are as follows: N-0: no supplemental phytase; N-3000: 3000 FTU unencapsulated phytase/kg (as-fed basis); E-3000: 3000 FTU encapsulated phytase/kg (as-fed basis); P-Min: NaH2 PO4 to NRC recommendations; MNR control fishmeal-based control diet. b Within each time period, mean values having different superscripts letters are significantly different (P<0.05). n = 3. c Thermal growth coefficient = (final body mass0.3333 − initial body mass0.3333 )/(Temperature × days). d Feed efficiency = gain in body mass (g)/feed consumption (g; as-fed).

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Table 5 Proteolytic enzyme activity of rainbow trout on days 0, 4 and 28 of feeding experimental diets. Treatmenta

Item

N-0 Trypsin Dayb

0 4 28

Chymotrypsinc Day 0 4 28

Pooled SEM N-3000

1.40 8.97y

1.46 8.99y

0.036 0.340y

0.030 0.406y

E-3000 2.45 ± 0.45 1.32 10.6xy 0.12 ± 0.018 0.028 0.361y

P-min

1.25 12.0x

0.030 0.574x

P-value

MNR

1.07 6.35z

0.14 1.0

0.434 0.007

0.038 0.223z

0.005 0.04

0.726 <0.001

Carboxypeptidase A 0 Day 4 28

40.3 266.6xy

34.9 234.2y

59.4 ± 8.3 23.4 246.9xy

29.5 287.1x

27.3 165.6z

4.9 19.1

0.318 0.001

Carboxypeptidase B Day 0 4 28

55.6 329.6y

59.9 353.0y

100.5 ± 15.2 45.8 368.6xy

54.6 411.7x

56.9 241.5z

5.4 34.2

0.451 0.021

a Treatment groups are as follows: N-0: no supplemental phytase; N-3000: 3000 FTU unencapsulated phytase/kg (as-fed basis); E-3000: 3000 FTU encapsulated phytase/kg (as-fed basis); P-Min: NaH2 PO4 to NRC recommendations; MNR control fishmeal-based control diet. b Within day 4 and 28 sampling periods, mean values having different superscripts letters are significantly different (P<0.05). n = 3. c Enzyme activity expressed as ␮M substrate hydrolyzed/min/mg soluble protein.

4. Discussion The MNR control diet is comprised mainly of animal-based protein sources, which are highly digestible and palatable, thus promoting feed intake and rapid growth. The sole plant protein included in this diet was maize gluten, which has been reported to be highly digestible (Sugiura, 1998), palatable and supports high growth rates in rainbow trout when used to replace a significant proportion of dietary fishmeal (Moyano et al., 1992). During the first two weeks of the study, fish fed the plant protein-based diets, irrespective of treatment, experienced an initial period of reduced feed intake and growth. Although not directly measured, there was increased feed wastage observed in these tanks, which is reflected in the reduced FE during this period. This initial adaptation period has been previously reported in a number of salmonid species

Fig. 2. Plasma inorganic phosphorus (Pi) concentration on days 28 and 56 of the feeding experiment. Plasma Pi concentrations were 112.9 ± 3.5 (mean ± SEM) at the start of the experiment.

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Table 6 The effect of diet on whole carcass dry matter, ash, phosphorus and protein on days 0, 28 and 56 of the experiment. Treatmenta

Item

Dry matter (g/kg) 0 Dayb 28 56

N-0

N-3000

E-3000

P-min

MNR

298.2x 302.5y

287.6x 291.9y

265.5 ± 0.18 281.5y 304.2y

283.6y 313.0x

280.4y 305.5y

20.5 ± 0.02 17.2z 18.6xz

19.7x 21.8x

Ash (g/kg, wet mass) Day 0 28 56

17.2z 17.6z

Phosphorus (g/kg, wet mass) 0 Day 28 56 Protein (g/kg, wet mass) Day 0 28 56

Pooled SEM

2.88y 2.91z

152x 154xy

18.8xy 19.1yz

3.10y 3.10yz

149xy 152y

3.33 ± 0.004 2.90y 3.12yz 142.0 ± 0.1 146yz 158x

3.37x 3.73x

145yz 160x

18.1xy 19.9y

3.07y 3.34y

143z 156xy

P-value

0.46 0.42

0.050 0.042

0.05 0.06

0.001 <0.001

0.011 0.012

0.001 <0.001

0.2 0.2

0.011 0.050

a Treatment groups are as follows: N-0: no supplemental phytase; N-3000: 3000 FTU unencapsulated phytase/kg (as-fed basis); E-3000: 3000 FTU encapsulated phytase/kg (as-fed basis); P-Min: NaH2 PO4 to NRC recommendations; MNR control fishmeal-based control diet. b Within day 28 and 56 sampling periods, mean values having different superscripts letters are significantly different (P<0.05). n = 3.

fed plant-based protein diets (Refstie et al., 1997, 1998) and is likely due to specific antinutritional factors in the plant protein sources employed (Tacon, 1997). The increased feed wastage during the initial stages of the experiment likely confounded some measured effects including values for N and P retention for the initial 28 days. Following this initial adaptation period, however, growth performance of fish fed plant protein-based diets, particularly those supplemented with unencapsulated phytase, approached that of fish receiving the fishmeal control diet. Rainbow trout fed plant protein-based diet supplemented with microbial phytase resulted in increased P ADC and P bioavailability, leading to increased growth, feed efficiency, tissue P levels and retention. To a lesser degree, phytase supplementation also increased the ADC of GE, as well as all other minerals in the current study. Although not measured, the increase in specific mineral ADC’s would likely be reflected as increased tissue mineral levels (Storebakken et al., 1998), particularly those that are not included in the mineral premix. Supplementation with microbial phytase has been reported to increase CP ADC in swine (Kemme et al., 1999) and poultry (Ravindran et al., 1999; Selle and Ravindran, 2007), resulting in an overall increase in N retention (Yi et al., 1996). Similar results have been observed in Atlantic salmon fed a soybean concentrate pre-treated with phytase (Storebakken et al., 1998; Sajjadi and Carter, 2004). In the current study, unencapsulated phytase induced a significant increase in CP digestibility resulting in an overall augmentation in N retention during the final 28 days of the study. Encapsulated phytase was without effect on these two parameters. Phytase is thought to reduce phytate-protein interactions, either with dietary proteins and/or proteolytic enzymes (Caldwell, 1992; Kies et al., 2006). In the current study, there were no differences in the activity of four Table 7 The effect of diet on ash and phosphorus of scales on days 0, 28, and 56 of the experiment. Item

Treatmenta

Pooled SEM

P-value

N-0

N-3000

E-3000

P-min

MNR

Dry matter (g/kg) 0 Dayb 28 56

196x 196x

187xy 187xy

147 ± 0.4 188xy 188xy

172y 172y

193x 193x

0.6 0.7

0.041 0.080

Ash (g/kg, DM)c Day 0 28 56

304z 279z

339xy 316y

376 ± 0.49 327y 306y

361w 369x

348wx 353x

0.5 0.6

<0.001 <0.001

0.16 0.21

<0.001 <0.001

Phosphorus (g/kg, DM) 0 Day 28 56

65.1z 55.5z

71.7xy 68.1y

8.05 ± 0.1 68.6z 60.8z

78.2wx 79.4w

75.7x 73.9x

a Treatment groups are as follows: N-0: no supplemental phytase; N-3000: 3000 FTU unencapsulated phytase/kg (as-fed basis); E-3000: 3000 FTU encapsulated phytase/kg (as-fed basis); P-Min: NaH2 PO4 to NRC recommendations; MNR control fishmeal-based control diet. b Within day 28 and 56 sampling periods, mean values having different superscripts letters are significantly different (P<0.05). n = 3. c Expressed as % DM.

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Table 8 The effect of diet on dry matter, ash and phosphorus of fish skin on days 0, 28 and 56 of the experiment. Treatmenta

Item

Pooled SEM

P-value

N-0

N-3000

E-3000

P-min

MNR

Dry matter (g/kg) 0 Dayb 28 56

324x 323x

408y 353xy

313 ± 0.9 328x 343xy

350xy 299x

344x 343xy

2.1 1.3

Ash (g/kg, DM) Day 0 28 56

103y 88.6z

121x 89.0z

130.5 ± 0.42 108y 89.8z

124x 117.3x

123x 100.7y

0.40 0.34

0.001 <0.001

68.6 68.6

86.1 75.0

62.5 ± 0.34 69.2 73.0

73.8 65.5

72.8 75.0

0.46 0.31

0.062 0.110

Phosphorus (g/kg, DM) 0 Day 28 56

0.046 0.039

a Treatment groups are as follows: N-0: no supplemental phytase; N-3000: 3000 FTU unencapsulated phytase/kg (as-fed basis); E-3000: 3000 FTU encapsulated phytase/kg (as-fed basis); P-Min: NaH2 PO4 to NRC recommendations; MNR control fishmeal-based control diet. b Within day 28 and 56 sampling periods, mean values having different superscripts letters are significantly different (P<0.05). n = 3.

Table 9 The effect of diet on dry matter, ash and phosphorus of fish vertebrae on days 0, 28 and 56 of the experiment. Treatmenta

Item

Dry matter (g/kg) 0 Dayb 28 56 Ash (g/kg, DM) Day

0 28 56

Phosphorus (g/kg, DM) Day 0 28 56

Pooled SEM

P-value

N-0

N-3000

E-3000

P-min

MNR

723y 592

821x 613

561 ± 1.9 650z 594

622z 634

774xy 539

2.0 1.4

<0.001 0.211

479y 397z

512xy 466x

531x 531v

517xy 499w

0.42 0.31

<0.001 <0.001

108x 103.7w

106x 97.8x

0.44 0.33

<0.001 <0.001

96.2z 75.5z

102y 93.5y

528.0 ± 0.8 507z 418y 108 ± 0.18 102y 80.9y

a Treatment groups are as follows: N-0: no supplemental phytase; N-3000: 3000 FTU unencapsulated phytase/kg (as-fed basis); E-3000: 3000 FTU encapsulated phytase/kg (as-fed basis); P-Min: NaH2 PO4 to NRC recommendations; MNR control fishmeal-based control diet. b Within day 28 and 56 sampling periods, mean values having different superscripts letters are significantly different (P<0.05). n = 3.

proteolytic enzymes as a result of phytase treatment, suggesting that interactions between phytate and dietary proteins, rather than between phytate and proteolytic enzymes. Interestingly, fish supplemented with mineral P tended to have higher levels of enzyme activity, although the reason for this is unknown. Feeding the plant protein-based diet for 28 days induced significantly higher levels of enzyme activity versus a fishmeal control. The increased proteolytic enzyme activity has been Table 10 The effect of diet on P intake and retention as a function of live weight gain (g/kg LWG) and calculated excretion of different forms of P during the final 28 days of the feeding study. Item

Treatmenta

Pooled SEM

P-value

6.26y 3.34y

0.49 0.09

0.002 <0.001

2.92y 0.83yz 2.28x

0.38 0.22 0.21

0.005 0.003 0.001

N-0

N-3000

E-3000

P-Min

MNR

P intake P retained

6.7y 2.92y

4.6z 3.03y

5.80y 3.12y

9.19x 3.74x

P waste (g/kg)b Total Solidc Dissolvedd

3.78y 2.41x 1.37y

1.57z 0.61z 0.94z

2.68y 1.20y 1.48y

5.45x 2.04x 3.41w

a Treatment groups are as follows: N-0: no supplemental phytase; N-3000: 3000 FTU unencapsulated phytase/kg (as-fed basis); E-3000: 3000 FTU encapsulated phytase/kg (as-fed basis); P-Min: NaH2 PO4 to NRC recommendations; MNR control fishmeal-based control diet. b Within individual columns, mean values having different superscripts letters are significantly different (P<0.05). n = 3. c Calculated using P ADC values from Table 3. d Calculated by difference Total P Waste − Solid P Waste.

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Fig. 3. Phosphorus retention (%; mean ± SEM) from days 1–28 and days 29–56 in fish fed different dietary regimes.

previously reported trout fed low levels of soybean meal (Haard et al., 1996) and is likely a result of compensatory pancreatic enzyme secretion, related to the presence of dietary trypsin inhibitor (Dabrowski et al., 1989). The enzyme activity in diets containing encapsulated phytase was significantly lower versus those supplemented with unencapsulated phytase, even prior to the steam pelleting process. This might suggest that a significant amount of phytase activity is lost during the encapsulation process; however, we have previously reported that the encapsulation process is

Fig. 4. Nitrogen retention (%; mean ± SEM) from days 1–28 and days 29–56 in fish fed different dietary regimes.

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efficient, with over 85% of the phytase activity remaining following encapsulation and microcapsule drying (Vandenberg and de la Noüe, 2001a,b). This observation may also be due to incomplete extractions of phytase from microcapsules during the phytase activity assay. Microcapsules remain stable in an acidic pH environment, particularly in the presence of calcium (Vandenberg and de la Noüe, 2001a,b), it is likely that phytase remained trapped within the microcapsules in the gastric region of the fish intestine and thus was not fully extracted, leading to incomplete enzyme recovery. This does not preclude the encapsulated phytase from hydrolyzing phytate, although the phytate would have to diffuse into the microcapsules in order to undergo hydrolysis. This would seem to be the case, given the increased P ADC in the plant protein-based diets containing encapsulated phytase versus unencapsulated phytase (Table 3). However, the efficacy of the encapsulated enzyme is likely reduced as a result of its limited distribution throughout the feed. Production of encapsulated phytase using an approach that produces smaller microcapsules or material with enhanced release, particularly in the gastric regions, may improve efficiency of encapsulated phytase. Ideally, a low pollution diet would provide optimal growth performance with limited P excretion in both the solid and dissolved fractions. Of particular interest is the reduction of dissolved forms of P, given that these are not easily removed from the effluent, whereas solid forms can be either settled or removed using mechanical means. All forms of waste P were lowest for the plant protein-based diet supplemented with unencapsulated phytase (N-3000), with extremely low levels of dissolved waste excreted from fish receiving this diet. Similar results have been observed in Japanese flounder fed a soybean meal supplemented with phytase (Sarker et al., 2006). The adequate growth rate of fish fed the unencapsulated phytasesupplemented plant protein diet along with the low levels of P excretion indicates that fish might meet their requirement at that level. The calculated level of P intake using the P digestibility values was 0.17 g available P/MJ digestible energy, which is below the estimated P requirement of 0.25 g available P/MJ digestible energy for larger trout (>50 g) (Rodehutscord, 1996). This group observed that the P requirement for maximal growth rate was lower than that for maximal tissue P levels, which corresponds with the observed reduction of tissue P levels versus mineral-supplemented controls in the present study, while maintaining adequate growth rates. Increased levels of phytase inclusion would likely further enhance the P availability of the plant protein-based diet, perhaps resulting in increased growth tissue mineralization rates, but likely at the expense of increased dissolved P excretion. 5. Conclusion The results of this study suggest that microbial phytase can effectively increase the apparent digestibility and bioavailability of a range of nutrients from plant protein-based diets for rainbow trout, resulting in increased growth performance and tissue mineralization. However, encapsulation of microbial phytase tended to diminish its ability to liberate P likely due to reduced interaction between the enzyme and dietary phytate-P. Further research is warranted with a new encapsulation and clarifies the issue regarding phytate-protein interactions, either with dietary proteins and/or proteolytic enzymes for this species. Acknowledgements This work was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC Strategic Projects Program) and BASF Canada Inc. Grant W. Vandenberg was the recipient of an NSERC Postgraduate Scholarship. The authors are grateful to Martin Feed Mills, BASF Canada, Finnfeeds International and Degussa-Hüls Canada Inc., who generously supplied dietary ingredients, and to Drs. R. Hardy and J. Babbit, who helped to source the low-P fishmeal. We thank Richard Prince and Pierre Castonguay for their help with the feed production, Jean Bricault, Francine Giguère and André Roy for their skilled analytical assistance, and Julie Mercier and the staff of the LARSA for their assistance with the feeding experiments. We thank Dr. D. Bureau, Fish Nutrition Laboratory, University of Guelph, for his helpful comments throughout the course of this study. References Adelizi, P.D., Rosati, R.R., Warner, K., Wu, Y.V., Muench, T.R., White, M.R., Brown, P.B., 1998. 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