ca4_the use of enzyme supplements in shrimp diets

8/3/2019 Ca4_the Use of Enzyme Supplements in Shrimp Diets

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THE USE OF ENZYME SUPPLEMENTS IN SHRIMP DIETS.

D. Allen Davis , W. Lyle Johnston and Connie R Arnold

Corresponding author

The University of Texas at AustinMarine Science Institute

Fisheries and Mariculture Laboratory750 Channel View Drive

Port Aransas, Texas, USA 78373-5015

Key Words: Penaeus vallnamei, exogenous enzymes, phytase

Symposium publication IV International Symp osium on Aquatic Nutrition, November 18-18, 1998.La Paz, B.C.S. , Mexico.

This document is not to be reproduced or di stributed witilout written conse nt of the corresponding author.


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2Abstract

The longlerm sustainability ofcommercial shrimp production is dependant on both economicand environmental constraints. Since the feed is one of he major costs associated with shrimp farmingand the initial source of pollutants, there are considerable pressures to reduce feed costs and minimizethe polluting effects of the feed. By adapting appropriate feed technologies and feeding strategies, thenutrient loading associated with supplemental feed systems can be minimized . A variety of feedsupplements, which have been demonstrated to increase animal production, could be applied to shrimpfeeds. Hence, the objectives of this research were to evaluate the use of a feed grade protease (FGP)in practical diets for shrimp feeds and preliminarily evaluation of the use of phytase to free phytinbound phosphorus .

Graded levels (0, 0 .2, 0.4 g/ 100g diet) of a feed grade protease were incorporated into acommercial shrimp feed and apparent protein digestibility values determined. Apparent proteindigestibility values were significantly improved from 65 .3% to 74.3% by the supplementation of theFGP at 0.4 g/IOOg diet, an increase of9% in APD over the basal diet. If this protein is available forassimilation and growth, the protein content of production diets could be reduced or less digestibleingredients could be incorporated into the diets without reductions in protein availability . To evaluatethe effects on growth and feed utilization an 8-week growth trial was conducted. Graded levels of aFGP were supplemented to practical diets containing either 15% or 30% protein . The supplementationof0.4 g FGPII 00 g diet resulted in a decrease in growth and feed utilization of the shrimp maintainedon this diet as compared to those offered the basal diet without enzyme supplementation. These resultswould indicate that high levels of this enzyme may negatively impact the performance of the shrimpand that lower levels had no significant influence on growth or feed utilization.

In the second component of this research a practical basal diet, previou sly demonstrated to bedeficient in available phosphorus, was used to evaluate the use of phytase enzyme to free phytatebound phosphorus. Although the basal diet was deficient in available phosphorus, phytasesupplementation to the diet resulted in a positive shift in weight gain and estimated feed conversionefficiency. This indicates that phytase may release phytate bound phosphorus warranting furtherinvestigation in practical shrimp feeds .

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3Introduction

Over the. past decade, Aquaculture has been the world's fastest growing food productionsystem, with total fish and crustacean production increas ing from 4.67 million metric tons (mmt) in1984 to 15 .80 mmt in 1995 (Tacon, A.G. 1. , 1997). Although, many species are grown utilizingextensive production systems, the industry is shifting towards intensive supplemental feed systemsand/or production of high value carnivorous species. Associated with this shift is a reliance oncompounded feeds, increasing the potential for environmental impact of the production system.

Nitrogen, phosphorus, and organic wastes from feeds are the major factors contributing toenvironmental pollution from aquaculture (Rijn and Shilo, 1989; Folke and Kautsky, 1991; Boyd andMusig, 1992) . As the initial source, manipulation of the feed is the mos t direct and effective means toreduce pollutants (Cho el aI, 199 1). Lo w pollution feeds must be designed to increase feed utilizationby the culture species and minimize nutrient losses to the environment. This objective can be ac hievedby increasing the digestibility of nutrients and reducing fecal waste.

Increasing digestibility of the feed will not only reduce environmental pollution, but w ill alsolower the total cost of production by lowering the nutrient expend iture per unit of production. Inaddition to selection of highly digest ible feed ingredients, the utilization of enzyme supplementsdesigned to enhance the digest ibility of feedstuffs has been successfully applied in terrestrial (Wenk,1992) and aquatic fe eds. Examples of the use of enzymes in aquatic animal feeds include: feedingcommon carp a soybean residue which has been pre-digested by Papain, a protease isolated from thelatex of Carica papaya (papaya) (Wong el ai, 1996); the inclusion of a exogenous multi-enzymemi xture supplemented to a canola based P. mOllodoll feed (Buchanan e/ a I, 1997); the supplementingofpractical striped bass Morolle saxa/ilis diets with a commercial phytase (Hughes and Soares, 1998);modification of digestive enzyme ratio (amylase/protease) in P. japollicus through di etary enzymesupplementat ion (Maugle el aI, 1983a); and the pretreat ment of soybean mea l with phytase prior touse in rainbow trout, Ollcorhynchus lIIykiss, diets (Cain and Garling, 1995). These studies indicate thatthe utilization of enzyme supplements in the feed or feed ingredients has the potential to improvedi etary nutrient utilization, activate endogenou s zymogen(s) and provide enzymes that are notnormally present in the digestive system of the culture animal. Consequently, the objectives of thisresearch were 1) to evaluate the effects of a feed grade protease (FGP) on apparent protein

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4digestibility of a commercial shrimp feed 2) to evaluate growth and feed utilization of uvenile shrimpoffered a practical diet formulated with varying levels of FGP 3) to evaluate growth and feedutilization of uvenile shrimp offered a phosphorus deficient diet supplemented with phytase.

Materials and MethodsDigestibility of a commer'cial diet supplemented with a general protease.

Apparent protein and dry matter digestibility values were determined based on the indirectmethodology utilizing chromic oxide as an inert marker. The basal diet consisted of (as gi l 00 g dryweight) 96.) commercial mash formulated for Pacific white shrimp, Penaells vallllamei, and obtainedfrom Rangen Inc. (Buhl, ID); 2.5, menhaden fish oil; 1.0, chromic oxide; and 0.4, diatomaceous earth.The test diets were then produced by replacing the diatomaceous earth in the basal diet with theappropriate level (0, 0.2 or 0.4 gi l 00 g) ofENZECO@Bromelain FG . ENZECO@Bromelain FG isa feed grade protease (FGP), containing proteolytic enzymes found in various species of theBromeliceae family. This FGP was selected because the protease remains entrapped within thepineapple stem fibers and hence should be less susceptible to leaching into the water. Dietaryingredients were homogenized in a food mixer (Hobart Corp., Troy, OH) for thirty minutes. Aftermixing, boiling water was blended into the mash until an appropriate consistency for pelletizing wasreached . Each diet was pelletized utilizing a meat grinder with a 3-mm die, then placed in a heatedoven 45 C) for 2 h followed by overnight drying (ambient air temperature) to reach a final moisturecontent of8-1 0%. Feeds were crumbled and sieved to the proper size for the shrimp, then stored underrefrigeration until needed. Protein content of he diet was confirmed to be 39.9% by Kjeldahl analysis.Digestibility trials followed similar methods as those reported by Davis and Arnold (1993) .

P. valll1amei (mean weight 15.4 g) were maintained in a semi-closed, recirculating systemconsisting of square tanks (75 L), circulation pump, sand filter, and biological filter. Environmentalparameters were maintained at: temperature, 26 C; salinity, 30 ppt; di ssolved oxygen, 6.2 ppm . Eachdiet was offered to four replicate tanks of shrimp (8 shrimp per tank) for a 17 -day conditioning periodfollowed by a three day collection period. During the collection period the following procedure wasfollowed. In the morning the tanks were siphoned and the feeding cycle initiated. Shrimp were allowedto feed for 35-45 minutes after which the feces was collected by hand siphoning into a collection sieve

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5(4811111) , followed by a rinse with distilled water. After the feces were collected, uneaten food wasremoved from tbe tank and the feeding process repeated . During the collection period, the firstcollection in the morning was discarded, with the following 4-5 collections/day pooled by tank overthe three-day period. Extracted fecal samples were oven dried (100 C) and stored at -15 C untilanalysis.

Total ammonia-nitrogen (TAN), nitrite-nitrogen, and pH were monitored bi-weekly byspectrophotometric methods (Spotte, 1979) and pH meter, and were maintained within acceptablelimits. Biochemical analyses of the test diets and each fecal sample were conducted in triplicate.Chromic ox.ide was measured by the wet acid digestion method (McGinns and Kasting, 1964) and totalnitrogen determined by micro-Kjeldahl method (Ma and Zuazago, 1942). One-way analysisofvariancewas utilized to determine significance (P < 0.05) of he treatment effects and Student-Newman-Keul'smultiple range test (Steel and Torrie, 1980) to determine differences among treatment means.Statistical analyses were performed using SAS System for Windows"!"M (v 6.11, SAS Institute Inc.,Cary, N .C.).

Evaluation of protease on growth and feed utilization.Following an eight day acclimation period, shrimp were hand graded to a uniform size (mean

initial weight 0.25 g/shrimp) and stocked in four replicate tanks per dietary treatment at a density of8 shrimp per tank. An eight week growth trial was then conducted to evaluate the effect ofENZECOBromelainFG on growth and apparent net protein retention for P. vallllal1lei .Two proteinlevels (15% and 30 % ) and four levels of the enzyme supplement ( 0, 0 I, 0.2, and 0.4 g FGP/I OOgfeed) were evaluated using a 2 x 4 factorial design. The test diets (Table I) were produced byreplacing non-nutritive filler in the basal diet with 0, 0.1, 0.2, and 0.4 g ENZECO BromelainFG/IOOg dry weight offeed. Prior to use, practical ingredients were ground with a laboratory hammermill using a size 24 screen (0 .609 mm diameter hole) . Dietary ingredients were homogenized in a foodmixer (Hobart Corp., Troy, OH) for thirty minutes. After mixing, boiling water was blended into themash until an appropriate consistency for pelletizing was reached. Each diet was pelletized utilizinga meat grinder with a 3-mm die and then placed in a heated oven 45C) for 2 h followed byovernight drying (ambient air temperature) to reach a final moisture content of 8-1 0% . Feeds werecrumbled and sieved to the proper size for the shrimp, then stored under refrigeration until needed.

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6The feeding trial was carried out in a semi-closed recirculating system consisting of32 square,

75 I tanks, circulation pump, sand filter, biological filter, and supplemental aeration . System make-upwater was exchanged with prefiltered 50I1m) ozone treated seawater at a rate of 4 IIhr. Salinity,temperature, and dissolved oxygen were measured daily. Water quality parameters (TAN, nitritenitrogen, and pH) were measured biweekly using spectrophotometric methods (Spotle, 1979) and apH meter. Photoperiod was controlled at 12 h light and 12 h dark. Water quality parameters measureddaily were as follows (means standard deviation): temperature, 27.3 1.2 C; dissolved oxygen,S .7 0.4 mglL; salinity, 30 2 ppt. TAN, nitrite-nitrogen, and pH levels were determined to be 0.028 0.034 ppm, 0.006 0.003 ppm, and 7.9 0.1, respectively.

At the end of the experiment, shrimp were weighed to obtain a final mean weight. A randomsample of three shrimp from each tank were collected, homogenized, and frozen for subsequentproximate analyses. All biochemical analyses were conducted in triplicate. Representative portions ofsamples were dried to a constant weight in an oven maintained at 90 C. Protein content wasdetermined by the micro-Kjeldahl method (Ma and Zuazago, 1942). Estimated feed efficiency (EFE)values were calculated for each treatment as the ratio of weight gain per unit of dry weight of feedoffered (gain!dry feed xl 00) . Protein conversion efficiency (PCE) was calculated as the grams ofprotein gain per grams of protein fed (protein gain! protein offered x 100). The data was analyzed bytwo-way ANaYA to determine significant differences due to the main affects and their interactions.The Student-Neuman Keul ' s multiple-range test (Steel and Torrie, 1980) was used to examinesignificant differences (P < 0.05) between treatments means.

Effects of phytase supplementation on the growth of shrimpA second feeding trial was designed as a preliminary evaluation of the capacity of phytase to

free phytate bound phosphorus. A basal diet , that has been demonstrated to be deficient in phosphorus(Davis and Arnold, 1998), and two replete diets utilizing feed grade phosphorus sources wereprepared utilizing the previously described procedures. The fourth diet consisted of the basal dietsupplemented with 0.25g1100g Natuphos (600 FTU/g, BASF Corporation, New Jersey) replacingfiller (Table 2) . The activity of the basal diet and the diet supplemented with Natuphos wereevaluated for phytase activity (FTU, defined as the quantity of enzyme which liberates I micro-mole

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7of inorganic phosphorus minute-' from 0.0051 mol L-' sodium phytate at pH 5.5 and 37 C) . Analyseswere conducted_by BASF Corporation (Wyandotte, Michigan) and indicated that the basal dietcontained 281 FTu/kg and the supplemented diet contained 1,919 FTU/kg .

Experimental procedures were as previously described, except that prior to beginning theexperiment the shrimp were offered the basal diet over a 7 -day pre-conditioning period. The test dietswere offered to four replicate groups of shrimp (8 shrimp per tank; mean initial weight ..Standarddeviation, 0.30 0.017g). During the 56-day growth trial the shrimp received a total of9 .8g dry feedper shrimp. Water temperature, dissolved oxygen and salinity were maintained at 28 .7 1.0 C, 5.9 0.5 ppm and 31 .6 2.0 ppt , respectively. Total ammonia-nitrogen, nitrite-nitrogen and pH weremeasured twice weekly and maintained at 0.04 0.02 ppm, 0.08 0.11 ppm and 7.8 0.22,respectively. At the conclusion of the growth trial, weight gain, survival, and EFE (based on offeredfeed) were determined. One-way analysis ofvariance was utilized to determine significance (P < 0.05)of the treatment effects and Dunnett's T test to determine significant differences from the control orbasal diet (Steel and Torrie, 1980).

Results and DiscussionThe digestibility trial was designed to initially evaluate the effects of a feed grade protease(FGP) on digestibility coefficients for P. vallnamei. Results from this trial are summarized in Table 3.

Although there were no significant differences in ADMD values, there was a general increase inADMD values corresponding with the supplementation of the FGP . This response is , in part, due toreplacement of inert filler with the FGP . Significant differences in APD values corresponded withincreases in the dietary supplement. For shrimp offered the basal diet supplemented with 0.4 g FGPthere was an increase of 9 % in APD observed over the basal diet.

These results would indicate that proteolytic enzymes may be limiting protein digestibility inshrimp maintained on a commercial shrimp feed and that increases in APD values can be obtainedthrough the use of dietary supplements. Maugle et aI, (1983a), demonstrated increased protease andzymogen activity in the hepatopancreas of shrimp, P. japollicllS, fed diets supplemented withmicroencapsulated bovine trypsin . Maugle et ai, (1983b), reported an increase in carbohydratedigestion by the addition of amylase to shrimp diets. If the supplementation of exogenous enzymes



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8results in increased enzyme activity and an increase in nutrient digestion, a greater percentage of thenutrients may be available for assimilation and growth. Hence, feed cost could be reduced byincorporating less expensive low-digestible ingredients and/or reducing the protein content of he diet.In the formulation of low pollution diets, reduced protein levels in the feed and increases in APDvalues could produce significant reductions in nitrogen entering the culture system. Although theseresults are encouraging, any adverse effects of he enzyme supplement on growth or nutrient retentionof the shrimp should be assessed.

To determine the effects ofFGP supplementation in a practical shrimp diet, two protein levels(15 and 30%) and four levels ofFGP (0.0, 0.1, 0.2, and 0.4 gil OOg diet) were evaluated. Results ofthis growth trial are summarized in Table 4. Two-way analysis of variance indicated that there weresignificant differences due to the main effects (protein and FGP level), but none due to theirinteractions for final weight, EFE, and PCE of the shrimp. Consequently the data was separated byprotein level for final analysis. There were no significant differences due to the main effects or theirinteractions for the survival data .

For shrimp fed the diet containing 15% protein, final weights ranged from 3.8 g to 3. 1 g forshrimp offered the basal diet and the diet containing 0.4 g FGPII 00 g , respectively . The remainingdiets produced intermediate weights and were not significantly different from the other diets. A similarresponse was observed for EFE and PCE values. With respect to the shrimp offered the 30% proteindiet, final weights ranged from 4.6 g to 3.9 g for shrimp offered the basal diet and the diet containing0.4 g FGP/ I00 g, respectively.Estimated feed efficiency values for shrimp maintained on the basal dietsupplemented with 0.4 g FGPII OOg diet was significantly lower than those observed for the basal dietand diets containing 0.1 and 0.2 g FGPII 00 g diet. Although PCE values were lowest for shrimpoffered the basal diet containing 0.4 g FGPII 00 g diet, there were no significant differences observedbetween dietary treatments.

The negative response at the highest level of supplementation would indicate an adverse effectof the enzyme on shrimp performance. The manufacturer recommended inclusion for ENZECOBromelain FG in shrimp diets is 0.2 g 11 OOg diet. Under the conditions of these experiments, thesupplementation of FGP at 0.1 or 0.2 gllOOg diet did no t result in a significant change in shrimpgrowth or feed utilization. At twice the recommended level (0.4 gFGP/ 100 g diet) fina l shrimp weight,

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9EFE, and PCE values were significantly lower than those observed for shrimp fed the basal diet. Thepoor response could be due to palatability changes in the diet or leeching of pre-digested nutrientsfrom the feed .

Buchanan et ai, (1997), demonstrated an improvement in weight gain by addition of a multienzyme supplement to prawn diets containing high level (64 gi l 00 g diet) of canola meal. Maugle etaI, (1983b) reported incremental increases in growth with increasing amylase supplements. In bothreports, increases in weight gain appear to be related to increased energy availability from thecarbohydrate component of the diet. The differences in response for the current growth trial could beattributed t species differences, dietary formulation, andlor enzyme function.

The second growth trial was designed to preliminarily evaluate the use of phytase to freephytate bound phosphorus. Phytate is a mineral salt of phytic acid which is found in a variety of feedingredients of plant origin. Phytate phosphorus is either unavailable to, or poorly utilized by shrimpand inhibits zinc availability (Davis et ai, 1993 , Civera and Guillaume, 1989). The supplementation ofphytase to the diet has been found to release phytate bound phosphorus in fish feeds (Ketola, 1994;Cain and Garling, 1995; Storebakken et aI, 1996; Jackson et aI, 1996; Hughes and Soares, 1998), buthas not been evaluated with shrimp.

In the present study a practical basal diet , which had been previously determined to be deficientin phosphorus (Davis and Arnold, 1998), was supplemented with either a commercial source ofphytase or replete levels of calcium phosphate originating from one of two feed grade phosphorussources. The results in terms of growth, survival, and EFE are presented in Table 5. Significantdifferences between dietary trea tments were observed for final biomass, percent weight gain, andEFCE values. Survival of shrimp offered the basal diet was poorest and survival of shrimp offered thebasal diet supplemented with phytase was intermediate; however, there were no significant differencesbetween dietary treatment detected. Weight gain for shrimp offered the basa l diet was poor whencompared to shrimp offered the other diets (530 vs 6.01 to 6.36 g). Percent weight gain wassignificantly improved by the addition of0.250 g PII OOg originating from Cefkaphos. Final biomass,a measurement that combines both survival and weight gain, indicated that both supplements ofphosphorus resulted in significant increases in biomass as compared to the basal diet. Similarly, EFEvalues for shrimp offered the two phosphorus supplements were significantly improved over shrimp

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10fed the basal diet, confirming that the basal diet was deficient in phosphorus.

In terms Gffinal biomass, percent weight gain, survival, and EFE values, the supplementationof phytate to the basal diet resulted in an improvement in these parameters. This response wasintermediate to those of the basal diet and replete diets. Although, this data is not statistically robust,the observed response would indicate that phytase may be releasing phytate phosphorus and/orpreventing it from binding with phosphorus originating from other ingredients. Consequently, furtherevaluations of the use of phytase in shrimp feeds is warranted.

SummaryUnder the reported conditions, the use of a FGP significantly improved APD of a commercial

shrimp feed containing 40% protein. However, the evaluation of the enzyme supplement in definedpractical shrimp diets, containing either 15% or 30% protein, did not result in improvements in growthor feed utilization. Based on these results, FGP does not appear to enhance growth and feed utilizationin a high quality shrimp feed . However, the use of phytase in a practical diet formulation that isdeficient in phosphorus appears to increase the availability of phosphorus in the diet, as demonstratedby increased growth and estimated feed conversion efficiencies.

Although, the results of the feeding trial with FGP did not result in improvements in feedutilization, the results with phytase were encouraging. Current literature indicates that, under certaincircumstances, enzyme supplements such as amylase, papain, trypsin, and multi-enzyme supplementshave produced positive results. In general, results appear best in diets containing low digestibleingredients and/or ingredients for which endogenous digestive enzymes are inadequate. Furtherresearch into the use of exogenous enzymes as feed supplements or as pretreatment of feedstuffs iswarranted.



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I ILiterature cited

Boyd, C.E. and Y. Musig. 1992. Shrimp pond effluents: observations of the nature of the problemon commercial farms. pp. 195-197. In J. Wyban, editors. Proceedings of the Special Sessionon Shrimp Farming. World Aquaculture Society. Baton Rouge, LA.

Buchanan, J., H.Z. Sarac, D. Poppi and RT. Cowan. 1997. Effects of enzyme addition to canola mealin prawn diets. Aquaculture 151: 29-35.

Cain, KD. and D.L. Garling. 1995. Pretreatment of soybean meal with phytase for salmonid diets toreduce phosphorus concentrations in hatchery effluents. The Progressive Fish-Culturist 57:114-119.

Cho, c.Y., J.D. Hynes, KR Wood and H.K Yoshida. 1991. Quantitation offish culture wastes bybiological (nutritional) and chemical (Iimnological) methods; the development ofhigh nutrientdense (HND) diets. Pages 37-50. In C.B. Cowey and C. Y. Cho, editors. Nutritional Strategiesand Aquaculture Waste. Proceedings of the First International Symposium on NutritionalStrategies in Management of Aquaculture Waste. University of Guelph, Guelph, Ontario,Canada, 1990; 275 p.

Civera, Rand J. Guillaume. 1989. Effect of sodium phytate on growth and tissue mineralization ofPenaeus japonicus and Penaeus vannamei juveniles. Aquaculture 77 :145-156.Davis, D.A. and C.R Arnold. 1998. Bioavailability offeed grade calcium phosphate incorporated into

practical diets for Penaeus vanl1amei. Aquaculture Nutrition 4:209-215.Davis, D .A. and C.R. Arnold. 1993. Evaluation offive carbohydrate sources for Penaells vannamei.

Aquaculture 114:285-292.Davis, D.A., A.L. Lawrence and D.M. Gatlin III. 1993. Dietary zinc requirement of Penaeus

val1namei and the effects ofphytic acid on zinc and phosphorus bioavailability. Journal of theWorld Aquaculture Society 24:40-47.

Folke, C. and N. Kautsky. 1991 . Ecological economic principles for aquaculture development. pp.207-222. In c.B. Cowey and c.Y. Cho (eds.). Nutritional Strategies and Aquaculture Waste.Proceedings of he First International Symposium on Nutritional Strategies in Management ofAquaculture Waste. University of Guelph, Guelph, Ontario, Canada, pp. 1990. 275



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12Hughes, K. Powers and J.H. Soares Jr. 1998. Efficacy ofphytase on phosphorus utilization in practical

diets fed to striped bass Marone saxalilis. Aquaculture Nutrition 4 :133-140.Jackson, L.S ., M.H . Li and E. H. Robinson. 1996. Use of microbial phytase in channel catfish

Jclalurlis p"nclailis diets to improve utilization ofphyate phosphorus. Journal of the WorldAquaculture Society 27 :309-313.

Ketola, H.G. 1994. Use ofenzymes in diets of rout to reduce environmental discharge ofphosphorus .World Aquaculture Society, Book ofAbstracts, 211 pp., World Aquaculture '94.

Ma , T.S. and G. Zuazago. 1942. Micro-Kjeldahl determination of nitrogen. A new indicator and animproved rapid method. Industrial and Engineering Chemistry 14 : 280-282 .

Maugle, P.D ., O. Deshimaru, T. Katayama, T. Nagatani and K. Simpson . 1983a. Effect ofmicroencapsulated amylase and bovine trypsin dietary supplements on growth and metabolismof shrimp. Bulletin of the Japanese Society of Scientific Fisheries 49 :1421-1427.

Maugle, P.D., O. Deshimaru, T. Katayama and K. Simpson. 1983b. The use of amylase supplementsin shrimp diets. Journal of the World Mariculture Society 14 :25-37.

McGinns, AJ . and R. Kasting. 1964. Colorimetric analysis of chromic oxide used to study foodutilization by phytophagous insects. Agric. Food Chem. 12 : 259-262.

Rijn, 1.V. and M. Shilo. 1989. Environmental factors in fish culture systems. pp. 163-178 . In M. Shiloand S. Sarig (eds.) . Fish Culture in Warm Water Systems: Problems and Trends . CRC Press,Boca Raton, Florida.

Spotte, S. 1979. Fish and invertebrate culture: water management in closed systems, 2nd ed. Wiley,New York, New York, USA.

Steel, R. G.D. and J.H. Torrie. 1980. Principles and procedures of statistics: a biometrics approach.McGraw-Hili, New York, New York, USA

Storebakken, T., K.D . Shearer and A.J. Roem. 1996. Availability of phosphorus, zinc and otherelements to Atlantic salmon fed fish meal, soy protein concentrate or phytase treated soyprotein concentrate based diets. Proceedings of the VII International Symposium on Nutritionand Feeding ofFish.

Tacon, A. G. J. 1997. Global trends in aquaculture and aquafeed production 1984-1995. InternationalAquafeed Directory and Buyers ' Guide J997/98. Turret T AI PLC, publisher. p 5-38.



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13

Wenk, c. 1992 . Enzymes in the nutrition of monogastric farm animals. pages 205-218. InBiotechnology in the feed industry. ProceedingsofAlitech's eighth annual symposium. AlltechTechnical Publications. Nicholasville, Kentucky. USA.

Wong, M.H. , L.Y. Tang, and F.S.L. Kwok. 1996. The use of enzyme-digested soybean residue forfeeding common carp. Biomedical and Environmental Sciences 9: 418-423.



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15Choline Chloride, 100.00; Inositol, 25.00; Vitamin A acetate (20,000 IU/g), 8.00; Vitamin D (400,000IU/g), 0.50; Vitamin E acetate (250 IU/g), 80.00; Menadione, 0.5; cellulose, 748.68.h Stay C, L-Ascorbyl-2-Polyphosphate, Hoffman-LaRouche, Inc, Nut ley, New Jersey, USA.I Feed grade calcium phosphate, BASF Corporation, Mount Olive, NJ, USA



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16Table 2. Composition of the test diets utilized to evaluate a dietary phytase supplement (gilOOg dryweight).

Basal Phytase 0.25% PAnchovy meal ' 20.0 20.0 20.0Soybean meal' 330 33 .0 330Fish solubles3 1.0 1.0 1.0Menhaden Fish oil' 3.8 3.8 3.8Wheat flour' 35.9 35.9 35.9Soy lecithin

6 1.3 1.3 1.3Trace Mineral premix' 0.5 0.5 0.5Vitamin premix' 2.0 2.0 2.0Vitamin C (25% active)9 0.1 0.1 0.1NaCI' 0.3 0.3 0.3Natuphos 10 0.25Cefkaphos 10 1.0Dynafos I IDiatomaceous earth' 2.1 1.85 1.0

Total phosphorus 0.98 1.35 1.23I Ralston Purina International, Checkerboard Square, St. Louis, Missouri, USA2 Solvent extracted, Producers Cooperative, Bryan, Texas, USA3 Zapata Haynie Corporation, Hammond, Louisiana, USA, Zapata Haynie Corporation, Reedville, Virginia, USA., United States Biochemical Corporation, Cleveland, Ohio, USA6 Aqualipid 95 , Central Soya Chemurgy Division, Fort Wayne, Indiana, USA

0.375% P20.03301.03.835.91.30.52.00.10.3

20250075

1.35

, Composition of trace-mineral premix (g kg' mix): Cobalt chloride, 0.04; Cupric sulfatepentahydrate, 5.50; Ferrous sulfate, 20.00; Magnesium sulfate heptahydrate, 283.98 ; Manganoussulfate monohydrate, 6.50; Potassium iodide, 0.67; Sodium selenite, 0 10; Zinc sulfate heptahydrate,

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17131.93; Alpha-cellulose, 551.28.

Composition ofvitamin premix (g kg- I mix) :Thiamin-HCI, 4.95; Riboflav in , 3.83; Pyridoxine-HCI,4.00; Ca-Pantothenate, 10.00; Nicotinic acid, 10.00; Biotin, 0.50; Folic acid, 4.00; Vitamin B12,0.05; Choline Chloride, 100.00; Inositol, 25.00; Vitamin A acetate (20,000 IV/g), 8.00; Vitamin D(400,000 IV/g), 0.50; Vitamin E acetate (250 IV/g), 80.00; Menadione, 0.5; cellulose, 748.68.

9 Stay C, L-Ascorbyl-2-Polyphosphate, Hoffman-La Roche, Inc ., Nutley, New Jersey, USA.10 Feed grade phosphorus source, primari ly monobasic calcium phosphate, BASF Corporation, MountOlive, New Jersey, USAI I Feed grade phosphorus source, primarily dibasic calcium phosphate, MallinckrodtFeed Ingredients,Mundelein, Illinois, USA



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Table 3. Apparent dry matter digestibility (ADMD) and protein digestibility (APD) values for P.val1l1amei offered a basal diet' and test dietsb containing varying levels of ENZECOBromelain FG .

ENZECO ADMD' APD'Bromelain FG

0 53 .2' 65.3x0.2 54 .2' 67.6'0.4 61.1 ' 74.3'PSEd 2 ..34 11.45

'Basal diet contained as gi l DOg dry weight: 96.1, commercial mash; 2.5, menhaden fish o il ; 1.0,chromic oxide and 0.4, diatomaceous earth.b Dietary treatments consisted of the basal diet modified to contain the supplement at the indicatedlevel, replacing diatomaceous earth.' Data represent the mean of four replicates. Mean values with the same letter are not significantlydifferent (P > 0.05) from each other.dPooled Standard Error

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19Table 4. Response ofP. VGIIIIGmei after 8 weeks of feeding diets containing various levels of proteinand feed grade protease (FGP) ' .

Protein FGP Final weight Survival EFE' PCE3(g/IOO diet) (g/ \00 diet) (g) (%) (%) (%)

IS a 3.8' 96.9' 35.8' 37.8'IS 0.1 3.4'" 100 ' 31. 9'" 36.0'"IS 0.2 3.5'" 96.9' 33 .0'" 34.7Y'IS 0.4 3.JY 96.9' 28 .7" 30.5"

PSE' 0.13 2.70 1.37 1.6730 a 4.6 ' 100' 44.3' 24.S'30 0.1 4.4' 96.9' 42.6' 24 .7'30 0.2 4.4' 100 ' 42.6' 24.6'30 0.4 3.9" 100' 37.0" 21.5'PSE 0.14 1.56 1.40 0.89

'Means of four replicates. Numbers within the same column and protein level with differentsuperscripts are significantly different (P < 0.05).2EFE, estimated feed efficiency = weight gain/feed offered x 100.3PCE, protein conversion efficiency = protein gaine/protei n offered x 100.'PSE, pooled standard error.

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20/20

Table S. Response ofP. val/name; to test diets at the conclusion of a 56 day growth trial.Supplementalphosphorus

oPhytase0.2S0-A0.375-BPSE

Biomass (g)

24.334 .8

44.844.5

3.5

Weight gain (%)

1736198721541924108

Survival (%)

56.2568 .7584.3887.508.60

'EFE, estimated food conversion efficiency = weight gain/feed offered ]00.A= CefkaphosB= Dynafos

EFE (%) I

35.941.542.] 45.5

1.6

20

Indicates a significant difference from the control (basal diet without supp]emental phosphorus) 1as determined by Dunnett's T test.

ca4_the use of enzyme supplements in shrimp diets

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