THE USE OF ENZYME SUPPLEMENTS IN SHRIMP DIETS.

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

The University of Texas at Austin Marine Science Institute Fisheries and Mariculture Laboratory 750 Channel View Drive Port Aransas, Texas, USA 78373-5015

Corresponding author

Key Words: Penaeus vallnamei, exogenous enzymes, phytase

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

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Abstract

The longlerm sustainability of commercial shrimp production is dependant on both economic and environmental constraints. Since the feed is one of the major costs associated with shrimp farming and the initial source of pollutants, there are considerable pressures to reduce feed costs and minimize the polluting effects of the feed. By adapting appropriate feed technologies and feeding strategies, the nutrient loading associated with supplemental feed systems can be minimized . A variety of feed supplements, which have been demonstrated to increase animal production, could be applied to shrimp feeds. 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 a commercial shrimp feed and apparent protein digestibility values determined . Apparent protein digestibility values were significantly improved from 65 .3% to 74.3% by the supplementation of the FGP at 0.4 g/IOOg diet, an increase of9% in APD over the basal diet . If this protein is available for assimilation and growth, the protein content of production diets could be reduced or less digestible ingredients could be incorporated into the diets without reductions in protein availability. To evaluate the effects on growth and feed utilization an 8-week growth trial was conducted. Graded levels of a FGP were supplemented to practical diets containing either 15% or 30% protein. The supplementation of 0.4 g FGPII 00 g diet resulted in a decrease in growth and feed utilization of the shrimp maintained on this diet as compared to those offered the basal diet without enzyme supplementation . These results would indicate that high levels of this enzyme may negatively impact the performance of the shrimp and that lower levels had no significant influence on growth or feed utilization . In the second component of this research a practical ba sal diet, previou sly demonstrated to be deficient in available phosphorus, was used to evaluate the use of phytase enzyme to free phytate bound phosphorus. Although the basal diet was deficient in available phosphorus, phyta se supplementation to the diet resulted in a positive shift in weight gain and estimated feed conversion efficiency. This indicates that phytase may release phytate bound pho sphorus warranting further investigation in practical shrimp feeds .

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Introduction
Over the. past decade, Aquaculture has been the world's fastest growing food production system, with total fish and crustacean production increasing from 4.67 milli o n metric tons (mmt) in 1984 to 15 .80 mmt in 1995 (Tacon, A.G. 1. , 1997). Although, many species are grown utilizing extensive production systems, the indu stry is shifting towards intensive supplemental feed systems and/or production of high value carnivorous species. Associated with this shift is a reliance on compounded feeds, increasing the potential for environmental impact of the production system. Nitrogen, phosphorus, and organic wastes from feeds are the major factors contributing to environmental pollution from aquaculture (Rijn and Shilo, 1989; Folke and Kautsky, 199 1; Boyd and Musig, 1992). As the initial source, manipulation of the feed is the most direct and effective means to reduce pollutants (Cho el aI, 199 1). Low pollution feeds must be designed to increase feed utilization by the culture species and minimize nutrient losses to the environment. This objective can be achi eved by increasing the digestibility of nutrients and reducing fecal waste. Increasing digestibility of the feed will not only reduce environmental pollution, but will also lower the total cost of productio n by lowering the nutrient expend iture per unit of productio n. In addition to selection of highly digestible feed ingredients, the utilizatio n of enzyme supplements designed to enhance the digest ibility of feedstuffs has been successfully applied in terrestrial (Wenk, 1992) and aquatic feeds. Examples of the use of enzymes in aquatic animal feeds include: feeding common carp a soybean residue which has been pre-digested by Papain, a protease isolated from the latex of Carica papaya (papaya) (Wong el ai, 1996); the inclusion of a exogenous multi-enzyme mixture supplemented to a canola based P. mOllodoll feed (Buchanan e/ aI, 1997); the supplementing of practical striped bass Morolle saxa/ilis diets with a commercial phytase (Hughes and Soares, 1998); modification of digestive enzyme ratio (amylase/protease) in P. japollicus throug h dietary enzyme supplementation (Maugle el aI, 1983a); and the pretreat ment of soybean meal with phytase prio r to use in rainbow trout, Ollcorhynchus lIIy kiss, diet s (Cain and Garling, 1995). These studies indicate that the utilization of enzyme supplements in the feed or feed ingredient s has the potential to improve dietary nutrient utilization, activate endogenou s zymogen(s) and provide enzymes that are not normally present in the digestive system of the culture animal. Consequently, the objectives of this research were 1) to evaluate the effects of a feed g rade protease (FGP) on apparent protein

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

Materials and Methods Digestibility of a commer'cial diet supplemented with a general protease. Apparent protein and dry matter digestibility values were determined based on the indirect methodology utilizing chromic oxide as an inert marker. The basal diet consisted of (as gil 00 g dry weight) 96.) commercial mash formulated for Pacific white shrimp, Penaells vallllamei, and obtained from 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 the appropriate level (0, 0.2 or 0.4 gil 00 g) ofENZECO@Bromelain FG. ENZECO@Bromelain FG is a feed grade protease (FGP), containing proteolytic enzymes found in various species of the Bromeliceae family. This FGP was selected because the protease remains entrapped within the pineapple stem fibers and hence should be less susceptible to leaching into the water. Dietary ingredients were homogenized in a food mixer (Hobart Corp., Troy, OH) for thirty minutes. After mixing, boiling water was blended into the mash until an appropriate consistency for pelletizing was reached . Each diet was pelletized utilizing a meat grinder with a 3-mm die, then placed in a heated oven 45 C) for 2 h followed by overnight drying (ambient air temperature) to reach a final moisture content of8-1 0%. Feeds were crumbled and sieved to the proper size for the shrimp, then stored under refrigeration until needed. Protein content of the 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 system

consisting of square tanks (75 L), circulation pump, sand filter, and biological filter. Environmental parameters were maintained at: temperature, 26 C; salinity, 30 ppt; di ssolved oxygen, 6.2 ppm . Each diet was offered to four replicate tanks of shrimp (8 shrimp per tank) for a 17 -day conditioning period followed by a three day collection period. During the collection period the following procedure was followed . In the morning the tanks were siphoned and the feeding cycle initiated . Shrimp were allowed to feed for 35-45 minutes after which the feces was collected by hand siphoning into a collection sieve

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(4811111) , followed by a rinse with distilled water. After the feces were collected, uneaten food was
removed from tbe tank and the feeding process repeated . During the collection period, the first collection in the morning was discarded, with the following 4-5 collections/day pooled by tank over the three-day period. Extracted fecal samples were oven dried (100 C) and stored at -15 C until analysis. Total ammonia-nitrogen (TAN), nitrite-nitrogen, and pH were monitored bi-weekly by spectrophotometric methods (Spotte, 1979) and pH meter, and were maintained within acceptable limits. Biochemical analyses of the test diets and each fecal sample were conducted in triplicate. Chromic ox. de was measured by the wet acid digestion method (McGinns and Kasting, 1964) and total i nitrogen determined by micro-Kjeldahl method (Ma and Zuazago, 1942). One-way analysis of variance was utilized to determine significance (P < 0.05) of the treatment effects and Student-Newman-Keul's multiple 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 of 8 shrimp per tank. An eight week growth trial was then conducted to evaluate the effect of ENZECO Bromelain FG on growth and apparent net protein retention for P. vallllal1lei . Two protein levels (15% and 30 % ) and four levels of the enzyme supplement ( 0, 0 I, 0.2, and 0.4 g FGP/I OOg feed) were evaluated using a 2 x 4 factorial design. The test diets (Table I) were produced by replacing non-nutritive filler in the basal diet with 0, 0.1, 0.2, and 0.4 g ENZECO Bromelain FG/I OOg dry weight offeed. Prior to use, practical ingredients were ground with a laboratory hammer mill using a size 24 screen (0 .609 mm diameter hole) . Dietary ingredients were homogenized in a food mixer (Hobart Corp., Troy, OH) for thirty minutes. After mixing, boiling water was blended into the mash until an appropriate consistency for pelletizing was reached . Each diet was pelletized utilizing a meat grinder with a 3-mm die and then placed in a heated oven

45C) for 2 h followed by

overnight drying (ambient air temperature) to reach a final moisture content of 8-1 0%. Feeds were crumbled and sieved to the proper size for the shrimp, then stored under refrigeration until needed .

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6
The 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-up water 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, nitrite nitrogen, and pH) were measured biweekly using spectrophotometric methods (Spotle, 1979) and a pH meter. Photoperiod was controlled at 12 h light and 12 h dark. Water quality parameters measured daily 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 random sample of three shrimp from each tank were collected, homogenized, and frozen for subsequent proximate analyses. All biochemical analyses were conducted in triplicate. Representative portions of samples were dried to a constant weight in an oven maintained at 90 C. Protein content was determined 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 feed offered (gain!dry feed xl 00). Protein conversion efficiency (PCE) was calculated as the grams of protein gain per grams of protein fed (protein gain! protein offered x 100). The data was analyzed by two-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 examine significant differences (P < 0.05) between treatments means.

Effects of phytase supplementation on the growth of shrimp A 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 were prepared utilizing the previously described procedures. The fourth diet consisted of the basal diet supplemented with 0.25g1100g Natuphos (600 FTU/g, BASF Corporation, New Jersey) replacing filler (Table 2). The activity of the basal diet and the diet supplemented with Natuphos were evaluated for phytase activity (FTU, defined as the quantity of enzyme which liberates I micro-mole

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7
of inorganic phosphorus minute-' from 0.0051 mol L-' sodium phytate at pH 5.5 and 37 C). Analyses were conducted_by BASF Corporation (Wyandotte, Michigan) and indicated that the basal diet contained 281 FTu/kg and the supplemented diet contained 1,919 FTU/kg. Experimental procedures were as previously described, except that prior to beginning the experiment the shrimp were offered the basal diet over a 7-day pre-conditioning period. The test diets were offered to four replicate groups of shrimp (8 shrimp per tank; mean initial weight ..Standard deviation, 0.30 0.017g). During the 56-day growth trial the shrimp received a total of9.8g dry feed per 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 were

measured 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 offered feed) were determined. One-way analysis of variance was utilized to determine significance (P < 0.05) of the treatment effects and Dunnett's T test to determine significant differences from the control or basal diet (Steel and Torrie, 1980).

Results and Discussion The 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 in ADMD values corresponding with the supplementation of the FGP. This response is, in part, due to replacement of inert filler with the FGP. Significant differences in APD values corresponded with increases in the dietary supplement. For shrimp offered the basal diet supplemented with 0.4 g FGP there was an increase of 9 % in APD observed over the basal diet. These results would indicate that proteolytic enzymes may be limiting protein digestibility in shrimp maintained on a commercial shrimp feed and that increases in APD values can be obtained through the use of dietary supplements. Maugle et aI, (1983a), demonstrated increased protease and zymogen activity in the hepatopancreas of shrimp, P. japollicllS, fed diets supplemented with microencapsulated bovine trypsin . Maugle et ai, (1983b), reported an increase in carbohydrate digestion by the addition of amylase to shrimp diets. If the supplementation of exogenous enzymes

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8 results in increased enzyme activity and an increase in nutrient digestion, a greater percentage of the nutrients may be available for assimilation and growth. Hence, feed cost could be reduced by incorporating less expensive low-digestible ingredients and/or reducing the protein content of the diet. In the formulation of low pollution diets, reduced protein levels in the feed and increases in APD values could produce significant reductions in nitrogen entering the culture system. Although these results are encouraging, any adverse effects of the enzyme supplement on growth or nutrient retention of 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 of this growth trial are summarized in Table 4 . Two-way analysis of variance indicated that there were significant differences due to the main effects (protein and FGP level), but none due to their interactions for final weight, EFE, and PCE of the shrimp. Consequently the data was separated by protein level for final analysis. There were no significant differences due to the main effects or their interactions for the survival data . For shrimp fed the diet containing 15% protein, final weights ranged from 3.8 g to 3. 1 g for shrimp offered the basal diet and the diet containing 0.4 g FGPII 00 g , respectively. The remaining diets produced intermediate weights and were not significantly different from the other diets. A similar response was observed for EFE and PCE values. With respect to the shrimp offered the 30% protein diet, final weights ranged from 4.6 g to 3.9 g for shrimp offered the basal diet and the diet containing 0.4 g FGP/ I 00 g, respectively . Estimated feed efficiency values for shrimp maintained on the basal diet supplemented with 0.4 g FGPII OOg diet was significantly lower than those observed for the basal diet and diets containing 0.1 and 0.2 g FGPII 00 g diet. Although PCE values were lowest for shrimp offered the basal diet containing 0.4 g FGPII 00 g diet, there were no significant differences observed between dietary treatments. The negative response at the highest level of supplementation would indicate an adverse effect of the enzyme on shrimp performance. The manufacturer recommended inclusion for ENZECO Bromelain FG in shrimp diets is 0.2 g 11 OOg diet. Under the conditions of these experiments, the supplementation of FGP at 0.1 or 0.2 gllOOg diet did not result in a significant change in shrimp growth or feed utilization. At twice the recommended level (0.4 gFGP/ 100 g diet) fina l shrimp weight,

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9
EFE, and PCE values were significantly lower than those observed for shrimp fed the basal diet. The poor response could be due to palatability changes in the diet or leeching of pre-digested nutrients from 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 gil 00 g diet) of canola meal. Maugle et

aI, (1983b) reported incremental increases in growth with increasing amylase supplements. In both
reports, increases in weight gain appear to be related to increased energy availability from the carbohydrate component of the diet. The differences in response for the current growth trial could be attributed t<;> species differences, dietary formulation, andlor enzyme function. The second growth trial was designed to preliminarily evaluate the use of phytase to free phytate bound phosphorus. Phytate is a mineral salt of phytic acid which is found in a variety of feed ingredients of plant origin. Phytate phosphorus is either unavailable to, or poorly utilized by shrimp and inhibits zinc availability (Davis et ai, 1993 , Civera and Guillaume, 1989). The supplementation of phytase 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), but has not been evaluated with shrimp. In the present study a practical basal diet, which had been previously determined to be deficient in phosphorus (Davis and Arnold, 1998), was supplemented with either a commercial source of phytase or replete levels of calcium phosphate originating from one of two feed grade phosphorus sources. The results in terms of growth, survival, and EFE are presented in Table 5. Significant differences between dietary treatments were observed for final biomass, percent weight gain, and EFCE values. Survival of shrimp offered the basal diet was poorest and survival of shrimp offered the basal diet supplemented with phytase was intermediate; however, there were no significant differences between dietary treatment detected . Weight gain for shrimp offered the basal diet was poor when compared to shrimp offered the other diets (530 vs 6.01 to 6.36 g). Percent weight gain was significantly improved by the addition of 0.250 g PII OOg originating from Cefkaphos. Final biomass, a measurement that combines both survival and weight gain, indicated that both supplements of phosphorus resulted in significant increases in biomass as compared to the basal diet. Similarly, EFE values for shrimp offered the two phosphorus supplements were significantly improved over shrimp

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10 fed the basal diet, confirming that the basal diet was deficient in phosphorus. In terms Gffinal biomass, percent weight gain, survival, and EFE values, the supplementation of phytate to the basal diet resulted in an improvement in these parameters. This response was intermediate 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/or preventing it from binding with phosphorus originating from other ingredients. Consequently, further evaluations of the use of phytase in shrimp feeds is warranted.

Summary Under 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 defined practical shrimp diets, containing either 15% or 30% protein, did not result in improvements in growth or feed utilization . Based on these results, FGP does not appear to enhance growth and feed utilization in a high quality shrimp feed . However, the use of phytase in a practical diet formulation that is deficient in phosphorus appears to increase the availability of phosphorus in the diet, as demonstrated by increased growth and estimated feed conversion efficiencies. Although, the results of the feeding trial with FGP did not result in improvements in feed utilization, the results with phytase were encouraging. Current literature indicates that, under certain circumstances, enzyme supplements such as amylase, papain, trypsin, and multi-enzyme supplements have produced positive results. In general , results appear best in diets containing low digestible ingredients and/or ingredients for which endogenous digestive enzymes are inadequate. Further research into the use of exogenous enzymes as feed supplements or as pretreatment of feedstuffs is warranted.

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II

Literature cited

Boyd, C.E. and Y. Musig. 1992. Shrimp pond effluents: observations of the nature of the problem on commercial farms. pp. 195-197. In J. Wyban, editors. Proceedings of the Special Session on 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 meal in prawn diets. Aquaculture 151: 29-35. Cain, KD. and D.L. Garling. 1995. Pretreatment of soybean meal with phytase for salmonid diets to reduce 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 by biological (nutritional) and chemical (Iimnological) methods; the development of high nutrient dense (HND) diets. Pages 37-50. In C.B. Cowey and C. Y. Cho, editors. Nutritional Strategies and Aquaculture Waste. Proceedings of the First International Symposium on Nutritional Strategies 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 of Penaeus 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 the
World 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.). N utritional Strategies and Aquaculture Waste. Proceedings of the First International Symposium on Nutritional Strategies in Management of Aquaculture Waste. University of Guelph, Guelph, Ontario, Canada, pp. 1990. 275

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12 Hughes, K. Powers and J.H. Soares Jr. 1998. Efficacy of phytase 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 World
Aquaculture Society 27 :309-313. Ketola, H.G. 1994. Use of enzymes in diets of trout to reduce environmental discharge of phosphorus. World Aquaculture Society, Book of Abstracts, 211 pp., World Aquaculture '94. Ma, T.S. and G. Zuazago. 1942. Micro-Kjeldahl determination of nitrogen. A new indicator and an improved rapid method . Industrial and Engineering Chemistry 14 : 280-282. Maugle, P .D ., O. Deshimaru, T. Katayama, T. Nagatani and K. Simpson. 1983a. Effect of microencapsulated amylase and bovine trypsin dietary supplements on growth and metabolism of 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 supplements in 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 food utilization 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. Shilo and 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 other elements to Atlantic salmon fed fish meal, soy protein concentrate or phytase treated soy protein concentrate based diets. Proceedings of the VII International Symposium on Nutrition and Feeding ofFish. Tacon, A. G. J. 1997. Global trends in aquaculture and aquafeed production 1984-1995. International Aquafeed 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 . In

Biotechnology in the feed industry. Proceedings of Alitech's eighth annual symposium. Alltech Technical Publications. Nicholasville, Kentucky. USA. Wong, M .H. , L.Y. Tang, and F.S.L. Kwok. 1996. The use of enzyme-digested soybean residue for feeding common carp. Biomedical and Environmental Sciences 9: 418-423.

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14
Table 1. Composition of test diets utilized to evaluate a feed grade protease (gll00g dry wt) .

15% protein
Menhaden fish meal' Soybean meal Fish solubles ' Menhaden fish oil ' Wheat flou~ d Soy lecithin ' Trace mineral premix Vitamin premix Stay C h Cefkaphos; Bromelin j Cellulose d
f
b

30% protein 8.50 17.00 1.00 2.80 64.98 1.30 0.50 3.00 0.12
0 AOO
OA

8.50 17.00 1.00 2.80 64.98 1.30 0.50 3.00 0.12

8.50 17.00 1.00 2.80 64 .98 1.30 0.50 3.00 0.12

0 AOO

8.50 17.00 1.00 2.80 64.98 1.30 0.50 3.00 0.12

0 AOO

17.00 35.50 1.00 3.80 36.98

1.30

17.00 35 .50 1.00 3.80 36.98

1.30

17.00 35.50 1.00 3.80 36.98

1.30

17.00 35 .50 1.00 3.80 36.98

1.30

0.50 3.00 0.12

OAOO

0.50 3.00 0.12

OAOO

0.50 3.00 0.12

OAOO

0.50 3.00 0.12

OAOO OA

oAOO
o
OA

0.1 0.3

0.2 0.2

o
0.4

0.1 0.3

0.2 0.2

'Special Select Hi, Zapata Haynie Corporation, Hammond, Louisiana, USA.

b

Solvent extracted, Producers Cooperative, Bryan, Texas, USA.

, Zapata Hayne Corporation, Reedville, Virginia, USA.

d

United States Biochemical Corporation, Cleveland, Ohio, USA.

, Aqualipid 95, Central Soya Chemurgy Division, Fort Wayne, Indiana,USA .

f

Composition of trace-mineral premix (g kg 1 mix): Cobalt chloride, 0.04; Cupric sulfate pentahydrate,

5.50; Ferrous sulfate, 20.00; Magnesium sulfate heptahydrate, 283 .98; Manganous sulfate
monohydrate, 6.50; Potassium iodide, 0.67; Sodium selenite, 0.10; Zinc sulfate heptahydrate, 131 .93; cellulose, 551 .28. Composition of vitamin premix (g kg' l mix) :Thiamin-HCI, 4.95; Riboflavin, 3.83; Pyridoxine-HCI,

4.00; Ca-Pantothenate, 10.00; N icotinic acid, 10.00; Biotin, 0.50; Folic acid, 4.00; Vitamin BI z, 0.05;
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15 Choline Chloride, 100.00; Inositol, 25 .00; Vitamin A acetate (20,000 IU/g), 8.00; Vitamin D (400,000 IU/g), 0.50; Vitamin E acetate (250 IU/g), 80.00; Menadione, 0.5; cellulose, 748.68.
h I

Stay C, L-Ascorbyl-2-Polyphosphate, Hoffman-LaRouche, Inc, Nutley, New Jersey, USA. Feed grade calcium phosphate, BASF Corporation, Mount Olive, NJ, USA

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16
Table 2. Composition of the test diets utilized to evaluate a dietary phytase supplement (gilOOg dry weight). Basal Anchovy meal ' Soybean meal' Fish solubles 3 Menhaden Fish oil' Wheat flour' Soy lecithin6 Trace Mineral premix' Vitamin premix' Vitamin C (25% active)9 NaCI' Natuphos
10 10

Phytase

0.25% P 20.0 330 1.0 3.8 35.9 1.3 0.5 2.0 0.1 0.3

0.375% P 20.0 330 1.0 3.8 35 .9 1.3 0.5 2.0 0.1 0.3

20.0 330 1.0 3.8 35.9 1.3 0.5 2.0 0.1 0.3

20.0 33 .0 1.0 3.8 35.9 1.3 0.5 2.0 0.1 0.3 0.25

Cefkaphos Dynafos
II

1.0 2025 2.1 1.85 1.0 0075

Diatomaceous earth'

Total phosphorus
I

0.98

1.35

1.23

1.35

Ralston Purina International, Checkerboard Square, St. Louis, Missouri, USA Solvent extracted, Producers Cooperative, Bryan, Texas, USA Zapata Haynie Corporation, Hammond, Louisiana, USA

2
3

, Zapata Haynie Corporation, Reedville, Virginia, USA. , United States Biochemical Corporation, Cleveland, Ohio, USA
6

Aqualipid 95, Central Soya Chemurgy Division, Fort Wayne, Indiana, USA

, Composition of trace-mineral premix (g kg ' mix): Cobalt chloride, 0.04; Cupric sulfate pentahydrate, 5.50; Ferrous sulfate, 20.00; Magnesium sulfate heptahydrate, 283.98 ; Manganous sulfate monohydrate, 6.50; Potassium iodide, 0.67; Sodium selenite, 0 10; Zinc sulfate heptahydrate,
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17 131.93; Alpha-cellulose, 551.28. Composition of vitamin premix (g kg- I mix) :Thiamin-HCI, 4.95; Riboflavin, 3.83; Pyridoxine-HCI, 4.00; Ca-Pantothenate, 10.00; Nicotinic acid, 10.00; Biotin, 0.50; Folic acid, 4.00; Vitamin B 12, 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 10

Stay C, L-Ascorbyl-2-Polyphosphate, Hoffman-La Roche, Inc., N utley, New Jersey, USA. Feed grade phosphorus source, primari ly monobasic calcium phosphate, BASF Corporation, Mount

Olive, New Jersey, USA

II

Feed grade phosphorus source, primarily dibasic calcium phosphate, Mallinckrodt Feed Ingredients,

Mundelein, Illinois, USA

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18

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 ENZECO

Bromelain FG. ENZECO Bromelain FG 0 0.2 0.4 PSE d ADMD' 53 .2' 54 .2' 61.1 ' 2 ..34 APD' 65.3 x 67.6' 74.3' 11.45

'Basal diet contained as gi l DOg dry weight: 96.1, commercial mash; 2.5, menhaden fish oil; 1.0, chromic oxide and 0.4, diatomaceous earth.
b

Dietary treatments consisted of the basal diet modified to contain the supplement at the indicated

level, replacing diatomaceous earth. ' Data represent the mean of four replicates. Mean values with the same letter are not significantly different (P > 0.05) from each other.
d

Pooled Standard Error

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19
Table 4. Response of P.
VGIIIIGmei

after 8 weeks of feeding diets containing various levels of protein

and feed grade protease (FGP) ' . Protein FGP Final weight (g) Survival EFE' PCE 3

(g/IOO diet) IS IS IS IS
PSE'

(g/ \00 diet)

(%)
96.9' 100' 96.9' 96.9' 2.70 100' 96.9' 100' 100' 1.56

(%)
35.8' 31. 9'" 33 .0'" 28 .7" 1.37 44.3' 42.6' 42.6' 37.0" 1.40

(%)
37.8' 36.0'" 34.7Y' 30.5" 1.67 24.S' 24 .7' 24.6' 21.5' 0.89

a
0.1 0.2 0.4

3.8' 3.4'" 3.5'" 3.JY 0.13

30 30 30 30
PSE

a
0.1 0.2 0.4

4.6' 4.4' 4.4' 3.9" 0. 14

'Means of four replicates. Numbers within the same column and protein level with different superscripts are significantly different (P < 0.05). 2EFE, estimated feed efficiency = weight gain/feed offered x 100. 3 PCE, protein conversion efficiency = protein gaine/protei n offered x 100. 'PSE, pooled standard error.

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20 Table S. Response of P. val/name; to test diets at the conclusion of a 56 day growth trial. Supplemental phosphorus Biomass (g) Weight gain (%) Survival (%) EFE (%) I

o
Phytase 0.2S0-A 0.375-B PSE

24.3 34.8 44.8 44.5 3.5

1736 1987 2154 1924 108

56.25 68.75 84.38 87.50 8.60

35.9
41.5

42.]

45.5
1.6

'EFE, estimated food conversion efficiency = weight gain/feed offered ] 00. A= Cefkaphos B= Dynafos Indicates a significant difference from the control (basal diet without supp]emental phosphorus) 1 as determined by Dunnett's T test.

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