INTRODUCTION. KORIN L. LESKE and CRAIG N. COON1. Department of Poultry Science, University of Arkansas, Fayetteville, Arkansas 72701

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1 A Bioassay to Determine the Effect of Phytase on Phytate Phosphorus Hydrolysis and Total Phosphorus Retention of Feed Ingredients as Determined with Broilers and Laying Hens KORIN L. LESKE and CRAIG N. COON1 Department of Poultry Science, University of Arkansas, Fayetteville, Arkansas ABSTRACT In order to accurately formulate diets for broilers and laying hens to meet phosphorus requirements without overfeeding, precise knowledge of an individual feed ingredient s contribution to the retainable phosphorus is needed. Seven feed ingredients, included as the sole source of phosphorus, were tested with and without the addition of 600 phytase units (FTU) phytase/kg diet, in a 5-d bioassay with d-old male broilers. Without addition of phytase, the amounts of phytate phosphorus hydrolyzed in corn, soybean meal, wheat, wheat midds, barley, defatted rice bran, and canola were 30.8, 34.9, 30.7, 29.1, 32.2, 33.2, and 36.7%, respectively. The addition of phytase increased (P 0.05) each value to 59.0, 72.4, 46.8, 52.2, 71.3, 48.0, and 55.8%, respectively. The addition of phytase increased total phosphorus retention from 34.8, 27.0, 16.0, 31.9, 40.3, 15.5, and 39.4% to 40.9, 58.0, 33.8, 43.4, 55.5, 26.5, and 45.7%, respectively. A similar bioassay was conducted with laying hens fed corn, soybean meal, and defatted rice bran. Without phytase addition, phytate phosphorus hydrolyzed in soybean meal, corn, and rice bran was determined to be 25.7, 23.0, and 36.1%, respectively, and was increased (P 0.05) to 62.4, 52.0, and 50.9%, respectively, with the addition of 300 FTU phytase/kg feed. Total phosphorus retention of soybean meal, corn, and rice bran increased from 36.8, 28.6, and 35.9% to 53.4, 44.7, and 43.0%, respectively, with the addition of phytase. (Key words: total phosphorus, phytate phosphorus, retention, broiler, layer) 1999 Poultry Science 78: INTRODUCTION Phosphorus from poultry wastes has become an environmental issue, as feed phosphorus not retained by the bird can ultimately enter and contaminate water supplies (Sharpley et al., 1993, 1994). Much of the phosphorus found in poultry diets is in the form of myo-inositol hexakis[dihydrogen phosphate] (IP6), an inositol ring with six phosphate groups commonly referred to as phytate phosphorus. Phytate phosphorus is considered to be poorly utilized by poultry. Ravindran et al. (1995a) reviewed the literature and made a comprehensive listing of feed ingredient phytate phosphorus contents, the factors that influence avian utilization of phytate phosphorus, and the effects phytate phosphorus can have on the availability of other nutrients in the diet. The authors noted wide disagreement on the availability (0 to 50%) of dietary phytate phosphorus to poultry. This wide range of values was attributed to the many factors that can influence phytate phosphorus availability, such as phytate source, age of birds, dietary calcium level, and differences in experimental protocol. Phytase enzymes have been shown to increase phosphorus availability and utilization in chickens (Nelson et al., 1971), turkeys (Ravindran et al., 1995b; Qian et al., 1996), and swine (Simons et al., 1990). Studies conducted to determine the effect of phytase on increasing plant phosphorus availability and reducing phosphorus excretion of poultry have measured growth parameters and performance (Nelson et al., 1971; Ballam et al., 1984; Nahashon et al., 1994). Digestibility or retention of dietary phytate phosphorus with and without phytase addition has been calculated by measuring phytate phosphorus in the diet and excreta (Nelson, 1976; Sooncharernying and Edwards, 1993). Other investigators have determined phosphorus equivalency values for added phytase based on performance parameters and retention (Denbow et al., 1995; Yi et al., 1996). Most studies have required at least a 2-wk period to determine the growth and bone parameter differences and most provided only indirect information about the phytase effects on phytate Received for publication October 13, Accepted for publication March 31, To whom correspondence should be addressed: ccoon@comp.uark.edu Abbreviation Key: FTU = phytase units: quantity of enzyme that liberates 1 mmd of inorganic phosphorus per minute from mol/l sodium phytate of ph 5.5 and 37 C. 1151

2 1152 hydrolysis and total phosphorus utilization of individual feed ingredients. The objective of this study was to evaluate a bioassay for the direct determination of phytase effects on phytate phosphorus utilization and the determination of the total phosphorus retention of individual feed ingredients. It is important to be able to efficiently and accurately quantify total phosphorus retention of feedstuffs, and to identify the factors affecting phytate and nonphytate phosphorus retention, so that phosphorus requirements can be more accurately met in diets and the ecological and economic costs of over-supplementation avoided. MATERIALS AND METHODS Broiler Hydrolysis of IP6 and Total Phosphorus Retention A bioassay, based on the bioassays conducted by Coon and Leske (1998) for inorganic phosphorus retention, was conducted to determine the effect of phytase on hydrolysis of IP6 and total phosphorus retention of seven cereal and legume grains: soybean meal, corn, wheat, wheat midds, barley, defatted rice bran, and canola meal. Corn, wheat, and barley were tested at 60% of the total diet. Soybean meal, canola meal, wheat midds, and defatted rice bran were tested at 30% of the total diet. The remainder of each diet consisted of a purified diet containing amino acids and vitamin and mineral mixtures to provide a complete balanced diet that contained no measurable additional phosphorus (Table 1). The diets were adjusted with each test ingredient to maintain approximately 19% protein and a ME content ranging from 2,900 to 3,200 kcal ME/kg. Because of its low phosphorus content, gelatin was used as a protein source. Synthetic amino acids were used to balance the protein and amino acid contents of the diets. Each diet contained amino acid levels that met or exceeded the levels recommended by NRC (1994). Celite,2 an acid insoluble ash marker, was added to all test diets at a level of 2%. Diets were analyzed by the bioassay with and without the addition of 600 phytase units (FTU) of phytase3/kg of diet. This design resulted in 14 treatments. Male broiler chicks (Ross Ross) were fed a standard starter diet until 22 d of age. At this point, 140 birds (10 chicks per test diet, mean weight = 809 g) were placed in individual cages. Broilers were acclimated to cages and test diets for 3 d prior to the initiation of the experiment. Individual stainless steel trays were placed under each cage for excreta collection. Excreta were collected for 48 h. Broilers had unlimited access to experimental diets and water during acclimation and excreta collection. Feed consumption and bird weights were recorded. Excreta were collected, frozen, freeze-dried, and ground for 2Celite Corp., Lompoc, CA BASF Corp., Mt. Olive, NJ Spectro Analytical Instruments Fitchburg, MA Analytical Software, Tallahassee, FL LESKE AND COON analysis. Diet and excreta samples were analyzed for acid insoluble ash using the dry ash and hydrochloric acid digestion technique of Scott and Balnave (1991). Grain, diet, and excreta phytate phosphorus was measured as IP6 using the ion-exchange chromatography method described by Bos et al. (1991). Total phosphorus was measured by an inductively coupled plasma (ICP) emission spectroscopic method. Duplicates for each sample were weighed (0.15 g) into porcelain crucibles and ashed at 600 C for 3 h. After cooling, 10 ml of a 50% hydrochloric acid was added to each sample crucible and the samples were allowed to digest overnight at room temperature. Samples were then brought to a slow boil on a hot plate for 2 h, with 50% hydrochloric acid added as necessary to maintain a volume of approximately 10 ml. The solution was cooled and quantitatively transferred to a 50-mL conical centrifuge tube. Crucibles were rinsed twice with 7 ml of a 5% hydrochloric acid solution to assure complete transfer. The total volume was increased to 40 ml with HPLC grade water, followed by centrifugation at 1,500 rpm for 10 min at room temperature. The supernatant was introduced to a SpectroAnalytical Axial Plasma ACP (SpectroFlame Modula E4 ) and measured at 215 nm for phosphorus content against prepared standards ranging from 1 to 100 ppm in phosphorus content. Laying Hen Hydrolysis of IP6 and Total Phosphorus Retention Three of the test ingredients used in the broiler experiment were also used in a laying hen trial. Sixty Hy- Line 36 laying hens at peak lay were randomly selected and placed in individual cages, 10 hens per experimental diet. The hens were acclimated to the cages and test diets for 3 d. Excreta were then collected for 72 h on stainless steel trays. Hens had unlimited access to water and feed during acclimation and excreta collection. Feed consumption, body weight, and egg production data were recorded. The test diets (Table 2) met or exceeded NRC (1994) recommended levels for laying hens consuming 80 g of feed per day, with the exception of phosphorus, which was fed at significantly lower levels to enhance retention. Test diets, formulated in a manner similar to that of the broiler diets, consisted of purified ingredients and contained no phytate or nonphytate phosphorus except for the phosphorus contained in the feed ingredient being tested. Enzyme diets consisted of the corresponding nonenzyme diet with 300 FTU phytase/kg of feed added. Acid insoluble ash marker was added to determine retention. Analysis of excreta and diets were as described for the broiler experiment. Statistical Analysis In the broiler and laying hen trials, birds that would not accept the highly synthetic diet were dropped from the statistical analysis of the data. Data were analyzed by the analysis of variance procedure of the Statistix 4.0 software. 5 Means were subjected to LSD (Least Significant Difference) test of Statistix 4.0 at the 5% rejection level.

3 PHYTATE PHOSPHORUS HYDROLYSIS AND PHOSPHORUS RETENTION 1153 TABLE 1. Diets used in broiler bioassay to determine hydrolysis of myo-inositol hexakis[dihydrogen phosphate] (IP6) and total phosphorus retention of feed ingredients Test feed ingredients Soybean Wheat Defatted Canola Ingredient meal Corn Wheat midds Barley rice bran meal (%) Test ingredient Corn oil Gelatin Starch Glucose Vitamin mix Trace mineral mix Cellulose Salt Choline chloride (50%) Limestone Potassium carbonate Magnesium sulfate Celite Biotin DL methionine L-lysine HCl L-glutamic acid L-leucine L-isoleucine L-valine L-tyrosine L-cystine L-arginine HCl L-threonine L-tryptophan L-phenylalanine L-histidine (HCl-H 2 O) Glycine/L-serine (50:50) Ethoxyquin Total Calculated nutrients Protein MEn, kcal/kg 3,200 3,200 2,900 2,950 2,955 3,200 2,900 Calcium TSAA Lysine Analyzed nutrients (as fed) Total P Phytate P Vitamin mix provided per kilogram of diet: vitamin A (retinyl A acetate), 6,600 IU; cholecalciferol, 2,200 IU; vitamin E (DL-a-tocopheryl acetate), 4.4 IU; vitamin B 12,13mg; riboflavin, 6.6 mg; niacin, 33 mg; calcium pantothenic acid, 11 mg; menadione sodium bisulfite, 4.4 mg; folic acid, 0.22 mg; pyridoxine HCl, 4 mg; thiamine mononitrate, 28 mg; biotin, 28 mg. 2Trace mineral mix provided per kg of diet: Cu, 3 mg; I, 1.1 mg; Fe, 12 mg; Mn, 75 mg; Zn, 60 mg. 3Celite Corp., Lompoc, CA RESULTS Analysis of Feed Ingredients The feed ingredient analyses for total and phytate phosphorus are summarized in Table 3. In most instances, values are similar to those determined by Nelson et al. (1968) and values listed in NRC (1994). However, a higher phytate phosphorus content (1.185%), and, consequently, total phosphorus content (1.322%), of wheat midds was found in this study. The NRC (1994) values for wheat midds phytate phosphorus content and total phosphorus content are 0.55 and 0.85%, respectively. Broiler Bioassay With no phytase supplementation, hydrolysis of IP6 (Table 4) of the feed ingredients ranged from 29.1% (wheat midds) to 36.7% (canola meal). Total phosphorus retention of feed ingredients with no phytase supplementation ranged from 15.5% (defatted rice bran) to 40.3% (Barley). Significant differences were observed among feed ingredients with no phytase supplementation in both broiler hydrolysis of IP6 and total phosphorus retention (P 0.05). The addition of phytase resulted in significant increases in the hydrolysis of IP6 for all of the feed ingredients studied. The addition of phytase also resulted in significant increases in the retention of total phospho-

4 1154 TABLE 2. Diets used in laying hen bioassay to determine hydrolysis of myo-inositol hexakis[dihydrogen phosphate] (IP6) and total phosphorus retention of feed ingredients Test feed ingredients Soybean Defatted Ingredient meal Corn rice bran (%) Test ingredient Corn oil Gelatin Starch Glucose Vitamin mix Trace mineral mix Cellulose Salt Choline chloride (50%) Limestone Sodium bicarbonate Celite Potassium carbonate DL methionine L-lysine HCl L-leucine L-isoleucine L-valine L-tyrosine L-cystine L-threonine L-tryptophan L-phenylalanine Biotin Ethoxyquin Total Calculated nutrients Protein MEn, kcal/kg 2,750 2,750 2,750 Calcium TSAA Lysine Analyzed nutrients (as fed) Total P Phytate P Vitamin mix provided per kilogram of diet: vitamin A (retinyla acetate), 9,921 IU; cholecalciferol, 3,307 IU; vitamin E (DL-a-tocopheryl acetate), 27 IU; vitamin B 12,16mg; riboflavin, 8 mg; niacin, 33 mg; calcium pantothenic acid, 18 mg; menadione sodium bisulfite, 4.4 mg; folic acid, 1.1 mg; pyridoxine HCl, 4 mg; thiamine mononitrate, 2.4 mg; biotin, 0.17 mg. 2Trace mineral mix provided per kg of diet: Cu, 3 mg; I, 1.1 mg; Fe, 3.5 mg; Mn, 54 mg; Zn, 50 mg. 3Celite Corp., Lompoc, CA LESKE AND COON 53.6 g/d (wheat midds) to g/d (corn). Significant differences in daily feed consumption were observed among feed ingredient diets, most likely due to palatability differences between the semi-synthetic diets. There were no significant effects of feed consumption on hydrolysis of IP6 or total phosphorus retention. Laying Hen Bioassay As in the broiler bioassay, significant differences in feed consumption were observed between test feed ingredients (Table 5). However, feed consumption was not a significant factor influencing hydrolysis of IP6 or total phosphorus retention. No significant effects of test feed ingredient on egg production were observed. Egg production across all treatments averaged 86.7%. With no phytase supplementation, hydrolysis of IP6 of soybean meal, corn, and defatted rice bran was determined to be 25.7, 23.0, and 36.1%, respectively. Total phosphorus retention of soybean meal, corn, and defatted rice bran was determined to be 36.8, 28.6, and 35.9%, respectively. The addition of phytase significantly (P 0.05) increased the hydrolysis of IP6 and total phosphorus retention of each of the three feed ingredients evaluated in laying hens. Laying hen hydrolysis of soybean meal, corn, and defatted rice bran IP6 with phytase addition was determined to be 62.4, 52.0, and 50.9%, respectively. Similarly, total phosphorus retention was increased to 53.4, 44.7, and 43.0%, respectively. DISCUSSION Sooncharenying and Edwards (1993) found IP6 retention of 3-wk-old broiler chicks on a corn-soybean diet with no phytase supplementation to be as high as 32%. This result agrees with the values for corn (30.8%) and soybean meal (34.9%) hydrolysis of IP6 determined in the present study. Simons et al. (1990) fed broilers a diet that contained corn, soybean meal, sorghum, and sunflower seed meal, but no feed grade phosphate, and determined the total phosphorus retention to be 49.8%. They observed that total phosphorus retention increased rus, with the exception of canola meal. Canola meal exhibited a significant increase in the hydrolysis of IP6 in response to phytase supplementation, but a nonsignificant change in total phosphorus retention. Hydrolysis of corn IP6 and total phosphorus retention of corn were increased from 30.8 and 34.8% with no phytase supplementation to 59.0 and 40.9%, respectively, with the addition of phytase. Phytase supplementation increased soybean meal hydrolysis of IP6 and total phosphorus retention from 34.9 and 27.0%, respectively, to 72.4 and 58.0%, respectively. Barley and soybean meal exhibited the largest hydrolysis of IP6 (71.3 and 72.4%) and total phosphorus retention (55.5 and 58.0%) with phytase supplementation. Daily feed consumption ranged from TABLE 3. Feed ingredient dry matter (DM) contents and phosphorus contents on a DM basis 1 Ingredient DM Total P Phytate P (%) Soybean meal Corn Rice bran Canola meal Barley Wheat Wheat midds Means are from triplicate analyses.

5 PHYTATE PHOSPHORUS HYDROLYSIS AND PHOSPHORUS RETENTION 1155 TABLE 4. Hydrolysis of myo-inositol hexakis[dihydrogen phosphate] (IP6) and total phosphorus retention by 3-wk-old broilers of seven feed ingredients with and without 600 phytase units (FTU) phytase as determined with a 2-d excreta collection Feed ingredient Phytase n Feed consumption per day Hydrolysis of IP6 Total P retention (g) (SD) (%) (SD) (%) (SD) SBM def b,wx b,y defg a,h a,h 4.3 Corn a b,wx b,wx ab a,i a,i 5.5 Rice bran cde b,wx b,z cde a,jk a,k 3.7 Canola meal cde b,w a,w cde a,ij a,i 7.8 Barley bcd b,wx b,w bc a,h a,h 5.3 Wheat defg b,wx b,z fg a,kf a,j 9.6 Wheat midds g b,x b,xy efg a,ijk a,i 5.8 a ghydrolysis of IP6 and Total P retention: means within a specific feed ingredient treatment and column with no common superscript differ significantly (P 0.05). h khydrolysis of IP6 and Total P retention: means within the added phytase (+) treatment and same column with no common superscript differ significantly (P 0.05). w zmeans within the no added phytase ( ) treatment and same column with no common superscript differ significantly (P 0.05). (56.5 and 64.5%, respectively) with the addition of phytase (250 units and 1,500 units/kg of diet, respectively). Results indicated that increasing the dietary phosphorus and calcium content with a dicalcium phosphate led to a decrease in percentage total phosphorus retention of the diet. Qian et al. (1997) found similar ranges of total phosphorus retention (50.9 to 68%) of a corn-soybean diet, with linear increases in phosphorus retention observed with supplemental phytase and cholecalciferol. Mohammed et al. (1991) calculated phytate phosphorus retention by 3- to 4-wk-old broilers of a corn-soybean diet to be 50.1%, with a total phosphorus retention of 41.5%. Sebastian et al. (1996) determined total phosphorus retention by 3-wk-old male broilers of a corn-soybean diet to be 51%. Retention of total phosphorus increased to 63.5% with the addition of phytase. Total phosphorus retention of the corn and soybean meal in this study was 34.8% and 27.0%, respectively, with no addition of phytase. These values are somewhat lower than the values reported by other researchers for corn-soybean diets; however, those diets also had additional phosphorus sources included in the form of highly retainable calcium phosphates. Broz et al. (1994) reported total phosphorus retention values of a cornsoybean diet with no added inorganic phosphates to be 44% as determined with 3-wk-old broilers. Total phosphorus retention was increased to 52% with the addition of phytase. The present study indicates that the feed ingredients utilized in the diet influenced phytate phosphorus hydrolysis and total phosphorus retention for broilers, and that the effect of supplementation with phytase was also feed ingredient dependent. We also observed that TABLE 5. Hydrolysis of myo-inositol hexakis[dihydrogen phosphate] (IP6) and retention of total phosphorus by laying hens of three feed ingredients with and without 300 phytase units (FTU) phytase as determined with a 3-d excreta collection Diet n Feed consumption per day Hydrolysis of IP6 Total P retention (g) SD (%) SD (%) SD Soybean meal abc b,y b,x 8.4 Soybean meal and phytase bc a,c ac 9.5 Corn c b,y b,y 4.4 Corn and phytase c ad ad 3.0 Rice bran ab b,x b,x 3.5 Rice bran and phytase a ad ad 6.1 a,bmeans within a specific feed ingredient treatment and column with no common superscript differ significantly (P 0.05). c,dmeans within the added phytase (+) treatment and same column with no common superscript differ significantly (P 0.05). x,ymeans within the no added phytase ( ) treatment and same column with no common superscript differ significantly (P 0.05).

6 1156 laying hens can hydrolyze a portion of IP6 (23% corn, 25.7% soybean meal, and 36.1% rice bran) from the feed ingredients studied without the addition of phytase (Table 5). In this study, laying hen hydrolysis of IP6 without phytase supplementation for both corn (23.0%) and soybean meal (25.7%) were greater than that reported by Nelson (1976), who determined laying hen hydrolysis of phytate phosphorus of a diet containing corn and soybean meal to be 8%. Nahashon et al. (1994) determined the total phosphorus retention of a cornsoybean diet to be approximately 18% as determined with Leghorn layers. In this study, slightly higher values were observed for corn (36.8%) and soybean meal (28.6%) total phosphorus retention. As seen with the broilers, the addition of phytase to soybean meal resulted in the highest IP6 hydrolysis values and highest total phosphorus retention values. Laying hen utilization of phytate phosphorus and retention of total phosphorus, both with and without phytase supplementation, was also found to be significantly affected by the feed ingredient. Van der Klis et al. (1997) determined laying hen ileal IP6 degradation of a diet containing corn soybean meal, hominy feed, and sunflower seed meal to be approximately 21%. Degradation of IP6 was increased to 71.7% with the addition of phytase at 500 FTU/kg diet. In a second experiment, the researchers found a corn-soybean-sunflower seed meal diet to have a laying hen ileal IP6 degradation of 10%, increased to 66% with the addition of 500 FTU phytase/kg of diet. No significant differences between laying hens and broilers were found in phosphorus retention of the corn diets (Table 4 and Table 5). However, compared to broilers, laying hen total phosphorus retention was found to be significantly greater for soybean meal with no phytase supplementation (P = ), defatted rice bran without phytase supplementation (P = ), and defatted rice bran with phytase supplementation (P = ). Hydrolysis of IP6 of soybean meal with no phytase supplementation by broilers was determined to be significantly greater than that of laying hens (P = ). Edwards (1983) also found breed and strain differences in phytate phosphorus utilization of a cornsoybean diet. The bioassay described provides a sensitive method for the determination of total phosphorus retention and phytate phosphorus hydrolysis of individual feed ingredients and can be modified in order to quantify the effect of other parameters (age of bird, calcium level, nonphytate phosphorus level) on phytate hydrolysis, total phosphorus retention, and phosphorus excretion. In this manner, it could be an important tool in the formulation of diets that take into consideration the phosphorus needs of the birds and also the amount of phosphorus that will be excreted from this diet. The addition of phytase to the diets of broilers and laying hens significantly increased phytate phosphorus hydrolysis and total phosphorus retention and can be used as a LESKE AND COON tool to more efficiently provide the birds with their phosphorus needs and reduce excreta phosphorus. ACKNOWLEDGMENTS The authors thank IMC-Agrico, Inc. and BASF Corp. for supporting the research related to the phytase effect on phosphorus retention. REFERENCES Ballam, G. C., T. S. Nelson, and L. K. Kirby, Effect of fiber and phytate source and of calcium and phosphorus level on phytate hydrolysis in the chick. Poultry Sci. 63: Bos, K. D., C. Verbeek, C. H. Peter van Eeden, P. Slump, and M.G.E. Wolters, Improved determination of phytate by ion-exchange chromatography. J. Agric. 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