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Supplemental Microbial Phytase Improves Zinc Utilization in Broilers
Supplemental Microbial Phytase Improves Zinc Utilization in Broilers Z. YI, E. T. KORNEGAY,! and D. M. DENBOW Department of Animal and Poultry Sciences, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061-0306 percentage of toe and tibia were not affected by adding Zn but were linearly improved by adding phytase (P < 0.10); however, the amount of ash in toe or tibia was increased by Zn (P < 0.05) and phytase (P < 0.01 for toe; not significant for tibia). The concentration and amount of Zn in toe and tibia were linearly increased by adding Zn and phytase (P < 0.001). The concentration of Zn in liver was increased by adding Zn (P < 0.10) but was not significantly improved by adding phytase. The amount of Zn retained in liver was linearly improved by adding Zn and phytase (P < 0.05). Nonlinear or linear response equations of the effects of Zn and phytase levels were generated and used to calculate the Zn equivalency values. The average function of Zn equivalency values (Y, milligrams per kilogram) of microbial phytase (X, units per kilogram of diet) was developed: Y = 0.20 + 0.0082X. The results indicate that approximately 0.9 mg of Zn was released per 100 U of phytase over the range of 150 to 600 U of phytase.
(Key words: broiler, phytase, zinc, utilization, equivalency value) 1996 Poultry Science 75:540-546
INTRODUCTION Phytate has complexing potential to form a wide variety of insoluble salts with cations such as Ca, Mg, Zn, and Cu (Vohra et al, 1965; Oberleas, 1973; Morris, 1986). Maddaiah et al (1964) and Reddy et al. (1982) reported that Zn has the strongest binding affinity with phytate. Phytate has been shown to impair the bioavailability of Zn in humans, rats, pigs, and chicks (Lease, 1966; Ferguson et al, 1989; Lonnerdal et al, 1989; Bobilya et al, 1991). Supplemental microbial phytase has been shown to be very effective for improving dietary phytate P bioavailability (Simons et al, 1990; Yi et al, 1994a,b; Denbow et al, 1995). A few studies also indicated that supplemental microbial phytase in the diets of young pigs improved the bioavailability and absorption or retention of Zn (Pallauf et al, 1992, 1994 a, b; Lei et al, 1993). Thiel et al (1993) found that the Zn
content of femur from chicks fed a diet containing 30 ppm Zn plus 700 U of phytase/kg of diet was equal to that of chicks fed a diet containing 39 ppm Zn without phytase. Roberson and Edward (1994) reported that adding phytase increased tibia Zn concentration but did not improve apparent Zn retention in broiler chicks. Biehl et al. (1995) found that the addition of 1,200 U of phytase/kg of diet to a glucose-soybean concentrate diet (13 ppm of Zn) increased growth rate by 40% and total tibia Zn by 107% in chicks. The objective of the present experiment was to investigate the influence of adding microbial phytase on Zn utilization in broilers fed a low Zn corn-soybean isolate diet and to calculate Zn equivalency values of phytase.
MATERIALS AND METHODS Birds, Diets, and Treatments The effect of microbial phytase on the utilization of Zn was investigated using 384 Peterson x Arbor Acres male broiler chicks fed corn-soybean isolate diets. The basal diet
Received for publication September 5, 1995. Accepted for publication December 6, 1995. !To whom correspondence should be addressed.
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ABSTRACT Day-old male broilers (n = 384) were used in a 21-d trial to investigate the effect of microbial phytase on the retention and utilization of Zn. A cornsoybean isolate basal diet containing 20 ppm Zn was fed alone and supplemented with 5, 10, or 20 ppm Zn as ZnS0 4 -7H 2 0 or with 150, 300, 450, or 600 U of phytase/ kg of diet. Total excreta were collected during Days 18 to 20. Toe, tibia, and liver samples were taken at the end of the experiment. Adding Zn and phytase to the low Zn basal diet linearly increased BW gain and feed intake of broilers (P < 0.01). The gain to feed ratio was not changed by adding Zn but was decreased by adding phytase (P < 0.01). The amount of DM retained was linearly increased by adding Zn and phytase (P < 0.10), but DM retained as a percentage of intake was only increased by adding Zn (P < 0.05). The amount of Zn retained per bird was linearly improved by adding Zn and phytase (P < 0.01). Zinc retained as a percentage of intake was linearly decreased by adding Zn but was linearly increased by adding phytase (P < 0.10). Ash
PHYTASE A N D Z I N C UTILIZATION TABLE 1. Percentage composition of basal diets
1
Percentage
Corn (8.5% CP) Soy isolate (92% CP) 2 Soybean oil Cornstarch 3 Limestone 4 Defluorinated phosphate 5 Vitamin premix 6 Trace mineral premix 7 Salt DL-Methionine Calculated nutrients CP Lysine Methionine + cystine Ca Total P Nonphytate P (nP) Ca:nP ratio
54.40 20.00 4.00 17.67 0.89 1.94 0.20 0.20 0.40 0.30 23.00 1.28 0.90 1.00 0.65 0.45 2.22
1
The assayed contents of Zn, total P, and Ca in the basal diet were 20 ppm, 0.61%, and 0.93%, respectively. Based on National Research Council (1994), the calculated phytate P in the basal diet was 0.21%. Three Zn levels of 5,10, and 20 ppm as ZnS0 4 -7H 2 0 and four phytase levels of 150, 300, 450, and 600 U / k g of diet were added to this basal diet. The assayed phytase activity in the basal diet and the diets with added four levels of phytase were 26, 173, 320, 443, and 631 U / k g of diet, respectively. 2 PP500E, Protein Technologies International, St. Louis, MO. Nonphytate P was assumed to make up 40% of total P in soy isolate, the same as reported for soy concentrate. 3 Food grade, National Starch and Chemical Co., Bridgewater, NJ 08807. "Limestone Dust Corp., Bluefield, VA 24605. SFine CDP, Southern Bag Corp., Valdosta, GA 31083. Supplied per kilogram of diet: retinyl acetate, 908 /ig; cholecalciferol, 66 ng; dl-a-tocopherol acetate, 26 mg; menadione sodium bisulfite, 0.75 mg; riboflavin, 7.5 mg; d-calcium pantothenate, 9.7 mg; niacin, 26.4 mg; cyanocobalamin, 0.011 mg; choline chloride, 1,012 mg; d-biotin, 0.31 mg; folic acid, 3.1 mg; thiamin-HCl, 8 mg; pyridoxine-HCl, 3.1 mg; ethoxyquin, 50 mg; and virginiamycin, 2.9 mg. 7 Supplied per kilogram of diet: manganese, 60 mg; iron, 80 mg; copper, 8 mg; iodine, 0.35 mg; and selenium, 0.15 mg.
met recommended (National Research Council, 1994) nutrient requirements except for that of Zn (Table 1). The calculated and assayed Zn content of the basal diet was 20 ppm. The assayed Zn contents of corn, soybean isolate, cornstarch, and defluorinated phosphate were 19, 38, 2.5, and 65 ppm, respectively. Eight dietary treatments were as follows: low Zn basal diet alone, three levels of Zn (5,10, and 20 ppm Zn as ZnSC^-TT^O)2 or four levels of Natuphos® phytase3 (150, 300,450, and 600 U / k g of diet) were added to the low Zn basal diet. A unit of phytase is defined as the quantity of enzyme that liberates 1 pimol of inorganic orthophosphate/min from 5.1 mM sodium
2
USP/FCC, Fisher Chemical, Fisher Scientific, Fair Lawn, NJ 07410. BASF Corp., Mount Olive, NJ 07828-1234. "Alternative Design, Manufacturing, and Supply, Inc., Siloam Springs, AR 72701. 5 Rolp 120-TF, Hydro, Research Triangle Park, NC 27709. 6 Model 5100 PC, Perkin-Elmer, Norwalk, CT 06859-0200. 3
phytate at pH 5.5 and 37 C (Engelen et al., 1994). The assayed dietary phytase activity was 26,173, 320,443, and 631 U / k g of diet, respectively, for the added phytase levels of 0, 150, 300, 450, and 600 U / k g of diet. The basal diet without added Zn or phytase was fed as a control. Defluorinated phosphate and ground limestone were used to maintain the desired P and Ca levels. The Ca: nonphytate P was 2.22:1 in all diets, as recommended by the National Research Council (1994). The assayed total P and Ca contents in the basal diets were 0.61 and 0.93%, respectively. Based on National Research Council studies (1994), the calculated phytate P in the basal diet was 0.21% and the nonphytate P was 0.45%.
Feeding and Management Broilers were randomly allotted on the day of hatch to 48 pens (eight birds per pen). They were housed in electrically heated, stainless steel starting batteries 4 in an environmentally controlled room. The eight treatments were randomly assigned to 48 pens (6 pens per treatment). The diets were fed in a mash form. Birds had ad libitum access to feed and deionized water produced by a reverse osmosis system. 5 Zinc content of the water was 0.3 ppm. The care and treatment of birds followed published guidelines (Consortium, 1988). Body weight and feed intake of the birds were recorded on a pen basis at weekly intervals during the 21-d experiment. Mortality was recorded daily.
Sampling and Analysis The total excreta of the birds were collected during Days 18 to 20. Feed intakes were recorded starting 24 h before the collection time and ending 24 h before the end of collection. The excreta were kept in plastic bags and dried in a stainless steel oven at 60 C and ground. All the birds were killed by cervical dislocation at the end of the experiment. Toe samples of the killed birds were obtained by severing the middle toe through the joint between the second and third tarsal bones from the distal end. The left middle toes of the birds within a pen were pooled, and the right middle toes from the same birds were pooled, yielding two samples of toes per pen. The composite samples were dried to a constant weight at 100 C and then ashed in a muffle furnace at 600 C for 4 h. Three killed birds from each pen were randomly selected and samples of liver and left tibia were collected. After the soft tissues were removed, tibia samples were dried to a constant weight at 100 C and then ashed in a muffle furnace at 600 C for 4 h. The ash from toes and tibias was solubilized with nitric and perchloric acid (5:3, vol/vol), and diets, excreta and liver were wet acid digested with the nitric and perchloric acid mixture (Association of Official Analytic Chemists, 1990). Zinc concentrations in diets, excreta, liver, toe, and tibia ash were analyzed with an atomic absorption spectrophotometer. 6
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Ingredients
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YI ET AL. TABLE 2. Effects of supplemental microbial phytase and zinc on performance (Days 1 to 21), apparent dry matter utilization (Days 18 to 20) and zinc retention of broilers 1
Diet
Zn added
Phytase added
(ppm)
(U/kg) 0 0 0 0 150 300 450 600
1 0 2 5 3 10 4 20 5 0 6 0 7 0 8 0 Pooled SEM
BW gain
Feed intake - (g/bird)
396 428 437 4432 398 431 440 440 s 11
530 600 605 6162 551 617 670 630« 20
Gain: feed
DM ]Retention
(g/kg)
(%)
747 714 721 722 724 700 660 7027
83.7 83.2 83.7 84.73 83.3 83.9 83.8 83.5 0.40
16
(g/bird) 75.9 87.2 87.6 91.1 4 77.6 86.1 86.1 85.68 3.5
Zn Retention
(%) 37.9 35.8 35.4 34.2 39.2 44.0 43.0 44.88 3.0
(mg/bird) 6.89 9.39 11.14 13.344 7.23 8.97 8.81 9.09» 0.95
!Six pens (eight birds per pen) per treatment mean. The values of apparent retention were based on total collection of excreta from Day 18 to 20. Zinc effect (linear, P < 0.01). 3 Zinc effect (linear and quadratic, P < 0.05). 4 Zinc effect (linear, P < 0.001). 5 Phytase effect (linear, P < 0.001). 6 Phytase effect (linear, 0.001; quadratic, P < 0.10). 7 Phytase effect (linear, 0.01; quadratic, P < 0.05). 8 Phytase effect (linear, 0.10). 9 Phytase effect (linear, P 0.01). 2
The data were subjected to analysis of variance by the General Linear Models (GLM) procedures of SAS® (SAS Institute, 1990). The model included diet and replicate with linear and quadratic contrast for effects of supplemental Zn or microbial phytase. Nonlinear and linear functions that best fit the data were derived for four Zn levels and for five phytase levels with the nonlinear model: Y = a(l -be _ l c X ) and linear model: Y = a + bX; where Y = the response measurements; and X = Zn (parts per million) added or phytase added (units per kilogram of diet). The nonlinear or linear response equations for added Zn and added phytase levels with higher r 2 values were set equal and solved as shown in the example below for amount of Zn retained in toe (Table 5): 159.7(1 - 0.5613e-00573X1) = 71.4 + rj.045X2 Xa = -17.45Ln(0.9851 - 0.0005X2); where Xi = Zn added (parts per million); and X2 = phytase added (units per kilogram of diet). For example, give phytase (X2) = 450 U / k g of diet, the Zn equivalency value (Xi) = -17.45 Ln(0.9851 - 0.0005 x 450) and Xi = 4.8 mg (Table 5). This result means that the amount of Zn retained in toe by 450 U of phytase/kg of diet is equal to that of 4.8 mg Zn as ZnSC>4-7H20. The resulting equations were used to calculate the Zn equivalency values of microbial phytase at 150, 300, 450, and 600 U / k g of diet for the various measurements. The use of a regression line (equation) generated from graded levels of Zn or phytase provides a more accurate means of estimating a response
than a single number. Also, the generation of a Zn equation from the equations for Zn and for phytase allows for the calculation of the Zn equivalency values of phytase for any point on the line. Further, the use of mathematical equations allows for the easy incorporation of this information into computer models.
RESULTS AND DISCUSSION Performance and Apparent Utilization Dry Matter and Retention of Zinc
of
Adding graded levels of Zn to the low Zn basal diet linearly (P < 0.01) increased BW gain and feed intake but did not influence gain to feed ratio of broilers (Table 2). In agreement, Dewar and Downie (1984), Hempe and Savage (1990), and Biehl et a\. (1995) reported that adding Zn (4 to 50 ppm) to the low Zn (0.9 to 13 ppm) diets of birds significantly increased growth performance. In our study, addition of graded levels of phytase to the basal diet linearly (P < 0.001) increased BW gain and feed intake but linearly decreased gain to feed ratio of broilers (P < 0.01). The improvement of BW gains resulting from the addition of phytase was primarily a result of increased feed intake. In agreement, Schoner et al. (1991), Yi et al. (1994b), and Denbow et al. (1996) observed improvements in BW gain and feed intake when phytase was added to the low P diets. (The P level was adequate in this study.) However, the gain to feed ratio was not affected by phytase in their studies. The decreased gain to feed ratio by adding phytase in our study may be due to the lower level of Zn in our diets. Even after the action of phytase, Zn in the diets may have not been adequate to meet the requirement of the birds.
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Statistical Analysis and Zinc Equivalency Values of Phytase
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PHYTASE A N D Z I N C U T I L I Z A T I O N TABLE 3. Effects of supplemental microbial phytase and zinc on ash and zinc contents in toes and tibias of broilers (Days 1 to 21)
Diet
Zn added
Phytase added
Toe ashi DM basis
(ppm)
(U/kg) 0 0 0 0 150 300 450 600
(%)
1 0 2 5 3 10 4 20 5 0 6 0 7 0 8 0 Pooled SEM J
12.5 12.7 12.7 12.7 13.1 12.9 13.6 13.27
Toe Zn 1 DM basis
(mg/toe) 15.3 18.0 17.7 18.83 16.6 17.5 19.0 17.48
0.21
(ppm) (Mg/toe)
(%)
71 88 113 1303 82 83 84 1049
36.5 36.9 36.8 36.7 37.4 37.4 37.4 37.810 0.53
5.3
8.8 12.5 15.6 19.33 10.3 10.7 11.7 14.4' 0.8
(mg/tibia) 570 611 633 636" 578 590 609 595 23
Tibia Zn2 DM basis
(ppm;1 (/ig/tibia) 125 146 208 2915 122 137 150 1519 7.8
195 247 356 507* 189 216 237 2309 17
mean. mean.
0.01). 0.05). < 0.05).
The amount of DM retained (Table 2) was linearly increased by adding Zn (P < 0.001) and phytase (P < 0.10), but DM retained as a percentage of intake was only linearly increased by adding Zn (P < 0.05). The amount of Zn retained (milligrams per bird) was linearly improved by adding Zn (P < 0.001) and phytase (P < 0.01); however, Zn retained as a percentage of intake was numerically decreased by adding Zn but was linearly increased by adding phytase (P < 0.10). In agreement, Thiel and Weigand (1992) reported that the addition of 800 U of phytase/kg of diet to a diet containing 27 ppm Zn increased Zn retention and decreased Zn excretion. However, Roberson and Edward (1994) reported that Zn retention was not influenced by adding 600 U of phytase/ kg of diet. This result may be related to the higher Zn contents (35 to 45 ppm) in their basal diet. However, the studies with Zn and phytase (Thiel and Weigand, 1992; Roberson and Edward, 1994) did not give the data of the total amount of Zn retained per bird.
Zinc Contents in Toe, Tibia, and Liver Ash percentage of toes and tibias (Table 3) was not affected by Zn levels, but were linearly increased by adding phytase (P < 0.05 for toes; P < 0.10 for tibias); however, the amount of ash (milligrams per toe or tibia) was linearly increased by Zn (P < 0.001 for toes; P < 0.05 for tibias) and phytase (P < 0.01 for toes; numerical for tibias). The concentration (parts per million, DM basis) and amount (micrograms per toe or tibia) of Zn in toe and tibia were linearly increased by adding Zn and phytase (P < 0.001). Adding 10 or 20 ppm Zn to the low Zn diets
resulted in an increase of Zn content by 60 to more than 100%. In the study of Biehl et al. (1995), adding 10 ppm Zn to the Zn deficient diet (13 ppm) for 12 d also increased total tibia Zn of birds by 107%. The magnitude of Zn content in toes and tibias indicates that bone Zn measurements are the most sensitive indicators for determining Zn utilization. This conclusion is also supported by the studies of Roberson and Edwards (1994) with chicks. They reported that adding 600 U of phytase/ kg of diet to the basal diet significantly increased bone Zn concentration (21 to 23 vs 23 to 25 m g / g ) , although it did not change Zn retention. Adding phytase increased the ash percentage of toes and tibias because it affected P and Ca utilization, which account for more than 50% of the ash (Qian et al, 1996). The amount of ash retained (milligrams per toe or tibia) is a good criterion for determining the effect of Zn and phytase. Both toe ash and tibia ash were linearly increased by adding Zn and phytase. The concentration of Zn in liver (parts per million) on a fresh or DM basis (Table 4) was linearly increased by adding Zn (P < 0.05 for fresh; P < 0.10 for DM) but did not show significant improvement from adding phytase. The amount of Zn retained (micrograms per bird) in the liver was linearly improved by adding Zn (P < 0.001) and phytase (P < 0.05). The Zn concentration of livers was reported by Schell and Kornegay (1994) and Cheng et al. (1995) to be a sensitive measurement to evaluate the bioavailability of Zn sources in young pigs. The Zn concentrations of livers of the birds in our study were increased by adding Zn and phytase, but the magnitude of increase by Zn addition was smaller than the increase in the Zn concentration of bones.
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Six pens (eight birds per pen) per treatment 2 Six pens (three birds per pen) per treatment 3 Zinc effect (linear, P < 0.001). 4 Zinc effect (linear, P < 0.05). 5 Zinc effect (linear, P < 0.001; quadratic, P 6 Zinc effect (linear, P < 0.001; quadratic, P 7 Phytase effect (linear, P < 0.05). 8 Phytase effect (linear, P < 0.01; quadratic, P 9 Phytase effect (linear, P < 0.001). "Phytase effect (linear, P < 0.10).
0.60
Tibia ash2 DM basis
544
YI ET Ah. TABLE 4. Effects of supplemental microbial phytase and zinc on the amount and concentration of zinc in liver of broilers (Days 1 to 21)1
Diet 1 2 3 4 5 6 7 8 Pooled SEM
Zn added
Phytase added
Zn fresh basis
(ppm)
(U/kg) 0 0 0 0 150 300 450 600
19.9 21.4 22.1 23.62 21.6 20.9 21.8 21.4
0 5 10 20 0 0 0 0
Zn retention
78.3 82.2 82.3 88.53 82.1 83.5 84.4 82.7 2.87
(/jg/bird) 202 221 270 2844 210 206 222 2445 16
— (ppm)i
0.81 per pen) per treatment mean. < 0.05). < 0.10). < 0.001). P < 0.05).
Zinc Equivalency Values of Phytase Nonlinear and linear response equations of birds fed graded levels of Zn or supplemental phytase were developed using treatment means of the various measurements (Table 5). The respective equations were solved and the derived equation for each of the measurements was used to calculate Zn equivalency values shown in Table 6. The addition of 150,300,450, and 600 U phytase/kg of diet to the low Zn corn-soybean isolate diets for broilers could release 1.4,2.7, 3.9, and 5.1 mg of Zn, respectively. The Zn equivalency equation, using averages of measurements
without BW gain and Zn in liver, was Y = 0.20 + 0.0082X (r2 = 0.99), where Y = Zn released (milligrams) and X = phytase (units per kilogram of diet). The results indicate that approximately 0.9 mg of Zn is released per 100 U of phytase over the range of 150 to 600 U of phytase. The basal diet was calculated to contain 0.21% phytate P (Table 1), and, thus, 11.3 mmol [2,100 mg x 0.287/648 molecular weight (MW)] of phytate. If we assume that one molecule of phytate complexes with one molecule of Zn, then 733 mg of Zn (11.3 mmol x 65 MW) could be combined with phytate. Thus, 18 mg of Zn (20 mg in basal diet - 2 mg from
TABLE 5. Nonlinear or linear response equations for the various measurements of broilers fed corn-soybean isolate diets with increased levels of zinc and supplemental phytase 1 Measurements Apparent DM utilization, mg/bird Apparent Zn retention, mg/bird Toe ash, mg/toe Zn in toe (DM basis), ppm Total Zn in toe, ng Tibia ash, mg/tibia Zn in tibia (DM basis), ppm Total Zn in tibia, /*g Total Zn in liver, ng Zn in liver (DM basis), ppm BW gain, g Apparent DM utilization, mg/bird Apparent Zn retention, mg/bird Toe ash, mg/toe Zn in toe (Dm basis), ppm Total Zn in toe, ^g Tibia ash, mg/tibia Zn in tibia (DM basis), ppm Total Zn in tibia, /xg Total Zn in liver, /tg Zn in liver (DM basis), ppm BW gain, g
Equation Zinc Y = 90.2(1 - 0.1570e^- 262ix ) Y = 15.6(1 - 0.5585e-o^ 7 3 x ) Y = 18.4(1 - 0.1710e-° 363ix ) 0573X ) Y = 159.7(1 • 0.5613e-° Y = 24.31(1 • 0.6405e-°-0570X) Y = 641.3(1 • 0.1128e-°-1628X) Y = 116.8 + 0.866X Y = 185.8 + 1.61X Y = 314.9(1 - 0.3698e-°° 71ix ) Y = 78.6 + 0.4797X Y = 443.1(1 - 0.1064e-°-2258X) Phytase Y = 89.1(1 -- 0.1579e-°0029X) Y = 9.85(1 -- 0.3153e-°0026X) Y = 18.3(1 -- 0.1712e-°-°°5iX) Y = 71.41 + 0.045X Y = 8.652 + 0.0084X Y = 599.6(1 - 0.0520e-°0038X) Y = 121.3 + 0.053X Y = 189.8 + 0.079X Y = 197.7 + 0.0641X ( Y = 83.6(1 -• 0.0637e-°° »5X) Y = 475.6(1 - 0.1748e-°0016X)
0.97 0.99 0.93 0.98 0.99 0.98 0.98 0.99 0.93 0.95 0.99 0.79 0.86 0.79 0.81 0.92 0.77 0.87 0.78 0.81 0.92 0.91
'Y = response measurement; X = Zn added (parts per million) or phytase added (units per kilogram of diet).
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'Six pens (three birds Zinc effect (linear, P 3 Zinc effect (linear, P 4 Zinc effect (linear, P 5 Phytase effect (linear, 2
Zn DM basis
PHYTASE AND ZINC UTILIZATION
545
TABLE 6. Calculated zinc equivalency values of microbial phytase in broilers fed corn-soybean isolate diets 1 Phytase Item
150 U / k g of diet
300 U / k g of diet
Apparent DM retention, g/bird Apparent Zn retention, mg/bird Toe ash retention, mg/toe Zn in toe, ppm DM Toe Zn retention, /ig/toe Tibia ash retention, mg/tibia Zn in tibia, ppm DM Tibia Zn retention, /ig/tibia Liver Zn retention, ^g/bird Zn in liver, ppm BW gain Mean 2 Mean (without BW gain) 3 Mean (without gain and Zn in liver) 4
1.3 1.5 1.9 1.6 1.4 1.2 1.4 1.0 1.1 7.8 1.6 2.0 2.0 1.4
2.7 2.9 3.9 3.2 3.0 2.1 2.4 1.7 2.4 9.8 4.0 3.5 3.4 2.7
450 U/kg of diet
600 U / k g of diet
4.0 3.9 5.6 4.8 4.8 2.6 3.3 2.5 3.9 10.3 7.9 4.9 4.6 3.9
5.3 4.6 7.0 6.6 6.7 2.9 4.2 3.2 5.5 10.4 19.0 6.9 5.6 5.1
(mg)
defluorinated phosphate) in the basal diet (Table 1) has the potential to totally complex with phytate. Therefore, the Zn equivalency values of phytase in this study are reasonable. Our findings are generally supported by the results of Biehl et al. (1995) and Thiel et al. (1993). Biehl et al. (1995) reported that using tibia Zn data and standardcurve methodology, the Zn equivalency values of 600 and 1,200 U of phytase/kg of diet were 3.8 and 5.5 mg Z n / k g diet, respectively, when phytase was added to the low Zn (13 ppm) basal diet. Thiel et al. (1993) found that the Zn concentration of femur from chicks fed a diet containing 30 mg Z n / k g of diet plus 700 U of phytase/kg of diet was equal to that of chicks fed a diet containing 39 mg Z n / k g of diet without phytase. The release of Zn is 2.0,3.5,4.9, and 6.9 mg, respectively for 150,300,450, and 600 U of phytase/kg of diet using the average calculated from all the measurements. The Zn equivalency equation based on average, including BW gain, was Y = 0.300 + 0.0107X (r2 = 0.99), where Y = Zn released (milligrams) and X - phytase (units per kilogram of diet). However, there was a very large increase of Zn equivalency values calculated with BW gain from 300 to 600 U of phytase/kg of diet. Adding 600 U of phytase/kg of diet could release 19 mg Zn, which is about 100% of the Zn content (20 m g / k g diet). This value was extremely high compared to the others. This high value may be due to the fact that phytase not only improved Zn utilization but also increased the utilization of other nutrients. Therefore, the BW gain values should be omitted from the average of the response measurements. When all BW gain values are removed, the release of Zn was 2.0,3.4,4.6, and 5.6 mg respectively for 150, 300, 450, and 600 U of phytase/kg of diet. Using average without BW gain, the
Zn equivalency equation was Y = 0.900 + 0.008X (r2 = 0.99), where Y = Zn released (milligrams) and X = phytase (units per kilogram of diet). Again, Zn equivalency values of phytase in the lower phytase levels (150 to 300 U / k g of diet) calculated from the measurement of Zn in liver were much higher than those from others. Although the response equation of phytase on the measurement of Zn in liver had a high r 2 (Table 5), the linear contrast was not significant (Table 4). Thus, the values calculated from Zn in livers should also be omitted from the average of the response measurements. A difference in mortality of the broilers in this study was not observed between the various treatments. The results of the present experiment indicate that microbial phytase is effective for improving Zn utilization in broilers fed a low Zn corn-soybean isolate diet. Approximately 0.9 mg of Zn was released per 100 U of phytase over the range of 150 to 600 U of phytase. The function of Zn equivalency values (Yj, milligrams per kilogram) of microbial phytase (Xj, units per kilogram of diet) is Y = 0.20 + 0.0082X over the range of 150 to 600 U of phytase.
ACKNOWLEDGMENTS Appreciation is expressed to BASF Corp., Mount Olive, NJ 07828-1234 for supplying phytase and for financial support, to Protein Technologies International, St Louis, MO 63125 for supplying soy isolate, to the John Lee Pratt Animal Nutrition Program for financial support, to Barbara Self for technical assistance, to Hao Qian, Jeng Cheng, and Max Wang for data collection, and to Cindy Hixon for preparation of the manuscript.
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'Equations for Zn and phytase see Table 5. Based on the means, the generated Zn equivalency equation is Y = 0.300 + 0.0107X (r2 = 0.99); where Y = Zn equivalency values of phytase (milligrams per kilogram of diet); X = added phytase (units per kilogram of diet). 3 The generated Zn equivalency equation is Y = 0.900 + 0.008X (r2 = 0.99); where Y and X are the same as above. 4 The generated Zn equivalency equation is Y = 0.20 + 0.0082X (r2 = 0.99); where Y and X are the same as above. 2
YI ET Ah.
546
This material is based o n w o r k s u p p o r t e d in p a r t b y the C o o p e r a t i v e State Research Service, USDA, u n d e r project N u m b e r 6129880.
REFERENCES
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Association of Official Analytic Chemists, 1990. Official Methods of Analysis. Association of Official Analytic Chemists, Washington DC. Biehl, R. R., D. H. Baker, and H. F. DeLuca, 1995. la-Hydroxylated cholecalciferol compounds act additively with microbial phytase to improve phosphorus, zinc and manganese in chicks fed soy-based diets. J. Nutr. 125: 2407-2416. Bobilya, D. J., M. R. Ellersieck, D. T. Gordon, and T. L. Veum, 1991. Bioavailability of zinc from nonfat dry milk, lowfat plain yogurt, and soy flour in diets fed to neonatal pigs. J. Agric. Food Chem. 39:1246-1251. Cheng, J., T. C. Schell, and E. T. Kornegay, 1995. Influence of dietary lysine on the utilization of zinc from zinc sulfate and a zinc lysine complex by young pigs. J. Anim. Sci. 73 (Suppl. 2):17. (Abstr.) Consortium, 1988. Guide for the Care and Use of Agricultural Animals in Agricultural Research and Teaching. Consortium For Developing a Guide for the Care and Use of Agricultural Animals in Agricultural Research and Teaching, Champaign, IL. Denbow, D. M., V. Ravindran, E. T. Kornegay, Z. Yi, and R. M. Hulet, 1995. Improving phosphorus availability in soybean meal for broilers by supplemental phytase. Poultry Sci. 74: 1831-1842. Dewar, W. A., and J. N. Downie, 1984. The zinc requirements of broiler chicks and turkey poults fed on purified diets. Br. J. Nutr. 51:467-477. Engelen, A. J., F. C , van der Heeft, P.H.G. Randsdorp, and E. L. C. Smit, 1994. Simple and rapid determination of phytase activity. J. AOAC 77:760-764. Ferguson, E. L., R. S. Gibson, L. U. Thompson, and S. Ounpuu, 1989. Dietary calcium, phytate, zinc, intake and the calcium, phytate, and zinc molar ratios of the diets of a selected group of East African children. Am. J. Clin. Nutr. 50:1450-1456. Hempe, J. M , and J. E. Savage, 1990. Autoclaved egg white as a protein source for chick diets low in zinc. Poultry Sci. 69: 959-965. Lease, J. G., 1966. The effect of autoclaving sesame meal on its phytic acid content and on the availability of its zinc to the chick. Poultry Sci. 45:237-241. Lei, X. G., P. K. Ku, E. R. Miller, D. E. Ullrey, and M. T. Yokoyama, 1993. Supplemental microbial phytase improves bioavailability of dietary zinc to weanling pigs. J. Nutr. 123:1117-1123. Lonnerdal, B., A.-S. Sandberg, B. Sandstrom, and C. Kunz, 1989. Inhibitory effects of phytic acid and other inositol phosphates on zinc and calcium absorption in suckling rats. J. Nutr. 119:211-214. Maddaiah, V. T., A. A. Kurnick, and B. L. Reid, 1964. Phytic acid studies. Proc. Soc. Exp. Biol. Med. 115:391-393. Morris, E. R, 1986. Phytate and dietary mineral bioavailability. Pages 57-76 in: Phytic Acid: Chemistry and Applications. E. Graf, ed. Pilatus Press, Minneapolis, MN. National Research Council, 1994. Nutrient Requirements of Poultry. 9th rev. ed. National Academy Press. Washington, DC.
Oberleas, D., 1973. Phytates. Pages 363-371 in: Toxicants Occurring Naturally in Foods. National Academy of Sciences, Washington, DC. Pallauf, J., D. Hohler, and G. Rimbach, 1992. Effect of microbial phytase supplementation to a maize-soya-diet on the apparent absorption of Mg, Fe, Cu, Mn and Zn and parameters of Zn-status in piglets. J. Anim. Physiol. Anim. Nutr. 68:1-9. Pallauf, J., G. Rimbach, S. Pippig, B. Schindler, D. Hohler, and E. Most, 1994a. Dietary effect of phytogenic phytase and an addition of microbial phytase to a diet based on field beans, wheat, peas and barley on the utilization of phosphorus and calcium, magnesium, zinc and protein in piglets. Z. Ernahrungswiss 33:128-135. Pallauf, J., G. Rimbach, S. Pippig, B. Schindler, and E. Most, 1994b. Effect of phytase supplementation to a phytate-rich diet based on wheat, barley and soya on the bioavailability of dietary phosphorus and calcium, magnesium, zinc and protein in piglets. Agribiol. Res. 47:39^18. Qian, H., E. T. Kornegay, and D. M. Denbow, 1996. Phosphorus equivalence of microbial phytase in turkey diets as influenced by calcium to P ratios and phosphorus levels. Poultry Sci. 75:69-81. Reddy, N. R., C. V. Balakrishnan, and D. K. Salunkhe, 1982. Phytates in legumes. Adv. Food Res. 28:1-92. Roberson, K. D., H. M. Edwards, Jr., 1994. Effects of 1, 25-dihydroxycholecalciferol and phytase on zinc utilization in broiler chicks. Poultry. Sci. 73:1312-1326. SAS Institute, 1990. SAS/STAT® User's Guide: Statistics. Version 6, 4th Edition. SAS Institute Inc., Cary, NC. Schell, T. C , and E. T. Kornegay, 1994. Comparison of Zn availability from ZnO, Zn-lysine, Zn-methionine and ZnS0 4 when fed at high concentrations to weanling pigs. J. Anim. Sci. (Suppl. 2):7. (Abstr.) Schoner, V. F. J., P. P. Hoppe, and G. Schwarz, 1991. Vergleich der effekte von mikrobieller phytase und anorganischem phosphat auf die leistungen und die retention von phosphoru, calcium und rohasche bei masthuhnerkuken in der anfangsmast. J. Anim. Physiol. Anim. Nutr. 66: 248-255. Simons, P.C.M., H.A.J. Versteegh, A. W. Jongbloed, P. A. Kemme, P. Slump, K. D. Bos, M.G.E. Wolters, R. F. Beudeker, and G. J. Verschoor, 1990. Improvement of phosphorus availability by microbial phytase in broilers and pigs. Br. J. Nutr. 64:525-540. Thiel, U., E. Weigand, P. P. Hoppe, and F. J. Schoner, 1993. Zinc retention of broiler chickens as affected by dietary supplementation of zinc and microbial phytase. Trace Elements in Man and Animals. M. Anker, D. Meissner, C. F. Mills, ed. TEMA 8:658-659. Thiel, U., and E. Weigand, 1992. Influence of dietary zinc and microbial phytase supplementation on Zn retention and Zn excretion in broiler chicks. Page 460 in: Proceedings of the World's Poultry Congress. Vol. 3. World's Poultry Science Association, Amsterdam, The Netherlands. Vohra, P., G. A. Gray, and F. H. Kratzer, 1965. Phytic acidmetal complexes. Proc. Soc. Exp. Biol. Med. 120:447^49. Yi, Z., E. T. Kornegay, M. D. Lindemann, and V. Ravindran, 1994a. Effectiveness of Natuphos® phytase for improving the bioavailabilities of phosphorus and other nutrients in soybean meal-based semi-purified diets for young pigs. J. Anim. Sci. 72(Suppl. 2):7. (Abstr.) Yi, Z., E. T. Kornegay, V. Ravindran, and D. M. Denbow, 1994b. Improving availability of corn and soybean meal P for broilers using Natuphos® phytase and calculation of replacement values of inorganic P by phytase. Poultry. Sci. 73(Suppl. 1):89. (Abstr.)
(Key words: broiler, phytase, zinc, utilization, equivalency value) 1996 Poultry Science 75:540-546
INTRODUCTION Phytate has complexing potential to form a wide variety of insoluble salts with cations such as Ca, Mg, Zn, and Cu (Vohra et al, 1965; Oberleas, 1973; Morris, 1986). Maddaiah et al (1964) and Reddy et al. (1982) reported that Zn has the strongest binding affinity with phytate. Phytate has been shown to impair the bioavailability of Zn in humans, rats, pigs, and chicks (Lease, 1966; Ferguson et al, 1989; Lonnerdal et al, 1989; Bobilya et al, 1991). Supplemental microbial phytase has been shown to be very effective for improving dietary phytate P bioavailability (Simons et al, 1990; Yi et al, 1994a,b; Denbow et al, 1995). A few studies also indicated that supplemental microbial phytase in the diets of young pigs improved the bioavailability and absorption or retention of Zn (Pallauf et al, 1992, 1994 a, b; Lei et al, 1993). Thiel et al (1993) found that the Zn
content of femur from chicks fed a diet containing 30 ppm Zn plus 700 U of phytase/kg of diet was equal to that of chicks fed a diet containing 39 ppm Zn without phytase. Roberson and Edward (1994) reported that adding phytase increased tibia Zn concentration but did not improve apparent Zn retention in broiler chicks. Biehl et al. (1995) found that the addition of 1,200 U of phytase/kg of diet to a glucose-soybean concentrate diet (13 ppm of Zn) increased growth rate by 40% and total tibia Zn by 107% in chicks. The objective of the present experiment was to investigate the influence of adding microbial phytase on Zn utilization in broilers fed a low Zn corn-soybean isolate diet and to calculate Zn equivalency values of phytase.
MATERIALS AND METHODS Birds, Diets, and Treatments The effect of microbial phytase on the utilization of Zn was investigated using 384 Peterson x Arbor Acres male broiler chicks fed corn-soybean isolate diets. The basal diet
Received for publication September 5, 1995. Accepted for publication December 6, 1995. !To whom correspondence should be addressed.
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ABSTRACT Day-old male broilers (n = 384) were used in a 21-d trial to investigate the effect of microbial phytase on the retention and utilization of Zn. A cornsoybean isolate basal diet containing 20 ppm Zn was fed alone and supplemented with 5, 10, or 20 ppm Zn as ZnS0 4 -7H 2 0 or with 150, 300, 450, or 600 U of phytase/ kg of diet. Total excreta were collected during Days 18 to 20. Toe, tibia, and liver samples were taken at the end of the experiment. Adding Zn and phytase to the low Zn basal diet linearly increased BW gain and feed intake of broilers (P < 0.01). The gain to feed ratio was not changed by adding Zn but was decreased by adding phytase (P < 0.01). The amount of DM retained was linearly increased by adding Zn and phytase (P < 0.10), but DM retained as a percentage of intake was only increased by adding Zn (P < 0.05). The amount of Zn retained per bird was linearly improved by adding Zn and phytase (P < 0.01). Zinc retained as a percentage of intake was linearly decreased by adding Zn but was linearly increased by adding phytase (P < 0.10). Ash
PHYTASE A N D Z I N C UTILIZATION TABLE 1. Percentage composition of basal diets
1
Percentage
Corn (8.5% CP) Soy isolate (92% CP) 2 Soybean oil Cornstarch 3 Limestone 4 Defluorinated phosphate 5 Vitamin premix 6 Trace mineral premix 7 Salt DL-Methionine Calculated nutrients CP Lysine Methionine + cystine Ca Total P Nonphytate P (nP) Ca:nP ratio
54.40 20.00 4.00 17.67 0.89 1.94 0.20 0.20 0.40 0.30 23.00 1.28 0.90 1.00 0.65 0.45 2.22
1
The assayed contents of Zn, total P, and Ca in the basal diet were 20 ppm, 0.61%, and 0.93%, respectively. Based on National Research Council (1994), the calculated phytate P in the basal diet was 0.21%. Three Zn levels of 5,10, and 20 ppm as ZnS0 4 -7H 2 0 and four phytase levels of 150, 300, 450, and 600 U / k g of diet were added to this basal diet. The assayed phytase activity in the basal diet and the diets with added four levels of phytase were 26, 173, 320, 443, and 631 U / k g of diet, respectively. 2 PP500E, Protein Technologies International, St. Louis, MO. Nonphytate P was assumed to make up 40% of total P in soy isolate, the same as reported for soy concentrate. 3 Food grade, National Starch and Chemical Co., Bridgewater, NJ 08807. "Limestone Dust Corp., Bluefield, VA 24605. SFine CDP, Southern Bag Corp., Valdosta, GA 31083. Supplied per kilogram of diet: retinyl acetate, 908 /ig; cholecalciferol, 66 ng; dl-a-tocopherol acetate, 26 mg; menadione sodium bisulfite, 0.75 mg; riboflavin, 7.5 mg; d-calcium pantothenate, 9.7 mg; niacin, 26.4 mg; cyanocobalamin, 0.011 mg; choline chloride, 1,012 mg; d-biotin, 0.31 mg; folic acid, 3.1 mg; thiamin-HCl, 8 mg; pyridoxine-HCl, 3.1 mg; ethoxyquin, 50 mg; and virginiamycin, 2.9 mg. 7 Supplied per kilogram of diet: manganese, 60 mg; iron, 80 mg; copper, 8 mg; iodine, 0.35 mg; and selenium, 0.15 mg.
met recommended (National Research Council, 1994) nutrient requirements except for that of Zn (Table 1). The calculated and assayed Zn content of the basal diet was 20 ppm. The assayed Zn contents of corn, soybean isolate, cornstarch, and defluorinated phosphate were 19, 38, 2.5, and 65 ppm, respectively. Eight dietary treatments were as follows: low Zn basal diet alone, three levels of Zn (5,10, and 20 ppm Zn as ZnSC^-TT^O)2 or four levels of Natuphos® phytase3 (150, 300,450, and 600 U / k g of diet) were added to the low Zn basal diet. A unit of phytase is defined as the quantity of enzyme that liberates 1 pimol of inorganic orthophosphate/min from 5.1 mM sodium
2
USP/FCC, Fisher Chemical, Fisher Scientific, Fair Lawn, NJ 07410. BASF Corp., Mount Olive, NJ 07828-1234. "Alternative Design, Manufacturing, and Supply, Inc., Siloam Springs, AR 72701. 5 Rolp 120-TF, Hydro, Research Triangle Park, NC 27709. 6 Model 5100 PC, Perkin-Elmer, Norwalk, CT 06859-0200. 3
phytate at pH 5.5 and 37 C (Engelen et al., 1994). The assayed dietary phytase activity was 26,173, 320,443, and 631 U / k g of diet, respectively, for the added phytase levels of 0, 150, 300, 450, and 600 U / k g of diet. The basal diet without added Zn or phytase was fed as a control. Defluorinated phosphate and ground limestone were used to maintain the desired P and Ca levels. The Ca: nonphytate P was 2.22:1 in all diets, as recommended by the National Research Council (1994). The assayed total P and Ca contents in the basal diets were 0.61 and 0.93%, respectively. Based on National Research Council studies (1994), the calculated phytate P in the basal diet was 0.21% and the nonphytate P was 0.45%.
Feeding and Management Broilers were randomly allotted on the day of hatch to 48 pens (eight birds per pen). They were housed in electrically heated, stainless steel starting batteries 4 in an environmentally controlled room. The eight treatments were randomly assigned to 48 pens (6 pens per treatment). The diets were fed in a mash form. Birds had ad libitum access to feed and deionized water produced by a reverse osmosis system. 5 Zinc content of the water was 0.3 ppm. The care and treatment of birds followed published guidelines (Consortium, 1988). Body weight and feed intake of the birds were recorded on a pen basis at weekly intervals during the 21-d experiment. Mortality was recorded daily.
Sampling and Analysis The total excreta of the birds were collected during Days 18 to 20. Feed intakes were recorded starting 24 h before the collection time and ending 24 h before the end of collection. The excreta were kept in plastic bags and dried in a stainless steel oven at 60 C and ground. All the birds were killed by cervical dislocation at the end of the experiment. Toe samples of the killed birds were obtained by severing the middle toe through the joint between the second and third tarsal bones from the distal end. The left middle toes of the birds within a pen were pooled, and the right middle toes from the same birds were pooled, yielding two samples of toes per pen. The composite samples were dried to a constant weight at 100 C and then ashed in a muffle furnace at 600 C for 4 h. Three killed birds from each pen were randomly selected and samples of liver and left tibia were collected. After the soft tissues were removed, tibia samples were dried to a constant weight at 100 C and then ashed in a muffle furnace at 600 C for 4 h. The ash from toes and tibias was solubilized with nitric and perchloric acid (5:3, vol/vol), and diets, excreta and liver were wet acid digested with the nitric and perchloric acid mixture (Association of Official Analytic Chemists, 1990). Zinc concentrations in diets, excreta, liver, toe, and tibia ash were analyzed with an atomic absorption spectrophotometer. 6
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Ingredients
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YI ET AL. TABLE 2. Effects of supplemental microbial phytase and zinc on performance (Days 1 to 21), apparent dry matter utilization (Days 18 to 20) and zinc retention of broilers 1
Diet
Zn added
Phytase added
(ppm)
(U/kg) 0 0 0 0 150 300 450 600
1 0 2 5 3 10 4 20 5 0 6 0 7 0 8 0 Pooled SEM
BW gain
Feed intake - (g/bird)
396 428 437 4432 398 431 440 440 s 11
530 600 605 6162 551 617 670 630« 20
Gain: feed
DM ]Retention
(g/kg)
(%)
747 714 721 722 724 700 660 7027
83.7 83.2 83.7 84.73 83.3 83.9 83.8 83.5 0.40
16
(g/bird) 75.9 87.2 87.6 91.1 4 77.6 86.1 86.1 85.68 3.5
Zn Retention
(%) 37.9 35.8 35.4 34.2 39.2 44.0 43.0 44.88 3.0
(mg/bird) 6.89 9.39 11.14 13.344 7.23 8.97 8.81 9.09» 0.95
!Six pens (eight birds per pen) per treatment mean. The values of apparent retention were based on total collection of excreta from Day 18 to 20. Zinc effect (linear, P < 0.01). 3 Zinc effect (linear and quadratic, P < 0.05). 4 Zinc effect (linear, P < 0.001). 5 Phytase effect (linear, P < 0.001). 6 Phytase effect (linear, 0.001; quadratic, P < 0.10). 7 Phytase effect (linear, 0.01; quadratic, P < 0.05). 8 Phytase effect (linear, 0.10). 9 Phytase effect (linear, P 0.01). 2
The data were subjected to analysis of variance by the General Linear Models (GLM) procedures of SAS® (SAS Institute, 1990). The model included diet and replicate with linear and quadratic contrast for effects of supplemental Zn or microbial phytase. Nonlinear and linear functions that best fit the data were derived for four Zn levels and for five phytase levels with the nonlinear model: Y = a(l -be _ l c X ) and linear model: Y = a + bX; where Y = the response measurements; and X = Zn (parts per million) added or phytase added (units per kilogram of diet). The nonlinear or linear response equations for added Zn and added phytase levels with higher r 2 values were set equal and solved as shown in the example below for amount of Zn retained in toe (Table 5): 159.7(1 - 0.5613e-00573X1) = 71.4 + rj.045X2 Xa = -17.45Ln(0.9851 - 0.0005X2); where Xi = Zn added (parts per million); and X2 = phytase added (units per kilogram of diet). For example, give phytase (X2) = 450 U / k g of diet, the Zn equivalency value (Xi) = -17.45 Ln(0.9851 - 0.0005 x 450) and Xi = 4.8 mg (Table 5). This result means that the amount of Zn retained in toe by 450 U of phytase/kg of diet is equal to that of 4.8 mg Zn as ZnSC>4-7H20. The resulting equations were used to calculate the Zn equivalency values of microbial phytase at 150, 300, 450, and 600 U / k g of diet for the various measurements. The use of a regression line (equation) generated from graded levels of Zn or phytase provides a more accurate means of estimating a response
than a single number. Also, the generation of a Zn equation from the equations for Zn and for phytase allows for the calculation of the Zn equivalency values of phytase for any point on the line. Further, the use of mathematical equations allows for the easy incorporation of this information into computer models.
RESULTS AND DISCUSSION Performance and Apparent Utilization Dry Matter and Retention of Zinc
of
Adding graded levels of Zn to the low Zn basal diet linearly (P < 0.01) increased BW gain and feed intake but did not influence gain to feed ratio of broilers (Table 2). In agreement, Dewar and Downie (1984), Hempe and Savage (1990), and Biehl et a\. (1995) reported that adding Zn (4 to 50 ppm) to the low Zn (0.9 to 13 ppm) diets of birds significantly increased growth performance. In our study, addition of graded levels of phytase to the basal diet linearly (P < 0.001) increased BW gain and feed intake but linearly decreased gain to feed ratio of broilers (P < 0.01). The improvement of BW gains resulting from the addition of phytase was primarily a result of increased feed intake. In agreement, Schoner et al. (1991), Yi et al. (1994b), and Denbow et al. (1996) observed improvements in BW gain and feed intake when phytase was added to the low P diets. (The P level was adequate in this study.) However, the gain to feed ratio was not affected by phytase in their studies. The decreased gain to feed ratio by adding phytase in our study may be due to the lower level of Zn in our diets. Even after the action of phytase, Zn in the diets may have not been adequate to meet the requirement of the birds.
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Statistical Analysis and Zinc Equivalency Values of Phytase
543
PHYTASE A N D Z I N C U T I L I Z A T I O N TABLE 3. Effects of supplemental microbial phytase and zinc on ash and zinc contents in toes and tibias of broilers (Days 1 to 21)
Diet
Zn added
Phytase added
Toe ashi DM basis
(ppm)
(U/kg) 0 0 0 0 150 300 450 600
(%)
1 0 2 5 3 10 4 20 5 0 6 0 7 0 8 0 Pooled SEM J
12.5 12.7 12.7 12.7 13.1 12.9 13.6 13.27
Toe Zn 1 DM basis
(mg/toe) 15.3 18.0 17.7 18.83 16.6 17.5 19.0 17.48
0.21
(ppm) (Mg/toe)
(%)
71 88 113 1303 82 83 84 1049
36.5 36.9 36.8 36.7 37.4 37.4 37.4 37.810 0.53
5.3
8.8 12.5 15.6 19.33 10.3 10.7 11.7 14.4' 0.8
(mg/tibia) 570 611 633 636" 578 590 609 595 23
Tibia Zn2 DM basis
(ppm;1 (/ig/tibia) 125 146 208 2915 122 137 150 1519 7.8
195 247 356 507* 189 216 237 2309 17
mean. mean.
0.01). 0.05). < 0.05).
The amount of DM retained (Table 2) was linearly increased by adding Zn (P < 0.001) and phytase (P < 0.10), but DM retained as a percentage of intake was only linearly increased by adding Zn (P < 0.05). The amount of Zn retained (milligrams per bird) was linearly improved by adding Zn (P < 0.001) and phytase (P < 0.01); however, Zn retained as a percentage of intake was numerically decreased by adding Zn but was linearly increased by adding phytase (P < 0.10). In agreement, Thiel and Weigand (1992) reported that the addition of 800 U of phytase/kg of diet to a diet containing 27 ppm Zn increased Zn retention and decreased Zn excretion. However, Roberson and Edward (1994) reported that Zn retention was not influenced by adding 600 U of phytase/ kg of diet. This result may be related to the higher Zn contents (35 to 45 ppm) in their basal diet. However, the studies with Zn and phytase (Thiel and Weigand, 1992; Roberson and Edward, 1994) did not give the data of the total amount of Zn retained per bird.
Zinc Contents in Toe, Tibia, and Liver Ash percentage of toes and tibias (Table 3) was not affected by Zn levels, but were linearly increased by adding phytase (P < 0.05 for toes; P < 0.10 for tibias); however, the amount of ash (milligrams per toe or tibia) was linearly increased by Zn (P < 0.001 for toes; P < 0.05 for tibias) and phytase (P < 0.01 for toes; numerical for tibias). The concentration (parts per million, DM basis) and amount (micrograms per toe or tibia) of Zn in toe and tibia were linearly increased by adding Zn and phytase (P < 0.001). Adding 10 or 20 ppm Zn to the low Zn diets
resulted in an increase of Zn content by 60 to more than 100%. In the study of Biehl et al. (1995), adding 10 ppm Zn to the Zn deficient diet (13 ppm) for 12 d also increased total tibia Zn of birds by 107%. The magnitude of Zn content in toes and tibias indicates that bone Zn measurements are the most sensitive indicators for determining Zn utilization. This conclusion is also supported by the studies of Roberson and Edwards (1994) with chicks. They reported that adding 600 U of phytase/ kg of diet to the basal diet significantly increased bone Zn concentration (21 to 23 vs 23 to 25 m g / g ) , although it did not change Zn retention. Adding phytase increased the ash percentage of toes and tibias because it affected P and Ca utilization, which account for more than 50% of the ash (Qian et al, 1996). The amount of ash retained (milligrams per toe or tibia) is a good criterion for determining the effect of Zn and phytase. Both toe ash and tibia ash were linearly increased by adding Zn and phytase. The concentration of Zn in liver (parts per million) on a fresh or DM basis (Table 4) was linearly increased by adding Zn (P < 0.05 for fresh; P < 0.10 for DM) but did not show significant improvement from adding phytase. The amount of Zn retained (micrograms per bird) in the liver was linearly improved by adding Zn (P < 0.001) and phytase (P < 0.05). The Zn concentration of livers was reported by Schell and Kornegay (1994) and Cheng et al. (1995) to be a sensitive measurement to evaluate the bioavailability of Zn sources in young pigs. The Zn concentrations of livers of the birds in our study were increased by adding Zn and phytase, but the magnitude of increase by Zn addition was smaller than the increase in the Zn concentration of bones.
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Six pens (eight birds per pen) per treatment 2 Six pens (three birds per pen) per treatment 3 Zinc effect (linear, P < 0.001). 4 Zinc effect (linear, P < 0.05). 5 Zinc effect (linear, P < 0.001; quadratic, P 6 Zinc effect (linear, P < 0.001; quadratic, P 7 Phytase effect (linear, P < 0.05). 8 Phytase effect (linear, P < 0.01; quadratic, P 9 Phytase effect (linear, P < 0.001). "Phytase effect (linear, P < 0.10).
0.60
Tibia ash2 DM basis
544
YI ET Ah. TABLE 4. Effects of supplemental microbial phytase and zinc on the amount and concentration of zinc in liver of broilers (Days 1 to 21)1
Diet 1 2 3 4 5 6 7 8 Pooled SEM
Zn added
Phytase added
Zn fresh basis
(ppm)
(U/kg) 0 0 0 0 150 300 450 600
19.9 21.4 22.1 23.62 21.6 20.9 21.8 21.4
0 5 10 20 0 0 0 0
Zn retention
78.3 82.2 82.3 88.53 82.1 83.5 84.4 82.7 2.87
(/jg/bird) 202 221 270 2844 210 206 222 2445 16
— (ppm)i
0.81 per pen) per treatment mean. < 0.05). < 0.10). < 0.001). P < 0.05).
Zinc Equivalency Values of Phytase Nonlinear and linear response equations of birds fed graded levels of Zn or supplemental phytase were developed using treatment means of the various measurements (Table 5). The respective equations were solved and the derived equation for each of the measurements was used to calculate Zn equivalency values shown in Table 6. The addition of 150,300,450, and 600 U phytase/kg of diet to the low Zn corn-soybean isolate diets for broilers could release 1.4,2.7, 3.9, and 5.1 mg of Zn, respectively. The Zn equivalency equation, using averages of measurements
without BW gain and Zn in liver, was Y = 0.20 + 0.0082X (r2 = 0.99), where Y = Zn released (milligrams) and X = phytase (units per kilogram of diet). The results indicate that approximately 0.9 mg of Zn is released per 100 U of phytase over the range of 150 to 600 U of phytase. The basal diet was calculated to contain 0.21% phytate P (Table 1), and, thus, 11.3 mmol [2,100 mg x 0.287/648 molecular weight (MW)] of phytate. If we assume that one molecule of phytate complexes with one molecule of Zn, then 733 mg of Zn (11.3 mmol x 65 MW) could be combined with phytate. Thus, 18 mg of Zn (20 mg in basal diet - 2 mg from
TABLE 5. Nonlinear or linear response equations for the various measurements of broilers fed corn-soybean isolate diets with increased levels of zinc and supplemental phytase 1 Measurements Apparent DM utilization, mg/bird Apparent Zn retention, mg/bird Toe ash, mg/toe Zn in toe (DM basis), ppm Total Zn in toe, ng Tibia ash, mg/tibia Zn in tibia (DM basis), ppm Total Zn in tibia, /*g Total Zn in liver, ng Zn in liver (DM basis), ppm BW gain, g Apparent DM utilization, mg/bird Apparent Zn retention, mg/bird Toe ash, mg/toe Zn in toe (Dm basis), ppm Total Zn in toe, ^g Tibia ash, mg/tibia Zn in tibia (DM basis), ppm Total Zn in tibia, /xg Total Zn in liver, /tg Zn in liver (DM basis), ppm BW gain, g
Equation Zinc Y = 90.2(1 - 0.1570e^- 262ix ) Y = 15.6(1 - 0.5585e-o^ 7 3 x ) Y = 18.4(1 - 0.1710e-° 363ix ) 0573X ) Y = 159.7(1 • 0.5613e-° Y = 24.31(1 • 0.6405e-°-0570X) Y = 641.3(1 • 0.1128e-°-1628X) Y = 116.8 + 0.866X Y = 185.8 + 1.61X Y = 314.9(1 - 0.3698e-°° 71ix ) Y = 78.6 + 0.4797X Y = 443.1(1 - 0.1064e-°-2258X) Phytase Y = 89.1(1 -- 0.1579e-°0029X) Y = 9.85(1 -- 0.3153e-°0026X) Y = 18.3(1 -- 0.1712e-°-°°5iX) Y = 71.41 + 0.045X Y = 8.652 + 0.0084X Y = 599.6(1 - 0.0520e-°0038X) Y = 121.3 + 0.053X Y = 189.8 + 0.079X Y = 197.7 + 0.0641X ( Y = 83.6(1 -• 0.0637e-°° »5X) Y = 475.6(1 - 0.1748e-°0016X)
0.97 0.99 0.93 0.98 0.99 0.98 0.98 0.99 0.93 0.95 0.99 0.79 0.86 0.79 0.81 0.92 0.77 0.87 0.78 0.81 0.92 0.91
'Y = response measurement; X = Zn added (parts per million) or phytase added (units per kilogram of diet).
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'Six pens (three birds Zinc effect (linear, P 3 Zinc effect (linear, P 4 Zinc effect (linear, P 5 Phytase effect (linear, 2
Zn DM basis
PHYTASE AND ZINC UTILIZATION
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TABLE 6. Calculated zinc equivalency values of microbial phytase in broilers fed corn-soybean isolate diets 1 Phytase Item
150 U / k g of diet
300 U / k g of diet
Apparent DM retention, g/bird Apparent Zn retention, mg/bird Toe ash retention, mg/toe Zn in toe, ppm DM Toe Zn retention, /ig/toe Tibia ash retention, mg/tibia Zn in tibia, ppm DM Tibia Zn retention, /ig/tibia Liver Zn retention, ^g/bird Zn in liver, ppm BW gain Mean 2 Mean (without BW gain) 3 Mean (without gain and Zn in liver) 4
1.3 1.5 1.9 1.6 1.4 1.2 1.4 1.0 1.1 7.8 1.6 2.0 2.0 1.4
2.7 2.9 3.9 3.2 3.0 2.1 2.4 1.7 2.4 9.8 4.0 3.5 3.4 2.7
450 U/kg of diet
600 U / k g of diet
4.0 3.9 5.6 4.8 4.8 2.6 3.3 2.5 3.9 10.3 7.9 4.9 4.6 3.9
5.3 4.6 7.0 6.6 6.7 2.9 4.2 3.2 5.5 10.4 19.0 6.9 5.6 5.1
(mg)
defluorinated phosphate) in the basal diet (Table 1) has the potential to totally complex with phytate. Therefore, the Zn equivalency values of phytase in this study are reasonable. Our findings are generally supported by the results of Biehl et al. (1995) and Thiel et al. (1993). Biehl et al. (1995) reported that using tibia Zn data and standardcurve methodology, the Zn equivalency values of 600 and 1,200 U of phytase/kg of diet were 3.8 and 5.5 mg Z n / k g diet, respectively, when phytase was added to the low Zn (13 ppm) basal diet. Thiel et al. (1993) found that the Zn concentration of femur from chicks fed a diet containing 30 mg Z n / k g of diet plus 700 U of phytase/kg of diet was equal to that of chicks fed a diet containing 39 mg Z n / k g of diet without phytase. The release of Zn is 2.0,3.5,4.9, and 6.9 mg, respectively for 150,300,450, and 600 U of phytase/kg of diet using the average calculated from all the measurements. The Zn equivalency equation based on average, including BW gain, was Y = 0.300 + 0.0107X (r2 = 0.99), where Y = Zn released (milligrams) and X - phytase (units per kilogram of diet). However, there was a very large increase of Zn equivalency values calculated with BW gain from 300 to 600 U of phytase/kg of diet. Adding 600 U of phytase/kg of diet could release 19 mg Zn, which is about 100% of the Zn content (20 m g / k g diet). This value was extremely high compared to the others. This high value may be due to the fact that phytase not only improved Zn utilization but also increased the utilization of other nutrients. Therefore, the BW gain values should be omitted from the average of the response measurements. When all BW gain values are removed, the release of Zn was 2.0,3.4,4.6, and 5.6 mg respectively for 150, 300, 450, and 600 U of phytase/kg of diet. Using average without BW gain, the
Zn equivalency equation was Y = 0.900 + 0.008X (r2 = 0.99), where Y = Zn released (milligrams) and X = phytase (units per kilogram of diet). Again, Zn equivalency values of phytase in the lower phytase levels (150 to 300 U / k g of diet) calculated from the measurement of Zn in liver were much higher than those from others. Although the response equation of phytase on the measurement of Zn in liver had a high r 2 (Table 5), the linear contrast was not significant (Table 4). Thus, the values calculated from Zn in livers should also be omitted from the average of the response measurements. A difference in mortality of the broilers in this study was not observed between the various treatments. The results of the present experiment indicate that microbial phytase is effective for improving Zn utilization in broilers fed a low Zn corn-soybean isolate diet. Approximately 0.9 mg of Zn was released per 100 U of phytase over the range of 150 to 600 U of phytase. The function of Zn equivalency values (Yj, milligrams per kilogram) of microbial phytase (Xj, units per kilogram of diet) is Y = 0.20 + 0.0082X over the range of 150 to 600 U of phytase.
ACKNOWLEDGMENTS Appreciation is expressed to BASF Corp., Mount Olive, NJ 07828-1234 for supplying phytase and for financial support, to Protein Technologies International, St Louis, MO 63125 for supplying soy isolate, to the John Lee Pratt Animal Nutrition Program for financial support, to Barbara Self for technical assistance, to Hao Qian, Jeng Cheng, and Max Wang for data collection, and to Cindy Hixon for preparation of the manuscript.
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'Equations for Zn and phytase see Table 5. Based on the means, the generated Zn equivalency equation is Y = 0.300 + 0.0107X (r2 = 0.99); where Y = Zn equivalency values of phytase (milligrams per kilogram of diet); X = added phytase (units per kilogram of diet). 3 The generated Zn equivalency equation is Y = 0.900 + 0.008X (r2 = 0.99); where Y and X are the same as above. 4 The generated Zn equivalency equation is Y = 0.20 + 0.0082X (r2 = 0.99); where Y and X are the same as above. 2
YI ET Ah.
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This material is based o n w o r k s u p p o r t e d in p a r t b y the C o o p e r a t i v e State Research Service, USDA, u n d e r project N u m b e r 6129880.
REFERENCES
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Association of Official Analytic Chemists, 1990. Official Methods of Analysis. Association of Official Analytic Chemists, Washington DC. Biehl, R. R., D. H. Baker, and H. F. DeLuca, 1995. la-Hydroxylated cholecalciferol compounds act additively with microbial phytase to improve phosphorus, zinc and manganese in chicks fed soy-based diets. J. Nutr. 125: 2407-2416. Bobilya, D. J., M. R. Ellersieck, D. T. Gordon, and T. L. Veum, 1991. Bioavailability of zinc from nonfat dry milk, lowfat plain yogurt, and soy flour in diets fed to neonatal pigs. J. Agric. Food Chem. 39:1246-1251. Cheng, J., T. C. Schell, and E. T. Kornegay, 1995. Influence of dietary lysine on the utilization of zinc from zinc sulfate and a zinc lysine complex by young pigs. J. Anim. Sci. 73 (Suppl. 2):17. (Abstr.) Consortium, 1988. Guide for the Care and Use of Agricultural Animals in Agricultural Research and Teaching. Consortium For Developing a Guide for the Care and Use of Agricultural Animals in Agricultural Research and Teaching, Champaign, IL. Denbow, D. M., V. Ravindran, E. T. Kornegay, Z. Yi, and R. M. Hulet, 1995. Improving phosphorus availability in soybean meal for broilers by supplemental phytase. Poultry Sci. 74: 1831-1842. Dewar, W. A., and J. N. Downie, 1984. The zinc requirements of broiler chicks and turkey poults fed on purified diets. Br. J. Nutr. 51:467-477. Engelen, A. J., F. C , van der Heeft, P.H.G. Randsdorp, and E. L. C. Smit, 1994. Simple and rapid determination of phytase activity. J. AOAC 77:760-764. Ferguson, E. L., R. S. Gibson, L. U. Thompson, and S. Ounpuu, 1989. Dietary calcium, phytate, zinc, intake and the calcium, phytate, and zinc molar ratios of the diets of a selected group of East African children. Am. J. Clin. Nutr. 50:1450-1456. Hempe, J. M , and J. E. Savage, 1990. Autoclaved egg white as a protein source for chick diets low in zinc. Poultry Sci. 69: 959-965. Lease, J. G., 1966. The effect of autoclaving sesame meal on its phytic acid content and on the availability of its zinc to the chick. Poultry Sci. 45:237-241. Lei, X. G., P. K. Ku, E. R. Miller, D. E. Ullrey, and M. T. Yokoyama, 1993. Supplemental microbial phytase improves bioavailability of dietary zinc to weanling pigs. J. Nutr. 123:1117-1123. Lonnerdal, B., A.-S. Sandberg, B. Sandstrom, and C. Kunz, 1989. Inhibitory effects of phytic acid and other inositol phosphates on zinc and calcium absorption in suckling rats. J. Nutr. 119:211-214. Maddaiah, V. T., A. A. Kurnick, and B. L. Reid, 1964. Phytic acid studies. Proc. Soc. Exp. Biol. Med. 115:391-393. Morris, E. R, 1986. Phytate and dietary mineral bioavailability. Pages 57-76 in: Phytic Acid: Chemistry and Applications. E. Graf, ed. Pilatus Press, Minneapolis, MN. National Research Council, 1994. Nutrient Requirements of Poultry. 9th rev. ed. National Academy Press. Washington, DC.
Oberleas, D., 1973. Phytates. Pages 363-371 in: Toxicants Occurring Naturally in Foods. National Academy of Sciences, Washington, DC. Pallauf, J., D. Hohler, and G. Rimbach, 1992. Effect of microbial phytase supplementation to a maize-soya-diet on the apparent absorption of Mg, Fe, Cu, Mn and Zn and parameters of Zn-status in piglets. J. Anim. Physiol. Anim. Nutr. 68:1-9. Pallauf, J., G. Rimbach, S. Pippig, B. Schindler, D. Hohler, and E. Most, 1994a. Dietary effect of phytogenic phytase and an addition of microbial phytase to a diet based on field beans, wheat, peas and barley on the utilization of phosphorus and calcium, magnesium, zinc and protein in piglets. Z. Ernahrungswiss 33:128-135. Pallauf, J., G. Rimbach, S. Pippig, B. Schindler, and E. Most, 1994b. Effect of phytase supplementation to a phytate-rich diet based on wheat, barley and soya on the bioavailability of dietary phosphorus and calcium, magnesium, zinc and protein in piglets. Agribiol. Res. 47:39^18. Qian, H., E. T. Kornegay, and D. M. Denbow, 1996. Phosphorus equivalence of microbial phytase in turkey diets as influenced by calcium to P ratios and phosphorus levels. Poultry Sci. 75:69-81. Reddy, N. R., C. V. Balakrishnan, and D. K. Salunkhe, 1982. Phytates in legumes. Adv. Food Res. 28:1-92. Roberson, K. D., H. M. Edwards, Jr., 1994. Effects of 1, 25-dihydroxycholecalciferol and phytase on zinc utilization in broiler chicks. Poultry. Sci. 73:1312-1326. SAS Institute, 1990. SAS/STAT® User's Guide: Statistics. Version 6, 4th Edition. SAS Institute Inc., Cary, NC. Schell, T. C , and E. T. Kornegay, 1994. Comparison of Zn availability from ZnO, Zn-lysine, Zn-methionine and ZnS0 4 when fed at high concentrations to weanling pigs. J. Anim. Sci. (Suppl. 2):7. (Abstr.) Schoner, V. F. J., P. P. Hoppe, and G. Schwarz, 1991. Vergleich der effekte von mikrobieller phytase und anorganischem phosphat auf die leistungen und die retention von phosphoru, calcium und rohasche bei masthuhnerkuken in der anfangsmast. J. Anim. Physiol. Anim. Nutr. 66: 248-255. Simons, P.C.M., H.A.J. Versteegh, A. W. Jongbloed, P. A. Kemme, P. Slump, K. D. Bos, M.G.E. Wolters, R. F. Beudeker, and G. J. Verschoor, 1990. Improvement of phosphorus availability by microbial phytase in broilers and pigs. Br. J. Nutr. 64:525-540. Thiel, U., E. Weigand, P. P. Hoppe, and F. J. Schoner, 1993. Zinc retention of broiler chickens as affected by dietary supplementation of zinc and microbial phytase. Trace Elements in Man and Animals. M. Anker, D. Meissner, C. F. Mills, ed. TEMA 8:658-659. Thiel, U., and E. Weigand, 1992. Influence of dietary zinc and microbial phytase supplementation on Zn retention and Zn excretion in broiler chicks. Page 460 in: Proceedings of the World's Poultry Congress. Vol. 3. World's Poultry Science Association, Amsterdam, The Netherlands. Vohra, P., G. A. Gray, and F. H. Kratzer, 1965. Phytic acidmetal complexes. Proc. Soc. Exp. Biol. Med. 120:447^49. Yi, Z., E. T. Kornegay, M. D. Lindemann, and V. Ravindran, 1994a. Effectiveness of Natuphos® phytase for improving the bioavailabilities of phosphorus and other nutrients in soybean meal-based semi-purified diets for young pigs. J. Anim. Sci. 72(Suppl. 2):7. (Abstr.) Yi, Z., E. T. Kornegay, V. Ravindran, and D. M. Denbow, 1994b. Improving availability of corn and soybean meal P for broilers using Natuphos® phytase and calculation of replacement values of inorganic P by phytase. Poultry. Sci. 73(Suppl. 1):89. (Abstr.)