Utilization of phytate phosphorus and calcium as influenced by microbial phytase, cholecalciferol, and the calcium: total phosphorus ratio in broiler diets

METABOLISM AND NUTRITION Utilization of Phytate Phosphorus and Calcium as Influenced by Microbial Phytase, Cholecalciferol, and the Calcium:Total Phosphorus Ratio in Broiler Diets H. QIAN, E. T. KORNEGAY,1 and D. M. DENBOW Department of Animal and Poultry Sciences, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061-0306 the presence or absence of supplemental phytase and D3. Dietary Ca:tP ratios between 1.1:1 to 1.4:1 appears critical to the efficient use of supplemental phytase and D3 for improving the utilization of phytate P and Ca. The addition of D3 in corn-soybean meal diets indicated a potential for improving the utilization of phytate P and Ca by increasing Ca and P retention by about 5 to 12% in birds, which led to an increase in toe ash content (P < 0.03). The enhanced phytate P utilization (P < 0.001) was also observed during assay of the phytase activity in the mixed diets with an addition of D3 and without added phytase. In summary, the findings of this study suggested that phytase, D3, and Ca:tP are important factors in degrading phytate and improving phytate P and Ca utilization in broilers.

(Key words: phytase, cholecalciferol, calcium:total phosphorus ratio, phytate, broiler) 1997 Poultry Science 76:37–46

utilization of P in turkey poults (Qian et al., 1996a), which was accompanied by a decrease in phytase activity of the diet. However, limited data are available examining the effect of Ca:tP ratios on the phytase efficacy in broilers. It has been suggested that cholecalciferol (D3) could improve the phytate P and Ca absorption by stimulating the hydrolysis of phytate (Shafey et al., 1990; Mohammed et al., 1991). Edwards et al. (1992) conducted two experiments in which the phytate P utilization was greatly enhanced by the addition of 5 to 10 mg of 1, 25-(OH)2D3/kg diet in the presence or absence of supplemental phytase; an interaction between phytase and 1, 25-(OH)2D3 was only found for the metabolizable energy value in one experiment. Mohammed et al. (1991) found that supplemental D3 dramatically increased phytate P digestibility and the retention of P and Ca of the chick. However, this improvement by D3 addition was also influenced by dietary Ca because the addition of D3 could not totally overcome the P depletion except by simultaneously lowering dietary Ca. The purpose of the present study was to investigate the influence of supplemental phytase, vitamin D3, and dietary Ca:tP ratios on the utilization of phytate P and

INTRODUCTION Supplemental dietary microbial phytase has been shown to increase the availability of phytate P for poultry and pigs fed a commercial corn-soybean meal diet. The P equivalency of microbial phytase for 1 g of nonphytate (n) P is reported to be 650 to 750 U of phytase in broilers (Schoner et al., 1991; Kornegay et al., 1996; Yi et al., 1996), and 520 to 700 U of phytase in turkey poults (Qian et al., 1996a; Ravindran et al., 1995). These P-equivalent values of phytase were achieved when dietary Ca:total P (tP) ratios were formulated using a 2.0:1 Ca:tP ratio. Furthermore, the equivalence of phytase for nP was influenced by dietary Ca:tP ratios and nP levels because these two factors could affect not only phytate P release and P absorption in the small intestines, but also phytase activity (Wise, 1983; Schoner et al., 1993; Qian et al., 1995). A Ca:tP ratio greater than 1.4:1 was reported to decrease the performance and

Received for publication September 21, 1995. Accepted for publication August 7, 1996. 1To whom correspondence should be addressed.

37

Downloaded from http://ps.oxfordjournals.org/ at New York University on April 27, 2015

ABSTRACT The present study was performed to evaluate the potential of microbial phytase and cholecalciferol (D3) for improving the utilization of phytate P and Ca and the influence of the Ca:total (t) P ratio in a corn-soybean meal diet fed to broilers from hatch to 21 d of age. A 4 × 4 × 2 factorial arrangement of treatments was used: 1.1, 1.4, 1.7, and 2.0:1 Ca:tP ratio; 0, 300, 600, and 900 U of phytase/kg of diet; and 66 and 660 mg of D3/kg of diet. Another four treatments were included: the four Ca:tP ratios with 6,600 mg of D3 addition, but without phytase. Added phytase linearly increased (P < 0.001) BW gain, feed intake, toe ash content, and P and Ca retention; these measurements were negatively influenced by widening the dietary Ca:tP ratio, and synergetically improved by addition of D3. Increasing the Ca:tP ratio decreased (P < 0.001) all measurements in

38

QIAN ET AL.

Ca. Furthermore, the interaction of these three factors in broilers was examined.

MATERIALS AND METHODS

2Natuphos BASF 3 Sigma P-3168;

Corp., Mount Olive, NJ 07828-1234. Sigma Chemical Co., St. Louis, MO

63178-9916. 4Titertek Multiskan MCC/340: serial number 1EEE-448, Flow Laboratories Inc., McLean, VA 22102.

TABLE 1. Composition of the basal diet1 Ingredients and analysis Soybean meal (48.5% CP) Corn (8.8% CP) Canola oil Limestone2 Deflourinated phosphate3 Vitamin premix4 Trace mineral premix5 Salt DL-methionine Cornstarch Calculated analysis CP Metabolizable energy, kcal/kg Calcium Total P Nonphytate P Lysine Methionine plus cystine

Percentage 37.10 57.39 2.0 0.601 0.69 0.20 0.20 0.40 0.20 1.219 23.04 3,025.00 0.561 0.510 0.27 1.32 0.93

1Dietary Ca:tP ratios were formulated at 1.1, 1.4, 1.7, and 2.0 with defluorinated phosphate and limestone supplied at the expense of cornstarch, and each Ca:tP ratio was supplemented with 0, 300, 600, and 900 U phytase/kg diet at cholecalciferol levels of 66 and 660 mg/kg diet. Phytase (Natuphos 5,000 U/g) was supplied by BASF Corp., Mount Olive, NJ 07828-1234. 2Limestone Dust Corp., Bluefield, VA 24605. 3Fine CDP, Southern Bag Corp., Valdosta, GA 31083. 4Supplied per kilogram of diet: retinyl acetate, 908 mg; cholecalciferol, 66 mg; dl-a-tocopheryl acetate, 26.5 mg; menadione sodium bisulfate complex, 0.75 mg; riboflavin, 7.5 mg; d-calcium pantothenic acid, 9.7 mg; niacin, 26.4 mg; cyanocobalamin, 11 mg; choline chloride, 1,013 mg; biotin, 0.31 mg; folic acid, 3.1 mg; thiamin·HCl, 8 mg; pyridoxine·HCl, 3.1 mg; ethoxyquin, 50 mg; virginiamycin, 2.9 mg. 5Supplied per kilogram of diet: manganese, 88 mg; zinc, 95 mg; iron, 100 mg; copper, 12.5 mg; iodine, 4 mg; selenium, 0.6 mg.

Downloaded from http://ps.oxfordjournals.org/ at New York University on April 27, 2015

Day-old Peterson × Arbor Acres male broiler chicks (n = 864) were used in a 21-d experiment to investigate the utilization of phytate P and Ca in corn-soybean meal diets as influenced by microbial phytase,2 D3 and dietary Ca:tP ratios. Three replicate pens (eight birds per pen) of a completely randomized design were used in a 4 × 4 × 2 factorial arrangement of treatments with 1.1, 1.4, 1.7, and 2.0:1, Ca:tP ratios, with 0, 300, 600, and 900 U phytase/kg of diet, and with 66 and 660 mg of D3/kg of diet. In addition, another four treatments included the four Ca:tP ratios with 6,600 mg of D3 added without supplemental phytase. The dietary P level was formulated at 0.27% of nonphytate P (0.51% tP) for all diets, which was below the current NRC (1994) recommendations to ensure maximum responses with phytase addition (Table 1). The dietary percentage of phytate P (0.24%) was calculated by using the data of NRC (1994) and was similar in all diets. The Ca:tP ratios were formulated by varying limestone at the expense of cornstarch. A unit of phytase activity is defined as the quantity of enzyme that liberates 1 mmol of inorganic P/min from 5.1 mM sodium phytate at pH 5.5 and 37 C. Supplemented microbial phytase activity in diets was determined using a modification of the method of Engelen et al. (1994). A suspension of supplemental phytase enzyme was extracted from about 5 g of dietary samples when mixed with 50 mL of 0.25 M, pH 5.5 buffer solution. Two milliliters of the suspension was cultured with 4 mL of 8.4 g/L sodium phytate solution3 at 37 C for 1 h, and then colored and stopped by a mixed color-stop solution of ammonium molybdate, ammonium vanadate, and nitric acid. The concentration of P released from sodium phytate by supplemental phytase was colorimetrically determined at 415 nm. At 1 d of age, chicks were wing-banded and randomly assigned to pens in electrically heated, raised wire-floored battery brooders in an environmentally controlled room. Chicks were exposed to continuous fluorescent light. All diets were provided for ad libitum consumption in mash form and birds had free access to water. Body weights and feed consumption were recorded on a pen basis at weekly intervals. The care and treatment of birds followed published guidelines (Consortium, 1988). During the 3rd wk (Days 18 to 20), all excreta were collected from each pen and stored in plastic bags at –20

C. Feed intake and production of excreta were measured quantitatively per pen over 3 consecutive d. After thawing, excreta were dried at 65 C and weighed. Excreta and diet samples were ground to pass a 1-mm sieve. Dry matter was determined according to AOAC (1990) procedures. Following a nitric-perchloric acid wet digestion, P concentrations were determined colorimetrically (AOAC, 1990) using the computer program “Microkinetics” and vertical photometer.4 Concentrations of Ca were determined using atomic absorption spectrophotometer. Apparent retention of P and Ca were calculated. On Day 21, all surviving birds were killed by cervical dislocation. Toe samples were obtained by severing the middle toe through the joint between the second and third tarsal bones from the distal end. The left and right middle toes of all birds within a pen were pooled, respectively, yielding two samples of toes per pen. The pooled samples were dried to a constant weight at 100 C and ashed in a muffle furnace at 600 C for 6 h. Toe ash was expressed as a percentage of dry weight. Data were analyzed by the General Linear Models procedure of SAS (SAS Institute, 1990) using pen as the experimental unit. Linear and quadratic effects of the Ca:tP ratio and supplemental phytase were tested with

39

UTILIZATION OF PHYTATE PHOSPHORUS AND CALCIUM TABLE 2. Weight gain of broilers fed corn-soybean meal diets with four phytase levels, four calcium:total phosphorus (Ca:tP) ratios and three cholecalciferol (D3) levels from hatch to 21 d of age1 Ca:tP ratio3 Added phytase2

1.1:1

1.4:1

1.7:1

2.0:1

Mean

(g/bird) 553 576 576 615 580

550 547 570 568 559

499 533 566 560 539

420 496 528 541 496

506 538 560 571 544

563 561 611 611 588

553 568 590 567 569

496 532 581 549 539

462 493 498 542 499

518 538 570 569 549

566

543

508

451

517

1Each

mean represents three pens (eight birds per pen). The root MSE were 23.3 for BW gain and the pooled SEM for a treatment mean would equal MSE/√n. Main effect: Ca:tP ratio and phytase (P < 0.001); D3 (P < 0.09). Ca:tP ratio × phytase interaction (P = 0.11). 2Ca:tP ratio effect: linear (P < 0.003) and quadratic (P < 0.001). 3Phytase effect: linear (P < 0.001) and quadratic (P = 0.11). 4Cat:tP ratio effect: linear (P < 0.07) and quadratic (P < 0.001). Phytase effect: linear (P < 0.001). 5Ca:tP ratio effect: linear (P < 0.09) and quadratic (P < 0.001). Phytase effect: linear (P < 0.01).

orthogonal polynomials. The linear and quadratic effects of D3 treatments without added phytase were tested after supplemental phytase treatments were deleted from the data set. Second order translog functions were developed for the 2 × 4 factorial arrangement of treatments of D3 and phytase levels or Ca:tP ratios with the model: LnY = a0 + a1D1 + a2LnX + a3(lnX)2 + a4D1LnX where Y = the response measurements; D1 = 0 at D3 = 66 mg/kg diet; and D1 = 1 at D3 = 660 mg/kg diet; X = added phytase, units per kilogram of diet, or X = Ca:tP ratios. For the phytase and Ca:tP ratio effect, the model was used as LnY = a0 + a1LnX1 + a2LnX2 + a3(LnX1)2 + a4(LnX2)2 + a5(LnX1LnX2) where Y = the response measurements; X1 = added phytase, units per kilogram of diet; X2 = Ca:tP ratios. The second order translog function was chosen because Driscoll (1994) demonstrated that this function is a flexible functional form than can provide second order approximations to any underlying function. This second order translog model was used to determine the response surfaces to a combination of tiered levels of phytase, D3, and various ratios of Ca:tP, and to evaluate the sensitivity of measurements to dietary Ca:tP ratios, D3 and supplemental phytase levels.

RESULTS Main effects of phytase and Ca:tP ratio on BW gain were observed. Body weight gain increased (P < 0.001) as phytase level increased, and decreased (P < 0.001) as the Ca:tP ratio became greater (Table 2). The addition of D3 from 66 to 660 mg of D3/kg of diet did not increase BW gain significantly (P < 0.09). The magnitude of the responses to phytase was greatest for the smallest Ca:tP ratio at both D3 levels. The increase in BW gain by phytase supplements was linear up to 900 U of phytase/ kg diet at 66 mg of D3/kg diet, whereas at 660 mg of D3/ kg diet, BW gain was linear up to 600 U of phytase and then reached a plateau. The detrimental effect of Ca:tP ratio was independent of phytase and D3 levels, and greatest for the largest ratio and at the lower D3 level with no added phytase. The maximum BW gains resulted when dietary Ca:tP ratio was reduced to 1.1:1 at each supplemental level of phytase or D3. Main effects of phytase, Ca:tP ratio, and D3 on feed intake were similar to those for BW gain (Table 3). Feed intake increased (P < 0.001) as the phytase level increased, decreased (P < 0.01) as the Ca:tP ratio became wider, and increased (P < 0.04) from 66 to 660 mg D3/kg. The magnitude of the response to added phytase was greater at the lower levels of phytase and the wider Ca: tP ratio in both levels of D3. Increasing the Ca:tP ratios decreased feed intake, the effect being independent of both phytase and D3 levels, because two-way interactions were not significant (P > 0.10). No effects were

Downloaded from http://ps.oxfordjournals.org/ at New York University on April 27, 2015

66 mg of D3/kg diet4 0 U/kg 300 U/kg 600 U/kg 900 U/kg Mean 660 mg of D3/kg diet5 0 U/kg 300 U/kg 600 U/kg 900 Mean 6,600 mg of D3/kg diet 0 U/kg

40

QIAN ET AL. TABLE 3. Feed intake of broilers fed corn-soybean meal diets with four phytase levels, four calcium:total phosphorus (Ca:tP) ratios and three cholecalciferol (D3) levels from hatch to 21 d of age1 Ca:tP ratio3 Added phytase2

1.1:1

1.4:1

1.7:1

2.0:1

Mean

(g/bird) 797 823 859 835 828

763 763 844 832 826

785 779 835 817 804

644 726 787 777 734

756 773 831 816 803

896 854 936 927 903

807 851 885 917 865

731 801 855 813 780

717 765 757 805 760

788 817 858 865 832

909

773

755

673

777

1Each

mean represents three pens (eight birds per pen). The root MSE were 38.9 for feed intake and the pooled SEM for a treatment mean would equal MSE/√n . Main effect: Ca:tP ratio (P < 0.001); phytase (P < 0.01); D3 (P < 0.04). 2Ca:tP ratio effect: linear (P < 0.03) and quadratic (P < 0.001). 3Phytase effect: linear (P < 0.002). 4Cat:tP ratio effect: linear (P < 0.04) and quadratic (P < 0.001). Phytase effect: linear (P = 0.13). 5Ca:tP ratio effect: quadratic (P < 0.001). Phytase effect: linear (P < 0.02).

observed on feed efficiency; the overall mean was 669 g BW gain/kg of feed intake. Increasing D3 to 6,600 mg/kg did not provide any additional benefit above 660 mg/kg for improving BW gain and feed intake (Tables 2 and 3). The influence of phytase addition, Ca:tP ratio, and D3 on toe ash percentage tended to parallel that of BW gains and feed intake (P < 0.001, 0.001, and 0.03, respectively) (Table 4). The effects of these three factors appeared independent because two- and three-way interactions were not significant. Supplemental phytase linearly increased toe ash percentage at each D3 level (P < 0.001) and Ca:tP ratio (P < 0.004). The magnitude of the response to supplemental phytase was greatest at the largest Ca:tP ratio at each level of D3. Optimum dietary Ca:tP ratio was 1.4:1 for toe mineralization at 66 mg of D3/kg diet, and 1.1:1 at 660 mg of D3/kg diet. The highest values for toe ash were obtained at the two lower Ca:tP ratios and at the higher levels of phytase. By comparing only means without added phytase, toe ash percentage linearly increased as the level of D3 increased for all Ca:tP ratios. The retention of P was linearly increased as added phytase increased (P < 0.001), was quadratically increased (P < 0.001) as the Ca:tP ratio became smaller, and was higher (P < 0.001) for 660 vs 66 mg D3/kg diet (Table 5). Two- or three-way interactions of these three factors were not significant. At Ca:tP ratios of 1.1:1 and 1.4:1, the magnitude of the response to added phytase was much larger for diets with 660 mg D3 than for those with 66 mg D3. By comparing only means without added phytase, the retention of P linearly increased (P < 0.001)

as the level of D3 increased. The maximum retention of P at both levels of D3 was achieved when dietary Ca:tP ratio was formulated at 1.1:1 with 600 to 900 U of phytase/kg of diet. Similar to P retention, the retention of Ca linearly increased (P < 0.001) as the level of phytase increased, was quadratically (P < 0.001) increased as the Ca:tP ratio became smaller, and was only slightly higher (P < 0.07) for 660 vs 66 mg of D3/kg (Table 6). The increase in Ca retention by supplemental phytase was more dramatic in combination with a small Ca:tP ratio and high D3 level. The two-way interactions were not significant, suggesting that the response to phytase, Ca:tP ratio, and D3 were independent. The optimum response to the three dietary factors was observed when dietary Ca:tP ratio was formulated at 1.1:1 with the supplementation of 900 U of phytase and 660 mg of D3/kg of diet. The supplemented phytase activity of the diet was linearly decreased (P < 0.001) as the Ca:tP ratio became wider at each phytase or D3 level (Table 7). The negative effect of the Ca:tP ratio was greater at lower levels of phytase supplementation, but was independent of D3 levels. The D3 also linearly increased (P < 0.001) the release of P from phytate in the absence of supplemental phytase in diets. At each phytase level, the assayed phytase activity was higher than the supplemental value. The increase in the determined values of phytase activity was greater at high levels of phytase and D3 supplementation. All two-way interactions between phytase Ca:tP ratios and D3 were significant. Coefficients, P values, and R2 values for second order translog equations of performance, toe ash content, and

Downloaded from http://ps.oxfordjournals.org/ at New York University on April 27, 2015

66 mg of D3/kg diet4 0 U/kg 300 U/kg 600 U/kg 900 U/kg Mean 660 mg of D3/kg diet5 0 U/kg 300 U/kg 600 U/kg 900 U/kg Mean 6600 mg of D3/kg diet 0 U/kg

41

UTILIZATION OF PHYTATE PHOSPHORUS AND CALCIUM TABLE 4. Toe ash content of broilers fed corn-soybean meal diets with four phytase levels, four calcium:total phosphorus (Ca:tP) ratios and three cholecalciferol (D3) levels from hatch to 21 d of age1,2 Ca:tP ratio4 Added phytase4

1.1:1

1.4:1

1.7:1

2.0:1

Mean

(%) 11.7 11.9 11.9 12.5 12.0

11.3 12.0 12.1 12.9 12.1

10.9 11.8 11.9 11.9 11.6

9.9 11.1 11.4 11.6 11.0

10.9 11.7 11.8 12.2 11.7

11.9 12.2 12.8 12.9 12.5

11.7 12.0 12.4 12.9 12.2

11.2 11.3 11.8 12.1 11.6

10.8 11.1 11.4 12.0 11.1

11.4 11.7 11.8 12.5 11.8

12.4

12.2

11.8

11.7

12.0

1Each

mean represents three pens (eight birds per pen). The root MSE were 0.43 for toe ash content and the pooled SEM for a treatment mean would equal MSE/√n . Main effect: Ca:tP ratio (P < 0.001); phytase (P < 0.001); D3 (P < 0.03). 2D effect: comparison of the three levels of D without phytase supplements: linear (P < 0.05). 3 3 3Ca:tP ratio effect: linear (P < 0.001) and quadratic (P < 0.001). 4Phytase effect: linear (P < 0.001). 5Cat:tP ratio effect: linear (P < 0.02) and quadratic (P < 0.001). Phytase effect: linear (P < 0.001). 6Ca:tP ratio effect: linear (P < 0.10) and quadratic (P < 0.001). Phytase effect: linear (P < 0.001).

TABLE 5. Phosphorus retention of broilers fed corn-soybean meal diets with four phytase levels, four calcium:total phosphorus (Ca:tP) ratios and three cholecalciferol (D3) levels from hatch to 21 d of age1,2 Ca:tP ratio4 Added phytase3

1.1:1

1.4:1

1.7:1

2.0:1

Mean

(%) 66 mg of D3/kg diet5 0 U/kg 300 U/kg 600 U/kg 900 U/kg Mean 660 mg of D3/kg diet6 0 U/kg 300 U/kg 600 U/kg 900 U/kg Mean 6600 mg of D3/kg diet 0 U/kg 1Each

54.6 58.3 59.3 59.6 57.9

54.2 57.9 59.3 59.1 57.6

52.0 54.2 58.2 58.7 55.6

50.9 52.8 51.8 56.1 52.9

52.9 55.8 57.0 58.4 56.0

58.5 61.5 61.8 68.0 61.0

55.5 57.9 61.1 63.0 60.8

53.4 54.3 60.1 59.7 56.9

54.3 54.4 55.2 55.1 54.7

55.4 57.0 59.6 61.4 58.4

61.3

59.3

57.2

54.8

58.2

mean represents three pens (eight birds per pen). The root MSE were 2.34 for phosphorus retention and the pooled SEM for a treatment mean would equal MSE/√n . Main effect: Ca:tP ratio (P < 0.001); phytase (P < 0.001); D3 (P < 0.001). 2D effect: comparison of the three levels of D without phytase supplements: linear (P < 0.003). 3 3 3Ca:tP ratio effect: linear (P < 0.05) and quadratic (P < 0.001). 4Phytase effect: linear (P < 0.001). 5Cat:tP ratio effect: quadratic (P < 0.007). Phytase effect: linear (P < 0.005). 6Ca:tP ratio effect: quadratic (P < 0.001). Phytase effect: linear (P < 0.001).

Downloaded from http://ps.oxfordjournals.org/ at New York University on April 27, 2015

66 mg of D3/kg diet5 0 U/kg 300 U/kg 600 U/kg 900 U/kg Mean 660 mg of D3/kg diet6 0 U/kg 300 U/kg 600 U/kg 900 U/kg Mean 6600 mg of D3/kg diet 0 U/kg

42

QIAN ET AL. TABLE 6. Calcium retention of broilers fed corn-soybean meal diets with four phytase levels, four calcium:total phosphorus (Ca:tP) ratios and three cholecalciferol (D3) levels from hatch to 21 d of age1,2 Ca:tP ratio4 Added phytase3

1.1:1

1.4:1

1.7:1

2.0:1

Mean

(%) 58.4 61.4 62.0 61.9 60.9

54.1 52.1 56.0 60.0 55.5

44.8 45.3 55.8 49.3 48.2

42.6 43.7 44.3 45.5 44.0

50.0 50.6 54.4 54.2 52.3

60.5 66.1 62.9 67.6 64.3

52.7 54.9 58.0 65.4 57.7

47.1 43.9 52.5 53.7 49.3

44.4 43.1 48.1 46.3 45.5

51.2 52.0 55.3 58.2 54.2

63.4

57.6

49.7

48.7

54.8

1Each

mean represents three pens (eight birds per pen). The root MSE were 3.13 for calcium retention and the pooled SEM for a treatment mean would equal MSE/√n . Main effect: Ca:tP ratio (P < 0.001); phytase (P < 0.001); D3 (P > 0.07). 2D effect: comparison of the three levels of D without phytase supplements: linear (P < 0.02). 3 3 3Ca:tP ratio effect: quadratic (P < 0.001). 4Phytase effect: linear (P < 0.001). 5Cat:tP ratio effect: quadratic (P < 0.001). Phytase effect: linear (P < 0.16). 6Ca:tP ratio effect: quadratic (P < 0.001). Phytase effect: linear (P < 0.02).

TABLE 7. Dietary phytase activity of the corn-soybean meal diets with four phytase levels, four calcium:total phosphorus (Ca:tP) ratios and three cholecalciferol (D3) levels1,2 Ca:tP ratio4 Added

phytase3

1.1:1

1.4:1

1.7:1

2.0:1

Mean

(U/kg diet) 66 mg of D3/kg diet5 0 U/kg 300 U/kg 600 U/kg 900 U/kg Mean 660 mg of D3/kg diet6 0 U/kg 300 U/kg 600 U/kg 900 U/kg Mean 6600 mg of D3/kg diet 0 U/kg 1Each

0 385 956 1,264 651

0 358 915 1,235 627

0 350 855 1,154 589

0 306 804 1,103 553

0 350 882 1,189 605

174 451 1,018 1,319 741

146 400 974 1,263 696

114 378 902 1,200 649

108 331 843 1,151 606

135 390 934 1,233 673

249

222

185

184

210

mean represents four replicates. The root MSE were 15.5 for phytase activity and the pooled SEM for a treatment mean would equal MSE/√n. Main effect: Ca:tP ratio (P < 0.001); phytase (P < 0.001); D3 (P < 0.001). D3 × phytase interaction (P < 0.001); D3 × Ca:tP ratio (P < 0.008); phytase × Ca:tP ratio (P < 0.001). 2D effect: comparison of the three levels of D without phytase supplements: linear (P < 0.001) and 3 3 quadratic (P < 0.001). 3Ca:tP ratio effect: linear (P < 0.03) and quadratic (P < 0.001). 4Phytase effect: linear (P < 0.001). 5Phytase effect: linear (P < 0.001). 6Phytase effect: linear (P < 0.001).

Downloaded from http://ps.oxfordjournals.org/ at New York University on April 27, 2015

66 mg of D3/kg diet5 0 U/kg 300 U/kg 600 U/kg 900 U/kg Mean 660 mg of D3/kg diet6 0 U/kg 300 U/kg 600 U/kg 900 U/kg Mean 6600 mg of D3/kg diet 0 U/kg

43

UTILIZATION OF PHYTATE PHOSPHORUS AND CALCIUM

TABLE 8. Second order translog functions for performance, toe ash content, and P and Ca retention of broilers fed corn-soybean meal diets with four Ca:total P (Ca:tP) ratios, four phytase and two cholecalciferol (D3) levels from hatch to 21 d of age Coefficients of equations Item

a0

a2

a3

a4

a5

P value

R2

6.1286 6.5167 2.2891 3.8515 3.6675

0.0156 0.0233 0.0204 0.0416 0.0316

0.0094 0.0040 0.0087 0.0077 0.0082

0.0032 0.0037 0.0032 0.0035 0.0060

–0.0018 0.0039 –0.0032 –0.0004 0.0008

0.001 0.023 0.001 0.001 0.101

0.283 0.216 0.272 0.239 0.071

6.3504 6.7420 2.4701 4.0493 4.1550

0.0217 0.0651 0.0346 0.0568 0.0525

0.0861 0.0425 0.1555 0.1213 –0.4163

–0.4227 –0.3441 –0.3995 –0.3511 –0.1940

–0.0278 –0.0732 –0.0549 –0.0382 –0.0403

0.001 0.001 0.001 0.001 0.001

0.461 0.458 0.350 0.283 0.543

6.2174 6.6244 2.3539 3.9016 3.9028

0.0028 –0.0016 0.0053 0.0091 0.0097

0.0308 –0.0484 0.0990 0.1210 –0.4215

0.0032 0.0037 0.0029 0.0035 0.0059

–0.4227 –0.3441 –0.3786 –0.3591 –0.2019

0.001 0.001 0.001 0.001 0.001

0.666 0.501 0.598 0.501 0.591

0.0138 0.0181 0.0043 –0.0038 –0.0026

1Model: LnY = a + a D + a LnX + a (LnX)2 + a D LnX. At 66 mg of D /kg diet, D = 0; At 660 mg of D /kg diet, D = 1. X = added phytase, U/ 0 1 1 2 3 4 1 3 1 3 1 kg diet; or X = Ca:tP ratios. 2Model: LnY = a + a LnX + a LnX + a (LnX )2 + a (LnX )2 + a (LnX LnX ). X = added phytase, U/kg diet. X = Ca:tP ratios. 0 1 1 2 2 3 1 4 2 5 1 2 1 2

P and Ca retention are shown in Table 8 for phytase and D3, Ca:tP ratio and D3, and phytase and Ca:tP ratio. Equations for phytase and D3 or Ca:tP ratios and D3 generally had low R2 values (< 0.5), whereas equations for phytase and Ca:tP ratio had relatively higher R2 (> 0.50) and low P values (P < 0.001). Nonlinear and linear response equations were also developed for the effects of phytase and Ca:tP ratios (equations not shown). For BW gain, toe ash content, and P retention, almost all equations had high R2 (mostly R2 > 0.90). It was observed that the phytase effect seemed more nonlinear, whereas the Ca:tP ratio effect was more linear to responses because nonlinear equations to phytase and linear equations to the ratio relatively had higher R2 and lower P value. Based on the R2 and P value of developed equations, BW gain, toe ash contents and P retention were found to be the most sensitive indicators to assess the effects of the three factors in broilers. In the evaluation of the negative effect of widening the Ca:tP ratio, the main effects of Ca:tP ratios on BW gain and toe ash contents were used. Narrowing the Ca: tP ratio from 2.0:1 to 1.4:1 in average resulted in an increase in the phytase efficacy of 11.1 and 12.2%, respectively, for the 66 and 660 mg of D3/kg diet, which was close to the change in phytase activity observed in diet (13.4 and 14.9%, respectively).

DISCUSSION The high content of phytate P in corn and soybean meal leads to limited availabilities of P, Ca, and trace minerals for poultry fed corn-soybean meal diets (Nelson et al., 1968; Reddy et al., 1980). Nelson et al. (1968) first reported that microbial preparation contain-

ing phytase greatly improved utilization of phytate P when supplemented in broiler diets. Phytase has also been reported to improve the utilization of phytate Ca (Simons et al., 1990; Schoner et al., 1994). Schoner et al. (1994) suggested that 500 U of phytase diet is equivalent to 0.35 to 0.45 g of Ca in diets fed to broilers. Phytic acid, a cation chelator, makes Ca unavailable for intestinal absorption. Phytase releases Ca from the insoluble salts of phytic acid, and potentially makes Ca available for absorption in birds. Recently, strong evidence indicates that microbial phytase is highly effective in degrading phytate (Simons et al., 1990; Schoner et al., 1991; Denbow et al., 1995). In our previous study, Kornegay et al. (1996) reported that 735 U of phytase/kg diet was equivalent to 1 g of nonphytate P for broilers fed corn-soybean meal diets, and that about 20 to 60% of phytate P was hydrolyzed by graded levels of supplemental phytase. Findings of the present study are in agreement with the results reported by Kornegay et al. (1996). Graded levels of phytase increased Ca and P retention by up to 18 and 14%, respectively, for 66 and 660 mg of D3/kg diet. That increased utilization of phytate Ca and P results in increased Ca and P retention is also supported by our findings of an increase in Ca, P, Zn, and Mg in bone ash and improved bone calcification and histological development (Qian et al., 1996b,c) when phytase was added to broiler and turkey poult diets. Calcium is thought to be a key factor influencing the activity of mucosal phytase in small intestines of poultry and rat (Bhandari, 1980; Wise, 1983). This effect was also observed in the present study, in which the activity of the microbial phytase added in the mixed diets was greatly decreased by a large Ca:tP ratio. The decrease in

Downloaded from http://ps.oxfordjournals.org/ at New York University on April 27, 2015

Phytase and D3 effect1 Weight gain, g/bird Feed intake, g/bird Toe ash content, % P retention, % Ca retention, % Ca:tP ratio and D3 effect1 Weight gain, g/bird Feed intake, g/bird Toe ash content, % P retention, % Ca retention, % Phytase and Ca:tP ratio effect2 Weight gain, g/bird Feed intake, g/bird Toe ash content, % P retention, % Ca retention, %

a1

44

QIAN ET AL.

The present study shows that the addition of D3 at the higher levels increased the toe ash content, perhaps by improving P and Ca retention of birds. The improved P and Ca retention were attributed to the enhanced P and Ca utilization of the Ca salt of phytic acid by D3 addition. The addition of D3 increased the utilization of phytate P and Ca in the presence or absence of supplemental phytase in diets in our study. This is supported by the report of Edwards (1993), who observed that the addition of 1,25-(OH)2D3 at 5 or 10 mg/kg diet increased the utilization of phytate P from 30 to 80% when the basal diet contained 27.5 mg of D3 with or without graded levels of phytase supplementation. The NRC (1994) recommends that requirement for D3 is 5 mg/kg diet for broilers. Edwards (1993) indicated that 3 to 5 mg/kg of 1,25-(OH)2D3 will give maximum biological responses in broilers fed diets either adequate or deficient in D3. Recently, however, addition of D3 or its derivatives at very high levels have shown a large increase in phytate P utilization (Shafey et al., 1990; Mohammed et al., 1991). Mohammed et al. (1991) also reported phytate P digestibility was about 50% when the diet contained normal amounts of inorganic P (0.45%), Ca (1.0%), and D3 (12.5 mg/kg diet). Increasing the addition of D3 to 1,250 mg/kg diet increased the phytate P utilization 59 to 77%. Increasing the addition of D3 from 66 to 660 to 6,600 mg/kg diet in our study enhanced P retention by 10% in the presence or absence of phytase supplementation. A large increase in the utilization of phytate P that resulted from very high levels of D3 addition is normally explained as increased intestinal phytase activity from the addition of D3 (Edwards, 1993). Pointillart et al. (1985, 1989) observed that D3 supplementation increased the level of phytase activity in pigs, which led to an improvement in phytate P utilization when diets were low or devoid of D3. However, supplementation of D3 at very high levels has not been shown to increase phytase activity in small intestines when diets contained adequate amounts of D3. In poultry, the results of studies by Shafey et al. (1990), Mohammed et al. (1991), and Edwards (1993) suggest that D3 or its derivatives have a potential in hydrolysis of phytate. Evidence in the present study tended to support this hypothesis. An increase in the release of P from phytate was observed by graded levels of D3 addition in diets when assaying the phytase activity in the mixed diet that contained no supplemental phytase and very limited vegetable-source phytase. In addition, a further increase in P release from phytate was also achieved when diets were simultaneously formulated with microbial phytase, which indicated some synergetic effect between D3 and phytase on hydrolysis of phytate. This synergetic effect was also observed in toe ash content, and P and Ca retention, although the two-way interaction between D3 and phytase was not significant, which suggested that both effects were independent of each other. More studies are needed to investigate the potential role of D3 supplementation in hydrolysis of

Downloaded from http://ps.oxfordjournals.org/ at New York University on April 27, 2015

the phytase activity as the Ca:tP ratio became greater could be explained as follows: 1) phytate P utilization of corn-soybean diets by broilers is influenced by Ca and P levels in the diet (Edwards and Veltmann 1983; Ballam et al., 1984); 2) extra Ca binds with phytate to form an insoluble complex that is less accessible to phytase; 3) the extra Ca, probably more importantly, could directly repress phytase activity by competing for the active sites of the enzymes (Wise, 1983; Pointillart et al., 1985). The negative effect is stronger at lower levels of supplemental phytase as well as at lower levels of available P because low levels of phytase released limited P from phytate that supplies a P-deficient environment leading to influencing phytase activity (Qian et al., 1995). The present study indicates that widening Ca:tP ratios from 1.4:1 to 2.0:1 reduced phytase activity by 13.4 and 14.9%, respectively, for the mixed diets of 66 and 660 mg of D3/ kg diet, which agreed well with our two previous studies (Qian et al., 1995). In agreement with results of the phytase activity of diets, a wider Ca:tP ratio negatively influenced the phytase efficacy in broilers and decreased all measurements at each phytase and D3 level. Schoner et al. (1991, 1993) also observed that the addition of Ca, thus widening the Ca:tP ratio, decreased all measurements whereas narrowing the Ca:tP ratio improved all measurements. In turkeys, widening the dietary Ca:tP ratios from 1.4:1 to 2.0:1 resulted in a decrease in the phytase efficacy by 7.4 and 4.9%, respectively, for 0.27 and 0.36% nP diets (Qian et al., 1996a). Results of the present broiler study suggest that phytase efficacy was decreased by 11.1 and 12.2%, respectively, for the diets with 66 and 660 mg of D3/kg diet when BW gain and toe ash content were considered as the criteria to response dietary Ca:tP ratios. The adverse effect of a wide Ca:tP ratio seemed to be independent of supplemental phytase and D3 because all two- and three-way interactions were not significant, which was consistent with our pig study (Qian et al., 1995). However, almost all two-way interactions between Ca:tP and phytase were significant, as observed using turkey poults (Qian et al., 1996a). The two-way interaction between Ca:tP and phytase was also observed in assay of phytase activity in the mixed diets. The meaning of this case is not clear and needs further research. Vitamin D enhances the enterocytes of the small intestine to transport P into the plasma compartment, which appears independent of intestinal Ca transport (Deluca et al., 1989; Edwards, 1993). As a result, dietary P absorption and retention is increased. Recent reports have indicated that the addition of D3 or its derivatives greatly increased phytate P utilization when the supplementation was at very high levels (Shafey et al., 1990; Mohammed et al., 1991). This improvement in the utilization of phytate P by D3 might result from an increase in the phytase activity in the small intestines of chicks (Pointillart et al., 1985).

UTILIZATION OF PHYTATE PHOSPHORUS AND CALCIUM

the Ca:tP ratio. Further studies are needed to determine the optimal Ca levels in the broiler diet containing supplemental phytase and D3. In addition, dietary Ca:tP ratio seems more critical than the amount of Ca or P alone in diets in the practical utilization of microbial phytase and D3 in broilers. Dietary Ca:tP ratios that are formulated in the range of 1.1:1 to 1.4:1 appear to provide the best efficacy of supplemental phytase and D3 in broilers. In summary, the effectiveness of microbial phytase for improving the utilization of phytate P and Ca by broilers is influenced by the Ca:tP ratio and D3 level. Results show that supplemental phytase improved BW gain, feed intake, toe ash content, and Ca and P retention of broilers fed a corn-soybean based diet; these improvements were negatively influenced by wide Ca:tP ratios, and positively influenced by higher levels of D3. High levels of D3 added to the diets resulted in an increase in the retention of P and Ca, which seemed independent of supplemental phytase but synergetic with it. Maximum responses to supplemental phytase were achieved when broiler chicks were fed diets with 600 to 900 U of phytase/kg diet, with dietary Ca:tP ratios of 1.1:1 to 1.4:1, and a D3 level of 660 mg/kg diet.

ACKNOWLEDGMENTS Appreciation is expressed to the United Soybean Board and the John Lee Pratt Animal Nutrition Program for financial support, to BASF Corp., Mount Olive, NJ 07828-1234 for phytase supply, to Barbara Self and Donald Conner, Jr. for technical assistance, and to Cindy Hixon for typing. This material is based on work supported in part by the Cooperative State Research Service, USDA, under project Number 6129880.

REFERENCES Association of Official Analytical Chemists, 1990. Official Methods of Analysis. 15th ed. Association of Official Analytical Chemists, Washington, DC. Ballam, G. C., T. S. Nelson, and L. K. Kirby, 1984. Effect of fiber and phytase source and of calcium and phosphorus level on phytate hydrolysis in the chick. Poultry Sci. 63: 333–338. Bhandari, S. D., 1980. Effect of phytate feeding with and without protein and vitamin D deficiencies on intestinal phytase activity in rat. Indian J. Biochem. Biophys. 17: 309–312. Consortium, 1988. Guide for the Care and Use of Agriculture Animals in Agricultural Research and Teaching. Consortium for Developing a Guide for the Care and Use of Agriculture Animals in Agricultural Research and Teaching, Champaign, IL. Deluca, H. F., 1989. Vitamin D, calcium, and metabolic bone disease. Page 109 in: Nutrition and the Origins of Disease. H. F. Deluca, ed. Academic Press, Inc., New York, NY. Denbow, D. M., V. Ravindran, E. T. Kornegay, B. B. Self, and R. M. Hulet, 1995. Improving phosphorus availability in soybean meal for broilers by supplemental phytase. Poultry Sci. 74:1831–1842.

Downloaded from http://ps.oxfordjournals.org/ at New York University on April 27, 2015

phytate, and its relationship with microbial and intestinal phytase. Findings in the present study also demonstrated that addition of D3 to corn-soybean diets for broilers increased Ca retention by 5 to 12% in the presence or absence of supplemental phytase. Calcium combined with phytic acid has low availability when it is present as the Ca salt of phytic acid. The addition of D3 has been shown to improve the utilization of phytate Ca, which may be due to degradation of phytic acid by D3. When broilers received diets of 1,25-(OH)2D3, the maximum bone ash was achieved for diets containing approximately two-thirds as much Ca as needed to obtain maximum bone ash when no 1,25-(OH)2D3 was present in the diet (Edwards et al., 1992). A vitamin D3 deficiency in laying hens, on the other hand, decreases Ca absorption, which leads to bone loss even with adequate dietary Ca (Ruschowski and Hart, 1992). Vitamin D repletion of vitamin D-deficient birds and pigs restores bone mineralization by improving the utilization of phytate Ca (Pointillart and Fontaine, 1986; Shafey et al., 1990). The efficacy of D3 in improving the utilization of phytate P is influenced by dietary Ca:tP ratios in the presence or absence of supplemental phytase. Widening dietary Ca:tP ratios reduced all the measurements and the phytase activity of the diets. Therefore, dietary Ca:tP ratios are critical to the efficacy of microbial phytase as well as D3 in the hydrolysis of phytic acid. A possible mechanism is that the solubility of phytate, a salt of phytic acid, determines the utilization of phytate P. Phytate with high solubility is more accessible to phytase and D3, whereas the solubility of phytate is, on the other hand, greatly influenced by Ca and P concentrations. This possibility has been indicated in vitro (Wise, 1983) and in broilers (Ballam et al., 1984; Edwards and Veltmann, 1983). This hypothesis is also supported by the recent study of Mohammed et al. (1991), who observed that the elevation of D3 alone dramatically increased phytate digestibility and the retention of P and Ca, but, this improvement could not totally overcome the P depletion due to a high phytate P, and that simultaneously lowering of dietary Ca and elevation of D3 in a phytate P diet restored all variables to the levels for the control. Similar results were reported in pigs (Pointillart and Fontaine, 1986; Pointillart et al., 1989) and in poultry (Shafey et al., 1990). The NRC (1994) sets the requirements of Ca and nP for 0- to 3-wk-old broilers to be 10 and 4.5 g/kg diet, respectively, to meet the optimal growth of birds. In the present study, nP levels in diets were below the requirement, and most Ca levels were also below the requirement to provide a more favorable situation for measuring the effect of phytase supplementation on increasing the bioavailabilities of the phytate P and Ca. As discussed above, supplementation of phytase and D3 dramatically increased the utilization of phytate P and Ca. However, on the other hand, the efficacy of phytase and D3 were influenced by dietary Ca and P levels, and

45

46

QIAN ET AL. mechanical and chemical traits of turkeys fed on soybean meal-based semi-purified diets high in phytate phosphorus. Br. J. Nutr. 76:263–272. Qian, H., H. P. Veit, E. T. Kornegay, V. Ravindran, and D. M. Denbow, 1996. Effects of supplemental phytase and phosphorus on histological and other tibial bone characteristics and performance of broilers fed semi-purified diets. Poultry Sci. 75:618–626. Ravindran, V., E. T. Kornegay, D. M. Denbow, Z. Yi, and R. M. Hulet, 1995. Response of turkey poults to tiered levels of Natuphos phytase added to soybean meal-based semipurified diets containing three levels of nonphytate phosphorus. Poultry Sci. 74:1843–1854. Reddy, N. R., S. K. Sathe, and D. K. Salunkhe. 1980. Phytates in legumes and cereals. Pages 1–9 in: Advances in Food Research. C. O. Chichester, E. M. Mrok, and G. F. Stewart, ed. Academic Press, New York, NY. Ruschkowski, S. R., and L. E. Hart, 1992. Ionic and endocrine characteristics of reproductive failure in calcium deficient and vitamin D deficient laying hens. Poultry Sci. 71: 1722–1732. SAS Institute, 1990. SAS/STAT User’s Guide: Statistics. Release 6.04. SAS Institute Inc., Cary, NC. Schoner, F. J., G. Schwarz, P. P. Hoppe, and H. Wiesche, 1994. Effects of microbial phytase on Ca-availability in broilers. Third Conference of Pig and Poultry Nutrition in Halle, Nov. 29–Dec. 1. Halle, Germany. Schoner, F. J., P. P. Hoppe, and G. Schwarz, 1991. Comparative effects of microbial phytase and inorganic phosphorus on performance and retention of phosphorus, calcium and crude ash in broilers. J. Anim. Physiol. Anim. Nutr. 66: 248–255. Schoner, F. J., P. P. Hoppe, G. Schwarz, and H. Wiesche, 1993. Effects of microbial phytase and inorganic phosphate in broiler chicken: Performance and mineral retention at various calcium levels. J. Anim. Physiol. Anim. Nutr. 69: 235–244. Shafey, T. M., M. W. McDonald, and R.A.E. Pym, 1990. Effects of dietary calcium, available phosphorus and vitamin D on growth rate, food utilization, plasma and bone constituents and calcium and phosphorus retention of commercial broiler strains. Br. Poult. Sci. 31:587–602. 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. Wise, A., 1983. Dietary factors determining the biological activities of phytate. Nutr. Abstr. Rev. 53:791–806. Yi, Z., E. T. Kornegay, V. Ravindran, and D. M. Denbow. 1996. Improving phytate phosphorus availability in corn and soybean meal for broilers using microbial phytase and calculation of phosphorus equivalency values of inorganic P by phytase. Poultry Sci. 75:240–249.

Downloaded from http://ps.oxfordjournals.org/ at New York University on April 27, 2015

Driscoll, P. J., 1994. When flexible forms are asked to flex too much. J. Agric. Resource Econ. 19:183–196. Edwards, H. M., Jr., 1993. Dietary 1,25-dihydroxycholecalciferol supplementation increases natu-ral phytate phosphorus utilization in chickens. J. Nutr. 123:567–577. Edwards, H. M., Jr., and J. R. Veltmann, Jr., 1983. The role of calcium and phosphorus in the etiology of tibial dyschondroplasia in young chicks. J. Nutr. 113:1568–1575. Edwards, H. M., Jr., M. A. Elliot, and S. Sooncharernying, 1992. Effect of dietary calcium on tibial dyschondroplasia. Interaction with light, cholecalciferol, 1,25-dihydroxycholecalciferol, protein, and synthetic zeolite. Poultry Sci. 71:2041–2055. 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. Int. 77(3):760–764. Kornegay, E. T., D. M. Denbow, Z. Yi, and V. Ravindran, 1996. Response of broilers to graded levels of microbial phytase added to maize-soybean meal-based diets containing three levels of nonphytate phosphorus. Br. J. Nutr. 75:839–852. Mohammed, A., M. J. Gibney, and T. G. Taylor, 1991. The effects of dietary levels of inorganic phosphorus, calcium and cholecalciferol on the digestibility of phytate-P by the chick. Br. J. Nutr. 66:251–259. Nelson, T. S., T. R. Shieh, R. J. Wodzinski, and J. H. Ware, 1968. The availability of phytate phosphorus in soybean meal before and after treatment with a mold phytase. Poultry Sci. 47:1842–1848. National Research Council, 1994. Nutrient Requirements of Poultry. 9th rev. ed. National Academy of Science, Washington, DC. Pointillart, A., and N. Fontaine, 1986. Effects of vitamin D on calcium regulation in vitamin D deficient pigs given a phytate-phosphorus diet. Br. J. Nutr. 56:661–669. Pointillart, A., A. Fourdin, A. Bourdeau, and M. Thomasset, 1989. Phosphorus utilization and hormonal control of calcium metabolism in pigs fed phytic phosphorus diets containing normal or high calcium levels. Nutr. Rep. Int. 40(3):517–527. Pointillart, A., A. Fourdin, M. Thomasset, and M. E. Jay, 1985. Phosphorus utilization, intestinal phosphatase and hormonal control of calcium metabolism in pigs fed phytic phosphorus: soybean or rapeseed diets. Nutr. Rep. Int. 32(1)155–167. Qian, H., and E. T. Kornegay, 1995. Characterization of the effects of varying Ca:P ratio on efficacy of phytase for replacing inorganic phosphorus in pig and poultry diets. J. Anim. Sci. 73(Suppl. 2)17. (Abstr.) Qian, H., E. T. Kornegay, and D. M. Denbow, 1996a. Phosphorus equivalence of microbial phytase in turkey diets as influenced by Ca:P ratios and P levels. Poultry Sci. 75:69–81. Qian, H., E. T. Kornegay, and H. P. Veit, 1996b. Effects of supplemental phytase and phosphorus on histological,