Influence of supplemental phytase on calcium and phosphorus utilization in laying hens

Research Notes Influence of Supplemental Phytase on Calcium and Phosphorus Utilization in Laying Hens1 R. W. GORDON, and D. A. ROLAND, SR.2 Department of Poultry Science and Alabama Agricultural Experiment Station, Auburn University, Alabama 36849-5416

(Key words: phytase, layer, calcium, phosphorus, phytate phosphorus) 1998 Poultry Science 77:290–294

(Pallauf et al., 1994; Roberson and Edwards, 1994; Biehl et al., 1995; Yi et al., 1996a), Mn (Biehl et al., 1995), Ca (Pallauf et al., 1994; Yi et al., 1996b) and Mg (Pallauf et al., 1994) when diets are supplemented with phytase. Apart from the influence of phytase on P liberation, however, very little research has evaluated the effects of phytase supplementation of laying hen diets. It is well known that P plays an important role in Ca metabolism. A portion of the phytase benefit observed in poultry fed NPP-deficient diets can be attributed not only directly to P, but also to the influence of P on improved Ca utilization. It is not known, however, whether there is a phytase influence on Ca metabolism separate from that associated with increased P liberation. Therefore, the following study was conducted to determine whether a non-P benefit on Ca metabolism is associated with phytase supplementation.

INTRODUCTION A number of studies have indicated that supplementing poultry diets with microbial phytase results in improved performance (Kiiskinen et al., 1994; Schwarz, 1994; van der Klis et al., 1996), particularly when dietary levels of nonphytate P (NPP) are low (Denbow et al., 1995; Gordon and Roland, 1997). Because phytic acid contains six phosphoric acid groups that are hydrolyzed by phytase (Heinzl, 1996), this improved performance has largely been attributed to the liberation of orthophosphates that can be absorbed. The increased P retention and reduced phytate P excretion that accompanies dietary inclusion of phytase (Edwards, 1993) is indicative of the relationship between phytase and phytate P liberation. In addition to containing P, phytic acid has a number of multivalent cations associated with the phosphoric acid residues (Kornegay, 1996). Work with broilers and pigs has shown increases in the availability of Zn

MATERIALS AND METHODS Fifteen hundred and thirty-six Hy-Line W-36 Phase 3 hens (58 wk of age) were randomly allocated to 1 of 12 dietary treatments (Table 1). Diets were fed in a factorial arrangement of three calcium levels (2.5, 2.8, and 3.1%)

Received for publication February 10, 1997. Accepted for publication October 20, 1997. 1Alabama Agricultural Experiment Station Journal Series Number 12-965736. 2To whom correspondence should be addressed: droland@ag. auburn.edu

Abbreviation Key: NPP = nonphytate phosphorus.

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observed during the 1st wk. Increasing NPP from 0.1 to 0.3% had not effect on egg specific gravity until Week 3, suggesting that the phytase benefit during Weeks 1 and 2 was related to improved Ca utilization. From Weeks 3 to 6, a significant P by phytase interaction was observed in which the magnitude of shell quality improvement was greatest when the 0.1% NPP diet was supplemented with phytase. This interaction was also observed from Weeks 3 to 6 for feed consumption and egg production and during Weeks 4 and 6 for egg weights. Phytase supplementation completely overcame the adverse effects associated with low dietary P and significantly reduced the impact of low dietary Ca on hen performance.

ABSTRACT A 6-wk study was conducted to determine the influence of supplemental phytase on Ca and P utilization in commercial laying hens. Diets were arranged factorially with three levels of dietary Ca (2.5, 2.8, and 3.1%), fed at two levels of nonphytate P (0.1 and 0.3% NPP) with and without supplemental phytase. Each diet was replicated eight times, with 16 hens per replicate. Criteria evaluated included egg specific gravity, feed consumption, egg production, egg weight, eggshell weight, bone quality, and body weight. Increasing dietary Ca significantly improved shell quality within 1 wk. A significant improvement in shell quality due to phytase supplementation was also

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RESEARCH NOTE TABLE 1. Ingredients and nutrient composition of experimental diets1 Diet Ingredients and composition

1

2 (%) 66.44 23.05 3.56 2.00 1.14 1.06 0.48 0.25 0.25 0.16 1.61

2,865 16.78 2.5 0.51 0.3 0.21 0.72 0.87

66.44 23.05 4.13 2.00 1.14 0.004 0.48 0.25 0.25 0.16 2.09 2,865 16.78 2.5 0.31 0.1 0.21 0.72 0.87

1Diets

1 and 2 were also fed with dietary Ca levels of 2.8 and 3.1%, with 0 or 300 U phytase/kg feed. To supply 300 units of phytase/kg feed, diets were supplemented with 0.0455% Natuphos. 2Provided per kilogram of diet: vitamin A, 8,000 IU (as retinyl acetate); cholecalciferol, 2,200 IU; vitamin E, 8 IU (as dl-a-tocopherol acetate); vitamin B12, 0.02 mg; riboflavin, 5.5 mg; D-calcium pantothenic acid, 13 mg; niacin, 36 mg; choline, 500 mg; folic acid, 0.5 mg; thiamin, 1 mg; pyridoxine, 2.2 mg; biotin, 0.50 mg; menadione sodium bisulfate complex, 2 mg. 3Provided per kilogram of diet: manganese, 65 mg; iodine, 1 mg; iron, 55 mg; copper, 6 mg; zinc, 55 mg; selenium, 0.3 mg. 4Dietary Ca and all P values were based on chemical analysis of the following ingredients (with their percentage analyzed Ca, total P and phytate P values, respectively): corn (0.02; 0.28; 0.20); soybean meal (0.24; 0.63; 0.44); ground limestone (38.0); large particle limestone (38.8); dicalcium phosphate (20.55; 18.95).

and two levels of NPP (0.1 and 0.3%) with and without phytase (Natuphos).3 All diets were considered to be Ca deficient based on Phase 2 data from this flock. Similarly, 0.1% NPP was considered deficient, but 0.3% NPP, based on Phase 1 and 2 data, was considered sufficient to meet the P requirement. Where supplemented, phytase was provided at 300 units/kg feed. Each treatment was replicated eight times with each replicate comprised of four adjoining cages housing four hens per cage. Diets were isonitrogenous (16.8% crude protein) and isocaloric (2,865 kcal ME/kg). Feed consumption, egg production, egg specific gravity, and egg weights were determined weekly for 6 wk. Egg specific gravity and egg weight values were determined from a 2-d collection of eggs per week. Egg specific gravity determination involved placing eggs in saline solutions varying in increments of 0.05 units as

3BASF Corp., 4Model 2780,

Mount Olive, NJ 07828-1234. Norland Corp., Fort Atkinson, WI 53538.

Yijklm = m + Ci + Pj + Ek + CPij + CEik + PEjk + CPEijk + Rl + Lm + eijklm where Yijklm is the individual observation; m is the experimental mean; Ci is the Ca level effect; Pj is the P level effect; Ek is the enzyme phytase effect, Rl is the cage row effect; and Lm is the cage level effect. Significance was based on a 5% probability level.

RESULTS AND DISCUSSION

Feed Consumption Phytase supplementation significantly increased feed consumption as early as Week 1. The effect of dietary P on feed consumption, however, was not observed until Week 2, indicating that the Week 1 phytase effect was not solely the result of liberated phytate P. A significant phytase by P interaction was observed during Weeks 3 through 6 and for the 6-wk average. The nature of this interaction during Weeks 3, 4, and 6 demonstrated that phytase supplementation increased consumption of diets containing 0.1% NPP, but had no effect when NPP levels were 0.3%. The interaction observed during Week 5 and for the 6-wk average revealed that supplemental phytase increased feed consumption at both levels of dietary P, but that the magnitude of that increase was greatest when diets contained 0.1% NPP. Previous work (Gordon and Roland, 1977) demonstrated that supplementing diets containing 0.1% NPP with phytase increased feed consumption to the level of hens consuming higher levels of dietary P. Increasing dietary P resulted in increased feed consumption when diets were not supplemented with phytase. Six-week average feed consumption of diets without supplemental phytase increased 8.9%, when dietary P was increased from 0.1 to 0.3%. During the final 3 wk of the study, feed consumption of the unsupplemented diet increased 13.4% as dietary P was increased from 0.1 to 0.3%. The negative effect of P deficiency on

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Corn Soybean meal (48%) Ground limestone (38.8% Ca) Large particle limestone (38.8% Ca) Poultry fat Dicalcium phosphate Salt Vitamin premix2 Mineral premix3 DL-Methionine Sand Nutrient composition4 ME, kcal/kg Crude protein Ca Total P Nonphytate P Sodium Methionine + Cystine Lysine

described by Strong (1989). During Week 4, eggshells were broken, washed, air-dried, and individually weighed. Shell membranes were not removed during the washing procedure. At the end of Week 6, 20 hens per treatment were killed at oviposition. After determining hen body weights, both tibiae were removed for measurement of bone mineral content and bone density. Bones were removed according to the direct excision method described by Orban et al. (1993). Each bone was scanned three times at mid-shaft using a Norland digital bone densitometer.4 This study utilized a randomized complete block design in which the main effects of Ca, P, and phytase level and their interactions were subjected to analysis of variance (Steel and Torrie, 1980) using the General Linear Models procedure of SAS (SAS Institute, 1986). The statistical model used was:

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TABLE 2. Influence of dietary Ca, phytase, and nonphytate P (NPP) on feed consumption (FC), egg production (EP), egg weight (EW), eggshell weight (ESW), bone mineral content (BMC), bone density (BD), BW, and egg specific gravity (ESG) FC

EP (%)

Ca 2.5% 2.8% 3.1% Phytase 0 U/kg 300 U/kg NPP 0.1% 0.3% Phytase × NPP 0 0.1% 300 0.1% 0 0.3% 300 0.3% SEM

(g/hen/d) *L1 89.6 88.8 88.1 *** 86.4 91.3 *** 86.6 91.0 *** 82.7c 90.5ab 90.1b 92.0a 0.256

73 74 74 *** 69 78 *** 70 77 *** 62b 77a 77a 78a 0.355

EW

ESW

ESG

(g) 62.6 62.4 62.7 62.5 62.7 62.4 62.8 62.1 62.6 62.8 62.7 0.113

***L 4.92 5.07 5.11 *** 4.92 5.10 *** 4.96 5.06 *** 4.80b 5.09a 5.02a 5.11a 0.055

***LQ1 1.0768 1.0784 1.0789 *** 1.0771 1.0790 * 1.0778 1.0783 *** 1.0764c 1.0791a 1.0779b 1.0788a 0.0001

BMC

BD

BW

(g/cm)

(g/cm2)

(kg)

0.149 0.153 0.153 *** 0.147 0.156 *** 0.146 0.158

0.228 0.236 0.235 *** 0.226 0.239 *** 0.223 0.242

1.50 1.50 1.47 *** 1.46 1.52

0.140 0.151 0.153 0.162 0.0013

0.213 0.233 0.238 0.245 0.0019

1.46 1.48 1.46 1.56 0.012

1.47 1.51

a–cValues

within a column with no common superscript differ significantly (P < 0.05). = linear effect; Q = quadratic effect. *P < 0.05. **P < 0.01. ***P < 0.001. 1L

feed consumption has also been demonstrated by others (Hartel, 1990; Frost and Roland, 1991). Increasing the P level of diets supplemented with phytase had no significant effect on feed consumption. There was a significant linear increase in feed consumption as dietary Ca decreased, indicating an apparent Ca appetite (Frost and Roland, 1991).

Egg Production A significant phytase by P interaction related to egg production was observed throughout the experiment (Table 2). When diets contained 0.1% NPP, egg production of unsupplemented hens significantly decreased. During Week 1, supplemented and unsupplemented hens consuming 0.1% NPP exhibited production rates of 78 and 75%, respectively (P = 0.08). By Week 6, the production of unsupplemented hens had fallen to 49%, whereas phytase-supplemented hens maintained production at 77%. The only indication of a phytase effect on egg production of hens consuming 0.3% NPP was a phytase by P interaction during Week 5 showing a phytase effect at the 0.3% NPP level but not of the magnitude observed when hens were consuming 0.1% NPP. Production was significantly increased when NPP levels of diets without supplemental phytase were increased from 0.1 to 0.3%. Increasing dietary P above 0.1% has also been shown to increase egg production by others (Hartel, 1990; Gordon and Roland, 1997). There was no evidence, however, of a P effect when diets were supplemented with phytase, nor were there indications that dietary Ca influenced egg production. Abdallah et al. (1993) reported increased egg

production as Ca levels were increased from 2.2 to 3.9%, but others (Frost and Roland, 1991; Clunies et al., 1992; Leeson et al., 1993), feeding Ca levels in the range of the present study, reported no effect.

Egg Weights, Body Weights, Bone Mineral Content, and Bone Density Supplemental phytase significantly increased egg weights during Weeks 4 and 6 when diets contained 0.1% NPP (Table 2). There was no phytase effect on egg weights associated with the 0.3% NPP diet. Increasing dietary NPP from 0.1 to 0.3% resulted in increased egg weights during Weeks 4 and 6 when diets were devoid of phytase, in agreement with the work of Hartel (1990). There was no evidence of a P effect on egg weights when phytase was included in the diet. Supplementing diets with phytase resulted in an increased bone mineral content and bone density of 6.1 and 5.8%, respectively, regardless of dietary P content (Table 2). Increasing NPP from 0.1 to 0.3% increased bone mineral content and bone density of 8.2 and 8.5%, respectively. Body weights were increased 4.4% by phytase inclusion, and increasing dietary P from 0.1 to 0.3% resulted in a 3.1% increase (P = 0.07).

Egg Specific Gravity and Eggshell Weights Typically, the most sensitive and commonly used indicators of Ca metabolism in laying hens relate to measurements of eggshell quality. Determination of egg specific gravity is useful because it is related to shell thickness and, therefore, CaCO3 deposition. Measuring

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Treatment

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RESEARCH NOTE

significantly increased shell weights but not relative to the 2.5% Ca/0.3% NPP diet without phytase. Based on the results from diets containing 2.8 and 3.1% Ca, however, 43 to 46% of the benefit from supplemental phytase appears to be related to something other than liberated phytate P. Based on these data, it is estimated that 32 to 54% of the increase in Ca utilization observed in this study was associated with factors other than the liberation of phytate P. At this time it is unknown what those factors are, but it is improbable that the phytate molecule would contain enough Ca to, upon hydrolysis, account directly for the increase. Clearly, further research is needed to identify specific factors involved in the increase in Ca utilization observed in this study. There is sufficient evidence from this study that phytase supplementation of diets containing 3.1% Ca or less (Ca-deficient diets) significantly increased Ca utilization.

REFERENCES Abdallah, A. G., R. H. Harms, and O. El-Husseiny, 1993. Performance of hens laying eggs with heavy or slight shell weight when fed diets with different calcium and phosphorus levels. Poultry Sci. 72:1881–1891. Biehl, R. R., D. H. Baker, and H. F. DeLuca, 1995. 1-a hydroxylated cholecalciferol compounds act additively with microbial phytase to improve phosphorus, zinc and manganese utilization in chicks fed soy-based diets. J. Nutr. 125:2407–2416. Clunies, M., D. Parks, and S. Leeson, 1992. Calcium and phosphorus metabolism and eggshell formation of hens fed different amounts of calcium. Poultry Sci. 71:482–489. Denbow, D. M., V. Ravidran, 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. Edwards, H. M., Jr., 1993. Dietary 1, 25-dihydroxycholecalciferol supplementation increases natural phytate phosphorus utilization in chickens. J. Nutr. 123:567–577. Frost, T. J., and D. A. Roland, Sr., 1991. The influence of various calcium and phosphorus levels on tibia strength and eggshell quality of pullets during peak production. Poultry Sci. 70:963–969. Gordon, R. W., and D. A. Roland, Sr., 1997. Performance of commercial laying hens fed various phosphorus levels, with and without supplemental phytase. Poultry Sci. 76: 1172–1177. Hartel, H., 1990. Evaluation of the dietary interaction of calcium and phosphorus in the high producing laying hen. Br. Poult. Sci. 31:473–494. Heinzl, W., 1996. Technical specifications of Natuphos. Pages 21–36 in: BASF Technical Symposium Phosphorus and Calcium Management in Layers. Atlanta, GA, January 23, 1996. Kiiskinen, T., J. Piironen, and T. Hakonen, 1994. Effects of supplemental microbial phytase on performance of broiler chickens. Agric. Sci. Finland 3:457–466. Kornegay, E. T., 1996. Effect of phytase on the bioavailability of phosphorus, calcium, amino acids and trace minerals in broilers and turkeys. Pages 39–68 in: BASF Technical Symposium Phosphorus and Calcium Management in Layers. Atlanta, GA, January 23, 1996.

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shell weights is a more labor-intensive method for evaluating Ca metabolism in layers, but can be used to confirm egg specific gravity data. Increasing dietary Ca in this study resulted in an increase in egg specific gravity (Table 2), as reported elsewhere (Ousterhout, 1980; Frost and Roland, 1991; Abdallah et al., 1993). This result was observed in Week 1 after only 3 d of treatment and continued through termination. No Ca-related interactions were observed. Although phytase supplementation increased egg specific gravity at both levels of NPP, a significant phytase by P interaction revealed that the phytase effect was greatest when NPP levels were lowest. Supplementing the 0.1% NPP diet with phytase also improved shell quality in a previous study (Gordon and Roland, 1997). However, this improvement was not observed at higher P levels, presumably because the adequate Ca levels (4%) of these diets prevented the Ca deficiency necessary to reveal the influence of liberated phytate P on Ca utilization and eggshell quality. Increasing dietary P significantly improved eggshell quality in the absence of supplemental phytase, but not in the presence of phytase. The improvement in eggshell quality associated with phytase supplementation could not be attributed solely to phytate P liberation. When the diet containing 2.5% Ca and 0.1% NPP was supplemented with phytase, egg specific gravity increased 0.0024 units. Similar increases of 0.0022 and 0.0034 units were observed when the 0.1% NPP diets with 2.8 and 3.1% Ca, respectively, were supplemented with phytase. Increasing dietary NPP from 0.1 to 0.3% also increased egg specific gravity. However, there was a significantly greater increase in egg specific gravity when the 0.1% NPP diet was supplemented with phytase. Increasing dietary P from 0.1 to 0.3% in diets containing 2.5, 2.8, and 3.1% Ca resulted in egg specific gravity increases of 0.0011, 0.0015, and 0.0016 units, respectively. Supplementing P-adequate diets with phytase increased egg specific gravity 0.0010, 0.0006, and 0.0012 units when dietary Ca levels were 2.5, 2.8, and 3.1%, respectively. When the phytase benefit associated with increased P availability was accounted for in the 0.1% NPP diet, the residual benefit was a similar 0.0013, 0.0007, and 0.0018 units for the 2.5, 2.8, and 3.1% Ca diets, respectively. Assuming that 0.3% NPP is adequate for these hens, as evidenced throughout the life of this flock, approximately 32 to 54% of the phytase benefit on eggshell quality in these studies appears to be attributable to something other than increased P availability. Similar results were observed when eggshell weights were evaluated. When phytase was included in the diet containing 2.8% Ca and 0.1% NPP, shell weights increased from 4.89 to 5.17 g per egg. When NPP was increased to 0.3% in the absence of phytase, shell weights only increased to 5.04 g. When dietary Ca was 3.1% and NPP 0.1%, supplemental phytase increased shell weights from 4.83 to 5.23 g. Increasing dietary NPP to 0.3% in the absence of phytase only increased shell weights to 5.06 g. Supplementing the 2.5% Ca/0.1% NPP diet with phytase

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GORDON AND ROLAND Schwarz, G., 1994. Phytase supplementation and waste management. In: BASF Technical Symposium, Arkansas Nutrition Conference, September 13, 1994. Steel, R.G.D., and J. H. Torrie, 1980. Principles and Procedures of Statistics. A Biometrical Approach. 2nd ed. McGraw-Hill Book Co., Inc., New York, NY. Strong, C. F., Jr., 1989. Relationship between several measures of shell quality and egg-breakage in a commercial processing plant. Poultry Sci. 68:1730–1733. van der Klis, J. D., H.A.J. Versteegh, and P.C.M. Simons, 1996. Natuphos in laying hen nutrition. Pages 71–82 in: BASF Technical Symposium Phosphorus and Calcium Management in Layers, Atlanta, GA, January 23, 1996. Yi, Z., E. T. Kornegay, and D. M. Denbow, 1996a. Supplemental microbial phytase improves zinc utilization in broilers. Poultry Sci. 75:540–546. Yi, Z., E. T. Kornegay, V. Ravidran, and D. M. Denbow, 1996b. Improving phytate phosphorus availability in corn and soybean meal for broilers using microbial phytase and calculation of phosphorus equivalency values for phytase. Poultry Sci. 75:240–249.

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Leeson, S., J. D. Summers, and L. Caston, 1993. Response of brown-egg strain layers to dietary calcium or phosphorus. Poultry Sci. 72:1510–1514. Orban, J. I., D. A. Roland, Sr., M. M. Bryant, and J. C. Williams, 1993. Factors influencing bone mineral content, density, breaking strength and ash as response criteria for assessing bone quality in chickens. Poultry Sci. 72:437–446. Ousterhout, L. E., 1980. Effects of calcium and phosphorus levels on egg weight and egg shell quality in laying hens. Poultry Sci. 59:1480–1484. Pallauf, J., G. Rimback, S. Pippig, B. Schindler, D. Ho¨hler, and E. Most, 1994. 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, calcium, magnesium, zinc and protein in piglets. Z. Erna¨hrungswiss 33:128–135. Roberson, K. D., and 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, 1986. SAS User’s Guide: Statistics. SAS Institute Inc. Cary, NC.