High Available Phosphorus Corn and Phytase in Layer Diets 1

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1 High Available Phosphorus Corn and Phytase in Layer Diets 1 N. Ceylan, 3 S. E. Scheideler, 2 and H. L. Stilborn 4 Department of Animal Sciences, University of Nebraska, Lincoln, Nebraska ABSTRACT High available phosphorus corn (HAP) developed NPP diets. A significant corn type NPP interaction effect using the low phytic acid 1-1 (lpal-1) allele of the corn LPA1 gene containing 0.27% P, with 0.17% nonphytate P (NPP), was compared to near isogenic normal corn (LPA1), which contained 0.23% P and 0.05% NPP. Five was observed for egg weight, such that within the HAP corn diets, egg weight decreased more markedly at the 0.25% NPP levels compared to the normal corn 0.25% NPP diets. Specific gravity was not affected by dietary levels of NPP from either HAPC or normal corn (0.40, treatment, but percent dry shell was improved at the 0.35, 0.30, 0.25 and 0.20% phytase units (FTU)/kg lower AP levels and with phytase treatment. Dietary NPP microbial phytase) were combined in a 2 5 factorial level and corn type had no significant effect on bone ash. Excreta levels of total phosphorus decreased significantly experiment for a total of 10 dietary treatments. Each dietary treatment was fed to eight replicate cages with five as dietary NPP decreased and were lower in the HAP corn excreta compared to normal corn excreta. Total P, Hy-Line W-36 hens per replicate cage from 20 to 40 wk Ca, Zn, Cu, and Mn retention were significantly affected of age. Feed consumption and egg production were not by NPP level and corn type. HAP corn reduced Ca, Zn, significantly affected by dietary NPP level or corn type. Cu, and Mn retention compared to normal corn; this negative effect was alleviated by phytase supplementation to Feed conversion ratio (g feed:g egg mass) was improved at the 0.35% NPP level (1.856) compared to the other levels HAP corn diets. HAP corn allowed less dicalcium phosphate supplementation in layer diets compared to normal of NPP 0.40, 0.30, 0.25, and 0.20% + phytase having feed conversion ratios of 1.872, 1.905, 1.930, and 1.898, corn while supporting equal egg production. Phytase supplementation respectively. Egg weight and egg mass decreased significantly as dietary NPP decreased; diets with 0.20% NPP plus phytase had equal egg mass to the 0.35 and 0.40% of low NPP diets had no significant positive effects on egg production parameters in either corn type diets. (Key words: high available phosphate corn, layers, phosphorus, phytase) 2003 Poultry Science 82: INTRODUCTION Most of the phosphorus contained in feed ingredients of plant origin occurs as phytic acid (Lott et al., 2000). The salts of phytic acid are described as phytates. In general, phytate phosphorus accounts for about twothirds of the total phosphorus present in feed ingredients of plant origin. The availability of phosphorus from plant feedstuffs is very low in poultry because birds have limited endogenous phytase activity. This situation poses three problems: 1) the need to add inorganic phosphorus supplements to poultry diets, 2) the excretion of large amounts of nonutilized phosphorus in manure, and 3) 2003 Poultry Science Association, Inc. Received for publication June 17, Accepted for publication January 23, Published with the approval of the Director as Paper Number 13727, Journal Series, Nebraska Agricultural Research Division. 2 To whom correspondence should be addressed: sscheideler1@ unl.edu. 3 Present address: Ankara University, Ankara, Turkey. 4 Present address: P. O. Box 71112, Des Moines, IA phytate limits availability of several other essential nutrients, such as protein, Ca, Cu, and Zn (Yi et al., 1996). Phytate can bind with Ca to form insoluble phytate complexes at intestinal ph. Insoluble phytates reduce both Ca and P availability (Kornegay, 2001). Phytate may also have a negative effect on the solubility of proteins and the function of pepsin because of potential ionic binding between phytate phosphate groups and amine groups (Kornegay, 2001). Phytase supplementation, which degrades phytate to release phosphorus and other nutrients, has been very beneficial in solving the aforementioned problems as shown in recent studies in broilers (Perney et al., 1993; Denbow et al., 1995; Sebestian et al., 1996, 1997; Yi et al., 1996), pullets ( Punna and Roland, 2000), and layers (Boling et al., 2000; Jalal and Scheideler, 2001). Phytase supplementation is rapidly becoming more commonplace in the poultry industry. In addition to phytase, another Abbreviation Key: FTU = phytase unit; HAP = high available phosphorus corn; NPP = nonphytate P. 789

2 790 CEYLAN ET AL. approach, which could be of benefit, is the use of low phytic acid or otherwise termed high available phosphorus (HAP) corn (Raboy et al., 2000 and Ertl et al., 1998) in poultry rations. HAP corn has low levels of phytate phosphorus (37% of total P) compared to normal corn, which contains an estimated 78% of the phosphorus bound in phytate form (NRC, 1994). Utilizing HAP corn in poultry diets would decrease levels of supplemental inorganic phosphorus and theoretically increase other nutrient utilization. Broilers have successfully been fed HAP corn with low levels of supplemental inorganic phosphorus, and studies have shown a significant reduction in fecal P while maintaining optimum performance (Waldroup et al., 2000; Yan et al., 2000). The objective of this experiment was to compare use of HAP corn to regular corn with and without phytase in the diets of young laying hens and their effects on phosphorus utilization and hen performance. MATERIALS AND METHODS Ten diets were fed to the Hy-Line W-36 strain of laying hens starting at 20 wk of age until 40 wk of age. Each dietary treatment was replicated eight times with five hens per replicate cage (480 cm 2 /hen) for a total of 80 cages. An automatic nipple drinker provided water ad libitum to each cage of hens, and feed was provided daily according to expected intake. Dietary treatments were arranged as a 2 5 factorial arrangement with two corn types (HAP and normal corn 5 ) and five levels of NPP (0.40, 0.35, 0.30, 0.25, 0.20% + phytase) for a randomized complete block design. Laying hen cages were blocked by row and level in a stacked cage unit (four high) with replication of each diet in each row (eight blocks) on both sides of the layer unit. The HAP corn was developed using the low phytic acid 1-1 (l pal-1) allele of corn LPA per gene (Raboy et al., 2000) containing 0.27% P with 0.17% NPP. The HAP corn was near isogenic to the normal corn (LPA 1), which contained 0.23% P and 0.05% NPP. Dietary phytase (Natuphos 600) 6 was added to both sources of corn at the lowest level of NPP supplementation (0.20%) at a level of 300 phytase units (FTU)/kg of feed. The normal corn sample was the isogenic equivalent of the HAP corn less the phytate gene allele alteration. The nutrient analysis of the normal and HAP corns were provided by Optimum Quality Grains, L.L.C., for diet formulation. HAP corn ME was predicted by Optimum Quality Grains, L.L.C., to be equivalent to normal corn ME at 3,420 kcal/kg. Diets (Table 1) were formulated to be isonitrogenous and isocaloric. Feed consumption and egg production were measured daily by cage. Biweekly, 1 d of egg production was kept for measurement of egg weight, specific gravity, and eggshell percent. Eggshell percent was measured on two eggs per cage by breaking the egg and separating the egg shell 5 Optimum Quality Grains, L.L.C., Des Moines, IA. 6 BASF Corporation, Wynadotte, MI. from the liquid content. The eggshells were then dried overnight for 24 h in a oven at 100 C. Dry shell weight is expressed as a percent of whole egg weight. All hens were weighed at the start of the trial and at 3-wk intervals thereafter to calculate an average hen weight per cage. At 28 and 40 wk of age, the diets were marked with chromic oxide as an undigestible marker to measure excreta phosphorus content and mineral retention for Ca, P, Cu, Zn, and Mn (Edwards and Gilllis, 1959; Scott et al., 1976; Kozloski et al., 1978). Chromic oxide marked diets (0.30%) were fed for 3 d and on the fourth day, and representative fecal samples were collected from each cage to determine nutrient retention. Diets and excreta were analyzed for Ca, P, Ca, Zn, and Mn by procedures established by the Association of Official Analytical Chemists (1984). Chromium in the diets and excreta were analyzed by the procedure described by Williams et al. (1962) using atomic absorption spectrophotometry. Diet samples from diets 5 and 10 were sent to BASF 6 for analysis of phytase activity approximately 1 yr after feed mixing. Results of the phytase assay indicated 190 to 210 FTU/kg activity 1 yr postmixing indicating some deterioration of predicted activity (300 FTU/kg) during sample storage. At 40 wk of age, two hens from each replicate cage were killed, and tibia samples from each hen were excised to determine fat-free bone ash (Garlich et al., 1982). Statistical analysis was performed by the general linear models procedure of SAS software, using PROC mixed (SAS Institute, 1995) testing for main effects of corn type and level of NPP and interaction effects of corn type phosphorus level, and corn type phytase (treatments 4 vs. 5 and 9 vs. 10). RESULTS Table 2 shows the dietary effects on average feed intake, NPP intake, egg production, feed conversion, and body weight gain during the experimental period. Overall diet effects along with the main and interaction effects are presented. Dietary treatments showed no significant effects on feed intake or egg production. Intake of NPP was directly affected (P < 0.01) by level of dietary NPP decreasing as level of dietary NPP decreased. There was an indication of a near significant interaction (P < 0.09) effect on egg production such that within the HAP corn treatments, egg production decreased at the low NPP levels (0.25 and 0.30%). This negative effect was not seen in the normal corn diets at the levels of NPP fed. Feed conversion showed a negative (P < 0.07) effect of reducing NPP to less than 0.35% in both the normal and HAP corn treatment groups. Supplementation of phytase alleviated this negative response in the low nonphytate phosphorus treatments (0.20% NPP). Body weight gain during the trial was not significantly affected by dietary treatment. Table 3 shows the effects of diet on egg weight, egg mass, specific gravity, and dry shell percent. Egg weight was affected by level of NPP (P < 0.001) as well as an observation of a significant corn source by NPP interac-

3 HIGH AVAILABLE P CORN AND PHYTASE 791 TABLE 1. Ingredients and nutrient composition of experimental diets Ingredient and composition Corn HAP corn Soybean meal Corn oil Tallow Calcium carbonate Oyster shell Dicalcium phosphate DL-Methionine Salt Lysine Vitamin premix Mineral premix Phytase Nutrient composition ME, kcal/kg 2,890 2,891 2,891 2,891 2,891 2,891 2,891 2,891 2,891 2,891 Crude protein, % Calcium, % Total phosphorus Calculated, % Analyzed, % Nonphytate P, % Methionine + cystine, % Lysine, % Cu, ppm analyzed Mn, ppm analyzed Zn, ppm analyzed High available phosphorus corn provided by Optimum Quality Grains, L.L.C., Des Moines, IA. 2 Vitamin premix provided Mn, 88 mg; Cu, 6.6 mg; Fe, 8.5 mg; Zn, 88 mg; Se, 0.30 mg; vitamin A, 6,600 IU; cholecalciferol, 2,805 IU; vitamin E, 10 IU; vitamin K, 2.0 mg per kg diet. 3 Mineral premix provided riboflavin, 4.4 mg; pantothenic acid, 6.6 mg; niacin 24.2, mg; choline, 110 mg; vitamin B 12, 8.8 mg; ethoxyquin, 1.1 mg per kilogram of diet. 4 Natuphos 600, provided 300 phytase units (FTU)/kg of feed. Diet tion. As the level of NPP decreased to less than 0.35%, egg weight decreased. This effect was more prominent in the HAP corn diets compared to normal corn diets as evidenced by the significant interaction (P < 0.05). Supplementation of phytase to the lowest phosphorus diets increased egg weight to values equal to the high NPP levels within the HAP corn source diets. Egg mass decreased as dietary NPP decreased, which was overcome by supplementation of phytase at the lowest level of NPP (0.20%). Specific gravity showed no overall significant effects of dietary treatments. Percent dry egg shell was influenced by NPP level, increasing as dietary NPP decreased (P < 0.02). Corn type had no effect on shell quality. Table 4 shows the effects of dietary treatments on bone ash, excreta phosphorus, and mineral retention. Bone ash was not significantly affected by any of the dietary treatments. Excreta phosphorus levels were determined at 28 and 40 wk of age and showed significant differences due to both level of NPP in the diet as well as type of corn fed. HAP corn significantly reduced overall excreta phosphorus at both 28 and 40 wk of age compared to the normal corn by an average of 13.4%. There was an overall increase in excreta phosphorus at 40 wk of age compared to the level at 28 wk of age. Excreta phosphorus decreased as the dietary NPP decreased (P < 0.001). Phosphorus retention was significantly affected by dietary phosphorus level at 28 wk of age, showing slight improvement at 0.35 and 0.30% NPP levels compared to lower retention at 0.40 and 0.25% NPP. At 40 wk of age, P retention was greatest in the high NPP (0.40%) and low NPP (0.20%) + phytase treatment groups compared to the other levels of NPP (0.25, 0.30, or 0.35% NPP). Type of corn did not significantly affect phosphorus retention at 28 wk of age; however, at 40 wk of age, lower overall phosphorus retention was measured in the HAP corn treatment groups compared to the normal corn treatments. Type of corn had an effect (P <.06) on calcium retention indicating that HAP corn had a slightly negative effect on calcium retention as well as the other measured mineral retention (Zn, P < ); (Cu, P < 0.001); and (Mn P < 0.001). Level of NPP also significantly influenced Zn, Cu, and Mn retention. As the level of NPP decreased from 0.35% to 0.30 and 0.25%, retention of Zn, Cu and Mn decreased. Supplementation of phytase improved retention of Zn, Cu and Mn to equal the 0.40% NPP treatment. DISCUSSION The use of HAP corn to reduce supplemental phosphorus from inorganic sources and phytate phosphorus from corn in layer diets achieved the goals of equal egg-laying performance in the laying hen and lowered excreta P when compared to normal corn use with higher levels of

4 792 CEYLAN ET AL. TABLE 2. Effects of dietary treatments on feed intake, egg production, feed conversion, and body weight gain in Hy-Line W-36 laying hens from 20 to 40 wk of age Nonphytate Feed NPP 1 Egg Feed Body weight phosphorus intake intake production conversion gain Diet Corn type (%) (g/hen/day) (mg/hen/day) (%) (g) (g) 1 Normal Normal Normal Normal Normal phytase HAP HAP HAP HAP HAP phytase SEM P-value NS 3 NS 0.11 NS NS Corn type Normal corn HAP corn P-value NS NS NS NS NS NPP level 0.40% % % % % + phytase P-value NS 0.01 NS 0.07 NS Interaction Corn type NPP level NS NS 0.09 NS NS Corn type phytase NS NS NS NS NS (diet 4 vs. 5 and 9 vs. 10) 1 NPP = nonphytate phosphorus. 2 HAP = high available phosphorus corn. 3 Nonsignificant P-value > TABLE 3. Effects of dietary treatments on egg weight, egg mass, specific gravity, and dry shell percentage in Hy-Line W-36 hens from 20 to 40 wk of age Egg Egg NPP 1 weight mass Specific Dry shell Diet Corn type (%) (g) (g/day per hen) gravity (%) 1 Normal Normal Normal Normal Normal phytase HAP HAP HAP HAP HAP phytase SEM P-value NS 0.02 Corn type Normal corn HAP corn P-value NS 2 NS NS NS NPP level 0.40% % % % % + phytase P-value Interaction Corn type NPP level 0.05 NS NS NS Corn type Phytase NS NS NS NS (Diet 4 vs. 5 and 9 vs. 10) 1 NPP = Nonphytate phosphorus. 2 HAP = high available phosphorus corn. 3 Nonsignificant P-value > 0.10.

5 HIGH AVAILABLE P CORN AND PHYTASE 793 TABLE 4. Effects of dietary treatments on bone ash, excreta phosphorus and retention of P, Ca, Zn, Cu, and Mn in Hy-Line W-36 laying hens Retention (%) Bone Excreta phosphorus (%) NPP 1 ash P P Ca Zn Cu Mn Diet Corn type (%) (%) 28 wk 40 wk 28 wk 40 wk 40 wk 40 wk 40 wk 40 wk 1 Normal Normal Normal Normal ( ) Normal 0.20 P + phytase ( )8.8 6 HAP ( )0.52 ( )0.22 ( ) HAP ( ) HAP ( )4.0 ( ) HAP ( ) ( ) HAP phytase SEM P-value NS Corn Type Normal corn HAP corn ( )10.7 P-value NS NS NPP Level 0.40% % % ( ) % ( ) ( ) % + phytase P-value NS NS Interaction Corn type NPP level NS NS NS NS Corn type phytase NS NS NS (Diet 4 vs. 5 and 9 vs. 10) 1 NPP = Nonphytate phosphorus. 2 HAP = high available phosphorus corn. 3 Nonsignificant P-value > 0.10.

6 794 CEYLAN ET AL. dicalcium phosphorus supplementation and phytate P. Feeding low levels of NPP (0.25 to 0.30%) had a negative effect on egg weight and egg mass produced compared to dietary levels of 0.35 to 0.40% NPP. Low levels of dietary NPP resulted in inadequate NPP intakes (<300 mg/d) compared to breeder recommendations (Hy-Line International 7 ) of 380 mg/day per hen (see Table 2). There is however disagreement among publications about the NPP requirement for layers. The 1994 NRC Nutrient Requirements of Poultry recommended 250 mg/d. This is considerably lower than breeder recommendations. In this study, while intakes of <300 mg/d adequately supported egg production, that level did not adequately support egg weight in the HAP corn group as evidenced by the significant interaction effect between corn type and NPP level. Supplementation of phytase at the lowest NPP level (0.20%) brought egg weight back equal to the control NPP level (0.40%). Intakes of less than 200 mg/d NPP were adequate when phytase was supplemented to either corn type at 300 FTU/kg feed. This positive effect of phytase on phosphorus availability has been previously reported by Jalal and Scheideler (2001). They reported a significant effect of NPP level and phytase supplementation on egg weight and egg mass in addition to a linear effect of NPP level on EP but their lowest levels of NPP fed were 0.10 and 0.15%, which may have been more deficient than levels fed in this study. The observed improvement in shell quality (measured as percentage of dry shell) as dietary phosphorus decreased has been previously reported (Damron and Harms, 1982). This is a positive benefit for a limited period of time as long as the bird s skeleton holds up. Industry practice has shown that if such a program is implemented too long, cage layer fatigue can result. Phosphorus retention decreased considerably between 28 and 40 wk of age, which is not unusual, as hens start to mobilize more skeletal stores of calcium for egg shell calcification as they age. Phosphorus retention decreased as dietary NPP decreased at 40 wk of age indicating that the adaptation mechanism that was functioning at 28 wk of age was no longer functioning efficiently at 40 wk of age. Scheideler and Sell (1987) reported similar decreases in phosphorus retention in the laying hen as she aged in addition to some negative retention values as also reported in this study. Adding phytase to the rations increased phosphorus retention in only the HAP corn types at both 28 and 40 wk of age. Dietary treatment had significant effects on calcium, zinc, manganese, and copper retention measured at 40 wk of age. A significant reduction in calcium retention occurred in hens fed the HAP corn compared to the normal corn in addition to a significant corn by P level interaction such that calcium utilization was lowest when HAP corn was fed at the highest phosphorus level. This indi- 7 Hy-Line Variety W-36 Commercial Management Guide A publication of Hy-Line International, P.O. Box 65190, West Des Moines, IA cated a type of negative interaction between the more highly available phosphorus form in HAP corn and calcium availability. Ertl et al. (1998) did not find such a negative interaction between HAP corn and Ca availability in chick studies. They reported improved Ca status in chicks as corn P availability improved. The negative effect was overcome with phytase supplementation to the HAP corn. This negative effect of HAP corn on cation retention was repeated in the Zn, Cu, and Mn retention data, indicating some type of negative factor (perhaps incomplete inositol phosphates or excess phosphate groups) having binding capacity for cations in the diet. This effect was highly significant for all cations tested. The corn type by NPP level interaction was also highly significant for cation retentions. It is interesting to note that the addition of phytase to the HAP corn diets low in NPP reversed this negative effect rendering the cations Zn, Cu, and Mn more available. Corn type by phytase interaction effects were significant such that cation utilization decreased in the normal corn with addition of phytase contrary to theory. The positive effect of phytase on cation utilization in HAP diets indicates some possible interaction occurring between either free phosphate groups or incomplete inositol phosphate left in the corn with potential binding sites available for cations. 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