METABOLISM AND NUTRITION

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1 METABOLISM AND NUTRITION The Effect of Different Levels of Nonphytate Phosphorus with and Without Phytase on the Performance of Four Strains of Laying Hens 1 K. Keshavarz 2 Department of Animal Science, Cornell University, Ithaca, New York ABSTRACT A4 7 factorial experiment was conducted during this phase of the experiment. The NPP regimen to determine the effect of different nonphytate phosphorus (NPP) regimens with and without phytase on performance of four strains of laying hens. The strains used were Babcock B300, DeKalb Delta White, Hy-Line W36, of % plus either level of phytase (T 6 and T 7 ) restored most of the traits for the entire experiment to the control level (T 1 ), except egg production (EP) and the nonprolapse mortality. Bone ash also remained significantly lower than the control group with this NPP regi- and ISA-White. The birds of T 1 (Treatment 1), control diet, were fed a diet with 0.45% NPP for the entire experiment (20 to 63 wk of age). The birds of T 2 were fed a traits were reduced, and nonprolapse mortality was inmen plus the higher level of phytase (T 7 ). Production NPP regimen of 0.25% for Phase 1 (20 to 35 wk of age), creased due to the use of a NPP regimen of % 0.2% for Phase 2 (36 to 51 wk of age), and 0.15% for Phase without phytase (T 2 ) for the entire experiment. The NPP 3 (52 to 63 wk of age). The birds of T 3 and T 4 were fed a regimen of % plus the lower level of phytase NPP regimen similar to T 2 plus 150 or 300 units phytase/ (T 3 ) restored all the traits except EP to the control level for the entire experiment. The only treatment that maintained kg diet, respectively. The birds of T 5 were fed a NPP performance of all the strains comparable to their controls regimen of 0.2, 0.1, and 0.1% for Phases 1, 2, and 3, respectively. The birds on T 6 and T 7 were fed a NPP regimen for the entire experiment was a NPP regimen of % plus the higher level of phytase (T 4 ). Total P similar to T 5, with 150 and 300 units phytase/kg diet, excretion of birds fed this NPP regimen was reduced by respectively. The criteria for evaluating the effect of dietary treatments were production performance, shell ally indicated that the higher level of phytase was more 55.6% as compared to the control group. The data gener- quality, bone ash, and total P excretion. Production traits effective than the lower level in restoring the performance were drastically reduced and mortality was drastically of birds fed the low-p diets to the control level. Numerous increased when the birds of T 5 were fed a diet with 0.1% interactions existed among strain diet for various traits NPP during Phase 2 (36 to 51 wk of age). Increasing the NPP level of this treatment from 0.1 to 0.45% during Phase 3 restored the production traits almost to the control level throughout the experiment, indicating the NPP requirement for maintaining production performance may be different among strains. (Key words: laying hen, phosphorus, phytase, egg production, phosphorus excretion) 2003 Poultry Science 82:71 91 INTRODUCTION Determination of the nonphytate P (NPP) requirement of laying hens and the potential of phytase to reduce this requirement have been the subject of numerous investigations in recent years. Based on the results of these investigations, diets with 0.15 to 0.2% NPP and in the absence of phytase (Keshavarz, 1986b; Gordon and Roland, 1997; Van der Klis et al., 1997; Boling et al., 2000a,b; Snow et al., 2000), and diets with 0.1% NPP in the presence of phytase (Gordon and Roland, 1998; Boling et al., 2000a,b) have been shown to be sufficient to maintain satisfactory egg production (EP) performance during the laying cycle. It has also been reported that such a beneficial effect can be obtained by adding 100 to 300 units of phytase/kg of layer s diet (Usayran and Balnave, 1995; Van der Klis et al., 1997; Gordon and Roland, 1998; Boling et al., 2000b). In a previous experiment, we were interested in reevaluating the NPP requirement of laying hens in a phasefeeding program and to determine to what extent this requirement can be reduced in the presence of phytase 2003 Poultry Science Association, Inc. Received for publication April 8, Accepted for publication August 5, Supported in part by ISA Babcock, P.O. Box 280, Ithaca, NY To whom correspondence should be addressed: kk33@cornell.edu. Abbreviation Key: EP = egg production; EM = egg mass; EW = egg weight; FC = feed conversion; FI = feed intake; NPP = nonphytate phosphorus; PS = percentage shell; SG = specific gravity; SW = shell weight; SWUSA = shell weight per unit surface area; T x = treatment. 71

2 72 KESHAVARZ (Keshavarz, 2000). The results of that experiment indicated that in the absence of phytase, a NPP regimen of 0.25, 0.2, and 0.15% which was used for the periods of 30 to 42 wk (0.25%), 42 to 54 wk (0.2%), and 54 to 66 wk of age (0.15%), respectively, resulted in a comparable performance to the control group, which was fed a NPP regimen of % for the same age periods. However, production performance for the entire experiment (30 to 66 wk of age), was significantly reduced when a NPP regimen of % in the absence of phytase for the above age period was used. All the signs of P deficiency attributed to feeding this NPP regimen were alleviated by inclusion of 300 units of phytase/kg diet. The results also indicated that phytase prevented the expression of the beneficial effect of low-p diets on several indices of shell quality. The results of the previous experiment raised a number of questions that warranted further investigation before proper recommendations could be made for the commercial application. These were 1) whether the information obtained regarding the NPP requirement with and without phytase for the strain used in the previous experiment (Babcock B300) is reproducible, 2) whether similar levels of NPP with and without phytase can be used satisfactorily for the three other commercial layer strains, 3) whether a lower level of phytase than the manufacturer 3 recommendation of 300 units/kg can be used satisfactorily in the layer diets in order to increase the economic incentive of using phytase rather than inorganic sources of P by the industry, and 4) whether the adverse effect of phytase in preventing the beneficial effect of low-p diets on several indices of shell quality as observed in a previous experiment (Keshavarz, 2000) is reproducible. It is worth noting that although numerous experiments have been conducted during recent years on the use of phytase in layer diets, to our knowledge, in none of these experiments was a direct strain comparison made to determine whether strain differences existed for the NPP requirement with and without phytase in the diet. The current experiment was conducted to find answers to the aforementioned questions. MATERIALS AND METHODS Two thousand one hundred 19-wk-old pullets consisting of four commercial strains (Babcock B300, DeKalb Delta White, Hy-Line W36, and ISA-White) were used in this experiment. The experiment started on March 1999 and continued up to February Hatching eggs for one strain (Babcock B300) were obtained from the breeder hatchery and were incubated at the Cornell University hatching facilities. Day-old chicks of the other strains were obtained from the breeders hatcheries. The eggs or the day-old chicks of all four strains belonged to the parent flocks, which were 30 to 40 wk of age. Although 3 BASF Corp., Mt. Olive, NJ. it was our intention that all four strains would have a similar age at the start of the experiment, it was not practical. Three strains had the same hatching date, whereas the Hy-Line chicks were 1 wk younger. Nevertheless, during both the growing and the laying periods, all the strains were treated on a similar age basis. Pullets were fed the Cornell chick starter diet (20.8% CP, 1.05% Ca, 0.44% NPP) from 1 d to 6 wk of age and the Cornell pullet grower diet (15.8% CP, 1.01% Ca, and 0.44% NPP) from 6 to 18 wk of age. At the end of 18 wk of age, 525 pullets from each strain (a total of 2100 pullets) were randomly selected and weighed in groups of five and transferred to a highrise, environmentally-controlled, three-deck cage laying house facility similar to housing used by the commercial industry in the northeastern United States. The pullets were housed five per deep-designed cage, measuring 38.1 cm across the front and 50.8 cm from front to back. The pullets of three adjacent cages were considered an experimental replicate and each dietary treatment was fed to five replicates per strain (75 pullets/treatment per strain). During wk 19, all the pullets were fed the Cornell layerbreeder diet (16.8% CP, 3.55% Ca, and 0.42% NPP). Based on the BW at the end of 18 wk of age, the treatment means for BW within each strain was kept similar at the start of the experiment. Pullets were fed the experimental diets from the beginning of 20 wk of age. The experiment was divided into three phases: Phase 1 (20 to 35 wk of age, 16-wk period), Phase 2 (36 to 51 wk of age, 16-wk period), and Phase 3 (52 to 63 wk of age, 12-wk period). The experimental design consisted of a 4 7 factorial arrangement of the treatments, with four strains and seven diets (Table 1). The composition of the positive control (T 1 ) is shown in Table 2. Other dietary treatments (T 2 to T 7 ) for the various phases of the experiment were formulated by reducing the level of mono-dicalcium phosphate and adjusting the limestone to maintain a similar Ca level in all the diets (3.8%). Builder sand was used to increase the weight of the diets to 100%. Phytase premix (Natuphos 600) 3 was added to the complete diets. Prior to mixing each new batch of the diets, a new shipment of Natuphos with determined phytase content was received from the manufacturer. 3 Based on the phytase activity of each premix, proper levels were added to the diets to provide 150 or 300 units phytase/kg diet. After mixing each batch of diet, a representative sample from each finished feed was saved and sent to the manufacturer of Natuphos (BASF) for determination of the phytase level in the finished feeds. The diets were isocaloric and isonitrogenous and, with the exception of NPP level and the presence or absence of phytase, contained similar levels of all the other nutrients to satisfy or exceed the NRC (1994) requirement. Records of daily EP and weekly feed intake (FI) were kept during the experiment and were summarized every 4 wk. Egg weight (EW), egg grades (according to the USDA grading system), and specific gravity (SG) were determined on all eggs produced during a 3-d period at the end of every 4 wk. Egg shell weight (SW), percentage

3 PHOSPHORUS REQUIREMENTS OF LAYING HENS 73 TABLE 1. Design of the experiment Nonphytate P (%) Phase 1 1 Phase 2 2 Phase 3 3 Phytase 4,5 Treatment (20 35 wk) (36 51 wk) (52 63 wk) (Units/kg diet) During Phase 1, the diets with calculated nonphytate P (NPP) contents of 0.45, 0.25, and 0.2%, corresponding to calculated total P contents of 0.668, 0.467, and 0.417% contained the determined total P levels of 0.679, 0.468, and 0.404%, respectively. 2 During Phase 2, the diets with calculated NPP contents of 0.45, 0.2, and 0.1% corresponding to calculated total P contents of 0.668, 0.417, and 0.321% contained the determined total P levels of 0.664, 0.417, and 0.327%, respectively. 3 During Phase 3, the diets with calculated NPP contents of 0.45, 0.15, and 0.1%, corresponding to calculated total P contents of 0.668, 0.368, and 0.321% contained the determined total P levels of 0.663, 0.359, and 0.298% respectively. 4 Natuphos 600 (BASF Corp., Mt. Olive, NJ) was used as the source of microbial phytase. 5 The diets T 3,T 4,T 6, and T 7 with calculated phytase activities of 150, 300, 150, and 300 units/kg of diet had average determined phytase activities of 175, 289, 181, and 297 units/kg of diet, respectively, for the entire experiment. shell (PS), and shell weight per unit surface area (SWUSA) were determined on a random sample of 10 eggs per replicate after measurement of SG and only at the ends of Phase 1 (35 wk of age), Phase 2 (51 wk of age), and Phase 3 (63 wk of age). Mortality records were kept during the experiment. BW was determined at the end of each phase. A digestion trial was conducted for 6 d immediately at the end of Phase 3 (i.e., during 64 wk of age) for determining total P excretion. Due to space limitations, it was not possible to conduct the digestion trial during Phases 1 to 3 of the experiment. At the start of the digestion trial, three hens from each replicate that had a hard shell in the uterus (through palpation) were selected and housed in one separate cage. Excreta was collected quantitatively every 3 d for 6 d for determining total P. Record of FI was kept during the digestion trial. At the end of the digestion trial, one bird from each replicate that was a part of the digestion trial was randomly selected and killed by CO 2 asphyxiation, and the left tibia was saved for measurement of tibia ash. Due to the abundant groups (4 strains 7 diets 5 replicates = 140 groups), only the birds of the control group (T 1 ), those fed a sequence of % NPP with and without 150 and 300 units phytase/kg diet (T 2,T 3, and T 4 ), as well as birds fed a sequence of % NPP plus 300 units phytase/kg diet (T 7 ), were used in the digestion trial. Salt solutions varying in SG from to 1.102, in increments of units, were used for measurement of SG. SW with membranes intact was measured after breaking the eggs and cleaning the shell from the adhering albumen and drying the shell in an oven at 100 C for 2 h. PS was determined by dividing the dried SW by EW. The SWUSA was determined by dividing the dried SW by the surface area of each egg. The surface area of each egg was determined according to the approach of Ousterhout (1980). Tibia ash was determined on a moisturefree, fat-free basis after ashing the tibia at 600 C overnight in a furnace. Total P content of feed and fecal samples were determined according to the method of Harris and Popat (1954). The data were analyzed by a two-way ANOVA (SAS, 1988), and means were compared according to the multiple-range test of Duncan (1955) when a significant probability value (P < 0.05) was detected. When the interaction among strain diet for a particular trait was significant, the data were analyzed as a one-way ANOVA, and the means were compared by the Duncan s multiple-range test. BW RESULTS The interactions between strain diet for BW were not significant during each phase or for the entire experiment. At the beginning of the experiment (18 wk of age), BW was not different among birds of various dietary treatments (Table 3). However, strain differences existed for BW. DeKalb had the heaviest and ISA-White had the lightest BW, whereas the BW of the other two strains were intermediate between DeKalb and ISA-White. At the end of Phase 1 (35 wk of age), BW was not influenced by dietary treatments; however, strain differences existed for BW. The BW gain was greater for Babcock and ISA- White (284 and 291 g/bird, respectively) than for DeKalb and Hy-Line (223 and 220 g/bird, respectively) during this phase of the experiment. As a result, BW of Babcock and DeKalb were not different from each other, but they were significantly heavier than the other two strains at 35 wk of age.

4 74 KESHAVARZ TABLE 2. Composition of the control diet Ingredient (%) Corn Soybean meal, dehulled DL-Methionine 0.07 Salt 0.33 Limestone 9.14 Monodicalcium phosphate 1.69 Vitamin mixture Mineral mixture Blended fat 1.00 Sand Natuphos 3 Calculated analysis 4 Energy (kcal ME/kg) 2,816 Protein (%) 16.5 Methionine (%) 0.34 TSAA (%) 0.62 Calcium (%) 3.80 Nonphytate P (%) 0.45 Total P (%) Vitamin mixture provided the following per kilogram of diet: vitamin A (retinyl acetate), 8,800 IU; cholecalciferol, 2,200 IU; DL-α-tocopheryl acetate, 11 IU; menadione sodium bisulfite, 2.2 mg; riboflavin, 4.4 mg; D-calcium pantothenate, 8.8 mg; nicotinic acid, 44 mg; pyridoxine hydrochloride, 2.2 mg; folic acid, 0.55 mg; d-biotin, 0.11 mg; thiamine hydrochloride, 2.5 mg; vitamin B 12, 6.6 µg; choline, 220 mg; ethoxyquin, 125 mg. 2 Mineral mixture provided the following per kilogram of diet: Mn, 60 mg; Zn, 50 mg; Fe, 30 mg; Cu, 5 mg; I, 1.06 mg; Se, 0.1 mg. 3 Natuphos 600 (BASF Corp., Mt. Olive, NJ) was used as the source of microbial phytase. 4 Based on tables of feed composition of NRC (1994). Diets had a significant effect on BW at the end of Phase 2 (51 wk of age). Body weight decreased significantly when NPP content of the diet reduced from the control level (0.45%) to 0.2%, and was decreased further when the NPP content of the diet was reduced to 0.1% (T 1 vs. T 2 and T 5, respectively). This information indicated that 0.1 and 0.2% NPP levels were not sufficient to maintain the BW during Phase 2 of the experiment. While both levels of phytase were effective in restoring the BW of birds fed 0.2% NPP to the control level, phytase levels were not effective in restoring the BW of birds fed 0.1% NPP. At 51 wk of age, Babcock had the heaviest and Hy- Line and ISA-White had the lightest BW, while the BW of DeKalb was intermediate between the BW of the heaviest and the lightest strains. Due to a very low EP and high mortality of birds on 0.1% NPP (T 5 ; see following sections), the use of this diet was discontinued from the beginning of Phase 3 (52 wk of age). However, the birds of this dietary treatment were fed the control diet (0.45% NPP) during Phase 3 to determine whether the adverse effect of feeding a low-p diet (0.1% NPP) on performance is permanent or whether a recovery can take place after increasing the NPP content of the diet. Therefore, the data of T 5 were not included in the statistical analysis for various traits during Phase 3 and for the entire experiment. Body weight at the end of Phase 3 (63 wk of age) and BW gain for the entire experiment were not significantly different among various diets. It is worth noting that changing the NPP content of T 5, from 0.1 to 0.45% at the beginning of Phase 3 resulted in a compensatory growth to the extent that the BW of birds on this dietary treatment were comparable to controls (T 1 ). This information indicated that the adverse effect of feeding a diet with 0.1% NPP level on BW is not permanent, and a complete recovery can take place after the birds are fed a diet with an adequate P level. At the end of the experiment (63 wk of age), BW of Babcock was significantly heavier than the other three strains. Although DeKalb had a BW that was significantly heavier than Hy-Line, the BW of these two strains were not significantly different from the BW of ISA-White. For the entire experiment, the BW gain was not significantly different either between Babcock and ISA-White or between DeKalb and Hy-Line; however, the BW gain of the former two strains were significantly greater than for the latter two strains. Overall, the BW data indicated that a NPP regimen of % without phytase or a NPP regimen of % with the lower level of phytase were adequate to support the BW and BW gain. EP The interaction between strain diet for EP was significant during Phases 1 and 2, indicating that strains responded differently to dietary treatments (Table 4). During Phase 1, while reducing the NPP level from the control level (0.45%) to 0.25 or 0.2% significantly reduced the EP of Babcock and ISA-White as compared to their controls (T 1 vs. T 2 and T 5 ), these NPP levels did not have a significant adverse effect on EP of DeKalb and Hy-Line. Both levels of phytase were effective in alleviating the adverse effect of 0.25 and 0.2% NPP on EP of ISA-White. The lower but not the higher level of phytase was effective in preventing the adverse effect of 0.25% NPP on EP of Babcock. Additionally, neither level of phytase was effective in alleviating the adverse effect of 0.2% NPP on EP of Babcock. This information indicated that the NPP requirement for EP during the early stages of the laying cycle is greater for Babcock than ISA-White, which are, in turn, greater than the requirement for DeKalb and Hy-Line. In Phase 2, EP of all the strains were significantly reduced due to the use of diets with 0.2 or 0.1% NPP as compared to their controls (T 1 vs. T 2 and T 5 ). Both levels of phytase were effective in restoring the EP of all the strains fed 0.2% NPP to their controls. However, the level of phytase that was required to restore the low-ep of birds fed 0.1% NPP to the control level was strain-related: the lower level with Babcock and Hy-Line, and the higher level with DeKalb and ISA-White. Unexpectedly, the higher level of phytase failed to restore the EP of Babcock fed 0.1% NPP to its control level. The EP information from Phases 1 and 2 indicated that the NPP requirement for maintaining EP during the early stages of the laying cycle varied among strains, and due to this, the levels of phytase required to restore their EP to the control levels are also strain-related. The interaction of strain diet for EP was not significant during Phase 3 and for the entire experiment. During

5 PHOSPHORUS REQUIREMENTS OF LAYING HENS 75 Phase 3, EP was significantly reduced due to the use of a diet with 0.15% NPP (T 2 ) and was restored to the control level only by the higher level of phytase. The EP of birds fed a NPP regimen of % plus both levels of phytase remained inferior to the control, although EP was significantly greater with the higher than with the lower level of phytase. Changing the NPP content of T 5 from 0.1 to 0.45% during Phase 3 increased EP considerably as compared to Phase 2 and was only slightly lower than the control group during Phase 3. Strain differences existed for EP during Phase 3; EP was significantly higher for Hy-Line than Babcock and ISA-White but was not significantly different than DeKalb. For the entire experiment (20 to 63 wk of age), EP of birds fed a NPP regimen of % was significantly lower than the control group and was restored to the control level only by the higher level of phytase (P > 0.05). The EP of birds fed a NPP regimen of % plus both levels of phytase remained significantly inferior to the control group. Strain differences existed for EP for the entire experiment; EP was not different among Babcock, DeKalb and Hy-Line, but the EP of ISA-White was significantly lower than DeKalb and Hy-Line. Overall, the EP data indicated that a NPP regimen of % with the higher level of phytase as adequate to result in a comparable EP as the control group for the entire experiment (P > 0.05). Egg Weight and Egg Grades The interaction between strain diet for EW was not significant during each phase or for the entire experiment (Table 5). EW was not influenced by the diets during Phases 1 and 3 and for the entire experiment. During Phase 2, EW was significantly reduced due to the use of 0.1% NPP (T 5 ) but was restored to the control when the diets contained either level of phytase. Similar to BW and EP, changing the NPP content of T 5 from 0.1 to 0.45% resulted in a compensatory EW response during Phase 3. Strain differences existed for EW during each phase of the experiment. For the entire experiment, EW was heaviest for ISA-White, lightest for Hy-Line, and intermediate for the other two strains. The effect of the treatments on egg grades, for the most part, was similar to their effect on EW and, for brevity, is described in the footnotes of Table 5. Egg Mass T6 There were significant interactions of strain diet in Phase 1, 2, and overall experiment. In Phase 1, and similar to EP, reducing the NPP from the control level (0.45%) to 0.25 or 0.2% did not have an adverse effect on egg mass (EM) of DeKalb and Hy-Line (Table 6). However, EM of ISA-White was significantly reduced by these two NPP levels but was not different from controls when the diets contained either level of phytase. With Babcock, EM was significantly reduced only due to decreasing the NPP to 0.25% and was restored to its control by both levels of phytase. However, EM of Babcock fed 0.2% NPP was not significantly different than its control. This was due to a slightly higher EW of this strain on 0.2% than on 0.25% NPP (Table 5). While the lower level of phytase had some beneficial effect on EM of Babcock fed 0.2% NPP, the EM of this strain remained significantly lower than its control with the higher level of phytase. This lower EM was the result of the lower EP and EW of Babcock fed 0.2% NPP with the higher rather than with the lower level of phytase (Tables 4 and 5). In Phase 2, whereas reducing the NPP level from the control level (0.45%) to 0.2% did not have an adverse effect on EM of DeKalb and Hy-Line, it significantly reduced the EM of Babcock and ISA-White. The EM of the latter two strains were restored to their controls by both levels of phytase. The EM of all the strains were significantly further reduced when the NPP level was reduced from 0.2 to 0.1% (T 2 vs. T 5 ). Although the lower level of phytase was adequate to restore the EM of Babcock and Hy-Line, the higher level of phytase was required for restoring the EM of DeKalb and ISA-White to their control levels. Similar to Phase 1, the higher level of phytase failed to restore the EM of Babcock fed 0.1% NPP level to its control level. This was due to a lower EP of Babcock fed 0.1% NPP with the higher rather than with the lower level of phytase (Table 4). In Phase 3, the EM was significantly reduced due to the use of a diet with 0.15% NPP and was restored to the control level only by the higher level of phytase. Neither level of phytase was effective in restoring the EM of birds fed 0.1% NPP to a level comparable to the control group. Similar to BW, EP, and EW, changing the NPP level from 0.1% to 0.45% increased the EM almost to a comparable level as the control group during Phase 3. The EM was not significantly different among the various strains during this phase of the experiment. For the entire experiment, the EM of all the strains were significantly reduced due to feeding a NPP regimen of % and were equivalent to controls when the diet contained either level of phytase. Although the EM of all the strains fed a NPP regimen of % plus phytase were not significantly different from their controls, the required level of phytase was strain related: the lower level with Babcock and Hy-Line, and the higher level with DeKalb and ISA-White. Similar to Phases 1 and 2, the higher level of phytase failed to restore the EM of Babcock fed the NPP regimen of % to its control level. This was due to a lower EP and EW of this group with the higher level of phytase (Tables 4 and 5). Overall, the EM data for the entire experiment indicated that, whereas a NPP regimen of % was not sufficient to produce EM equal to control group for any strain, adding the lower level of phytase to this NPP regimen was effective in restoring the EM of all the strains to their control groups (P > 0.05). Furthermore, a NPP regimen of % plus the higher or the lower level of phytase (depending on the strain) restored the EM of all the strains to their control levels.

6 76 KESHAVARZ TABLE 3. The effect of different levels of nonphytate P (NPP) with and without phytase (Phy) on BW of four commercial strains of laying hens (18 to 63 wk of age) Body weight at 18 wk of age Body weight at 35 wk of age Body weight at 51 wk of age (g) (g) (g) DeKalb DeKalb DeKalb Delta Hy-line Delta Hy-Line Delta Hy-Line Diet Babcock White W36 ISA-White Babcock White W36 ISA-White Babcock White W36 ISA-White 1) % NPP 1 1,247 1,286 1,251 1,174 1,549 1,550 1,413 1,472 1,600 1,585 1,515 1,515 2) % NPP 1,247 1,287 1,251 1,184 1,507 1,508 1,472 1,472 1,550 1,518 1,429 1, Phy 1,253 1,287 1,252 1,188 1,546 1,507 1,502 1,495 1,593 1,541 1,504 1, Phy 1,252 1,294 1,252 1,190 1,541 1,494 1,479 1,486 1,602 1,537 1,532 1,524 5) % NPP 4 1,241 1,286 1,259 1,178 1,513 1,504 1,471 1,467 1,499 1,383 1,382 1, Phy 1,242 1,296 1,263 1,189 1,543 1,534 1,484 1,470 1,567 1,522 1,472 1, Phy 1,246 1,283 1,254 1,186 1,521 1,484 1,502 1,460 1,567 1,516 1,494 1,477 SEM ) % NPP 1,239 1,496 1,554 a 2) % NPP 1,243 1,490 1,498 c Phy 1,245 1,513 1,538 ab Phy 1,247 1,500 1,549 a 5) % NPP 1,241 1,489 1,406 d Phy 1,247 1,508 1,514 bc Phy 1,243 1,492 1,514 bc Babcock 1,247 b 1,531 a 1,568 a DeKalb Delta White 1,288 a 1,511 a 1,515 b Hy-Line W36 1,255 b 1,475 b 1,476 c ISA-White 1,184 c 1,475 b 1,484 c Diet effect < Strain effect < < < Diet strain effect a d Means in each column with no common superscript differ significantly (P < 0.05). 1 Percentage NPP used during Phase 1 (20 to 35 wk), Phase 2 (36 to 51 wk), and Phase 3 (52 to 63 wk). 2 Natuphos phytase (BASF Corp., Mt. Olive, NJ) was used at 150 units/kg diet during the experiment. 3 Natuphos phytase was used at 300 units/kg diet during the experiment. 4 Birds of this dietary treatment were fed 0.1% NPP up to the end of Phase 2 (51 wk of age). Due to high mortality and low egg production, the NPP content of this diet was increased to the control level (0.45%) during Phase 3 of the experiment (52 to 63 wk of age). 5 Not included in the statistical analysis. 6 Data not shown. Feed Intake No interactions of strain diet were detected in Phases of 1 to 3 or overall. In Phase 1, FI was reduced only due to the use of 0.2% NPP (T 5 ) and restored to the control level by the lower level of phytase (Table 7). In Phase 2, FI was significantly reduced only for hens fed 0.1% NPP (T 5 ) and was restored to the control by both levels of phytase. In Phase 3, FI was significantly reduced only for hens fed 0.15% NPP (T 2 ) and was restored to the control by both levels of phytase. Increasing the NPP of T 5 from 0.1% to the control level (0.45%) resulted in a compensatory increase in FI. For the entire experiment, FI was lower only for hens fed a NPP regimen of % (T 2 ) and was restored to the control by both levels of phytase. Furthermore, both levels of phytase were effective in maintaining the FI of birds fed a NPP regimen of % comparable to the control group. Strain differences existed for FI during each phase of the experiment. For the entire experiment, FI was highest for Babcock, and in decreasing order, DeKalb, ISA-White, and Hy-Line had lower FI. Overall, the FI data indicated that a NPP regimen of % was not sufficient to maintain the FI for the entire experiment. However, both levels of phytase restored the FI of birds of this NPP regimen or even a lower NPP regimen of % to the control group. Feed Conversion There were significant interactions of strain diet for feed conversion (FC) in all phases and in the overall experiment. In Phase 1, and similar to EP and EM, reducing the NPP level from the control level (0.45%) to 0.25 or

7 PHOSPHORUS REQUIREMENTS OF LAYING HENS 77 TABLE 3. (continued) Body weight at 63 wk of age (g) Body weight gain, 18 to 63 wk (g) DeKalb DeKalb Delta Hy-Line ISA- Delta Hy-Line ISA- Diets Babcock White W36 White Babcock White W36 White 1) % NPP 1 1,535 1,500 1,428 1, ) % NPP 1,525 1,480 1,424 1, Phy 1,561 1,494 1,470 1, Phy 1,584 1,485 1,480 1, ) % NPP 4 (1,680) 5 (1,653) (1,654) (1,507) Phy 1,542 1,535 1,479 1, Phy 1,551 1,526 1,509 1, SEM ) % NPP 1, ) % NPP 1, Phy 1, Phy 1, ) % NPP (1,623) Phy 1, Phy 1, Babcock 1,550 a 301 a DeKalb Delta White 1,503 b 214 b Hy-Line W36 1,465 c 211 b ISA-White 1,478 bc 293 a Diet effect Strain effect < < Diet strain effect % did not produce an adverse effect on FC of DeKalb and Hy-Line (Table 8). On the other hand, the FC of ISA- White was significantly impaired by these two NPP levels and was restored to the control level by both levels of phytase. The FC of Babcock was significantly impaired by 0.25% NPP and was restored to its control by both levels of phytase. While the lower level of phytase slightly improved the FC of Babcock fed 0.2% NPP diet, the FC of this strain remained significantly inferior to its control group with the higher level of phytase. This was mainly due to the lower EM of this strain fed 0.2% NPP with the higher rather than with the lower level of phytase (Table 6). Similar to EP and EM, the FC information indicated that strain differences existed with regard to NPP requirement for maintaining the FC at the optimum level during the early stages of the laying cycle. In Phase 2, the use of 0.2% NPP (T 2 ), significantly impaired only the FC of Babcock, which was ameliorated by both levels of phytase. FC of all the strains were significantly impaired due to reducing the NPP level to 0.1%, and both levels of phytase restored the FC to their controls. In Phase 3, FC of all the strains were significantly impaired due to the use of 0.15% NPP, and both levels of phytase were effective in restoring the FC of all the strains to their control levels. During this phase and similar to EM, the FC of all the strains fed a NPP level of 0.1% plus phytase were comparable to their controls (T 1 vs. T 6 and T 7 ), although the required level of phytase was strainrelated: the higher level with DeKalb, but the lower level with the other three strains. The FC of Babcock fed the higher level of phytase remained significantly inferior to its control and this was due to its lower EM (Table 6). Changing the NPP content of T 5 from 0.1% to the control level (0.45%) during Phase 3 failed to improve the FC of various strains to comparable levels as their controls. This was due to use of the all FI of the birds of T 5 during Phase 3 in calculating the FC, while a good proportion of this feed was used for compensatory growth (Table 3). For the entire experiment, the FC of all the strains fed a NPP regimen of % were significantly inferior to their controls but were restored by both levels of phytase (P > 0.05). Similar to EM, although the FC of all the strains fed a NPP regimen % plus phytase were comparable to their controls, the required level of phytase was strain-related: the lower level with Babcock and Hy-Line, but the higher level with the other two strains. Similar to EM, the FC of Babcock fed a NPP regimen of % plus the higher level of phytase remained inferior to its control mainly due to its lower EM. Overall, the FC data for the entire experiment indi-

8 78 KESHAVARZ TABLE 4. The effect of different levels of nonphytate P (NPP) with and without phytase (Phy) on egg production of four commercial strains of laying hens (20 to 63 wk of age) Egg production (%) Egg production (%) Egg production (%) Overall egg production (%) Phase 1, 20 to 35 wk of age Phase 2, 36 to 51 wk of age Phase 3, 52 to 63 wk of age 20 to 63 wk of age DeKalb- DeKalb DeKalb DeKalb Delta Hy-Line ISA- Delta Hy-Line ISA- Delta Hy-Line ISA- Delta Hy-Line ISA- Diet Babcock White W36 White Babcock White W36 White Babcock White W36 White Babcock White W36 White 1) % NPP a 82.6 abcd 81.6 bcd 82.2 bcd 82.9 a 84.2 a 80.8 abc 82.4 ab ) % NPP 80.2 cd 78.8 d 80.5 cd 72.5 e 73.0 de 76.6 bcde 73.5 de 73.0 de Phy ab 80.6 cd 81.4 bcd 81.7 bcd 78.8 abcde 77.6 abcde 81.0 abc 80.1 abcd Phy bcd 83.7 abc 81.7 bcd 82.2 bcd 77.9 abcde 82.3 ab 80.9 abc 81.9 ab ) % NPP Phy cd 80.1 cd 80.4 cd 72.3 e 46.4 f 35.3 g 43.7 f 34.9 g (73.6) 5 (66.9) (72.7) (63.6) Phy 82.3 bcd 81.1 bcd 81.8 bcd 80.3 cd 79.0 abcde 72.8 e 79.3 abcde 74.2 cde Phy 80.6 cd 81.7 bcd 81.1 bcd 78.6 d 73.5 de 79.8 abcde 77.9 abcde 76.4 bcde SEM ) % NPP a 80.3 a 2) % NPP d 69.0 e Phy b 77.6 bc Phy ab 78.9 ab 5) % NPP (69.2) Phy c 75.0 d Phy b 76.0 cd Babcock bc 76.1 ab DeKalb Delta ab 76.7 a Hy-Line W a 77.2 a ISA-White c 74.6 b Diet effect < < < < Strain effect < Diet strain effect a e Means in each column with no common superscript differ significantly (P < 0.05). 1 Percentage of NPP used during Phase 1 (20 to 35 wk), Phase 2 (36 to 51 wk), and Phase 3 (52 to 63 wk). 2 Natuphos phytase (BASF Corp., Mt. Olive, NJ) was used at 150 units/kg of diet during the experiment. 3 Natuphos phytase was used at 300 units/kg of diet during the experiment. 4 Birds of this dietary treatment were fed 0.1% NPP up to the end of Phase 2 (51 wk of age). Due to high mortality and low egg production, the NPP content of this diet was increased to the control level (0.45%) during Phase 3 of the experiment (52 to 63 wk of age). 5 Not included in the statistical analysis. 6 Data not shown.

9 PHOSPHORUS REQUIREMENTS OF LAYING HENS 79 TABLE 5. The effect of different levels of nonphytate P (NPP) with and without phytase (Phy) on egg weight of four commercial strains of laying hens (20 to 63 wk of age) 1 Egg weight (g) Egg weight (g) Egg weight (g) Overall egg weight (g) Phase 1, 20 to 35 wk of age Phase 2, 36 to 51 wk of age Phase 3, 52 to 63 wk of age 20 to 63 wk of age DeKalb- DeKalb- DeKalb- DeKalb- Delta Hy-Line ISA- Delta Hy-Line ISA- Delta Hy-Line Delta Hy-Line ISA- Diet Babcock White W36 White Babcock White W36 White Babcock White W36 ISA-qcWhite Babcock White W36 White 1) % NPP ) % NPP Phy Phy ) % NPP (63.4) 6 (63.8) 6 (63.1) 6 (62.2) Phy Phy SEM ) % NPP 53.7 a 58.2 a 61.7 a 57.5 a 2) % NPP 54.0 a 59.2 a 61.9 a 58.0 a Phy 54.0 a 58.4 a 61.4 a Phy 54.0 a 58.6 a 61.3 a 57.7 a 5) % NPP 54.2 a 56.9 b (63.6) Phy 54.1 a 59.1 a 62.3 a 58.3 a Phy 53.7 a 58.1 a 61.2 a 57.4 a Babcock 54.2 b 58.6 b 62.2 b 58.0 b DeKalb Delta White 54.0 b 58.2 b 61.2 c 57.6 b Hy-Line W c 56.2 c 59.7 d 55.5 c ISA-White 56.2 a 60.4 a 63.3 a 59.8 a Diet effect Strain effect < < < < Diet strain effect a d Means in each column with no common superscript differ significantly (P < 0.05). 1 Neither the interaction of strain diet nor the diet main effect on egg grades (large and above or medium and below) were significant for the entire experiment. However, strain differences existed for egg grades for the entire experiment. The percentage of large and above large-sized eggs for the entire experiment was significantly higher for ISA-White than Hy-Line, whereas for the other two strains they were intermediate and not significantly different from each other. On the other hand, the percentage of medium plus below medium-sized eggs were significantly lower for ISA-White than for Hy-Line, whereas for the other two strains, they were intermediate and not significantly different from each other (data not shown). 2 Percentage of NPP used during Phase 1 (20 to 35 wk), Phase 2 (36 to 51 wk), and Phase 3 (52 to 63 wk). 3 Natuphos phytase (BASF Corp., Mt. Olive, NJ) was used at 150 units/kg of diet during the experiment. 4 Natuphos phytase was used at 300 units/kg of diet during the experiment. 5 Birds of this dietary treatment were fed 0.1% NPP up to the end of Phase 2 (51 wk of age). Due to high mortality and low egg production, the NPP content of this diet was increased to the control level (0.45%) during Phase 3 of the experiment (52 to 63 wk of age). 6 Not included in the statistical analysis. 7 Data not shown.

10 80 KESHAVARZ TABLE 6. The effect of different levels of nonphytate P (NPP) with and without phytase (Phy) on egg mass of four commercial strains of laying hens (20 to 63 wk of age) Egg mass (g/hen per d) Egg mass (g/hen per d) Egg mass (g/hen per d) Phase 1, 20 to 35 wk of age Phase 2, 36 to 51 wk of age Phase 3, 52 to 63 wk of age Overall egg mass (g/hen per d) DeKalb- DeKalb- DeKalb- DeKalb- Delta Hy-Line ISA- Delta Hy-Line ISA- Delta Hy-Line Delta Hy-Line ISA- Diet Babcock White W36 White Babcock White W36 White Babcock White W36 ISA-White Babcock White W36 White 1) % NPP abc 44.6 abcdefg 41.7 jkl 46.3 ab 47.7 abcd 49.0 abc 45.6 cdef 49.9 ab ab 46.4 abc 44.4 bcdef 47.2 a 2) % NPP 43.8 defghij 42.6 ghijkl 41.2 kl 40.7 l 42.8 f 45.5 cdef 42.1 f 45.0 def hij 41.7 ghi 39.6 ij 38.3 j Phy abcd 43.6 efghij 42.1 hijkl 45.6 abcde 45.9 abcd 45.1 cdef 45.7 cdef 48.6 abcd abcde 43.8 cdefg 43.8 cdefg 45.9 abcd Phy cdefgh 45.2 abcdef 41.9 ijkl 46.6 a 45.9 cdef 47.7 abc 45.4 cdef 50.2 a abcdef 45.9 abcd 43.7 defg 47.4 a 5) % NPP cdefghi 43.1 fghijk 41.8 ijkl 40.4 l 27.1 g 20.1 i 23.7 h 20.2 i (46.7) 5 (42.5) (45.8) (40.9) Phy 45.2 abcdef 44.2 bcdefgh 42.5 ghijkl 45.2 abcdef 47.4 abcde 42.9 f 44.9 def 45.1 cdef abcdef 42.5 fgh 42.9 efg 44.1 cdefg Phy 43.1 fghijk 43.6 efghij 41.7 jkl 44.4 bcdefg 43.6 ef 46.0 cdef 43.5 ef 46.2 bcdef efgh 44.3 bcdef 42.6 efgh 44.9 abcdef SEM ) % NPP a ) % NPP d Phy b Phy ab ) % NPP (44.0) Phy c Phy bc 43.6 Babcock a 44.2 DeKalb Delta a 44.1 Hy-Line W a 42.8 ISA-White a 44.6 Diet effect < < < < Strain effect < Diet strain effect a l Means in each column with no common superscript differ significantly (P < 0.05). 1 Percentage of NPP used during Phase 1 (20 to 35 wk), Phase 2 (36 to 51 wk), and Phase 3 (52 to 63 wk). 2 Natuphos phytase (BASF Corp., Mt. Olive, NJ) was used at 150 units/kg of diet during the experiment. 3 Natuphos phytase was used at 300 units/kg of diet during the experiment. 4 Birds of this dietary treatment were fed 0.1% NPP up to the end of Phase 2 (51 wk of age). Due to high mortality and low egg production, the NPP content of the diet was changed to the control regimen (0.45% NPP) during Phase 3 of the experiment (52 to 63 wk of age). 5 Not included in the statistical analysis. 6 Data not shown.

11 PHOSPHORUS REQUIREMENTS OF LAYING HENS 81 TABLE 7. The effect of different levels of nonphytate P (NPP) with and without phytase (Phy) on feed intake of four commercial strains of laying hens (20 to 63 wk of age) Feed intake (g/hen per d) Feed intake (g/hen per d) Feed intake (g/hen per d) Overall feed intake (g/hen per d) Phase 1, 20 to 35 wk of age Phase 2, 36 to 51 wk of age Phase 3, 52 to 63 wk of age 20 to 63 wk of age DeKalb- DeKalb- DeKalb- DeKalb- Delta Hy-Line ISA- Delta Hy-Line ISA- Delta Hy-Line ISA- Delta Hy-Line ISA- Diet Babcock White W36 White Babcock White W36 White Babcock White W36 White Babcock White W36 White 1) % NPP ) % NPP Phy Phy ) % NPP (116.6) 5 (113.4) (108.1) (108.5) Phy Phy SEM ) % NPP 97.1 a a a a 2) % NPP 96.1 abc a 96.0 b 97.8 b Phy 96.9 ab a a a Phy 96.7 ab a a a 5) % NPP 95.5 c 82.0 b (111.7) Phy 96.4 abc a a 99.6 a Phy 96.0 bc a a a Babcock 98.8 a a a a DeKalb Delta White 98.2 a b a b Hy-Line W c 91.5 c 96.0 c 93.3 d ISA-White 97.4 b 99.1 b 99.9 b 99.9 c Treatment effect < < < < Diet effect < < Strain effect < < < < Diet strain effect a c Means in each column with no common superscript differ significantly (P < 0.05). 1 Percentage of NPP used during Phase 1 (20 to 35 wk), Phase 2 (36 to 51 wk), and Phase 3 (52 to 63 wk), respectively. 2 Natuphos phytase (BASF Corp., Mt. Olive, NJ) was used at 150 units/kg of diet during the experiment. 3 Natuphos phytase was used at 300 units/kg of diet during the experiment. 4 Birds of this dietary treatment were fed 0.1% NPP up to the end of Phase 2 (51 wk of age). Due to high mortality and low egg production, the NPP content of the diet was changed to the control regimen (0.45% NPP) during Phase 3 of the experiment (52 to 63 wk of age). 5 Not included in the statistical analysis. 6 Data not shown.

12 82 KESHAVARZ TABLE 8. The effect of different levels of nonphytate P (NPP) with and without phytase (Phy) on feed conversion of four commercial strains of laying hens (20 to 63 wk of age) Feed conversion (g/g) Feed conversion (g/g) Feed conversion (g/g) Overall feed conversion (g/g) Phase 1, 20 to 35 wk of age Phase 2, 36 to 51 wk of age Phase 3, 52 to 63 wk of age 20 to 63 wk of age DeKalb- DeKalb- DeKalb- DeKalb- Delta Hy-Line ISA- Delta Hy-Line ISA- Delta Hy-Line ISA- Delta Hy-Line ISA- Diet Babcock White W36 White Babcock White W36 White Babcock White W36 White Babcock White W36 White 1) % NPP efgh 2.23 cdef 2.15 defg 2.13 fgh 2.19 defgh 2.14 ef 2.05 f 2.03 f 2.37 defgh 2.39 defgh 2.08 h 2.28 fgh 2.22 efgh 2.23 efgh 2.11 h 2.12 h 2) % NPP 2.27 abcd 2.30 abc 2.21 cdefg 2.37 a 2.47 c 2.29 cdef 2.20 ef 2.29 cdef 3.32 a 2.90 b 2.75 bc 3.58 a 2.54 a 2.41 bc 2.31 cde 2.53 ab Phy efgh 2.26 bcde 2.17 efgh 2.16 efgh 2.28 cdef 2.30 cdef 2.05 f 2.11 ef 2.52 cdefg 2.52 cdefg 2.21 gh 2.45 cdefg 2.28 def 2.34 cde 2.13 gh 2.20 efgh Phy cdefg 2.18 defgh 2.18 defgh 2.11 h 2.26 cdef 2.16 def 2.09 f 2.08 f 2.55 cdef 2.41 defg 2.27 fgh 2.33 efgh 2.32 cde 2.22 efgh 2.17 fgh 2.15 gh 5) % NPP cdef 2.26 bcde 2.19 defgh 2.35 ab 3.30 b 3.95 a 3.32 b 4.16 a (2.50) 5 (2.72) (2.39) (2.68) Phy 2.18 defgh 2.23 cdef 2.12 gh 2.17 efgh 2.26 cdef 2.40 cde 2.06 f 2.24 cdef 2.67 bcd 2.75 bc 2.39 defgh 2.46 cdefg 2.32 cde 2.41 cd 2.15 fgh 2.26 efg Phy 2.29 abcd 2.23 cdef 2.19 defgh 2.18 defgh 2.45 cd 2.26 cdef 2.19 cdef 2.19 cdef 2.72 bc 2.59 cde 2.39 defgh 2.39 defgh 2.44 abc 2.33 cde 2.23 efgh 2.23 efgh SEM ) % NPP ) % NPP Phy Phy ) % NPP (2.56) Phy Phy Babcock DeKalb-Delta White Hy-Line W ISA-White Diet effect < < < < Strain effect < < < Diet strain effect < a h Means in each column with no common superscript differ significantly (P < 0.05). 1 Percentage of NPP used during Phase 1 (20 to 35 wk), Phase 2 (36 to 51 wk), and Phase 3 (52 to 63 wk). 2 Natuphos phytase (BASF Corp., Mt. Olive, NJ) was used at 150 units/kg of diet during the experiment. 3 Natuphos phytase was used at 300 units/kg of diet during the experiment. 4 Birds of this dietary treatment were fed 0.1% NPP up to the end of Phase 2 (51 wk of age). Due to high mortality and low egg production, the NPP content of the diet was changed to the control regimen (0.45% NPP) during Phase 3 of the experiment (52 to 63 wk of age). 5 Not included in the statistical analysis. 6 Data not shown.

13 PHOSPHORUS REQUIREMENTS OF LAYING HENS 83 cated that, although a NPP regimen of % was not adequate to support the FC of any strain, both levels of phytase were effective in restoring the FC of all the strains to their control groups. Furthermore, a NPP regimen of % plus a higher or a lower level of phytase (depending on the strain) restored the FC of all the strains to their control levels. Specific Gravity There were strain diet interactions only in Phases 2 and 3. In Phase 1, reducing the NPP from the control level (0.45%) to 0.25 or 0.2% significantly improved the SG [T 1 vs. T 2 and T 5 (Table 9)]. However, SG of the birds fed these two NPP levels were reduced to the control in the presence of phytase. This probably was due to increasing the NPP content of the diets by phytase, which, in turn, overcame the expression of the beneficial effect of the low-p diets on SG. This phenomenon was observed in a previous report also (Keshavarz, 2000). Strain differences existed for SG; it was highest for Babcock and ISA- White, lowest for Hy-Line, and intermediate for DeKalb. In contrast to Phase 1, reducing the NPP from the control level to 0.2% in Phase 2 reduced the SG of all the strains, although the differences were not significant only between DeKalb and its control group. Here again, both levels of phytase restored the lower SG of all the strains fed 0.2% NPP level to their control groups. When the NPP was further reduced to 0.1% (T 5 ) with the exception of ISA-White, the SG of the other three strains were not different than their controls. The adverse effect of 0.1% NPP on SG of ISA-White was alleviated by both levels of phytase. In Phase 3, whereas reducing the NPP from the control level (0.45%) to 0.15% did not have a significant effect on SG of Babcock and DeKalb, the SG of Hy- Line was significantly increased, and in contrast, the SG of ISA-White was significantly decreased (T 1 vs. T 2 ). The higher SG of Hy-Line was restored to its control by both levels of phytase, whereas the lower SG of ISA-White was restored to its control by the higher level of phytase. The SG of eggs on a diet with 0.1% NPP plus both levels of phytase were not significantly different than their controls for any strain. Changing the NPP of T 5 from 0.1 to 0.45% in Phase 3 did not appear to have an effect on SG of Babcock, but it increased the SG of DeKalb and Hy-Line and reduced the SG of ISA-White as compared to their controls in Phase 3. The observation of SG responses due to feeding low-p diets during Phases 1 to 3 are examples of the complexity of the effect of low-p diets on SG, particularly in an experiment involving multi-strain hens. For the entire experiment, SG was not influenced by different NPP regimens or presence or absence of phytase in the diets. However, strain differences existed for SG. The SG was not different between Babcock and ISA-White or between DeKalb and Hy-Line, but the SG of the former two strains were significantly higher than the latter two strains. SW, PS, and SWUSA In contrast to SG, which was measured on all eggs for 3 consecutive d of production every 28 d throughout the experiment, SW, PS, and SWUSA were measured on 10 eggs per replicate and only at the end of Phase 1 (35 wk), Phase 2 (51 wk), and Phase 3 (63 wk) of the experiment. Consequently, due to a considerably larger sample size and repeated measurements every 4 wk, it appeared that SG data more accurately represented the effect of dietary treatments on shell quality than the other indices of shell quality. Due to this and for brevity, the effects of dietary treatments on the other indices of shell quality are described in the footnotes of Table 9. Mortality No interaction of strain diet on mortality were detected. Birds which died during the experiment were divided into two groups: prolapse- and nonprolapse-related mortality (Table 10). Prolapse mortality were those birds that died or were culled due to an obviously protruded oviduct together with bleeding. All of the other birds that died during the experiment were referred to as nonprolapse mortality. Prolapse mortality during Phase 1 plus Phase 2 (20 to 51 wk of age) or for the entire experiment (20 to 63 wk of age) were not significantly different among the diets. However, during both of these periods, strain differences existed with regard to prolapse mortality; prolapse mortality was significantly higher for Babcock than for DeKalb and Hy-Line, whereas for ISA-White it was intermediate and not significantly different than the other three strains. The nonprolapse mortality was significantly increased due to reducing the NPP regimen from the control to a sequence of % during 20 to 51 wk of age and for the entire experiment (20 to 63 wk of age). Both levels of phytase were effective in ameliorating the adverse effect of this NPP regimen on nonprolapse mortality during these periods (T 1 vs. T 3 and T 4 ). Further reducing the NPP regimen to a sequence of % (T 5 ) increased the nonprolapse mortality drastically during 20 to 51 wk of age. Most of this mortality happened during Phase 2, when birds were consuming a diet with 0.1% NPP. The adverse effect of this NPP regimen on nonprolapse mortality was ameliorated by the lower level, and uncharacteristically, not by the higher level of phytase, as compared to the control during 20 to 51 wk of age. For the entire experiment, the nonprolapse mortality of hens fed a NPP regimen of % plus both levels of phytase still remained significantly higher than the control group (T 1 vs. T 6 and T 7 ). The nonprolapse mortality was significantly higher for Babcock and De- Kalb than for Hy-Line and was intermediate for ISA- White, which was not significantly different than the other three strains during 20 to 51 wk of age. For the entire experiment (20 to 63 wk of age), the nonprolapse mortality was significantly lower for Hy-Line than for the other three strains, which were not different from each other. Overall, a NPP regimen of % plus