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Effect of dietary phosphorus and phytase levels on the reproductive performance of large white turkey breeder hens1
Effect of Dietary Phosphorus and Phytase Levels on the Reproductive Performance of Large White Turkey Breeder Hens1 J. L. Godwin,* J. L. Grimes,*,2 V. L. Christensen,* and M. J. Wineland* *Department of Poultry Science, North Carolina State University, Raleigh, North Carolina 27695-7608
(Key words: turkey breeder hen, phosphorus, phytase, reproductive performance) 2005 Poultry Science 84:485–493
with a mean BW of 10.8 kg in 1996 for LW hens also at 30 wk of age (Applegate and Lilburn, 1996). Turkey industry surveys indicate that the range of available P (aP) levels currently fed to turkey breeder hens is 0.45 to 0.55% (Kuhl, 1993, 1997). Breeder recommendations range from 0.4 to 0.55% aP (Hybrid Turkeys, 1996; BUTA, 1997; Nicholas Turkey Breeder Farms, 1997). Increased emphasis on the effect of animal-derived P in the environment, especially from poultry litter applications to farm land (Moore and Miller, 1994; Martin, 1997; Sauer et al., 1999), warrants additional information on the effect of dietary P level fed to current LW turkey hens. The objective of this study was to provide additional information on the ability of the modern LW turkey hen to maintain reproductive performance using less supplemental P with or without dietary phytase enzyme.
INTRODUCTION Previous National Research Council reports (NRC, 1950, 1971) indicated a nonphytate P dietary requirement of 0.75% for laying turkey hens. However, the recommended level was subsequently lowered to 0.35% (NRC, 1984; 1994). Manley et al. (1980) reported that caged Broad Breasted White (BBW) turkey hens fed diets with 0.3% total P perform as well as hens fed diets with 0.4 or 0.5% total P. However, the modern turkey hen has been selected for increased BW (Marini, 2003), which might have an effect on its P requirement. For example, mean BW of Large White (LW) turkey breeder hens was 8.6 kg at 30 wk of age in 1976 (Thomason et al., 1976) compared
2005 Poultry Science Association, Inc. Received for publication July 21, 2004. Accepted for publication October 27, 2004. 1 The use of trade names implies neither endorsement of the products named nor criticism of similar products not mentioned by the North Carolina Agricultural Research Service or the North Carolina Cooperative Extension Service. 2 To whom correspondence should be addressed: jesse_grimes @ncsu.edu.
Abbreviation Key: aP = available phosphorus; BBW = Broad Breasted White; BSW = Beltsville Small White; E = enzyme; HH = hen housed; HP = available P = 0.55%; HPE = HP + E; LP = available P = 0.17%; LPE = LP + E; LW = Large White; MP = available P = 0.35%; MPE = MP + E; TCA = trichloroacetic acid.
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tion, hen-day egg production, settable eggs, cumulative settable eggs, hens out of lay, and hen mortality and for biweekly performance for egg fertility, hatchability of all eggs, hatchability of fertile eggs, egg weight loss, conductance, conductance constant (k), and embryonic mortality. Egg weight, eggshell thickness, egg components, and albumen and yolk P were measured monthly. At 62 wk of age, hen tibia P, plasma P, total fecal P, and water-soluble fecal P were determined. Decreasing dietary P resulted in no decreases in reproductive performance for turkey breeder hens to 62 wk. Additionally, decreased dietary P resulted in decreased total fecal P and water-soluble fecal P. Feeding turkey breeder hens dietary phytase enzyme resulted in significantly fewer hens going out of lay; however, this was not reflected in hen housed egg production. It was concluded that phosphorus could be lowered in turkey breeder hen diets, compared with current surveyed industry levels, without impairing reproductive performance.
ABSTRACT An experiment was conducted to determine the effect of dietary P levels and dietary phytase enzyme (E) inclusion on Large White turkey breeder hen reproductive performance from 31 to 62 wk of age. Hens were placed in a curtain-sided house with 48 pens (10 birds per pen; 8 pens per treatment) at 31 wk and were fed a breeder ration with treatments as follows: HP, dietary available P = 0.55%; HPE, HP + E; MP, dietary available P = 0.35%; MPE, MP + E; LP, dietary available P = 0.17%; and LPE, LP + E. Feed and water were available ad libitum for 28 wk of lay. Diets were fed in mash form, and all other nutrients were formulated to meet or exceed NRC requirements. All hens were photostimulated in January (31 wk) with 15.5 h of light daily. Production data were recorded on a pen basis. Individual bird BW and feed consumption, by pen, were determined at monthly intervals from 31 to 62 wk. Hens were observed for weekly reproductive performance for hen housed egg produc-
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TABLE 1. Feed ingredients for 3 basal diets HP
MP
Corn Soybean meal (45% protein) Limestone Dicalcium phosphase Poultry fat DL-Methionine Salt Minerals (TM-90)2 Vitamins (NCSU-90)3 Selenium supplement4 Rendox (45 g) Sand Total
64 22 6.5 2.2 4.0 0.1 0.4 0.1 0.1 0.1 0.01 100
64 22 7.0 1.3 4.0 0.1 0.4 0.1 0.1 0.1 0.01 0.4 100
64 22 7.5 0.3 4.0 0.1 0.4 0.1 0.1 0.1 0.01 0.4 100
Calculated analysis Protein, % ME, kcal/kg Calcium, % Phosophorus (available, %) Lysine, % Methionine, %
16.1 2,971 2.9 0.55 0.89 0.39
16.1 2,971 2.9 0.35 0.89 0.39
16.1 2,971 2.9 0.17 0.89 0.39
Analyzed Crude protein, % Ca, % P (total), %
16.9 3.5 0.7
16.4 4.0 0.5
LP
16.3 3.5 0.3
1 HP, available P = 0.5%; MP, available P = 0.3%, LP, available P = 0.17%, HPE, MPE, and LPE were formulated by adding phytase enzyme (Alltech, Inc., Nichloasville, KY), to HP, MP, LP, respectively, at 1 kg/ ton of feed. 2 Mineral composition (in g/kg of diet): zinc sulfate, 120,; manganese sulfate, 120; copper sulfate, 10; calcium iodate, 2.5; colbalt sulfate, 1.0. 3 Vitamins in amounts per kilogram of diet; vitaminA, 26,400 IU; vitamin D3, 8,3000 KU; vitamin E 132 IU; vitamin B12, 79.2 µg; riboflavin 26.4.
MATERIALS AND METHODS All birds used in this study were handled using methods approved by the North Carolina State University Institutional Animal Care and Use Committee. The facilities used in this experiment consisted of a curtain-sided house containing 48 pens (9 m2 per pen). Pen floors were covered with clean pine shavings. Caked litter was removed as necessary throughout the trial. Four hundred eighty Hybrid EURO FP turkey breeder hen poults were brooded in 12 pens until 3 wk of age. The hens were then randomly distributed to all 48 pens (10 poults per pen). Hens were fed grower diets as reported by Crouch et al. (2002). At 31 wk of age, the hens were fed a typical cornsoybean meal diet meeting all NRC (1994) nutrient recommendations except for P (Table 1). The dietary treatments included 3 levels of P and 2 levels of phytase enzyme (E; 0 and 1 kg/ton of feed). The feed treatments were as follows: HP, aP = 0.55%; HPE, HP + enzyme; MP, aP = 0.35%; MPE MP + enzyme; LP, aP = 0.17%; and LPE, LP + enzyme. The phytase enzyme product (Allzyme Phytase3) is derived from Aspergillus niger and contained 11.27 phytase units (PTU)/g. The feed was provided as mash to prevent possible degradation of the enzyme dur-
3
Alltech, Inc., Nicholasville, KY.
ing pelleting. Diets were analyzed by the North Carolina Department of Agriculture feed laboratory (Raleigh, NC) to determine CP, Ca, and total P on an as-fed basis. Hens received feed and water ad libitum and were photostimulated in January (31 wk) with 15.5 h of light/d. Hens were inseminated biweekly with pooled semen from same strain breeder males reared in a separate building and managed according to the primary breeder recommendations. Hens that could not be everted for biweekly insemination were removed from the study and defined as out of lay. Significant treatment effects were not observed at 20 wk of lay; therefore, all supplemental P was removed from LP and LPE to create basal diets in which all aP was supplied only by the corn and soybean meal. Feed consumption, by pen, and individual BW were recorded at monthly intervals from 31 until 62 wk of age. Hen housed (HH) and hen-day egg production, settable eggs, cumulative settable eggs were determined weekly by pen. Egg hatchability, egg fertility, and hatchability of fertile eggs were measured biweekly by pen. This provided 14 hatches over 28 wk of lay. Eggs were collected 5 times a day and classified as settable, double yolk, misshapen (crowded), cracked, or soft shell. Settable eggs were sanitized and stored in an egg cooler at approximately 13°C and 70% RH. All settable eggs from each week of lay were set in trays and incubated by pen in a common incubator for each set date on a biweekly basis. Eggs were incubated for 24 d at 37.5°C and 53% RH and then transferred into a hatcher operating at 37.2°C and 65% RH. All eggs that failed to hatch after 28 d of incubation were broken open and examined macroscopically to determine egg fertility and age at embryonic death. All unhatched eggs were categorized as follows: infertile, wk 1 dead, wk 2 dead, wk 3 dead, wk 4 dead, internal pip, external pip, cracked, and rotten. There was also a category for cull poults. Percentages for each category were calculated for each setting of eggs. Three eggs from each pen from the last day of each 2-wk production period were weighed to the nearest gram initially and at transfer (25 d) to the hatcher; subsequent poults were also weighed. Egg weight loss (%), conductance, and conductance constant (k) were measured as reported by Christensen and Nestor (1994). At 4, 20, and 28 wk of lay, 3 eggs from each pen were selected at random, numbered, weighed, and then broken out to determine shell weight, albumen and yolk weight, and albumen and yolk P content. Relative shell, albumen, and yolk weight (%) were calculated. The yolk and albumen were separately bagged and frozen until P analysis (see Appendix). The shells from each egg were then washed to remove any residue and then dried. A dry weight of the shell was then recorded. Three additional eggs per pen were collected every 4 wk to determine egg weight and eggshell thickness. Once the egg was broken and emptied of contents, the shells were used to measure shell thickness at the top, middle, and bottom of the egg using a micrometer. The mean of the 3 measurements was used for shell thickness. After 28 wk of lay, blood from the brachial vein and the right tibias (post euthana-
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Ingredient (%)
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sia) were collected from 2 hens per pen for plasma and tibia P analysis (see Appendix). Fresh fecal samples were collected over a 5-d period from each pen by treatment to measure total fecal P and water-soluble reactive P (see Appendix). The design of this study was a randomized complete block design with a 2 × 3 factorial arrangement of treatments (2 levels of phytase and 3 levels of P). The experimental period was 31 to 62 wk of age. The house design was in quadrant formation with each quadrant representing a block of 12 pens. Dietary treatments were randomly assigned to 2 pens in each block. The pen served as the experimental unit. Each parameter was regressed on enzyme, dietary P, and enzyme × dietary P (SAS Institute, 1992). Percentage data were divided by 100 and subjected to arc sin transformation of the square root before analysis; however, actual percentage means are presented. Differences among treatment means were partitioned using the least squares means procedure of SAS (SAS Institute, 1992). Statements of significance are based on P ≤ 0.05 unless otherwise stated.
RESULTS There were no differences in hen reproductive performance due to feed treatments. Hen feed consumption was 265 g/hen per day during the first 2 wk of egg production and was increased to 308 g/hen per day by 14 wk of lay (Figure 1). Hen BW was 11.0 kg at photostimulation, 12.7 kg during wk 6 of lay, 11.7 kg during wk 12 of lay, and 12.0 kg at 28 wk of lay (Figure 2). Mean HH (Figure 3) and hen-day (not shown) egg productions ranged from 64 to 70% and 67 to 72% for the different treatments groups, respectively, at 3 wk of lay with a
subsequent steady decline to the end of the egg production period. By the end of the 28 wk of lay, fewer hens fed dietary phytase went out of production than hens fed diets without phytase (Table 2; 62 vs. 86 out of 480 total). However, this result was not reflected in HH egg production. Egg fertility was 90% or higher for lay from wk 1 through 18 and then began to decline (Figure 4). Egg hatchability (not shown) and hatchability of fertile eggs (Figure 5) responded in a similar manner. Poult weight and embryonic mortality were not different over the 28 wk of lay due to treatment (data not shown). There was no effect of dietary phytase enzyme on egg weight or eggshell thickness; therefore, only dietary P level means are presented (Figure 6). Egg weight increased while shell thickness decreased during lay (Figure 6). There were few and inconsistent differences for the conductance and the conductance constant (k) of the eggshell due to dietary treatment (data not shown). Ending plasma and tibia P were not affected by dietary P, enzyme levels, or an interaction of the 2 (Table 2). Total fecal P and water-soluble fecal P decreased as dietary P decreased (Table 2). There were no treatment effects on absolute or relative weight of egg yolk, albumen, or shell or on yolk or albumen P concentration (Table 3). However, egg weight of eggs used in egg component analyses increased from 80 g at 4 wk of lay to 93 g at 20 and 28 wk of lay. Yolk weight increased from 24.2 g at 4 wk of lay to 32.0 g at 28 wk of lay. Albumen weight increased from 48.2 g at 4 wk of lay to 55.6 g at 20 wk of lay and then decreased to 53.3 g at 28 wk of lay. Shell weight was greatest at 20 wk of lay (8.2 g) compared with at 4 (7.7 g) or 28 (7.8 g) wk of lay. Relative yolk weight increased from 30.2 to 34.4% from 4 to 28 wk of lay. Relative albumen weight decreased from 60.2 and 59.6% at 4 and 20
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FIGURE 1. Feed consumption of turkey breeder hens fed dietary treatments over 28 wk of lay. HP, available P = 0.5%; MP, available P = 0.3%; LP, available P = 0.17%; HPE, MPE, and LPE were formulated by adding phytase enzyme (Alltech, Inc., Nicholasville, KY) to HP, MP, and LP, respectively, at 1 kg/ton of feed. For feed consumed, SEM = 5.62.
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wk of lay to 57.2% at 28 wk of lay. Relative shell weight (%) decreased from 9.6% at 4 wk of lay to 8.4% at 28 wk of lay. Yolk P concentration was not affected by diet but was greater at 28 wk of lay (664 ± 25 mg/100 mL) than at 4 (584 ± 62 mg/100 mL) or 20 (593 ± 45 mg/100 mL) wk of lay. Albumen P concentration (17.94 ± 4.82 mg/ 100 mL) was not affected by dietary treatments or hen age.
DISCUSSION The results of this study agree with previous reports utilizing similar type hens. Wilcox et al. (1961) reported
that the removal of supplemental P from a practical type diet, with dietary P levels similar to this study, did not appear to influence egg production, egg fertility, or hatchability of fertile eggs for Broad Breasted Bronze, Broad White, or Beltsville Small White (BSW) turkeys. However, further decreases in dietary P through the use of purified diets did result in decreased egg production and hatchability of fertile eggs. Miller et al. (1976) provided diets containing 0.1 or 0.4% supplemental P (0.45 or 0.75% total P) to individually caged BBW turkey breeder hens for a 16-wk lay period. No differences were reported for egg production, egg fertility, or hatchability of fertile eggs.
FIGURE 3. Hen housed egg production (%) of turkey breeder hens fed dietary treatments over 28 wk of lay. HP, available P = 0.5%; MP, available P = 0.3%; LP, available P = 0.17%; HPE, MPE, and LPE were formulated by adding phytase enzyme (Alltech, Inc., Nicholasville, KY) to HP, MP, and LP, respectively, at 1 kg/ton of feed. For hen housed egg production, SEM = 0.81.
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FIGURE 2. Body weights of turkey breeder hens fed dietary treatments over 28 wk of lay. HP, available P = 0.5%; MP, available P = 0.3%; LP, available P = 0.17%; HPE, MPE, and LPE were formulated by adding phytase enzyme (Alltech, Inc., Nicholasville, KY) to HP, MP, and LP, respectively, at 1 kg/ton of feed. For hen body weights, SEM = 0.14.
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TURKEY BREEDER HENS AND DIETARY PHOSPHORUS TABLE 2. Plasma P, tibia P, total fecal P, water-soluble P, and hens out of lay for turkey breeders after 28 wk of lay
Diet
Plasma P (mg/dL)
Tibia P (%)
Total fecal P (%)
Fecal water-soluble P (%)
Hens out of Lay (%)
HP MP LP
6.50 7.64 8.09
14.7 15.3 15.0
2.03a 1.36b 1.04b
0.19a 0.12b 0.06c
33.9 38.5 37.4
E− E+
7.75 7.12
15.1 14.8
1.50b 1.40a
0.11 0.13
40.9a 32.3b
X SEM
7.42 1.56
14.9 0.5
1.47a 0.14a
0.12 0.02
36.6 2.9
Means within a column with no common superscripts are significantly different (P ≤ 0.05). HP, available P = 0.55%, MP, available P = 0.35%; LP, available P = 0.17%; E (phytase enzyme, Alltech, Inc., Nichloasville, KY) was (+) or was not (−) added to HP, MP, and LP at 1 kg/ton of feed. a-c 1
White turkey breeder hens maintained reproductive performance on diets calculated to contain 0.17% aP and 2.9% Ca (analyzed Ca was 3.5 to 4.1% Ca) compared with hens fed an NRC (0.35% aP) or industry level diet (0.55% aP) with or without enzyme even with the withdrawal of all supplemental P from LP and LPE diets at 20 wk. In previous research conducted to demonstrate the need for practical turkey breeder hen diets to be supplemented with inorganic P, caged BSW hens were used. Sewell et al. (1972), using caged BSW turkey hens, reported decreased egg production and hatchability of fertile eggs, but not egg fertility, when fed a practical diet with supplemental P removed. Ferguson et al. (1974) when feeding similar diets to caged BSW turkey hens observed a reduction in egg production, egg weight, and hatchability of fertile eggs but no effect on fertility. Portal and Ferguson (1974) fed diets with 0.38% total P (all from plant source) or the basal diet supplemented with inorganic P to contain 0.75% total P to caged BSW turkey
FIGURE 4. Fertility of turkey breeder hens fed dietary treatments over 28 wk of lay. HP, available P = 0.5%; MP, available P = 0.3%; LP, available P = 0.17%; HPE, MPE, and LPE were formulated by adding phytase enzyme (Alltech, Inc., Nicholasville, KY) to HP, MP, and LP, respectively, at 1 kg/ton of feed. For fertility, SEM = 1.01.
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Waldroup et al. (1974) fed a factorial arrangement of diets to caged Nicholas turkey breeder hens. The diets contained 2.25 or 3.50% Ca and 0.1, 0.2, 0.3, or 0.4% inorganic P. At the lower level of Ca, 0.3% inorganic P was required to maintain maximum egg production. However, at 3.50% dietary Ca, the inorganic P requirement appeared to be no greater than 0.2%. Egg fertility was lowest at 0.1% inorganic P regardless of Ca level. There were no effects on hatchability of fertile eggs or shell thickness due to dietary inorganic P level. Manley et al. (1980) reported that caged BBW turkey hens fed diets with 0.3% total phosphorus (P) performed as well as hens fed diets with either 0.4 or 0.5% total P. Slaugh et al. (1989), using individually caged Medium White hens, reported that BW, egg production, egg weight, hatchability of fertile eggs, and egg specific gravity were not affected by decreasing the aP from 0.7, 0.5, and 0.3 to 0.15%. Retained P and egg fertility were decreased in hens fed the lowest P level. These reports agree with the current study in which Large
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breeder hens. Hens fed the 0.75% total P had greater egg production and increased hatchability of fertile eggs compared with hens fed the basal diet. Egg fertility was not affected by dietary P level. Potter et al. (1974) fed 0.64 or 0.82% P to BSW hens but observed no differences in reproductive performance due to dietary P. Comparison
of LW and BSW turkey hens with respect to dietary P needs might not be relevant for the purposes of this study. In the current study, egg weights were similar regardless of dietary treatment throughout the 28 wk of lay, even after removal of all supplemental P from the LPE and LP diets. The increase in egg weight with increasing
FIGURE 6. Egg weight and shell thickness of turkey breeder hens fed dietary treatments over 28 wk of lay. HP, available P = 0.55%; MP, available P = 0.35%; LP, available P = 0.17%; st = shell thickness. For egg weight, SEM = 0.60; for shell thickness, SEM = 0.03.
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FIGURE 5. Hatchability of fertile eggs of turkey breeder hens fed dietary treatments over 28 wk of lay. HP, available P = 0.5%; MP, available P = 0.3%; LP, available P = 0.17%; HPE, MPE, and LPE were formulated by adding phytase enzyme (Alltech, Inc., Nicholasville, KY) to HP, MP, and LP, respectively, at 1 kg/ton of feed. SEM for hatchability of fertile eggs = 1.57. There were significant (P < 0.05) treatment effects at 16 to 17 wk of lay; however, this effect was not consistent throughout the trial and was considered by the authors to be an artifact.
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TURKEY BREEDER HENS AND DIETARY PHOSPHORUS TABLE 3. Egg components of turkey breeder hens at 4, 20, and 28 wk of lay Diet1
Egg (g)
4 weeks of lay HP 80.2 MP 79.2 LP 80.7
Yolk (g)
Yolk (%)
Yolk P2
Albumen (g)
Albumen (%)
Albumen P2
Shell (g)
Shell (%)
23.9 24.0 24.6
29.8 30.3 30.6
602 571 579
48.8 47.7 48.3
60.6 60.2 59.8
16.73 13.29 15.74
7.7 7.6 7.8
9.6 9.6 9.6
E− E+
79.2 81.0
24.5 23.9
30.9 29.5
601 567
47.2 49.3
59.6 60.8
14.99 15.51
7.6 7.8
9.6 9.7
X SEM
80.1b 1.5
24.2c 0.8
30.2c 10.7
584b 62
48.2c 1.0
60.2a 4.2
15.25 4.15
7.7b 0.2
9.6a 0.7
29.6 29.2 29.2
32.0 31.8 31.6
607 575 598
55.2 56.0 55.7
59.6 59.8 59.6
19.73 20.10 17.37
8.1 8.1 8.2
8.8 8.6 8.8
20 weeks of lay HP 92.7 MP 93.5 LP 93.6 92.4 94.1
29.2 29.4
31.6 32.0
595 592
55.1 56.2
59.6 59.7
21.06 17.07
8.1 8.2
8.7 8.7
X SEM
93.2a 1.1
29.3b 0.6
31.8b 9.0
593b 45
55.6a 1.2
59.6a 2.8
19.07 4.90
8.2a 0.2
8.7b 1.1
31.8 32.6 31.8 32.0 32.0
34.0 34.6 34.4 34.6 34.2
654 664 676 563 655
53.7 53.3 53.0 52.4 54.2
57.6 56.5 57.5 56.6 57.8
19.09 15.25 24.14 19.35 19.63
7.8 8.0 7.7 7.8 7.9
8.4 8.4 8.4 8.4 8.4
32.0a 0.8
34.4a 4.9
664a 25
53.3b 1.3
57.2b 3.0
19.49 5.42
7.8b 0.2
8.4c 0.6
28 weeks of lay HP 93.0 MP 94.2 LP 92.2 E− 92.6 E+ 93.7 X SEM
93.2a 1.2
Means within a column without common superscripts are significantly different (P ≤ 0.05). HP, available P = 0.55%, MP, available P = 0.35%; LP, available P = 0.17%; E (phytase enzyme, Alltech, Inc., Nichloasville, KY) was (+) or was not (−) added to HP, MP, and LP at 1 kg/ton of feed. 2 Units are milligrams of P per 100 ml of liquid yolk and albumen, respectively. a-c 1
age observed in this study agrees with Reidy et al. (1994) who reported that the weight of eggs from commercial turkey breeders increased 11% between the onset of lay and 24 wk of production. The values for the egg components agree with the values reported by Reidy et al. (1994). The changes in egg component weights agrees with observations of Applegate and Lilburn (1996) who reported that as hens aged, egg weight increased along with relative yolk weight at the expense of albumen. The range for plasma P was 5.50 to 9.52 mg/dL P, which agrees with findings made by Ledoux et al. (1995) who reported plasma P levels for 15-wk-old turkey hens to be 7.55 to 8.24 mg/dL P. The wider range for plasma P in this study might be attributed to the time of day when the blood was sampled from hens. Past studies have shown that plasma P levels fluctuate due to daily egg production status (Miller et al., 1977; Reichmann and Conner; 1977). Woodard and Mather (1964) also noted that in turkey plasma, P level remains higher for a longer period than in chickens, which may be due to the longer shell formation. Tibial P content in this study ranged from 14.6 to 15.3% P agreeing with reports made by Slaugh et al. (1989) that femur P increased from 15.8 to 16.4% as the level of P increased from 0.15 to 0.70% aP. Our values for yolk P agree with the value for turkey yolk P reported by Cunningham and Lee (1978) and for yolk and albumen P for chicken eggs reported by Cotterill et al. (1977) and Naber (1979). The decrease in fecal P with decreasing dietary P in this study agrees with the report by Slaugh et al. (1989)
in which fecal P level decreased from 3.10 to 1.93% as the level of P decreased from 0.70 to 0.15% aP. As expected, hens fed 0.7% total P (0.55% aP) excreted feces with significantly greater P content than those fed lower dietary P levels. Gordon and Roland (1997) observed that hens fed 0.1% aP with phytase supplementation excreted 25% less P than those consuming the same diet without phytase. Water-soluble P content of the turkey hen manure ranged from 0.06 to 0.19%. Hens fed LPE or LP excreted lower water-soluble fecal P levels than all other treatments. These findings suggest that higher levels of dietary P increase water-soluble P. The only effect of dietary E over the 28 wk of lay was for the number of hens that went out of lay. By 28 wk of lay, 41% of the hens not fed dietary phytase had gone out of production, whereas 32% of the hens fed phytase had gone out of production. This was a difference of 24 total hens with half of this difference occurring after 20 wk of lay. It has been proposed that the improvements in growth rate and feed conversion with the addition of phytase to the diet are due to the ability of phytase to release and make available for absorption minerals and trace elements complexed with phytic acid (Simons et al., 1990). The increased availability of some nutrients may be a determining factor for keeping hens in production longer. However, this difference was not reflected in HH egg production. Van der Klis et al. (1997) reported that production performance of turkey breeder hens fed diets supplemented with phytase was comparable to hens receiving supplemental inorganic P. Simons et al. (1990)
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E− E+
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ACKNOWLEDGMENTS This study was funded, in part, by the North Carolina Agriculture Foundation, Alltech, Inc. (Nicholasville, KY), and Hybrid Turkeys (Kitchener, Ontario, Canada).
REFERENCES Applegate, T. J., and M. S. Lilburn. 1996. Independent effects of hen age and egg size on incubation and poult characteristics in commercial turkeys. Poult. Sci. 75:1210–1216. Association of Official Analytical Chemists. 1984. Official Methods of Analysis. 14th ed. Association of Official Analytical Chemists, Washington, DC BUTA. 1997. British United Turkeys of America Parent Stock Management Guide. British United Turkeys of America, Lewisburg, WV. Christensen, V. L., and K. E. Nestor. 1994. Changes in functional qualities of turkey eggshells in strains selected for increased egg production or growth. Poult. Sci. 73:1458–1464. Cotterill, O. J., W. W. Marion, and E. C. Naber. 1977. A nutrient re-evaluation of shell eggs. Poult. Sci. 56:1927–1934. Crouch, A. N., J. L. Grimes, V. L. Christensen, and K. K. Krueger. 2002. Effect of physical feed restriction during rearing on Large White turkey breeder hens: 2. reproductive performance. Poult. Sci. 81:16–22. Cunningham, F. E., and H. W. Lee. 1978. A study of turkey egg yolk 1. composition and electrophoretic separation of components. J. Food Biochem. 2:251–257. Ferguson, T. M., C. E. Sewell, Jr, and R. L. Atkinson. 1974. Phosphorus levels in the turkey breeder diet. Poult. Sci. 53:1627–1629. Goldenberg, A., and A. Fernandez. 1966. Simplified method for the estimation of inorganic phosphorus in body fluids. Am. Chem. J. 12:871–882. Gordon, R. W., and D. A. Roland, Sr. 1997. Performance of commercial laying hens fed various phosphorus levels, with and without supplemental phytase. Poult. Sci. 76:1172–1177. Hybrid Turkeys. 1996. Hybird Feeding Programs. Hybrid Turkeys, Inc., Kitchner, Ontario, Canada.
Kuhl, H. J., Jr. 1993. An industry survey of nutrient levels fed currently. Pages 19–28 in Third International Symposium on Turkey Reproduction. Department of Poultry Science, North Carolina State University, Raleigh, NC. Kuhl, H. J., Jr. 1997. An industry survey of nutrient levels fed currently to turkey breeder hens. Pages 57–65 in Fourth International Symposium on Turkey Reproduction. Department of Poultry Science, North Carolina State University, Raleigh, NC. Ledoux, D. R., K. Zyla, and T. L. Veum. 1995. Substitution of phytase for inorganic phosphorus for turkey hens. J. Appl. Poult. Res. 4:157–163. Manley, J. M., R. A. Voitle, and R. H. Harms. 1980. The influence of dietary calcium and phosphorus on egg production and hatchability of turkey breeder hens. Poult. Sci. 59:2077–2079. Marini, P. J. 2003. A concise history of turkey breeder restriction programs. Pages 1–3 in Fifth International Symposium on turkey reproduction. Department of Poultry Science, North Carolina State University, Raleigh, NC. Martin, J. H., Jr. 1997. The clean water act and animal agriculture. J. Environ. Qual. 26:1198–1203. Miller, D. H., J. W. Bradley, and T. M. Ferguson. 1976. Reproductive performance of Broad Breasted White turkeys in relation to dietary phosphorus. Poult. Sci. 55:2481–2483. Miller, E. R., R. H. Harms, and H. R. Wilson. 1977. Cyclic changes in serum phosphorus of laying hens. Poult. Sci. 56:586–589. Moore, P. A., and D. M. Miller. 1994. Decreasing phosphorus in poultry litter with aluminum, calcium, and iron amendments. J. Environ. Qual. 23:325–330. Naber, E. C. 1979. The effect of nutrition on the composition of eggs. Poult. Sci. 58:518–528. National Research Council. 1950. Nutrient Requirements of Domestic Animals. Vol. 1. Nutrient Requirements for Poultry. National Academy Press, Washington, DC. National Research Council. 1971. Nutrient Requirements of Domestic Animals. Vol. 1. Nutrient Requirements for Poultry. National Academy Press, Washington, DC. National Research Council. 1984. Nutrient Requirements of Poultry. 8th rev. ed. National Academy Press, Washington, DC. National Research Council. 1994. Nutrient Requirements of Poultry. 9th rev. ed. National Academy Press, Washington, DC. Nicholas Turkey Breeding Farms. 1997. Nicholas Management Guidelines. Nicholas Turkey Breeding Farms, Sonoma, CA. Portal, C., and T. M. Ferguson. 1974. Reproductive performance of caged turkeys as affected by age, bird density and dietary phosphorus. Poult. Sci. 53:2218–2221. Potter, L. M., A. T. Leighton, Jr, and A. B. Chu. 1974. Calcium, phosphorus and Nopgro as variables in diets of breeder turkeys. Poult. Sci. 53:15–22. Reichmann, I. G., and J. K. Conner. 1977. Influence of dietary calcium and phosphorus on metabolism and production in laying hens. Br. Poult. Sci. 18:633–640. Reidy, R. R., J. L. Atkinson, and S. Leeson. 1994. Strain comparisons of turkey egg components. Poult. Sci. 73:388–395. SAS Institute. 1992. SAS User’s Guide. Version 6.08. SAS Institute Inc., Cary, NC. Sauer, T. J., T. C. Daniel, P. A. Moore, Jr., K. P. Coffey, D. J. Nicholas, and C. P. West. 1999. Poultry litter and grazing animal waste effects on runoff water quality. J. Environ. Qual. 28:860–865. Self-Davis, M. L., and P. A. Moore. 2000. Determining watersoluble phosphorus in animal manure. Pages 74–76 in Methods of Phosphorus Analysis for Soils, Sediments, Residuals, and Waters. G. M. Pierztnski, ed. http://www.soil. ncsu.edu/sera17/publications/sera17-2/pm_cover.htm Sewell, C. E., Jr., R. S. Atkinson, J. R. Couch, and T. M. Ferguson. 1972. The effect of supplemental phosphorus on the reproductive performance of turkey hens and the subsequent effect upon the poults. Poult. Sci. 51:792–796.
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reported that addition of phytase to feed containing 4.5 g/kg of total P results in levels of feed conversion and performance that are as good or better than the levels achieved using rations supplemented with inorganic P to a total P level of 7.5 g/kg. If hens fed phytase are more likely to remain in production, one might expect to measure an increased HH egg production with a larger number of hens than used in this study. On the other hand, this observation could be an artifact with no real biological effect. A field trial with typical industry size breeder houses should ascertain this possible effect. Feeding low dietary P (0.17% aP) resulted in turkey breeder hen reproductive performance comparable with performance of hens fed dietary P levels used by surveyed members of the turkey industry (0.55% aP) or of hens fed NRC-recommended levels (0.35% aP) and agrees with previous reports for LW or similar type hens. Feeding hens dietary phytase resulted in little of no effect on hens in production because hens were able to perform adequately with no supplemental P. Therefore, the turkey industry might be able to lower dietary P without impairing reproductive performance while reducing total and water-soluble fecal P.
TURKEY BREEDER HENS AND DIETARY PHOSPHORUS
APPENDIX A modified phosphorus assay was used to analyze for inorganic phosphate content in various samples through colorimetric determination using a microplate reader. This assay was modified from the original method reported by Goldenberg and Fernandez (1966). The method uses 2 stable reagents and requires a minimum number of steps. Blood was collected into heparin tubes. The tubes were centrifuged at 1,500 rpm for 15 min. Then 0.2 mL of plasma sample and 0.8 mL of 5% trichloroacetic acid (TCA) reagent were added to bullet tubes. The tubes were then vortexed and allowed to set for 20 min. The tubes were then centrifuged at 2,200 rpm for 30 min. The supernatant was used for final P analysis. Tibial P was determined by using samples that were fat free and moisture free using the method described by the Association of Official Analytical Chemists (1984). Tibia were cleaned of soft tissue, measured for length and weight, and then cut into pieces with a band saw.
The tibias were dried overnight at 100°C, and then fat was extracted with chloroform. Yolk, albumen, and total P fecal samples were dried for 12 h at 100°C. All samples except for plasma were then ground and ashed in a muffle furnace at 600°C. These samples were then digested with HCl before final P analysis. Water-soluble reactive P fecal samples were prepared as reported by Self-Davis and Moore (2000). Fecal samples for water-soluble P were dried overnight at 100°C, ground, and then mixed with distilled water (1 g in 20 mL). This mixture was shaken for 1 h, centrifuged at 2,200 rpm for 30 min, and then filtered using 0.45 millapore filter paper. The resulting filtered solution was used for final P analysis. The standard for the final P analysis was 0.0878 g of dry KH2PO4 mixed into 100 mL of distilled water. A range of 0 to 20% solution of this mixture was used to develop the standard curve. Processed samples (10 µL), standards (10 µL of appropriate dilution), and a blank sample (10 µL distilled water) were placed in separate microtiter plate wells along with 240 µL of TCA reagent and 25 µL of molybdate. A Spectra Max microplate reader using Softmax software program (Spectramax 250, Molecular Devices Corporation, Sunnyvale, CA) was used to measure the absorbance of the sample and to plot the results against a standard curve to calculate the phosphorus concentration.
TCA Reagent This solution contained 125 g of TCA, 37.5 g of ferrous ammonium sulfate, and 12.5 g of thiourea brought to 1,000 mL with distilled water.
Molybdate Reagent Concentrated H2SO4 (45 mL) was added to 200 mL of distilled water in a 500-mL flask set in an ice bath. Ammonium molybdate (22 g) was dissolved into 200 mL of distilled water in an acid-washed beaker. The ammonium molybdate solution was slowly added to the H2SO4 solution set in ice bath. After cooling, the volume was brought to 500 mL.
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Simons, P. C. M., H. A. J. Versteegh, A. W. Jongbloed, P. Slump, K. D. Bos, M. G. E. Wol Beudeker, and G. J. Verchoor. 1990. Improve phosphorus availability by microbial phytase and pigs. Br. J. Nutr. 64:525–540. Slaugh, B. T., N. P. Johnston, and J. D. Pattern. 1989. Research Note: Effect of dietary phosphorus levels on performance of turkey breeder hens. Poult. Sci. 68:319–322. Thomason, D. M., A. T. Leighton, Jr, and J. P. Mason, Jr. 1976. A study of certain environmental factors and mineral chelation on the reproductive performance of young and yearling turkey hens. Poult. Sci. 55:1343–1355. Van der Klis, J. D., H. A. J. Versteegh, P. C. M. Simons, and A. K. Klies. 1997. The efficacy of phytase in corn-soybean mealbased diets for laying hens. Poult. Sci. 76:1535–1542. Waldroup, P. W., J. F. Maxey, and L. W. Luther. 1974. Studies on calcium and phosphorus requirements of caged turkey breeder hens. Poult. Sci. 53:886–888. Wilcox, R. A., C. W. Carlson, C. W. Kohlmeyer, and G. F. Gastler. 1961. Studies on the phosphorus requirement of turkey breeder hens with evidence for unknown factors needed for embryonic development and normal egg production. Poult. Sci. 40:1540–1546. Woodard, A. W., and F. B. Mather. 1964. The timing of ovulation and movement of the ovum through the oviduct, pigmentation and shell deposition in Japanese quail. Poult. Sci. 43:1427–1432.
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(Key words: turkey breeder hen, phosphorus, phytase, reproductive performance) 2005 Poultry Science 84:485–493
with a mean BW of 10.8 kg in 1996 for LW hens also at 30 wk of age (Applegate and Lilburn, 1996). Turkey industry surveys indicate that the range of available P (aP) levels currently fed to turkey breeder hens is 0.45 to 0.55% (Kuhl, 1993, 1997). Breeder recommendations range from 0.4 to 0.55% aP (Hybrid Turkeys, 1996; BUTA, 1997; Nicholas Turkey Breeder Farms, 1997). Increased emphasis on the effect of animal-derived P in the environment, especially from poultry litter applications to farm land (Moore and Miller, 1994; Martin, 1997; Sauer et al., 1999), warrants additional information on the effect of dietary P level fed to current LW turkey hens. The objective of this study was to provide additional information on the ability of the modern LW turkey hen to maintain reproductive performance using less supplemental P with or without dietary phytase enzyme.
INTRODUCTION Previous National Research Council reports (NRC, 1950, 1971) indicated a nonphytate P dietary requirement of 0.75% for laying turkey hens. However, the recommended level was subsequently lowered to 0.35% (NRC, 1984; 1994). Manley et al. (1980) reported that caged Broad Breasted White (BBW) turkey hens fed diets with 0.3% total P perform as well as hens fed diets with 0.4 or 0.5% total P. However, the modern turkey hen has been selected for increased BW (Marini, 2003), which might have an effect on its P requirement. For example, mean BW of Large White (LW) turkey breeder hens was 8.6 kg at 30 wk of age in 1976 (Thomason et al., 1976) compared
2005 Poultry Science Association, Inc. Received for publication July 21, 2004. Accepted for publication October 27, 2004. 1 The use of trade names implies neither endorsement of the products named nor criticism of similar products not mentioned by the North Carolina Agricultural Research Service or the North Carolina Cooperative Extension Service. 2 To whom correspondence should be addressed: jesse_grimes @ncsu.edu.
Abbreviation Key: aP = available phosphorus; BBW = Broad Breasted White; BSW = Beltsville Small White; E = enzyme; HH = hen housed; HP = available P = 0.55%; HPE = HP + E; LP = available P = 0.17%; LPE = LP + E; LW = Large White; MP = available P = 0.35%; MPE = MP + E; TCA = trichloroacetic acid.
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tion, hen-day egg production, settable eggs, cumulative settable eggs, hens out of lay, and hen mortality and for biweekly performance for egg fertility, hatchability of all eggs, hatchability of fertile eggs, egg weight loss, conductance, conductance constant (k), and embryonic mortality. Egg weight, eggshell thickness, egg components, and albumen and yolk P were measured monthly. At 62 wk of age, hen tibia P, plasma P, total fecal P, and water-soluble fecal P were determined. Decreasing dietary P resulted in no decreases in reproductive performance for turkey breeder hens to 62 wk. Additionally, decreased dietary P resulted in decreased total fecal P and water-soluble fecal P. Feeding turkey breeder hens dietary phytase enzyme resulted in significantly fewer hens going out of lay; however, this was not reflected in hen housed egg production. It was concluded that phosphorus could be lowered in turkey breeder hen diets, compared with current surveyed industry levels, without impairing reproductive performance.
ABSTRACT An experiment was conducted to determine the effect of dietary P levels and dietary phytase enzyme (E) inclusion on Large White turkey breeder hen reproductive performance from 31 to 62 wk of age. Hens were placed in a curtain-sided house with 48 pens (10 birds per pen; 8 pens per treatment) at 31 wk and were fed a breeder ration with treatments as follows: HP, dietary available P = 0.55%; HPE, HP + E; MP, dietary available P = 0.35%; MPE, MP + E; LP, dietary available P = 0.17%; and LPE, LP + E. Feed and water were available ad libitum for 28 wk of lay. Diets were fed in mash form, and all other nutrients were formulated to meet or exceed NRC requirements. All hens were photostimulated in January (31 wk) with 15.5 h of light daily. Production data were recorded on a pen basis. Individual bird BW and feed consumption, by pen, were determined at monthly intervals from 31 to 62 wk. Hens were observed for weekly reproductive performance for hen housed egg produc-
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TABLE 1. Feed ingredients for 3 basal diets HP
MP
Corn Soybean meal (45% protein) Limestone Dicalcium phosphase Poultry fat DL-Methionine Salt Minerals (TM-90)2 Vitamins (NCSU-90)3 Selenium supplement4 Rendox (45 g) Sand Total
64 22 6.5 2.2 4.0 0.1 0.4 0.1 0.1 0.1 0.01 100
64 22 7.0 1.3 4.0 0.1 0.4 0.1 0.1 0.1 0.01 0.4 100
64 22 7.5 0.3 4.0 0.1 0.4 0.1 0.1 0.1 0.01 0.4 100
Calculated analysis Protein, % ME, kcal/kg Calcium, % Phosophorus (available, %) Lysine, % Methionine, %
16.1 2,971 2.9 0.55 0.89 0.39
16.1 2,971 2.9 0.35 0.89 0.39
16.1 2,971 2.9 0.17 0.89 0.39
Analyzed Crude protein, % Ca, % P (total), %
16.9 3.5 0.7
16.4 4.0 0.5
LP
16.3 3.5 0.3
1 HP, available P = 0.5%; MP, available P = 0.3%, LP, available P = 0.17%, HPE, MPE, and LPE were formulated by adding phytase enzyme (Alltech, Inc., Nichloasville, KY), to HP, MP, LP, respectively, at 1 kg/ ton of feed. 2 Mineral composition (in g/kg of diet): zinc sulfate, 120,; manganese sulfate, 120; copper sulfate, 10; calcium iodate, 2.5; colbalt sulfate, 1.0. 3 Vitamins in amounts per kilogram of diet; vitaminA, 26,400 IU; vitamin D3, 8,3000 KU; vitamin E 132 IU; vitamin B12, 79.2 µg; riboflavin 26.4.
MATERIALS AND METHODS All birds used in this study were handled using methods approved by the North Carolina State University Institutional Animal Care and Use Committee. The facilities used in this experiment consisted of a curtain-sided house containing 48 pens (9 m2 per pen). Pen floors were covered with clean pine shavings. Caked litter was removed as necessary throughout the trial. Four hundred eighty Hybrid EURO FP turkey breeder hen poults were brooded in 12 pens until 3 wk of age. The hens were then randomly distributed to all 48 pens (10 poults per pen). Hens were fed grower diets as reported by Crouch et al. (2002). At 31 wk of age, the hens were fed a typical cornsoybean meal diet meeting all NRC (1994) nutrient recommendations except for P (Table 1). The dietary treatments included 3 levels of P and 2 levels of phytase enzyme (E; 0 and 1 kg/ton of feed). The feed treatments were as follows: HP, aP = 0.55%; HPE, HP + enzyme; MP, aP = 0.35%; MPE MP + enzyme; LP, aP = 0.17%; and LPE, LP + enzyme. The phytase enzyme product (Allzyme Phytase3) is derived from Aspergillus niger and contained 11.27 phytase units (PTU)/g. The feed was provided as mash to prevent possible degradation of the enzyme dur-
3
Alltech, Inc., Nicholasville, KY.
ing pelleting. Diets were analyzed by the North Carolina Department of Agriculture feed laboratory (Raleigh, NC) to determine CP, Ca, and total P on an as-fed basis. Hens received feed and water ad libitum and were photostimulated in January (31 wk) with 15.5 h of light/d. Hens were inseminated biweekly with pooled semen from same strain breeder males reared in a separate building and managed according to the primary breeder recommendations. Hens that could not be everted for biweekly insemination were removed from the study and defined as out of lay. Significant treatment effects were not observed at 20 wk of lay; therefore, all supplemental P was removed from LP and LPE to create basal diets in which all aP was supplied only by the corn and soybean meal. Feed consumption, by pen, and individual BW were recorded at monthly intervals from 31 until 62 wk of age. Hen housed (HH) and hen-day egg production, settable eggs, cumulative settable eggs were determined weekly by pen. Egg hatchability, egg fertility, and hatchability of fertile eggs were measured biweekly by pen. This provided 14 hatches over 28 wk of lay. Eggs were collected 5 times a day and classified as settable, double yolk, misshapen (crowded), cracked, or soft shell. Settable eggs were sanitized and stored in an egg cooler at approximately 13°C and 70% RH. All settable eggs from each week of lay were set in trays and incubated by pen in a common incubator for each set date on a biweekly basis. Eggs were incubated for 24 d at 37.5°C and 53% RH and then transferred into a hatcher operating at 37.2°C and 65% RH. All eggs that failed to hatch after 28 d of incubation were broken open and examined macroscopically to determine egg fertility and age at embryonic death. All unhatched eggs were categorized as follows: infertile, wk 1 dead, wk 2 dead, wk 3 dead, wk 4 dead, internal pip, external pip, cracked, and rotten. There was also a category for cull poults. Percentages for each category were calculated for each setting of eggs. Three eggs from each pen from the last day of each 2-wk production period were weighed to the nearest gram initially and at transfer (25 d) to the hatcher; subsequent poults were also weighed. Egg weight loss (%), conductance, and conductance constant (k) were measured as reported by Christensen and Nestor (1994). At 4, 20, and 28 wk of lay, 3 eggs from each pen were selected at random, numbered, weighed, and then broken out to determine shell weight, albumen and yolk weight, and albumen and yolk P content. Relative shell, albumen, and yolk weight (%) were calculated. The yolk and albumen were separately bagged and frozen until P analysis (see Appendix). The shells from each egg were then washed to remove any residue and then dried. A dry weight of the shell was then recorded. Three additional eggs per pen were collected every 4 wk to determine egg weight and eggshell thickness. Once the egg was broken and emptied of contents, the shells were used to measure shell thickness at the top, middle, and bottom of the egg using a micrometer. The mean of the 3 measurements was used for shell thickness. After 28 wk of lay, blood from the brachial vein and the right tibias (post euthana-
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Ingredient (%)
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sia) were collected from 2 hens per pen for plasma and tibia P analysis (see Appendix). Fresh fecal samples were collected over a 5-d period from each pen by treatment to measure total fecal P and water-soluble reactive P (see Appendix). The design of this study was a randomized complete block design with a 2 × 3 factorial arrangement of treatments (2 levels of phytase and 3 levels of P). The experimental period was 31 to 62 wk of age. The house design was in quadrant formation with each quadrant representing a block of 12 pens. Dietary treatments were randomly assigned to 2 pens in each block. The pen served as the experimental unit. Each parameter was regressed on enzyme, dietary P, and enzyme × dietary P (SAS Institute, 1992). Percentage data were divided by 100 and subjected to arc sin transformation of the square root before analysis; however, actual percentage means are presented. Differences among treatment means were partitioned using the least squares means procedure of SAS (SAS Institute, 1992). Statements of significance are based on P ≤ 0.05 unless otherwise stated.
RESULTS There were no differences in hen reproductive performance due to feed treatments. Hen feed consumption was 265 g/hen per day during the first 2 wk of egg production and was increased to 308 g/hen per day by 14 wk of lay (Figure 1). Hen BW was 11.0 kg at photostimulation, 12.7 kg during wk 6 of lay, 11.7 kg during wk 12 of lay, and 12.0 kg at 28 wk of lay (Figure 2). Mean HH (Figure 3) and hen-day (not shown) egg productions ranged from 64 to 70% and 67 to 72% for the different treatments groups, respectively, at 3 wk of lay with a
subsequent steady decline to the end of the egg production period. By the end of the 28 wk of lay, fewer hens fed dietary phytase went out of production than hens fed diets without phytase (Table 2; 62 vs. 86 out of 480 total). However, this result was not reflected in HH egg production. Egg fertility was 90% or higher for lay from wk 1 through 18 and then began to decline (Figure 4). Egg hatchability (not shown) and hatchability of fertile eggs (Figure 5) responded in a similar manner. Poult weight and embryonic mortality were not different over the 28 wk of lay due to treatment (data not shown). There was no effect of dietary phytase enzyme on egg weight or eggshell thickness; therefore, only dietary P level means are presented (Figure 6). Egg weight increased while shell thickness decreased during lay (Figure 6). There were few and inconsistent differences for the conductance and the conductance constant (k) of the eggshell due to dietary treatment (data not shown). Ending plasma and tibia P were not affected by dietary P, enzyme levels, or an interaction of the 2 (Table 2). Total fecal P and water-soluble fecal P decreased as dietary P decreased (Table 2). There were no treatment effects on absolute or relative weight of egg yolk, albumen, or shell or on yolk or albumen P concentration (Table 3). However, egg weight of eggs used in egg component analyses increased from 80 g at 4 wk of lay to 93 g at 20 and 28 wk of lay. Yolk weight increased from 24.2 g at 4 wk of lay to 32.0 g at 28 wk of lay. Albumen weight increased from 48.2 g at 4 wk of lay to 55.6 g at 20 wk of lay and then decreased to 53.3 g at 28 wk of lay. Shell weight was greatest at 20 wk of lay (8.2 g) compared with at 4 (7.7 g) or 28 (7.8 g) wk of lay. Relative yolk weight increased from 30.2 to 34.4% from 4 to 28 wk of lay. Relative albumen weight decreased from 60.2 and 59.6% at 4 and 20
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FIGURE 1. Feed consumption of turkey breeder hens fed dietary treatments over 28 wk of lay. HP, available P = 0.5%; MP, available P = 0.3%; LP, available P = 0.17%; HPE, MPE, and LPE were formulated by adding phytase enzyme (Alltech, Inc., Nicholasville, KY) to HP, MP, and LP, respectively, at 1 kg/ton of feed. For feed consumed, SEM = 5.62.
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wk of lay to 57.2% at 28 wk of lay. Relative shell weight (%) decreased from 9.6% at 4 wk of lay to 8.4% at 28 wk of lay. Yolk P concentration was not affected by diet but was greater at 28 wk of lay (664 ± 25 mg/100 mL) than at 4 (584 ± 62 mg/100 mL) or 20 (593 ± 45 mg/100 mL) wk of lay. Albumen P concentration (17.94 ± 4.82 mg/ 100 mL) was not affected by dietary treatments or hen age.
DISCUSSION The results of this study agree with previous reports utilizing similar type hens. Wilcox et al. (1961) reported
that the removal of supplemental P from a practical type diet, with dietary P levels similar to this study, did not appear to influence egg production, egg fertility, or hatchability of fertile eggs for Broad Breasted Bronze, Broad White, or Beltsville Small White (BSW) turkeys. However, further decreases in dietary P through the use of purified diets did result in decreased egg production and hatchability of fertile eggs. Miller et al. (1976) provided diets containing 0.1 or 0.4% supplemental P (0.45 or 0.75% total P) to individually caged BBW turkey breeder hens for a 16-wk lay period. No differences were reported for egg production, egg fertility, or hatchability of fertile eggs.
FIGURE 3. Hen housed egg production (%) of turkey breeder hens fed dietary treatments over 28 wk of lay. HP, available P = 0.5%; MP, available P = 0.3%; LP, available P = 0.17%; HPE, MPE, and LPE were formulated by adding phytase enzyme (Alltech, Inc., Nicholasville, KY) to HP, MP, and LP, respectively, at 1 kg/ton of feed. For hen housed egg production, SEM = 0.81.
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FIGURE 2. Body weights of turkey breeder hens fed dietary treatments over 28 wk of lay. HP, available P = 0.5%; MP, available P = 0.3%; LP, available P = 0.17%; HPE, MPE, and LPE were formulated by adding phytase enzyme (Alltech, Inc., Nicholasville, KY) to HP, MP, and LP, respectively, at 1 kg/ton of feed. For hen body weights, SEM = 0.14.
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TURKEY BREEDER HENS AND DIETARY PHOSPHORUS TABLE 2. Plasma P, tibia P, total fecal P, water-soluble P, and hens out of lay for turkey breeders after 28 wk of lay
Diet
Plasma P (mg/dL)
Tibia P (%)
Total fecal P (%)
Fecal water-soluble P (%)
Hens out of Lay (%)
HP MP LP
6.50 7.64 8.09
14.7 15.3 15.0
2.03a 1.36b 1.04b
0.19a 0.12b 0.06c
33.9 38.5 37.4
E− E+
7.75 7.12
15.1 14.8
1.50b 1.40a
0.11 0.13
40.9a 32.3b
X SEM
7.42 1.56
14.9 0.5
1.47a 0.14a
0.12 0.02
36.6 2.9
Means within a column with no common superscripts are significantly different (P ≤ 0.05). HP, available P = 0.55%, MP, available P = 0.35%; LP, available P = 0.17%; E (phytase enzyme, Alltech, Inc., Nichloasville, KY) was (+) or was not (−) added to HP, MP, and LP at 1 kg/ton of feed. a-c 1
White turkey breeder hens maintained reproductive performance on diets calculated to contain 0.17% aP and 2.9% Ca (analyzed Ca was 3.5 to 4.1% Ca) compared with hens fed an NRC (0.35% aP) or industry level diet (0.55% aP) with or without enzyme even with the withdrawal of all supplemental P from LP and LPE diets at 20 wk. In previous research conducted to demonstrate the need for practical turkey breeder hen diets to be supplemented with inorganic P, caged BSW hens were used. Sewell et al. (1972), using caged BSW turkey hens, reported decreased egg production and hatchability of fertile eggs, but not egg fertility, when fed a practical diet with supplemental P removed. Ferguson et al. (1974) when feeding similar diets to caged BSW turkey hens observed a reduction in egg production, egg weight, and hatchability of fertile eggs but no effect on fertility. Portal and Ferguson (1974) fed diets with 0.38% total P (all from plant source) or the basal diet supplemented with inorganic P to contain 0.75% total P to caged BSW turkey
FIGURE 4. Fertility of turkey breeder hens fed dietary treatments over 28 wk of lay. HP, available P = 0.5%; MP, available P = 0.3%; LP, available P = 0.17%; HPE, MPE, and LPE were formulated by adding phytase enzyme (Alltech, Inc., Nicholasville, KY) to HP, MP, and LP, respectively, at 1 kg/ton of feed. For fertility, SEM = 1.01.
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Waldroup et al. (1974) fed a factorial arrangement of diets to caged Nicholas turkey breeder hens. The diets contained 2.25 or 3.50% Ca and 0.1, 0.2, 0.3, or 0.4% inorganic P. At the lower level of Ca, 0.3% inorganic P was required to maintain maximum egg production. However, at 3.50% dietary Ca, the inorganic P requirement appeared to be no greater than 0.2%. Egg fertility was lowest at 0.1% inorganic P regardless of Ca level. There were no effects on hatchability of fertile eggs or shell thickness due to dietary inorganic P level. Manley et al. (1980) reported that caged BBW turkey hens fed diets with 0.3% total phosphorus (P) performed as well as hens fed diets with either 0.4 or 0.5% total P. Slaugh et al. (1989), using individually caged Medium White hens, reported that BW, egg production, egg weight, hatchability of fertile eggs, and egg specific gravity were not affected by decreasing the aP from 0.7, 0.5, and 0.3 to 0.15%. Retained P and egg fertility were decreased in hens fed the lowest P level. These reports agree with the current study in which Large
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breeder hens. Hens fed the 0.75% total P had greater egg production and increased hatchability of fertile eggs compared with hens fed the basal diet. Egg fertility was not affected by dietary P level. Potter et al. (1974) fed 0.64 or 0.82% P to BSW hens but observed no differences in reproductive performance due to dietary P. Comparison
of LW and BSW turkey hens with respect to dietary P needs might not be relevant for the purposes of this study. In the current study, egg weights were similar regardless of dietary treatment throughout the 28 wk of lay, even after removal of all supplemental P from the LPE and LP diets. The increase in egg weight with increasing
FIGURE 6. Egg weight and shell thickness of turkey breeder hens fed dietary treatments over 28 wk of lay. HP, available P = 0.55%; MP, available P = 0.35%; LP, available P = 0.17%; st = shell thickness. For egg weight, SEM = 0.60; for shell thickness, SEM = 0.03.
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FIGURE 5. Hatchability of fertile eggs of turkey breeder hens fed dietary treatments over 28 wk of lay. HP, available P = 0.5%; MP, available P = 0.3%; LP, available P = 0.17%; HPE, MPE, and LPE were formulated by adding phytase enzyme (Alltech, Inc., Nicholasville, KY) to HP, MP, and LP, respectively, at 1 kg/ton of feed. SEM for hatchability of fertile eggs = 1.57. There were significant (P < 0.05) treatment effects at 16 to 17 wk of lay; however, this effect was not consistent throughout the trial and was considered by the authors to be an artifact.
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TURKEY BREEDER HENS AND DIETARY PHOSPHORUS TABLE 3. Egg components of turkey breeder hens at 4, 20, and 28 wk of lay Diet1
Egg (g)
4 weeks of lay HP 80.2 MP 79.2 LP 80.7
Yolk (g)
Yolk (%)
Yolk P2
Albumen (g)
Albumen (%)
Albumen P2
Shell (g)
Shell (%)
23.9 24.0 24.6
29.8 30.3 30.6
602 571 579
48.8 47.7 48.3
60.6 60.2 59.8
16.73 13.29 15.74
7.7 7.6 7.8
9.6 9.6 9.6
E− E+
79.2 81.0
24.5 23.9
30.9 29.5
601 567
47.2 49.3
59.6 60.8
14.99 15.51
7.6 7.8
9.6 9.7
X SEM
80.1b 1.5
24.2c 0.8
30.2c 10.7
584b 62
48.2c 1.0
60.2a 4.2
15.25 4.15
7.7b 0.2
9.6a 0.7
29.6 29.2 29.2
32.0 31.8 31.6
607 575 598
55.2 56.0 55.7
59.6 59.8 59.6
19.73 20.10 17.37
8.1 8.1 8.2
8.8 8.6 8.8
20 weeks of lay HP 92.7 MP 93.5 LP 93.6 92.4 94.1
29.2 29.4
31.6 32.0
595 592
55.1 56.2
59.6 59.7
21.06 17.07
8.1 8.2
8.7 8.7
X SEM
93.2a 1.1
29.3b 0.6
31.8b 9.0
593b 45
55.6a 1.2
59.6a 2.8
19.07 4.90
8.2a 0.2
8.7b 1.1
31.8 32.6 31.8 32.0 32.0
34.0 34.6 34.4 34.6 34.2
654 664 676 563 655
53.7 53.3 53.0 52.4 54.2
57.6 56.5 57.5 56.6 57.8
19.09 15.25 24.14 19.35 19.63
7.8 8.0 7.7 7.8 7.9
8.4 8.4 8.4 8.4 8.4
32.0a 0.8
34.4a 4.9
664a 25
53.3b 1.3
57.2b 3.0
19.49 5.42
7.8b 0.2
8.4c 0.6
28 weeks of lay HP 93.0 MP 94.2 LP 92.2 E− 92.6 E+ 93.7 X SEM
93.2a 1.2
Means within a column without common superscripts are significantly different (P ≤ 0.05). HP, available P = 0.55%, MP, available P = 0.35%; LP, available P = 0.17%; E (phytase enzyme, Alltech, Inc., Nichloasville, KY) was (+) or was not (−) added to HP, MP, and LP at 1 kg/ton of feed. 2 Units are milligrams of P per 100 ml of liquid yolk and albumen, respectively. a-c 1
age observed in this study agrees with Reidy et al. (1994) who reported that the weight of eggs from commercial turkey breeders increased 11% between the onset of lay and 24 wk of production. The values for the egg components agree with the values reported by Reidy et al. (1994). The changes in egg component weights agrees with observations of Applegate and Lilburn (1996) who reported that as hens aged, egg weight increased along with relative yolk weight at the expense of albumen. The range for plasma P was 5.50 to 9.52 mg/dL P, which agrees with findings made by Ledoux et al. (1995) who reported plasma P levels for 15-wk-old turkey hens to be 7.55 to 8.24 mg/dL P. The wider range for plasma P in this study might be attributed to the time of day when the blood was sampled from hens. Past studies have shown that plasma P levels fluctuate due to daily egg production status (Miller et al., 1977; Reichmann and Conner; 1977). Woodard and Mather (1964) also noted that in turkey plasma, P level remains higher for a longer period than in chickens, which may be due to the longer shell formation. Tibial P content in this study ranged from 14.6 to 15.3% P agreeing with reports made by Slaugh et al. (1989) that femur P increased from 15.8 to 16.4% as the level of P increased from 0.15 to 0.70% aP. Our values for yolk P agree with the value for turkey yolk P reported by Cunningham and Lee (1978) and for yolk and albumen P for chicken eggs reported by Cotterill et al. (1977) and Naber (1979). The decrease in fecal P with decreasing dietary P in this study agrees with the report by Slaugh et al. (1989)
in which fecal P level decreased from 3.10 to 1.93% as the level of P decreased from 0.70 to 0.15% aP. As expected, hens fed 0.7% total P (0.55% aP) excreted feces with significantly greater P content than those fed lower dietary P levels. Gordon and Roland (1997) observed that hens fed 0.1% aP with phytase supplementation excreted 25% less P than those consuming the same diet without phytase. Water-soluble P content of the turkey hen manure ranged from 0.06 to 0.19%. Hens fed LPE or LP excreted lower water-soluble fecal P levels than all other treatments. These findings suggest that higher levels of dietary P increase water-soluble P. The only effect of dietary E over the 28 wk of lay was for the number of hens that went out of lay. By 28 wk of lay, 41% of the hens not fed dietary phytase had gone out of production, whereas 32% of the hens fed phytase had gone out of production. This was a difference of 24 total hens with half of this difference occurring after 20 wk of lay. It has been proposed that the improvements in growth rate and feed conversion with the addition of phytase to the diet are due to the ability of phytase to release and make available for absorption minerals and trace elements complexed with phytic acid (Simons et al., 1990). The increased availability of some nutrients may be a determining factor for keeping hens in production longer. However, this difference was not reflected in HH egg production. Van der Klis et al. (1997) reported that production performance of turkey breeder hens fed diets supplemented with phytase was comparable to hens receiving supplemental inorganic P. Simons et al. (1990)
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E− E+
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ACKNOWLEDGMENTS This study was funded, in part, by the North Carolina Agriculture Foundation, Alltech, Inc. (Nicholasville, KY), and Hybrid Turkeys (Kitchener, Ontario, Canada).
REFERENCES Applegate, T. J., and M. S. Lilburn. 1996. Independent effects of hen age and egg size on incubation and poult characteristics in commercial turkeys. Poult. Sci. 75:1210–1216. Association of Official Analytical Chemists. 1984. Official Methods of Analysis. 14th ed. Association of Official Analytical Chemists, Washington, DC BUTA. 1997. British United Turkeys of America Parent Stock Management Guide. British United Turkeys of America, Lewisburg, WV. Christensen, V. L., and K. E. Nestor. 1994. Changes in functional qualities of turkey eggshells in strains selected for increased egg production or growth. Poult. Sci. 73:1458–1464. Cotterill, O. J., W. W. Marion, and E. C. Naber. 1977. A nutrient re-evaluation of shell eggs. Poult. Sci. 56:1927–1934. Crouch, A. N., J. L. Grimes, V. L. Christensen, and K. K. Krueger. 2002. Effect of physical feed restriction during rearing on Large White turkey breeder hens: 2. reproductive performance. Poult. Sci. 81:16–22. Cunningham, F. E., and H. W. Lee. 1978. A study of turkey egg yolk 1. composition and electrophoretic separation of components. J. Food Biochem. 2:251–257. Ferguson, T. M., C. E. Sewell, Jr, and R. L. Atkinson. 1974. Phosphorus levels in the turkey breeder diet. Poult. Sci. 53:1627–1629. Goldenberg, A., and A. Fernandez. 1966. Simplified method for the estimation of inorganic phosphorus in body fluids. Am. Chem. J. 12:871–882. Gordon, R. W., and D. A. Roland, Sr. 1997. Performance of commercial laying hens fed various phosphorus levels, with and without supplemental phytase. Poult. Sci. 76:1172–1177. Hybrid Turkeys. 1996. Hybird Feeding Programs. Hybrid Turkeys, Inc., Kitchner, Ontario, Canada.
Kuhl, H. J., Jr. 1993. An industry survey of nutrient levels fed currently. Pages 19–28 in Third International Symposium on Turkey Reproduction. Department of Poultry Science, North Carolina State University, Raleigh, NC. Kuhl, H. J., Jr. 1997. An industry survey of nutrient levels fed currently to turkey breeder hens. Pages 57–65 in Fourth International Symposium on Turkey Reproduction. Department of Poultry Science, North Carolina State University, Raleigh, NC. Ledoux, D. R., K. Zyla, and T. L. Veum. 1995. Substitution of phytase for inorganic phosphorus for turkey hens. J. Appl. Poult. Res. 4:157–163. Manley, J. M., R. A. Voitle, and R. H. Harms. 1980. The influence of dietary calcium and phosphorus on egg production and hatchability of turkey breeder hens. Poult. Sci. 59:2077–2079. Marini, P. J. 2003. A concise history of turkey breeder restriction programs. Pages 1–3 in Fifth International Symposium on turkey reproduction. Department of Poultry Science, North Carolina State University, Raleigh, NC. Martin, J. H., Jr. 1997. The clean water act and animal agriculture. J. Environ. Qual. 26:1198–1203. Miller, D. H., J. W. Bradley, and T. M. Ferguson. 1976. Reproductive performance of Broad Breasted White turkeys in relation to dietary phosphorus. Poult. Sci. 55:2481–2483. Miller, E. R., R. H. Harms, and H. R. Wilson. 1977. Cyclic changes in serum phosphorus of laying hens. Poult. Sci. 56:586–589. Moore, P. A., and D. M. Miller. 1994. Decreasing phosphorus in poultry litter with aluminum, calcium, and iron amendments. J. Environ. Qual. 23:325–330. Naber, E. C. 1979. The effect of nutrition on the composition of eggs. Poult. Sci. 58:518–528. National Research Council. 1950. Nutrient Requirements of Domestic Animals. Vol. 1. Nutrient Requirements for Poultry. National Academy Press, Washington, DC. National Research Council. 1971. Nutrient Requirements of Domestic Animals. Vol. 1. Nutrient Requirements for Poultry. National Academy Press, Washington, DC. National Research Council. 1984. Nutrient Requirements of Poultry. 8th rev. ed. National Academy Press, Washington, DC. National Research Council. 1994. Nutrient Requirements of Poultry. 9th rev. ed. National Academy Press, Washington, DC. Nicholas Turkey Breeding Farms. 1997. Nicholas Management Guidelines. Nicholas Turkey Breeding Farms, Sonoma, CA. Portal, C., and T. M. Ferguson. 1974. Reproductive performance of caged turkeys as affected by age, bird density and dietary phosphorus. Poult. Sci. 53:2218–2221. Potter, L. M., A. T. Leighton, Jr, and A. B. Chu. 1974. Calcium, phosphorus and Nopgro as variables in diets of breeder turkeys. Poult. Sci. 53:15–22. Reichmann, I. G., and J. K. Conner. 1977. Influence of dietary calcium and phosphorus on metabolism and production in laying hens. Br. Poult. Sci. 18:633–640. Reidy, R. R., J. L. Atkinson, and S. Leeson. 1994. Strain comparisons of turkey egg components. Poult. Sci. 73:388–395. SAS Institute. 1992. SAS User’s Guide. Version 6.08. SAS Institute Inc., Cary, NC. Sauer, T. J., T. C. Daniel, P. A. Moore, Jr., K. P. Coffey, D. J. Nicholas, and C. P. West. 1999. Poultry litter and grazing animal waste effects on runoff water quality. J. Environ. Qual. 28:860–865. Self-Davis, M. L., and P. A. Moore. 2000. Determining watersoluble phosphorus in animal manure. Pages 74–76 in Methods of Phosphorus Analysis for Soils, Sediments, Residuals, and Waters. G. M. Pierztnski, ed. http://www.soil. ncsu.edu/sera17/publications/sera17-2/pm_cover.htm Sewell, C. E., Jr., R. S. Atkinson, J. R. Couch, and T. M. Ferguson. 1972. The effect of supplemental phosphorus on the reproductive performance of turkey hens and the subsequent effect upon the poults. Poult. Sci. 51:792–796.
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reported that addition of phytase to feed containing 4.5 g/kg of total P results in levels of feed conversion and performance that are as good or better than the levels achieved using rations supplemented with inorganic P to a total P level of 7.5 g/kg. If hens fed phytase are more likely to remain in production, one might expect to measure an increased HH egg production with a larger number of hens than used in this study. On the other hand, this observation could be an artifact with no real biological effect. A field trial with typical industry size breeder houses should ascertain this possible effect. Feeding low dietary P (0.17% aP) resulted in turkey breeder hen reproductive performance comparable with performance of hens fed dietary P levels used by surveyed members of the turkey industry (0.55% aP) or of hens fed NRC-recommended levels (0.35% aP) and agrees with previous reports for LW or similar type hens. Feeding hens dietary phytase resulted in little of no effect on hens in production because hens were able to perform adequately with no supplemental P. Therefore, the turkey industry might be able to lower dietary P without impairing reproductive performance while reducing total and water-soluble fecal P.
TURKEY BREEDER HENS AND DIETARY PHOSPHORUS
APPENDIX A modified phosphorus assay was used to analyze for inorganic phosphate content in various samples through colorimetric determination using a microplate reader. This assay was modified from the original method reported by Goldenberg and Fernandez (1966). The method uses 2 stable reagents and requires a minimum number of steps. Blood was collected into heparin tubes. The tubes were centrifuged at 1,500 rpm for 15 min. Then 0.2 mL of plasma sample and 0.8 mL of 5% trichloroacetic acid (TCA) reagent were added to bullet tubes. The tubes were then vortexed and allowed to set for 20 min. The tubes were then centrifuged at 2,200 rpm for 30 min. The supernatant was used for final P analysis. Tibial P was determined by using samples that were fat free and moisture free using the method described by the Association of Official Analytical Chemists (1984). Tibia were cleaned of soft tissue, measured for length and weight, and then cut into pieces with a band saw.
The tibias were dried overnight at 100°C, and then fat was extracted with chloroform. Yolk, albumen, and total P fecal samples were dried for 12 h at 100°C. All samples except for plasma were then ground and ashed in a muffle furnace at 600°C. These samples were then digested with HCl before final P analysis. Water-soluble reactive P fecal samples were prepared as reported by Self-Davis and Moore (2000). Fecal samples for water-soluble P were dried overnight at 100°C, ground, and then mixed with distilled water (1 g in 20 mL). This mixture was shaken for 1 h, centrifuged at 2,200 rpm for 30 min, and then filtered using 0.45 millapore filter paper. The resulting filtered solution was used for final P analysis. The standard for the final P analysis was 0.0878 g of dry KH2PO4 mixed into 100 mL of distilled water. A range of 0 to 20% solution of this mixture was used to develop the standard curve. Processed samples (10 µL), standards (10 µL of appropriate dilution), and a blank sample (10 µL distilled water) were placed in separate microtiter plate wells along with 240 µL of TCA reagent and 25 µL of molybdate. A Spectra Max microplate reader using Softmax software program (Spectramax 250, Molecular Devices Corporation, Sunnyvale, CA) was used to measure the absorbance of the sample and to plot the results against a standard curve to calculate the phosphorus concentration.
TCA Reagent This solution contained 125 g of TCA, 37.5 g of ferrous ammonium sulfate, and 12.5 g of thiourea brought to 1,000 mL with distilled water.
Molybdate Reagent Concentrated H2SO4 (45 mL) was added to 200 mL of distilled water in a 500-mL flask set in an ice bath. Ammonium molybdate (22 g) was dissolved into 200 mL of distilled water in an acid-washed beaker. The ammonium molybdate solution was slowly added to the H2SO4 solution set in ice bath. After cooling, the volume was brought to 500 mL.
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Simons, P. C. M., H. A. J. Versteegh, A. W. Jongbloed, P. Slump, K. D. Bos, M. G. E. Wol Beudeker, and G. J. Verchoor. 1990. Improve phosphorus availability by microbial phytase and pigs. Br. J. Nutr. 64:525–540. Slaugh, B. T., N. P. Johnston, and J. D. Pattern. 1989. Research Note: Effect of dietary phosphorus levels on performance of turkey breeder hens. Poult. Sci. 68:319–322. Thomason, D. M., A. T. Leighton, Jr, and J. P. Mason, Jr. 1976. A study of certain environmental factors and mineral chelation on the reproductive performance of young and yearling turkey hens. Poult. Sci. 55:1343–1355. Van der Klis, J. D., H. A. J. Versteegh, P. C. M. Simons, and A. K. Klies. 1997. The efficacy of phytase in corn-soybean mealbased diets for laying hens. Poult. Sci. 76:1535–1542. Waldroup, P. W., J. F. Maxey, and L. W. Luther. 1974. Studies on calcium and phosphorus requirements of caged turkey breeder hens. Poult. Sci. 53:886–888. Wilcox, R. A., C. W. Carlson, C. W. Kohlmeyer, and G. F. Gastler. 1961. Studies on the phosphorus requirement of turkey breeder hens with evidence for unknown factors needed for embryonic development and normal egg production. Poult. Sci. 40:1540–1546. Woodard, A. W., and F. B. Mather. 1964. The timing of ovulation and movement of the ovum through the oviduct, pigmentation and shell deposition in Japanese quail. Poult. Sci. 43:1427–1432.
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