Effects of 1,25-Dihydroxycholecalciferol and Phytase on Zinc Utilization in Broiler Chicks1

Effects of 1,25-Dihydroxycholecalciferol and Phytase on Zinc Utilization in Broiler Chicks1 KEVIN D. ROBERSON2 and HARDY M. EDWARDS, JR.3 Department of Poultry Science, Livestock-Poultry Building, The University of Georgia, Athens, Georgia 30602

1994 Poultry Science 73:1312-1326

INTRODUCTION The NRC (1984) estimates the zinc requirement for broiler chickens to be 40 ppm, which is based upon a corn-soybean meal diet. Young et al. (1958) found that 40 ppm zinc from zinc chloride added to an isolated soybean protein-based diet containing 15 ppm zinc resulted in maximum

Received for publication December 15, 1993. Accepted for publication April 21, 1994. Supported by state and Hatch funds allocated to the Georgia Agricultural Experiment Stations of The University of Georgia. Continental Grain Co., East St. Louis, IL 62202. 3 To whom correspondence should be addressed.

body weight in 10-d-old chicks. When the growth response was plotted against the log of the amount of zinc in the diet, about 25 or 30 ppm added zinc was needed to maximize body weight. Edwards et al. (1958) showed that 40 ppm zinc from zinc chloride provided equivalent 10-d gain to 160 ppm added zinc in chicks fed an isolated soybean protein diet. Roberson and Schaible (1958) found that 30 ppm zinc from zinc sulfate supplemented to an isolated soybean protein-based diet containing 10 ppm zinc increased growth. The protein source affects the zinc requirement of the chick (Ziegler et al, 1961). When casein was fed, the requirement was 5 to 20 ppm. However, the

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ABSTRACT Studies were conducted with corn-soybean meal diets to evaluate the effects of phytate phosphorus utilization on zinc absorption and retention in broiler chicks. In the first two experiments, zinc-65 was used to determine zinc absorption. Experiment 1 was a 2 x 2 factorial with 0 or 5 /ig/kg dihydroxycholecalciferol and 0 or 40 ppm supplemental zinc. In Experiment 2, 5 n g / k g 1,25-dihydroxycholecalciferol [l,25-(OH)2D3] or 750 units/kg phytase or both were added to a diet containing 35 ppm zinc. The diets in Experiment 3 were similar to Experiment 2 except that 600 units/kg phytase was fed. Experiment 4 was similar to Experiment 3 except that dietary phosphorus was decreased by .15%. There were no treatment effects on body weight in Experiments 1 and 2. Zinc absorption was higher in zinc-deficient birds in Experiment 1, but there were no other effects on zinc-65 absorption or retention. Body weight was increased by l,25-(OH) 2 D 3 in Experiments 3 and 4 and by phytase in Experiment 4. Phytate phosphorus retention was increased by phytase and l,25-(OH) 2 D 3 and was increased additively when both sources were fed. Dietary l,25-(OH) 2 D 3 increased zinc retention at times during Experiments 3 and 4, but this response was inconsistent. Phytase did not affect zinc retention. Phytase plus l,25-(OH) 2 D 3 increased zinc retention synergistically in Experiment 3. Bone zinc was increased by l,25-(OH) 2 D 3 and phytase, and there was an additive effect in Experiment 3. Plasma zinc and alkaline phosphatase were not affected. The results suggest that supplemental zinc may be decreased in a corn-soybean meal diet when phytate phosphorus utilization is enhanced. (Key words: 1,25-dihydroxycholecalciferol, phytase, phytate phosphorus, tibial dyschondroplasia, zinc)

1,25-DIHYDROXYCHOLECALCIFEROL, PHYTASE, AND ZINC

4

Seaboard Farms, Athens, GA 30601.

retention in chicks fed a diet containing 27 ppm zinc. Excretion of zinc was decreased by about 12%. However, in a recent balance study with pigs, Lei et al. (1993) observed no effect of dietary phytase on zinc retention. Supplementation of 1,25-dihydroxycholecalciferol [l,25(OH) 2 D 3 ] at 5 ^ g / k g has been shown to increase phytate phosphorus retention in broiler chicks (Edwards, 1993). Dietary l,25-(OH) 2 D 3 also prevents tibial dyschondroplasia in broilers (Edwards, 1989, 1990; Edwards et al, 1992; Rennie et al, 1993), regardless of the calcium to phosphorus ratio in the diet (Whitehead, 1992) or housing conditions (Roberson and Edwards, 1993a). It has also been s u g g e s t e d that p h y t a s e decreases the incidence of tibial dyschondroplasia in broilers (Scheideler et al, 1992). The birds in those studies were raised on litter and fed various amounts of phosphorus with or without 500 units/kg phytase and .85 or 1.00% calcium. The increase in phytate phosphorus retention when phytase or l,25-(OH) 2 D 3 is fed at adequate levels may eliminate the need for supplemental zinc in the trace mineral mix because the amount of zinc in corn-soybean meal diets is approximately the same as the estimated requirement for zinc by the NRC (1984). Hence, studies were conducted to evaluate the effects of improved phytate phosphorus utilization on zinc absorption and retention in broilers.

MATERIALS AND METHODS Experiments 1 and 2 A total of 64 Peterson x Arbor Acres dayold broiler cockerels obtained from a commercial hatchery 4 were utilized in each experiment. In Experiment 1, two levels of l,25-(OH) 2 D 3 (0 or 5 /xg/kg) were fed to chicks receiving a corn-soybean meal diet with 0 or 40 ppm supplemental zinc from zinc oxide. Hence, Experiment 1 was conducted as a 2 x 2 factorial arrangement. The basal diet was analyzed to contain 42 p p m zinc and is listed in Table 1. The diets were low in calcium and high in phosphorus and chloride, which has been reported previ-

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requirement increased to approximately 28 ppm when isolated soybean protein was fed. Addition of 100 p p m zinc from zinc chloride to corn-soybean meal diets containing 36 to 43 ppm zinc increased 28-d body weight. Recently, the zinc requirement was estimated to be 13 to 15 ppm when autoclaved egg white was used as the protein source (Hempe and Savage, 1990). Watkins and Southern (1993) reported that the addition of 5 ppm zinc from zinc carbonate to a corn-soybean meal diet containing 35 ppm zinc increased 5- to 15-d gain by 21 g, which was equivalent to the gain of birds consuming 50 ppm additional zinc. Different zinc requirements are due to various amounts of phytate phosphorus in feed. Phytate is known to reduce bioavailability of zinc in animal diets (O'Dell and Savage, 1960). Phytic acid present in isolated soybean meal has been shown to reduce zinc-65 availability in chickens (Edwards, 1966). Removal of phytic acid from soybean products enhances zinc absorption and bioavailability in rats (Lonnerdal et al, 1988; Zhou et al, 1992). Bioavailability of zinc to chicks is increased by dietary zeolite (Watkins and Southern, 1993), presumably by its effect on phytate phosphorus metabolism (Edwards, 1988). Phytase is an enzyme that hydrolyzes phytate to inositol and inorganic phosphate. Incubation of feedstuffs with phytase has been shown to increase availability of phytate phosphorus (Nelson et al, 1968) and, thus, decrease the dietary requirement for zinc in chickens (Rojas and Scott, 1969). Supplementation of phytase directly to the diet also results in more available phosphorus in broilers (Nelson et al, 1971; Simons et al, 1990; Edwards, 1993) and pigs (Simons et al, 1990; Jongbloed et al, 1992; Cromwell et al, 1993). Edwards (1993) found that total phosphorus excretion was decreased when 600 units/kg phytase was fed with 0 or . 1 % added phosphorus to the basal diet. Thiel and Weigand (1992) reported that 800 units/kg phytase will increase zinc

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ROBERSON AND EDWARDS, JR.

TABLE 1. Composition of the basal diet

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At 6 d of age, the chicks were dosed with a solution containing .2 /iCi of zinc-65 by Ingredient Percentage inserting a pipette into the crop. The amount of radioactive zinc in the chicks was Ground yellow corn 56.85 Soybean meal (dehulled) 35.00 counted at 0, 4, 6, and 8 d from oral Poultry fat (stabilized) 5.00 inoculation using a whole body counter 7 Dicalcium phosphate monohydrate 1.86 (Suso and Edwards, 1968). A multichannel (reagent grade) analyzer 8 was used in Experiment 1 to Calcium carbonate (food grade) .28 determine the amount of zinc-65 activity in Iodized sodium chloride .45 DL-methionine (98%) .20 the peak between 500 and 1,350 channels. A Vitamin premix1 .25 spectrometer 9 was used in Experiment 2 2 Mineral premix .06 because the multichannel analyzer was not Selenium concentrate .05 available. Standard and background counts Witamin premix provided in milligrams per kilo- were determined each time the radiation in gram of diet (except as noted): vitamin A (as all-trans the chicks was counted. retinol acetate), 5,500 IU; cholecalciferol, 1,100 IU; At 14 d of age, the chicks were weighed vitamin E (dl-a-tocopherol acetate), 11 IU; riboflavin, 4.4; calcium pantothenate, 12; nicotinic acid, 44; choline by pen and feed efficiency was determined. chloride, 220; vitamin B12, 9 ^g; vitamin B6, 3.0; The birds were killed by asphyxiation with menadione (as menadione sodium bisulfite), 1.1; C 0 and mixed together so that examina2 thiamin (as thiamin mononitrate), 2.2; folic acid, 3; tion of each bird for tibial dyschondroplasia biotin, .3; and ethoxyquin, 125. was conducted at random. The right tibia of 2 Trace mineral premix provided in milligrams per kilogram of diet: MnS0 4 H 2 0, 185; FeS04-7H20, 400; each chick was cut longitudinally at the CuS04-5H20, 31; and KI, .4 in Experiment 1 and proximal end and scored for tibial dyschonCa(I03)2, 5 in Experiments 2 to 4. droplasia (Edwards and Veltmann, 1983). The severity scores of tibial dyschondroplasia ranged from 0 (no lesion) to 3 (severe lesion). The data were analyzed by ANOVA ously to induce tibial dyschondroplasia in using the General Linear Models (GLM) chickens (Edwards and Veltmann, 1983). procedure of SAS® (Helwig and Council, Experiment 2 utilized the same basal diet 1979). Regression analysis of the percentage as the previous experiment. However, the dose retained by days after original dosing diet was analyzed to have 32 ppm zinc. The gave intercept and slope values for each basal diet was supplemented with 750 chick. The intercept may be interpreted to units/kg phytase {Aspergillus ficuum) or 5 be zinc-65 absorption and retention is Mg/kg l,25-(OH) 2 D 3 or both for a total of reflected as the slope. The slope may be used to calculate the biological half-life of four treatments. In each experiment, the birds were zinc-65 (Suso and Edwards, 1968). Treatment means were separated by Duncan's housed in an electrically heated battery multiple range test (Duncan, 1955) with a 5 brooder with wire mesh floors and were 5% level of probability. provided ad libitum access to feed and water. Four pens of four chicks per pen were used for each treatment. Lighting was Experiments 3 and 4 continuous in the room and battery A total of 240 day-old male broiler chicks brooder. Plastic tubes 6 were placed over the were obtained from the same hatchery and fluorescent lights to prevent exposure to utilized in each experiment. In Experiment light wavelengths in the ultraviolet range. 3, Peterson x Arbor Acres chicks were used. Ross x Ross chicks were used in Experiment 4. The housing and lighting conditions were the same as in Experiments 1 and 2. Six pens 5 Petersime Incubator Co., Gettysburg, OH 54328. of 10 chicks were housed per treatment. 6 Arm-a-lite®, Thermoplastic Processes, Stirling, NJ The same basal diet was used in Experi07980. ments 3 and 4 as in the previous two 7 Metrix, Inc., Denver, CO 80206. experiments. The diet provided a model to 8 Spectrum 88, The Nucleus, Inc., Oak Ridge, TN test the ability of phytase to prevent the 37830. 9 development of tibial dyschondroplasia. Baird-Atomic 530, Cambridge, MA 02138.

1,25-DIHYDROXYCHOLECALCIFEROL, PHYTASE, AND ZINC

was collected every 4 d in Experiment 1, whereas chromic oxide was added at .1% as a marker in Experiment 2. Chromic oxide was measured by the procedure described by Brisson (1956). The data were analyzed by ANOVA using the GLM procedure of SAS® (Helwig and Council, 1979). Significant difference among treatment means were separated by Duncan's multiple range test (Duncan, 1955) with a 5% level of probability. RESULTS Experiments 1 and 2

There were no dietary effects on body weight or gain:feed in Experiments 1 and 2 (Tables 2 and 3). Absorption of zinc-65 was increased in Experiment 1 when zinc was not added to the diet. Addition of phytase or l,25-(OH)2D3 to the diet did not effect zinc-65 absorption. However, zinc-65 absorption was increased marginally by l,25-(OH)2D3 in Experiment 1 (P < .130) and Experiment 2 (P < .186). There were no effects on the rate of turnover of zinc-65. The incidence and severity of tibial dyschondroplasia was decreased by l,25-(OH)2D3 supplementation in Experiment 1. There were zinc by l,25-(OH)2D3 interactions for severity of tibial dyschondroplasia (P = .654) and percentage severe lesions (P = .006) due to greater severity when zinc was supplemented to the diet. The number of Number 3 scores was decreased when zinc was not added to the diet. The incidence of tibial dyschondroplasia was reduced marginally (P < .109) in Experiment 2 by dietary l,25-(OH)2D3. Phytase marginally increased (P < .085) the incidence of tibial dyschondroplasia. Birds fed phytase only had a significantly higher level of tibial dyschondroplasia than the chicks fed only supplemental l,25-(OH)2D3. The severity of tibial dyschondroplasia was decreased by dietary l,25-(OH)2D3. Phytase 10 TECHNICON Autoanalyzer Methodology, Tech- did not affect the severity of tibial dyschondroplasia. Even though percentage nicon Corp., Tarrytown, NY 10591. n Model 5000, Perkin-Elmer Corp., Norwalk, CT Number 3 scores were eliminated by 06859. l,25-(OH)2D3 in Experiment 2, this was a "Technical Bulletin No. 104, Sigma Chemical Co., marginal (P < .161) response. St. Louis, MO 63178-9916.

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Zinc was analyzed to be 35 ppm in the basal diet. The design of Experiment 3 was similar to that of Experiment 2 except that phytase was added at 600 units/kg. Experiment 4 was the same as Experiment 3 except that dietary phosphorus was decreased by about .15%. This diet provided enough phosphorus to prevent rickets. Therefore, any body weight response to enhanced phytate phosphorus utilization would presumably be due to increased utilization of zinc. Blood was obtained by cardiac puncture at 16 d in Experiment 3 and 15 d in Experiment 4 for plasma analyses. Plasma calcium1" (Section N-31) and phosphorus (Section N-46)10 were analyzed immediately after withdrawal of the blood and centrifugation. The plasma was stored at 0 C and later analyzed for zinc by flame atomic absorption spectroscopy.11 Plasma alkaline phosphatase was measured in Experiment 4 after storage using pnitrophenyl phosphate as the substrate and the sample and substrate were incubated with 2-amino-2-methyl-l-propanol buffer for 15 min at 37 C 1 2 Alkaline phosphatase activity is stable when the sample is stored frozen (Connolly, 1953). At 16 d of age, the birds were killed by C0 2 asphyxiation and mixed together. The right tibia was examined for tibial dyschondroplasia as described previously. The left tibia was excised and analyzed for bone ash on a fat-free, dry basis (Association of Official Agricultural Chemists, 1955). The ash from the bones of each pen of birds was solubilized and measured for zinc content by atomic absorption spectroscopy.11 Excreta were collected every 4 d of Experiment 3 and every 5 d in Experiment 4. The feed and dried excreta were analyzed for calcium (Hill, 1955), phosphorus (O'Neill and Webb, 1970), phytate phosphorus (Common, 1940), and zinc (Jones, 1972), and retention values were calculated (Edwards and Gillis, 1959). Total excreta

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ROBERSON AND EDWARDS, JR. TABLE 2. Effects of dietary zinc and 1,25-dihydroxycholecalciferol [l,25-(OH)2D3] on performance, zinc-65 metabolism, and the development of tibial dyschondroplasia in broiler chicks, Experiment 1 1,25(OH) 2 D 3i

Zinc

Tibial dyschondroplasia

Zinc-65

Score

No. 3 1

feed

Absorpt ion Retention

Incidence

(g) 423 378 387 410 21

(&g) .756 .734 .722 .712

58 a l9b 77a 8»> 9

1.88ab 1.12b<: 2.67* .25c .39

8b 6b 52* 0b

.019

(intercept) (slope) 33.6" -.65 -.86 39.0a 21.1 b -.52 24.4b -.55 .17 2.6

401 399

.745 .717

36.3" 22.8b

-.76 -.54

43 39

1.50 1.46

7b 26a

405 394

.739 .723

27.4 31.7

-.59 -.71

68* 14b

2.28a ,68b

30a 3b

dt 1 1 1

.925 .158 .615 .387 .126 .737

.206 .485 .584

.001 .130 .700

(%)

(%)

.662 .001 .142

8

.029 .004 .006

.918 .002 .054

^ M e a n s within each column with no common superscript differ significantly (P < .05) Percentage of birds that scored Number 3 (severe lesion) at the end of the experiment.

TABLE 3. Effects of phytase and 1,25-dihydroxycholecalciferol [l,25-(OH)2D3] on performance, zinc-65 metabolism, and the development of tibial dyschondroplasia in broiler chicks, Experiment 2

Phytase (mg/kg) 0 0 750 750 Pooled SE

14-d BW

feed

Absorption Retention

(Mg/kg) 0 5 0 5

(g) 323 380 334 344 22

(g:g) .700 .706 .695 .685 .032

(intercept) 28.9 34.3 31.2 36.3 3.7

(slope) -1.34 -1.74 -1.40 -1.46 .17

352 341

.703 .690

31.6 33.7

-1.54 -1.43

330 362

.698 .696

30.0 35.3

Main effects Phytase 0 mg/kg 750 m g / k g l,25-(OH) 2 D 3 0 Mg/kg 5 Mg/kg ANOVA Source of variation Phytase l,25-(OH) 2 D 3 Phytase x 1,25(OH) 2 D 3 ab

Tibial dyschondroplasia

Zinc-65

1,25(OH) 2 D 31

-1.37 -1.60 — Probability

Incidence

Score

(%) 40 ab 14b 64a

No. 3 1

(%) 1.88a .50b 1.92a 1.12*b

14

.42

14 0 25 0 13

27 53

1.19 1.52

7 12

52 28

1.90a 81 b

20 0

4 2 ab

df 1 1

.626 .699 .181 .946

.570 .186

.517 .206

.085 .109

.442 .024

.698 .161

1

.268 .814

.964

.343

.994

.501

.698

' Means within each column with no common superscript differ significantly (P < .05). Percentage of birds that scored Number 3 (severe lesion) at the end of the experiment.

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(mg/kg) (Mg/kg) 0 0 0 5 40 0 40 5 Pooled SE Main effects Zinc 0 mg/kg 40 m g / k g l,25-(OH) 2 D 3 0 Mg/kg 5 Mg/kg ANOVA Source of variation Zinc l,25-(OH) 2 D 3 Zinc x l,25-(OH) 2 D 3

14-d BW

1,25-DIHYDROXYCHOLECALCIFEROL, PHYTASE, AND ZINC

Experiments 3 and 4

phosphorus retention. This effect appeared to be a synergistic effect at 4 and 12 d of age. Phytate phosphorus retention was increased similarly by phytase and l,25-(OH) 2 D 3 in Experiment 4 and there was no additive response except at 10 d. Although phytate phosphorus retention increased in both studies when phytase was added to the diet, there was no effect of phytase on total phosphorus retention. At 16 d of age, total phosphorus retention was increased by l,25-(OH) 2 D 3 in Experiment 3. Although total phosphorus retention was increased by l,25-(OH) 2 D 3 supplementation alone at 5 d in Experiment 4, there was no consistent effect. Calcium retention was increased by 4 d of age by l,25-(OH) 2 D 3 in Experiment 3 (Table 6) and phytase increased calcium retention by 5 d of age in Experiment 4 (Table 7). There was a significant phytase main effect at 16 d in Experiment 3 due to a s y n e r g i s t i c effect of p h y t a s e a n d l,25-(OH) 2 D 3 . 1,25-Dihydroxycholecalciferol alone and both sources together consistently improved calcium retention. Zinc retention was also increased in Experiment 3 when phytase and l,25-(OH) 2 D 3 were added to the diet. There was a significant l,25-(OH) 2 D 3 response at 16 d of age. Zinc retention was increased by l,25-(OH) 2 D 3 supplementation in Experiments 4 at 5 and 10 d. However, there was no effect at 15 d of age. DISCUSSION The small number of birds used in Experiments 1 and 2 (16 per treatment) prevented many responses from being significant. Body weight was increased by dietary l,25-(OH) 2 D 3 when 60 birds per treatment were used in Experiments 3 and 4. Phytase also increased body weight significantly in Experiment 4 and marginally in Experiment 3. Because adequate phosphorus was provided in Experiment 4 to prevent rickets and a decrease in feed intake, the body weight response is presumed to be increased zinc utilization. Watkins and Southern (1993) reported that an additional 5 ppm of zinc increased the body weight gain in chicks fed a cornsoybean meal diet containing 35 ppm zinc.

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Body weight increased when 1,25(OH) 2 D 3 was added to the diet in Experiment 3 (Table 4). Phytase and l,25-(OH) 2 D 3 independently increased body weight in Experiment 4 (Table 5). The addition of both sources did not further increase body weight in Experiment 3 above the weight of birds consuming only l,25-(OH) 2 D 3 . Body weight was not increased when both were added in Experiment 4. Feed efficiency was not affected by the diets in these studies. Bone ash was increased consistently by dietary l,25-(OH) 2 D 3 (Tables 4 and 5). Phytase increased bone ash when dietary phosphorus was lowered in Experiment 4. There was no additive effect on bone ash, which resulted in a significant interaction in Experiment 4. Bone zinc was increased by both phytase and l,25-(OH) 2 D 3 . There was an additive response in Experiment 3. In Experiment 4, l,25-(OH) 2 D 3 was more effective than phytase at increasing the accumulation of zinc into the bone. There was not an additive effect in Experiment 4. Plasma calcium and zinc were not affected in these studies (Tables 4 and 5). Phytase alone decreased plasma dialyzable phosphorus in Experiment 3, but there was no effect in Experiment 4. There were no effects on plasma alkaline phosphatase activity (Table 5). However, l,25-(OH) 2 D 3 did marginally (P < .079) reduce alkaline phosphatase activity. Supplementation of l,25-(OH) 2 D 3 clearly decreased the incidence and severity of tibial dyschondroplasia in Experiment 3 (Table 4). There was no effect of phytase on tibial dyschondroplasia in this experiment. However, tibial dyschondroplasia was reduced in Experiment 4 only when both l,25-(OH) 2 D 3 and phytase were in the diet (Table 5). Phytate phosphorus utilization was increased by 4 d of age with phytase and by 8 d of age when l,25-(OH) 2 D 3 was fed in E x p e r i m e n t 3 (Table 6). H o w e v e r , l,25-(OH) 2 D 3 increased phytate phosphorus retention by 5 d of age in Experiment 4 (Table 7). Phytase was more effective than l,25-(OH) 2 D 3 at increasing phytate phosphorus retention in Experiment 3. Addition of both phytase and l,25-(OH) 2 D 3 to the diet produced an additional increase in phytate

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l,25-(OH) 2 D 3

df 1 1 1

.732 .738

.737 .733

428 441

423b 446"

.664 .704 .474

.730 .744 .734 .731 .012

.246 .044 .207

(g:g)

(g)

409b 436 ab 447* 446a 11

feed

.069 .470 .348

11.3 11.0

21.8 b 25.8*

38.6b 40.3*

.001 .001 .697

11.5 10.8

22.4b 25.2*

39.6 39.4

.536 .001 .659

11.5 11.1 11.6 10.5 .4

(mg/g) 20.5C 23.0b 24.3b 27.3 a .6

(%)

6.70 6.30

.29

Vmg/ U L ;a 6.94 6.01b 6.45»b 6.59*

DPI

Plasma

.184 .875 .076

6.48 6.52 Probability

Calcium

38.7^ 38.6b 40.5" 40.2" .2

Zinc

Ash

Bone

^Means within each column with no common superscript differ significantly (P < .05). !DP = Dialyzable phosphorus. Percentage of birds that scored Number 3 (severe lesion) at the end of the experiment.

(units/kg) (Mg/kg) 0 0 600 0 0 5 600 5 Pooled SE Main effects Phytase 0 units/kg 600 units/kg l / 25-(OH) 2 D 3 0 jig/kg 5 pg/kg ANOVA Source of variation Phytase l,25-(OH) 2 D 3 Phytase x l,25-(OH) 2 D 3

Phytase

16-d BW

TABLE 4. Effects of phytase and 1,25-dihydroxycholecalciferol [l,25-(OH)2D3] on performance, plasma a tibial dyschondroplasia in broiler chicks. Experiment 3

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(Mg/kg) 0 0 5 5

(units/kg)

df 1 1 1 .155 .001 .038

.037 .001 .486

24.1b 28.5*

36.6b 37.9"

.755 .742

456 477

.604 .522 .308

25.6" 27.0"

37.0 37.4

(mg/g) 23.1c 25.0^ 28.0* 29.0" .6

(%) 36.1c 37.0t> 38.0* 37.8a .2

.754 .745

Zinc

Ash

465 466

.866 .149 .002

(g:g) .751 .760 .758 .729 .001

(g)

429c 483* 509" 456^ 15

feed

Bone

.914 .922 .408

10.8 10.7

10.8 10.7

10.9 10.6 10.6 10.8 .3

Calcium

DP1

Plasma Zinc

119 111

.536 .594

.693

4.35 111 4.25 119 • Probability

4.33 4.27

.4 .5 .8

- (Mg/dL) 4.42 114 4.27 109 4.24 124 4.26 113 .15 12

"-^Means within each column with no common superscript differ significantly (P < .05). J DP = Dialyzable phosphorus. Percentage of birds that scored Number 3 (severe lesion) at the end of the experiment.

Main effects Phytase 0 units/kg 600 units/kg l,25-(OH) 2 D 3 0 Mg/kg 5 >*g/kg ANOVA Source of variation Phytase l,25-(OH) 2 D 3 Phytase x l,25-(OH) 2 D 3

0 600 0 600 Pooled SE

l,25-(OH) 2 D 3

Phytase

16-d BW

TABLE 5. Effects of phytase and 1,25-dihydroxycholecalciferol [l,25-(OH)2D3] on performance, plasma and (ALP), and the development of tibial dyschondroplasia, Experime

from http://ps.oxfordjournals.org/ at Michigan State University on January 27, 2015

l,25-(OH) 2 D 3

a_d

df 1 1 1

.001 .001 .614

.001 .001 .161 .001 .001 .342

37b 44a

36 b 44a

39b 44a

49 52

.001 .259 .375

35b 46o

35b 45 a

38= 50" 1

42b

32d

48 a

32c 40 b 38b 51 a 2

16 d

35>>

32 d 45b 37 c 50^ 1

12 d

Phytate P

8 d

43 b 58 a

42b 55 a 43b 61 a 3

4 d

61 63

62 62

62 61 63 63 2

.691 .444 .565

4 d

54 55

54 55

.419 .534 .103

54 54 53 56 1

53 55

.610 .059 .179

.622 .033 .188

51b 53 a

58 57

56 54 60 60 2

(%1

4 d

.789 .046 .576

Cal

58 59

57b 56b 59 ab 61 a 1

8 d

.381 .028 .279

55b 57b 60a 60" Pro bability -

53 a 1

55 a 1

52 52

5 2 ab

543b

54 54

51 b 51b

16 d

54 ab 53b

12 d

Phosphorus 8 d

Means within each column with no common superscript differ significantly (P < .05).

(units/kg) (fg/kg) 0 0 0 600 5 0 5 600 Pooled SE Main effects Phytase 0 units/kg 600 units/kg l,25-(OH) 2 D 3 0 /tg/kg 5 /»g/kg ANOVA Source of variation Phytase l,25-(OH) 2 D 3 Phytase x 1,25-(OH) 2 D 3

Phytase

TABLE 6. Effects of phytase and 1,25-dihydroxycholecalciferol [l,25-(OH)2D3] on the retentions of p and zinc in broiler chicks at various ages, Experiment 3

from http://ps.oxfordjournals.org/ at Michigan State University on January 27, 2015

l,25-(OH) 2 D 3

df 1 1 1 .059 .008 .325

32b 43*

43 b 57*

19b 32*

.001 .001 .204

33 41

41b 59*

20^ 35"

.004 .026 .278

26 b 37* 41* 44* 4

15 d

33c 54b 49b 65* 2

10 d

10b 31" 26* 37* 5

5 d

39 42

40 42

2

.253 .179 .193

38 b 41*b 44* 42*b

5 d

50 52

50 51

49 51 52 51 2

.660 .276 .422

10 d

Phosphorus

48 49

49 49

48 48 50 49 1

f°'l

42

b

42b 48*

38b 46* 46* 49* 2

5 d

761 280 1 945

.004 .033 .110

47* Probability

15 d

'"Means within each column with no common superscript differ significantly (P < .05).

(units/kg) (^g/kg) 0 0 600 0 0 5 600 5 Pooled SE Main effects Phytase 0 units/kg 600 units/kg l,25-(OH) 2 D 3 0 /tg/kg 5 Mg/kg ANOVA Source of variation Phytase l,25-(OH) 2 D 3 Phytase x l,25-(OH) 2 D 3

Phytase

Phytate P

TABLE 7. Effects of phytase and 1,25-dihydroxycholecalciferol [l,25-(OH)2Dj] on the retentions of p and zinc in broiler chicks at various ages, Experiment 4

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reliable to estimate the retention of zinc in chicks under the conditions of our studies. The increase in b o n e ash w h e n l,25-(OH) 2 D 3 is fed has been reported previously (Edwards, 1989, 1990; Roberson and Edwards, 1993a,b), even when dietary calcium is fed near the NRC (1984) estimated requirement (Edwards et al, 1992). Phytase does not increase bone ash unless high levels are added to a diet with low phosphorus (Edwards, 1993). However, bone (tibia) zinc was increased by both l,25-(OH) 2 D 3 and phytase in these studies. Tibia zinc was increased in chicks by dietary zeolite even though zeolite did not affect bone ash (Watkins and Southern, 1993). Soares et al. (1987) reported that dietary l,25-(OH) 2 D 3 enhances the accumulation of zinc in the bones of young female rats. Bone zinc was the most sensitive variable for measuring zinc utilization in Experiments 3 and 4. Halpin et al. (1986) found that tibia manganese was an efficient indicator of manganese absorption in chicks. The bone zinc data in our studies verify the body weight data that shows l,25-(OH) 2 D 3 is more effective than phytase at enhancing zinc utilization in chicks. The bone data also indicate that maximum zinc utilization is achieved when both phytase and l,25-(OH) 2 D 3 are fed. The plasma calcium data agree with other work that states that plasma calcium is not affected by phytase (Edwards, 1993) or l,25-(OH) 2 D 3 (Rennie et al, 1993; Roberson and Edwards, 1993b). Plasma dialyzable phosphorus was decreased by phytase addition alone only when the higher phosphorus level was fed. Edwards (1993) observed a linear decrease in plasma dialyzable phosphorus when increasing levels of phytase was supplemented to a diet with no added phosphorus. However, a low level of phytase had no effect when .2% phosphorus was added to the basal diet. The lack of an effect of l,25-(OH) 2 D 3 on plasma phosphorus (Rennie et al, 1993) and dialyzable phosphorus (Edwards, 1993; Roberson and Edwards, 1993a,b) has been reported. There were no effects of l,25-(OH) 2 D 3 or phytase on plasma zinc. Again, this indicates that the level of zinc in the diet is close to the dietary requirement. Appar-

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The absence of any effect on feed efficiency was expected. Improvements in gain:feed for broilers have only been accomplished when phytase was supplemented to a diet with no added inorganic phosphorus (Simons et al, 1990; Edwards, 1993). The addition of 5 fig/kg l,25-(OH) 2 D 3 or less did not affect feed efficiency in previous studies (Edwards, 1990). The absorption of zinc-65 ranged from 21 to 39% in these studies. This is similar to previous studies using similar techniques with young White Leghorn chicks (Suso and Edwards, 1968). The lack of an effect of l,25-(OH) 2 D 3 or phytase on zinc65 absorption may have been due to the experimental diet. The diet used in these studies was high in phosphorus and low in c a l c i u m . A l o w - c a l c i u m , h i g h phosphorus diet may inhibit factors that enhance zinc utilization (Watkins and Southern, 1992). This was particularly evident in Experiment 2. The basal diet was analyzed to contain higher total and phytate phosphorus levels in Experiment 2 man in Experiment 1. The response of zinc-65 absorption to l,25-(OH) 2 D 3 supplementation in a zinc-deficient diet was less in Experiment 2 than the first experiment. However, zinc utilization was clearly improved in Experiments 3 and 4 by l,25-(OH) 2 D 3 and phytase. The marginal responses to l,25-(OH) 2 D 3 observed in the zinc studies indicates that more replication was needed to more accurately determine the effects of phytate utilization on zinc absorption. Mineral retention is usually increased when phytase or l,25-(OH) 2 D 3 is fed (Edwards, 1993). The results of Experiment 2 differ from the findings of Thiel and Weigand (1992) in which 800 units/kg phytase increased zinc retention in chicks. The authors did not state the dietary calcium or phosphorus levels in the report. It is likely that lower phosphorus levels were fed in that study. Although there was no effect on zinc-65 retention in our studies, the slope of the regression line tended to be more negative when l,25-(OH) 2 D 3 was fed. This would indicate a slightly shorter biological half-life of zinc-65 and is contradictory to the zinc retention data in Experiments 3 and 4. It seems that the use of zinc-65 may not be

1,25-DIHYDROXYCHOLECALCIFEROL, PHYTASE, AND ZINC

likely due to l,25-(OH)2D3 supplementation and the lack of an effect when l,25-(OH)2D3 was fed alone demonstrates the variation sometimes encountered between experiments evaluating tibial dyschondroplasia. The amount of tibial dyschondroplasia found in pens of birds consuming only l,25-(OH)2D3 ranged from 0 to 30% in this experiment. There was about a 20% incidence of calcium rickets in Experiment 2 (data not shown), which was eradicated by l,25-(OH)2D3 supplementation. The incidence of calcium rickets in the basal diet was less than 2% in all other experiments. The birds in Experiment 2 did not grow as well as the chicks in the other experiments. There were significant zinc by l,25-(OH)2D3 interactions for severity of tibial dyschondroplasia and percentage of severe lesions in Experiment 1 due to greater tibial dyschondroplasia severity when zinc was supplemented to the diet without l,25-(OH)2D3. The number of Number 3 scores was decreased when zinc was not added to the diet. Zinc has not been previously associated with tibial dyschondroplasia. However, alkaline phosphatase is involved in the calcification process of epiphyseal cartilage (Poole et al, 1989). Experiment 1 was only a small study and the overall incidence of tibial dyschondroplasia was not affected by the zinc level of the diet. It does not seem likely that zinc is playing a significant role in the etiology of tibial dyschondroplasia. It is clear that phytase increases phytate phosphorus utilization. Cholecalciferol may also increase phytate phosphorus utilization when fed at very high levels (Mohammed et al, 1991). However, cholecalciferol does not prevent tibial dyschondroplasia when fed at 2,000 ICU/ kg (Elliot, 1992). The increased utilization of phytate phosphorus by dietary l,25-(OH)2D3 was reported recently (Edwards, 1993). When l,25-(OH)2D3 was added at 5 MgAg t o a diet containing low phosphorus and 75 units/kg phytase, phytate phosphorus retention was increased more than the addition of the increases observed when each source was supplemented individually. This synergistic effect was seen in

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ently, plasma zinc is not as sensitive an indicator of changes in zinc utilization as bone zinc or weight gain. However, Watkins and Southern (1993) observed an increase in plasma zinc when zeolite was added to a corn-soybean meal diet that was deficient in zinc. In those studies, many more plasma samples were analyzed per treatment. The negative effect of zeolite on phosphorus retention (Edwards, 1988) may have influence the plasma zinc response as well. Plasma alkaline phosphatase, a zinccontaining enzyme, was not affected by increased zinc utilization in other studies when dietary zinc was fed at 35 ppm (Watkins and Southern, 1993). A zinc deficiency will decrease alkaline phosphatase levels in the tibia (Starcher and Kratzer, 1963; Lease, 1972). Lei et al (1993) reported an increase in both plasma alkaline phosphatase and zinc in pigs fed 1,350 units/kg phytase and a diet devoid of added phosphorus or zinc. Plasma alkaline phosphatase was not affected by dietary l,25-(OH)2D3, which agrees with a previous report (Roberson and Edwards, 1993b). However, there was a marginal decrease in plasma alkaline phosphatase with l,25-(OH)2D3. Because phosphorus was added at .2% rather than the higher level used in previous studies (.35%), the chicks may have been borderline deficient in phosphorus in Experiment 4. Alkaline phosphatase is decreased back down from elevated levels when a ricketic situation is rectified (Motzok, 1950). The ability of l,25-(OH)2D3 to prevent tibial dyschondroplasia when broilers are fed a low-calcium, high-phosphorus diet is well known (Edwards, 1989, 1990; Edwards et al, 1992; Rennie et al, 1993; Roberson and Edwards, 1993a,b). This effect was less evident in Experiment 4 when dietary phosphorus was lowered. Phytase alone had absolutely no effect on the development of tibial dyschondroplasia regardless of the phosphorus level of the diet. However, Scheideler et al. (1992) reported a decrease in tibial dyschondroplasia at 3 and 9 wk of age in broilers raised on litter when phytase was added at 500 units/kg and available phosphorus was fed at various levels. The decrease in tibial dyschondroplasia in Experiment 4 is

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Supplementation of l,25-(OH) 2 D 3 has been shown to increase the absorption of calcium-47 (Edwards, 1989) and the retent i o n of c a l c i u m ( E d w a r d s , 1993). 1,25-Dihydroxycholecalciferol is known to enhance calcium absorption in the intestine by stimulating calcium-binding protein (Wasserman et al, 1982). Calcium retention was consistently increased by l,25-(OH) 2 D 3 in Experiments 3 and 4. Phytase did not increase calcium retention in previous studies (Edwards, 1993) when fed alone or in combination with l,25-(OH) 2 D 3 . However, phytase enhanced calcium retention at 5 and 10 d in Experiment 4. There appeared to be a synergistic increase in calcium retention at 16 d in Experiment 3. Calcium availability would be expected to increase as phosphorus becomes more available. Zinc retention was increased when both l,25-(OH) 2 D 3 and phytase were fed in Experiment 3, which resulted in an overall l,25-(OH) 2 D 3 response at 16 d. When lower levels of phosphorus were fed in Experiment 4, zinc retention was increased early (5 and 10 d) by l,25-(OH) 2 D 3 , but was not affected at 15 d. Thiel and Weigand (1992) reported increased zinc retention when 800 units/kg phytase was fed to chicks consuming a diet containing 27 ppm zinc. The fact mat there was only a 5% or less difference in zinc retention in these studies again indicates that the level of zinc in a corn-soybean meal diet (- 35 ppm) is close to the requirement for broilers (NRC, 1984) and may be adequate if the phytic acid in the diet is utilized more efficiently. The results of these studies indicate that significant financial savings can be achieved by decreasing the amount of phosphorus in the diet when phytase or l,25-(OH) 2 D 3 is added at proper levels. Maximum benefit is achieved when both are supplemented to the diet. Commercial production of phytase should make it economical in the future. The level of calcium in the diet can be decreased by one third when l,25-(OH) 2 D 3 is fed (Edwards et al, 1992). This offers more potential for financial savings and even higher phytate phosphorus utilization as the level of calcium in the diet affects phytate phosphorus utilization (Nelson, 1967).

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Experiment 3 at 4 and 12 d. Tanka and DeLuca (1974) suggested that 1,25(OH) 2 D 3 may be a phosphate transport hormone and generally stimulates the transport of phosphate at many points in the body. Hence, l,25-(OH) 2 D 3 may be working with phytase to transport the phosphate hydrolyzed to the bone by transport systems in the mucosa and blood. Phytate phosphorus retention was very low at 5 d in Experiment 4. The retention values for calcium, total phosphorus, and zinc were also low. The analyzed values for chromic oxide in the excreta were lower at 5 d than at 10 and 15 d. There was also considerable variation at 5 and 15 d for phytate phosphorus retention in this experiment. Published values for phytate retention vary widely (Nelson, 1976; Edwards and Veltmann, 1983; Edwards, 1993) and have been reported to be as low as 2.8% (Matyka et al, 1990). Regardless of the percentage retentions, the relative relationship of the effects of phytase and l,25-(OH) 2 D 3 on phytate utilization compared to the basal diet remained consistent. Although phytate phosphorus retention was obviously improved, total phosphorus retention was not affected by phytase or l,25-(OH) 2 D 3 individually. The addition of both sources increased total phosphorus retention at 16 d in Experiment 3, which resulted in the significant l,25-(OH) 2 D 3 main effect. However, there was no effect in Experiment 4 even with the lower dietary phosphorus level. In a previous study, the retention of total phosphorus was increased when 600 units/kg phytase was added to a diet with no added phosphorus (Edwards, 1993). It seems that an available phosphorus level lower than .3% needs to be fed to decrease phosphorus excretion with phytase or 1,25(OH) 2 D 3 . Simons et al (1990) minimized manure phosphorus with 375 units/kg added to a diet with no added phosphorus from dicalcium phosphate. They reported that 1,000 units/kg phytase supplemented to a diet with no added inorganic phosphorus and low calcium would maximize phosphorus availability and support growth and feed conversion equal to a diet containing normal calcium and phosphorus levels.

1,25-DIHYDROXYCHOLECALCIFEROL, PHYTASE, AND ZINC

The growth data in these studies indicates that zinc may be eliminated from the trace mineral premix if l / 25-(OH) 2 D 3 or phytase is fed at an efficacious level. This assumes that additional zinc would not be required during higher stress conditions such as diseases. Further investigation is required to address this question. However, it seems that zinc and probably other cationic trace minerals such as manganese and iron can at least be decreased in the diet as a result of enhanced phytate phosphorus utilization.

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