Phosphorus Availability

Phosphorus Availability. 1. The Effect of Chick Age and Vitamin D Metabolites on the Availability of Phosphorus in Defluorinated Phosphate1 J. H. SOARES, JR., M. R. SWERDEL, and E. H. BOSSARD Department of Poultry Science, University of Maryland, College Park, Maryland 20742 (Received for publication December 7, 1977)

INTRODUCTION A l t h o u g h n u m e r o u s r e p o r t s have been m a d e on t h e availability of p h o s p h o r u s in defluorinated p h o s p h a t e ( D F P ) , t h e r e is still considerable controversy as t o p h o s p h o r u s ' biological availability from this source t o chicks. Relative availabilities of 92% or less have been r e p o r t e d by Edwards ( 1 9 6 8 ) , Gillis et al. ( 1 9 5 7 ) , a n d Dilworth and Day ( 1 9 6 4 ) . Whereas variable results have been o b t a i n e d by Nelson and Walker ( 1 9 6 4 ) ( 8 1 - 1 0 5 % ) using tricalcium p h o s p h a t e as a standard, Wilcox et al. ( 1 9 5 4 ) (75—101%) using dibasic calcium p h o s p h a t e as a s t a n d a r d and M o t z o k et al. ( 1 9 5 6 ) also using tricalcium p h o s p h a t e as standard. More recently, Twining and Twining ( 1 9 7 2 ) r e p o r t e d t h a t D F P was equal t o dicalcium p h o s p h a t e dihydrate w h e n fed at o p t i m u m calcium and phosp h o r u s levels (.45% Avg. P a n d . 9 % Ca). Several a u t h o r s have indicated t h a t vitamin D 3 level influences p h o s p h o r u s availability (Nelson, 1 9 6 7 ; M o t z o k 1 9 6 5 ; a n d Fritz et al, 1 9 6 8 ) . Recently, Wasserman and Taylor ( 1 9 7 3 ) and DeLuca ( 1 9 7 4 ) have shown t h a t phosp h o r u s absorption is an active m e c h a n i s m requiring vitamin D or, m o r e specifically, t h e h o r m o n e l , 2 5 - ( O H ) 2 - D 3 . Charles and Ernst

1 Scientific Article No. A2381. Contribution No. 5396 of the Maryland Agricultural Experiment Station (Department of Poultry Science).

1978 Poultry Sci 57:1305-1312

( 1 9 7 4 ) , McLoughlin a n d Soares ( 1 9 7 5 ) , and Sunde ( 1 9 7 5 ) have s h o w n t h a t 2 5 - O H - D 3 is m o r e effective t h a n D 3 in improving eggshell calcification a n d / o r b o n e mineralization. Finally, several r e p o r t s have indicated b u t n o t shown conclusively t h a t t h e chick a n d p o u l t can utilize various p h o s p h o r u s (particularly organic) sources m o r e efficiently with age (Nelson, 1 9 6 7 ; A n d r e w s et al., 1 9 7 2 ) . T h e p u r p o s e of this s t u d y is t o re-evaluate t h e bioavailability of p h o s p h o r u s in commercial defluorinated p h o s p h a t e s and d e t e r m i n e if age (0 t o 3 weeks vs. 4 t o 7 weeks) a n d vitamin D form ( D 3 vs. 2 5 - O H - D 3 or 1 a - O H - D 3 ) have a n y significant effect o n t h e utilization of this dietary source of p h o s p h o r u s .

MATERIALS AND METHODS Experiment 1. Three h u n d r e d fifty-seven day-old A r b o r Acre male chicks were r a n d o m l y allotted t o 17 dietary t r e a t m e n t s . Five treatm e n t g r o u p s served as t h e standard curve with total p h o s p h o r u s levels of . 4 1 , . 5 1 , . 6 1 , .71 a n d . 8 1 % . T h e corn-soybean meal basal (Table 1) contained . 4 1 % t o t a l p h o s p h o r u s . T h e remaining 12 t r e a t m e n t groups were arranged in a 3 x 2 x 2 factorial design with t h e t h r e e forms of vitamin D, t w o levels of p h o s p h o r u s and t w o sources of D F P . Each diet was replicated 3 times w i t h 7 chicks per replication.

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Dietary t r e a t m e n t s were fed for 3 weeks.

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ABSTRACT Four experiments were conducted with 0 to 3- and 4 to 7-week-old broiler chicks to study the effects of age and vitamin D form on the phosphorus availability in several samples of defluorinated phosphate (DFP). All birds were housed in raised, wire floored cages. A corn-soybean meal ration was used as the basal diet throughout the experiment. Monobasic sodium phosphate was used as the standard (assumed to be 100% available) and the calcium/phosphorus ratio was maintained at 1.25 to 1. The youngest age group (0 to 3 weeks) appeared to utilize the phosphorus from DFP virtually 100%, but the older birds could utilize only 82-90% of this phosphorus. Approximate calculations indicate that 25-OH-D3 and 1 a-OH-D, are 2 to 2.5 and 4.5 times as active as pure D 3 (cholecalciferol) on a weight basis, respectively. The values obtained for phosphorus availability were relatively consistent whether the assessment was based on growth, toe ash, or biological index, although toe ash was generally the most reproducible parameter used. Phosphorus availability from most current commercial sources of DFP is quite high even though feed grade sources for poultry vary considerably in particle size.

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TABLE 1 .—Basal and preliminary diet composition

Ingredient

Basal

Preliminary diet

Corn, yellow Fat, HE Fa Soybean meal Salt DL-methionine Choline CI (25%) Vitamin mix*5 Mineral mix c Se mix Cellulose Limestone NaH,P04-HjO Test DFP Dicalcium phosphate

52.33 6.0 36.34 .386 .193 .091 .20 .05 .05 1.77 1.37

52.33 6.0 36.34 .386 .192 .091 .20 .05 .05 .69 1.37

Total P, % Total Ca, %

1.08 .41 .51

.61 .86

a Proctor and Gamble Company, St. Louis, MO, hydrolyzed edible fat.

Provides in mg/kg of diet as follows: .1 biotin, 2.2 menadione, 2.0 folic acid, 5.0 vitamin B 6 , 4.8 vitamin B 2 , 3.0 vitamin B , , 50 a-tocopheryl acetate, 22.0 Ca pantothenate, 26.5 niacin, .009 vitamin B, 2 , 100 ethoxyquin, and 1625 IU vitamin A. Provides in mg/kg of diet as follows: 60 manganese, 25 iron, 2 copper, 1.2 iodine, 18 zinc.

Calcium and phosphorus ratio was calculated to be 1.25/1 in all experiments. Feed consumption, weight gains, toe ash, and a biological index (BI) were used to assess phosphorus availability. Biological index was calculated as follows: BI = % Ash x Feed efficiency X BW

3/4

(kg)

A standard curve was established for each of these parameters from which relative availability was calculated. Toe ash was determined on the middle toe of the right leg of two birds per replicate. The procedure was as described by Fritz et al. (1968). Monobasic sodium phosphate was used as the comparative standard in this and all successive studies. All diets were analyzed for phosphorus by the procedure described by Kraml (1966) as adapted for autoanalysis. Experiment 2. Day-old Arbor Acre male chicks (of the same hatch as used in Experiment 1) were reared on the basal diet for 28 days at which time the birds were weighed and all those birds whose weight was outside one standard deviation of the mean were discarded. Vitamin D 3 supplementation was as described previously. The remaining birds were randomly allotted to 11 dietary treatments in three replications per treatment and 7 birds per pen. The standard curve was established using 5 levels of sodium phsophate (.41, .51, .61, .71, and .81% total P). Cholecalciferol (D 3 ), 25OH-D 3 , and 1 a-OH-D 3 were again used as the vitamin D 3 sources as previously described. The two sources of DFP used previously were supplemented to the basal to provide .2% or .61% total P. The birds were on the test diets from 4—7 weeks of age. All parameters measured were as previously described. Experiment 3. Day-old Arbor Acre male chicks were randomly allotted to 18 treatment groups each of which was replicated 3 times with 8 chicks per replicate. Six treatment groups with incremental levels of monobasic sodium phosphate were used as the standard curve with total phosphorus ranging from .45—.80%. Three sources of supplemental inorganic phosphate were tested at two levels of supplementation (.1 and .3% P) and two forms of vitamin D (D 3 and 1 a-OH-D 3 ). All conditions and parameters used in previous experiments were monitored as before. Experiment 4. Three-hundred and sixty Arbor Acre male broiler chicks 28 days of age were randomly allotted to 30 dietary treat-

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Each test source of phosphate supplemented the basal corn-soybean meal diet to supply .1 and .3% phosphorus (P). Vitamin D 3 supplementation as crystalline cholecalciferol dissolved in corn oil was adjusted to be equivalent to the NRC requirement level of 150 IU/kg of diet in all experiments. The two vitamin D 3 metabolites, 25-OH-D 3 and 1 a-OH-D 3 , were estimated to be 2.5 and 4.4 times the potency of cholecalciferol (D 3 ) according to the estimates of DeLuca (1974) and Haussler et al. (1968). Therefore, the appropriate reduction in the rate of supplementation of these two metabolites was made to yield approximately equal vitamin D 3 activity in all treatments. The metabolites were stored at —50 C until used. Generally, crystalline metabolite was dissolved in ether evaporated under nitrogen and redissolved in purified corn oil before each experiment. This was then added to the diets at the rate described. The diets were stored at approximately IOC during the experiment.

PHOSPHORUS AVAILABILITY: CHICK AGE AND VITAMIN D FORM

RESULTS AND DISCUSSION Experiment 1. Weight gains, percent toe ash, and BI are reported in Table 2. Large variations were obtained for all three parameters for any given sample. The values obtained with 25-OHD3 supplementation were generally lower when weight gains were measured, while percent toe ash values were much more consistent within dietary phosphorus level. Although standard curves for all parameters were quite linear

(r>.90), availability data (Table 3) for the two levels of dietary phosphorus within each sample varied significantly when weight gain was the parameter of evaluation. This effect was not observed with percent toe ash. No major differences were observed between the two phosphorus sources. Vitamin D sources, however, did affect phosphorus availability with a tendency for lower values to be derived from the addition of 25-OH-D 3 to the diet and when weight gain was the parameter. It appears from these data the 0 to 3 week-old broiler chicks utilize at least 90% of the phosphorus in defluorinated phosphate based on the standard monobasic sodium phosphate. This is similar to the value reported by Fritz (1974). Experiment 2. In Experiment 2, the finisher stage chicks (4 to 7 weeks old) appeared to utilize phosphorus from both DFP samples well (Table 4). Again, however, feeding the metabolite 25-OH-D3 reduced performance of the birds. Vitamin D 3 generally proved to be as good a source of vitamin D in this study as any of the levels fed, although feeding 1 o>OH-D3 resulted in nearly equal performance. It is possible that a feedback mechanism is activated when the circulating levels of 25-OH-D 3 reach a

TABLE 2.—Weight gain, toe ash, and biological index of broiler chicks fed vitamin D3 metabolites and defluorinated phosphate (Experiment 1) P

Weight gain

Toe ash

(%)

(%)

(g)

D3 series Sample A Sample A Sample B Sample B

.51 .71 .51 .71

163 412 204 445

25-OH-D3 series Sample A Sample A Sample B Sample B

.51 .71 .51 .71

"147 352 134 336

l-a-OH-D 3 series Sample A Sample A Sample B Sample B

.51 .71 .51 .71

193 405 180 408

Pooled SE

17.54

BI d

33.3a 42.8bc 29.4 a 10.5 C

6.78 10.29 6.68 11.48

.75 a 1.52 b .84 a 1.85 b

.995 a 4.943 c 1.328 a 4.726bc

53.7 a 17.5 b 12.4 a 35.0 b

7.02 9.56 6.63 10.85

.52 a 1.57 ab .25 a .79 b

.893 a 3.133b .790 a 3.546 b c

24.0 a 23.6 b c 17.3 a 32.9 b c

6.91 10.09 6.77 10.99

.77 a 1.16 b .81a .89 b

1.165 a 3.720 b c 1.086 a 4.151bc

.464

.369

' ' c Values expressed as means : SD. Means with different superscripts within the same column are significantly different at P<.05. Biological index.

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ments which were replicated twice with 6 chicks per replicate. The phosphate standard curve consisted of 6 treatment groups with total phosphorus ranging from .41—.71%. The remaining groups consisted of six sources of DFP fed in combination with either D 3 or 1 a-OH-D 3 at two levels of supplementation (.1 and .2% P). Vitamin D levels, environmental conditions, and all parameters were as previously described in Experiment 2. Statistical Analysis. Analysis of variance was performed on the data by the method of Sokal and Rohlf (1973). Means were separated for statistical significance by the Student-NewmanKeuls method (P<0.05).

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TABLE 3 .—Available phosphorus (%) in defluorinated (Experiment 1)

phosphate

Parameter measured Toe ash

Bie

89.8 90.3 88.7 99.4 92.1 ± 4.9

86a 118b 92ab 114b 102.5 + 15.9

P

Weight gain

A A B B

.51 .71 .51 .71

77b 100cd 90bc 106 d 93.3 ± 12.7

25-OH-D3 series DFP sample A DFP sample A DFP sample B DFP sample B Mean ± SD

.51 .71 .51 .71

76b 89bc 72 a 87bc 81 ± 8.3

92.3 84.8 88.2 94.5 90 + 4.3

1 a-OH-D3 series DFP sample A DFP sample A DFP sample B DFP sample B Mean ± SD

.51 .71 .51 .71

87bc

91.0 89.0 90.0 96.0 91.5 ± 3.1

D 3 series DFP sample DFP sample DFP sample DFP sample Mean ± SD

('")

83

99cd

92 + 8.2

Pooled SE

ND f

3.65

89ab 100ab 87a 106 b 95.5 ±9.0 5.61

' ' c ' Means with different superscripts within the same column are significantly different at P<.05. f

Biological index. Cannot be calculated since data were pooled.

TABLE 4.—Weight gain, toe ash, and biological index of broilers fed defluorinated phosphate (Experiment 2) Weight gain

Toe ash

(g)

(%)

BIC

584 632

37.7bd 30.6 b

11.36 11.76

.37 d .88

5.508 5.804

25-OH-D3 series Sample A Sample B

417 441

65.0 a 47.0 a

11.35 11.37

.50 .45

3.876 4.066

1 a-OH-Dj series Sample A Sample B

442 569

83. l a 2.6 b

11.23 12.00

.25 .45

4.086 5.138

Sample A Sample B

Pooled SE

29.51

.302

a,b Means within the same column bearing different superscripts are significantly different at P<.05. Biological index. d

Mean ± SD.

.405

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99cd c

84 a 92ab 82a 98ab 89 ± 7.4

PHOSPHORUS AVAILABILITY: CHICK AGE AND VITAMIN D FORM TABLE 5.-Available phosphorus (%) defluorinated phosphate fed to 4—7 week-old broilers (Experiment 2)

of

Toe ash

(%) D 3 series Sample A Sample B Mean

104 c 110 c 107

25-OH-D3 series Sample A Sample B Mean 1 a-OH-D3 :series Sample A Sample B Mean Pooled SE

87 92 89.5

80 a 84*b 82

87 87 87

84ab bc

85 94 89.5

101 92.5

3.69

4.51

BI d

107 a llla 109 82b 84b 83 85b 101a 93 6.20

a

' ' c Means within the same column bearing different superscripts are significantly different at P<.05.

Experiment 4. Since n o significant differences were observed b e t w e e n levels of phos-

Biological index.

TABLE 6.—Weight gain, toe ash, and biological index of chicks (0—3 weeks) fed defluorinated phosphate and various forms of vitamin D3 (Experiment 3) Weight gain

Toe ash

(%)

BI d

(%)

(g)

D 3 series Sample A Sample A Sample B Sample B Sample C Sample C

.51 .71 .51 .71 .51 .71

155 a 282bc 166 a 307c 150 a 280bc

7.32 a b 9 94ab 7.59 a b 9.28 a b 7.14 a 9.60 a b

1.164^ 2.544b<: 1.160 a b 2.862 c .983 a 2.606 b c

1 a-OH-Dj series Sample A Sample A Sample B Sample B Sample C Sample C

.51 .71 .51 .71 .51 .71

210ab 345 c 218ab 344 e 187 a 364 c

6.73 a 9 #94ab 7.32 a b 10.68 b 7.16 a 10.61 b

l^O^ 3.029 c 1.502 a b 3.262 c 1.362 a b 3.514 c

Pooled SE

20.13

.699

' ' Means within the same column bearing different superscripts are significantly different at P<.05. Biological index.

.214

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certain p o i n t which results in a shut-off of l , 2 5 - ( O H ) 2 - D 3 p r o d u c t i o n . In earlier w o r k in o u r l a b o r a t o r y (McLoughlin a n d Soares, 1 9 7 5 ) with laying hens, this effect was n o t observed. It is also quite possible t h a t t h e broiler chick does n o t utilize 2 5 - O H - D 3 as effectively as indicated earlier or t h a t significant losses in activity are occurring. Calculated from t h e d a t a o b t a i n e d from all D 3 sources, t h e average p h o s p h o r u s availability of D F P t o these finisher chicks a p p e a r e d t o be slightly lower (Table 5) t h a n t o t h e 0 t o 3 week-old chicks. Experiment 3. F u r t h e r studies of t h e effects of 1 a - O H - D 3 o n p h o s p h o r u s availability of D F P are shown in Tables 6 and 7. These results s h o w t h a t 1 CK-OH-D3 is equal t o or slightly b e t t e r in t e r m s of broiler p e r f o r m a n c e t h a n vitamin D 3 . This w o u l d indicate t h a t 1 a-OHD 3 is at least 4.5 times (on a weight basis) as effective as crystalline cholecalciferol in prom o t i n g g r o w t h and b o n e d e v e l o p m e n t . Generally t h e values o b t a i n e d for p h o s p h o r u s availability are 100% or m o r e of t h e standard monobasic sodium phosphate.

Parameter measured Weight gain

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TABLE 7.—Available phosphorus (%) of defluorinated phosphate fed to starting chicks and as affected by vitamin D 3 form (Experiment 3) Parameter m e a s u r e d Weight gain

P

T o e ash

A A B B C C

1 a-OH-D 3 series D F P sample A DFP sample A DFP sample B D F P sample B DFP sample C D F P sample C Mean ± SD

(%)

.51 .71 .51 .71 .51 .71

98 99 102 104 91 98 9 9 . 7 a ± 2.7

106 100 110 94 104 97 1 0 1 . 8 ± 5.9

102 95 101 101 98 96 9 8 . 8 ± 2.9

.51 .71 .51 .71 .51 .71

115 112 118 112 108 117 1 1 3 . 7 b ± 3.7

99 100 107 107 104 106 1 0 3 . 8 ± 3.5

104 104 110 107 106 111 107 ± 3.0

Pooled SE

4.04

5.26

3.95

a,b Means with different superscripts are significantly different at P<.05. Biological index.

TABLE 8.—Weight gain, toe ash, and biological index of broilers fed defluorinated supplemented diets from 4—7 weeks of age (Experiment 4)

D 3 series DFP sample DFP sample DFP sample DFP sample DFP sample DFP sample Mean + SD

A B C D E F

1 a-OH-Dj series DFP sample A DFP sample B DFP sample C DFP sample D DFP sample E DFP sample F Mean ± SD Pooled SE Biological index.

phosphate

Weight gain

Toe ash

(g)

<%)

654 671 706 685 613 683 669 ± 32

11.73 11.27 10.64 10.70 12.02 11.12 11.25 : .55

5.893

626 724 577 674 683 645 654 : 51

11.06 10.83 11.26 11.72 10.85 11.79 11.25

5.428 5.920 5.110 5.710 5.242 5.998 5.568 : .365

20.1

.70

BI a

.42

5.745 5.731 5.647 5.722 5.862 5.767 ± .093

.21

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D 3 series D F P sample D F P sample D F P sample DFP sample D F P sample DFP sample Mean ± SD

Bic

PHOSPHORUS AVAILABILITY: CHICK AGE AND VITAMIN D FORM

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TABLE 9 .—Available phosphorus (%) of defluorinated phosphate as affected by vitamin D3 form (Experiment 4) Parameter measured Weight gain

Toe ash

BI a

(%) A B C D E F

1 a-OH-Dj series DFP sample A DFP sample B DFP sample C DFP sample D DFP sample E DFP sample F Mean ± SD Pooled SE

84 81 82 82 79 81 82 ± 2.7

90 89 84 85 90 86 87 ± 2.7

83 83 83 83 83 83 83 + .0

80 83 78 81 81 81 81 + 1.6

86 85 87 89 86 90 87 ± 1.9

82 83 80 84 81 83 82 : 1.5

.03

.02

.02

Biological index.

p h o r u s s u p p l e m e n t a t i o n within samples, t h e data presented in Tables 8 and 9 are c o m b i n e d averages of t h e t w o levels of p h o s p h o r u s supplem e n t a t i o n fed. Again, as in E x p e r i m e n t 2, it was observed t h a t t h e D F P p h o s p h o r u s was n o t as available t o 4 t o 7 week-old birds as t o t h e starter chicks. Average p h o s p h o r u s availability ranged from 82—87%, values s o m e w h a t lower t h a n those o b t a i n e d in E x p e r i m e n t 2. This lower availability could be due t o reduced physiological need or r e q u i r e m e n t since t h e rate of skeletal d e v e l o p m e n t was slowing. Recycling of p h o s p h o r u s does n o t seem t o be a major factor since all birds were reared in cages with raised wire floors. It is possible a feedback m e c h a n i s m could be activated here as discussed earlier. It logically would produce a stronger response mechanism t h a n seen in t h e younger chicks because of t h e relatively lower need of p h o s p h o r u s at this age. No significant differences were observed w h e n 1 a - O H - D 3 was t h e vitamin D3 source in t h e diet. This confirms o u r earlier e x p e r i m e n t s indicating t h a t 1 a - O H - D 3 is a b o u t 4.5 times as active as crystalline cholecalciferol. Therefore, these studies indicate t h a t 25OH-D3 and 1 a - O H - D 3 are a p p r o x i m a t e l y 2.0 and 4.5 times (on a weight basis) as active as

crystalline D 3 w h e n fed to growing broiler chicks. F u r t h e r m o r e , finisher broilers (4 to 7 weeks old) utilize p h o s p h o r u s less efficiently (82—90%), possibly because of a r e d u c e d req u i r e m e n t for p h o s p h o r u s as c o m p a r e d t o 0 t o 3-week-old chicks which a p p a r e n t l y utilize 100% of this p h o s p h o r u s source. Little variation in p h o s p h o r u s availability a m o n g samples was observed even t h o u g h particle size varied from 15—94% of t h e particles passing t h r o u g h a 60-mesh screen. These studies indicate t h a t 25-OH-D3 used was n o t as effective in p r o m o t ing p h o s p h o r u s utilization in chicks as earlier studies (McLoughlin and Soares, 1 9 7 5 ) with laying hens. It is possible t h a t a strong feedback m e c h a n i s m is in effect here or t h a t significant losses in activity occur during t h e 3-week assay period.

REFERENCES Andrews, T. L., B. L. Damron, and R. H. Harms, 1972. Utilization of plant phosphorus by the turkey poult. Poultry Sci. 51:1248-1252. Charles, O. W., and R. A. Ernst, 1974. Effect of age, calcium level and vitamin D metabolites on eggshell quality of SCWL. Poultry Sci. 53:1908. DeLuca, H. E., 1974. Vitamin D: The vitamin and the hormone. Fed. Proc. 33:2211-2219. Dilworth, B. C , and E. J. Day, 1964. Phosphorus

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D 3 series DFP sample DFP sample DFP sample DFP sample DFP sample DFP sample Mean ± SD

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SOARES ET AL. Utilization of phosphorus from various phosphate supplements by chicks. Poultry Sci. 35:627—649. Nelson, T. S., 1967. The utilization of phytate phosphorus by poultry — A review. Poultry Sci. 46:862-871. Nelson, T. S., and A. C. Walker, 1964. The biological evaluation of phosphorus compounds — A summary. Poultry Sci. 4 3 : 9 4 - 9 8 . Sokal, R. R., and F. J. Rohlf, 1973. Page 210-211 in Introduction to biostatistics. D. Kennedy and R. B. Park, ed. W. H. Freeman and Company, San Francisco. Sunde, M. L., 1975. What about 25-hydroxycholecalciferol for poultry? Proc. Dist. Feed Res. Council 30:53-62. Twining, P. V., Jr., and P. F. Twining, 1972. Utilization of defluorinated phosphorus sources by the broiler chicken. Feedstuffs 83:32—32. Wassermann, R. H., and A. N. Taylor, 1973. Intestinal absorption of phosphate in the chick: Effect of vitamin D 3 and other parameters. J. Nutr. 103:586-599. Wilcox, R. A., C. W. Carlson, W. Kohlmeyer, and G. F. Gastler, 1954. The availability of phosphorus from difference sources for poults fed purified diets. Poultry Sci. 33:1010-1014.

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availability studies with feed grade phosphates. Poultry Sci. 43:1039-1044. Edwards, H. M., Jr., 1968. A re-examination of the nutritional role of vitamin D. Page 77—83 in Proc. Georgia Nutr. Conf. Fritz, J. C , T. Roberts, J. W. Boehne, and E. L. Hove, 1968. Factors affecting the chick's requirement for phosphorus. Poultry Sci. 47:307-320. Fritz, J. C , 1974. Phosphorous requirements of poultry. Page 96—102 in Proc. Maryland Nutr. Conf. Gillis, M. B., K. W. Keane, and R. A. Collins, 1957. Comparative metabolism of phytate and inorganic P32 by chicks and poults. J. Nutr. 6 2 : 1 3 - 2 6 . Haussler, M. R., J. F. Myrde, and A. W. Norman, 1968. The association of a metabolite of vitamin D 3 with intestinal mucosa chromatin in vivo. J. Biol. Chem. 243:4055-4064. Kraml, M., 1966. A semi-automated determination of phospholipids. Clin. Chim. Acta 13:442-448. McLoughlin, C. P., and J. H. Soares, Jr., 1975. The effects of various forms of calcium and vitamin D on eggshell quality. Poultry Sci. 54:1793 (Abstr.). Motzok, I., 1965. Factors affecting the utilization of calcium and phosphorus from soft phosphate by chicks. Poultry Sci. 44:1261 - 1 2 7 0 . Motzok, I., D. Arthur, and H. D. Branion, 1956.