Eggshell Pimpling in Young Hens as Influenced by Dietary Vitamin D31

Eggshell Pimpling in Young Hens as Influenced by Dietary Vitamin D 3 ' R. GOODSON-WILLIAMS, D. A. ROLAND, SR., 2 and J. A. MCGUIRE3 Poultry Science Department, Alabama Agricultural Experiment Station, Auburn University, Alabama 36849 (Received for publication May 19, 1986)

1987 Poultry Science 66:1980-1986 INTRODUCTION

Egg shell pimpling (occurrence of small calciferous deposits on the egg shell surface), which has plagued egg producers for many years, was noted as early as 1907 (Romanoff and Romanoff, 1949). Several researchers have investigated this problem in recent years. Ball et al. (1973) postulated that pimpling was brought about by the presence of foreign mate rial on the shell membrane prior to initiation of shell secretion. Roland et al. (1975) found that egg shell pimples contained calcium and classified the pimples into two types: first those attached only to the exterior surface of the shell and second those attached between the shell membrane and the exterior surface. They were also able to create artificial pimples by the introduction of calcium carbonate into the uterus of hens. Ball et al.(1974) dispelled the hypothesis that pimpling was caused by common infectious diseases of poultry, by demonstrating pimpling in pathogen-free flocks.

Alabama Agricultural Experiment Station Journal Number 12-861009. 2 To whom correspondence should be addressed. 3 Present address: Research Data Analysis, Alabama Agricultural Experiment Station, Auburn University, AL 36849.

Farmer and Roland (1982) showed that factors such as cage density, photoperiod, dietary phosphorus, sodium bicarbonate, and aureomycin did not significantly influence egg shell pimpling. Goto et al. (1982) reported a significant decrease in carbonic anhydrase concentration of uterus and kidney from hens laying excessively pimpled egg shells compared with concentrations of hens laying smooth shelled eggs. Roland and Bushong (1979) found that pimpling could be reduced by forced molting. However, the incidence of pimpling gradually increased during the postmolt period in a manner similar to that which would be expected during the hen's first year of lay. Arafa et al. (1982) showed that pimpling scores increased as the hen aged, which results agreed with those of Roland et al. (1975). Work by Goodson-Williams et al. (1986) using the older hen as a model demonstrated that the severity of pimpling could be reduced or increased by altering levels of dietary vitamin D 3 (D3). However, these results gave no indication as to whether pimpling could be prevented from occurring by feeding lower levels of vitamin D3 to young flocks. Although the important role of vitamin D in calcium metabolism has been well established, research concerning the influence of D 3 on egg shell pimpling is lacking. Excessive levels of

1980

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ABSTRACT An investigation of the relationship between dietary level of vitamin D, and egg shell pimpling was conducted using 450 Single Comb White Leghorn hens. Hens were approximately 4 wk into lay at the initiation of the study and were maintained at one of nine treatment levels (0, 138, 275, 550, 1,100, 2,200, 22,000, 44,000 and 88,000 ICU vitamin D3/kg) for 9 mo. Egg production, egg specific gravity, egg pimple score, egg weight, and feed consumption were determined weekly for the first 6 wk and monthly thereafter. Electrolyte concentrations of serum and tissue samples were determined at termination of the experiment. Egg pimple scores increased in a linear pattern (P= .05) when regressed on vitamin D3 level of the diet at each monthly test period following Month 1. Egg specific gravity and egg production values responded quadratically, with declines at the high levels of vitamin D 3 supplementation. Feed consumption was significantly depressed with 0 vitamin D supplementation but rates were not significantly different in remaining treatments. Egg weights, as well as sodium and potassium levels of tissue and serum, were not significantly influenced by dietary treatment. Magnesium levels increased in a linear fashion (P=£.05) in both uterine and isthmic tissues, with increased vitamin D 3 . These^data_inc^^MJhaJj[hejiegree_o£egg shell pimpling in young hens was altered by manipulating the dietary level of vitamin D3. (Key wor~ds7~egg shell^iiality71)impring7 rough shells, vitamin D3)

VITAMIN D, AND EGG SHELL PIMPLING

on 2 sides and 1 end; 4 = more than 10 pimples total; 5 = large pimple or pimple hole. At the end of the 9-mo period, blood was obtained via anterior heart puncture within 1 to 2 min of oviposition from 20 hens per treatment. Hens were killed by cervical dislocation and the uterus-isthmus region of the oviduct was removed. Serum, uterine, and isthmic calcium, as well as magnesium and sodium content were determined by Atomic Absorption Spectrophotometer (Perkin-Elmer model 460 PerkinElmer Corp., Norwalk, CT) with lanthanum oxide as the sequestering agent as described by Gimblet et al. (1967). Data for each variable were regressed on D 3 level of the diet. Both linear and quadratic regressions were tested using statistical procedures of the SAS Institute Inc. (1979) with significance level of P=£.05. A semilog plotting of the data was necessary to facilitate demonstration of relationships that exist due to the large expanse of the values on the x axis. RESULTS AND DISCUSSION

MATERIALS AND METHODS

Four hundred and fifty individually caged Single Comb White Leghorn hens approximately 4 wk into production were used. Dietary treatments were randomly assigned to five replicates of 10 birds each. Treatments consisted of nine levels of D3 (0, 138, 275, 550, 1,100, 2,200, 22,000, 44,000, and 88,000 ICU/kg). Diet composition and nutrient profile are shown in Table 1. A vitamin-mineral premix devoid of D 3 was prepared and added to each diet at .5%. The D 3 level of each diet was obtained by the replacement of a quantity of corn with the same quantity of a prepared vitamin D 3 supplement. Vitamin D 3 supplements were prepared by adding a calculated quantity of a commercial vitamin D 3 concentrate (6,810,000 ICU/lb) to corn, which provided a high (1, 834,895 ICU/kg) and a low (74,536 ICU/kg) supplement. The study was conducted for a 9-mo period. Egg production, egg specific gravity, egg weight, egg pimple score, and feed consumption were determined weekly for the first 6 wk and monthly thereafter. At each time period a 3-day sampling of eggs from each hen was collected and used for egg variable measurements. All eggs were scored for pimpling by the scoring method similar to that described by Farmer and Roland, (1982), where; 0 = no pimples; 1 = pimples on 1 side; 2 = pimples on 2 sides; 3 = pimples

The averages for egg shell pimpling scores are shown in Figure 1. There was a significant TABLE 1. Basal diet composition and calculated analysis Ingredient Corn, yellow Soybean meal (48.5% protein) Alfalfa meal (17% protein) Dicalcium phosphate (18.5% P, 22.0% Ca) Limestone Salt Vitamin-mineral premix (devoid of vitamin D 3 ) ' Total Calculated Analysis: Protein, % Metabolizable energy (kcal/kg) Calcium, % Phosphorus, % Methionyl + cysteinyl, % Lysine, %

Percent 66.72 20.89 1.00 2.04 8.50 .35 .50

100.00 16.00 2,809 3.75 .70 .55 .82

'Vitamin premix provided per kilogram of diet: vitamin A, 8,000 USPU; vitamin E, 8.0 IU; vitamin B 1 2 , .02 mg; riboflavin, 5.5 mg; pantothenic acid, 13.0 mg; niacin, 36.0 mg; choline, 500 mg; folic acid, .50 mg; thiamine, 1.0 mg; pyridoxine, 2.2 mg; biotin, .05 mg; menadione, 2.0 mg; manganese, 65.0 mg; iodine, 1.0 mg; iron, 55.0 mg; copper, 6.0 rag; zinc, 55.0 mg; and cobalt, .20 mg.

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vitamin D3 have been shown to cause soft tissue calcification in monkeys, pigs, and rats, (Hunt et al., 1972, Chineme et al., 1976). In the typical layer diet, D 3 is supplied at four to eight times the National Research Council recommendation of 500 ICU/kg. Vitamin D 3 is also a fat soluble vitamin and has the potential to be stored in the hen's body. If excessive levels of D3 are provided to a young hen for extended periods, the pimpling problem may be accentuated due to increased mobilization of calcium and possibly increased storage of D3. Therefore, it is conceivable that a relationship between D 3 in the diet and the degree of pimpling is possible, particularly in circumstances where high levels of D3 are fed for extended periods of time. The study reported herein was designed to investigate two questions: 1) could pimpling be alleviated by feeding lower levels of D 3 throughout the laying cycle, and 2) could feeding high levels of D3 during the early stages of the laying cycle accentuate pimpling as the hen ages?

1981

1982

GOODSON-WILLIAMS ET AL.

1.75

-

1.65

•

1

1.15

-

1.05

s

Ui

.95 .85 (

S*

*

Y = .906 + .I5X r =.56

S

-

s

1 ' ! ' 1

C)

' 1 '

138 550 2 2 0 0 Vitamin

44,000

O3 (ICU/kg)

FIGURE 1. Mean pimpling scores for a 9-mo period as affected by dietary level of vitamin D3(0, 138, 275, 550, 1,100, 2,200, 22,000, 44,000, or 88,000 ICU/kg). The letter r stands for correlation coefficient.

increase in egg shell pimpling with increased D 3 level. This relationship was evident as early as the 6th week of the experiment (y = .147 + .08 x; r = .40). Table 2 gives the monthly pimple scores for each treatment, including the significance levels for linear regression and correlation values at each time period. The increase in pimpling over time is apparent at all levels, however, large increases were noted earliest with the higher levels of D 3 .

TABLE 2. Influence of dietary vitamin D 3 on egg pimpling: pimple score Months 1 to 9 Vitamin D 3 level

1

2*

.24 .25 .26 .30 .28 .26 .32 .33 .28

.17 .26 .35 .37 .37 .30 .57 .37 .41

3*

4*

5*

6*

7*

8*

9*

1.22 1.58 1.82 1.98 2.16 1.82 2.48 1.78 2.18

1.68 1.86 1.78 2.16 2.00 1.92 2.40 2.12 2.24

1.58 1.90 2.04 2.58 2.50 2.24 2.78 2.56 2.48

(ICU/kg) 0 138 275 550

1,100 2,200 22,000 44,000 88,000 Significance level

r2

.46 .75 .65 .95

1.11 .88

1.15 .93 .90

.53 .89 .84

1.26 1.16 1.14 1.40 1.32 1.26

.99

.90

1.44 1.30 1.72 1.66 1.60 2.04 1.66 1.86

1.18 1.20 1.78 1.66 1.38 2.00 1.68 1.84

.003

.002

.0003

.0001

.0003

.001

.004

.0008

.42

.45

.51

.57

.52

.48

.41

.48

'Significant linear effect within column (P<.05).

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1.25

£

1.45

1

1.35

1

mple Score

1.55

Mean egg production values exhibited a quadratic relationship when regressed on D 3 level of the diet (Figure 2). Production was greatly decreased in those hens receiving a diet devoid of D 3 and slightly reduced at the highest levels. Table 3 shows the percentage egg production values at each monthly period. By Month 7, production had fallen to 23% for those hens receiving 0 D 3 but began to climb slowly in Months 8 and 9. At each time period except Month 1, the quadratic regression was significant. By the 4th week egg production ranged from 82.3 to 91.4 and demonstrated a significant linear increase, with r = .32. The quadratic relationship first appeared in Week 5, (y =73.6 + 8.47 x - 1.27 x2; r = .55) and was also present at Week 6 (y = 77.6 + 7.73 x - 1.12 x 2 , r = .48). Egg production declined in the two highest levels during the last 3 monthly periods. A significant quadratic relationship was also observed for egg specific gravity values for the overall monthly average (Figure 3). By Week 3, egg specific gravity had developed a significant linear pattern (r = .45), which was also present at Week 4 (r = .55). A significant quadratic pattern was observed in Weeks 5 and 6, with r = .75 and .78, respectively. This quadratic pattern remained significant from Months 2 through 7 (Table 4). The linear pattern reappeared again at Month 9 with egg specific gravity values of 1.0729 and 1.0800 forO and 88,000 ICU/kg of vitamin D 3 , respectively. Correlation

VITAMIN D, AND EGG SHELL PIMPLING

Y = 51.5 + 2I.0X-3.4X r =.87

UJ

I ' I ' 1 0

138 550 2200

44,000

Vitamin O3 UCU/kg) FIGURE 2. Mean hen day (HD) egg production for a 9-mo period as influenced by vitamin D 3 level of the diet (0, 138,275,550, 1,100, 2,200, 22,000, 44,000, or 88,000 ICU/kg). The latter r stands for correlation coefficient.

coefficients for each time period with significant quadratic patterns were much greater than for the linear relationships. There were no significant differences in egg weights of hens receiving various levels of vitamin D 3 . Egg weights ranged from mean of 56 g across treatments at the initiation of the experiment to a mean of 63-g by the end of the 9th month. Mean egg weight for all treatments was 60.5 g. Mean feed consumption of hens having no D 3 supplementation was significantly reduced

TABLE 3. Influence of dietary level of vitamin D 3 on egg production, Months 1 to 9 Vitamin D 3 level

1

2*

3*

4*

90 90 89 92 93 91 94 93 92

72 84 89 90 92 87 90 87 90

59 89 89 89 90 88 90 84 86

42 86 81 84 85 85 83 80 79

5*

1,100 2,200 22,000 44,000 88,000

7*

8*

Q*

28 79 77 72 81 77 78 73 70

23 80 79 76 83 78 78 71 70

36 78 74 68 79 75 77 66 68

54 70 67 65 68 66 66 59 59

- (% hen day

(ICU/kg)

0 138 275 550

6*

Significance level

0001

r2

67

,0001 85

54 81 83 79 83 81 84 75 70

.0001

.0001

0001

0001

.82

.85

89

83

'Significant quadratic effect within column (P<.05).

.002 51

.0001

.49

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(101 g/day) (Figure 4). Feed consumption ranged from 106 to 113 g/day for the remaining treatments, with no significant differences. Serum calcium values demonstrated a quadratic relationship when regressed on D 3 levels of the diet (Figure 5). Values tended to increase in a linear fashion for those hens fed up to 1,100 ICU/kg, after which values were lower but within a 1.2 mg/100 mL range. This significant quadratic pattern was also observed with serum magnesium (y = 2.93 + .30 x - .08 x2, r = .40). There were no significant patterns observed for serum sodium or potassium, which ranged from 268.1 to 315.1, and from 16.8 to 20.7 m, respectively. The calcium contents of both the uterus and the isthmus were not significantly different among the vitamin D3 treatments, and ranged from .126 to .143% and from .199 to .233% for uterus and isthmus, respectively. However, uterine and isthmic magnesium values demonstrated a linear relationship (Figure 6). As the D 3 level of the diet increased, the presence of magnesium in these reproductive tissues increased. This finding is in agreement with Aikawa (1971) who reported increased intestinal absorption and decreased renal loss of magnesium following large doses of vitamin D. This resulted in a net positive magnesium balance, which was sequestered into bony or soft tissue. The results of this study indicate that the level of D 3 in the diet has a direct effect on the degree of egg shell pimpling. Even though D 3 may not be the only factor involved, these data reveal it

Q X

0.

1983

1984

GOODSON-WILLIAMS ET AL. TABLE 4. Influence of dietary level of vitamin D 3 on egg specific gravity, Months 1 to 91

Vitamin D 3 level 0 138 275 550

Significance level

3**

4**

r **

6**

J* *

8

9*

83.5 85.1 84.1 85.8 86.4 85.6 84.2 84.8 87.2

79.4 84.6 83.9 86.7 86.2 85.2 84.1 84.3 87.3

68.4 81.8 80.5 81.6 82.9 82.5 81.3 81.3 84.9

76.6 81.5 81.7 82.0 83.1 82.2 81.4 81.0 84.5

71.0 82.1 81.9 83.3 83.7 82.6 81.5 81.8 84.7

72.4 79.0 79.5 82.0 81.6 80.8 78.9 79.5 83.1

68.6 78.2 79.5 80.4 80.4 79.7 78.7 78.6 81.3

80.2 79.1 79.5 81.2 79.7 79.7 77.4 79.0 81.6

72.9 75.8 76.6 78.4 78.5 78.3 75.5 75.5 80.0

.02 .33

r2 1

2**

.0001

.0001

.81

.87

.01 .59

.0001

.002

.0001

.84

.64

.81

.02 .33

To obtain true specific gravity multiply value by 1 0 ~ 3 and add 1.0.

'Indicates significant linear effect within column (P<.05). * "Indicates significant quadratic effect within column (P=S.05).

to be of measurable importance. Findings for egg pimple score, egg specific gravity, egg production, and egg weights are in agreement with Goodson-William et al. (1986). Those authors also found a significant linear pattern for serum calcium values that differs from the findings in this study. In this study, a significant quadratic curve was noted in serum calcium after 9 mo on the respective treatments. In the study by

84

Goodson-Williams et al. (1986), serum calcium was measured after only 10 wk of the dietary treatments. Therefore, it appears that some physiological adjustment to excessive D 3 is made in serum calcium after longer feeding periods. The mechanism for greater egg shell pimpling with higher levels of D 3 may involve increases in levels of available calcium at the site of shell secretion. Roland et al. (1975) showed that pimples attached to the shell were approximately 32% calcium, whereas the crystalline material associated with the uterus and isthmus of hens

•

83

• •

82

y*^~

81

V* /

80

* \

•

•

79 Y = 74.9 - 4.I7X - . 5 9 X * r

77

=.77

76 75 <

(r3

I

1 Con sump

/

78

">•

TJ

1 ' 1' 1

138 550 2200 Vitamin

' 1'

44,000

D3 (ICU/kg)

FIGURE 3. Mean egg specific gravity for a 9-mo period as influenced by vitamin D3 level of the diet (0, 138, 275, 550, 1,100, 2,200, 22,000, 44,000, or 88,000 ICU/kg). To obtain true specific gravity multiply by 10-3 and add 1.0. The letter r stands for correlation coefficient.

if

114 112 110 108 106 104102 100 0

138 550 2200

44,000

Vitamin D3 (ICU/kg) FIGURE 4. Mean feed consumption for a 9-mo period as affected by vitamin D-, level of the diet (0, 138, 275, 550. 1,100, 2,200, 22,000. 44,000, or 88,000 ICU/kg).

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1,100 2,200 22,000 44,000 88,000

1*

VITAMIN D, AND EGG SHELL PIMPLING .024

1985

• •,

.023

A*

wt.)

.021

(•/. dry

.022

.020

• Ay

.019

&/

t

.018

V

sf

Y = 25.6 + 3.7X-.5IX r = .2S

0

I38 55O220O

-M-l-

44,000

.017 .016-

s ,' /

.0l5,f'

/

s

/

/ • —Cr-

.014

Isthmus Y».014 +.00181X1 r ».69 Uterus Y=.015 + .0015 (X) r ' .66

Vitamin 0 3 (ICU/kg)

FIGURE 5. Influence of vitamin D, (0. 138. 275, 550, 1,100, 2,200, 22,000, 44,000, or 88,000 ICU/kg) on serum calcium at oviposition after 9 mo on treatment. The letter r stands for correlation coefficient.

laying pimpled egg shells was 14 and 24% calcium, respectively. Those findings lend credence to the hypojth£sisJh^tjre^terjranjities of calcium at the tissue level, created by excessive~TJJ7Tead tolncreased egg Ishell pimpling. Several researchers (Wasserman and Corradino, 1973; Cohen et al., 1978) have associated increasedjibsorption of calcium caused by vitamin D 3 withTT^/italrrilnTT^ ciujTT-bjndingJprotein (CaBP). However, with one exception (Corradino and Wasserman, 1968) researchers have shown no relationship between vitaminj) 3 andCaBPin the uterus of laying hens (Barr etal., 1983;Nysefa/., 1984). With increasing levels of dietary vitamin D 3 , the herTappears tojbsorb~and~transport calcium to ffiellterus fastel-lharTshe can use it. This may be clue to the" look of a corresponding increase in synthesis of CaBP in the uterus. However, because of many uncertainties concerning the role of CaBP in calcium absorption and transport (Bar and Hurwitz, 1984) it is difficult to postulate a relationship between vitamin D 3 , CaBP, and soft-tissue calcification. It is currently accepted that the pathogenesis of vitamin D3-induced soft-tissue calcification involves biochemical and morphological degeneration, which precede the deposition of the mineral (Scarpelli, 1965, Kirui etal., 1981). However, it is not known if these degenerative changes are a consequence of the vitamin per se or hypercalcemia (Kirui etal., 1981). Reports by Roland et al. (1975) and Arafa et al. (1982), indicating that pimpling increases with age, pro-

i

1 i 1

138 950 2200

i

1 i

44,000

Vitomin D3 (ICU/kg)

FIGURE 6. Effect of vitamin D, (0, 138, 275, 550, 1,100, 2,200, 22,000, 44,000, or 88,000 ICU/kg) on magnesium content (dry weight) of the uterus and isthmus at oviposition following 9 mo of dietary treatments. The letter r stands for correlation coefficient.

vide additional support for the theory that feeding excessive levels of vitamin D 3 may lead to the accumulation of vitamin D 3 in the hen's body and accelerate or accentuate pimpling problems in the aging hen. The data obtained for magnesium content of the uterus provide evidence that D 3 is somehow affecting magnesium metabolism in the hen. The positive magnesium balance resulting from high levels of vitamin D, as reported by Aikawa (1971), may also affect other tissues of the hen. Becausesome pimpling was observed on eggs from hens fed no vitamin D 3 , it is apparent that dietary D 3 is not the only factor involved._However, the significant linear effect of D 3 on pimpling indicates that D 3 is of considerable importance. REFERENCES Aikawa, J. K., 1971. The relationship of magnesium to disease in domestic animals and in humans. Charles C. Thomas, Springfield, IL. Arafa, A. S.. F. M. Hassanien, and R. H. Harms, 1982. Relationship between age of hens, egg specific gravity and time of day to pimpling of egg shells. Poultry Sci. 61:385-387. Ball, R. F., J. F. Hill, R. J. Mackin, and V. Logan, 1974. Evidence that the common rough or pimply egg shell condition is not disease related. Poultry Sci. 53:840842. Ball, R. F., R. J. Mackin, R. J. Hill, and A. J. Wyatt, 1973. The nature and probable cause of rough egg

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S

-M-J-

s /

1986

GOODSON-WILLIAMS ET AL. mental temperature and carbonic anhydrase activity of certain body tissues. Poultry Sci. 61:364-366. Helwig, J. T., and K. A. Council, 1979. SAS User's Guide. SAS Institute Inc., Cary, NC. Hunt, R. D., F. C. Garcia, and R. J. Walsh, 1972. A comparison of the toxicity of ergocalciferol and cholecalciferol in Rhesus monkeys. J. Nutr. 102:975986. Kirui, N. A., S. E. Weisbrode, and O. R. Kindig, 1981. The role of dietary calcium on the development of soft tissue mineralization due to 1,25-Dihydroxy vitamin D3 intoxication in rats. Virchows Arch. B Cell Pathol. 37:251-263. Nys, Y., and X. De Laage, 1984. Effects of suppression of eggshell calcification and of l,25(OH)2 D3 on Mg2 + , Ca 2+ and Mg2 + HC03-ATPASE, alkaline phosphatase, carbonic anhydrase and CaBP levels-1. The laying hen uterus. Comp. Biochem. Physiol. 78A:833-838. Roland, D. A., Sr., J. B. Thompson, R. A. Voitle, and R. H. Harms, 1975. Studies on the cause, prevention and artificial creation of pimpled egg shells. Poultry Sci. 54:1485-1491. Roland, D. A., Sr., and R. D. Bushong, 1979. Bodychecked, misshapen and pimpled eggs as influenced by force molting. Poultry Sci. 59:955-959. Romanoff, A. L., and A. J. Romanoff, 1949. The Avian Egg. John Wiley and Sons, Inc., New York, NY. Scarpelli, D., 1965. Experimental nephrocalcinosis. A biochemical and morphologic study. Lab Invest. 4:123-141. Wasserman, R. H., and R. Corradino, 1973. Vitamin D, calcium and protein synthesis. Vitam. Horm. 31:43— 103.

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shell laid by two lines of White Leghorns. Poultry Sci. 52:500-506. Bar, A., andH. Hurwitz, 1984. Egg shell quality, medullary bone ash, intestinal calcium and phosphorus absorption and calcium-binding protein in phosphorus-deficient hens. Poultry Sci. 63:1975-1979. Bar, A., J. Rosenberg, and S. Hurwitz, 1983. The lack of relationships between vitamin D3, metabolites and calcium binding protein in the eggshell gland of laying birds. Comp. Biochem. Physiol. 78:75-79. Chineme, C. N., L. Krook, and W. G. Pond, 1976. Bone pathology in hypervitaminosis D: An experimental study in young pigs. Cornell Vet. 66:387^112. Cohen, A., A. Bar, U. Eisner, and S. Hurwitz, 1978. Calcium absorption, calcium-binding protein and egg shell quality in laying hens fed hydroxylated vitamin D derivatives. Poultry Sci. 57:1646-1651. Corradino, R., and R. H. Wasserman, 1968. Vitamin D 3 induction of a calcium-binding protein in the uterus of laying hens. Arch. Biochem. Biophys. 125:378380. Farmer, M., and D. A. Roland, Sr., 1982. The influence of dietary ingredients, photoperiod and cage density in egg shell pimpling. Poultry Sci. 61:495-502. Gimblet, E. G., A. F. Manney, and R. Bonsnes, 1967. Determination of calcium and magnesium in serum, urine, diet and stool by atomic absorption spectrophotometry. Clin. Chcm. 13:204. Goodson-Williams, R., D. A. Roland, Sr., and J. A. McGuire, 1986. Effects of feeding graded levels of vitamin D 3 on egg shell pimpling in aged hens Poultry Sci. 65:1556-1560. Goto, K., G. C. Harris, Jr., and P. W. Waldroup, 1982. Relationship between pimpling of egg shells, environ-