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Potential Role of 1,25 Dihydroxycholecalciferol in Egg Shell Calcification1
Potential Role of 1,25 Dihydroxycholecalciferol in Egg Shell Calcification 1 J. H. SOARES, JR. and M. A. OTTINGER Department of Poultry Science, University of Maryland, College Park, Maryland 20742 E. G. BUSS The Pennsylvania State University, University Park, Pennsylvania 18802 (Received for publication February 17, 1987)
1988 Poultry Science 67:1322-1328 INTRODUCTION
Although it has been previously reported that egg shell quality can be improved by feeding 25-hydroxycholecalciferol (25-OH-D3) instead of cholecalciferol to laying hens (Charles and Ernest, 1974; Sunde, 1975; Roland and Harms, 1976; McLoughlin and Soares, 1976; Polin and Ringer 1977; Abdulrahim et al., 1979; Hamilton, 1980), the effectiveness of 25-OH-D3 is controversial. Relatively few studies have evaluated the effects of the more active 1 hydroxylated vitamin D metabolites on egg shell quality. Sunde et al. (1978) briefly mention that these metabolites have a positive effect on egg shell quality. Soares (1979) reported that laOH-D3, a synthetic analogue of 1,25 dihydroxycholecalciferol (l,25(OH)2D3), can improve egg shell quality in laying hens. However,
'Scientific Article Number A-4590, Contribution Number 7586 of the Maryland Agricultural Experiment Station, Department of Poultry Science.
Abdulrahim et al. (1979) found no effect of la-OH-D 3 or l,25(OH)2D3 on egg shell weight when fed to young hens. Bar and coworkers (Bar et al., 1984; Bar and Hurwitz, 1987) have shown that plasma l,25(OH)2D3 was greater in laying hens with good egg shell quality and was reduced with age. Recently Narbaitz et al. (1987) have shown that feeding l,25(OH)2D3 to hens results in no abnormal changes in ultrastructural development of egg shells. It is also clear that level is critical when feeding D 3 metabolites, as toxic effects can be seen with as little as 7 to 10 |xg 1,25(OH)2D3/kg diet (Sunde et al., 1978; Soares et al., 1983). The active metabolic form of vitamin D 3 is l,25(OH)2D3 and its role as an essential component for maintaining calcium homeostasis has been reviewed by several workers (DeLuca, 1980; Soares, 1984; Norman, 1987). Furthermore, plasma l,25(OH)2D3 has been shown to be influenced by ovulatory state (Castillo et al., 1978; Abe et al, 1982; Kaetzel and Soares, 1985; Nys et al., 1986). The ovulating avian with its extraordinary demand of calcium for egg shell production has been shown to have
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ABSTRACT Several experiments were conducted to determine if circulating 1,25 dihydroxycholecalciferol (l,25(OH)2D3) concentrations correlated with the laying hens' ability to calcify egg shells. Total Ca and 1,25(OH)2D3 concentrations in plasma of hens selected to produce high quality egg shells averaged greater than 34% of concentrations found in egg shells of the hens' counterparts producing poor quality egg shells. Immature female chickens had low (60 to 117 pg/mL) levels of circulating l,25(OH)2D3; concentrations were similar for immature females later classified to be either poor or good egg shell calcifiers. However, differences in 1,25(OH)2D3 concentrations became progressively greater with age in the two groups and reached statistical significance (P<.05) at 56 to 64 wk of age. Feeding l a , hydroxycholecalciferol (la-OH-D 3 ) or D 3 to laying hens for 22 wk resulted in significantly improved egg shell quality (~ 7%) for those fed la-OH-D 3 . No differences in egg weight, egg production, or egg mass were observed. Tibial calcification and breaking strengths were significantly improved by feeding la-OH-D 3 . Hens producing eggs with superior egg shell quality have higher concentrations of circulating 1,25(OH)2D3 than similar hens producing poor egg shells. Dietary supplementation of 1 hydroxylated vitamin D metabolites may be useful in improving egg shell quality. (Key words: egg shell, calcification, laying hens, 1,25 dihydroxycholecalciferol)
EGG SHELL CALCIFICATION
MATERIALS AND METHODS
Experiment 1. In this experiment, two lines of Single Comb White Leghorn (SCWL) hens (Buss etal., 1977; Grunder etal., 1980) selected for at least seven generations for ability to produce differences in egg shell quality were compared for circulating levels of l,25(OH)2D3. These two lines (thick and thin) were bred and reared at the Department of Poultry Science, Pennsylvania State University. Hens were maintained in individual suspended cages and fed a complete nutritionally balanced diet (15% protein, 3.5% calcium, .45% available phosphorus, and 2,860 kcal ME/kg). A total of 20 hens, 7 mo of age, 10 from each line, were blood sampled via the brachial vein at 6 and 20 h post-
ovipositioning (PO). The same birds were also sampled at 12 mo of age at 5 and 18 h PO. Sampling times within the ovulatory cycles were selected to correspond to periods when calcification of shell was low or nonexistent (5 to 6 h PO) and when it was near maximum (18 to 20 hPO). After collection of the blood sample, serum was obtained by centrifugation at 2,000 X g for 20 min. The serum was then stored in 4-cc glass vials and frozen at -40 C until analyzed. Radioreceptor Binding Assay for l,25(OH)2D3. The methodology for extracting vitamin D metabolites from hen serum involved dilution of 1 mL of serum with 5 mL of isotonic saline incubated with approximately 3,000 disintegrations per min 1,25(OH)2D3 (1,25-(26,273 H) (OH)2D3) (160 Ci/mmol, New England Nuclear Co., Wilmington, DE) and extracted with mefhanol-methylenechloride as described by Lambert et al. (1978). Further extraction of the lipid fraction was carried out using the method of Mallon et al. (1980) and Hollis et al. (1981). Purification of the steroids was by Sephadex LH-20 column chromatography (Lambert etal., 1983) and HPLC (Kaetzel and Soares, 1985). Preparation of chick cytosol binding protein was by the method of Lambert et al. (1978). The interassay coefficient of variation was 8%, with the lower limit of sensitivity 4 pg/tube. Specific binding averaged 40% and nonspecific binding, 5%. Serum Total Calcium. Serum samples were diluted 1:250 in .5N HC1 containing .2% lanthanum oxide. Total calcium was then assayed by atomic absorption spectrophotometry using a Perkin-Elmer spectrophotometer, Model #503 (Perkin Elmer, Norwalk, CT). Shell Quality. The percentage egg shell of total egg weight was determined by first weighing whole eggs within 12 h of ovipositioning. Shells were then removed with membranes intact, washed with tap water, and dried overnight at room temperature. If shell membranes were removed, this was accomplished by peeling the membranes off the freshly broken shell under a stream of water. The air-dried shells were weighed and expressed as a percentage of total egg weight. In addition, egg specific gravity was determined by weighing the intact eggs in air and in distilled water. Experiment 2. Twenty-five hens were selected from a commercial line of SCWL layers that were individually caged, housed in a building with forced ventilation and automatic water-
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much higher levels of circulating 1,25(OH)2D3 than most other animals (Bar and Norman, 1981; Kaetzel and Soares, 1984, 1985). Estrogens have been shown to stimulate the vitamin D activation process in vitro (Baksi and Kenny, 1977; Kenny and Baksi, 1978) and in vivo (Kaetzel and Soares, 1984; Holbrook and Soares, 1985). The ability to produce and utilize 1,25(OH)2D3 is related to the capacity of a laying hen to synthesize adequate amounts of egg shell. In one of the few published reports on the subject, Bar et al. (1984) indicated that although calcium-binding protein levels were significantly lower in uterine tissue from hens producing thin egg shells, they did not observe significantly lower circulating levels of 1,25(OH)2D3 in these hens. Bar and Hurwitz (1987) reported that reduced egg shell quality in "aged" hens is associated with a lower ability to maintain calcium homeostasis with dietary calcium restriction. The authors had, however, observed that plasma concentrations of 1,25(OH)2D3 were higher in hens laying eggs with thick shells than similar hens producing thin egg shells (unpublished observations). Therefore, four experiments were undertaken with laying hens to determine if the levels of 1,25(OH)2D3 circulating in blood are related to the quality of egg shells produced. Three experiments were conducted with laying hens to study the relationship of l,25(OH)2D3 and other parameters to the hen's ability to produce either thick or thin-shelled eggs. A fourth experiment was conducted to determine if feeding the analogue of the vitamin D hormone, la-OH-D 3 , to laying hens improved egg shell quality.
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The method used was previously described by Holbrook and Soares (1985). In order to determine the effects of D 3 steroids on skeletal calcification, the right tibia was removed after each hen was sacrificed by cervical dislocation, cleaned of all flesh, dried at 100 C, digested in nitric acid (Brown et al., 1976), and analyzed for calcium by atomic absorption spectrophotometry. The phalange of the middle toe on the right leg was used to determine percentage toe ash (Soares et al., 1978). Bones were ashed at 500 C for 12 h in a muffle furnace. In addition liver, kidney, and uteri (based on wet weight) were analyzed for calcium. The latter measurements were used to determine if abnormal calcification of soft tissue had occurred. Calcium concentrations of soft tissues were assayed by digesting a preweighed sample in nitric acid (Brown et al., 1976) and determining calcium by atomic absorption spectrophotometry as previously described. Statistical Analyses. Student-Newman-Keuls separation of means was performed with ANOVA using P<.05 as the maximal error chance for determining significance (Snedecor
TABLE 1. Laying hen basal diet composition (Experiment 4) Ingredient
(%) Yellow corn Soybean meal (49% protein) Defluorinated phosphate Limestone Corn oil 1 Sodium chloride Vitamin premix 2 Mineral premix 3 DL-Methionine Calcium Available phosphorus 1
70.618 19.070 2.270 6.500 .900 .500 .050 .050 .042 3.26 .52
Crystalline vitamin D3 or la-OH-D 3 added.
2
Vitamin premix supplied in milligrams per kilogram diet: riboflavin, 4.3; pyridoxine HC1, 4.4; calcium pantothenate, 4.4; menadione, 2.2; niacin, 11; folacin 1.1; vitamin B I 2 , .004; ethoxyquin, 125; d,la-tocopheryl acetate, 22; retinyl acetate, 4,000 I.U.; and glucose to make .050%. 3 Mineral premix supplied in milligrams per kilogram diet: MgS0 4 , 300; ZnSO„, 168; MnSO„ -H,0, 250; K I 0 3 , 10; CuSO,, 10; KCl, 2,500; Fe citrate, 200;NajSeO 3 , .2.
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ing, and provided 16 h of light/day. Feed was supplied ad libitum; it was commercially formulated to provide adequate amounts of all known nutrients (15% protein, 4.0% calcium, .44% available phosphorus, .16% sodium, and 2,829 kcal ME/kg). Blood samples were taken by venipuncture at 18, 32, and 56 wk of age and serum was extracted as previously described. Serum was analyzed for l,25(OH)2D3 as described above. Initial blood samples were taken when the pullets were showing signs of sexual maturity (18 wk) and before the onset of egg production. Egg shell quality measurements, previously described, were determined over a 4-day period at 32 and 56 wk of age. A working group of 25 individually caged hens were selected such that 13 and 12 birds were classified, respectively, as good or poor shell producers at 56 wk of age. "Good" egg shell quality was defined as eggs with specific gravity greater than 1.082 and percentage shell greater than 8.5. Eggs with "poor" egg shell quality had specific gravity less than 1.082 and percentage shell less than 8.2. All hens were sampled at approximately 20 h PO and each was confirmed to have a partially calcified egg in the uterus at that time. Experiment 3. Using a commercial strain of SCWL hens, an additional experiment was conducted to compare circulating levels of l,25(OH)2D3 at 8 and 64 wk of age with the ability (as determined at 64 wk of age) to form egg shells. In this way, circulating levels of 1,25(OH)2D3 in sexually immature pullets were compared with l,25(OH)2D3 levels in these birds late in their first production cycle. Sample collection, storage, and analyses of 1,25(OH)2D3 were as described above. Management and feeding of hens was as described in Experiment 2. Experiment 4. In this experiment, SCWL hens were fed a corn-soybean basal diet (Table 1) supplemented with either vitamin D 3 (20 (jug/ kg) or la-OH-D 3 (5 (Jig/kg). Each dietary treatment was fed to 20 individually caged hens at 40 wk of age. All birds were given ad libitum access to water and maintained in a 16 h:8 h light/dark photoperiod. The experimental period was 22 wk, covering the months from January to June. Daily egg production was summarized monthly throughout the experiment. Egg shell quality measurements were determined by periodic samplings of all eggs laid on 4 consecutive days. Egg shell breaking strength was determined by an Instron Model 1132 pressure analyzer (Instron Corp., Canton, MA 02021).
EGG SHELL CALCIFICATION
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TABLE 2.Mean (± SE) serum calcium and 1,25 dihydroxycholecalciferol (l,25(OH)2D3) concentrations in two populations of hens selected for the ability to produce thick or thin-shelled eggs Age
Variable Serum calcium, mg/dL
7 mo 12 m o
l,25(OH)2D3, pg/mL
7 mo 12 m o
6 20 5 18 6 20 5 18
7 mo 12 mo x
Thick
Thin
35.3 ± 1.8a 34.2 + 1.4a 31.6 ± .9a 32.5 ± .la .8a 33.4 + 333 309 319 244 313
+ ± ± ± ±
10.4 ± 10.1 ± 10.3 ±
32a 27 a 5a 60 a 18a ,16a .22a ,15a
29.5 : 24.3 : 29.3 : 30.7: 28.5 : 209 235 279 214 232
± ± ± ±
8.8 ± 8.6 ± 8.7 ±
1.4b 2.9a 6.6a 1.4b 29b 32a 84a 13 a
16L .12u .09b .10 u
a,bMeans with different superscripts within a row are significantly different (P<.05). 1
hPO = Hours Postoviposition.
and Cochran, 1967) in Experiment 1. Students t tests were performed in Experiments 2, 3, and 4. RESULTS AND DISCUSSION
Data in Table 2 show that there were significant differences between overall mean percentages of shell in the two selected strains of hens. Average serum total calcium was significantly higher in the hens from the thick shell line. This is in agreement with previous work reported by Grander etal. (1980; 1983) and Tsang and Grander (1985). However, after 5 mo more of egg production, serum calcium had declined in the thick line hens, so that there were no significant differences between calcium levels in the two strains of hens. Concentrations of serum l,25(OH)2D3 at 7 and 12 mo of age for the two lines of hens are given in Table 2. Similar to the observed blood calcium concentrations, l,25(OH)2D3 was, on the average, significantly higher in the serum of hens from the thick line than in the thin line over the experimental period. Although it is possible that the improved shell calcification observed was directly due to increased circulating l,25(OH)2D3 and its subsequent effects on calcium homeostasis, it has been suggested (Bar et al., 1984) that increased need for calcium by the hens in the thick line causes increased production of l,25(OH)2D3. Elevated 1,25(OH)2D3 concentrations induce increased absorption and retention of calcium and
raise serum calcium (DeLuca, 1980; Soares, 1984; Norman, 1987). Elevated l,25(OH)2D3 could also result in a greater number of receptors binding this sterol, allowing more efficient cellular translocation of calcium ion. Increased number binding affinity of 1,25(OH)2D3 receptors produced by the vitamin D hormone in rats has been shown for kidney and, to a lesser extent intestine, by Costa and Feldman (1986). It is equally possible that hens producing poor shells are not utilizing as much calcium, and this feed back reduces plasma l,25(OH)2D3. In Experiment 2, a commercial line of SCWL laying hens was observed from 18 through 56 wk of age. At preovulation (18 wk) and peak production (32 wk) there were no significant differences between circulating levels of l,25(OH)2D3 (Table 3) of hens classified as good or poor egg shell quality producers at the end of the experiment. There were also no differences in egg shell quality observed at peak production. At the termination of this experiment (56 wk), there were significant differences in the circulating level of 1,25(OH)2D3 measured and in egg shell quality. Although percentage shell with membranes averaged 9.4 and 8.0 for the good and poor shell quality groups respectively, there were no significant differences in egg weight. Therefore, differences in egg size could not be the cause for the alterations in shell quality. Average serum l,25(OH)2D3 concentrations at 56 wk of age were significantly greater in the
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% Shell
h/PO 1
SOARES ET AL.
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TABLE 3. Mean (± SEM) egg shell quality and circulating 1,25 dibydroxycholecalciferol (l,25(OH)2D2) levels (pg/mL) in laying bens segregated according to good or poor shell quality at 56 weeks of age (Experiment 2) Poor
Parameter
Weeks of age
Good
Egg weight, g % Shell Specific gravity l,25(OH) 2 D 3 l,25(OH)jD 3 l,25(OH) 2 D 3
56 56 56 56 32 18
65.6 9.4 1.084 236 289 117
66.4 8.0 1.075 137 223 114
± 1.4 ± .16* ± .002* ± 39* ± 60* ± 28a
+ 1.4 ± .17 b ± 002b + 32 b ± 43* ± 16*
a,b'Means ; in the same row with different superscripts are significantly different (P<.05).
assay variability. Furthermore, the poor shell quality hens snowed lower concentrations of 1,25(OH)2D3 at peak egg production (32 wk) and a greater decline of vitamin D hormone concentrations as shell quality deteriorated with age (52 wk).
TABLE 4. Mean (± SEM) serum 1,25 dibydroxycholecalciferol (l,25(OH)%Dz) levels at 8 and 64 weeks and egg quality in bens at 64 weeks of age producing eggs with good or poor shell quality (Experiment 3) l,25(OH) 2 D 3
Shell quality group
8 wk
Good Poor
60.6 ± 9.4* 65.8 ± 8 . 6 *
64 wk (pg/mL) 305 + 19.0* b 242 + 18.4
Egg weight
% Shell
Specific gravity
(g) 61.2 ± 1.19 60.6 + 1.43
9.4+ .10* 7.8 ± , l l b
1.088 ± .0006* 1.075 + .001 l b
a,b Means with different superscripts within a column are significantly different (P<.05).
TABLE 5. Comparison of shell quality parameters (x + SE) in laying hens fed vitamin D3 or la-hydroxycholecalciferol (la-OH-D3) (Experiment 4) Data stage
Parameter
Initial
Egg weight, g Breaking straight, kg % Shell
Final
Body weight, g Feed consumption, g/hen/day Egg weight, g % Shell (membranes) % Shell (no membranes) Shell breaking strength, kg Egg production, per hen/day
Vitamin D,
la-OH-D,
61.5 ± .90 1.39 ± .017 9.4 + .12 1,739 101.6 62.6 8.8 8.2 .73
±4 ± 3.84 ± 1.03 ± .llb + .16 b .01
Means within rows with different superscripts are significantly different (P<.05).
60.5 ± .85 1.40 + .027 9.5 ± .11 1,714 96.0 62.1 9.0 8.8
±4 ± 1.51 ± 1.08 ± .14* ± .16*
.70:
.01
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good shell quality hens than in the poor shell producers. This is consistent with the results of Experiment 1. The average values for l,25(OH)2D3 were lower than previously observed and probably reflect the effects of both physiological changes in biosynthesis as well as
EGG SHELL CALCIFICATION
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TABLE 6. Tissue calcification (x ± SE) in laying hens fed vitamin D3 or la-hydroxycholecalciferol (la-OH-D}) (Experiment 4) Parameter
Vitamin D 3
la-OH-D 3
Toe ash, % Tibia calcium, % Tibia phosphorus, % Tibia breaking strength, kg Kidney calcium, Mg/g Liver calcium, Mg/g Uterus calcium, Mg/g
17.4 ± .25 a 15.2 ± .40 b 7.5+ .18b 25.7 ± 1.03 b 142 + 9.3 a 76 ± 4.5 a 273 + 16.9 a
18.0 ± 16.8+ 8.2 ± 33.5 ± 150 ± 82 ± 271 ±
.26' .63 1 .27' 2.21 ! 7.5 a 3.6 a 11.4 a
'Means within a row with different superscripts are significantly different (P<.05).
centrations in kidney, liver, tibia, or uterus in the two experimental groups. This indicates that abnormal calcification of soft tissue was not occurring in these birds and therefore the diet did not contain excessive vitamin D 3 metabolite. Data reported here show an association of the active vitamin D metabolite l,25(OH)2D3 with improved egg shell and bone calcification in the laying hen. Based on these data, supplementation of la-OH-D 3 or l,25(OH)2D3 in practical hen diets may offer the possibility of improving shell quality in hens with poor ability to do so. ACKNOWLEDGMENTS
The authors wish to thank Timothy Allen and Patricia Madara for their assistance in the laboratory analysis of the data presented in this manuscript and M. Uskokovic, Hoffmann-LaRoche, Inc., and the Upjohn Co. for supply of vitamin D metabolites. The assistance of Carole Dingess, Annette Gonzales, and Terri Ferrara in the preparation of this manuscript is greatly appreciated. REFERENCES Abdulrahim, S. M., M. B. Patel, and J. McGinnis, 1979. Effects of vitamin D 3 and D 3 metabolites on production parameters and hatchability of eggs. Poultry Sci. 58:858-863. Abe, E., H. Horikawa, T. Masumara, M. Sugahara, M. Kubota, and T. Suda, 1982. Disorders of cholecalciferol metabolism in old egg-laying hens. J. Nutr. 112:436-446. Baksi, S. N., and A. D. Kenny, 1977. Vitamin D 3 metabolism in immature quail: Effects of ovarian hormones. Endocrinology 101:1216-1220. Bar, A., and S. Hurwitz, 1987. Vitamin D metabolism and Calbindin (calcium-binding protein) in aged laying hens. J. Nutr. 117:1775-1779. Bar, A., and A. W. Norman, 1981. Studies on the mode
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Results from Experiment 3 (Table 4) show that circulating levels of 1,25(OH)2D3 in female chickens were not different at a preovulatory stage of development (8 wk of age), but they were clearly different when there were measurable differences in egg shell quality (64 wk of age). Data from female chickens at 8 wk of age are in agreement with the observations made at 18 wk of age in Experiment 2. As these data were obtained from pullets before sexual development, the values are similarly low but less variable than those in pullets 18 wk of age in Experiment 2. Changes in plasma 1,25(OH)2D3 observed in hens laying eggs with different egg shell quality were not detectable at immaturity or when hens had good egg shell quality. This indicates that even when a hen is genetically limited in its ability to adequately calcify egg shells, differences in plasma l,25(OH)2D3 may not be expressed until age and physiologically induced modifiers of vitamin D metabolism and calcium homeostasis are activated. Apparently plasma l,25(OH)2D3 in immature pullets is not a good indicator for predicting future egg shell quality. There were no symptoms of vitamin D insufficiency after 22 wk of feeding laying hens (Experiment 4) a corn-soybean diet containing only la-OH-D 3 as the vitamin D source. Body weights, egg weight, egg production, and egg shell breaking strength were similar to those of controls fed vitamin D 3 (Table 5). Percentage egg shell (less membranes), however, was significantly greater for hens fed the la-OH-D 3 than for the hens fed vitamin D 3 . Similarly, percentage toe ash, tibia calcium phosphorus, and breaking strength (Table 6) were all significantly improved by feeding la-OH-D 3 . No differences were observed between calcium con-
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SOARES ET AL. 1983. Assay for multiple vitamin D metabolites. Pages 99—124 in: Assay of Calcium-regulating Hormones. D. D. Bikle, ed. Springer-Verlag, New York, NY. Lambert, P. W., D. O. Taft, S. F. Hodgson, E. A. Lindmark, B. J. Witrak, and B. A. Roos, 1978. An improved method for the measurement of 1,25(OH)2D3 in human plasma. Endocr. Res. Commun. 5:293-310. 1976. Mallon, J. P., J. G. Hamilton, C. Nauss-Karol, C.J. Ashley, D. S. Matuszewski, C. A. Tratnyek, G. F. Bryce, and O. N. Miller, 1980. An improved competitive protein binding assay for 1,25-dihydroxyvitamin D. Arch. Biochem. Biophys. 201(l):277-285. McLoughlin, C. P., and J. H. Soares, Jr., 1976. A study of the effect of 25-OH-D3 and calcium source on egg shell quality. Poultry Sci. 55:1400-1410. Narbaitz, R. C , P. W. Tsang, A. A. Grander, and J. H. Soares, Jr., 1987. Scanning electron microscopy of thin and soft shells induced by feeding calcium and vitamin D deficient diets to laying hens. Poultry Sci. 66:341-347. Norman, A. W., 1987. Studies on the vitamin D endocrine system in the avian. J. Nutr. 117:797-807. Nys, Y., T. M. N'Guyen, J. Williams, and R. J. Etches, 1986. Blood levels of ionized calcium, inorganic phosphorus, 1,25-dihydroxycholecalciferol and gonadal hormones in hens laying hard-shelled and shell-less eggs. J. Endocrinol. 111:151-157. Polin, D., andR. K. Ringer, 1977. 25-hydroxy-D3, vitamin D 3 and graded levels of phosphorus: Effect on egg production and shell quality. Feedstuffs 49(44):40-41. Roland, D. A., Sr., and R. H. Harms, 1976. The lack of response of 25-OH-D3 on egg shell quality or other criteria in laying hens. Poultry Sci. 55:1983-1985. Snedecor, G. W., and W. G. Cochran, 1967. Analysis of Variance. Pages 273-275 in: Statistical Methods. 6th ed. The Iowa State Univ. Press, Ames, IA. Soares, J. H., Jr., 1979. Investigations on feeding vitamin D 3 steroids to laying hens to improve egg shell quality. Poultry Sci. 58:1109. (Abstr.) Soares, J. H., Jr., 1984. Calcium metabolism and its control: A review. Poultry Sci. 63:2075-2083. Soares, J. H., Jr., D. M. Kaetzel, J. T. Allen, and M. R. Swerdel, 1983. Toxicity of a vitamin D steroid to laying hens. Poultry Sci. 62:24—29. Soares, J. H., Jr., M. R. Swerdel, andE. H. Bossard, 1987. Phosphorus availability. 1. The effect of chick age and vitamin D metabolites on the availability of phosphorus in defluorinated phosphate. Poultry Sci. 57:1305-1312. Sunde, M. L., 1975. What about 25-hydroxycholecalciferol for poultry? Proc. Dist. Feed Res. Counc. 3:53-61. Sunde, M. L., C. M. Turk, and H. F. DeLuca, 1978. The essentiality of vitamin D metabolites for embryonic chick development. Science 200:1067-1069. Tsang, C.P.W., and A. A. Grander, 1985. Prepubertal plasma estradiol and total calcium levels in two strains of White Leghorns in relation to egg shell quality. Arch. Gefluegelkd. 49:12-15.
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of action of calciferol. XXXIV. Relationship of the distribution of 25-hydroxyvitamin D3 metabolites to gonadal activity and egg shell formation in the quail. Endocrinology 109:950-955. Bar, A., J. Rosenberg, and S. Hurwitz, 1984. The lack of relationships between vitamin D 3 metabolites and calcium binding protein in the eggshell gland of laying birds. Comp. Biochem. Physiol. 78B(l):75-79. Brown, E. D., W. Chan, and J. C. Smith, Jr., 1976. Vitamin A metabolism during the repletion of the zinc deficient rats. J. Nutr. 106:563-568. Buss, E. G., R. M. Leach, Jr., and J. T. Stout, 1977. Egg shell quality for chickens in selected lines F,'s and F 2 's. Poultry Science. 56:1699-1700. Castillo, L., Y. Tanaka, H. F. DeLuca, and M. L. Sunde, 1978. Synergistic effect of progesterone, testosterone and estradiol in the stimulation of chick renal 25-hydroxy-vitamin D3-l-hydroxylase. Endocrinology 103:2035-2039. Charles, O. W., and R. A. Ernst, 1974. Effect of age, calcium level and vitamin D metabolites on egg shell quality of SCWL. Poultry Sci. 53:12. (Abstr.) Costa, E. M., and D. Feldman, 1986. Homologous up-regulation of the 1,25(OH) vitamin D 3 receptor in rats. Biochem. Biophys. Res. Commun. 137:742-747. DeLuca, H. F., 1980. Some new concepts emanating from a study of the metabolism and function of vitamin D. Nutr. Dev. 38:169-182. Grander, A. A., R. B. Guyer, E. G. Buss, and C. O. Claggett, 1980. Effect of estradiol on calcium and calcium binding in serum of thick vs. thin-shell lines of chickens. Poultry Sci. 59:2776-2781. Grander, A. A., K. G. Hollands, andC.P.W. Tsang, 1983. Plasma estrogen, calcium and shell quality in two strains of White Leghorns. Poultry Sci. 62:1294-1296. Hamilton, R.M.G., 1980. The effects of dietary phosphorus, vitamin D 3 and 25-OH-D3 levels on feed intake, production performance and egg shell quality in two strains of force-molted White Leghorns. Poultry Sci. 59:598-604. Holbrook, J. T., and J. H. Soares, Jr., 1985. The effects of estradiol and 1,25(OH)2D3 on skeletal metabolism in female Japanese quail. Nutr. Res. 5:729-736. Hollis, B. W., B. A. Roos, and P. W. Lambert, 1981. Vitamin D in plasma: Quantification by a nonequilibrium ligand binding assay. Steroids 37(6):609-619. Kaetzel, D. M., Jr., and J. H. Soares, Jr., 1984. The effects of dietary cholecalciferol steroids and gonadal hormone implantation on plasma 1,25-dihydroxycholecalciferol and bone calcification in preovulatory and senescent female Japanese quail. Nutr. Res. 4:621-632. Kaetzel, D. M., Jr., and J. H. Soares, Jr., 1985. Effect of dietary calcium stress on plasma vitamin D3 metabolites in the egg-laying Japanese quail. Poultry Sci. 64:1121-1127. Kenny, A. D., and S. N. Baksi, 1978. Stimulation of renal vitamin D endocrine system of physiological doses of estradiol in Japanese quail. Fed Proc. 37:926. (Abstr.) Lambert, P. W., I. Y. Fu, D. M. Kaetzel, andB. W. Hollis,
1988 Poultry Science 67:1322-1328 INTRODUCTION
Although it has been previously reported that egg shell quality can be improved by feeding 25-hydroxycholecalciferol (25-OH-D3) instead of cholecalciferol to laying hens (Charles and Ernest, 1974; Sunde, 1975; Roland and Harms, 1976; McLoughlin and Soares, 1976; Polin and Ringer 1977; Abdulrahim et al., 1979; Hamilton, 1980), the effectiveness of 25-OH-D3 is controversial. Relatively few studies have evaluated the effects of the more active 1 hydroxylated vitamin D metabolites on egg shell quality. Sunde et al. (1978) briefly mention that these metabolites have a positive effect on egg shell quality. Soares (1979) reported that laOH-D3, a synthetic analogue of 1,25 dihydroxycholecalciferol (l,25(OH)2D3), can improve egg shell quality in laying hens. However,
'Scientific Article Number A-4590, Contribution Number 7586 of the Maryland Agricultural Experiment Station, Department of Poultry Science.
Abdulrahim et al. (1979) found no effect of la-OH-D 3 or l,25(OH)2D3 on egg shell weight when fed to young hens. Bar and coworkers (Bar et al., 1984; Bar and Hurwitz, 1987) have shown that plasma l,25(OH)2D3 was greater in laying hens with good egg shell quality and was reduced with age. Recently Narbaitz et al. (1987) have shown that feeding l,25(OH)2D3 to hens results in no abnormal changes in ultrastructural development of egg shells. It is also clear that level is critical when feeding D 3 metabolites, as toxic effects can be seen with as little as 7 to 10 |xg 1,25(OH)2D3/kg diet (Sunde et al., 1978; Soares et al., 1983). The active metabolic form of vitamin D 3 is l,25(OH)2D3 and its role as an essential component for maintaining calcium homeostasis has been reviewed by several workers (DeLuca, 1980; Soares, 1984; Norman, 1987). Furthermore, plasma l,25(OH)2D3 has been shown to be influenced by ovulatory state (Castillo et al., 1978; Abe et al, 1982; Kaetzel and Soares, 1985; Nys et al., 1986). The ovulating avian with its extraordinary demand of calcium for egg shell production has been shown to have
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ABSTRACT Several experiments were conducted to determine if circulating 1,25 dihydroxycholecalciferol (l,25(OH)2D3) concentrations correlated with the laying hens' ability to calcify egg shells. Total Ca and 1,25(OH)2D3 concentrations in plasma of hens selected to produce high quality egg shells averaged greater than 34% of concentrations found in egg shells of the hens' counterparts producing poor quality egg shells. Immature female chickens had low (60 to 117 pg/mL) levels of circulating l,25(OH)2D3; concentrations were similar for immature females later classified to be either poor or good egg shell calcifiers. However, differences in 1,25(OH)2D3 concentrations became progressively greater with age in the two groups and reached statistical significance (P<.05) at 56 to 64 wk of age. Feeding l a , hydroxycholecalciferol (la-OH-D 3 ) or D 3 to laying hens for 22 wk resulted in significantly improved egg shell quality (~ 7%) for those fed la-OH-D 3 . No differences in egg weight, egg production, or egg mass were observed. Tibial calcification and breaking strengths were significantly improved by feeding la-OH-D 3 . Hens producing eggs with superior egg shell quality have higher concentrations of circulating 1,25(OH)2D3 than similar hens producing poor egg shells. Dietary supplementation of 1 hydroxylated vitamin D metabolites may be useful in improving egg shell quality. (Key words: egg shell, calcification, laying hens, 1,25 dihydroxycholecalciferol)
EGG SHELL CALCIFICATION
MATERIALS AND METHODS
Experiment 1. In this experiment, two lines of Single Comb White Leghorn (SCWL) hens (Buss etal., 1977; Grunder etal., 1980) selected for at least seven generations for ability to produce differences in egg shell quality were compared for circulating levels of l,25(OH)2D3. These two lines (thick and thin) were bred and reared at the Department of Poultry Science, Pennsylvania State University. Hens were maintained in individual suspended cages and fed a complete nutritionally balanced diet (15% protein, 3.5% calcium, .45% available phosphorus, and 2,860 kcal ME/kg). A total of 20 hens, 7 mo of age, 10 from each line, were blood sampled via the brachial vein at 6 and 20 h post-
ovipositioning (PO). The same birds were also sampled at 12 mo of age at 5 and 18 h PO. Sampling times within the ovulatory cycles were selected to correspond to periods when calcification of shell was low or nonexistent (5 to 6 h PO) and when it was near maximum (18 to 20 hPO). After collection of the blood sample, serum was obtained by centrifugation at 2,000 X g for 20 min. The serum was then stored in 4-cc glass vials and frozen at -40 C until analyzed. Radioreceptor Binding Assay for l,25(OH)2D3. The methodology for extracting vitamin D metabolites from hen serum involved dilution of 1 mL of serum with 5 mL of isotonic saline incubated with approximately 3,000 disintegrations per min 1,25(OH)2D3 (1,25-(26,273 H) (OH)2D3) (160 Ci/mmol, New England Nuclear Co., Wilmington, DE) and extracted with mefhanol-methylenechloride as described by Lambert et al. (1978). Further extraction of the lipid fraction was carried out using the method of Mallon et al. (1980) and Hollis et al. (1981). Purification of the steroids was by Sephadex LH-20 column chromatography (Lambert etal., 1983) and HPLC (Kaetzel and Soares, 1985). Preparation of chick cytosol binding protein was by the method of Lambert et al. (1978). The interassay coefficient of variation was 8%, with the lower limit of sensitivity 4 pg/tube. Specific binding averaged 40% and nonspecific binding, 5%. Serum Total Calcium. Serum samples were diluted 1:250 in .5N HC1 containing .2% lanthanum oxide. Total calcium was then assayed by atomic absorption spectrophotometry using a Perkin-Elmer spectrophotometer, Model #503 (Perkin Elmer, Norwalk, CT). Shell Quality. The percentage egg shell of total egg weight was determined by first weighing whole eggs within 12 h of ovipositioning. Shells were then removed with membranes intact, washed with tap water, and dried overnight at room temperature. If shell membranes were removed, this was accomplished by peeling the membranes off the freshly broken shell under a stream of water. The air-dried shells were weighed and expressed as a percentage of total egg weight. In addition, egg specific gravity was determined by weighing the intact eggs in air and in distilled water. Experiment 2. Twenty-five hens were selected from a commercial line of SCWL layers that were individually caged, housed in a building with forced ventilation and automatic water-
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much higher levels of circulating 1,25(OH)2D3 than most other animals (Bar and Norman, 1981; Kaetzel and Soares, 1984, 1985). Estrogens have been shown to stimulate the vitamin D activation process in vitro (Baksi and Kenny, 1977; Kenny and Baksi, 1978) and in vivo (Kaetzel and Soares, 1984; Holbrook and Soares, 1985). The ability to produce and utilize 1,25(OH)2D3 is related to the capacity of a laying hen to synthesize adequate amounts of egg shell. In one of the few published reports on the subject, Bar et al. (1984) indicated that although calcium-binding protein levels were significantly lower in uterine tissue from hens producing thin egg shells, they did not observe significantly lower circulating levels of 1,25(OH)2D3 in these hens. Bar and Hurwitz (1987) reported that reduced egg shell quality in "aged" hens is associated with a lower ability to maintain calcium homeostasis with dietary calcium restriction. The authors had, however, observed that plasma concentrations of 1,25(OH)2D3 were higher in hens laying eggs with thick shells than similar hens producing thin egg shells (unpublished observations). Therefore, four experiments were undertaken with laying hens to determine if the levels of 1,25(OH)2D3 circulating in blood are related to the quality of egg shells produced. Three experiments were conducted with laying hens to study the relationship of l,25(OH)2D3 and other parameters to the hen's ability to produce either thick or thin-shelled eggs. A fourth experiment was conducted to determine if feeding the analogue of the vitamin D hormone, la-OH-D 3 , to laying hens improved egg shell quality.
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SOARES ET AL.
The method used was previously described by Holbrook and Soares (1985). In order to determine the effects of D 3 steroids on skeletal calcification, the right tibia was removed after each hen was sacrificed by cervical dislocation, cleaned of all flesh, dried at 100 C, digested in nitric acid (Brown et al., 1976), and analyzed for calcium by atomic absorption spectrophotometry. The phalange of the middle toe on the right leg was used to determine percentage toe ash (Soares et al., 1978). Bones were ashed at 500 C for 12 h in a muffle furnace. In addition liver, kidney, and uteri (based on wet weight) were analyzed for calcium. The latter measurements were used to determine if abnormal calcification of soft tissue had occurred. Calcium concentrations of soft tissues were assayed by digesting a preweighed sample in nitric acid (Brown et al., 1976) and determining calcium by atomic absorption spectrophotometry as previously described. Statistical Analyses. Student-Newman-Keuls separation of means was performed with ANOVA using P<.05 as the maximal error chance for determining significance (Snedecor
TABLE 1. Laying hen basal diet composition (Experiment 4) Ingredient
(%) Yellow corn Soybean meal (49% protein) Defluorinated phosphate Limestone Corn oil 1 Sodium chloride Vitamin premix 2 Mineral premix 3 DL-Methionine Calcium Available phosphorus 1
70.618 19.070 2.270 6.500 .900 .500 .050 .050 .042 3.26 .52
Crystalline vitamin D3 or la-OH-D 3 added.
2
Vitamin premix supplied in milligrams per kilogram diet: riboflavin, 4.3; pyridoxine HC1, 4.4; calcium pantothenate, 4.4; menadione, 2.2; niacin, 11; folacin 1.1; vitamin B I 2 , .004; ethoxyquin, 125; d,la-tocopheryl acetate, 22; retinyl acetate, 4,000 I.U.; and glucose to make .050%. 3 Mineral premix supplied in milligrams per kilogram diet: MgS0 4 , 300; ZnSO„, 168; MnSO„ -H,0, 250; K I 0 3 , 10; CuSO,, 10; KCl, 2,500; Fe citrate, 200;NajSeO 3 , .2.
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ing, and provided 16 h of light/day. Feed was supplied ad libitum; it was commercially formulated to provide adequate amounts of all known nutrients (15% protein, 4.0% calcium, .44% available phosphorus, .16% sodium, and 2,829 kcal ME/kg). Blood samples were taken by venipuncture at 18, 32, and 56 wk of age and serum was extracted as previously described. Serum was analyzed for l,25(OH)2D3 as described above. Initial blood samples were taken when the pullets were showing signs of sexual maturity (18 wk) and before the onset of egg production. Egg shell quality measurements, previously described, were determined over a 4-day period at 32 and 56 wk of age. A working group of 25 individually caged hens were selected such that 13 and 12 birds were classified, respectively, as good or poor shell producers at 56 wk of age. "Good" egg shell quality was defined as eggs with specific gravity greater than 1.082 and percentage shell greater than 8.5. Eggs with "poor" egg shell quality had specific gravity less than 1.082 and percentage shell less than 8.2. All hens were sampled at approximately 20 h PO and each was confirmed to have a partially calcified egg in the uterus at that time. Experiment 3. Using a commercial strain of SCWL hens, an additional experiment was conducted to compare circulating levels of l,25(OH)2D3 at 8 and 64 wk of age with the ability (as determined at 64 wk of age) to form egg shells. In this way, circulating levels of 1,25(OH)2D3 in sexually immature pullets were compared with l,25(OH)2D3 levels in these birds late in their first production cycle. Sample collection, storage, and analyses of 1,25(OH)2D3 were as described above. Management and feeding of hens was as described in Experiment 2. Experiment 4. In this experiment, SCWL hens were fed a corn-soybean basal diet (Table 1) supplemented with either vitamin D 3 (20 (jug/ kg) or la-OH-D 3 (5 (Jig/kg). Each dietary treatment was fed to 20 individually caged hens at 40 wk of age. All birds were given ad libitum access to water and maintained in a 16 h:8 h light/dark photoperiod. The experimental period was 22 wk, covering the months from January to June. Daily egg production was summarized monthly throughout the experiment. Egg shell quality measurements were determined by periodic samplings of all eggs laid on 4 consecutive days. Egg shell breaking strength was determined by an Instron Model 1132 pressure analyzer (Instron Corp., Canton, MA 02021).
EGG SHELL CALCIFICATION
1325
TABLE 2.Mean (± SE) serum calcium and 1,25 dihydroxycholecalciferol (l,25(OH)2D3) concentrations in two populations of hens selected for the ability to produce thick or thin-shelled eggs Age
Variable Serum calcium, mg/dL
7 mo 12 m o
l,25(OH)2D3, pg/mL
7 mo 12 m o
6 20 5 18 6 20 5 18
7 mo 12 mo x
Thick
Thin
35.3 ± 1.8a 34.2 + 1.4a 31.6 ± .9a 32.5 ± .la .8a 33.4 + 333 309 319 244 313
+ ± ± ± ±
10.4 ± 10.1 ± 10.3 ±
32a 27 a 5a 60 a 18a ,16a .22a ,15a
29.5 : 24.3 : 29.3 : 30.7: 28.5 : 209 235 279 214 232
± ± ± ±
8.8 ± 8.6 ± 8.7 ±
1.4b 2.9a 6.6a 1.4b 29b 32a 84a 13 a
16L .12u .09b .10 u
a,bMeans with different superscripts within a row are significantly different (P<.05). 1
hPO = Hours Postoviposition.
and Cochran, 1967) in Experiment 1. Students t tests were performed in Experiments 2, 3, and 4. RESULTS AND DISCUSSION
Data in Table 2 show that there were significant differences between overall mean percentages of shell in the two selected strains of hens. Average serum total calcium was significantly higher in the hens from the thick shell line. This is in agreement with previous work reported by Grander etal. (1980; 1983) and Tsang and Grander (1985). However, after 5 mo more of egg production, serum calcium had declined in the thick line hens, so that there were no significant differences between calcium levels in the two strains of hens. Concentrations of serum l,25(OH)2D3 at 7 and 12 mo of age for the two lines of hens are given in Table 2. Similar to the observed blood calcium concentrations, l,25(OH)2D3 was, on the average, significantly higher in the serum of hens from the thick line than in the thin line over the experimental period. Although it is possible that the improved shell calcification observed was directly due to increased circulating l,25(OH)2D3 and its subsequent effects on calcium homeostasis, it has been suggested (Bar et al., 1984) that increased need for calcium by the hens in the thick line causes increased production of l,25(OH)2D3. Elevated 1,25(OH)2D3 concentrations induce increased absorption and retention of calcium and
raise serum calcium (DeLuca, 1980; Soares, 1984; Norman, 1987). Elevated l,25(OH)2D3 could also result in a greater number of receptors binding this sterol, allowing more efficient cellular translocation of calcium ion. Increased number binding affinity of 1,25(OH)2D3 receptors produced by the vitamin D hormone in rats has been shown for kidney and, to a lesser extent intestine, by Costa and Feldman (1986). It is equally possible that hens producing poor shells are not utilizing as much calcium, and this feed back reduces plasma l,25(OH)2D3. In Experiment 2, a commercial line of SCWL laying hens was observed from 18 through 56 wk of age. At preovulation (18 wk) and peak production (32 wk) there were no significant differences between circulating levels of l,25(OH)2D3 (Table 3) of hens classified as good or poor egg shell quality producers at the end of the experiment. There were also no differences in egg shell quality observed at peak production. At the termination of this experiment (56 wk), there were significant differences in the circulating level of 1,25(OH)2D3 measured and in egg shell quality. Although percentage shell with membranes averaged 9.4 and 8.0 for the good and poor shell quality groups respectively, there were no significant differences in egg weight. Therefore, differences in egg size could not be the cause for the alterations in shell quality. Average serum l,25(OH)2D3 concentrations at 56 wk of age were significantly greater in the
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% Shell
h/PO 1
SOARES ET AL.
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TABLE 3. Mean (± SEM) egg shell quality and circulating 1,25 dibydroxycholecalciferol (l,25(OH)2D2) levels (pg/mL) in laying bens segregated according to good or poor shell quality at 56 weeks of age (Experiment 2) Poor
Parameter
Weeks of age
Good
Egg weight, g % Shell Specific gravity l,25(OH) 2 D 3 l,25(OH)jD 3 l,25(OH) 2 D 3
56 56 56 56 32 18
65.6 9.4 1.084 236 289 117
66.4 8.0 1.075 137 223 114
± 1.4 ± .16* ± .002* ± 39* ± 60* ± 28a
+ 1.4 ± .17 b ± 002b + 32 b ± 43* ± 16*
a,b'Means ; in the same row with different superscripts are significantly different (P<.05).
assay variability. Furthermore, the poor shell quality hens snowed lower concentrations of 1,25(OH)2D3 at peak egg production (32 wk) and a greater decline of vitamin D hormone concentrations as shell quality deteriorated with age (52 wk).
TABLE 4. Mean (± SEM) serum 1,25 dibydroxycholecalciferol (l,25(OH)%Dz) levels at 8 and 64 weeks and egg quality in bens at 64 weeks of age producing eggs with good or poor shell quality (Experiment 3) l,25(OH) 2 D 3
Shell quality group
8 wk
Good Poor
60.6 ± 9.4* 65.8 ± 8 . 6 *
64 wk (pg/mL) 305 + 19.0* b 242 + 18.4
Egg weight
% Shell
Specific gravity
(g) 61.2 ± 1.19 60.6 + 1.43
9.4+ .10* 7.8 ± , l l b
1.088 ± .0006* 1.075 + .001 l b
a,b Means with different superscripts within a column are significantly different (P<.05).
TABLE 5. Comparison of shell quality parameters (x + SE) in laying hens fed vitamin D3 or la-hydroxycholecalciferol (la-OH-D3) (Experiment 4) Data stage
Parameter
Initial
Egg weight, g Breaking straight, kg % Shell
Final
Body weight, g Feed consumption, g/hen/day Egg weight, g % Shell (membranes) % Shell (no membranes) Shell breaking strength, kg Egg production, per hen/day
Vitamin D,
la-OH-D,
61.5 ± .90 1.39 ± .017 9.4 + .12 1,739 101.6 62.6 8.8 8.2 .73
±4 ± 3.84 ± 1.03 ± .llb + .16 b .01
Means within rows with different superscripts are significantly different (P<.05).
60.5 ± .85 1.40 + .027 9.5 ± .11 1,714 96.0 62.1 9.0 8.8
±4 ± 1.51 ± 1.08 ± .14* ± .16*
.70:
.01
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good shell quality hens than in the poor shell producers. This is consistent with the results of Experiment 1. The average values for l,25(OH)2D3 were lower than previously observed and probably reflect the effects of both physiological changes in biosynthesis as well as
EGG SHELL CALCIFICATION
1327
TABLE 6. Tissue calcification (x ± SE) in laying hens fed vitamin D3 or la-hydroxycholecalciferol (la-OH-D}) (Experiment 4) Parameter
Vitamin D 3
la-OH-D 3
Toe ash, % Tibia calcium, % Tibia phosphorus, % Tibia breaking strength, kg Kidney calcium, Mg/g Liver calcium, Mg/g Uterus calcium, Mg/g
17.4 ± .25 a 15.2 ± .40 b 7.5+ .18b 25.7 ± 1.03 b 142 + 9.3 a 76 ± 4.5 a 273 + 16.9 a
18.0 ± 16.8+ 8.2 ± 33.5 ± 150 ± 82 ± 271 ±
.26' .63 1 .27' 2.21 ! 7.5 a 3.6 a 11.4 a
'Means within a row with different superscripts are significantly different (P<.05).
centrations in kidney, liver, tibia, or uterus in the two experimental groups. This indicates that abnormal calcification of soft tissue was not occurring in these birds and therefore the diet did not contain excessive vitamin D 3 metabolite. Data reported here show an association of the active vitamin D metabolite l,25(OH)2D3 with improved egg shell and bone calcification in the laying hen. Based on these data, supplementation of la-OH-D 3 or l,25(OH)2D3 in practical hen diets may offer the possibility of improving shell quality in hens with poor ability to do so. ACKNOWLEDGMENTS
The authors wish to thank Timothy Allen and Patricia Madara for their assistance in the laboratory analysis of the data presented in this manuscript and M. Uskokovic, Hoffmann-LaRoche, Inc., and the Upjohn Co. for supply of vitamin D metabolites. The assistance of Carole Dingess, Annette Gonzales, and Terri Ferrara in the preparation of this manuscript is greatly appreciated. REFERENCES Abdulrahim, S. M., M. B. Patel, and J. McGinnis, 1979. Effects of vitamin D 3 and D 3 metabolites on production parameters and hatchability of eggs. Poultry Sci. 58:858-863. Abe, E., H. Horikawa, T. Masumara, M. Sugahara, M. Kubota, and T. Suda, 1982. Disorders of cholecalciferol metabolism in old egg-laying hens. J. Nutr. 112:436-446. Baksi, S. N., and A. D. Kenny, 1977. Vitamin D 3 metabolism in immature quail: Effects of ovarian hormones. Endocrinology 101:1216-1220. Bar, A., and S. Hurwitz, 1987. Vitamin D metabolism and Calbindin (calcium-binding protein) in aged laying hens. J. Nutr. 117:1775-1779. Bar, A., and A. W. Norman, 1981. Studies on the mode
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Results from Experiment 3 (Table 4) show that circulating levels of 1,25(OH)2D3 in female chickens were not different at a preovulatory stage of development (8 wk of age), but they were clearly different when there were measurable differences in egg shell quality (64 wk of age). Data from female chickens at 8 wk of age are in agreement with the observations made at 18 wk of age in Experiment 2. As these data were obtained from pullets before sexual development, the values are similarly low but less variable than those in pullets 18 wk of age in Experiment 2. Changes in plasma 1,25(OH)2D3 observed in hens laying eggs with different egg shell quality were not detectable at immaturity or when hens had good egg shell quality. This indicates that even when a hen is genetically limited in its ability to adequately calcify egg shells, differences in plasma l,25(OH)2D3 may not be expressed until age and physiologically induced modifiers of vitamin D metabolism and calcium homeostasis are activated. Apparently plasma l,25(OH)2D3 in immature pullets is not a good indicator for predicting future egg shell quality. There were no symptoms of vitamin D insufficiency after 22 wk of feeding laying hens (Experiment 4) a corn-soybean diet containing only la-OH-D 3 as the vitamin D source. Body weights, egg weight, egg production, and egg shell breaking strength were similar to those of controls fed vitamin D 3 (Table 5). Percentage egg shell (less membranes), however, was significantly greater for hens fed the la-OH-D 3 than for the hens fed vitamin D 3 . Similarly, percentage toe ash, tibia calcium phosphorus, and breaking strength (Table 6) were all significantly improved by feeding la-OH-D 3 . No differences were observed between calcium con-
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