Effects of Increased Vitamin D3 on Calcium Retention and Egg Shell Calcification

Effects of Increased Vitamin D 3 on Calcium Retention and Egg Shell Calcification J. D. GARLICH AND R. D. WYATT Department of Poultry Science, North Carolina State University, Raleigh, N.C. 27607 (Received for publication November 26, 1970)

HE minimum requirement for vitamin D 3 by the laying hen has been established but the effect of increased amounts have been only partially investigated. The minimum requirement is stated as 500 International Chick Units (I.C.U.) per kg. of feed (NAS-NRC 1966). Murphy et al. (1936) fed up to 1,550 I.C.U. per kg. of feed using fish oil as a source of vitamin D 3 and concluded that the requirement level for maximum egg production and satisfactory shell quality was 780 I.C.U. per kg. However, values for percent shell increased with increasing vitamin D 3 up to the highest amount fed. Couch et al. (1947) fed up to 760 I.C.U. per kg. of feed and concluded that whereas the vitamin D 3 requirement for maximum egg production for layers in their first year of production was between 380 and 760 I.C.U. per kg., the requirement for hens in their second year of production was at least 760 I.C.U. per kg. Bragg and Stephenson (1964) reported a significant interaction between dietary calcium and vitamin D 3 as related to increased shell as per cent of the egg. Dietary calcium content ranged from 2.25 to 3.75 percent and vitamin D 3 from 300 to 1,000 I.C.U. (it may be presumed that this amount of vitamin D 3 was added per

Paper number 334S of the Journal Series of the North Carolina State University Agricultural Experiment Station, Raleigh, North Carolina. The use of trade names in this publication does not imply endorsement by the North Carolina Agricultural Experiment Station of the products named, nor criticism of similar ones not mentioned.

pound of feed although this was not stated in their report). The 1960 NAS-NRC publication listed the D 3 requirement for laying hens as 225 I.C.U. per pound of feed. Berg et al. (1951) reported that the decline in egg shell thickness with the increasing age of the layer was not prevented by periodically increasing vitamin D 3 from 990 to 1,485 to 1,980 I.C.U. per kg. of feed over three consecutive three-month periods of egg production. The effects of greater amounts of vitamin D 3 have not been investigated. Recent reports support the hypothesis that calcium absorption and egg shell formation by laying hens would be enhanced by dietary levels of vitamin D 3 in excess of the minimum level recommended by the NAS-NRC. Wasserman and Taylor (1966) reported that vitamin D 3 was necessary for the production of the calcium binding protein in the intestine of the chick. Corradino et al. (1968) reported that vitamin D 3 was responsible for the production of a calcium binding protein in the shell gland of the laying hen. Ebel et al. (1969) demonstrated that both the production of intestinal calcium binding protein and intestinal absorption of calcium showed a continual increase with injected doses of vitamin D 3 from 10 to 105 I.C.U. per chick. A dose of 102 I.C.U. per chick was considered to be near the physiological amount in their experiments. The objectives of the following experiments were to determine whether or not dietary levels of vitamin D 3 of 2,400 to 39,000 I.C.U. per kg. of feed would im-

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VITAMIN D, CA AND EGG SHELL

prove calcium retention and enhance egg shell calcification as compared to 600 I.C.U. per kg. of feed. MATERIALS AND METHODS

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Registered trade name, "Fixtdee," Dawe's Laboratories, Chicago, Illinois.

TABLE 1.—Basal diet Ingredients Corn, yellow Soybean meal (49 % protein) Soya oil Choline citrate DL-methionine NaCl CaHP0 4 -2H 2 0 Trace mineral mix1 Vitamin mix2 Cr 2 0 3 CaCOa Vit. D3 pre-mix Cornstarch

% 55.85 27.70 3.00 0.20 0.30 0.37 3.06 0.07 0.04 0.30 Variable Variable Variable

J I n mg./lOO g. diet: MnS0 4 -H 2 0, 20; FeS0 4 •7H 2 0, 20; CuS0 4 -5H 2 0, 1.6; ZnS0 4 -5H 2 0, 30; Ca(I0 3 ) 2 -6H 2 0, 0.6. 2 In mg./lOO g. diet: d-alpha-tocopherol acetate 1.2, inositol 25, niacin 6, Ca-pantothenate 3, thiamine HC1 1.5, riboflavin 1.5, pyridoxine-HCl 0.8, folic acid 0.6, biotin 0.06, menadione 0.4, BHT 10, B n 0.003, and vit. A 500 IU.

quirement but rather to evaluate the effects of vitamin D 3 fed at many times this level. The 600 I.C.U per kg. level of vitamin D 3 provided a 20% margin of safety which might be necessary to accommodate individual bird variations in the requirement. The dietary calcium was supplied by U.S.P quality calcium carbonate. Three percent vegetable oil was added to the diet to prevent dustiness and ingredient separation. Calcium analysis of feed taken from the bottom of the feeders after five days of feeding indicated no settling out of the calcium carbonate. Calcium in the diet and excreta was determined by atomic absorption spectrophotometry2 after wet ashing with nitric acid followed by perchloric acid (Johnson and Ulrich, 1959). Egg shell calcium was determined by atomic absorption spectrophotometry after dissolving the shells in hydrochloric acid. Calcium retention was determined by use of the chromic oxide indicator method of Hill and Anderson (1958). A representative sample of excreta, uncontaminated by feed, was collected for periods of five consecutive days in experiment 1 and six days in experiment 2. Feed and excreta chromium were determined according to the following modification of the method of Hill and Anderson (1958). (1) Heating of the digest was continued for 3 minutes after the production of the yellow-orange color of chromate. (2) After chilling and diluting the digest with 60 ml. of water, 20 ml. of concentrated sulfuric acid were added. (3) The chromate concentration was determined from the optical density at a wave length of 444 m[A. A known concentration of sodium chromate in 20% sulfuric acid was used as a standard. The values for the percentage of calcium retained refer to the percentage of dietary 2 Perkin-Elmer Technical Bulletin (1968).

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The chickens were kept in individual laying cages in a room with continuous lighting and a temperature of 21 ± 1°C. The following observations were made: individual feed consumption, body weight, egg production, egg weight, shell calcium, and calcium retention. The basal diet is shown in Table 1. The vitamin D 3 source was a commercial premix stabilized by the addition of antioxidants. The premix contained 3,600 I.C.U. of vitamin D 3 activity per gram according to the manufacturer's analysis of the lot.1 The basal diet contained 600 I.C.U. of vitamin D 3 per kg. rather than the S00 I.C.U. per kg. amount which is stated by NAS-NRC (1966) to be the requirement because the objectives of the experiments were not to redetermine the minimum re-

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J. D. GARLICH AND R. D. WYATT

Statistical analysis of the data were performed using the t-test for significance. The percentage retention values were analyzed statistically after an arcsin transformation of the data (Snedecor, 1950). In the first experiment 12 pullets were fed a control diet containing 600 I.C.U. of vitamin D 3 per kg. of feed while another 12 pullets received periodic increases of dietary vitamin D 3 over a period of 17 weeks as shown in Tables 2 and 3. The experi-

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calcium consumed which was retained by the hen for storage, deposited in the egg yolk and albumen, or used in egg shell formation. Calcium balance was calculated using the average of values taken during the collection period. In experiment 1 a value of 0.05 5 g. of calcium was assumed to be contained in the yolk plus albumen of the egg. In experiment 2 a value of 0.060 grams of calcium per egg contents was assumed because of the greater egg size (Hurwitz and Griminger, 1960). These assumed values- were used in the calculation of calcium balance because the amount of calcium contained in the egg albumen and yolk was not determined by analysis. The birds were consisered to be in balance when the value was 0 ± 0.09 g. calcium per bird per day when consuming the 2% calcium diet and 0 ± 0.17 g. calcium per day when consuming the 3.3% calcium diet. This range represents approximately 5 percent of the daily calcium intake and was considered the limit of accuracy. In experiment 1 the pullets in each group used for calcium retention studies were selected on the basis of their high rate of egg production at the beginning of the experiment. In each of the two groups, five of the seven remained in satisfactory egg production during the entire experiment. The remaining two in each group decreased their production to one or two eggs per week during the course of the experiment and other pullets were chosen as replacements.

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VITAMIN D, CA AND EGG SHELL TABLE 3.—Experiment 1. The effects of increased dietary vitamin Ds on calcium metabolism by pullets: Results from pullets on calcium balance trials1 1-4 4 3.31

S-6 5 3.21

7-8 8 1.99

9-11 9 2.03

11 2.14

12-14 12 2.14

Control: Dietary Vit. D 3 , I.C.U./kg. No. of pullets Eggs/bird/day Shell Ca, g./egg Feed consumed,2 g./bird/day Ca retention, % 5 Calcium balance, g./bird/day

600 7 0.92 2.14 97 64.9a 3 + 2.2 +0.07

600 7 0.80 2.18 100 62.1 +0.18

600 7 0.78 1.75 90 57.5 +0.26

600 5 0.68 1.66 88 77.9 +0.21

)00 7 0.66 1.67 78 74.8b'+1.7 +0.12

600 7 0.72 1.64 83 71.5 + 3.2 +0.30

Experimental: Dietary Vit. D3> I.C.U./kg. No. of pullets Eggs/bird/day Shell Ca, g./day Feed consumed,2 g./bird/day Ca retention, % Calcium balance,5 g./bird/day

600 7 0.92 2.11 93 66.2c"±2.8 +0.09

2,400 7 0.76 2.19 96 63.6 +0.21

2,400 6 0.87 1.77 94 71.6 +0.32

9,600 6 0.70 1.70 94 57.5 +0.13

9,600 7 0.74 1.72 76 79.7d 4 +1.5 -0.02

38,400 7 0.88 1.71 94 79.9d 4 +1.7 +0.40

1 The results shown were obtained during either the last 5 days of the period or days 3 to 7 at the beginning of the period. 2 Moisture-free weight. 3 a, b The two means are significantly different, P < 0 . 0 5 . 4 c, d The means are significantly different, P < 0 . 0 1 . 5 The calcium content of the yolk plus albumen was assumed to be 55 mg. per egg.

mental periods varied from 2 to 3 weeks in length. Eggs for shell calcium determinations and excreta for calcium retention determinations were collected on days 3 through 7 after changing the vitamin D 3 content of the diet. The allowance of two days for the vitamin to develop its effectiveness seemed adequate after consideration of the report of Ebel et al. (1969) that vitamin D 3 increases calcium transport in the chick intestine within 20 hours after administration and the report of Turk and McGinnis (1964) that vitamin D 3 deficient hens respond with increased egg shell thickness within two days after administration of the vitamin. Determinations of egg shell calcium and calcium retention were also done on samples taken during the last 5 days of the experimental period. The calcium content of the diets was also varied in order to assess the effects of vitamin D 3 levels at low calcium intakes. For the first 6 weeks all the pullets received a diet con-

taining 3.3 ± 0 . 1 % calcium. During weeks 7 through 14 the diet contained 2.0 ± 0.15% calcium. The actual calcium content by analysis is shown in Tables 2 and 3. During weeks 15 through 17 the dietary calcium content was 3.3%. The experiment was initiated when the pullets were 28 weeks of age and had reached peak production. The pullets were a commercial Single Comb White Leghorn strain. In the second experiment two groups of 6 Leghorn hens in their second year of production were fed a diet containing 600 I.C.U. of vitamin D 3 per kg. for three weeks. The control group continued on the diet for an additional two weeks while the experimental group received a diet containing 39,000 I.C.U. of vitamin D 3 per kg. of diet. Observations were taken during the last six days of the first period of three weeks duration and during the last six days of the second period of two weeks duration.

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Weeks included in period Week of observation Dietary Ca, %

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J. D. GARLICH AND R. D. WYATT

RESULTS AND DISCUSSION

in week 5 even though the calcium content of the diet was the same. The decrease in shell calcification could be due to the age of the pullet or may have been a consequence of the previous dietary treatment. The decline in egg shell calcification with increasing age of the layer is well documented (Berg et al., 1951). Experiment 2 was conducted with hens in their second year of egg production. The calcium content of the diet was 3.2%. The initial average egg shell calcium content was 1.901.97 grams per egg. Table 4 shows that an increase in the vitamin D 3 content of the diet from 600 to 39,000 I.C.U. per kg. did not improve egg shell calcification by old hens. Table 3 contains the results of the pullets in experiment 1 which were used in the calcium balance trials. Average egg weight, egg production, feed consumption, and shell calcium values for these pullets are similar to the values obtained by averaging the data of all the pullets in the experiment (Table 2). There was not a significant improvement in either the percentage of calcium retained or calcium balance as a result of the increased dietary intake of vitamin D 3 . The pullets remained in calcium balance throughout the course of the experiment despite the reduction in calcium intake after the decrease in the calcium content of the diet from 3.2 to approximately 2.0%. This was possible for 3 reasons: (1) a decreased rate of egg production, (2) decreased deposition of calcium in the egg shell, and (3) an increased percentage of ingested calcium retained. In the control group the value of 74.8% retention observed in week 11 is significantly greater than the 64.9% observed during week 4 (P < 0.05). In the experimental group the 79.7 and 79.9% retention obtained during weeks 11 and 12 were significantly greater than the 66.2% retention obtained during week 4 (P < 0.01). The range of individ-

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If amounts of dietary vitamin D 3 greatly in excess of the presently stated requirements were to increase calcium transport in the intestine and shell gland then an increase in egg shell calcification could be expected. An increase in the amount of vitamin D 3 dependent calcium binding protein in the intestine and shell gland could be expected to improve the efficiency of calcium transport in the intestine and shell gland. Therefore the effect of vitamin D 3 might be most apparent when the pullets consumed a diet low in calcium (2.0% as compared to 3.3% dietary calcium). Table 2 shows the values for shell calcium obtained in experiment 1. An increase in dietary vitamin D 3 from 600 to 2,400 I.C.U. per kg. did not increase the shell calcium content of eggs from pullets fed a diet containing 3.2% calcium. When the dietary calcium level was reduced from 3.2 to 1.99% the amount of calcium deposited in the egg shell decreased significantly. However, the 2,400 I.C.U. per kg. amount of dietary vitamin D 3 did not prevent or lessen the decrease in egg shell calcium content. Further periodic increases in vitamin D 3 from 2,400 up to 38,400 I.C.U. per kg. of feed did not significantly improve egg shell calcification by pullets fed the low calcium diet. Shell calcium content increased after dietary calcium was increased from 2.14 to 3.31% in week IS. This increase was significant only for the control group. The increase in shell calcium content was maximal by the second week after increasing dietary calcium. The final average shell calcium content was the same for both the control group and the group of pullets fed the diet containing 38,400 I.C.U. of vitamin D 3 per kg. The final values of 1.85 to 1.89 grams of calcium per shell at week 17 were significantly less (P < 0.01) than the values of 2.18 g. calcium per shell observed

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VITAMIN D, CA AND EGG SHELL TABLE 4.—Experiment 2. Effects of increased dietary vitamin D% on calcium metabolism by hens Control Period l Dietary Vit. D 3 ,1.C.U./kg. No. of hens Egg production, % Egg weight, g. Shell calcium, g./egg Feed consumed,4 g./hen/day Calcium retention, % Calcium balance,6 g./hen/day 2 3 4 6

600 6 90 68.7 3 + 1.2 1.90 + 0.07 90.4+5.4 57.2 + 1.2 + 0 . 1 6 + 0.06

Experimental Period 2

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600 6 73 69.5 + 3.3 1.88 + 0.08 98.9 + 8.7 54.1 + 2.1 + 0 . 4 8 + 0.07

Period 1

Period 2

600 6 83 73.1 + 1.5 1.97 + 0.05 89.5 + 5.3 56.2 + 3.1 + 0 . 1 5 + 0.13

39,000 6 80 71.9 + 1.8 2.00 + 0.06 105.0 + 3.1 56.0+1.6 + 0 . 4 9 + 0.07

Period 1 was 3 weeks. Observations made during the last 6 days of the 3rd week. Period 2 was 2 weeks. Observations made during the last 6 days of the 2nd week. All values shown are the mean + standard error of the mean. Moisture-free weight. The calcium content was 3.20%. The calcium content of the yolk plus albumen was assumed to be 60 mg. per egg.

ual values for the experimental group was 74 to 85% retention. The relationship of an increase in the percentage of calcium retained with a decrease in calcium intake has also been reported by Common (1943) and Hurwitz and Griminger (1962). Table 4 shows that increased dietary vitamin D 3 did not improve percentage calcium retention or calcium balance by old hens in their second year of production. Percentage calcium retention values averaged 56% with a range of 46 to 67%. These values are in accord with Hurwitz and Griminger (1962) who reported that layers consuming 3 g. of calcium per day, and in 80% production retained 50-55% of the ingested calcium. The results of these experiments do not support the original hypothesis that amounts of dietary vitamin D 3 greatly in excess of the present NAS-NRC (1966) requirement of 500 I.C.U. per kg. of feed would increase egg shell calcification. These results are in agreement with the report of Berg et al. (1951) who observed no benefit from amounts of vitamin D 3 up to 1980 I.C.U. per kg. of feed. Our results indicate that even greater amounts of vitamin D 3 are without benefit. The reports by Murphy et al. (1963) and Couch et al. (1947) indicated benefi-

cial effects from amounts of vitamin D 3 in excess of 500 I.C.U. per kg. of feed. Their results might have reflected the use of unstable preparations of vitamin D 3 . Our results did not indicate an interaction between dietary vitamin D 3 and calcium as reported by Bragg and Stephenson (1964). The shell gland of the laying hen also produces a vitamin D 3 dependent calcium binding protein (Corradino et al., 1968). Increased dietary vitamin D 3 could be expected to increase the amount of the shell gland's calcium binding protein and consequently increase calcium transport and egg shell calcification. Our experiments showed that egg shell calcification was not increased by increased amounts of vitamin D 3 . Possibly the amount of dietary vitamin D 3 required to significantly influence calcium transport is greater than the amounts used in our experiments. An alternate explanation is that vitamin D 3 dependent calcium transport was not the factor limiting egg shell calcification. SUMMARY Two experiments were conducted with Single Comb White Leghorns to determine whether or not amounts of dietary vitamin D 3 greatly in excess of the currently stated requirement would improve the efficiency

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ACKNOWLEDGMENTS

The authors thank Mrs. Carole Morris for technical assistance. The authors are grateful to Dawe's Laboratories, Inc., 4800 South Richmond Avenue, Chicago, Illinois 60632, for the gift of vitamin D 3 . REFERENCES Berg, R. G. F. Bearse and V. L. Miller, 1951. The effect of periodically increasing the level of calcium and/or vitamin D on the performance of laying pullets. Poultry Sci. 30: 799-804. Bragg, D. B., and E. L. Stephenson, 1964. The use of radioactive tracer in studying the effect of calcium and vitamin D 3 and season on egg shell quality and performance of white leghorn hens. Poultry Sci. 43 : 1304.

Common, R. H., 1943. Observations on the mineral metabolism of pullets. VII. J. Agr. Res. 33 : 213-220. Corradino, R. A., R. H. Wasserman, M. H. Pubols and S. I. Chang, 1968. Vitamin D 3 induction of a calcium-binding protein in the uterus of the laying hen. Arch. Biochem. Biophys. 125:378380. Couch, J. R., L. E. James and R. M. Sherwood, 1947. The effect of different levels of manganese and different amounts of vitamin D in the diet of hens and pullets. Poultry Sci. 26: 30-37. Ebel, J. G., A. N. Taylor and R. H. Wasserman, 1969. Vitamin D-induced calcium-binding protein of intestinal mucosa. Amer. J. Clin. Nutr. 22: 431-436. Hill, F. W., and D. L. Anderson, 1958. Comparison of metabolizable energy and productive energy determinations with growing chicks. J. Nutr. 64: 587-603. Hurwitz, S., and P. Griminger, 1960. Observations on the calcium balance of laying hens. J. Agric. Sci. 54: 373-377. Hurwitz, S., and P. Griminger, 1962. Estimation of calcium and^ phosphorus requirement in laying hens by balance techniques. J. Sci. Food Agric. 13: 185-191. Johnson, C. M., and A. Ulrich, 1959. II. Analytical methods for use in plant analysis. Bulletin 766, California Agricultural Experiment Station, University of California. Murphy, R. R., J. E. Hunter and B. C. Knandel, 1936. The vitamin D-requirements of growing chicks and laying hens. Bulletin 334, The Pennsylvania State College, Pennsylvania. National Academy of Sciences, National Research Council, Publication 1345, 1966. Nutrient requirements of poultry. Printing and Publishing Office, 2101 Constitution Ave., Washington, D.C. Snedecor, G. S. Statistical Methods, Fourth edition. The Iowa State University Press, 1950. Turk, J. L., and J. S. McGinnis, 1964. Effects of a vitamin D deficiency in mature S. C. W. L. hens. Poultry Sci. 4 3 : 1372. Wasserman, R. A., and A. N. Taylor, 1966. Vitamin D3-induced calcium-binding protein in chick intestinal mucosa. Science, 152: 791-793.

AUGUST 29-SEPTEMBER 3. 2 2ND ANNUAL MEETING AMERICAN INSTITUTE OF BIOLOGICAL SCIENCES, COLORADO STATE UNIVERSITY, FORT COLLINS

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of calcium utilization and increase egg shell calcification. The criteria used were grams of shell calcium per egg, percentage calcium retention, and calcium balance. The first experiment was conducted to investigate the effects of levels of dietary vitamin D 3 of 600 to 38,400 I.C.U. per kg. on the calcium metabolism of birds in their first year of production while consuming a diet containing 3.2 or 2.0% calcium. The second experiment was conducted to compare the effects of vitamin D 3 fed at 600 or 39,000 I.C.U. per kg. of diet on the calcium metabolism of hens in their second year of egg production. In neither experiment was there a beneficial effect of vitamin D 3 in excess of 600 I.C.U. per kg. These results suggest that dietary vitamin D 3 was not the limiting factor in egg shell calcification when the diet contained 600 I.C.U. per kg. and that additional dietary vitamin D 3 has no beneficial effect on egg shell calcification.