The Effect of Dietary Supplementation with 1,25-Dihydroxycholecalciferol or Vitamin C on the Characteristics of the Tibia of Older Laying Hens

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1 The Effect of Dietary Supplementation with 1,25-Dihydroxycholecalciferol or Vitamin C on the Characteristics of the Tibia of Older Laying Hens S. NEWMAN and S. LEESON1 Department of Animal and Poultry Science, University of Guelph, Guelph, Ontario, Canada, N1G 2W1 ABSTRACT Seventy-two-week-old Leghorn hens were fed a conventional corn-soybean meal diet, or comparable diets either devoid of all supplemental vitamins, or with additions of 5 mg/kg 1,25- dihydroxycholecalciferol [1,25(OH) 2 D 3 ], or 100 ppm vitamin C. The diets were fed for up to 30 d, with periodic observation on bone characteristics of selected birds. With a vitamin-deficient diet it took 30 d to realize significant reductions (P < 0.05) in bone breaking strength and these birds had less bone ash as early as 15 d (P < 0.05), although bone calcium content was not affected. Adding 1,25(OH) 2 D 3 to the diet caused an increase in bone breaking strength after 15 d (P < 0.05) in the vitamin-deficient birds, although no difference was seen after 30 d. This increase in bone strength was associated with increase in bone cross-sectional area. Vitamin C generally had little effect on bone characteristics of the bird. These results suggest that there is little bone remodeling of older laying hens in response to short-term feeding of 1,25(OH) 2 D 3 or vitamin C. (Key words: layer, cholecalciferol, vitamin C, skeleton, tibia) 1999 Poultry Science 78:85 90 INTRODUCTION Recent studies involved with possible solutions to poor shell quality have examined the effects of two dietary vitamin supplements, namely 1,25- dihydroxycholecalciferol [1,25(OH) 2 D 3 ], and ascorbic acid (vitamin C). Because depletion of structural calcium present in the skeleton to augment the requirements for shell production in high producing hens is likely the cause of osteoporosis in aging birds (Abe et al., 1982; Newman and Leeson, 1997), it is possible that a treatment that improves shell characteristics might also have an impact on bone parameters of the hen. However, there are few reports on the effect of vitamin status on bone integrity of older laying hens. Bone integrity of older hens is important because of concerns over bird welfare and also the potential for bone contamination of processed fowl meat. It has been suggested that the efficiency of the hydroxylation reactions necessary to convert cholecalciferol (vitamin D 3 ), the usual form of the vitamin provided in laying hen diets, to 1,25(OH) 2 D 3 may be reduced in aged hens (Frost et al., 1990; Elaroussi et al., 1994). 1,25(OH) 2 D 3 is required for the regulation of calcium absorption and excretion, and can initiate the mobilization of calcium reserves from the bones to provide adequate calcium for the production of eggshells if needed (Abe et al., 1982). If 1,25(OH) 2 D 3 is unavailable for increased intestinal absorption and reduced excretion of calcium in the kidney, the only alternative source of the mineral will be the skeleton, which can be utilized in response to increased parathyroid hormone levels in the blood. Investigations into the ability of older hens to metabolize or respond to 1,25(OH) 2 D 3 have shown that although there may be similar levels of production of 1,25(OH) 2 D 3 in the kidney of young and aged hens, when aged hens are challenged with calcium restriction, their shell quality and bone strength deteriorate more rapidly (Bar and Hurwitz, 1987). This finding indicates that the actual 25-hydroxylation activity in the liver of old hens may not be affected by age, but rather that the body may be less able to adjust the production level of 1,25(OH) 2 D 3 to suit the birds requirements (Bar and Hurwitz, 1987; Elaroussi et al., 1994). Although aged hens showed elevated levels of 1,25(OH) 2 D 3 when fed a low (0.8%) calcium diet, the speed and magnitude of the adaptive response was greater in 22-wk-old birds than in older birds. Abe et al. (1982) and Elaroussi et al. (1994) found that older hens had decreased renal 25(OH)D 3-1-hydroxylase levels than younger birds. In addition to the decreasing accumulation of 1,25(OH) 2 D 3, Abe et al. (1982) reported lowered blood plasma levels of 1,25(OH) 2 D 3, which suggests an increasing rate of Received for publication January 5, Accepted for publication September 1, To whom correspondence should be addressed: SLEESON@aps.uoguelph.ca 85 Abbreviation Key: 1,25(OH) 2 D 3 vitamin D 3 = cholecalciferol. = 1,25-dihydroxycholecalciferol;

2 86 degradation of the compound in the system of these older birds. Although the focus of most 1,25(OH) 2 D 3 studies involving hens has been on shell quality, trends indicate that tibial mass is increased in birds fed a diet containing 3 to 5 mg/kg of 1,25(OH) 2 D 3, and is an indication of increasing mineralization (Frost et al., 1990; Tsang et al., 1990). Toxicity, indicated by morbidity or mortality, was detected when levels of 7 mg/kg were fed (Tsang et al., 1990). To date, no studies have shown clear, significant effects of 1,25(OH) 2 D 3 on the mechanical properties of the Leghorn skeleton. In addition to the observed changes in eggshell properties, vitamin C may play some role in improving bone properties (Orban et al., 1993). This improvement may be attributed to increased calcium absorption, or possibly to the role that vitamin C plays in the development of bone tissue. Ascorbic acid is an essential cofactor in the formation of collagen and formation of the extracellular matrix (Leeson et al., 1995; Newman and Leeson, 1997). The present study was designed to determine the effect of short-term supplementation with either 5 mg/kg of 1,25(OH) 2 D 3 or 100 ppm of ascorbic acid on the mechanical and structural properties of the tibia of aged hens. In addition, the effects of these two supplements on variables relating to eggshell quality were investigated. The diets were compared to a control diet containing all appropriate vitamins, including vitamin NEWMAN AND LEESON D 3 but without supplemental ascorbic acid, and a control diet without the addition of a vitamin premix, which was therefore deficient in vitamin D 3 and devoid of ascorbic acid. MATERIALS AND METHODS One hundred and ninety-two, 72-wk-old Single Comb White Leghorn hens of four strains were used in the trial. All of the hens had been involved in a previous trial to determine the effects of diet and strain on the performance of Leghorn hens. Birds were housed in individual cages, measuring cm (width, length, and height). The trial was conducted in an environmentally controlled room maintained at 21 C and birds were provided with 14 h of constant light at 100 lx each day. The experiment was a randomized complete block design, with diet as the treatment, and blocking on strain. Forty-eight birds were assigned to each diet. The birds were allotted to the diets such that 12 birds per treatment were from each of the 4 strains that had been tested in the previous trial. The 48 birds were then randomly assigned to 12 groups of 4 cages each. For the calculation of feed intake, each group of four birds in adjacent cages with a common feeder was considered an experimental unit; conversely, for the bone parameters and egg data, each individually caged bird was treated as an experimental unit during the statistical evaluation of the data. TABLE 1. Percentage composition of experimental diets 1 Ingredients C C V C + D C + AA Corn Soybean meal, 48% CP Barley Limestone Animal-vegetable fat Calcium phosphate, 20% P Vitamin premix Mineral premix Iodized salt, 0.015% Kl DL-methionine Alpha floc, cellulose ,25(OH) 2 D 3 premix Ascorbic acid premix Analyzed composition Calcium, % Phosphorus, % Calculated composition ME, kcal/kg 2,785 2,785 2,785 2,785 Crude protein, % Lysine, % Methionine, % Calcium, % Available phosphorus, % Explanation of codes: C = control diet; C V = control diet minus supplemental vitamins; C + D = control diet plus 5 mg/kg 1,25-dihydroxycholecalciferol [1,25(OH) 2 D 3 ]; C + AA = control diet plus 100 mg/kg ascorbic acid. 2Provides per kilogram of diet: retinyl acetate, 2,750 mg; cholecalciferol, 40 mg; a-tocopheryl acetate, 11 mg; riboflavin, 9.0 mg; pantothenic acid, 11.0 mg; vitamin B 12,13mg; niacin, 26 mg; choline, 900 mg; vitamin K, 1.5 mg; folic acid, 1.5 mg; biotin, 0.25 mg; ethoxyquin, 125 mg; manganese, 55 mg; zinc, 50 mg; copper, 5 mg; iron, 30 mg; and selenium, 0.1 mg. Diets

3 DIET AND BONE STRENGTH 87 TABLE 2. Mechanical properties of the left tibia from hens fed corn-soybean meal diets with modified vitamin contents (n = 16) Modulus of Day Diet 1 Force Stress elasticity (N) (MPa) (GPa) 5 C C V C + D C + AA SD C b C V ab C + D a C + AA ab SD C a ab 21.1 ab C V b a 19.9 a C + D a b 22.9 b C + AA a ab 20.5 ab SD a,bmeans within columns and days with no common superscript Birds were fed one of four diets; either the control diet (C), the control diet minus supplemental vitamins (C V), the control diet supplemented with 5 mg/kg of 1,25(OH) 2 D 3 (C+D), or the control diet supplemented with 100 ppm of ascorbic acid (C+AA) (Table 1). Ascorbic acid was of a stable form provided by Hoffmann La Roche.2 Birds were sampled after 5, 15, or 30 d on each diet. The control diet was formulated to meet the nutrient requirements of laying hens (Leeson and Summers, 1997). The C V diet was formulated by substituting the vitamin premix portion of the diet with cellulose, a non-nutritive filler. Calculated values for the vitamin content of the C V diet indicated that it was deficient in vitamin A (0% of requirement), vitamin D 3 (0%), vitamin K (0%), riboflavin (27%), folic acid (31%), and biotin (75%) (Leeson and Summers, 1997). Sufficient vitamin E, choline, pantothenate, niacin, thiamine, and pyridoxine were supplied by the other ingredients in the ration. The quantities of 1,25(OH) 2 D 3 and ascorbic acid required to reach the desired vitamin levels in the final C+D and C+AA diets were prepared by initial premixing with corn. Feed intake was measured at Day 5, 15, and 30. Following weighing, four birds per strain per diet were randomly selected and euthanatized by cervical dislocation. The birds were then dissected and both tibia (used in all tests) were dissected and air-dried for 7 d. Physical bone characteristics were determined by the three-point bending test commonly used to assess bone strength in poultry (Crenshaw et al., 1981). Bones were then frozen 2Hoffmann La Roche, Cambridge, ON, Canada, N1R 5X9. 3Model 4204, Instron Corp., Canton, MA at 12 C. After thawing, the outer dimensions of the bone were determined at two points midshaft using vernier calipers, once in the plane perpendicular to the direction of the applied force, and once parallel to the force. The bending test was conducted using an Instron Universal Testing Machine3 fitted with a 1 kn load cell. Crosshead movement was at 5 mm/min and paper feed rate at 100 mm/min. Bones were placed dorsal side up on supports 7 cm apart. Ultimate breaking force and deflection were determined for each tibia. Following bone breakage, wall thickness was determined, again perpendicular and parallel to the applied force. Force (Newtons), stress (megapascals), and modulus of elasticity (gigapascals) were determined. Bone fragments were fat extracted and ashed at 600 C for 12 h and then calcium level determined by atomic absorption spectrophotometry (AOAC, 1980). Egg production for each bird was electronically recorded at approximately 1330 h daily. On Days 4, 5, 14, 15, 29, and 30 of the trial, all eggs were collected for estimates of weight and eggshell characteristics. Data were subjected to ANOVA using the General Linear Models procedure (SAS Institute, 1991) to assess the effect of dietary treatment on response variables relating to egg production, shell properties, and the mechanical (force, stress, and elasticity) and structural (area, percentage ash, and percentage Ca) properties of the tibia. Data were analyzed for each of the three time periods (Day 5, 15, and 30) separately. Means of response variables having a significant F test for diet differences (P < 0.05) were separated using least significant differences (Cochran and Cox, 1957). The effect of strain of hen on the mechanical properties of TABLE 3. Estimated cross-sectional area and composition of the left tibia from hens fed corn-soybean meal diets with modified vitamin contents Estimated Bone Bone Day Diet 1 area ash calcium (cm 2 ) (%) (% of ash) 5 C C V C + D C + AA SD C c 56.2 a 39.2 C V ab 54.1 b 40.0 C + D a 56.9 a 40.5 C + AA bc 57.4 a 39.9 SD C a 39.0 C V b 39.3 C + D a 38.9 C + AA a 38.3 SD a cmeans within columns and days with no common superscripts

4 88 TABLE 4. Egg production and feed intake of hens fed corn-soybean meal based diets with modified vitamin contents Egg Feed Day Diet 1 n production n intake (%) (g/bird/d) 0 to 5 C C V C + D C + AA SD to 15 C C V C + D C + AA SD to 30 C a a C V b b C + D a a C + AA a a SD a,bmeans within columns and days with no common superscript the tibia was assessed in a similar fashion to determine the impact of blocking on the precision of the model. RESULTS No differences were observed in any of the response variables associated with the mechanical properties or the cross-sectional area of the tibia from hens sampled on Day 5 of the trial (Tables 2 and 3). After 15 d, control birds had significantly lower tibia breaking force than the hens fed 1,25(OH) 2 D 3, and this was accompanied by a significantly smaller cross-sectional area value for these bones. Birds fed 1,25(OH) 2 D 3 had bones that were stronger in terms of ultimate breaking force than those of the control birds, accompanied by greater crosssectional area measurements relative to hens on either the control or ascorbic acid-supplemented diet after 15 d of treatment. Stress values were not significantly different for any treatment, suggesting that the difference in ultimate strength is a result of differing amounts of bone material and not due to changes in the mineralization of the bone matrix. Percentage bone ash values for the vitamin-deficient birds were significantly lower than for any other treatment group at 15 and 30 d (Table 3). This reduction in mineral content did not affect bone strength, although as this measure considers the total bone, not just the cortical bone, it may be an indication of depletion of medullary bone calcium reserves. After 30 d, neither supplemental vitamin C nor 1,25(OH) 2 D 3 had any effect on breaking force, stress, or elasticity of the tibia when compared to the control birds. Force was significantly reduced in the bones of birds fed a diet without supplemental vitamins compared to all other treatments, and both the stress and elasticity of the bones were lower than for the NEWMAN AND LEESON 1,25(OH) 2 D 3 birds, although there was no significant difference between bones of birds receiving vitamindeficient diets and control or ascorbic acid diets with respect to these two parameters. Tibia ash for the vitamin-deficient birds was significantly lower than with any other treatment. The percentage of calcium present in the mineral component of the tibia was not significantly different for any of the treatments during any of the time periods tested. Egg production and feed intake values were similar for all hens except those consuming the vitamindeficient diet (Table 4). Although the production level was similar to that of the birds fed the control diet for the first 15 d of the trial, by the conclusion of the 30-d period the egg production for these hens had dropped to well below the rate observed for all other treatment groups. This decline in productivity was accompanied by a significant reduction in the feed intake of the group during the last 15 d of the experiment. Alteration of the vitamin content of the diets had minor effects on other egg parameters (Table 5). Ascorbic acid initially caused a significant reduction in egg weight and deformation values after 5 d on the test diet, although no significant difference between the eggs from these hens and the hens fed the control diet were observed by the end of the 30-d test period. The vitamin-deficient hens laid eggs with signs of reduced shell quality after 15 d on the test diet, and by the conclusion of the experiment, the thickness of the shell was significantly decreased, and the deformation values recorded for these eggs were elevated well beyond those that were observed for the control hens. Supplemental 1,25(OH) 2 D 3 had no effect on the eggshell properties observed during the trial. TABLE 5. Egg weight, deformation, and shell thickness of hens fed corn-soybean meal diets with modified vitamin contents Egg Egg Shell Day Diet 1 n weight deformation thickness (g) (mm) (mm) 5 C a 26.3 a 0.33 C V a 25.9 ab 0.33 C + D a 25.2 ab 0.33 C + AA b 24.1 b 0.34 SD C ab 24.0 a 0.35 a C V a 34.1 b 0.29 b C + D a 22.8 a 0.35 a C + AA b 25.1 a 0.34 a SD C b 0.33 a C V a 0.24 b C + D b 0.34 a C + AA b 0.32 a SD a,bmeans within columns and days with no common superscript

5 DIET AND BONE STRENGTH 89 TABLE 6. Force, stress, elasticity, estimated cross-sectional area and percentage ash of left tibia from hens of four different strains 1 Modulus of Bone Bone Strain Force Stress elasticity Area ash calcium (N) (MPa) (GPa) (cm 2 ) (%) (% ash) DeKalb c a b Hy-Line a b a Shaver b b b H + N b b c SD a cmeans within columns with no common superscript 1Main effect of strain, averaged over three time periods and four diets (n = 48). The four strains of birds used in the trial had significantly different values for ultimate breaking force, modulus of elasticity, and estimated cross-sectional area (Table 6). The contrast in ultimate breaking force is likely linked to the accompanying changes observed in the cross-sectional area, with the stronger bones having the greatest area. However, when stress is considered, there were no significant differences among the four strains of birds. Bone strength measured as force was less in the DeKalb birds and these birds also demonstrated increased brittleness as measured by modulus of elasticity. The percentage ash and percentage calcium of the tibia were not significantly different for any of the strains tested. By blocking on strain during the statistical analysis of the data, any variation that might have been contributed to the results by these strain differences should be minimized. DISCUSSION The results of the present experiment indicate that when adequate D 3 is supplied in the diet, 1,25(OH) 2 D 3 has limited effects on bone strength and composition parameters. Hens fed 1,25(OH) 2 D 3 had significantly greater values for estimated tibia area and breaking force than did the control birds at Day 15. This result possibly suggests that the metabolite does have some impact on bone characteristics, although in this study the effect declined over time. Higher levels of inclusion may have shown a greater effect; however, amounts exceeding the 5 mg/kg level have been shown to cause increased mortality (Tsang et al., 1990). These results are in partial agreement with those of Frost et al. (1990), who reported that 1,25(OH) 2 D 3 may have an effect on increasing tibia weight and breaking strength. In addition, Tsang et al. (1990) found an increase in tibial weight expressed as a percentage total body weight in response to 1,25(OH) 2 D 3. Although the diet devoid of supplemental vitamins was deficient in several vitamins, the decreased bone quality observed in these birds was probably related to the absence of vitamin D 3 in the diet. The pattern of decline observed in production and shell quality are typical of those that have been observed by other researchers investigating vitamin D 3 deficiencies in laying hens (Shen et al., 1981, 1982). Diets devoid of the other vitamins, such as vitamin A, vitamin B 12, and riboflavin, apparently have little effect on egg production or shell thickness, even after several weeks of deficiency. In a series of trials conducted by Squires and Naber (1992, 1993) and Naber and Squires (1993), it was shown that feeding a diet deficient in of any one of these vitamins (A, B 12, and riboflavin) fails to significantly affect shell thickness or egg production after a period exceeding 8 wk. Because shell quality was not affected by these deficiencies, it is unlikely that such deficiencies would be a contributing factor in the observed decline in mechanical and structural properties of the tibia. In the present study, even when birds were fed a diet containing no supplemental vitamin D 3, their mechanical bone properties were maintained at a level that was equivalent to that of the birds supplemented with 1,25(OH) 2 D 3 for a period of 15 d (Table 2). However, the first signs of skeletal depletion are evident in the significantly reduced percentage ash values for this group of hens (Table 3). Because the ash values reflect the status of the whole bone, low values were probably an indication that medullary bone was being resorbed at a faster rate in order to supply sufficient calcium to maintain shell synthesis. Ruschkowski and Hart (1992) observed a similar depletion in tibia ash in birds fed a diet devoid of vitamin D 3 for 8 wk. The reduction in ultimate breaking force observed by Day 30 suggests the beginning of the resorption of cortical bone tissue, although the other parameters associated with this bone compartment were not significantly different from those of the birds fed the control diet (Table 2). Although the tibia of the deficient birds were significantly more elastic than those of the 1,25(OH) 2 D 3 -supplemented hens, the value was still in the range expected for an aged hen, and therefore this result is of limited biological significance. In the present study, ascorbic acid at 100 ppm was ineffective in altering the properties of the tibia (Table 2). However, some limited improvements in skeletal properties have been reported by our laboratory (Leeson et al., 1995) and other researchers when this vitamin is fed. For example, 100 ppm was shown to improve bone strength in developing turkey poults (Leeson et al, 1995). A linear increase in bone mineral content and density

6 90 was reported by Orban et al. (1993) when hens were fed increased (1,000 to 3,000 ppm) levels of ascorbic acid, although no changes were observed in structural properties of the cortical bone. This effect may be due to the deposition of the additional mineral in the medullary bone instead of the cortical tissue (Orban et al., 1993). This result could explain the effectiveness of the treatment in the younger turkey (Leeson et al., 1995), but not in the older hen. Ascorbic acid supplementation improved eggshell deformation values (Table 5) and reduced egg weight by Day 5 of treatment compared to the other treatments, but not at 30 d. These effects are possibly linked, as a decrease in egg size without a concurrent decrease in shell weight will likely cause an increase in shell quality (Al Batshan et al., 1994). Results from the present experiment suggest that short term vitamin supplementation of a diet that meets the NRC (1994) recommendations for the laying hen, with either 5 mg/kg of 1,25(OH) 2 D 3 or 100 ppm of ascorbic acid will not improve the mechanical or structural properties of the tibia of the Leghorn hen. The inability of vitamin supplementation to influence bone strength may be due to the general inability of the older hen to form new bone (Whitehead, 1994). In a study by Hudson (1992) fluorochrome labeling was used to determine the mineral apposition rate of immature (20- wk-old) and mature (39.5-wk-old) hens. Although mineral accumulation was detected in cortex, periosteum, and haversian systems of the younger birds, no active bone mineralization sites were detected in the structural bone of the mature birds. If bone formation is not occurring in the older birds, then any increased calcium uptake may simply be used to meet the demands of shell production, thus maintaining, but not improving, the status of the skeleton. ACKNOWLEDGMENTS This work was supported by the Poultry Industry Centre, Arkell, Ontario and the Ontario Ministry of Agriculture Food and Rural Affairs. The 1,25 (0H) 2 D 3 and vitamin C were donated by Hoffmann La-Roche, Canada, Cambridge, Ontario. REFERENCES Abe, E., H. Horikawa, T. Masumura, M. Sugahara, M. Kubota, and T. Suda, Disorders of cholecalciferol metabolism in old egg-laying hens. J. Nutr. 112: Al Batshan, H. A., S. E. Scheideler, B. L. Black, J. D. Garlich, and K. E. Anderson, Duodenal calcium uptake, femur ash and eggshell quality decline with age and increase following molt. Poultry Sci. 73: Association of Official Analytical Chemists, Official Methods of Analysis. 3rd ed. Association of Official Analytical Chemists, Washington, DC. Bar, A., and S. 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Poultry Sci. 71: SAS Institute, SAS User s Guide. SAS Institute Inc., Cary, NC. Shen, H., J. D. Summers, and S. Leeson, Egg production and shell quality of layers fed various levels of vitamin D 3. Poultry Sci. 60: Shen, H., J. D. Summers, and S. Leeson, Influence of a Vitamin D deficiency on egg shell, membrane, and egg shell weight. Poultry Sci. 61: Squires, M. W., and E. C. Naber, Vitamin profiles of eggs as indicators of nutritional status in the laying hen: vitamin B 12 study. Poultry Sci. 71: Squires, M. W., and E. C. Naber, Vitamin profiles of eggs as indicators of nutritional status in the laying hen: vitamin A study. Poultry Sci. 72: Tsang, C.P.W., A. A. Grunder, and R. Narbaitz, Optimal dietary level of 1a,25-dihydroxycholecalciferol for eggshell quality in laying hens. Poultry Sci. 69: Whitehead, C. C., Nutritional factors and bone structure in laying hens. Pages in: Proceedings of the Ninth European Conference, Glasgow, UK., Vol. 2. Walker and Connell Ltd., Darvel, UK.