Effect of Source and Level of Vitamin D on Live Performance and Bone Development in Growing Broilers 1

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1 2003 Poultry Science Association, Inc. Effect of Source and Level of Vitamin D on Live Performance and Bone Development in Growing Broilers 1 C. A. Fritts and P. W. Waldroup 2 Poultry Science Department, University of Arkansas, Fayetteville, Arkansas Primary Audience: Nutritionists, Veterinarians, Production Managers, Researchers SUMMARY Cholecalciferol (VIT-D3) and 25-hydroxycholecalciferol (25-OH-D3) were each used to provide 125, 250, 500, 1,000, 2,000, or 4,000 IU/kg of vitamin D activity in a nutritionally complete cornsoybean meal diet. Each treatment was assigned to four pens of 60 male broilers (Cobb 500) grown in an open-sided house with sidewall curtains. At 21 and 42 d, BW and feed conversion were determined. Samples of birds (8 per pen) were killed at the same ages; the right tibia was subjected to bone ash determination, and incidence and severity of tibial dyschondroplasia (TD) were determined on the left tibia. At 21 and 42 d, the BW of birds fed the 25-OH-D3 were significantly greater than those of birds fed the VIT-D3. At 21 d, vitamin D in excess of 500 IU/kg appeared necessary to maximize BW regardless of source. At 42 d, approximately 1,000 IU/kg was needed to maximize BW of birds fed VIT-D3, whereas no significant difference in BW was noted among birds fed the various levels of 25-OH-D3. Bone ash at 21 and 42 d was significantly greater for birds fed the 25-OH-D3 as compared to those fed the VIT-D3. Approximately 2,000 IU/kg of VIT-D3 was needed for maximum bone ash, whereas there were no significant differences in bone ash content of birds fed from 250 to 4,000 IU/kg from 25-OH-D3. The incidence and severity of TD was significantly lower for birds fed 25-OH-D3 and was reduced by increasing levels of vitamin D regardless of source. Results of the study show that 25-OH-D3 is more metabolically potent on a per unit basis than VIT-D3 for support of BW, tibia ash, and reduction in incidence and severity of TD. The differences were observed primarily at lower levels of vitamin D; at typical industry levels, few differences were observed between the two sources. Use of the 25-OH-D3 may allow for supplementation with lower levels or may provide a greater margin of safety. Key words: vitamin D, broiler, tibial dyschondroplasia, 25-hydroxycholecalciferol 2003 J. Appl. Poult. Res. 12:45 52 DESCRIPTION OF PROBLEM It has long been known that vitamin D is needed for proper absorption of calcium [1, 2, 3]. The NRC [4] recommends that broiler diets be fortified with a minimum of 200 IU/kg of vitamin D 3. However, commercial broiler diets are typically fortified with this vitamin at 10 to 20 times above the NRC recommendations [5]. Vitamin D in excess of NRC recommendations 1 Published with approval of the Director, Arkansas Agricultural Experiment Station, Fayetteville, AR Mention of a trade name, proprietary product, or specific equipment does not constitute a guarantee or warranty by the University of Arkansas and does not imply its approval to the exclusion of other products that may be suitable. 2 To whom correspondence should be addressed: Waldroup@uark.edu.

2 46 has been shown to reduce the incidence of field rickets and tibial dyschondroplasia (TD), which are still observed in commercial situations with heavy, fast-growing broilers [6, 7, 8, 9]. Vitamin D is generally provided to poultry by supplementation of the diet with crystalline forms of cholecalciferol (VIT-D3). This metabolite must undergo additional changes in the liver to become 25-hydroxycholecalciferol (25-OH- D3) and in the kidney to become 1,25-dihydroxycholecalciferol, considered the active metabolite [10]. In 1995, 25-OH-D3 was given generally recognized as safe (GRAS) status for use as a vitamin D source in poultry diets [11]. Compared to comparable levels of vitamin D 3, this isomer has been shown to improve body weight gain, feed efficiency, bone ash, and breast meat yield and to reduce the incidence of TD and rickets [6, 12, 13, 14, 15, 16, 17]. The objective of the present study was to evaluate the effects of source and level of vitamin D in broiler diets on live performance and bone development of broilers grown under conditions approximating commercial poultry production. MATERIALS AND METHODS Experimental Diets and Birds JAPR: Research Report All procedures used during the study were approved by the University of Arkansas Animal Care Committee. Diets based on corn and soybean meal were formulated for starter (0 to 21 d) and grower (21 to 42 d) periods, containing the minimum crude protein content suggested by NRC [4] with a minimum of 110% of the suggested amino acid levels. Calcium and nonphytate phosphorus were provided at levels as suggested by NRC [4]. All diets were fortified with a vitamin premix that provided adequate amounts of all vitamins except for vitamin D. A complete trace mineral mix was provided with all minerals provided in sulfate form. Compositions of starter and grower diets are shown in Table 1. By using aliquots of a common mix of starter or grower diets, 12 experimental diets were prepared with a 2 6 factorial arrangement of treatments. A commercially available cholecalciferol product [18] was used to provide 125, 250, 500, 1,000, 2,000, or 4,000 IU/kg vitamin D (VIT- D3). A commercial source of 25-hydroxycholecalciferol [19] was used to provide 3.125, 6.25, 12.5, 25, 50, or 100 µg/kg (25-OH-D3), calculated to be equal to the levels provided by the VIT-D3 based on the conversion of µg of cholecalciferol to 1 IU [4]. The sources of vitamin D activity were blended with a portion of the basal diet in a pharmaceutical-grade V-mixer prior to adding to the final mixer to enhance distribution in the diet. All diets were pelleted with steam; starter diets were crumbled. Pelleting temperatures were not recorded. Each of the 12 experimental treatments was assigned to four replicate pens of 60 male broilers. Samples of mixed feeds were retained for analysis of crude protein, calcium, phosphorus [20], and vitamin D [21] activity. Day-old male chicks of a commercial broiler strain [22], originating from a breeder flock that were fed VIT-D3 as a source of vitamin D, were obtained from a local hatchery and randomly assigned to pens in a steel truss poultry house of commercial design. The house had a 1-m sidewall curtain [23] with two outside rows of 12 pens each and two inside rows of 12 pens each. Inside and outside rows served as blocks in the experimental design. Sixty chicks were randomly allocated to each of 48 pens (5.2 m 2 ). Previously used litter, top-dressed with new softwood shavings, served as bedding over concrete floors. Each pen was equipped with one automatic water fount and two tube-type feeders. Birds were provided ad libitum access to feed and water during the study with 23 h of light and 1 h of darkness. One 9-watt fluorescent light suspended 198 cm over the litter provided supplemental light in each pen. Live Performance and Bone Development Measurements Mean body weights by pen were taken at 21 and 42 d of age. Feed consumption was determined at the same ages. Mortality was checked twice daily; weights of birds that died were used to adjust the feed conversion ratio (FCR; total feed consumed divided by weight of live birds plus dead birds). At 21 and 42 d, eight birds per pen were killed by CO 2 inhalation, and the right tibia was removed for ash determination on dry fat-free bones as described by AOAC [24]. The left tibia was examined for incidence and sever-

3 FRITTS AND WALDROUP: VITAMIN D SOURCES AND BROILER PERFORMANCE 47 TABLE 1. Composition (g/kg) and nutrient analysis of diets Ingredient 0 to 21 d 21 to 42 d Yellow corn Soybean meal (48% CP) Poultry oil Dicalcium phosphate Ground limestone DL-Methionine (98%) Feed-grade salt Coban-60 A Vitamin premix B Trace mineral mix C Choline chloride (60%) D L-Threonine L-Lysine HCl (98%) Nutrient analysis E ME, kcal/kg 3, , Crude protein, % Crude protein, % (Analyzed) Calcium, % Calcium, % (Analyzed) Total P, % Total P, % (Analyzed) Nonphytate P, % Methionine, % Lysine, % TSAA, % A Elanco Animal Health Division of Eli Lilly & Co., Indianapolis IN. B Provided per kilogram of diet: 8,800 IU vitamin A; 20 IU vitamin E; mg vitamin B 12 ; 8 mg riboflavin; 50 mg niacin; 15 mg pantothenic acid; 465 mg choline; 2 mg vitamin K; 1 mg folic acid; 2 mg thiamin; 2.5 mg pyridoxine; 0.1 mg D- biotin; 135 mg ethoxyquin; 0.1 mg Se. C Provided per kilogram of diet: Mn (from MnSO4 H20), 100 mg; Zn (from ZnSO4 7H2O), 100 mg; Fe (from FeSO4 7H2O), 50 mg; Cu (from CuSO4 5H20), 10 mg; I from Ca(IO3)2 H20), 1 mg. D Provided 236 mg/kg supplemental choline. E Calculated from NRC (1994) adjusted for crude protein and moisture contents of ingredients unless otherwise noted. ity of TD, using the scoring system of Edwards and Veltmann [25]. Statistical Analysis Pen means were the experimental unit for live production and bone data. Data were subjected to ANOVA as a factorial arrangement of treatments with vitamin source, level, and outside versus inside pens as the main effects; all possible interactions among and between the main effects were evaluated using the general linear models procedure of SAS software [26]. Mortality data were transformed to n + 1 prior to analysis; means are presented as natural numbers. Significant differences among or between means were separated by repeated t-tests using the least squares means option of SAS software. Statements of significance were based on P 0.05 unless otherwise noted. After ANOVA, the data were subjected to nonlinear regression using the PROC NLIN of SAS software [26] to estimate the vitamin D needs from the two sources as described by Robbins et al. [27], Yu and Morris [28], and Yan et al. [29]. However, this analysis did not result in a good fit as indicated by R 2 values. Vitamin D Analysis RESULTS Analyses of the diets for vitamin D activity indicated that the diets were in reasonable agreement with calculated values (Table 2). Therefore, the results of the present study could be used with confidence in evaluating possible differences in performance related to vitamin D sources.

4 48 JAPR: Research Report TABLE 2. Calculated and analyzed levels of vitamin D in test diets A 0 to 21 d 21 to 42 d Source of Expected Found Expected Found vitamin D B (µg/kg) (µg/kg) (µg/kg) (µg/kg) VIT-D < <5 VIT-D VIT-D VIT-D VIT-D VIT-D OH-D < <2 25-OH-D OH-D OH-D OH-D OH-D A Assays conducted by Monsanto Animal Nutrition, Naperville, IL. B VIT-D3 = cholecalciferol; 25-OH-D3 = 25-hydroxycholecalciferol. Live Performance There were no significant differences between inside or outside pens, nor was there any interaction of pen location with source or level of vitamin D. Therefore, pen location was eliminated as a variable. At 21 and 42 d, the body weights of birds fed the 25-OH-D3 were significantly greater than those of birds fed VIT-D3 (Table 3). The level of vitamin D in the diet also influenced body weight. At 21 and 42 d, vitamin D greater than 500 IU/kg appeared necessary for maximum body weight. However, at 42 d the interaction of source and level of vitamin D influenced BW. When VIT-D3 was used to provide the vitamin D source, approximately 1,000 to 2,000 IU/kg was needed to maximize body weight, but when 25-OH-D3 was used to provide the vitamin D source, there were no significant differences in BW among chicks fed any of the levels of vitamin D. Feed conversion, expressed as grams of feed required per gram of gain, was not significantly affected by dietary treatments at 21 d (Table 3). At 42 d, birds that had been fed diets containing the 25-OH-D3 had significantly poorer feed conversion than those fed the diets with VIT-D3. A part of this difference may be attributed to the greater body weights of birds fed the diets supplemented with 25-OH-D3. The poultry industry uses various factors to adjust for differences in feed conversion related to body weight, but no attempt was made in this study to do so as these values are generally arbitrary. Feed conversion was significantly influenced by level of vitamin D, but the response followed no consistent trend related to level of vitamin D. No significant interaction between source and level of vitamin D was observed for feed conversion. Mortality during the study was not significantly affected by source or level of vitamin D, with no interaction between source and level of vitamin D (Table 3). No trends in mortality were noted in relation to source or level of vitamin D. Bone Development Bone ash at 21 and 42 d was significantly increased in birds fed diets supplemented with 25-OH-D3 as compared to those fed diets supplemented with VIT-D3 (Table 4). The level of vitamin D also influenced bone ash at 21 and 42 d. However, a significant interaction between source and level of vitamin D at 21 and 42 d was also observed. Approximately 2,000 IU/kg of VIT-D3 was needed to maximize bone ash at 21 and 42 d. When 25-OH-D3 was provided as the vitamin D source, there were no significant differences among chicks fed 250 IU/kg to 4,000 IU/kg. The incidence of TD (percentage of birds having TD scores greater than zero) in broilers was significantly influenced by source of vitamin D at 21 and 42 d (Table 4). Birds fed diets supplemented with 25-OH-D3 had a significantly lower incidence of TD than did birds

5 FRITTS AND WALDROUP: VITAMIN D SOURCES AND BROILER PERFORMANCE 49 TABLE 3. Effects of source and level of vitamin D on body weight, feed conversion, and mortality (means of four replicate pens of 60 male broilers) Body weight (g) Feed:gain ratio C Mortality (%) D Vitamin D Vitamin D level source A (IU/kg) B 21 d 42 d 0 21 d 0 42 d 0 21 d 0 42 d Source of vitamin D Vit-D3 784 b 2,548 b b OH-D3 806 a 2,608 a a Level of vitamin D b 2,523 b ab ab 2,509 b a b 2,576 ab c , a 2,603 a bc , a 2,614 a ab , a 2,645 a bc Source level Vit-D ,461 cd Vit-D ,421 d Vit-D ,487 bcd Vit-D3 1, ,596 ab Vit-D3 2, ,664 a Vit-D3 4, ,659 a OH-D ,584 ab OH-D ,597 ab OH-D ,666 a OH-D3 1, ,610 a OH-D3 2, ,564 abc OH-D3 4, ,630 a Source of variance Probability > F Source of D Level of D Source level SEM a d Within comparisons, means with common superscripts do not differ significantly (P 0.05). A Vit-D3 = cholecalciferol; 25-OH-D3 = 25-hydroxycholecalciferol. B Levels of 25-hydroxycholecalciferol were calculated to be equal to similar levels of cholecalciferol based on conversion of µg of cholecalciferol to 1 IU. C Grams of feed required per gram of gain, adjusted for mortality. D Coefficient of variation adjusted for transformed means. Natural numbers presented in table. fed diets supplemented with VIT-D3. Levels of vitamin D had no significant effect on incidence of TD at 21 d, but at 42 d vitamin D levels significantly influenced TD scores. Overall, birds fed diets with 2,000 or 4,000 IU/kg had a significantly lower incidence of TD at 42 d than those fed lower levels. Incidence of TD at the lower levels of vitamin D did not appear to be influenced by source of vitamin D; however, at the higher levels of vitamin D supplementation, the use of 25-OH-D3 appeared to be more effective in reducing the incidence of TD than did VIT-D3. The severity of tibial dyschondroplasia, expressed as percentage of birds having a TD score of 3, was influenced by source and level of vitamin D with a significant interaction between source and level (Table 4). The severity of TD in birds fed diets supplemented with 25-OH-D3 was significantly lower than that of birds fed VIT-D3 at 21 d and was lower (P = 0.10) at 42 d. Increasing the level of vitamin D reduced the severity of TD, with 25-OH-D3 appearing to be more effective at lower levels of supplementation than VIT-D3. DISCUSSION The present study was conducted to compare VIT-D3 and 25-OH-D3 as sources of vitamin D activity when fed to male broilers at levels

6 50 JAPR: Research Report TABLE 4. Effects of source and level of vitamin D on tibia ash and the incidence and severity of tibial dyschondroplasia (TD) in male broilers Tibia ash C (%) TD incidence D (%) TD severity E (%) Vitamin D Vitamin D level source A (IU/kg) B 21 d 42 d 21 d 42 d 21 d 42 d Source of vitamin D Vit-D b b a a a OH-D a a b b b Level of vitamin D c b a ab a c b a ab a d b a a ab 1, bc a a abc ab 2, ab a b 9.38 c bc 4, a a b bc 7.81 c Source level Vit-D bcd d abc ab ab Vit-D e e abc ab abc Vit-D e e ab a a Vit-D3 1, d cd a ab a Vit-D3 2, abc ab bc 9.38 c bcd Vit-D3 4, a ab cd bc 6.25 d 25-OH-D cd bc ab bc a 25-OH-D ab a abc bc ab 25-OH-D a a cd 6.25 c cd 25-OH-D3 1, a a cd 3.13 c 9.38 d 25-OH-D3 2, ab ab de 9.38 c cd 25-OH-D3 4, a a e 8.33 c 9.38 d Source of variance Probability > F Source of D Level of D Source level SEM a d Within comparisons, means with common superscripts do not differ significantly (P 0.05). A Vit-D3 = cholecalciferol; 25-OH-D3 = 25-hydroxycholecalciferol. B Levels of 25-hydroxycholecalciferol were calculated to be equal to similar levels of cholecalciferol based on conversion of µg of cholecalciferol to 1 IU. C Percentage of ash in dry, fat-free bone. D Percentage with TD score greater than 0 (no apparent TD) based on system of Edwards and Veltmann [25]. E Percentage with TD score of 3 (most severe). ranging from below NRC recommendations to those commonly fed in commercial formulations on performance and bone development. Most research trials using 25-OH-D3 have been conducted using battery trials with very few trials conducted using floor pens to demonstrate typical stresses observed in commercial situations [11]. Broilers fed diets supplemented with 25- OH-D3 had a significant increase in body weight compared to those fed VIT-D3. This result is consistent with previous studies conducted supplementing broiler diets with 25-OH-D3 compared to diets supplemented with vitamin D3 [6, 14, 30]. Birds fed 25-OH-D3 had similar BW at all levels of vitamin D supplementation; however, broilers fed VIT-D3 required at least 1,000 IU/kg to maximize body weight. Feed conversion was higher for the birds fed 25-OH-D3 versus VIT-D3. Most trials show very little difference or improved FCR when 25-OH-D3 is fed to broilers [16, 31]. No differences were observed in mortality at 21 or 42 d. This result is in agreement with a study conducted by Yarger et al. [16]. Source and level of vitamin D influenced bone ash at 21 and 42 d. The incidence and severity of TD was significantly influenced by diets supplemented with 25-OH-D3. Birds fed

7 FRITTS AND WALDROUP: VITAMIN D SOURCES AND BROILER PERFORMANCE 51 diets with 25-OH-D3 had a lower incidence and severity of TD. Roberson [31] found that addition of 25-OH-D3 does not prevent the incidence of TD in broilers. However, our findings are in agreement with other studies in which the incidence and severity of TD are influenced by addition of 25-OH-D3 [8, 9,17, 32, 33]. The findings of the present study demonstrate that 25-OH-D3 is more metabolically potent on a per unit basis than cholecalciferol for support of BW and bone ash and for reducing the incidence and severity of TD. The differences were primarily observed at lower levels of vitamin D supplementation. At levels typically used by the poultry industry, few differences were observed among birds fed the two sources. However, use of 25-OH-D3 may allow for supplementation with lower levels of vitamin D or may provide a greater margin of safety under stress conditions. CONCLUSIONS AND APPLICATIONS 1. Cholecalciferol (VIT-D3) and 25-OH-D3 were compared as sources of vitamin D in diets for broiler chickens. 2. At 21 and 42 d, the BW and tibia ash of birds fed 25-OH-D3 were significantly greater than those of birds fed VIT-D3. 3. The incidence and severity of TD were significantly lower for birds fed 25-OH-D3 and were reduced by increasing levels of vitamin D, regardless of source. 4. The differences in performance between birds fed 25-OH-D3 and those fed VIT-D3 were observed primarily at levels lower than typically used in the poultry industry. At higher usage levels typical of current usage, few differences were observed. 5. The use of 25-OH-D3 as a source of vitamin D may allow for supplementation with lower levels of vitamin D or may provide a greater margin of safety. REFERENCES AND NOTES 1. McChesney, E. W., and N. J. Giacomino Studies of calcium and phosphorus metabolism in the chick. III. Some time relationships in the action of vitamin D. J. Nutr. 29: Waldroup, P. W., C. B. Ammerman, and R. H. Harms The relationship of phosphorus, calcium, and vitamin D 3 in the diet of broiler-type chicks. Poult. Sci. 42: Norman, A. W Studies on the vitamin D endocrine system in the avian. J. Nutr. 117: National Research Council Nutrient Requirements of Poultry. 9th rev. ed. National Academy Press, Washington, DC. 5. BASF Corporation Vitamins, One of the Most Important Discoveries of the Century. 5th rev. ed. BASF Corporation, Mount Olive, NJ. 6. McNutt, K. W., and M. R. Haussler Nutritional effectiveness of 1,25-dihydroxycholecalciferol in preventing rickets in chicks. J. Nutr. 103: Lofton, J. T., and J. H. Soares, Jr The effects of vitamin D on leg abnormalities in broilers. Poult. Sci. 65: Edwards, H. M., Jr The effect of dietary cholecalciferol, 25-hydroxycholecalciferol and 1,25-dihydroxycholecalciferol on the development of tibial dyschondroplasia in broiler chickens in the absence and presence of disulfiram. J. Nutr. 119: Edwards, H. M., Jr Efficacy of several vitamin D compounds in the prevention of tibial dyschondroplasia in broiler chickens. J. Nutr. 120: Collins, E. D., and A. W. Norman Vitamin D. Pages in Handbook of Vitamins. L. J. Machlin, ed. Marcel Dekker, New York. 11. Ward, N. E Research examines use of 25-OH vitamin D3 in broiler diets. Feedstuffs 67(30): Sunde, M. L What about 25-Hydroxycholecalciferol for poultry? Proc. Distillers Feed Res. Counc. 30: McNaughton, J. L., E. J. Day, and B. C. Dilworth The chick s requirement for 25-hydroxycholecalciferol and cholecalciferol. Poult. Sci. 56: Cantor, A. H., and W. L. Bacon Performance of caged broilers fed vitamin D3 and 25-hydroxyvitamin D 3. Poult. Sci. 57: (Abstr.) 15. Soares, J. H., M. R. Swerdel, and E. H. Bossard Phosphorus availability 1. The effect of chick age and vitamin D metabolites on the availability of phosphorus in defluorinated phosphate. Poult. Sci. 57: Yarger, J. G., C. A. Saunders, J. L. McNaughton, C. L. Quarles, B. W. Hollis, and R. W. Gray Comparison of dietary 25-hydroxycholecalciferol and cholecalciferol in broiler chickens. Poult. Sci. 74: Mitchell, R. D., and H. M. Edwards, Jr The effects of ultraviolet light and cholecalciferol and its metabolites on the development of leg abnormalities in chickens genetically selected for high or low incidence for tibial dyschondroplasia. Poult. Sci. 76: Alpharma, Fort Lee, NJ. 19. Hy-D, Monsanto Animal Nutrition, Naperville, IL. 20. Analyzed by Agricultural Diagnostic Laboratory, University of Arkansas, Fayetteville AR. 21. Analyzed by Monsanto Animal Nutrition, Naperville, IL.

8 52 JAPR: Research Report 22. Cobb 500, Cobb-Vantress Inc., Siloam Springs, AR. 23. Polylite Super, Southwestern Sales Company, Rogers, AR. Curtains had an ultraviolet inhibition rating of 70%. 24. AOAC Vitamin D3 in poultry feed supplements. Method Pages in Official Methods of Analysis. 15th ed. Association of Official Analytical Chemists, Arlington, VA. 25. Edwards, H. M., Jr., and J. R. Veltmann, Jr The role of calcium and phosphorus in the etiology of tibial dyschondroplasia in young chicks. J. Nutr. 113: SAS Institute SAS User s Guide: Statistics. Version 6.03 Edition. SAS Institute Inc., Cary, NC. 27. Robbins, K. R., H. W. Norton, and D. H. Baker Estimation of nutrient requirements from growth data. J. Nutr. 109: Yu, S., and J. G. Morris Chloride requirements of kittens for growth is less than current recommendations. J. Nutr. 129: Yan, F., J. K. Kersey, and P. W. Waldroup Phosphorus requirements of broiler chicks three to six weeks of age as influenced by phytase supplementation. Poult. Sci. 80: Yarger, J. G., J. L. McNaughton, C. L. Quarles, B. W. Hollis, and R. W. Gray Safety of 25-hydroxycholecalciferol as a source of cholecalciferol in poultry rations. Poult. Sci. 74: Roberson, K. D Hydroxycholecalciferol fails to prevent tibial dyschondroplasia in broiler chicks raised in battery brooders. J. Appl. Poult. Res. 8: Rennie, J. S., and C. C. Whitehead Effectiveness of dietary 25- and 1-hydroxycholecalciferol in combating tibial dyschondroplasia in broiler chickens. Br. Poult. Sci. 37: Zhang, X., G. Liu, G. R. McDaniel, and D. A. Roland Responses of broiler lines selected for tibial dyschondroplasia incidence to supplementary 25-hydroxycholecalciferol. J. Appl. Poult. Res. 6: Acknowledgments Financial support, contribution of materials, and vitamin D assays were graciously provided by Monsanto Animal Nutrition, Naperville, IL.