Foot Ash as a Means of Quantifying Bone Mineralization in Chicks

2006 Poultry Science Association, Inc. Foot Ash as a Means of Quantifying Bone Mineralization in Chicks A. R. Garcia and N. M. Dale 1 Department of Poultry Science, University of Georgia, Athens 30602-2772 Primary Audience: Nutritionists, Researchers, Veterinarians SUMMARY Three studies were conducted to evaluate foot ash as an alternative method to quantify the degree of bone mineralization in broiler chicks. In Experiment 1, the objective was to evaluate whether degree of bone mineralization would be reflected by foot ash in broiler chickens at 14 d of age. Birds receiving 0.24, 0.32, or 0.40% of dietary available phosphorus (AP) demonstrated significant differences in foot ash (10.93, 13.46, and 15.45%, respectively). In Experiment 2, we determined that the time required for a foot sample to dry to a constant weight at 105 C was 48 h. In Experiment 3, the objectives were to compare the response of bone mineralization to dietary phosphorus levels as reflected by foot ash and tibia bone ash and to evaluate the effect of fat extraction on the determination of these parameters. Broiler chicks were fed graded levels of AP (0.25, 0.30, 0.35, and 0.40%) from 1 to 14 d of age. Significantly different responses between each dietary AP level could be detected by any method used. A significant linear response was observed between dietary AP and percentage of extracted or unextracted tibia or foot ash, with determination coefficients above 90% found for all 4 methods used. Results confirmed that the dietary phosphorus levels affected bone mineralization and that the degree of mineralization could reliably be reflected by foot ash. The assay was found to be as reliable as the tibia bone ash in reflecting degree of bone mineralization in chicks during the first 14 d of age. Fat extraction did not affect the reliability of either assay. Key words: foot ash, tibia bone ash, bone mineralization, phosphorus, broiler 2006 J. Appl. Poult. Res. 15:103 109 DESCRIPTION OF PROBLEM The need to quantify bone mineralization has long been recognized by researchers in poultry nutrition and physiology. At the present time, and for most of the past century, the most popular assay for this purpose is that of tibia bone ash as described by the AOAC [1]. This wellknown assay has been successfully used in innumerable studies but has the disadvantage of being very labor intensive and, thus, slow and expensive. As early as 1855, Fremy [2] conducted studies to determine the ash content of bones of numerous animal species. In the following decades, bone ash was used to quantify skeletal abnormalities in rachitic children [3] and horses [4] as well as in other species. Determination of bone ash was thus a well-established technique for describing skeletal abnormalities long before the need arose for a means to quantify bone mineralization in poultry. Some of the first uses of bone ash as a parameter in nutrition research are described in the reports of Bethke et al. [5] and Dutcher [6] in work with rats in 1923 and 1925, respectively. 1 Corresponding author: ndale@uga.edu

104 Still, in the early 1920s [7] and as late as 1926 [8], body weight gain and death were often the principal response criteria in poultry research on antirachitic factors. In the late 1920s, however, a series of 3 studies on rickets in chickens by Heuser and Norris at Cornell University [9, 10, 11] firmly established tibia bone ash as the most popular parameter for quantifying bone mineralization. Upon review of these and other papers from the period, however, it is evident that no effort was made to compare the efficacy of this assay with other less tedious procedures. Instead, when researchers began to focus attention on the antirachitic properties of cod liver oil [9, 10] and direct sunlight [11] and needed an assay to quantify bone mineralization, a procedure (bone ash) was adopted that had been reported in the scientific literature for almost 80 yr. However, it is clear from reviewing these papers that the bone ash assay was simply adopted and never critically tested or compared with other possible assays. Very small numbers of chicks (as few as 3/treatment) were often used in these studies. Because differences in response were often great, it was correctly observed that tibia (or tibia + femur) ash was, in fact, a useful parameter. Thus, although the tibia bone ash assay has served researchers very well for almost a century, it has never been demonstrated to be a superior or simpler and less time-intensive procedure. During the early 1940s, several studies explored the use of toe ash as a means of quantifying bone mineralization and found it to be equally acceptable to the existing tibia ash assay [12, 13]. The toe ash assay has been used sporadically since then [14, 15], and Yoshida and Hoshii [16] have reported a high degree of correlation with tibia bone ash. More recently, Ravindran et al. [17], in studies on the biological availability of phosphorus, found toe ash plus body weight to be slightly more sensitive than tibia ash and vastly easier to obtain. The tips or heads are those portions of the bone most sensitive to differences in mineralization because they include the growth plate. Thus, it can be hypothesized that the entire foot, with 17 individual bones [18], as opposed to a tibia or toe, might well be the appendage of choice in bone mineralization tests. Such an assay was initially evaluated at this laboratory [19, 20] and found to give encouraging results. It is obvious JAPR: Research Report that use of the dried whole foot; rather than the dried, cleaned, fat-extracted tibia; would constitute a far less labor-intensive assay. The question remains as to whether possible differences in skin, scales, flesh, and fat might introduce an unacceptable artifact into the results of a foot ash assay. A series of studies was conducted to refine an assay for foot ash and to evaluate whether this assay might prove reliable in reflecting degree of bone mineralization in chicks. Should these studies prove successful, the assay might also prove to be of special value in diagnosing leg problems in the field where the traditional tibia ash assay is far too slow. MATERIALS AND METHODS Experiment 1 This experiment was conducted to determine if the foot ash assay would be sensitive enough to detect differences in bone mineralization in response to varying levels of dietary phosphorus. Ninety 1-d-old broiler chickens were randomly assigned to 3 dietary treatments containing 0.24, 0.32, and 0.40% available phosphorus (AP) These levels were designed to cover a range from a severe deficiency to a marginally adequate level. Treatments contained 3 replicates of 10 birds each. Nutrient levels for all dietary treatments were based on those used in commercial broiler starter diets [21] varying only in AP concentrations (Table 1). The birds were allocated to battery brooders and fed the experimental diets ad libitum from 1 to 14 d of age. Body weight and feed intake were recorded, and body weight gain and feed conversion were calculated at the end of the experiment. The chickens were euthanized by CO 2 asphyxiation, and the foot samples were obtained by severing the foot at the tibiometatarsal joint, labeled, dried for 60 h at 105 C, weighed, and placed in porcelain crucibles to be ashed at 600 C overnight. Experiment 2 Because ash determination is based on the dry weight of a sample before ashing, the objective of this study was to determine the minimum time required to obtain an adequately dried foot for the foot ash assay. Because muscle, tendons, skin, and adipose tissue are not removed, a satis-

GARCIA AND DALE: FOOT ASH IN CHICKS 105 Table 1. Composition (as fed-basis) of the experimental diets, Experiment 2 0.24% AP 0.32% AP 0.40% AP Ingredient (%) (%) (%) Corn 57.78 57.31 56.82 Soybean meal 36.61 36.68 36.76 Animal fat 2.05 2.23 2.41 Limestone 2.04 1.88 1.73 Dicalcium phosphate 0.53 0.91 1.29 Salt 0.47 0.47 0.47 DL-Methionine 0.20 0.20 0.20 Vitamin mix 1 0.25 0.25 0.25 Mineral mix 2 0.07 0.07 0.07 Contents by calculation TME n, kcal/kg 3,025 3,025 3,025 Protein, % 22 22 22 Available phosphorus, % 0.24 0.32 0.40 Calcium, % 0.95 0.95 0.95 1 Vitamin mix provided the following (per kg of diet): thiamin mononitrate, 2.4 mg; nicotinic acid, 44 mg; riboflavin, 4.4 mg; D-Ca pantothenate, 12 mg; vitamin B 12 (Cobalamin), 12.0 g; pyridoxine HCL, 4.7 mg; D-biotin, 0.11 mg; folic acid, 5.5 mg; menadione sodium bisulfate complex, 3.34 mg; choline chloride, 220 mg; cholecalciferol, 1,100 IU; transretinyl acetate, 5,500 IU; all-rac-tocopherol acetate, 11 IU; ethoxyquin, 125 mg. 2 Trace mineral mix provides the following (per kg of diet): manganese (MnSO 4 H 2 O), 60 mg; iron (FeSO 4 7H 2 O),30mg; zinc (ZnO), 50 mg; copper (CuSO 4 5H 2 O), 5 mg; iodine (ethylene diamine dihydroiodide), 0.15 mg; selenium (sodium selenite), 0.3 mg. factory drying time could be markedly extended. Thirty feet were obtained from 16-d-old broiler chickens. All birds had been fed a diet adequate in all nutrients (Tables 1 and 2) containing 0.45% AP and 0.90% calcium. No symptoms of disease were observed. The feet were severed at the tibiometatarsal joint, labeled, weighed, and placed in a drying oven at 105 C. At 6, 12, 24, 36, 48, 60, 72, and 90 h, all the feet were removed from the drying oven to record their weight and immediately returned to continue drying. After 90 h, all feet were ashed at 600 C Table 2. Composition (as fed-basis) of the diets deficient in available phosphorus (AP), Experiment 3 0.25% AP 0.30% AP 0.35% AP 0.40% AP Ingredient (%) (%) (%) (%) Corn 57.94 57.71 57.50 57.29 Soybean meal 36.58 36.62 36.65 36.69 Animal fat 2.00 2.08 2.16 2.23 Limestone 1.84 1.67 1.50 1.33 Dicalcium phosphate 0.65 0.93 1.20 1.47 Salt 0.47 0.47 0.47 0.47 DL-Methionine 0.20 0.20 0.20 0.20 Vitamin mix 1 0.25 0.25 0.25 0.25 Mineral mix 2 0.07 0.07 0.07 0.07 Contents by calculation TME n, kcal/kg 3,025 3,025 3,025 3,025 Protein, % 22 22 22 22 Available phosphorus, % 0.25 0.30 0.35 0.40 Calcium, % 0.95 0.95 0.95 0.95 1 Vitamin mix provided the following (per kg of diet): thiamin mononitrate, 2.4 mg; nicotinic acid, 44 mg; riboflavin, 4.4 mg; D-Ca pantothenate, 12 mg; vitamin B 12 (Cobalamin), 12.0 g; pyridoxine HCL, 4.7 mg; D-biotin, 0.11 mg; folic acid, 5.5 mg; menadione sodium bisulfate complex, 3.34 mg; choline chloride, 220 mg; cholecalciferol, 1,100 IU; transretinyl acetate, 5,500 IU; all-rac-tocopherol acetate, 11 IU; ethoxyquin, 125 mg. 2 Trace mineral mix provides the following (per kg of diet): manganese (MnSO 4 H 2 O), 60 mg; iron (FeSO 4 7H 2 O),30mg; zinc (ZnO), 50 mg; copper (CuSO 4 5H 2 O), 5 mg; iodine (ethylene diamine dihydroiodide), 0.15 mg; selenium (sodium selenite), 0.3 mg.

106 Table 3. Growth performance and foot ash percentage of broiler chickens fed graded levels of available phosphorus (AP) from 1 to 14 d of age, Experiment 1 1 Weight Feed Foot AP 2 gain conversion ash (%) (g/chick) (kg:kg) (%) 0.24 253 c 1.35 a 10.9 c 0.32 283 b 1.32 ab 13.5 b 0.40 315 a 1.28 b 15.5 a Pooled SEM 9.0 0.12 0.30 a c Means within a column with no common superscript differ significantly (P < 0.05). 1 Means represent 6 pens of 5 chicks each. 2 AP = available phosphorus. overnight, and the ash percentage was calculated considering the weight at each time the samples were removed. Experiment 3 The objective of this experiment was to compare the results of the foot ash and the tibia bone ash assays and to investigate whether fat extraction would affect the ability of these assays to detect differences in bone mineralization as a response to dietary phosphorus. One hundred sixty broiler chickens that were 1 d old were randomly assigned among 4 dietary treatments that consisted of graded levels of AP (0.25, 0.30, 0.35, and 0.40%). The diets were formulated to be isocaloric and isonitrogenous, and varied only in the concentrations of AP mentioned above. Nutrient requirements were based on those used in commercial broiler starter diets [21] (See Table 2). The birds were housed in battery brooders and received the experimental diets ad libitum from 1 to 14 d of age. At the end of the experiment, body weight gain and feed conversion were calculated. For the tibia and foot ash assays, the chickens were euthanized with CO 2, and both JAPR: Research Report tibias and feet from each bird per pen were removed and labeled to preserve the identity of the 4 samples. All tibias were boiled for approximately 5 min to loosen the muscle and connective tissue, which were removed along with the fibulas, leaving the lateral condyle and the ossified tibial cartilage [18]. The right tibias were used for determining the percentage of ash according to the AOAC method [1] that includes a fat extraction procedure. The left tibias were cleaned from adhering tissue, dried, and ashed without fat extraction. The left feet were subjected to the same fat extraction procedure as the right tibias. The right feet were assayed for foot ash as described for Experiment 1. Statistical Analysis Body weight gain, feed conversion, and percentage of bone ash were analyzed by the AN- OVA procedure of SAS [22], followed by the Tukey s test for comparison of treatment means. Bone ash data obtained from each method used in Experiment 3 were regressed to supplemental phosphorus intake and fitted to a linear model using Proc Reg of SAS [22]. RESULTS AND DISCUSSION In experiment 1 (Table 3), as dietary AP increased, significantly greater weight gains were observed, whereas feed conversion decreased with each increase in AP. There was a significant increase in the percentage of foot ash with increasing AP, which followed a significantly linear response (r 2 = 0.90). Therefore, foot ash effectively reflected the response in bone mineralization to different dietary phosphorus levels, and it could potentially be used to quantify bone mineralization status in chickens. The calculated percentage of foot ash for each drying time is presented in Table 4. The Table 4. Percentage of foot ash obtained after different drying times, Experiment 2 Drying time (h) 0 6 12 24 36 48 60 72 90 FA 1 (%) 4.52 d 11.11 c 14.13 b 14.74 a 14.86 a 14.90 a 14.91 a 14.93 a 14.94 a SEM 2 0.04 0.13 0.14 0.15 0.15 0.15 0.15 0.15 0.15 a d Means within a row with no common superscript differ significantly (P < 0.05). 1 FA = foot ash. 2 n = 30 feet, r 2 = 0.38, linear trend, P < 0.0001.

GARCIA AND DALE: FOOT ASH IN CHICKS 107 Table 5. Growth performance and foot ash percentage of broiler chickens fed graded levels of available phosphorus (AP) from 1 to 14 d of age, Experiment 3 1 Weight Feed AP 2 gain conversion (%) (g/chick) (kg:kg) 0.25 241 b 1.40 0.30 285 a 1.30 0.35 283 a 1.33 0.40 310 a 1.28 Pooled SEM 14.0 0.16 r 2 0.48 0.17 Linear trend P-value 0.003 0.11 a c Means within a column with no common superscript differ significantly (P < 0.05). 1 Means represent 4 pens of 10 chicks each. 2 AP = available phosphorus. percentage of foot ash increased significantly from 0 to 24 h of drying, indicating that moisture content of the feet had yet to reach a plateau. No significant increases were observed thereafter. However, at 24 h the sample was not completely dry, because the ash percentage difference between 24 and 90 h drying was 0.2%. The dry weight did not become virtually constant until after 48 h. Thus, for diagnostic purposes where speed is necessary, a 24-h drying period would be adequate, but for research purposes, a 48-h drying period is necessary. In Experiment 3, the chicks fed 0.25% AP had significantly lower weight gain (Table 5) than the chicks of the other treatments. No sig- nificant differences in feed conversion were observed at any dietary AP level. The percentage ash (Table 6) obtained with all 4 assay procedures conducted herein was significantly responsive to dietary phosphorus levels, and significant differences could be detected at each dietary level of AP. The ash percentage obtained by all the assays followed a significantly linear response, and determination coefficients obtained by each method were above 90%. Fat extraction did not affect the sensitivity of either tibia or foot ash assays. Numerically, the greatest bone ash percentage was observed in the lipid-free tibia ash assay, for which the response ranged from 31.8 to 41.5% for 0.25 to 0.40% dietary AP, respectively. This finding is not surprising because the percentage of ash is highly dependent on the amount of organic matter burned. For the unextracted tibia, the percentage of ash at each phosphorus levels was slightly lower than for the lipid-free tibia and ranged from 29.6 to 39.1%. When fat was extracted from the feet, a slightly higher percentage of ash was also observed. However, this result did not affect the sensitivity of the assay. For the fat-extracted foot ash assay, results ranged from 11.7 to 16.3%, whereas for the unextracted foot ash, values ranged from 11.6 to 15.7%. The effect of fat extraction in reducing variation between samples was first noted in the early 1920s by Bethke et al. [5] when femur and tibia of rats were analyzed. Table 6. Comparison between foot and tibia ash with or without fat extraction, Experiment 3 1 Foot Foot Tibia Tibia AP 2 ash NFE 3 ash FE 4 ash NFE 3 ash FE 4 (%) (%) (%) (%) (%) 0.25 11.6 d 11.7 d 29.6 d 31.8 d 0.30 12.6 c 12.8 c 32.6 c 34.8 c 0.35 14.6 b 15.1 b 36.9 b 39.5 b 0.40 15.7 a 16.3 a 39.1 a 41.5 a Pooled SEM 0.25 0.32 0.51 0.66 r 2 0.92 0.90 0.90 0.93 Parameter estimates regression lines Intercept 8.631 ± 0.532 8.881 ± 0.426 23.638 ± 0.890 25.661 ± 1.148 Slope 0.040 ± 0.003 0.036 ± 0.003 0.082 ± 0.006 0.085 ± 0.008 P-value < 0.0001 < 0.0001 < 0.0001 < 0.0001 a d Means within a column with no common superscript differ significantly (P < 0.05). 1 Means represent 4 pens of 10 chicks each. 2 AP = available phosphorus. 3 NFE = nonfat extracted. 4 FE = fat extracted.

108 Ever since, the lipid extraction procedure has been part of the tibia bone ash method. However, under the present conditions, fat extraction did not affect the sensitivity of the assays, because significant differences between dietary phosphorus levels could be detected by using the unextracted foot or tibia ash method. Bone ash measurements from older birds may be more variable due to higher lipid content, although that hypothesis is still subject to evaluation. In numerous reports, it has been demonstrated that alternative methods to assess bone mineralization are highly correlated to tibia ash, particularly toe ash [12, 13, 14, 15, 17]. Foot ash might represent bone mineralization status more accurately than toe ash because a greater JAPR: Research Report number of bones are being included in the determination. This, however, was not addressed in the current studies. A high degree of agreement between tibia and foot ash should not be surprising and has been previously suggested [19, 20, 23]. Further evaluation of how responsive foot ash might be to other factors influencing bone mineralization, such as calcium or vitamin D, is still necessary. Nonetheless, the use of foot ash as a criterion would facilitate research on feed ingredients or feed additives that influence bone mineralization. Moreover, potential exists for its use in assessing bone mineralization disorders under field situations in a more rapid and expeditious manner, because results can be obtained within 36 h after the samples arrive at the laboratory. CONCLUSIONS AND APPLICATIONS 1. The foot ash assay effectively reflects differences in bone mineralization from different levels of dietary phosphorus and is at least as precise as the tibia bone ash assay. 2. Fat extraction did not affect the sensitivity of the tibia or foot ash assay. This finding drastically decreases the time required to conduct these assays. 3. The simplicity of the foot ash assay should encourage its use in a variety of applications including research and diagnostics. REFERENCES AND NOTES 1. Association of Official Analytical Chemists. 2000. Pages 61 62 in Official Methods of Analysis of the Association of Official Analytical Chemists. Vol. 2. 17th ed. Assoc. Off. Anal. Chem., Washington, DC. 2. Fremy, E. 1855. Recherches chimiques sur les os. Ann. Chim. Phys. 214:47 109. 3. Brubacher, H. 1890. Ueber den Gelhalt an anorganishen Stoffen, besonders and Kalk, in den Knochen und Organen normaler und rhachitisher kinder. Z. Biol. 27:517 549. 4. McCrudden, F. H. 1910. Studies of bone metabolism, especially the pathological process, etiology and treatment of osteomalacia. Arch. Intern. Med. 5:596 630. 5. Bethke, R. M., H. Steenbock, and M. T. Nelson. 1923. Fatsoluble vitamins. XV. Calcium and phosphorus relations to growth and composition of blood and bone with varying vitamin intake. J. Biol. Chem. 58:71 103. 6. Dutcher, R. A., M. Creighton, and H. A. Rothrock. 1925. Vitamin studies. XI. Inorganic blood phosphorus and bone ash in rats fed on normal, rachitic, and irradiated rachitic diets. J. Biol. Chem. 66:401 407. 7. Hart, E. B., H. Steenbock, S. Lepkovsky, and J. G. Halpin. 1923. The nutritional requirements of baby chicks. III. The relation of light to the growth of the chicken. J. Biol. Chem. 58:33 42. 8. Mussehl, F. E., R. Hill, and J. A. Rosenbaum. 1926. The antirachitic properties of irradiated feedstuffs. Poult. Sci. 6:25 30. 9. Heuser, G. F., and L. C. Norris. 1926. Rickets in chicks. I. Variations in the antirachitic potency of different brands of cod liver oil. Poult. Sci. 6:9 17. 10. Heuser, G. F., and L. C. Norris. 1927. Rickets in chicks. II. Variations in the antirachitic potency of different grades of cod liver oil. Poult. Sci. 6:94 98. 11. Heuser, G. F., and L. C. Norris. 1929. Rickets in chicks. III. The effectiveness of mid-summer sunshine and irradiation from a quartz mercury vapor arc in preventing rickets in chicks. Poult. Sci. 8:89 98. 12. Baird, F. D., and M. J. MacMillan. 1942. Use of toes rather than tibiae in A.O.A.C. chick method of vitamin D determination. J. Assoc. Off. Agric. Chem. 25:518 524. 13. Evans, R. J., and J. S. Carver. 1944. The toe ash as a measure of calcification in chicks. Poult. Sci. 23:351 352. 14. Fritz, J. C., and T. Roberts. 1968. Use of toe ash as a measure of calcification in the chick. J. Assoc. Off. Anal. Chem. 51:519 594. 15. Potter, L. M. 1988. Bioavailability of phosphorus from various phosphates based on body weight and toe ash measurements. Poult. Sci. 67:96 102. 16. Yoshida, M., and H. Hoshii. 1983. Relationship between ash contents of the tibia bone and the toe of chicks. Jpn. Poult. Sci. 20:51 54.

GARCIA AND DALE: FOOT ASH IN CHICKS 109 17. Ravindran, V., E. T. Kornegay, L. M. Potter, B. O. Ogunabameru, M. K. Welten, J. H. Wilson, and M. Potchanakorn. 1995. An evaluation of various response criteria in assessing biological availability of phosphorus for broilers. Poult. Sci. 74:1820 1830. 18. Lucas, A. M., and P. R. Stettenheim. 1972. Avian Anatomy: Integument. Part I. US Gov. Printing Off., Washington, DC. 19. Méndez, A., and N. Dale. 1998. Foot ash as a parameter to assay bone mineralization. Poult. Sci. 77(Suppl. 1):40. (Abstr.) 20. Méndez, A., N. Dale, and M. Garcia. 1998. Comparison of parameters to assay bone mineralization. Poult. Sci. 77(Suppl. 1):176. (Abstr.) 21. Agri-Stats, Fort Wayne, IN. 22. SAS Institute Inc. 1990. SAS STAT User s Guide Release 6.08. SAS Inst. Inc., Cary, NC. 23. Dale, N., and A. R. Garcia. 2004. Evaluation of foot ash as an alternative to tibia bone ash for quantifying bone mineralization. Abstr. 19 in Int. Poult. Sci. Forum. SPSS, Tucker, GA.