2005 Poultry Science Association, Inc.
Comparison of Methods to Evaluate Bone Mineralization1 F. Yan, C. A. Keen, K. Y. Zhang, and P. W. Waldroup2 Poultry Science Department, University of Arkansas, Fayetteville, Arkansas 72701
Primary Audience: Nutritionists, Lab Managers, Researchers SUMMARY Extraction of lipid from tibias of broilers in the Association of Official Analytical Chemistsrecommended procedure is often a rate-limiting step in evaluation of bone mineralization. In addition, environmental concerns may limit the use of solvents to extract lipids. A study was conducted to compare various alternative methods of evaluating bone mineralization in young (0 to 21 d) broiler chicks. Male chicks of a commercial broiler strain were fed diets ranging from deficit to surfeit in supplemental phosphorus. At 21 d, all surviving birds were killed. From each bird, the toes were removed from the right leg, and the foot (from the tibiotarsal junction) was removed from the left leg. After drying, these were ashed with no lipid extraction. Tibias from the right leg were taken and subjected to lipid extraction prior to ashing; tibias from the left leg were ashed without lipid extraction. Estimates of phosphorus requirements by nonlinear regression using each of the measurements resulted in similar values. A high correlation existed between each of the alternative measures and values from extracted tibias, with unextracted tibia ash appearing to have the highest relationship (R2 = 0.95) followed by foot ash (R2 = 0.92) and toe ash (R2 = 0.88). Considerations for using any of these alternative methods are the greater amount of organic matter that is burned off in ashing, which might create problems with alarm systems in laboratories, possible differences in lipid metabolism due to nutrition, disease, or other factors, and effect of age of the chick. Key words: phosphorus, bone ash, toe ash, alternative methods 2005 J. Appl. Poult. Res. 14:492–498
DESCRIPTION OF PROBLEM Tibia ash has long been considered a response criterion to Ca and P concentrations in poultry diets [1, 2]. The tibia is the fastest growing bone in the body and is considered as the most sensitive to deficiencies in Ca or P [3]. Nelson and Walker [4] reported that the zone of proliferation in the developing tibia of the young chick is especially sensitive to nutritional defi1
ciencies and is quickly influenced by a P deficiency. Use of tibia ash is recommended in the official Association of Official Analytical Chemists [5] assay for vitamin D sources. The procedure typically followed [5] extracts the fat from the bone prior to ashing and is done to minimize the effects of differences in rate of growth among or between comparative groups. This extraction step has always been one of the rate-limiting factors in bone ash determination, and with the
Published with approval of the Director, Arkansas Agricultural Experiment Station, Fayetteville, AR 72701. 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:
[email protected].
YAN ET AL.: EVALUATION OF BONE MINERALIZATION growing environmental concerns, the use of solvents to extract the fat may be limited in the future. Toe ash has long been used as a means of evaluating bone development and has been shown by a number of authors to be at least equivalent to tibia ash in sensitivity to changes in dietary Ca and P concentrations [6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16]. In some of the early studies on use of toe ash the lipid was extracted from the toes, but most studies have used unextracted toes. In many of these studies, only 1 toe, usually the middle toe, was used for ashing. Dale and Garcia [17] suggested the use of foot ash as an alternative method for evaluating mineralization, primarily to provide for a larger sample size. Some laboratories have modified the tibia ash procedure to eliminate the lipid extraction process [18]. Because of the recent upsurge in interest in P use in broilers, due in large measure to the commercial introduction of phytase enzymes and the growing concerns over the influence of P in chicken litter on eutrophication of surface waters, it has become essential to reduce the time needed to obtain information regarding bone development under different nutritional strategies. Therefore, in the process of evaluating various P sources, we compared alternative methods of evaluating response to P concentrations in diets for young (0 to 21 d) male broilers, including 1) lipid-extracted tibias, 2) unextracted tibias, 3) toe ash, and 4) foot ash.
MATERIALS AND METHODS A basal diet, meeting current industry nutrient standards except for nonphytate P, was formulated using corn and soybean meal as intact protein sources (Table 1). Sufficient space was provided for the variable ingredients (phosphate source, limestone, and sand). This diet was calculated to contain approximately 0.12% nonphytate P from corn and soybean meal. An amount of the basal diet sufficient to provide feed for the experimental period was mixed and divided into aliquots. One aliquot (NEG) was fortified with limestone to provide 0.90% Ca with the balance of the variable weight as sand. A second aliquot was supplemented with sufficient bone meal (BM; 24.96% Ca, 16.64% P) to provide a total of 0.45% nonphytate P (0.33% P from the BM). Levels of limestone and sand were ad-
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TABLE 1. Composition (g/kg) and calculated analysis of low P test diet Ingredient
Low P basal
Yellow corn Soybean meal Poultry oil Vitamin premix1 Sodium chloride DL-Methionine Trace minerals2 L-Lysine HCl (98%) L-Threonine L-Tryptophan Variable3 Total
604.09 323.09 29.47 5.00 4.68 2.43 1.00 0.62 0.31 0.14 29.17 1,000.00
ME, kcal/kg CP, % Ca, % Total P, % Nonphytate, P % Methionine, % TSAA, % Lysine, % Threonine, % Tryptophan, %
3,063.00 21.04 0.90 0.36 0.12 0.58 0.95 1.24 0.87 0.25
1
Provided per kilogram of diet: vitamin A (from vitamin A acetate) 7,714 IU; cholecalciferol 2,204 IU; vitamin E (from DL-α-tocopheryl acetate) 16.53 IU; vitamin B12 0.013 mg; riboflavin 6.6 mg; niacin 39 mg; pantothenic acid 10 mg; menadione (from menadione dimethylpyrimidinol) 1.5 mg; folic acid 0.9 mg; choline 1,040 mg; thiamin (from thiamin mononitrate) 1.54 mg; pyridoxine (from pyridoxine HCl) 2.76 mg; D-biotin 0.066 mg; ethoxyquin 125 mg; Se 0.1 mg. 2 Provided per kilogram of diet: Mn (from MnSO4ⴢH2O) 100 mg; Zn (from ZnSO4ⴢ7H2O) 100 mg; Fe (from FeSO4ⴢ7H2O) 50 mg; Cu (from CuSO4ⴢ5H2O) 10 mg; I from Ca(IO3)2 ⴢH2O), 1 mg. 3 Contained variable amounts of bone meal, dicalcium phosphate, ground limestone, or sand.
justed as needed to provide for a total of 0.90% Ca. The third aliquot was supplemented with dicalcium phosphate (DCP; 20.0% Ca, 18.55% P) to provide a total of 0.45% nonphytate P (0.33% P from the DCP) with limestone added to provide a total of 0.90% Ca. Blends of the NEG with BM or with DCP were made to provide final test diets with additions of 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, and 0.33% total P from each of the 2 test products. Along with the NEG diet, this resulted in a total of 15 dietary treatments. Each of these was fed to 6 replicate pens of 5 male broilers of a commercial strain [19] maintained in electrically heated battery brood-
JAPR: Research Report
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TABLE 2. Calcium and P assays of test diets (means and SD from quadruplicate analyses from 2 different laboratories) Analyzed (%) Calculated P added (%)1 0 0.05 0.10 0.15 0.20 0.25 0.30 0.33
Calculated (%)
Ca
Total P
Total P
Ca
Mean
SD
0.36 0.41 0.46 0.51 0.56 0.61 0.66 0.69
0.90 0.90 0.90 0.90 0.90 0.90 0.90 0.90
1.052 1.088 1.032 1.081 1.031 0.967 1.051 0.975
0.034 0.101 0.031 0.076 0.088 0.116 0.092 0.065
2
Mean 0.386 0.430 0.471 0.525 0.581 0.670 0.695 0.726
SD 0.010 0.008 0.013 0.041 0.020 0.057 0.040 0.036
(0.044) (0.085) (0.139) (0.195) (0.284) (0.309) (0.340)
1
Amount added to diet calculated to contain 0.12% nonphytate P. Value in parentheses represents increase over basal diet.
2
ers from hatch to 21 d of age. Care and management of the birds followed accepted guidelines [20]. Samples of the mixed feeds were sent to 2 laboratories, specializing in feed analysis for Ca and P analysis. This report will focus primarily upon evaluation of the response criteria to P levels with difference between P sources to be reported in a separate communication. Measurements included beginning and ending pen weight, feed consumed during the study, and weight of birds that died, which was used to adjust feed conversion. At 21 d of age, all surviving birds were killed by CO2 asphyxiation as recommended by the American Veterinary Medical Association [21]. On the right leg, all the toes (right, left, middle, and back) of all surviving birds were removed and dried for 24 h at 105°C, and toe ash determined on pooled
samples of toes from each pen. The right tibia was removed and cleaned of adhering tissue; fat was removed by extraction with ether and alcohol and dried, and ash content determined on pooled samples of all bones from each pen as described by the Association of Official Analytical Chemists [5]. On the left leg, the left tibia was removed, cleaned of adhering tissue, and dried, and pooled samples of all bones within a pen ashed without fat extraction. The left foot was removed at the tibiotarsal joint and dried for 24 h at 105°C, and ash content determined on pooled samples of all feet within a pen. Data were analyzed as described in References and Notes [22].
RESULTS AND DISCUSSION Results of assays for Ca and P in test diets are shown in Table 2. The results indicated Ca
TABLE 3. Effect of P level on performance of broilers Added P (%)1
21-d BW (g)
Feed conversion (kg:kg)
Mortality (%)
0 0.05 0.10 0.15 0.20 0.25 0.30 0.33 P>F SEM CV
257e 297e 455d 506c 558b 579ab 592ab 595a
