The Determination of Retainable Phosphorus, Relative Biological Availability, and Relative Biological Value of Phosphorus Sources for Broilers

The Determination of Retainable Phosphorus, Relative Biological Availability, and Relative Biological Value of Phosphorus Sources for Broilers C. N. Coon,1 S. Seo,2 and M. K. Manangi Department of Poultry Science, University of Arkansas, Fayetteville 72701 P determined at 0.45% NPP was 62.41% for DCP, 63.58% for DF-1, and 59.25% for DF-2. The relationship of ARV and RBA were similar in that DCP was 6% higher in ARV at the breakpoint compared with DF-1 and the RBA of DF-1 was 71 and 91% from tibia weight and tibia breaking force, respectively, compared with the bone parameters from chicks fed DCP. The DF-1 phosphate had 3 and 7% higher ARV at the breakpoint and 0.45% NPP, respectively, compared with DF-2. The RBA of DF-2 was 59 and 80% from tibia weight and bone-breaking force. The ARV of phosphate sources were independent of an arbitrary reference. The ARV for P sources provide retainable P information for industry-based feed formulation that can reduce excess P in poultry waste. The excreta P data from broilers fed increasing levels of DCP indicates that the data are best described statistically with a 1-slope brokenline regression, 2-slope broken-line regression, or polynomial regression.

Key words: retainable phosphorus, relative biological availability, phosphorus retention bioassay, nonphytate phosphorus requirement, broiler 2007 Poultry Science 86:857–868

percentage tibia ash after chicks or poults are fed varying amounts of test phosphates in a P-deficient bioassay diet for a 2- to 3-wk period. The BV for feed phosphates are usually expressed as a relative BV (RBV) by comparing the BV of a test P source to a reference standard food grade or USP P source. Numerous reference standards have been used, including potassium phosphates, sodium phosphates, and mono-, di-, and tricalcium phosphates. Practical and purified diets have been used in these types of assays (Coon et al., 2001). The relative biological availability (RBA) of different P sources has also been reported. In this type of study, bone strength, bone ash weight, or percentage bone ash of broilers or turkeys that are fed various sources of test P are compared with the same bone parameters of poultry fed a standard P source (Waibel et al., 1984; Coffey et al., 1994; Axe, 1998; Fernandes et al., 1999). The RBA of a standard P source is given a value of 100%, and the biological availability of test P is estimated as a relative percentage to the standard using a slope ratio method. Sullivan (1966) reported a triple response index method for evaluating test P when determining the RBV by using data of bone ash weight, BW

INTRODUCTION Knowledge of various P retention from concentrated feed P sources, as well as the percentage retainable nonphytate phosphorus (NPP) and phytate phosphorus (PP) from feedstuffs in poultry is of economic and ecologic importance. Phosphorus in feedstuffs is presently identified as total P (TP) and NPP; a limited amount of information exists regarding the actual biological availability or retention of the different P types that account for a majority of dietary NPP. Using poultry bioassays, feed P sources have been given a biological value (BV) instead of being expressed on a percentage available or retainable P basis. The BV of a feed or standard phosphate is determined by measuring weight gain, feed conversion, and

©2007 Poultry Science Association Inc. Received May 31, 2006. Accepted January 10, 2007. 1 Corresponding author: [email protected] 2 Present address: Department of Natural Science, Albany State University, Albany, GA 31705.

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ABSTRACT A 10- to 21-d chick bioassay was conducted to determine the absolute retention value (ARV) for 2 different defluorinated phosphates (DF-1 and DF-2) and a reagent grade dicalcium phosphate (DCP). The total and test P in excreta regressed on feed P levels were subjected to general straight-line (linear), 1-slope brokenline, 2-slope broken-line, and polynomial regression methods to find the best analysis model. The relative biological availability (RBA) and relative biological value (RBV) for P from the 2 different defluorinated phosphates (DF-1 and DF-2) were obtained by the slope ratio method using 3 different bone measurements (% tibia ash, tibia breaking force, tibia weight) and RBV calculated using percentage tibia ash, weight gain, and feed/gain. The DCP was used as reference standard for RBA and RBV. The ARV measured at the breakpoints for test P by 2slope analysis were determined to be 82.99% for DCP, 76.34% for DF-1, and 70.30% for DF-2. The ARV of test

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MATERIALS AND METHODS Husbandry and Diets Eight hundred Cobb × Ross male 1-d-old broiler chicks were fed a corn soybean starter diet (21.8% CP, 3,065 kcal of ME/kg, 1.00% Ca, and 0.45% NPP) until 10 d of age. All chicks were individually weighed at 10 d of age, and 264 chicks were selected from the midsection weight range with mean BW and SD of 286 ± 11 g. The chicks were randomly assigned to individual metabolic cages in an environmentally controlled room with 24-h lighting, and feed and water were provided for ad libitum consumption. Twenty-four individual chicks were fed a semipurified corn soybean basal diet (Table 1) with no added P to serve as negative control for the feeding experiment. Two hundred forty chicks were offered 1 of 30 different test diets (8 individual chicks per/diet) that consisted of the basal diet plus 10 levels of 3 different P sources. Phosphorus levels for each of the 3 sources were supple-

Table 1. Phosphorus basal diet Ingredient

(%)

Corn, ground Teklad Premix1 Cellulose2 Soybean meal, dehulled, 48% CP Limestone2 Total

35.0 33.0 6.25 25.0 0.75 100.0

Calculated value CP, % ME, kcal/kg TSAA, % Lysine, % Analyzed value (as-is basis) Calcium, % Phytate phosphorus, % Nonphytate phosphorus, % Total phosphorus, %

20.4 3,215 0.93 1.15 0.43 0.18 0.14 0.32

1 Teklad Premix, Harlan Teklad, Madison, WI) provided per kilogram of complete diet: egg white solids (spray-dried), 44.55 g; gelatin, 22.11 g; DL-methionine, 2.046 g; glucose monohydrate, 172.08 g; corn oil, 69.828 g; ethoxyquin (antioxidant), 10 mg; celite, 10.00 g; sodium chloride, 3.05 g; potassium citrate (monohydrate), 2.03 g; magnesium oxide, 610 mg; ferric citrate, 710 mg; manganese sulfate, 320 mg; zinc carbonate, 200 mg; cupric carbonate, 70 mg; potassium iodate, 10 mg; sodium selenite, 1 mg; choline bitartrate, 2.03 g; niacin, 60 mg; vitamin B12 (0.1% trituration), 50 mg; calcium pantothenate, 30 mg; thiamin HCl, 20 mg; menadione sodium bisulfate complex, 20 mg; pyridoxine HCl, 20 mg; riboflavin, 20 mg; folic acid, 10 mg; biotin, 3 mg; vitamin E acetate (500 U/g), 100 mg; vitamin A palmitate (500,000 U/g), 20 mg; vitamin D3 (500,000 U/g), 10 mg. 2 The calcium phosphate sources were supplemented as a replacement for cellulose and limestone in all the experimental diets.

mented at the expense of cellulose. The 3 P sources tested were 2 commercial DF samples and a reagent grade dicalcium phosphate (C129-12, Fisher Scientific, Suwanee, GA) for comparison (Table 2). The NPP, TP, and Ca for the test diets for each P source ranged from 0.15 to 0.85%, 0.33 to 1.03%, and 0.44 to 1.42%, respectively, on an asis basis (Table 2). The test P inclusion levels were selected to provide adequate intake data above and below the NRC (1994) recommended level (0.45% NPP). The limestone was adjusted in each formulation for each level of test P to provide a 1.34 to 1.38:1 ratio of Ca to TP. The 10 levels of test P fed in the experimental diets ranged from 0.01 to 0.71% for the 3 sources (Table 3). Mortality was recorded daily. Birds were weighed at 10, 15, and 21 d of age, whereas feed consumption was recorded for the 10- to 13-d acclimation period, 13- to 15-d excreta collection period, and for the additional 6-d performance period. Acid insoluble ash (Celite, Celite Corp., Lompoc, CA) was added to the feed and used as a marker. Chicks were acclimated to the cages and diets for 3 d prior to excreta collection on d 15. After excreta collection, the birds were fed the test diets for an additional 6 d (21 d of age) to provide more time for the P levels to affect performance and bone parameters. Birds had unlimited access to feed and water for the entire duration of the experiment. Feed consumption and weight gain were recorded during the entire 11-d experiment (i.e., d 10 to 21).

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gain, and feed efficiency. The RBV data for test P sources was also reported in relative numerical terms in comparison with a reference standard. The biological availability of feedstuff P determined with these assays has been shown to vary depending upon the standard, source of P, molecular formula, and the age and species of poultry utilized in the bioassay (Ammerman et al., 1995; Axe, 1998). Leske and Coon (1999) reported the retention of PP, NPP, and TP in 6 feedstuffs by using a 5-d feeding assay with an acid insoluble ash marker that included the option of adding phytase to the feedstuff. Van der Klis and Versteegh (1999) measured the retention of P by 3-wk-old male broilers for feedstuffs with a total excreta collection method when feeding a synthetic diet in which the test feedstuff provided the vast majority of the P. Diets were standardized with the test ingredient to contain 0.18% calculated retainable P (RP), and all diets contained 0.5% Ca. Leske and Coon (2002) determined the absolute retention value (ARV) of PP, NPP, and TP with a 5-d bioassay for reagent grade monocalcium phosphate (MCP) and 3 feed grade mono/dicalcium phosphate (DCP) samples. The researchers utilized the RP values for formulating broiler diets and evaluated RP requirements in a 42-d floor pen feeding study. Phosphorus retention values provide nutritionists valuable information to meet the poultry P requirements in a similar way that digestibility or availability values are used in amino acid nutrition. The development of ARV for P will provide an opportunity to formulate more cost-effective diets and help minimize the amount of P in excreta. The objectives of this study were 1) to determine the retention values of 2 feed grade defluorinated phosphates (DF-1 and DF-2) and a reagent grade DCP, 2) to determine the relationship of ARV with RBA and RBV of the latter P sources, and 3) to determine the optimum regression model for describing the relationship of feed P with excreta P for broilers.

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RETAINABLE PHOSPHORUS BIOASSAY Table 2. Total P (TP), nonphytate P (NPP), retainable P (RP), and calcium levels for defluorinated phosphate 1 (DF-1), defluorinated phosphate 2 (DF-2), and reagent grade dicalcium phosphate (DCP) experimental diets1 Added P source Basal DCP

DF-1

Total P level (mg/g)3

Total RP level2 (mg/g)

Total Ca level (mg/g)3

DM (%)

1.4 (1.4) 1.51 (1.4) 2.01 (2.0) 2.51 (2.5) 3.51 (3.6) 4.51 (4.4) 5.01 (5.3) 5.51 (6.1) 6.51 (7.2) 7.51 (7.9) 8.51 (9.0) 1.51 (1.4) 2.01 (2.1) 2.51 (2.8) 3.51 (3.3) 4.51 (5.4) 5.01 (6.1) 5.51 (6.3) 6.51 (8.0) 7.51 (9.0) 8.51 (9.7) 1.51 (1.1) 2.01 (2.4) 2.51 (2.8) 3.51 (3.9) 4.51 (5.0) 5.01 (5.1) 5.51 (7.1) 6.51 (7.9) 7.51 (8.9) 8.51 (10.3)

3.2 (3.2) 3.31 (3.1) 3.81 (3.6) 4.31 (4.3) 5.31 (5.3) 6.31 (6.0) 6.81 (6.9) 7.31 (7.6) 8.31 (8.8) 9.31 (9.4) 10.31 (10.5) 3.31 (3.1) 3.81 (3.6) 4.31 (4.4) 5.31 (5.1) 6.31 (6.3) 6.81 (6.8) 7.31 (7.3) 8.31 (8.8) 9.31 (9.6) 10.31 (10.0) 3.31 (2.7) 3.81 (3.9) 4.31 (3.9) 5.31 (5.1) 6.31 (5.9) 6.81 (5.6) 7.31 (7.8) 8.31 (8.4) 9.31 (9.1) 10.31 (10.7)

1.94 2.30 2.65 2.90 3.50 3.94 4.12 4.31 4.54 4.69 4.84 2.31 2.68 3.03 3.63 4.10 4.24 4.40 4.64 4.81 4.96 2.10 2.47 2.84 3.25 3.58 3.77 3.83 3.99 3.98 3.93

4.3 (4.3) 4.44 (4.5) 5.14 (5.3) 5.84 (5.9) 7.24 (7.1) 8.64 (10.2) 9.34 (9.0) 10.04 (10.3) 11.44 (11.8) 12.84 (12.4) 14.24 (15.7) 4.45 (5.0) 5.14 (4.7) 5.84 (5.4) 7.25 (8.2) 8.64 (8.7) 9.34 (9.1) 10.05 (9.8) 11.45 (11.5) 12.85 (12.7) 14.25 (13.3) 4.44 (3.5) 5.16 (4.7) 5.84 (5.1) 7.20 (6.7) 8.64 (7.9) 9.34 (7.8) 10.04 (10.0) 11.44 (11.5) 12.85 (13.2) 14.24 (14.4)

90 89.5 89.1 88.9 88.7 89.0 89.3 89.8 89.6 89.4 89.4 89.8 89.5 89.7 89.3 90.2 89.8 89.7 89.7 89.9 89.8 90.2 90.2 90.2 89.9 89.9 90.3 90.0 90.1 90.5 90.1

1 DCP = 18.26% P, 23.14% Ca. DF-1 = feed grade, 18.2% P, 30.4% Ca, 4.53% Na, 53.5% citrate solubility. DF2 = feed grade, 17.9% P, 30.8% Ca, 5.43% Na, 78.3% citrate solubility. 2 The values of RP were obtained by determining the retention of TP with acid insoluble ash marker on asis basis. 3 The TP, NPP, and total Ca contents in the basal diet are analyzed values on as-is basis. The calculated values for each experimental diet are based on adding increments of each analyzed P source with the analyzed values from the basal on as-is basis. The NPP was estimated based on calculated TP (using the analyzed basal and analyzed additions of test P) and analyzed PP (NPP = TP − PP). The values in parentheses are analyzed values on as-is basis.

Animal Care All procedures were carried out in accordance with Animal Use Protocol No. 03008 for the experiment, which was approved by the University of Arkansas Institutional Animal Care and Use Committee.

Excreta and Bone Preparation The 3-wk-old broiler chicks were euthanized (CO2 asphyxiation) and the right tibia collected. The tibias were cleaned, tested for bone-breaking force, and extracted with ether prior to being ashed. Excreta samples were collected and stored at −20°C until they could be subsequently freeze-dried. The excreta samples were freezedried and ground to pass through a 5-mm screen in preparation for analysis.

Analysis Methods Tibias were analyzed for bone-breaking force by the shear force measurement as outlined by Wilson (1991),

utilizing an Instron Universal Testing Machine (Model 1123, Instron Corp., Canton, MA). Following the shear test, the tibias were cut lengthwise and defatted in refluxing petroleum ether in a Soxhlet apparatus for 48 h. The defatted tibia samples were oven-dried and ashed in ceramic crucibles for 24 h at 600°C. Ash content was determined on dry, fat-free tibia and expressed as grams of ash/bone and as a percentage of the defatted tibia weight. Total P and Ca were determined in each test P source, feed, and excreta by an inductively coupled plasma emission spectroscopic method as described by Leske and Coon (2002). Citrate solubility in neutral ammonium citrate and sodium analysis were determined for the 2 commercial DF samples by the method of Sullivan et al. (1992) and by inductively coupled plasma analysis, respectively. The CP was determined by AOAC procedure (990.03; 1995) and moisture determined by AOAC procedure (934.01; 1990). Diet and excreta PP was measured as IP6 (inositol hexaphosphate) using the ion-exchange chromatography method described by Bos et al. (1991).

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DF-2

Total NPP level (mg/g)3

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COON ET AL. Table 3. The effects of adding different levels of test P on BW for 21-d broiler chicks, and BW gain (BWG), feed intake, and feed:gain for 10- to 21-d broiler chicks1

P source Basal DCP

DF-1

Pooled SEM Source effect Level effect

0 0.11 0.61 1.10 2.10 3.10 3.60 4.10 5.10 6.10 7.10 0.11 0.61 1.10 2.10 3.10 3.60 4.10 5.10 6.10 7.10 0.11 0.61 1.10 2.10 3.10 3.60 4.10 5.10 6.10 7.10 DCP DF-1 DF-2 0.11 0.61 1.10 2.10 3.10 3.60 4.10 5.10 6.10 7.10

21-d BW (g)

10- to 21-d BWG (g)

795.8 791.2ghijk 794.5fghijk 795.2fghijk 826.1bcdefghik 822.5cdefghij 847.7abcdef 838.6cdefghij 847.4abcdef 843.7abcdefg 834.4abcdefgh 794.4fghijk 776.2jkl 788.8hijk 781.0ijk 855.9abcde 844.6abcdef 873.0abc 879.4ab 869.9abcd 885.7a 725.0l 819.4defghij 831.5bcdefghi 839.3abcdefgh 857.2abcde 760.3kl 836.1abcdefgh 840.3abcde 814.1efghij 821.8cdefghi 19.1 822.4 833.1 818.3 769.2 796.7 805.9 815.0 844.2 818.8 845.2 864.6 839.9 843.8

510.2 509.9efghi 507.4efghi 511.8defghi 540.2abcdefgh 542.8abcdefgh 558.8abcdef 531.1defghi 562.7abcde 533.8bcdefghi 552.7abcdef 508.1efghi 494.9hij 503.5fghi 496.4ghij 566.6abcd 563.5abcde 590.4ab 593.4a 528.0cdefghi 573.7abc 443.8j 535.9bcdefghi 545.3abcdefgh 549.7abcdef 566.7abcd 479.0ij 543.8abcdefgh 548.3abcdefgh 531.0cdefghi 535.1bcdefghi 20.43 535.0 541.8 529.6 486.3 512.7 520.9 528.3 558.0 535.1 556.2 568.3 531.0 553.0

Source of variation Source Level Source × level

10- to 21-d feed intake (g) 756.6 750.1 749.3 750 745.2 752.8 791.5 756.2 754.9 765.7 765.1 709.2 726.6 733.5 697.5 780.4 795.8 803.6 798.5 782.8 791.7 655.0 770.7 794.0 773.1 801.2 727.9 773.3 790.7 797.7 750.5 35.9 755.0 767.2 780.8 711.4 748.9 777.2 753.3 778.0 803.2 778.6 782.5 770.9 769.5

10- to 21-d feed/gain 1.49 1.47 1.48 1.48 1.38 1.39 1.41 1.43 1.34 1.37 1.39 1.39 1.47 1.45 1.41 1.38 1.48 1.36 1.35 1.24 1.38 1.52 1.44 1.55 1.47 1.42 1.64 1.42 1.46 1.50 1.40 0.06 1.42b 1.42b 1.48a 1.46ab 1.47ab 1.50a 1.42ab 1.40b 1.51a 1.41b 1.39b 1.46ab 1.39b

P-value 0.1045 <0.0001 0.0009

0.3017 <0.0001 0.0214

0.3125 0.2579 0.5400

0.0079 0.0371 0.7876

Means within column with differing superscripts are significantly different (P < 0.05). Values are means of 8 replicate birds per level of test P for dicalcium phosphate (DCP) and 2 different defluorinated phosphates (DF-1 and DF-2). For basal, the values are means of 24 replicate birds. Dietary nonphytate P, total P, retainable P, and Ca values are shown in Table 2 for all experimental diets. a–l 1

The NPP of the diet and excreta was determined by the difference between TP and PP. Phosphorus sources, diets, and excreta samples were analyzed for acid insoluble ash using the dry ash and hydrochloric acid digestion technique of Scott and Balnave (1991). The total RP for each test diet (basal P plus inclusion level of test P) was determined by measuring TP and acid insoluble ash marker in feed and excreta and using the equation of Scott and Balnave (1991). The RP ranged from 0.194% for the basal diet alone to 0.496% for the highest level of DF-1 inclusion plus the basal diet.

Retention of the 3 P sources was described as ARV calculated for all levels of inclusion by subtracting the dietary TP of basal from the dietary TP of test P diet and also subtracting TP in excreta for chicks fed only the basal diet from excreta TP of chicks fed added test P. The ARV of each test P is the percentage of TP from each test P retained. The ARV of each test P inclusion level is determined by using the measured acid insoluble ash marker in the feed and excreta along with the determined test P in the feed and excreta with the equation of Scott and Balnave (1991). The ARV of the 3 sources was deter-

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DF-2

Test P level (mg/g)

RETAINABLE PHOSPHORUS BIOASSAY

Figure 1. The comparison of tibia percentage bone ash of 21-d-old broilers fed 3 different P sources using 1-slope broken-line analysis. The general equation of the broken-line regression is Y = L + U(XXR − R), where L is the ordinate and R is the abscissa of the break point, XXR means × less than R, and U is the slope of the line for XXR. P sources: DCP = dicalcium phosphate; DF-1 and DF-2 = 2 different defluorinated phosphates.

The retention data from chicks fed diets containing DCP, DF-1, and DF-2 were compared with a 1-slope broken-line and 2-slope regression analysis to determine break points and slopes for excreta P with P in the feed. The 1-slope and 2-slope broken-line regression models for estimating the slope of lines and the point at which the lines intersect have been described by Leske and Coon (2002). The data of BW, BWG, feed/gain, and bone parameters were analyzed by 2-way ANOVA (SAS Institute, 1989) with means compared by LSD tests. The bone parameters were analyzed with 1-slope and 2-slope brokenline regression to obtain the break point and slope for each P source. All linear, 1-slope broken-line, 2-slope broken-line, and polynomial regressions for ARV, RBP, and RBV were determined with dry matter test P. The abscissa or x-axis test P-values determined at breakpoints for TP and PP excretion as well as for bone parameters can be converted to dietary test P on an as-is basis by multiplying determined breakpoint test P-values by 0.90.

BV index (P availability) = (10- to 21-d body weight gain/ 10) + 21-d tibia ash % + 10 × gain/feed); RBV (P availability) = (P availability of P source/ P availability of reference source) × 100. Retention data for the DCP source were subjected to ANOVA (SAS Institute, 1989), linear (straight-line; Dellaert et al., 1990), 1-slope broken-line (Robbins et al., 1979), 2-slope broken-line (Leske and Coon, 2002), and polynomial (Wedekind and Baker, 1990) regression analysis to find the best model for describing the relationship of excreta P with feed P. The P-values and the coefficient of determination (for linear and polynomial regression) were compared with the different models. The polynomial, 1-slope, and 2-slope broken-line models were compared against a linear straight-line model for statistical significance (α = 0.05) by adopting the method of Draper and Smith (1981) for testing a general linear hypothesis.

Figure 2. The comparison of tibia bone ash weight of 21-d-old broilers fed 3 different P sources using 1-slope broken-line analysis. The general equation of the broken-line regression is Y = L + U(XXR − R), where L is the ordinate and R is the abscissa of the break point, XXR means × less than R, and U is the slope of the line for XXR. P sources: DCP = dicalcium phosphate; DF-1 and DF-2 = 2 different defluorinated phosphates.

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mined at the regression breakpoint and also at the NRC (1994) suggested dietary level of 0.45% dietary NPP for 1 to 21-d-old broilers. The relative biological phosphorus availability (RBPA) and RBV were determined for DF-1 and DF-2 using the reagent grade dicalcium phosphate as a standard. The RBPA was determined by comparing the slope ratio of bone ash weight, bone ash percentage, and bone strength of the test samples to the standard by the method of Nelson and Walker (1964). The bone parameters were regressed on P added for each source of DF. The values obtained for the broilers fed the standard were used as a common intercept in regression equations to estimate P availability. The slopes of the linear portion (prior to breakpoint) for a broken-line regression model of the bone data were used for the slope ratio analysis (Sullivan et al., 1994). The slopes of the bone responses for each P source were divided by the slopes for the bone responses for broilers fed reference standard (DCP) to give an estimate of the RBPA. The RPBA for the DCP standard was given a value of 100%. A triple response bioassay procedure and mathematical formula used to calculate the RBV index of feed P sources for poultry was also utilized to compare the 2 DF with the DCP reference standard. The triple response bioassay was developed by Sullivan (1966) and consists of 3 response criteria (tibia ash percentage, weight gain, and gain:feed) combined to compute a BV. The BV (actually an index) or P availability was determined for each of the P sources by averaging the BV for the 3 smallest inclusion levels of test phosphorus added to the basal diets. The RBV was determined only for the 3 smallest levels of added P for each of the sources because these P levels were deficient for BW, BW gain (BWG), feed/ gain, and all bone measurements for each of the P sources. In order for RBV of different P sources to have comparative value, the P levels utilized for RBV determinations must be deficient, and the differences in RBV indicate improved P availability. The equations used were as follows:

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RESULTS Average BW for 21-d-old broilers, broiler BWG from d 10 to 21, feed intake, and feed/gain are presented in Table 3. The main effects of P levels on BW (<0.0001), BWG (<0.0001), and feed:gain (P = 0.0371) were significant, but the main effect for source was only significant for feed:gain (P = 0.0079). The main effect of source for BW was P = 0.1045. There was a significant interaction for Table 4. The effect of adding different test P levels on percentage bone ash, bone-breaking force and bone ash weight of 21-d-old broilers1

Basal DCP

DF-1

DF-2

Bone ash (%)

Bone-breaking force (kg)

Bone ash weight (g)

0 0.11 0.61 1.10 2.10 3.10 3.60 4.10 5.10 6.10 7.10 0.11 0.61 1.10 2.10 3.10 3.60 4.10 5.10 6.10 7.10 0.11 0.61 1.10 2.10 3.10 3.60 4.10 5.10 6.10 7.10

40.4 46.5ef 47.7de 45.7efg 50.4bc 53.1a 52.1abc 52.4abc 52.4abc 51.7abc 52.2abc 41.1ij 45.7efg 45.7efg 40.1j 50.2bcd 52.1abc 51.4abc 51.2abc 52.4ab 52.4ab 42.7hi 43.7ghi 44.2fgh 49.8cd 51.1abc 52.2abc 51.2abc 52.5ab 52.1abc 53.1a 0.94

8.1 8.0efg 10.8de 11.2de 18.7abc 18.3bc 17.9bc 18.8abc 20.5ab 18.3bc 19.8ab 7.1efg 10.0def 12.2d 16.7g 20.8ab 19.8ab 20.1ab 22.0a 18.7abc 19.6ab 7.3fg 10.3def 12.1d 15.9c 18.4bc 18.2bc 19.4abc 17.9bc 19.9ab 18.6abc 1.14

0.48 0.46i 0.58fgh 0.64ef 0.81cd 0.84bc 0.85bc 0.85bc 0.91ab 0.83bc 0.87bc 0.49hi 0.54ghi 0.62efg 0.72i 0.86bc 0.87bc 0.93ab 0.98a 0.85bc 0.87bc 0.46i 0.58fgh 0.61fg 0.72de 0.85bc 0.81cd 0.89abc 0.89abc 0.88bc 0.85bc 0.04

DCP DF-1 DF-2 0.11 0.61 1.10 2.10 3.10 3.60 4.10 5.10 6.10 7.10

50.6 48.3 49.3 43.2 45.5 45.1 46.7 51.7 52.1 51.6 52.3 52.1 52.6

16.4 15.7 15.9 7.6 10.4 11.8 13.4 19.0 18.7 19.4 20.1 19.0 19.3

0.78 0.74 0.75 0.47 0.56 0.62 0.66 0.85 0.84 0.89 0.93 0.85 0.87

<0.0001 <0.0001 <0.0001

0.6248 <0.0001 <0.0001

Pooled SEM Source effect

Level effect

Source of variation Level Source × level

P-value 0.4481 <0.0001 <0.0001

a–j Means within column with differing superscripts are significantly different (P < 0.05). 1 Values are means of data collected from 8 replicate birds per level of test P for dicalcium phosphate (DCP) and 2 different defluorinated phosphates (DF-1 and DF-2). The values for the basal are means of data collected from 24 replicate birds.

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P source

Test P level (mg/g)

source and level on BW (P = 0.0009) and BWG (P = 0.0214). The BW and BWG of 21-d-old broilers varied according to the level and source of P. The broilers fed 3.60 mg of test P of DCP/g of diet produced the highest BW among the DCP-fed broilers, and the BW was significantly higher than broilers fed the lowest level of DCP test P (0.11). There were no significant differences in BWG, feed intake, and feed/gain for the broilers fed the DCP test P diets. Broilers fed 3.1 mg of test P of DF-1/g of diet had a significantly heavier BW and BWG than broilers fed the 4 smallest inclusion levels of DF-1 test P. The BW and BWG of broilers fed higher levels of test P from DF-1 were not significantly different compared with broilers fed 3.1 mg of DF-1 test P/g of diet. Broilers fed 7.1 mg of test P of DF-1/g of diet produced the largest BW of all 30 groups, and the broilers fed 5.1 mg of test P of DF1/g of diet produced the highest BWG for all 30 test groups. The BW of broilers fed the highest level of DF-1 was significantly larger than BW of broilers fed 4.1 mg of test P of DCP/g of diet and also larger than BW of broilers fed the 2 highest levels of DF-2 test P. Broilers fed 5.1 mg of test P of DF-1/g of diet had a significantly higher BWG than broilers fed the DCP test diets containing 0.11, 0.61, 1.1, 4.1, and 6.1 mg of test P/g of diet; had increased BWG compared with broilers fed DF-1 test diets containing 0.11, 0.61, 1.1, 2.1, and 6.1 mg of test P/ g of diet; and had increased BWG compared with broilers fed DF-2 test diets containing 0.11, 0.61, 3.6, 6.1, and 7.1 mg of test P/g of diet. Broilers fed DF-2 test diets did not respond equally to broilers fed DF-1 treatments. This is because the lowest level of added test P (0.11) produced significantly lower BW and BWG than the broilers fed the next higher level of 0.61 DF-2 test P. The broilers fed 0.11 DF-2 also had significantly lower BW and BWG than broilers fed 0.11 test P from DCP and DF-1. There were no significant differences in BW and BWG for broilers fed the range of test P concentrations from 0.61 to 7.1 mg of DF-2 test P/g of diet. Broilers fed 3.6 mg/g of DF-2 test P did not respond in a manner that was consistent with other treatments that had similar nutrient profiles. The analyzed P and Ca for this diet was similar to the calculated values (Table 2), and the lower response could not be explained. The broilers fed the DCP and DF-1 test P sources with the highest weight gain had 4.54 and 4.64 mg/g of diet of RP, respectively (Table 2). The broilers fed DF-2 did not retain the test P equal to broilers fed the DF-1 and DCP test P because no broiler groups fed DF-2 retained above 4 mg of RP/g of diet. The main effects of P levels on percent tibia ash, tibia ash weight, and tibia breaking force from the 21-d-old broilers were significant (P < 0.0001; Table 4). The main effect of P source was significantly different for percentage bone ash (<0.0001), but source main effects for bone ash weight and bone-breaking force were not significant. There was a significant (P < 0.0001) source and level interaction for each of the 3 bone parameters tested. The highest significant responses for the 3 bone parameters are different for each P source. The addition of 3.1 mg of test

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P/g of feed produced the highest percentage bone ash of 53.1 for chicks fed the DCP source, whereas chicks required 6.1 and 7.1 mg of added test P/g of feed to produce 52.4 and 53.1% bone ash when fed DF-1 and DF-2, respectively. The largest amount of percentage bone ash for broilers fed 3.1 mg of DCP test P/g of diet was significantly more than broilers fed the 4 smallest inclusion levels of test P from DCP. The percentage bone ash for broilers fed 6.1 mg of DF-1 test P/g of diet was significantly increased compared with broilers fed the 5 smallest inclusion levels of test P from DF-1. The percentage bone ash for broilers fed 7.1 mg of DF-2 test P/g of diet was significantly better than the 4 smallest inclusion levels of test P from DF-2. The largest bone ash weight of 0.91, 0.98, and 0.89 g for 21-d-old chicks fed the DCP, DF-1, and DF-2 diets, respectively, were each from chicks fed 5.1 mg of added test P/g of feed. The largest amount of bone-breaking force of 20.5, 22.0, and 19.9 kg was for chicks fed 5.1 mg of added test P/g of feed with the DCP and DF-1 P sources compared with 6.1 mg of test P/g of feed for chicks fed the DF-2 diet. The 1-slope broken-line regression analysis for each of the 3 bone parameters show a linear response with different breakpoints and slopes for the 3 P sources (Table 5; Figures 1 to 3). The breakpoint (3.10 mg of added P: 52.3% bone ash) and slope (2.15% bone ash increase/mg of

Figure 4. The 1-slope broken-line analysis of phytate P (PP) in excreta for comparing 3 different P sources. The general equation of the brokenline regression is Y = L + U(XXR − R), where L is the ordinate and R is the abscissa of the break point, XXR means × less than R, and U is the slope of the line for XXR. P sources: DCP = dicalcium phosphate; DF-1 and DF-2 = 2 different defluorinated phosphates.

added test P) for percentage bone ash from chicks fed the DCP source showed a lower response to test P compared with chicks fed DF-1 and DF-2 (Table 5, Figure 1). The slope of percentage bone ash increase per milligram of added test P was better for the chicks fed DF-2 compared with DF-1 P diets. Chicks only needed 3.3 mg of added P in DF-2 P diets compared with 4.4 mg in DF-1 P diets to reach the breakpoint. The breakpoint (2.36 mg of added P: 0.86 g of bone ash weight) and slope (0.17 g of bone ash increase/mg of added P) of bone ash weight for chicks fed diets with DCP showed a better response to test P than broilers fed DF-1 or DF-2 (Table 5; Figure 2). The slope of tibia ash weight increase per milligram of added test P was better for the chicks fed DF-1 compared with DF-2 P diets, and the chicks only needed 3.47 mg of added P compared with 3.77 mg to reach the breakpoint. The regression response for increased bone-breaking force with added P was the highest for chicks fed DCP diets with a breakpoint of 2.27 mg of added P and 18.9 kg of bone-breaking force with a slope of 5.24 kg of increased bone-breaking force per mg of added P (Table 5; Figure 3). The slope of 4.76 kg of increased bone-breaking force per mg of added P for chicks fed DF-1 was higher than

Table 5. A comparison of 1-slope broken-line regression analysis for percentage bone ash, bone ash weight, and bone-breaking force for broilers fed 3 different P sources1 Item

Breakpoint and slope for percentage bone ash

Breakpoint and slope for bone ash weight

Breakpoint and slope for bone-breaking force

DCP DF-1 DF-2

3.10 ± 0.13:52.3 ± 0.76:2.15 ± 0.67 4.4 ± 1.67:52.0 ± 1.09:2.40 ± 0.76 3.3 ± 0.40:52.2 ± 0.29:3.11 ± 0.37

2.36 ± 0.01:0.86 ± 0.19:0.17 ± 0.02 3.47 ± 0.02:0.90 ± 0.36:0.12 ± 0.02 3.77 ± 0.02:0.88 ± 0.30:0.10 ± 0.01

2.27 ± 0.45:18.9 ± 0.25:5.24 ± 0.75 2.80 ± 0.40:20.2 ± 0.24:4.76 ± 0.66 2.72 ± 0.28:18.7 ± 0.19:4.21 ± 0.47

1 The general equation of the 1-slope broken-line regression is Y = L + U(XXR − R), where L is the ordinate and R is the abscissa of the breakpoint, XXR means X less than R, and U is the slope of the line for XXR. For percentage bone ash, bone ash weight, and bone-breaking force, the break point values are determined from the mean values of bone data collected from 8 replicate birds per level for 10 different levels of test P. For breakpoint of percentage bone ash (%) the values represent mg/g of test P ± SE: break point for percentage bone ash ± SE: slope ± SE. For breakpoint of bone ash weight (mg/g) the values represent mg/g of test P ± SE: break point for bone ash weight ± SE: slope ± SE. For breakpoint of bonebreaking force (kg) the values represent mg/g of test P ± SE: break point for bone-breaking force ± SE: slope ± SE.

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Figure 3. The comparison of bone-breaking force of tibia for 21-dold broilers fed 3 different P sources using 1-slope broken-line analysis. The general equation of the broken-line regression is Y = L + U(XXR − R), where L is the ordinate and R is the abscissa of the break point, XXR means × less than R, and U is the slope of the line for XXR. P sources: DCP = dicalcium phosphate; DF-1 and DF-2 = 2 different defluorinated phosphates.

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COON ET AL. Table 6. Relative bioavailability (%) for 3 P sources determined from bone data and relative biological value (RBV) index from a triple response method Relative biological availability1 Item Dicalcium phosphate DF-1 DF-2

RBV2

Bone ash weight

Percentage bone ash

Bone-breaking force

Triple response

100 71 59

100 112 145

100 91 80

100 99.3 ± 2.0 98.9 ± 2.4

1 For the 2 defluorinated phosphates (DF-1 and DF-2), values are the estimates obtained by dividing the slopes of the bone responses for each P source by the slopes for the bone responses for broilers fed reference standard (dicalcium phosphate). 2 For DF-1 and DF-2, values are the means of 24 RBV (n = 24) for the lowest 3 levels of P tested; ± pooled SEM.

5). The ARV of P from DF-1 was the highest at 63.6% when determined at the dietary NPP requirement levels of 0.45% (NRC, 1994). The ARV for P from DCP and DF-2 were slightly less at 62.4 and 59.3%, respectively, measured at the same 0.45% dietary NPP levels. The ARV for the 3 sources of P decreased by 20.06, 12.7, and 11.0 percentage points for chicks fed DCP, DF-1, and DF-2, respectively, when ARV was determined at NRC (1994) suggested levels for dietary NPP compared with ARV determined at excreta P breakpoints using regression analysis. The P from DF-1 has the highest ARV of 85.6 and 71.2% using breakpoints for 1-slope broken-line regression with 1.59 mg of added P/g feed and NRC (1994) suggested NPP levels of 0.45%, respectively. The ARV for DCP and DF-2 were 83.0 and 75.9% at breakpoints of 1.38 and 1.52 mg of added P/g of feed and 0.45% dietary NPP, respectively (Table 9; Figure 6). The slope of the regression line following the breakpoint that best describes excreta P related to added P indicates that chicks fed DF-2 are excreting a higher level of TP (3.33 mg of TP/mg of added P) compared with chicks fed dietary DCP and DF-1. The chicks fed DCP and DF-1 were excreting P at a rate of 2.72 and 2.71 mg of TP/mg of added P, respectively. The ARV for the 3 P sources decreased by 17.8, 14.4, and 16.3 percentage points for chicks fed DCP, DF-1, and DF-2, respectively, when ARV was determined at the higher dietary P levels suggested by NRC (1994) compared with the breakpoints using 1-slope broken-line regression. The data from the broilers fed the DCP standard were evaluated to determine the best model to use for express-

Table 7. A comparison of phytate P (PP) disappearance using 1-slope broken-line regression analysis for 3 P sources1

Item

Breakpoints and slope for PP

Disappearance (%) for PP at breakpoints

DCP DF-1 DF-2

2.7 ± 0.14:7.08 ± 0.15:1.43 ± 0.01 3.6 ± 0.10:5.62 ± 1.09:0.68 ± 0.27 2.22 ± 0.27:4.22 ± 1.25:0.57 ± 0.45

33.7 40.5 37.9

1 DCP = dicalcium phosphate; DF-1 and DF-2 = 2 different defluorinated phosphates. The general equation of the 1-slope broken-line regression is Y = L + U(XXR − R), where L is the ordinate and R is the abscissa of the breakpoint, XXR means X less than R, and U is the slope of the line for XXR. The break point values for PP disappearance were determined from the mean PP disappearance values of excreta samples collected from 8 replicate birds per level for 10 different levels of test P. For breakpoint of PP (mg/g) the values represent mg/ g of test P ± SE: break point for PP ± SE: slope ± SE.

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the slope of 4.21 kg of increased bone-breaking force per mg of added P for chicks fed DF-2. The RBA of P from DF-1 was superior to DF-2 for 2 of the 3 bone parameters (Table 6). The RBA of P from bone ash weight, percentage bone ash, and bone-breaking force for chicks fed DF-1 was 71, 112, and 91% compared with 59, 145, and 81%, respectively, for chicks fed DF-2. The slope for percentage bone ash was better for chicks fed DF-1 and DF-2 compared with chicks fed the standard (DCP), thus producing a RBA of 112 and 145%, respectively. The RBV determined with the triple response method indicates the DF-1 P source was 0.4 units higher than RBV for broilers fed DF-2 (Table 6). Statistical differences cannot be determined for the RBA and RBV values because the bone values are composed of different levels of P divided by the response for DCP broilers. The broilers fed DCP test P excreted more PP per mg of test P than the DF-1 or DF-2 fed broilers (Table 7; Figure 4). The broilers fed DCP test P had a breakpoint of 7.08 mg/g of excreta PP: 2.7 mg/g of test P. The retention for the PP at breakpoint for the 1-slope broken-line regression was 33.7%, whereas the retention of PP for broilers fed DF-1 and DF-2 was 40.5 and 37.9%, respectively. The DCP has the highest ARV (83%) for test P at the breakpoint for 2-slope broken-line regression with 1.59 mg of added P/g of feed compared with an ARV of 76.3 and 70.3% for DF-1 and DF-2 at breakpoints of 2.37 and 2.14 mg of added P/g of feed, respectively (Table 8; Figure

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RETAINABLE PHOSPHORUS BIOASSAY Table 8. The comparison of total P (TP) and test P retention from best fitted 2-slope broken-line regression analysis for 3 different P sources1

Item DCP DF-1 DF-2

Alpha, gamma, and slopes for total P 2.69 0.62 1.22 1.09 0.27 1.33

± ± ± ± ± ±

1.91:4.74 0.52:2.72 1.39:5.51 0.35:2.71 2.10:5.28 0.55:3.33

± ± ± ± ± ±

0.34: 0.09 0.48: 0.14 0.56: 0.20

Alpha, gamma, and slopes for test P 0.06 0.64 −0.13 1.25 −0.41 1.48

± ± ± ± ± ±

0.40:1.59 0.55:3.02 0.49:2.37 0.40:3.00 0.39:2.14 0.32:3.67

± ± ± ± ± ±

0.28: 0.09 0.46: 0.17 0.27: 0.14

Retention (%) at gamma of TP and test P

Retention (%) for test P at NPP (0.45%)

67.11: 82.99

62.41

65.15: 76.34

63.58

59.83: 70.30

59.25

ing the relationship of excreta P and feed P. The P-values and coefficients of determination were compared with different models as seen in Table 10. Though R2 values for polynomial and straight-line models were close to 1, there exists a significant (P ≤ 0.05) difference between these 2 models. The differences between the observed data from predicted values and the trend lines of residuals for the 4 different models were plotted in Figure 7. The residuals of 3 models (1-slope broken-line, 2-slope broken-line, and polynomial) were very similar and close to the zero line when these models were fitted to analyze the measured data of this experiment, whereas the residuals from the straight-line linear regression did not distribute with constant variance. The P-values from the 3 models (1-slope broken-line, 2-slope broken-line, and polynomial) were 0.233 × 10−6, 0.658 × 10−6 and 0.236 × 10−5, respectively, and were significantly different (P ≤ 0.05) than the straight-line linear model.

DISCUSSION The RP level of 4.54 mg/g of feed for DCP and 4.64 mg/g of feed for DF-1 that produced maximum gain

Figure 5. Two-slope broken-line analysis for test P in excreta of 15d-old broilers fed 3 different P sources. The general equation of the 2slope broken-line regression is Y = α + (X ≤ γ) × (β1 × X) + (X > γ) × (β1 × γ + β2 × (X − γ), where α is the ordinate and γ is the abscissa of the break point, and β1 and β2 are the slopes of the 2 lines. P sources: DCP = dicalcium phosphate; DF-1 and DF-2 = 2 different defluorinated phosphates.

during the bioassay is the same amount of available P suggested by NRC (1994) for broilers during the first 3 wk of age. The NRC (1994) suggested that available P requirements are based on all of the NPP being available and all PP being unavailable. The BW, BWG, bone ash weight, percentage bone ash, and bone-breaking force data indicate the test P from DCP and DF-1 are more biologically available than the test P from DF-2. The RBV obtained in the present study for DF-1 and DF-2 were close to the reference standard (DCP). Sullivan et al. (1992) reported the average RBV determined with female turkeys in 21-d bioassay using 3 response criteria such as weight gain, tibia ash, and gain:feed ratio for 18 DF samples as 90.8% when compared with dicalcium phosphate as the reference standard. Axe (1998) indicated the relative bioavailability of DF values ranged from 85 to 91%. Sullivan et al. (1994) has suggested that a difference of 3 to 5 points for RBV were required for statistical differences based on conducting approximately 30 bioassays. In the present study, if the RBA for percentage tibia ash were excluded, the average RBA for DF-1 and DF-2 were 81 and 69.5%, respectively, compared with the RBA from DCP. Studies of the literature also indicate that P in DFP is slightly less available than that in commercial dicalcium phosphate for the chick (Peeler, 1972; Nelson et al., 1990). Coffey et al. (1994) reported the overall biological availability of P in DF for the chick as 83% of that in monosodium phosphate (MSP). The differences from the biological availability or utilization of different phosphates can be attributed to type, source, and particle size of phosphates, the reference standard, species, age of the birds, and the levels of Ca and P in the testing diet (Axe, 1998). Based on the premise that the biological availability of a P source is directly related to its solubility within the gastrointestinal tract of the chick, Sullivan et al. (1992) compared different solvents for measuring in vitro solubility of P sources for the purpose of developing a quick test that would be correlated to the biological availability of a P source in chicks. Sullivan et al. (1992) reported that in vitro citrate solubility for DF produced the highest correlation to RBV and RBA for chicks fed DF sources.

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1 The general equation of the 2-slope broken-line regression is Y = α + (X ≤ γ) × (β1 × X) + (X > γ) × (β1 × γ + β2 × (X − γ)), where α is the ordinate and γ is the abscissa of the breakpoint, and β1 and β2 are the slopes of the 2 lines. For column 2, the values represent mg/g of test P ± SE: break point for TP excretion (mg/g) ± SE: slope, β1 ± SE: slope, β2 ± SE. For column 3, the values represent mg/g of test P ± SE: break point for test P excretion (mg/g) ± SE: slope, β1 ± SE: slope, β2 ± SE. For columns 2 and 3, values are determined from the mean values of excreta data from 8 replicate birds per level for 10 different levels of TP and test P, respectively.

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Table 9. The comparison of parameters from best fitted 1-slope broken-line regression analysis for 3 different P sources1

Item

Breakpoint and slope for TP

Breakpoint and slope for test P

Retention (%) for TP at break point

DCP DF-1 DF-2

4.48 ± 0.84:4.94 ± 0.46:2.72 ± 0.22 4.72 ± 0.44:5.17 ± 0.23:2.71 ± 0.18 4.56 ± 0.73:4.96 ± 0.35:3.33 ± 0.19

1.38 ± 0.23:0.45 ± 0.13:3.02 ± 0.09 1.59 ± 0.40:0.67 ± 0.22:2.96 ± 0.16 1.52 ± 0.35:0.50 ± 0.16:3.67 ± 0.14

68.9 69.4 65.5

Retention (%) for test P at break point

Retention (%) for test P at NPP (0.45%)

83.0 85.6 75.9

65.2 71.2 59.6

1 TP = total P. The general equation of the one-slope broken-line regression is Y = L + U(XXR − R), where L is the ordinate and R is the abscissa of the breakpoint, XXR means X less than R, and U is the slope of the line for XXR. For column 2, the values represent mg/g of test P ± SE: break point for TP excretion (mg/g) ± SE: slope ± SE. For column 3, the values represent mg/g of test P ± SE: break point for test P excretion (mg/g) ± SE: slope ± SE. For both columns 2 and 3, values are determined from the mean values of excreta data from 8 replicate birds per level for 10 different levels of TP and test P, respectively.

Figure 6. One-slope broken-line analysis for test P in excreta of 15d-old broilers fed 3 different P sources. The general equation of the broken-line regression is Y = L + U(XXR − R), where L is the ordinate and R is the abscissa of the break point, XXR means × less than R, and U is the slope of the line for XXR. P sources: DCP = dicalcium phosphate; DF-1 and DF-2 = 2 different defluorinated phosphates.

slope and 2-slope broken-line regression analysis, the values at break points for percentage TP retention are much higher for DCP and DF-1 compared with DF-2 (Tables 8 and 9). The percentage TP retention values determined with all test P sources were lower than the percentage ARV for the test P sources because the percentage TP retention includes the retention of PP and NPP from the basal diet plus test P source. The percentage ARV for the test P is much higher because the P in the basal diet and P in excreta from basal diet-fed birds are subtracted from the P from the feed and excreta from birds fed test P. Previous research reported by Leske and Coon (2002) showed a maximum 67.6% TP retention for a corn soybean basal with 0.06% added P from MCP using a 5-d bioassay with an AIA marker. The maximum P retention from MCP from the bioassay was much higher at 98% compared with the lower percentage retention of TP in the diet. The researchers reported that PP and NPP retention in the corn soybean basal diet were 32.3 and 65.5%, respectively. The values for bone ash weight and bone-breaking force, used in determining RBA, follow the same trend of having higher values for DCP and DF-1 compared with DF-2, whereas the RBV values for DF-1 and DF-2 were close to DCP (Table 6). The RBA for percentage tibia ash was higher for DF-1- and DF-2-fed broilers compared with DCP-fed broilers. The low percentage bone ash values for the 3 smallest inclusion levels of DF-2 and a low percentage bone ash value for the smallest inclusion level of DF-1 produced a larger slope for both of the DF samples compared with slope of percentage bone ash increase from broilers fed DCP. The lower percentage bone ash in tibia of broilers fed the small inclusion levels of DF compared with the percentage bone ash of tibia from broilers fed the same amount of test P from DCP show the test P is less available from DF compared with P from DCP. The slope of percentage bone ash increase was not an accurate method for determining RBA in the present study compared with using the slope of bone ash weight and bone-breaking force. The RBV and RBA for P sources are based on comparing the chick utilization of the P source to a standard P source. These values are not appropriate estimates of available P when formulating poultry diets. Previous research by Leske and Coon (2002) showed that RP values from re-

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The researchers stated that the in vitro solubility test for DF should only be used as a screening tool and additional assay information would be needed to evaluate P sources. Sullivan et al. (1992) suggested that a citrate solubility for DF below 55 may indicate an unacceptable P source for use in chick starter diets. Tom Sullivan (1991, University of Nebraska, Lincoln, unpublished data) also reported that DF samples with a higher Na content tended to have a higher RBV for 21-d-old turkey poults. The DF1 and DF-2 from the present experiment produced in vitro citrate solubility values of 53.5 and 78.3% with Na levels of 4.53 and 5.43%, respectively (Table 2). The higher Na levels and citrate solubility values for DF-2 did not correlate to a higher biological P availability for chicks compared with DF-1 in this study. Coffey et al. (1994) also showed that DF with different solubilities in neutral ammonium citrate did not consistently influence the availability of P from DF for chicks and pigs. The concept of RP is based on the amount of TP retained from NPP and PP and is a value that is calculated and reported as percentage TP retention (Leske and Coon, 2002). The TP retention or ARV for test P determined in the present study for DCP, DF-1, and DF-2 provide a direct measurement of P that could be retained at a given dietary P level. The fact that the present study utilized a minimum of 10 points for dietary P gives strength to develop a good fit using regression curves. Based on 1-

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RETAINABLE PHOSPHORUS BIOASSAY Table 10. The comparison of P-values and coefficients of determination from 3 different regression analyses compared to linear analyses for predicting TP in excreta from broilers fed DCP diet1 Item

Linear

Polynomial

P-value R2

— 0.9494

0.236 × 10−5 0.9686

1-slope broken-line

2-slope broken-line

0.233 × 10−6

0.658 × 10−6

P-values and R2 were significantly different (P ≤ 0.05) from linear (straight line) model.

1

reported that increased serum phosphate enhances urinary excretion and that parathyroid hormone decreases it. Manangi and Coon (2006a) indicated that the P threshold for broilers could be supplied by feeding various combinations of RP from feed ingredients, feed phosphates, or P released from added dietary phytase enzymes. An unexpected observation was the effect of different dietary P sources on PP excretion. The only difference in formulation was that the diets containing DCP had to be adjusted with additional limestone compared with the broilers fed DF-1 and DF-2. Although the dietary Ca to TP is the same for the different P sources, the Ca from the limestone may be more soluble than the Ca from the DF. The soluble Ca may attach to PP, forming a PP complex that limits endogenous PP hydrolysis in the gut. Recent research by Manangi and Coon (2006b) observed that limestone with a small particle size had a higher solubility in weak HCl and produced lower in vitro phytate hydrolysis with added phytase compared with limestone with larger particles and lower solubility. In summary, the relative bioavailability of P determined by the slope ratio method using the tibia ash weight and tibia breaking force compared closely to the ranking of TP retention determined with the balance method for the test P sources. The relative bioavailability of P and TP retention of DF-1 was slightly better than the relative bioavailability of P and TP retention of DF2 in this study. The excretion of P in relation to dietary P is best expressed by 1-slope broken-line, 2-slope brokenline, and polynomial regression analysis other than straight-line linear models. Phytate P disappearance was 4.2 to 6.8% higher at breakpoint for broilers fed DF compared with broilers fed DCP. The retention of phosphorus was approximately 10 to 15% higher for test P sources when measured at the breakpoint determined with a 1slope or 2-slope broken-line regression model compared with the P retention measured at the 0.45% NPP level. The RP obtained at the breakpoint for maximum P retention in the present study is approximately half the dietary RP needed for optimum bone development as shown by Leske and Coon (2002) or when compared with the suggested NRC (1994) requirements of 0.45% for available P in the broiler starter period.

ACKNOWLEDGMENTS Figure 7. The plot of residual from the measured total P in excreta of dicalcium phosphate (DCP) source and the predicted data from 4 different regression models.

The authors are indebted to IMC Feed Ingredients for financially supporting this research. The authors also are

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agent grade monocalcium phosphate, often used as a standard P source by researchers conducting RBV or RBA assays, was significantly less than 100 available when fed at levels needed to provide available P suggested by NRC (1994). The researchers suggested RP values for feedstuffs need to be established for each P source to utilize the information for feed formulation. The present study was conducted to determine the possibility of using ARV in comparing actual chick utilization of different P sources to have an independent value that could be utilized in the formulation of dietary P levels. The 1- and 2-slope broken-line regression methodology adopted in the present study shows the chicks inability to retain the dietary P efficiently beyond the regression break point. This is indicative of a P physiological threshold and the chicks fed the higher levels of P are unable to use the P efficiently regardless of dietary P source (DCP, DF-1, or DF-2) as observed from the results of the present study. Manangi and Coon (2006a) recently utilized colostomized broilers and showed that increasing P intake produced increasing P levels in the plasma that eventually plateau with adequate P intake. The researchers indicate the P physiological threshold occurs after the plasma P plateaus and broilers increase P excretion in urine and feces with additional P intake. The researchers suggested the P loss in the urine and feces was directly related to the dietary P level and that the broilers were showing a biological P threshold that was substantially below the suggested NRC (1994) requirement for available P. Berner and Shike (1988) indicated that P balance in animals and humans is regulated by the intestinal tract, kidneys, and the bones. The authors suggest that the regulation of P reabsorption in the renal tubules of animals is dependent upon serum P levels and to a lesser degree, parathyroid hormone. The authors

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indebted to Cobb-Vantress for providing Cobb 500 male broiler chicks for the research.

REFERENCES

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