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Citric acid improves phytate phosphorus utilization in crossbred and commercial broiler chicks
Citric Acid Improves Phytate Phosphorus Utilization in Crossbred and Commercial Broiler Chicks K. A. Rafacz-Livingston, C. Martinez-Amezcua, C. M. Parsons,1 D. H. Baker, and J. Snow Animal Sciences Laboratory, 1207 West Gregory Drive, University of Illinois, Urbana, Illinois 61801 poor growth and severe leg problems. Chick weight gain and tibia ash were significantly increased (P < 0.05) by CA in both types of chicks. In experiment 2, the same 2 × 2 × 2 factorial treatment arrangement was again used except that the NPP levels were 0.18 and 0.28%. Tibia ash was increased significantly (P < 0.05) with the addition of CA in both breeds of chicks; response was greater at 0.18% NPP than at 0.28% NPP. In experiment 3, graded levels of CA (0, 1, 2, 3, and 4%) were evaluated in commercial chicks fed diets containing 0.18% NPP. Tibia ash increased linearly (P < 0.05) as CA increased from 0 to 4%. The average increase in bone ash resulting from 3% CA supplementation in experiments 2 and 3 was 41%. These results indicate that CA markedly improved phytate P utilization in NHC and Ross × Ross commercial broiler chicks.
(Key words: citric acid, phosphorus, crossbred chick, broiler chick) 2005 Poultry Science 84:1370–1375
INTRODUCTION Phosphorus is an essential mineral needed for structural and metabolic growth and development. Cereal grains and oilseeds have a high content of P present in the form of phytate. The P in this form is generally thought to be poorly used by monogastric animals due to low phytase activity found in the digestive tract (Cromwell, 1992). As a result, it is necessary to supplement most monogastric diets with P to meet the requirements of the animals. Phosphorus is expensive, however, and the inefficient use of phytate P can also cause environmental problems. Several feed additives have been investigated to determine their efficiency for increasing P use and decreasing P excretion by poultry and swine. Studies have shown that citric acid (CA) improves weight gain and tibia ash in New Hampshire × Columbian (NHC) crossbred chicks when added to a P-deficient corn-soybean meal diet but not when added to a P-deficient dextrose-casein-phytate– free diet (Boling et al., 2000b). Additionally, CA does
2005 Poultry Science Association, Inc. Received for publication January 14, 2005. Accepted for publication May 5, 2005. 1 To whom correspondence should be addressed: [email protected].
not improve weight gain or bone ash in chicks fed a Padequate corn-soybean meal diet (Boling-Frankenbach et al., 2001). These results suggest that CA can improve the use of phytate P per se. Researchers at the University of Maryland have also reported increased bone ash from CA supplementation to chick and poult diets (Angel et al., 2001a,b). In more recent work, however, Shellem and Angel (2002) found that CA supplementation depresses feed intake in commercial broiler chicks and that the apparent effect of CA on P use may be due to its effects on feed intake and not on P use per se. They concluded that the increased bone ash from CA might be due to reduced feed intake, which yields smaller chicks and smaller bones that contain higher concentrations of ash. No response to CA has been observed in laying hens (Boling et al., 2000a) or pigs (Boling et al., 2000b) fed diets deficient in nonphytate P (NPP). All of our previous work with CA has been with NHC chicks, and we have observed no depression in feed intake from CA. Based on the study by Shellem and Angel (2002), it seems that commercial broiler chicks may respond dif-
Abbreviation Key: CA = citric acid, NHC = New Hampshire × Columbian; NPP = nonphytate phosphorus.
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ABSTRACT Previous research in our laboratory has shown that citric acid (CA) improves phytate P utilization in New Hampshire × Columbian (NHC) crossbred chicks fed a P-deficient corn-soybean meal diet. The current study was conducted to determine if CA is also effective in commercial broiler chicks (Ross × Ross). In 3 experiments, 4 replicate groups of 5 male NHC chicks and male commercial chicks were fed corn-soybean meal diets varying in CA and nonphytate P (NPP) from 8 to 22 d of age. In experiment 1, a 2 × 2 × 2 factorial treatment arrangement was used to evaluate the effect of 2 levels of CA (0 and 3%) and NPP (0.13 and 0.28%) in NHC chicks and commercial chicks. The commercial chicks, but not the NHC chicks, fed the 0.13% NPP diet had to be removed from the experiment after 3 to 5 d due to very
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CITRIC ACID AND PHOSPHORUS TABLE 1. Composition of the phosphorus deficient corn-soybean meal basal diets used in experiments 1, 2, and 3 Amount (%) Ingredient
Experiment 2
Experiment 3
47.685 40.930 5.000 4.440 — 1.650 0.400 0.200 0.150 0.200 0.100 0.025 —
47.840 41.210 5.000 3.225 — 1.650 0.400 0.200 0.150 0.200 0.100 0.025 0.220
47.575 39.600 5.000 2.960 1.773 1.800 0.400 0.200 0.150 0.200 0.100 0.042 0.220
23.70 3.25 0.75 0.41 0.13 1.34 0.94
23.80 3.24 0.75 0.46 0.18 1.34 0.95
23.00 3.19 0.80 0.46 0.18 1.30 0.90
Corn Soybean meal, dehulled Soybean oil Cornstarch Silica flour Limestone Salt Vitamin mix1 Mineral mix2 DL-Met Choline chloride (60%) Bacitracin-MD premix3 KH2PO4 Composition, calculated: Protein (%) TME (kcal/g) Ca (%) Total P (%), analyzed Nonphytate P (%) Lys (%) Met + Cys (%)
1 Provided per kilogram of diet: retinyl acetate, 4,400 IU; cholecalciferol, 25µg; DL-α-tocopheryl acetate, 11 IU; vitamin B12, 0.01 mg; riboflavin 4.41 mg; D-pantothenic acid, 10 mg; niacin, 22mg; menadione sodium bisulfite, 2.33 mg. 2 Provided as milligrams per kilogram of diet: manganese, 75 from MnSO4ⴢH2O; iron, 75 from FeSO4ⴢH2O; zinc, 75 from ZnO; copper, 5 from CuSO4ⴢH2O; iodine, 0.75 from ethylene diamine dihydroiodide; selenium, 0.1 from Na2SeO3. 3 Provided 13.75 mg of bacitracin methylene disalacylate per kilogram of complete diet.
ferently than NHC chicks to dietary CA supplementation. The objective of our study was to determine if CA improves phytate P use in NHC and commercial (Ross × Ross) chicks without compromising growth or feed consumption.
MATERIALS AND METHODS General Procedures The University Committee on Laboratory Animal Care approved all experimental procedures. Chicks were housed in thermostatically controlled starter battery cages with raised wire floors in an environmentally controlled room where continuous light was provided daily. From 0 to 7 d posthatch, chicks were fed a nutritionally complete corn-soybean meal starter diet (NRC, 1994). On 8 d posthatch, following overnight feed withdrawal, chicks were weighed, wing-banded, and assigned to treatment groups such that the mean initial weights were similar among treatments. Four replicate groups of 5 chicks were fed their experimental diets ad libitum from 8 to 22 d after hatch in all experiments. The P-deficient basal diets for all experiments are shown in Table 1. The Ca level (0.75 to 0.80%) in the experimental diets was below NRC (1994) recommendations of 1.0% to prevent decreased growth
2
Dry product from Archer Daniels Midland, Decatur, IL.
and anorexia due to high a Ca to P ratio (Biehl et al., 1995). Total P contents of the basal diets in each experiment were analyzed using the AOAC (1980) procedure. Total analyzed P contents of the basal diets in experiments 1, 2, and 3 were 0.41, 0.46, and 0.46, respectively. At the end of each experiment, chicks were euthanized by CO2 inhalation, and the right tibia was collected for bone ash determination. Tibias were pooled by replicate pens and autoclaved, and all adhering tissue was removed. Bones were dried for 24 h at 100°C, weighed, and then dry-ashed for 24 h in a muffle furnace at 600°C. Ash weights of tibias were expressed as a percentage of dry bone weight (Chung and Baker, 1990) and as milligrams per tibia.
Experiment 1 It was our objective to evaluate the effect of CA on phytate P use in a P-deficient corn-soybean meal diet fed to male NHC and male Ross × Ross commercial broiler chicks. A 2 × 2 × 2 factorial treatment arrangement was used to test 0 and 4% CA2 at 2 deficient levels of NPP (0.13 and 0.23%) in NHC and commercial chicks. The CA was included in the diets at 4%, based on the work by Boling et al. (2000b) and Snow et al. (2004). The level of 0.1% supplemental P was included to compare the responses in growth performance and tibia ash from CA to those from P. The basal diet contained 0.75% Ca and 0.13% NPP (Table 1). Citric acid and P (KH2PO4) were added in place of cornstarch.
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Experiment 1
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Experiment 2 In experiment 1, the 0.13% NPP basal diet resulted in severe negative effects on growth performance in the commercial broiler chicks, indicating that the 0.13% NPP level was too low for commercial birds. Therefore, the NPP level of the basal diet was increased to 0.18% in this experiment by adding KH2PO4. A 2 × 2 × 2 factorial arrangement was used to evaluate 0 and 3% CA at NPP levels of 0.18 and 0.28% in NHC and commercial broiler chicks (Ross × Ross). Dietary additions of P (KH2PO4) and CA replaced cornstarch.
Experiment 3
Experiment 2 Similar to results of experiment 1, growth performance and feed consumption were greater in commercial broiler chicks compared with NHC chicks. Tibia ash was also higher in commercial chicks (Table 3). Addition of 3% CA generally had no consistent effect on growth performance. However, there was a significant breed × CA interaction for weight gain and feed intake due to CA having a general positive effect on these parameters in NHC chicks fed 0.28% NPP but a negative effect only in commercial chicks fed 0.28% NPP. As expected, dietary P supplementation increased growth and feed intake with the response being larger in commercial than NHC chicks (breed × P interaction, P < 0.05). Tibia ash increased significantly (P < 0.05) with the addition of CA and P in both breeds, and the response was greater at 0.18% NPP than at 0.28% NPP (CA × P interaction, P < 0.05). There was no significant breed × CA interaction, indicating that the tibia ash response to CA was similar in both breeds of chicks.
Experiment 3 Statistical Analysis Data were analyzed using the general linear model procedures of SAS (SAS Institute, 1990). The results of experiment 1 were analyzed as a completely randomized design due to early termination of some treatments. Experiment 2 was analyzed as a 2 × 2 × 2 factorial. Experiment 3 was initially analyzed as a completely randomized design. In addition, a linear regression analysis was used to determine significant linear effects of CA. Differences among individual treatment means were assessed using the least significant difference test (Carmer and Walker, 1985) if the F-value for the ANOVA was significant (P < 0.05).
RESULTS Experiment 1 The commercial broiler chicks fed 0.13% NPP were removed from the experiment after 3 to 5 d due to very
A linear (P < 0.05) increase in weight gain and feed intake was observed in chicks supplemented with up to 3% of CA (Table 4). Gain:feed ratio was significantly increased (P < 0.05) by 2 to 4% CA and by 0.05% NPP when compared with the basal diet treatment. Tibia ash increased linearly (P < 0.05) with additions of CA up to 4%. The response in tibia ash to 0.05% supplemental P was intermediate to that obtained from 2 and 3% CA. The positive control diet yielded growth performance and tibia ash values greater (P < 0.05) than those obtained from the other treatments.
DISCUSSION Improvement of P use has become a primary concern of animal producers due to increasing concerns about P pollution. As a result, several feed additives have been investigated to determine if they will increase P use and decrease P excretion by poultry and swine. Previous research from our laboratory has shown that CA additions
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The objective of this experiment was to determine if P use would be increased in commercial broiler chicks by levels of CA that were lower than those fed in experiments 1 and 2. Therefore, graded levels of CA (0, 1, 2, 3, and 4%) were fed to determine if a linear response to CA would be observed in commercial chicks (Ross × Ross). Two additional treatments consisting of 0.05% supplemental P with and without 4% CA were included to better compare the response of CA to that from P. Thus, results of the first 2 experiments indicated that CA yielded positive responses in tibia ash and P use that were significant but were less than those obtained from 0.10% supplemental P. An eighth treatment was included as a positive control with 0.32% supplemental NPP from KH2PO4 (0.45% total NPP) and 0.21% supplemental Ca from limestone (1.0% total Ca). The Ca in the basal diet was increased to 0.80% to ensure that Ca would not be limiting in any of the first 7 treatments. Citric acid and P (KH2PO4) were added in place of cornstarch and silica flour to keep diets isocaloric. The determined gross energy of CA (2,400 kcal/g) and the TMEn of cornstarch were used for the isocaloric ingredient substitutions.
poor growth and severe leg problems, and those data were not included in the statistical analysis. As expected, growth performance and feed consumption were greater for the commercial broilers than the NHC chicks (Table 2). Chick weight gain and tibia ash were increased (P < 0.05) by CA in NHC chicks fed 0.13 or 0.23% NPP and by CA in commercial chicks fed 0.23% NPP. Feed intake was increased (P < 0.05) by CA in NHC chicks fed the 0.13% NPP diet and was numerically increased in both types of chicks fed 0.23% NPP. Gain:feed ratio was not influenced by CA supplementation in NHC chicks fed 0.13 or 0.23% NPP. However, a significant improvement in gain:feed ratio (P < 0.05) from CA was observed in commercial chicks fed 0.23% NPP.
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CITRIC ACID AND PHOSPHORUS TABLE 2. Effects of citric acid (CA) in New Hampshire × Columbian (NHC) crossbred and Ross × Ross commercial (RR) chicks fed a P-deficient corn-soybean meal diet, experiment 11 Dietary treatment
Breed
NPP2 (%)
Weight gain (g)
Feed intake (g)
Gain:feed (g:kg)
Tibia ash (mg/tibia)
Tibia ash (%)
Basal (B) B + 4% CA B + 0.10% P3 B + 4% CA +0.10% P3 B + 0.10% P3 B + 4% CA +0.10% P3 Pooled SEM
NHC NHC NHC NHC RR RR
0.13 0.13 0.23 0.23 0.23 0.23
247e 305d 334cd 354c 432b 480a 10
380d 445c 486b 499b 564a 593a 11
648d 685cd 688c 710c 767b 808a 13
265e 402d 462c 580a 399d 533b 14
28.4c 34.0b 35.3b 39.0a 35.4b 40.9a 0.8
Means within a column with no common superscript are significantly different (P < 0.05) as a result of least significant difference means comparison. 1 Values are means for 4 replicate groups of 5 chicks per treatment during the period 8 to 22 d after hatching. The commercial chicks on the basal and basal + 4% CA treatments were removed from the experiment after 3 to 5 d due to poor growth and leg problems. 2 NPP = nonphytate P. 3 Inorganic P source was potassium phosphate monobasic. a-e
also observed 69 and 40% mortality when Cobb 500 chicks were fed 0.10 and 0.15% NPP levels in corn-soybean meal diets. They suggested a minimum of 0.25% NPP to diminish high mortality rates when phytase is not supplemented. Increasing the NPP level to 0.18% in experiments 2 and 3 of our study improved growth performance and caused no health or leg problems in commercial or NHC chicks. Thus, our results indicate that a diet containing only 0.18% NPP can be fed to commercial broiler chicks in P evaluation studies from 8 to 22 d of age after they have been fed a sufficient NPP diet from 0 to 7 d. Our results clearly show that CA is markedly efficacious in improving P use in commercial broiler chicks fed P-deficient corn-soybean meal diets. Thus, CA produced large and significant increases in bone ash in all 3 experiments. The magnitude of the bone ash responses to CA in NHC and commercial chicks is similar to that observed
TABLE 3. Effects of citric acid (CA) in New Hampshire × Columbian (NHC) crossbred and Ross × Ross commercial (RR) chicks fed a P-deficient corn-soybean meal diet, experiment 21 Dietary treatment
Breed
NPP2 (%)
Basal (B) B + 3% CA B + 0.10% P3 B + 3% CA +0.10% P3 Basal (B) B + 3% CA B + 0.10% P3 B + 3% CA +0.10% P3 Pooled SEM Source of variation
NHC NHC NHC NHC RR RR RR RR
0.18 0.18 0.28 0.28 0.18 0.18 0.28 0.28
Breed CA NPP Breed × CA Breed × NPP CA × NPP Breed × CA × NPP
Weight gain (g) 271d 300cd 315c 329c 401b 414b 532a 503a 11
Feed intake (g) 417e 453d 462d 483d 525c 567b 666a 635a 12
Gain:feed (g:kg) 649c 663c 682c 680c 764ab 733b 799a 793a 18
Tibia ash (mg/tibia) 324d 419c 500b 559a 346d 463bc 573a 576a 17
Tibia ash (%) 35.4f 38.2e 41.6cd 43.0bc 34.5f 40.6d 43.9ab 45.1a 0.7
Probabilities 0.001 NS 0.001 0.016 0.001 NS NS
0.001 NS 0.001 0.026 0.001 NS NS
0.001 NS 0.003 NS NS NS NS
0.045 0.001 0.001 NS NS NS NS
0.007 0.001 0.001 NS NS 0.004 NS
a-f Means within a column with no common superscript are significantly different (P < 0.05) as a result of least significant difference means comparison. 1 Values are means for 4 replicate groups of 5 chicks per treatment from 8 to 19 d after hatching. 2 NPP = nonphytate P. 3 Inorganic P source was potassium phosphate monobasic.
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up to 6% improve weight gain and tibia ash in NHC chicks fed a P-deficient corn-soybean meal diet (Boling et al., 2000b; Boling-Frankenbach et al., 2001; Snow et al., 2004). An initial study by Angel et al. (2001a) indicated that CA also improves P use in commercial broiler chicks. A later study by Shellem and Angel (2002), however, reported that the apparent effects of CA are actually due to depressed feed intake and do not result from effects on P use per se (i.e., chicks fed CA were smaller and had smaller tibia bones with increased ash content). Thus, the primary objective of our study was to evaluate and compare the effects of CA in NHC and commercial broiler chicks. In experiment 1, the NPP level of 0.13% was too low for commercial broiler chicks, and this treatment was terminated early because of high morbidity and mortality as well as severe leg problems. Waldroup et al. (2000)
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RAFACZ-LIVINGSTON ET AL. TABLE 4. Effects of citric acid (CA) in Ross × Ross commercial chicks fed a P-deficient corn-soybean meal diet, experiment 31 Dietary treatment2 Basal (B) B + 1% CA B + 2% CA B + 3% CA B + 4% CA B + 0.05% P4 B + 4% CA +0.05% P4 Positive control Pooled SEM
NPP3 (%)
Weight gain (g)
Feed intake (g)
Gain:feed (g:kg)
Tibia ash (mg/tibia)
Tibia ash (%)
0.18 0.18 0.18 0.18 0.18 0.23 0.23 0.45
291e 347de 387cd 449b 431bc 435bc 430bc 564a 19
441d 494c 513c 580b 554b 568b 577b 709a 14
658c 703bc 754ab 774ab 778ab 767ab 746abc 796a 30
298g 334fg 371ef 443cd 457c 401de 506b 786a 16
30.6e 32.2e 32.7de 36.8c 39.5b 35.0cd 40.5b 46.9a 0.8
in previous studies from our laboratory with NHC chicks (Boling et al., 2000b; Boling-Frankenbach et al., 2001; Snow et al., 2004). Feed intake generally was not negatively affected by CA concentrations in our experiments (in NHC or commercial broiler chicks); however, this has not been the case in reports by Angel et al. (2001a) and Shellem and Angel (2002) for commercial broiler chicks. In contrast, feed intake was often increased by CA in our study, particularly in NHC chicks. In instances when feed intake was increased by CA in our study, the responses in tibia ash to CA were generally much greater than that which could be explained by increased feed intake (i.e., the percentage increases in tibia ash from CA were much greater than the percentage increases in feed intake). Our experiments suggest that 3 to 4% CA can release or spare between 0.05 and 0.1% P. In experiment 3, 3% CA increased bone ash by 49% (from 298 to 443 mg/ tibia), which is greater than the 35% increase obtained from 0.05% P. The tibia ash response from 0.05% P was intermediate to that obtained from 2 and 3% CA. These responses to CA are slightly higher than those observed by Snow et al. (2004), who generally obtained tibia ash increases of 18 to 46% when NHC chicks were supplemented with 3 to 4% CA. The mechanism by which CA improves phytate P use is still unclear. It has been speculated that CA may decrease the pH of the digesta in the small intestine, thereby inhibiting phytic acid from chelating minerals and forming insoluble phytate salts, which are resistant to hydrolysis by endogenous phytase enzymes (Cosgrove, 1980; Ravindran et al., 1995; Maenz et al., 1999; Applegate et al., 2003). Boling et al. (2000b) inferred that CA could potentially chelate or bind Ca to prevent the formation of insoluble Ca phytate complexes. Citric acid was not effective in increasing P use in laying hens that are fed very high dietary levels of Ca (Boling et al., 2000a), supporting the latter theory. In summary, the results of our study clearly show that CA improved P use in commercial broiler chicks as well as in NHC chicks. Depressions in chick weight gain and
feed intake by CA were generally not observed in either NHC or commercial broiler chicks. Thus, the effects of CA on P use and tibia ash in our study were not due to depressed feed intake and smaller tibia bones as hypothesized by Shellem and Angel (2002).
REFERENCES Angel, R., T. J. Applegate, M. Christman, and A. S. Dhandu. 2001a. Non-phytate phosphorus sparing effect of phytase and citric acid when fed to poults. Poult. Sci. 80(Suppl. 1):134. (Abstr.) Angel, R., A. S. Dhandu, T. J. Applegate, and M. Christman. 2001b. Phosphorus sparing effect of phytase, 25-hydroxycholecalciferol, and citric acid when fed to broiler chicks. Poult. Sci. 80(Suppl. 1):133–134. (Abstr.) Applegate, T. J., R. Angel, and H. L. Classen. 2003. Effect of dietary calcium, 25-hydroxycholecalciferol, or bird strain on small intestinal phytase activity in broiler chickens. Poult. Sci. 82:1140–1148. Association of Official Analytical Chemists. 1980. Official Methods of Analysis. 13th ed. Association of Official Analytical Chemists, Washington, DC. Biehl, R. R., D. H. Baker, and H. F. DeLuca. 1995. 1α-hydroxylated cholecalciferol compounds act additively with microbial phytase to improve phosphorus, zinc, and manganese utilization in chicks fed soy-based diets. J. Nutr. 125:2407–2416. Boling, S. D., M. W. Douglas, J. L. Snow, C. M. Parsons, and D. H. Baker. 2000a. Citric acid does not improve phosphorus utilization in laying hens fed a corn-soybean meal diet. Poult. Sci. 79:1335–1337. Boling, S. D., D. M. Webel, I. Mavromichalis, C. M. Parsons, and D. H. Baker. 2000b. The effects of citric acid on phytate phosphorus utilization in young chicks and pigs. J. Anim. Sci. 78:682–689. Boling-Frankenbach, S. D., J. L. Snow, C. M. Parsons, and D. H. Baker. 2001. The effect of citric acid on the calcium and phosphorus requirements of chicks fed corn-soybean meal diets. Poult. Sci. 80:783–788. Carmer, S. G., and W. M. Walker. 1985. Pairwise multiple comparisons of treatment means in agronomic research. J. Agron. Educ. 14:19–26. Chung, T. K., and D. H. Baker. 1990. Phosphorus utilization in chicks fed hydrated sodium calcium aluminosilicate. J. Anim. Sci. 68:1992–1998.
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a-g Means within a column with no common superscript are significantly different (P < 0.05) as a result of least significant difference means comparison. 1 Values are means for 4 replicate groups of 5 chicks per treatment during the period 8 to 22 d after hatching. 2 All diets contained 0.75% Ca except the positive-control diet, which contained 1% Ca. 3 NPP = nonphytate P. 4 Inorganic P source was potassium phosphate monobasic.
CITRIC ACID AND PHOSPHORUS Cosgrove, D. J. 1980. Inositol Phosphates. Their chemistry, Biochemistry, and Physiology. Elsevier Scientific Publishing, New York. Cromwell, G. L. 1992. The biological availability of phosphorus for pigs. Pig News Info. 13:75–78. Maenz, D. D., C. M. Engele-Schaan, R. W. Newkirk, and H. L. Classen. 1999. The effect of minerals and mineral chelators on the formation of phytase-resistant and phytase-susceptible forms of phytic acid in solution and in a slurry of canola meal. Anim. Feed Sci. Technol. 81:177–192. National Research Council. 1994. Nutrient Requirements of Poultry. 9th rev. ed. National Academy Press, Washington, DC. Ravindran, V., W. L. Bryden, and E. T. Kornegay. 1995. Phytate: Occurrence, bioavailability and implications in poultry nutrition. Poult. Avian Biol. Rev. 6:125–143.
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SAS Institute. 1990. SAS/STAT User Guide: Statistics. Release 6.04 ed. SAS Institute Inc., Cary, NC. Shellem, T., and R. Angel. 2002. Is the effect of citric acid on apparent phosphorus availability mediated primarily through feed consumption changes? Poult. Sci. 81(Suppl. 1):94. (Abstr.) Snow, J. L., D. H. Baker, and C. M. Parsons. 2004. Phytase, citric acid, and 1α-hydroxycholecalciferol improve phytate phosphorus utilization in chicks fed a corn-soybean meal diet. Poult. Sci. 83:1187–1192. Waldroup, P. W., J. H. Kersey, E. A. Saleh, C. A. Fritts, F. Yan, H. L. Stillborn, R. C. Crum, Jr., and V. Raboy. 2000. Nonphytate phosphorus requirement and phosphorus excretion of broiler chicks fed diets composed of normal or high available phosphorus corn with or without microbial phytase. Poult. Sci. 79:1451–1459.
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(Key words: citric acid, phosphorus, crossbred chick, broiler chick) 2005 Poultry Science 84:1370–1375
INTRODUCTION Phosphorus is an essential mineral needed for structural and metabolic growth and development. Cereal grains and oilseeds have a high content of P present in the form of phytate. The P in this form is generally thought to be poorly used by monogastric animals due to low phytase activity found in the digestive tract (Cromwell, 1992). As a result, it is necessary to supplement most monogastric diets with P to meet the requirements of the animals. Phosphorus is expensive, however, and the inefficient use of phytate P can also cause environmental problems. Several feed additives have been investigated to determine their efficiency for increasing P use and decreasing P excretion by poultry and swine. Studies have shown that citric acid (CA) improves weight gain and tibia ash in New Hampshire × Columbian (NHC) crossbred chicks when added to a P-deficient corn-soybean meal diet but not when added to a P-deficient dextrose-casein-phytate– free diet (Boling et al., 2000b). Additionally, CA does
2005 Poultry Science Association, Inc. Received for publication January 14, 2005. Accepted for publication May 5, 2005. 1 To whom correspondence should be addressed: [email protected].
not improve weight gain or bone ash in chicks fed a Padequate corn-soybean meal diet (Boling-Frankenbach et al., 2001). These results suggest that CA can improve the use of phytate P per se. Researchers at the University of Maryland have also reported increased bone ash from CA supplementation to chick and poult diets (Angel et al., 2001a,b). In more recent work, however, Shellem and Angel (2002) found that CA supplementation depresses feed intake in commercial broiler chicks and that the apparent effect of CA on P use may be due to its effects on feed intake and not on P use per se. They concluded that the increased bone ash from CA might be due to reduced feed intake, which yields smaller chicks and smaller bones that contain higher concentrations of ash. No response to CA has been observed in laying hens (Boling et al., 2000a) or pigs (Boling et al., 2000b) fed diets deficient in nonphytate P (NPP). All of our previous work with CA has been with NHC chicks, and we have observed no depression in feed intake from CA. Based on the study by Shellem and Angel (2002), it seems that commercial broiler chicks may respond dif-
Abbreviation Key: CA = citric acid, NHC = New Hampshire × Columbian; NPP = nonphytate phosphorus.
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ABSTRACT Previous research in our laboratory has shown that citric acid (CA) improves phytate P utilization in New Hampshire × Columbian (NHC) crossbred chicks fed a P-deficient corn-soybean meal diet. The current study was conducted to determine if CA is also effective in commercial broiler chicks (Ross × Ross). In 3 experiments, 4 replicate groups of 5 male NHC chicks and male commercial chicks were fed corn-soybean meal diets varying in CA and nonphytate P (NPP) from 8 to 22 d of age. In experiment 1, a 2 × 2 × 2 factorial treatment arrangement was used to evaluate the effect of 2 levels of CA (0 and 3%) and NPP (0.13 and 0.28%) in NHC chicks and commercial chicks. The commercial chicks, but not the NHC chicks, fed the 0.13% NPP diet had to be removed from the experiment after 3 to 5 d due to very
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CITRIC ACID AND PHOSPHORUS TABLE 1. Composition of the phosphorus deficient corn-soybean meal basal diets used in experiments 1, 2, and 3 Amount (%) Ingredient
Experiment 2
Experiment 3
47.685 40.930 5.000 4.440 — 1.650 0.400 0.200 0.150 0.200 0.100 0.025 —
47.840 41.210 5.000 3.225 — 1.650 0.400 0.200 0.150 0.200 0.100 0.025 0.220
47.575 39.600 5.000 2.960 1.773 1.800 0.400 0.200 0.150 0.200 0.100 0.042 0.220
23.70 3.25 0.75 0.41 0.13 1.34 0.94
23.80 3.24 0.75 0.46 0.18 1.34 0.95
23.00 3.19 0.80 0.46 0.18 1.30 0.90
Corn Soybean meal, dehulled Soybean oil Cornstarch Silica flour Limestone Salt Vitamin mix1 Mineral mix2 DL-Met Choline chloride (60%) Bacitracin-MD premix3 KH2PO4 Composition, calculated: Protein (%) TME (kcal/g) Ca (%) Total P (%), analyzed Nonphytate P (%) Lys (%) Met + Cys (%)
1 Provided per kilogram of diet: retinyl acetate, 4,400 IU; cholecalciferol, 25µg; DL-α-tocopheryl acetate, 11 IU; vitamin B12, 0.01 mg; riboflavin 4.41 mg; D-pantothenic acid, 10 mg; niacin, 22mg; menadione sodium bisulfite, 2.33 mg. 2 Provided as milligrams per kilogram of diet: manganese, 75 from MnSO4ⴢH2O; iron, 75 from FeSO4ⴢH2O; zinc, 75 from ZnO; copper, 5 from CuSO4ⴢH2O; iodine, 0.75 from ethylene diamine dihydroiodide; selenium, 0.1 from Na2SeO3. 3 Provided 13.75 mg of bacitracin methylene disalacylate per kilogram of complete diet.
ferently than NHC chicks to dietary CA supplementation. The objective of our study was to determine if CA improves phytate P use in NHC and commercial (Ross × Ross) chicks without compromising growth or feed consumption.
MATERIALS AND METHODS General Procedures The University Committee on Laboratory Animal Care approved all experimental procedures. Chicks were housed in thermostatically controlled starter battery cages with raised wire floors in an environmentally controlled room where continuous light was provided daily. From 0 to 7 d posthatch, chicks were fed a nutritionally complete corn-soybean meal starter diet (NRC, 1994). On 8 d posthatch, following overnight feed withdrawal, chicks were weighed, wing-banded, and assigned to treatment groups such that the mean initial weights were similar among treatments. Four replicate groups of 5 chicks were fed their experimental diets ad libitum from 8 to 22 d after hatch in all experiments. The P-deficient basal diets for all experiments are shown in Table 1. The Ca level (0.75 to 0.80%) in the experimental diets was below NRC (1994) recommendations of 1.0% to prevent decreased growth
2
Dry product from Archer Daniels Midland, Decatur, IL.
and anorexia due to high a Ca to P ratio (Biehl et al., 1995). Total P contents of the basal diets in each experiment were analyzed using the AOAC (1980) procedure. Total analyzed P contents of the basal diets in experiments 1, 2, and 3 were 0.41, 0.46, and 0.46, respectively. At the end of each experiment, chicks were euthanized by CO2 inhalation, and the right tibia was collected for bone ash determination. Tibias were pooled by replicate pens and autoclaved, and all adhering tissue was removed. Bones were dried for 24 h at 100°C, weighed, and then dry-ashed for 24 h in a muffle furnace at 600°C. Ash weights of tibias were expressed as a percentage of dry bone weight (Chung and Baker, 1990) and as milligrams per tibia.
Experiment 1 It was our objective to evaluate the effect of CA on phytate P use in a P-deficient corn-soybean meal diet fed to male NHC and male Ross × Ross commercial broiler chicks. A 2 × 2 × 2 factorial treatment arrangement was used to test 0 and 4% CA2 at 2 deficient levels of NPP (0.13 and 0.23%) in NHC and commercial chicks. The CA was included in the diets at 4%, based on the work by Boling et al. (2000b) and Snow et al. (2004). The level of 0.1% supplemental P was included to compare the responses in growth performance and tibia ash from CA to those from P. The basal diet contained 0.75% Ca and 0.13% NPP (Table 1). Citric acid and P (KH2PO4) were added in place of cornstarch.
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Experiment 1
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Experiment 2 In experiment 1, the 0.13% NPP basal diet resulted in severe negative effects on growth performance in the commercial broiler chicks, indicating that the 0.13% NPP level was too low for commercial birds. Therefore, the NPP level of the basal diet was increased to 0.18% in this experiment by adding KH2PO4. A 2 × 2 × 2 factorial arrangement was used to evaluate 0 and 3% CA at NPP levels of 0.18 and 0.28% in NHC and commercial broiler chicks (Ross × Ross). Dietary additions of P (KH2PO4) and CA replaced cornstarch.
Experiment 3
Experiment 2 Similar to results of experiment 1, growth performance and feed consumption were greater in commercial broiler chicks compared with NHC chicks. Tibia ash was also higher in commercial chicks (Table 3). Addition of 3% CA generally had no consistent effect on growth performance. However, there was a significant breed × CA interaction for weight gain and feed intake due to CA having a general positive effect on these parameters in NHC chicks fed 0.28% NPP but a negative effect only in commercial chicks fed 0.28% NPP. As expected, dietary P supplementation increased growth and feed intake with the response being larger in commercial than NHC chicks (breed × P interaction, P < 0.05). Tibia ash increased significantly (P < 0.05) with the addition of CA and P in both breeds, and the response was greater at 0.18% NPP than at 0.28% NPP (CA × P interaction, P < 0.05). There was no significant breed × CA interaction, indicating that the tibia ash response to CA was similar in both breeds of chicks.
Experiment 3 Statistical Analysis Data were analyzed using the general linear model procedures of SAS (SAS Institute, 1990). The results of experiment 1 were analyzed as a completely randomized design due to early termination of some treatments. Experiment 2 was analyzed as a 2 × 2 × 2 factorial. Experiment 3 was initially analyzed as a completely randomized design. In addition, a linear regression analysis was used to determine significant linear effects of CA. Differences among individual treatment means were assessed using the least significant difference test (Carmer and Walker, 1985) if the F-value for the ANOVA was significant (P < 0.05).
RESULTS Experiment 1 The commercial broiler chicks fed 0.13% NPP were removed from the experiment after 3 to 5 d due to very
A linear (P < 0.05) increase in weight gain and feed intake was observed in chicks supplemented with up to 3% of CA (Table 4). Gain:feed ratio was significantly increased (P < 0.05) by 2 to 4% CA and by 0.05% NPP when compared with the basal diet treatment. Tibia ash increased linearly (P < 0.05) with additions of CA up to 4%. The response in tibia ash to 0.05% supplemental P was intermediate to that obtained from 2 and 3% CA. The positive control diet yielded growth performance and tibia ash values greater (P < 0.05) than those obtained from the other treatments.
DISCUSSION Improvement of P use has become a primary concern of animal producers due to increasing concerns about P pollution. As a result, several feed additives have been investigated to determine if they will increase P use and decrease P excretion by poultry and swine. Previous research from our laboratory has shown that CA additions
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The objective of this experiment was to determine if P use would be increased in commercial broiler chicks by levels of CA that were lower than those fed in experiments 1 and 2. Therefore, graded levels of CA (0, 1, 2, 3, and 4%) were fed to determine if a linear response to CA would be observed in commercial chicks (Ross × Ross). Two additional treatments consisting of 0.05% supplemental P with and without 4% CA were included to better compare the response of CA to that from P. Thus, results of the first 2 experiments indicated that CA yielded positive responses in tibia ash and P use that were significant but were less than those obtained from 0.10% supplemental P. An eighth treatment was included as a positive control with 0.32% supplemental NPP from KH2PO4 (0.45% total NPP) and 0.21% supplemental Ca from limestone (1.0% total Ca). The Ca in the basal diet was increased to 0.80% to ensure that Ca would not be limiting in any of the first 7 treatments. Citric acid and P (KH2PO4) were added in place of cornstarch and silica flour to keep diets isocaloric. The determined gross energy of CA (2,400 kcal/g) and the TMEn of cornstarch were used for the isocaloric ingredient substitutions.
poor growth and severe leg problems, and those data were not included in the statistical analysis. As expected, growth performance and feed consumption were greater for the commercial broilers than the NHC chicks (Table 2). Chick weight gain and tibia ash were increased (P < 0.05) by CA in NHC chicks fed 0.13 or 0.23% NPP and by CA in commercial chicks fed 0.23% NPP. Feed intake was increased (P < 0.05) by CA in NHC chicks fed the 0.13% NPP diet and was numerically increased in both types of chicks fed 0.23% NPP. Gain:feed ratio was not influenced by CA supplementation in NHC chicks fed 0.13 or 0.23% NPP. However, a significant improvement in gain:feed ratio (P < 0.05) from CA was observed in commercial chicks fed 0.23% NPP.
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CITRIC ACID AND PHOSPHORUS TABLE 2. Effects of citric acid (CA) in New Hampshire × Columbian (NHC) crossbred and Ross × Ross commercial (RR) chicks fed a P-deficient corn-soybean meal diet, experiment 11 Dietary treatment
Breed
NPP2 (%)
Weight gain (g)
Feed intake (g)
Gain:feed (g:kg)
Tibia ash (mg/tibia)
Tibia ash (%)
Basal (B) B + 4% CA B + 0.10% P3 B + 4% CA +0.10% P3 B + 0.10% P3 B + 4% CA +0.10% P3 Pooled SEM
NHC NHC NHC NHC RR RR
0.13 0.13 0.23 0.23 0.23 0.23
247e 305d 334cd 354c 432b 480a 10
380d 445c 486b 499b 564a 593a 11
648d 685cd 688c 710c 767b 808a 13
265e 402d 462c 580a 399d 533b 14
28.4c 34.0b 35.3b 39.0a 35.4b 40.9a 0.8
Means within a column with no common superscript are significantly different (P < 0.05) as a result of least significant difference means comparison. 1 Values are means for 4 replicate groups of 5 chicks per treatment during the period 8 to 22 d after hatching. The commercial chicks on the basal and basal + 4% CA treatments were removed from the experiment after 3 to 5 d due to poor growth and leg problems. 2 NPP = nonphytate P. 3 Inorganic P source was potassium phosphate monobasic. a-e
also observed 69 and 40% mortality when Cobb 500 chicks were fed 0.10 and 0.15% NPP levels in corn-soybean meal diets. They suggested a minimum of 0.25% NPP to diminish high mortality rates when phytase is not supplemented. Increasing the NPP level to 0.18% in experiments 2 and 3 of our study improved growth performance and caused no health or leg problems in commercial or NHC chicks. Thus, our results indicate that a diet containing only 0.18% NPP can be fed to commercial broiler chicks in P evaluation studies from 8 to 22 d of age after they have been fed a sufficient NPP diet from 0 to 7 d. Our results clearly show that CA is markedly efficacious in improving P use in commercial broiler chicks fed P-deficient corn-soybean meal diets. Thus, CA produced large and significant increases in bone ash in all 3 experiments. The magnitude of the bone ash responses to CA in NHC and commercial chicks is similar to that observed
TABLE 3. Effects of citric acid (CA) in New Hampshire × Columbian (NHC) crossbred and Ross × Ross commercial (RR) chicks fed a P-deficient corn-soybean meal diet, experiment 21 Dietary treatment
Breed
NPP2 (%)
Basal (B) B + 3% CA B + 0.10% P3 B + 3% CA +0.10% P3 Basal (B) B + 3% CA B + 0.10% P3 B + 3% CA +0.10% P3 Pooled SEM Source of variation
NHC NHC NHC NHC RR RR RR RR
0.18 0.18 0.28 0.28 0.18 0.18 0.28 0.28
Breed CA NPP Breed × CA Breed × NPP CA × NPP Breed × CA × NPP
Weight gain (g) 271d 300cd 315c 329c 401b 414b 532a 503a 11
Feed intake (g) 417e 453d 462d 483d 525c 567b 666a 635a 12
Gain:feed (g:kg) 649c 663c 682c 680c 764ab 733b 799a 793a 18
Tibia ash (mg/tibia) 324d 419c 500b 559a 346d 463bc 573a 576a 17
Tibia ash (%) 35.4f 38.2e 41.6cd 43.0bc 34.5f 40.6d 43.9ab 45.1a 0.7
Probabilities 0.001 NS 0.001 0.016 0.001 NS NS
0.001 NS 0.001 0.026 0.001 NS NS
0.001 NS 0.003 NS NS NS NS
0.045 0.001 0.001 NS NS NS NS
0.007 0.001 0.001 NS NS 0.004 NS
a-f Means within a column with no common superscript are significantly different (P < 0.05) as a result of least significant difference means comparison. 1 Values are means for 4 replicate groups of 5 chicks per treatment from 8 to 19 d after hatching. 2 NPP = nonphytate P. 3 Inorganic P source was potassium phosphate monobasic.
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up to 6% improve weight gain and tibia ash in NHC chicks fed a P-deficient corn-soybean meal diet (Boling et al., 2000b; Boling-Frankenbach et al., 2001; Snow et al., 2004). An initial study by Angel et al. (2001a) indicated that CA also improves P use in commercial broiler chicks. A later study by Shellem and Angel (2002), however, reported that the apparent effects of CA are actually due to depressed feed intake and do not result from effects on P use per se (i.e., chicks fed CA were smaller and had smaller tibia bones with increased ash content). Thus, the primary objective of our study was to evaluate and compare the effects of CA in NHC and commercial broiler chicks. In experiment 1, the NPP level of 0.13% was too low for commercial broiler chicks, and this treatment was terminated early because of high morbidity and mortality as well as severe leg problems. Waldroup et al. (2000)
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RAFACZ-LIVINGSTON ET AL. TABLE 4. Effects of citric acid (CA) in Ross × Ross commercial chicks fed a P-deficient corn-soybean meal diet, experiment 31 Dietary treatment2 Basal (B) B + 1% CA B + 2% CA B + 3% CA B + 4% CA B + 0.05% P4 B + 4% CA +0.05% P4 Positive control Pooled SEM
NPP3 (%)
Weight gain (g)
Feed intake (g)
Gain:feed (g:kg)
Tibia ash (mg/tibia)
Tibia ash (%)
0.18 0.18 0.18 0.18 0.18 0.23 0.23 0.45
291e 347de 387cd 449b 431bc 435bc 430bc 564a 19
441d 494c 513c 580b 554b 568b 577b 709a 14
658c 703bc 754ab 774ab 778ab 767ab 746abc 796a 30
298g 334fg 371ef 443cd 457c 401de 506b 786a 16
30.6e 32.2e 32.7de 36.8c 39.5b 35.0cd 40.5b 46.9a 0.8
in previous studies from our laboratory with NHC chicks (Boling et al., 2000b; Boling-Frankenbach et al., 2001; Snow et al., 2004). Feed intake generally was not negatively affected by CA concentrations in our experiments (in NHC or commercial broiler chicks); however, this has not been the case in reports by Angel et al. (2001a) and Shellem and Angel (2002) for commercial broiler chicks. In contrast, feed intake was often increased by CA in our study, particularly in NHC chicks. In instances when feed intake was increased by CA in our study, the responses in tibia ash to CA were generally much greater than that which could be explained by increased feed intake (i.e., the percentage increases in tibia ash from CA were much greater than the percentage increases in feed intake). Our experiments suggest that 3 to 4% CA can release or spare between 0.05 and 0.1% P. In experiment 3, 3% CA increased bone ash by 49% (from 298 to 443 mg/ tibia), which is greater than the 35% increase obtained from 0.05% P. The tibia ash response from 0.05% P was intermediate to that obtained from 2 and 3% CA. These responses to CA are slightly higher than those observed by Snow et al. (2004), who generally obtained tibia ash increases of 18 to 46% when NHC chicks were supplemented with 3 to 4% CA. The mechanism by which CA improves phytate P use is still unclear. It has been speculated that CA may decrease the pH of the digesta in the small intestine, thereby inhibiting phytic acid from chelating minerals and forming insoluble phytate salts, which are resistant to hydrolysis by endogenous phytase enzymes (Cosgrove, 1980; Ravindran et al., 1995; Maenz et al., 1999; Applegate et al., 2003). Boling et al. (2000b) inferred that CA could potentially chelate or bind Ca to prevent the formation of insoluble Ca phytate complexes. Citric acid was not effective in increasing P use in laying hens that are fed very high dietary levels of Ca (Boling et al., 2000a), supporting the latter theory. In summary, the results of our study clearly show that CA improved P use in commercial broiler chicks as well as in NHC chicks. Depressions in chick weight gain and
feed intake by CA were generally not observed in either NHC or commercial broiler chicks. Thus, the effects of CA on P use and tibia ash in our study were not due to depressed feed intake and smaller tibia bones as hypothesized by Shellem and Angel (2002).
REFERENCES Angel, R., T. J. Applegate, M. Christman, and A. S. Dhandu. 2001a. Non-phytate phosphorus sparing effect of phytase and citric acid when fed to poults. Poult. Sci. 80(Suppl. 1):134. (Abstr.) Angel, R., A. S. Dhandu, T. J. Applegate, and M. Christman. 2001b. Phosphorus sparing effect of phytase, 25-hydroxycholecalciferol, and citric acid when fed to broiler chicks. Poult. Sci. 80(Suppl. 1):133–134. (Abstr.) Applegate, T. J., R. Angel, and H. L. Classen. 2003. Effect of dietary calcium, 25-hydroxycholecalciferol, or bird strain on small intestinal phytase activity in broiler chickens. Poult. Sci. 82:1140–1148. Association of Official Analytical Chemists. 1980. Official Methods of Analysis. 13th ed. Association of Official Analytical Chemists, Washington, DC. Biehl, R. R., D. H. Baker, and H. F. DeLuca. 1995. 1α-hydroxylated cholecalciferol compounds act additively with microbial phytase to improve phosphorus, zinc, and manganese utilization in chicks fed soy-based diets. J. Nutr. 125:2407–2416. Boling, S. D., M. W. Douglas, J. L. Snow, C. M. Parsons, and D. H. Baker. 2000a. Citric acid does not improve phosphorus utilization in laying hens fed a corn-soybean meal diet. Poult. Sci. 79:1335–1337. Boling, S. D., D. M. Webel, I. Mavromichalis, C. M. Parsons, and D. H. Baker. 2000b. The effects of citric acid on phytate phosphorus utilization in young chicks and pigs. J. Anim. Sci. 78:682–689. Boling-Frankenbach, S. D., J. L. Snow, C. M. Parsons, and D. H. Baker. 2001. The effect of citric acid on the calcium and phosphorus requirements of chicks fed corn-soybean meal diets. Poult. Sci. 80:783–788. Carmer, S. G., and W. M. Walker. 1985. Pairwise multiple comparisons of treatment means in agronomic research. J. Agron. Educ. 14:19–26. Chung, T. K., and D. H. Baker. 1990. Phosphorus utilization in chicks fed hydrated sodium calcium aluminosilicate. J. Anim. Sci. 68:1992–1998.
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a-g Means within a column with no common superscript are significantly different (P < 0.05) as a result of least significant difference means comparison. 1 Values are means for 4 replicate groups of 5 chicks per treatment during the period 8 to 22 d after hatching. 2 All diets contained 0.75% Ca except the positive-control diet, which contained 1% Ca. 3 NPP = nonphytate P. 4 Inorganic P source was potassium phosphate monobasic.
CITRIC ACID AND PHOSPHORUS Cosgrove, D. J. 1980. Inositol Phosphates. Their chemistry, Biochemistry, and Physiology. Elsevier Scientific Publishing, New York. Cromwell, G. L. 1992. The biological availability of phosphorus for pigs. Pig News Info. 13:75–78. Maenz, D. D., C. M. Engele-Schaan, R. W. Newkirk, and H. L. Classen. 1999. The effect of minerals and mineral chelators on the formation of phytase-resistant and phytase-susceptible forms of phytic acid in solution and in a slurry of canola meal. Anim. Feed Sci. Technol. 81:177–192. National Research Council. 1994. Nutrient Requirements of Poultry. 9th rev. ed. National Academy Press, Washington, DC. Ravindran, V., W. L. Bryden, and E. T. Kornegay. 1995. Phytate: Occurrence, bioavailability and implications in poultry nutrition. Poult. Avian Biol. Rev. 6:125–143.
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SAS Institute. 1990. SAS/STAT User Guide: Statistics. Release 6.04 ed. SAS Institute Inc., Cary, NC. Shellem, T., and R. Angel. 2002. Is the effect of citric acid on apparent phosphorus availability mediated primarily through feed consumption changes? Poult. Sci. 81(Suppl. 1):94. (Abstr.) Snow, J. L., D. H. Baker, and C. M. Parsons. 2004. Phytase, citric acid, and 1α-hydroxycholecalciferol improve phytate phosphorus utilization in chicks fed a corn-soybean meal diet. Poult. Sci. 83:1187–1192. Waldroup, P. W., J. H. Kersey, E. A. Saleh, C. A. Fritts, F. Yan, H. L. Stillborn, R. C. Crum, Jr., and V. Raboy. 2000. Nonphytate phosphorus requirement and phosphorus excretion of broiler chicks fed diets composed of normal or high available phosphorus corn with or without microbial phytase. Poult. Sci. 79:1451–1459.
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