The Effect of Citric Acid on the Calcium and Phosphorus Requirements of Chicks Fed Corn-Soybean Meal Diets

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1 The Effect of Citric Acid on the Calcium and Phosphorus Requirements of Chicks Fed Corn-Soybean Meal Diets S. D. Boling-Frankenbach, 1 J. L. Snow, C. M. Parsons, 2 and D. H. Baker Department of Animal Sciences, University of Illinois, Urbana, Illinois ABSTRACT Data previously reported from our laboratory levels of citric acid (0, 2, 4, or 6%) on P utilization, and indicated that supplementation of a corn-soybean meal diet with citric acid improves P utilization in chicks. The four experiments reported herein were conducted to further evaluate the effects of citric acid on Ca and P utilization for chicks fed a corn-soybean meal diet. Diets in all experiments were fed to chicks from 8 to 21 or 22 d of age. The first experiment evaluated the effect of 6% citric acid on the Ca requirement of chicks. A Ca-deficient basal diet [23% CP, 0.54% Ca, 0.45% available P (AP)] containing 0 to 0.7% supplemental Ca in 0.1% increments was fed with or without 6% citric acid. The results indicated that citric acid did not significantly affect the Ca requirement. A second experiment evaluated different results indicated that 4 and 6% citric acid produced the largest responses in growth and tibia ash. Experiments 3 and 4 were then conducted to determine whether 4 or 6% citric acid would reduce the level of supplemental P required. Dietary treatments were a P-deficient basal diet (23% CP, 1.0 or 1.3% Ca, 0.20% AP) supplemented with 0 to 0.25% inorganic P with or without 4 or 6% citric acid. When diets contained citric acid, weight gain and tibia ash were maximized at lower AP levels than when diets contained no citric acid. The results of this study indicate that citric acid increases P utilization in corn-soybean meal diets and reduces the AP requirement by approximately 0.10% of the diet. (Key words: citric acid, calcium, phosphorus, chicks) 2001 Poultry Science 80: INTRODUCTION Phytase is a commercial means of improving P utilization by poultry (BASF, 1992), but it is has some limitations because it is heat labile and is not always cost effective. Therefore, alternative methods for increasing P utilization need consideration. One alternative may include organic acids, as very early studies have indicated that citric acid significantly improved phytate-p utilization in rats (Shohl, 1937; Pileggi et al., 1956). Recent work from our laboratory has indicated that citric acid is also very efficacious in improving P utilization in chicks (Boling et al., 2000b). That work showed that supplementation of a P-deficient corn-soybean meal (SBM) diet [0.10% available P (AP)] with 6% citric acid resulted in a 43% increase in tibia ash and a 22% increase in weight gain in chicks compared to those consuming diets with no added citric acid. The tibia ash response observed in the latter study was of the same magnitude as that observed with addition of 1,450 U phytase/kg 2001 Poultry Science Association, Inc. Received for publication July 31, Accepted for publication January 15, Present address: Purina Mills, Inc., 1401 S. Hanley Road, St. Louis, MO To whom correspondence should be addressed: poultry@uiuc.edu. to a P-deficient diet (Biehl et al., 1995). The previous experiments clearly indicated beneficial effects of citric acid on P utilization in chicks. The primary objective of the current study was to determine if citric acid would reduce the AP requirement or amount of supplemental P needed in a corn-sbm diet for chicks. Also, because citric acid has long been known to be a chelator for Ca (Greenberg and Greenberg, ; Hastings et al., 1934), we determined whether citric acid affects Ca utilization or the Ca requirement in chicks. MATERIALS AND METHODS General Procedures Male chicks resulting from a cross of New Hampshire male and Columbian females were used in all experiments. All housing, handling, and euthanization procedures were approved by the University of Illinois Committee on Laboratory Animal Care. 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 Days 0 to 7 posthatch, chicks were fed a nutritionally complete Abbreviation Key: AP = available P; SBM = soybean meal. 783

2 784 BOLING-FRANKENBACH ET AL. corn and SBM starter diet (NRC, 1994) containing 23% CP. On Day 8 posthatch, following an overnight feed withdrawal, chicks were weighed, wing-banded, and assigned to treatment groups so that mean initial weights were similar among the treatment groups. Four replications of four chicks per treatment were fed each experimental diet ad libitum from Days 8 to 21 (Experiment 1) or Days 8 to 22 (Experiments 2, 3, and 4) after hatching. At the end of each experiment, chicks were euthanized by CO 2 inhalation, and right tibiae were collected for bone ash determination. Tibiae were pooled by replicate cages and autoclaved, and adhering tissue was removed. Bones were dried for 24 h at 100 C, weighed, and then dry-ashed for 24 h in a 600 C muffle furnace. Ash weight was expressed as a percentage of dry bone weight or as milligrams per chick. Experiment 1 It was the objective of this experiment to evaluate the effect of 6% citric acid on the Ca requirement of chicks. This information was also considered necessary for designing the later experiments to estimate the effect of citric acid on the AP requirement, because we needed to know the optimal dietary Ca level required in the presence of citric acid. Limestone was added to a corn- SBM basal diet (Table 1) (23% CP, 0.54% Ca, 0.45% AP) in 0.1% increments to attain total Ca levels of 0.54 to 1.24%. Each level of Ca was fed with 0 or 6% citric acid to compose 16 dietary treatments in an 8 2 factorial design. Limestone and citric acid 3 were added at the expense of cornstarch. Experiment 2 In Experiment 1, 6% citric acid resulted in some negative effects on growth performance, suggesting that the level may be too high for diets containing 0.45% AP. Therefore, a second experiment was conducted to further evaluate lower citric acid levels in an attempt to determine the optimal level of citric acid in corn-sbm diets containing deficient and adequate levels of AP. A 2 4 factorial experiment was designed in which two levels of AP (0.20 or 0.45%) were fed with four levels of citric acid (0, 2, 4, or 6%). The basal diet is shown in Table 1. Dietary additions of P (KH 2 PO 4 ) and citric acid were made at the expense of cornstarch. Experiment 3 This chick study was conducted to determine if 6% citric acid would reduce the level of supplemental P required in a corn-sbm diet. Dietary treatments were a basal P-deficient diet (Table 1) (23% CP, 1.3% Ca, 0.20% AP) supplemented with 0, 0.05, 0.10, 0.15, 0.20, or 0.25% 3 Archer Daniels Midland, Decatur, IL TABLE 1. Composition of the basal diet fed to chicks, Experiments 1 to 4 Ingredient Experiment 1 1 Experiments 2 to 4 2 (%) Cornstarch to 100 to 100 Corn Soybean meal Soybean oil Limestone Dicalcium phosphate Salt Mineral premix Vitamin premix DL-Methionine Choline chloride Antibiotic Calculated to contain 23% CP, 0.54% Ca, and 0.45% available P. 2 Calculated to contain 23% CP, 1.3% Ca, and 0.20% available P. This same diet was used in Experiment 4, except that the Ca level was lowered to 1% by reducing the limestone from 2.8 to 2.0% and by adding 0.8% cornstarch. 3 Provided as milligrams per kilogram of diet: manganese, 75 from manganese oxide; iron, 75 from iron sulfate; zinc, 75 from zinc oxide; copper, 5 from copper sulfate; iodine, 0.35 from ethylene diamine dihydroiodide; selenium, 0.2 from sodium selenite. 4 Provided per kilogram of diet: vitamin A (as retinyl A acetate), 4,400 IU; cholecalciferol (as activated animal sterol), 1,000 IU; vitamin E (as DL-α-tocopheryl acetate), 11 IU; vitamin B 12, 0.01 mg; riboflavin, 4.41 mg; d-pantothenic acid, 10 mg; niacin, 22 mg; menadione sodium bisulfite, 2.33 mg. 5 Flavomycin, which contains 4.4 g bambermycin/kg, was added at a level of 0.05% to diets in Experiments 1 and 2; bacitracin methylene diasalicylate was used in Experiment 3, included at 0.025% of the diet. inorganic P (KH 2 PO 4 ) in the absence or presence of 6% citric acid (6 2 factorial design). The Ca level was set at 1.3% to ensure that it would not be limiting, based on the results of Experiment 1. Citric acid and KH 2 PO 4 were supplemented at the expense of cornstarch to the basal diet. Experiment 4 This experiment was similar to Experiment 3, except that 4% citric acid was evaluated and the dietary Ca level was lowered to 1% by reducing the limestone from 2.8 to 2.0% and adding 0.8% cornstarch. The purpose of these changes was to determine if a lower level of 4% citric acid would reduce the AP requirement. We also endeavored to evaluate citric acid in diets containing a Ca level that is typically fed commercially. The eight dietary treatments were a P-deficient diet (23% CP, 1.0% Ca, 0.2% AP) supplemented with 0, 0.10, 0.15, or 0.20% inorganic P (KH 2 PO 4 ) in the absence or presence of 4% citric acid (4 2 factorial design). Citric acid and KH 2 PO 4 were again added in place of cornstarch in the basal diet. Statistical Analysis Data were subjected to ANOVA using the general linear models procedure of SAS software (SAS Institute, 1990). The results of Experiments 1, 2, 3, and 4 were analyzed as 8 2, 2 4, 6 2, and 4 2 factorial treatment

3 CITRIC ACID FOR CHICKS 785 TABLE 2. Effects of Ca and citric acid on chicks consuming a Ca-deficient corn-soybean meal diet, Experiment 1 1 Dietary Ca Tibia ash 2 Supplemental Total Citric acid Weight gain 2 Gain:feed 2 (%) (%) (%) (g) (g/kg) (%) (mg) cdef 663 bcd 39.5 hi 473 gh bcd 673 abcd 41.8 efg 548 ef abc 694 abc 41.3 fg 544 ef bcd 969 ab 42.1 defg 564 e ab 694 abc 43.0 cde 616 cd abc 700 ab 45.2 ab 654 bc a 706 a 46.1 a 699 ab a 705 a 45.9 a 711 a g 610 f 38.3 i 398 j g 616 ef 38.6 i 409 ij fg 652 de 40.6 gh 455 hi ef 679 abcd 41.2 fg 482 gh def 654 cde 42.0 efg 508 fg bcde 682 abcd 43.6 cd 564 e def 649 def 42.7 cdef 561 e bcd 650 de 44.1 bc 579 de Pooled SEM a j Means within a column with no common superscript differ significantly (P < 0.05). 1 Data are means of four replications of four chicks during 8 to 22 d after hatching. 2 Significant Ca and citric acid main effects (P < 0.05) and significant quadratic effect of Ca (P < 0.05). designs, respectively. Differences among individual treatment means were assessed using the least significant difference test (Carmer and Walker, 1985). Linear regression analyses were also used to determine significant linear and quadratic effects. Broken-line regression analyses (Robbins et al., 1979) were also used in some experiments to estimate Ca and AP requirements. RESULTS AND DISCUSSION Growth performance and tibia ash were increased (quadratic, P < 0.05) by Ca supplementation in Experiment 1 (Table 2). Growth responses to Ca were not greatly affected by citric acid, with growth being statistically maximized by 0.2% supplemental Ca in the absence of citric acid and 0.3 to 0.4% supplemental Ca in the presence of citric acid. Tibia ash (% or mg/chick) was maximized by approximately 0.5 to 0.6% supplemental Ca in the presence or absence of citric acid. Broken-line regression analysis (r 2 = 0.99) indicated that the total Ca requirement for tibia ash (mg/chick) was 1.2% in the presence or absence of citric acid. The latter results indicated that citric acid did not have a major effect on Ca availability or the Ca requirement. Sifri et al. (1977) also reported that citric acid did not have any effect on Ca metabolism in chicks. In contrast to Ca results in our study, addition of 6% citric acid resulted in depressions in weight gain, gain:feed, and tibia ash at almost all dietary Ca levels. The reason for the negative response is unknown and was not observed in our earlier study (Boling et al., 2000b). It is important to note, however, that the diets used in the current experiment were not deficient in AP, as was the case in our earlier study. Thus, the results of this experiment suggested that 6% citric acid may be too high for diets containing adequate AP. Consequently, Experiment 2 was conducted to evaluate lower levels of citric acid. As expected, increasing AP from 0.2 to 0.45% increased growth and tibia ash in Experiment 2 (Table 3). An increasing response (linear, P < 0.05) in weight gain and tibia ash was also observed from increasing levels of supplemental citric acid in chicks fed diets containing 0.20% AP. The latter responses were in agreement with our earlier experiments (Boling et al., 2000b) with chicks fed diets containing 0.10% AP and 0 to 6% citric acid. Also in agreement with our earlier experiments, no consistent gain:feed response to citric acid occurred in the current experiment, indicating that citric acid supplementation generally increased feed intake. The increase in feed intake from citric acid was probably due mainly to increasing P availability in the diet; however, part of the response might also have been due to decreased dietary energy when citric acid was added in place of cornstarch. The ME n of citric acid for chicks is unknown, but it is probably less than that of cornstarch. Although citric acid supplementation did generally increase feed intake, the resulting increase in basal diet AP intake was not nearly large enough to produce the large growth and bone ash responses observed herein. Moreover, citric acid supplementation produced no significant decrease in gain:feed ratio for chicks fed diets containing 0.45% AP (Table 3) when weight gains were similar across all diets. The latter results further indicate that the increased feed intake from citric acid in the P deficient diets (0.20% AP) was due mainly to increased weight gains and not due to lower dietary ME n. In contrast to diets containing 0.2% AP, no significant improvements in growth performance or tibia ash were observed when any level of citric acid (0 to 6%) was included in a diet containing adequate P (0.45% AP), resulting in an

4 786 BOLING-FRANKENBACH ET AL. TABLE 3. Effects of available P (AP) level and citric acid level on chicks consuming a P-deficient cornsoybean meal diet, Experiment 2 1 Tibia ash 2 AP Citric acid Weight gain 2 Gain:feed 2 (%) (%) (g) (g/kg) (%) (mg) d 658 c 31.6 d 321 f c 676 abc 33.3 d 369 ef b 661 bc 37.4 c 429 e b 652 c 40.1 b 512 d a 700 a 44.9 a 761 a a 679 abc 45.2 a 718 ab a 691 ab 44.7 a 693 bc a 679 abc 43.7 a 634 c Pooled SEM a f Means within a column with no common superscript differ significantly (P < 0.05). 1 Data are means of four replications of four chicks during 8 to 22 d after hatching. 2 Significant main effect of AP and citric acid and AP citric acid interaction (P < 0.05) for weight gain and tibia ash. Significant main effect of AP (P < 0.05) for gain:feed. Significant linear effect (P < 0.05) for citric acid in the 0.20% AP diets for weight gain and tibia ash. AP citric acid interaction. Thus, the negative effect of citric acid in a 0.45% AP diet observed in Experiment 1 did not occur in this experiment. Therefore, 6% citric acid was chosen for Experiment 3, because it yielded the largest response in growth and tibia ash in chicks fed the P-deficient (0.2% AP) diet. In Experiment 3, birds responded quadratically (P < 0.05) to increasing P for all criteria measured (Table 4). Addition of citric acid to diets containing the lower AP levels (0.2 to 0.3%) increased (P < 0.05) weight gain and tibia ash but had no significant effect at the higher AP levels. The latter responses resulted in an AP citric acid interaction (P < 0.05). When no citric acid was present, the maximum response in weight gain and tibia ash was observed with 0.20% added P (0.40% AP). In diets containing 6% citric acid, weight gain was maximized at 0.05% added P (0.25% AP), and tibia ash was maximized at 0.10 to 0.15% added P (0.30 to 0.35% AP). Broken-line regression analysis (r 2 = 0.99) indicated that the AP requirement for tibia ash was 0.41% in the absence of citric acid and 0.32% in the presence of citric acid. These results indicated that 6% citric acid was releasing approximately 0.10% AP from the corn and SBM, and this in turn decreased the AP requirement by about 0.10%. Similar results were obtained with 4% citric acid in diets containing 1% Ca in Experiment 4 (Table 5). Increasing the AP from 0.2 to 0.40% yielded quadratic increases (P < 0.05) in weight gain, gain:feed ratio, and tibia ash when diets contained no citric acid; a quadratic increase in tibia ash also occurred when diets contained 4% citric acid. Supplementation of the 0.20% AP diet with 4% citric acid increased growth and tibia ash, but this addition had no effect at the higher AP levels of 0.35 and 0.40%. The latter again resulted in an AP citric acid interaction (P < 0.05). In diets containing no citric TABLE 4. Effects of available P (AP) level and 6% citric acid for chicks consuming a P-deficient cornsoybean meal diet, Experiment 3 1 Dietary AP Tibia ash 2 Supplemental Total Citric acid Weight gain 2 Gain:feed 2 (%) (%) (%) (g) (g/kg) (%) (mg) g 669 cde 32.3 g 317 g f 656 e 36.7 f 429 f e 665 de 40.3 de 495 ef cde 681 bcde 42.5 bc 563 cde abc 695 abc 45.3 a 662 a a 711 a 45.4 a 636 ab de 680 bcde 38.9 e 473 f bcd 686 abcd 41.0 cd 545 de bcde 678 bcde 42.7 b 572 bcd bcd 683 bcd 43.9 ab 605 abcd ab 697 ab 44.0 ab 621 abc ab 700 ab 43.8 ab 619 abc Pooled SEM a g Means within a column with no common superscript differ significantly (P < 0.05). 1 Data are means of four replications of four chicks during 8 to 22 d after hatching. 2 Significant main effect of AP and citric acid for weight gain and tibia ash and significant main effect of AP for gain:feed (P < 0.05). Significant AP citric acid interaction (P < 0.05) for weight gain and tibia ash. Significant quadratic effect (P < 0.05) of AP for all three parameters.

5 CITRIC ACID FOR CHICKS 787 TABLE 5. Effects of available P (AP) level and 4% citric acid for chicks consuming a P-deficient cornsoybean meal diet, Experiment 4 1 Dietary AP Tibia ash 2 Supplemental Total Citric acid Weight gain 2 Gain:feed 2 (%) (%) (%) (g) (g/kg) (%) (mg) e 643 c 33.2 f 363 e bcd 676 abc 40.0 d 536 c a 688 a 42.2 ab 613 a ab 686 a 42.5 a 607 a cd 667 abc 38.3 e 486 d cd 678 ab 41.9 abc 564 abc d 676 abc 42.0 ab 588 ab abc 692 a 42.2 ab 609 a Pooled SEM a f Means within a column with no common superscripts are significantly different (P < 0.05). 1 Data are means of four replications of four chicks during the period 8 to 22 d after hatching. 2 Significant main effect of AP and citric acid for weight gain and tibia ash and significant main effect of AP for gain:feed (P < 0.05). Significant AP citric acid interaction (P < 0.05) for weight gain and tibia ash. Significant quadratic effect (P < 0.05) of AP for all three parameters. acid, weight gain and tibia ash were maximized with 0.15% added P (0.35% total AP). When diets contained 4% citric acid, weight gain was statistically maximized at 0.2% AP and tibia ash was maximized at 0.30% AP. These results agreed with Experiment 3 in that 4% citric acid increased the availability of P in the corn-sbm basal diet and reduced the amount of needed supplemental P by approximately 0.10%. The mechanism by which citric acid increases the availability of P in a corn-sbm diet is unknown. Some early studies (Hamilton and Dewar, 1937; Day, 1940; Pileggi et al., 1956) proposed that effects of citrate on P may be due indirectly to its ability to bind or complex with Ca. For example, Pileggi et al. (1956) suggested that the antirachitogenic effects of citric acid in rats were due to citric acid binding Ca and reducing the inhibitory effects of Ca on intestinal phytic acid hydrolysis. It can be speculated that perhaps citric acid, a strong chelator of Ca, may complex with Ca and decrease its binding to phytate, thereby making the phytate more soluble or less stable and, in turn, more susceptible to endogenous phytate. This hypothesis is supported by our recent work with laying hens (Boling et al., 2000a) that showed no response to citric acid in a corn-sbm laying hen diet containing a high level of Ca (3.8%). Another possible mode of action of citric acid is its effect on intestinal ph. However, citric acid would not be expected to have a large effect on intestinal ph because it is an organic acid that is metabolized rapidly. Furthermore, an earlier study (Boling et al., 2000b) showed that 6% citric acid, 6% Na citrate, or a 1:1 combination of the two produced similar responses in growth and bone ash. The results of the current study confirm our earlier study (Boling et al., 2000b) that dietary citric acid improves phytate P utilization in chicks. Our current study has further quantified the effect of citric acid to being equivalent to approximately 0.10% AP and that citric acid has no major effect on Ca availability or the Ca requirement. Thus, citric acid provides another means for increasing P utilization and decreasing P excretion in chicks fed phytate containing corn-sbm diets. Phytase and hydroxylated vitamin D 3 compounds have already been shown to have the potential for replacing up to 0.2% AP when used in combination (Biehl et al., 1995; Mitchell and Edwards, 1996; Biehl and Baker, 1996). The use of citric acid in combination with phytase and hydroxylated vitamin D 3 compounds warrants future investigation. The combination of all three of these additives could possibly totally eliminate the need for inorganic P supplementation of corn-sbm chick diets. REFERENCES BASF, Use of Natuphos in pigs and poultry. From research and practical experience. Ed. 30. BASF Corporation, Mount Olive, NJ. Biehl, R. R., and D. H. 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