The Relationship of Calcium Intake, Source, Size, Solubility In Vitro and In Vivo, and Gizzard Limestone Retention in Laying Hens 1 BINGFAN ZHANG and CRAIG N. COON2 Department of Animal Science, University of Minnesota, St. Paul, Minnesota 55108 ABSTRACT A 10-d trial was conducted to investigate the relationship of Ca particle size (CPS), dietary Ca level (DCL), limestone source (LS), limestone solubility in vitro (LST) and in vivo (LSV), and limestone retention in the gizzard (LRG). A total of 120 molted Leghorn hens, 88 wk of age, were randomly assigned into a 2 3 4 factorial arrangement of treatments with LS (A and B), DCL (1.95, 3.72, and 5.32%), and CPS of each limestone source (average U.S. Screen Nos. 5, 8, 14, and 27). The in vitro solubility of the four respective particle sizes were 29.8, 45.8, 49.3, and 63.1% for Source A; and 36.3, 54.8, 57.7, and 67.6% for Source B. The limestone retention in the gizzard of Source A was greater than that of Source B. The limestone retention in the gizzard was increased as in vitro solubility decreased (P < 0.001) or dietary Ca level increased (P < 0.001). The in vivo solubility of the limestone was decreased as dietary Ca level increased (P < 0.001). The in vivo solubility was negatively correlated (P < 0.05) with in vitro solubility for Source A when Ca was fed at 3.72%. The data showed that larger particle size limestone (> 0.8 mm) with lower in vitro solubility (30 to 50%) was retained in the gizzard for a longer time, which increased the in vivo solubility (94% maximum). The results support the concept that larger particle size or lower in vitro solubility may increase Ca retention for layers. (Key words: limestone source, solubility in vitro and in vivo, calcium retention, dietary calcium level, limestone retention in gizzard) 1997 Poultry Science 76:1702 1706 INTRODUCTION A prolonged retention of large particle Ca supplements in the gizzard has been suggested to make Ca more available during the period of shell formation throughout the night (Scott et al., 1971; Roland et al., 1973). The retention of Ca in the gizzard of laying hens for improving shell quality may be dependent upon particle size, porosity of the Ca source, and the overall in vivo solubility of the Ca source. The potential in vivo solubility of Ca particles has been estimated by using various in vitro solubility methods (Cheng and Coon, 1990a). In 8-wk layer feeding studies, Cheng and Coon (1990b) reported that the eggshell quality and bone status were more closely related with limestone in vitro solubility than particle size. The researchers indicated a potential difference in Ca retention for layers when two Ca sources of the same particle size with different in vitro solubilities were compared. Rao and Roland (1989) conducted 25-h collection studies for hens fed by intubation and reported that the Received for publication November 21, 1996. Accepted for publication July 30, 1997. 1Published as Paper Number 971165804, Scientific Journal Series, Minnesota Agricultural Experiment Station. 2To whom correspondence should be addressed. percentage of Ca solubilized in the digestive system decreased as Ca intake increased and that larger particle Ca supplements had a higher in vivo solubility but were less soluble under in vitro conditions. The researchers found no relationship of Ca solubility in vitro with the Ca solubility in vivo when two Ca sources of the same particle size but with different in vitro solubilities were compared (Rao and Roland, 1990). The conflicting results between the 8-wk feeding studies and the intubation experiments may have resulted from the failure to simulate actual feeding conditions in the shortterm studies. The passage rate of Ca particles for hens intubated with Ca after a feed withdrawal period may not reflect actual feeding conditions because the amount of Ca particles retained in the gizzard may be dependent upon the type and amount of Ca consumed following the intubation period. The present study was conducted to determine the effect of dietary Ca level, particle size, and in vitro limestone solubility on in vivo limestone solubilization and limestone retention in the gizzard of laying hens under ad libitum feeding conditions. MATERIALS AND METHODS A total of 120 Leghorn hens, 88 wk of age (molted at 64 wk of age), were randomly assigned into a 2 3 4 factorial arrangement with two limestone sources (A 1702
LIMESTONE EVALUATION FOR LAYERS 1703 TABLE 1. Composition of the experimental diets Ingredients Diet 1 Diet 2 Diet 3 (%) Corn 55.06 64.30 72.14 Soybean meal, dehulled (48.5% CP) 24.83 22.84 21.75 DL-methionine 0.05 0.04 0.03 Limestone 13.31 9.12 4.49 Salt 0.19 0.19 0.19 Vitamin mixture 1 0.05 0.05 0.05 Mineral mixture 2 0.04 0.04 0.04 Choline chloride, 50% 0.26 0.26 0.26 Vegetable oil 5.13 2.13 0.00 DiCal phosphate 0.90 0.87 0.84 Na 2 HPO 4 0.18 0.18 0.18 Calculated composition Dietary Ca 5.32 3.72 1.95 ME n, kcal/kg 2,870 2,870 2,870 CP 16.5 16.5 16.5 Methionine 0.32 0.32 0.32 TSAA 0.58 0.58 0.58 Nonphytate phosphorus 0.30 0.30 0.30 Analyzed Ca in diet Limestone source A 5.29 3.71 1.92 Limestone source B 5.37 3.81 1.99 1Vitamin mixture provides in milligrams per kilogram of diet: vitamin A, 5,500 IU; vitamin E, 25 IU; menadione, 1.45 mg; cholecalciferol, 1,100 IU; riboflavin, 5.4 mg; pantothenic acid, 23 mg; nicotinic acid, 55 mg; vitamin B 12, 9.9 mg; vitamin B 6, 9.5 mg; thiamine, 5.4 mg; folic acid, 1.8 mg; biotin, 0.28 mg. 2Trace mineral mixture provides in milligrams per kilogram of diet: Mn, 68; Zn, 61; Fe, 120; Cu, 7; I, 0.7; Se, 0.3. and B), three dietary Ca levels (1.95, 3.72, and 5.32% Ca), and four limestone particle sizes (average U.S. Standard Screen Nos. 5, 8, 14, and 27). Three isocaloric and isonitrogenous corn-soybean test diets containing 16.5% CP and 2,870 kcal ME/kg were utilized for feeding layers the three levels of Ca (Table 1). The four particle sizes from the two limestone sources were included for each test level of dietary Ca. Hens were housed in individual layer cages in an environmentally controlled room. The room temperature was set at 23 C. Individual feed intake was taken in a 7-d acclimation period and a following 3-d excreta collection period. Hens consumed corresponding experimental diets ad libitum for both of the periods. Hens were exposed to a lighting regimen of 16 h light (L):8 h dark (D). The mixed diets were provided just before the initiation of the dark hours at 1900 h. The excreta was collected for each hen and the experimental diets were withdrawn and weighed after 72 h. Limestone in the feed residuals for each hen after the 72-h period was washed out, dried, and weighed to determine the actual Ca intake during the experiment. Hens were euthanatized 5 h after feed withdrawal by carbon dioxide asphyxiation and the digestive tracts were removed and frozen at 20 C until analysis. Limestone in the feed residuals, excreta, and gizzard content was separated using the method described by Rao and Roland (1989). The Ca content of feed was analyzed using atomic absorption (AOAC, 1990). Limestone solubility in vivo was calculated as (LM intake LM excreted) 100/LM intake, and expressed on a percentage basis. The in vitro solubility of the limestone used in present study was determined by the method described by Zhang and Coon (1997). Briefly, a 2.0-g limestone sample was poured into a 400-mL beaker containing 200 ml of 0.2 N HCl solution that was warmed at 42 C until the temperature of the solution became constant (about 15 min) in a water bath oscillating at 80 Hz. After allowing 10 min for reaction, the undissolved limestone was filtered onto a preweighed Whatman ashless filter paper (No. 41), and weighed after drying in a 60 C oven for 20 h. The in vitro solubility of limestone was expressed as the percentage weight loss. Data were analyzed using the General Linear Models procedure of SAS (1988, Version 6.03), and difference between treatments were tested using Duncan s multiple range test. The correlation between variables were analyzed by the correlation procedure of SAS (SAS Institute, 1988). RESULTS AND DISCUSSION The in vitro solubility of the four particle sizes (average U.S. Standard Screen Nos. (SN) of 5, 8, 14, and 27) were 29.8, 45.8, 49.3, and 63.1% for Source A; and 36.3, 54.8, 57.7, and 67.6% for Source B, respectively. The larger particle size had lower in vitro solubility when the comparison was made within the same source. Source A was less soluble than Source B when the size was identical. The amount of limestone retained in the gizzard was significantly affected by limestone source, size, Ca dietary intake, and all levels of their interaction (Table 2). The amount of limestone retained in the gizzard was increased with increasing particle size for
1704 ZHANG AND COON TABLE 2. The main and interaction effects of limestone source, dietary Ca level, and limestone particle size on the limestone in vivo solubility and the retention of limestone in gizzard Limestone Solubility Solubility retention in Effects Sources Diet Ca Size 1 in vitro in vivo gizzard (%) (%) (g) Source A 81.5 5.90 a B 81.2 3.81 b Diet Ca (DCL, %) 1.95 88.2 a 2.77 b 3.72 78.6 b 5.98 a 5.32 77.3 b 5.81 a Size 5 84.4 a 7.87 a 8 84.0 a 6.24 b 14 79.1 b 4.52 c 27 78.0 b 0.79 d Source size A 5 86.3 a 9.70 a 8 82 ab 7.99 b 14 78.1 bc 5.28 cd 27 79.5 bc 0.64 f B 5 82.5 ab 6.04 c 8 86.0 a 4.49 de 14 80.0 bc 3.75 e 27 76.5 c 0.94 f Source DCL size A 1.95 5 29.8 93.9 a 4.70 de 8 45.8 92.3 ab 4.05 ef 14 49.3 83.0 b 2.15 f 27 63.1 87.9 ab 1.13 fg 3.72 5 29.8 84.8 b 15.37 a 8 45.8 79.0 bc 11.77 b 14 49.3 77.8 bc 5.49 de 27 63.1 76.5 bc 0.68 fg 5.32 5 29.8 80.3 bc 9.02 c 8 45.8 74.7 c 8.16 cd 14 49.3 73.6 c 8.20 cd 27 63.1 74.0 c 0.10 g B 1.95 5 36.3 84.1 b 4.00 ef 8 54.8 90.7 ab 2.87 ef 14 57.7 88.4 ab 2.19 f 27 67.6 85.5 ab 1.09 fg 3.72 5 36.3 82.5 bc 3.94 ef 8 54.8 84.0 b 4.25 e 14 57.7 74.4 c 4.71 de 27 67.6 69.4 c 1.63 fg 5.32 5 36.3 80.9 bc 10.19 bc 8 54.8 83.2 b 6.35 d 14 57.7 77.1 bc 4.36 de 27 67.6 74.7 c 0.10 g ANOVA root MSE 6.97 1.63 Source of variation Probabilities Source NS *** Size * *** Diet Ca (DCL, %) ** *** Source DCL NS *** Source size * *** DCL size NS *** Source DCL size NS *** a gwithin mean effects or interaction, means with no common superscript differ significantly (P < 0.05). 1Average U.S. Screen numbers 5, 8, 14, and 27 correspond to the range of sieve diameters 3.3 to 4.7, 2.0 to 2.8, 1.0 to 2.0, and 0.5 to 0.8 mm, respectively. *P < 0.05. **P < 0.01. ***P < 0.001. the same dietary Ca level and source. In general, the amount retained in the gizzard was increased when dietary Ca level was increased. However, a decrease was observed when the Ca level exceeded 3.72% for the two largest sizes of Source A. This decreased retention in the gizzard was not a result of an increase in the in vivo Ca solubilization, as the in vivo solubility was lower for hens fed the 5.32% than those fed 3.72% Ca diet. These results suggested that larger limestone could be expelled from the gizzard (pushed out by the followed ingestion
LIMESTONE EVALUATION FOR LAYERS 1705 TABLE 3. Unweighted least squares linear regressions of in vivo solubility Combined data of Sources A and B Regression 2 303.1 17.83 <0.0001 0.629 Residual 21 17.0 Constant 102.6 4.248 24.15 <0.0001 Diet Ca, % 3.277 0.611 5.36 <0.0001 In vitro solubility 0.183 0.069 2.63 0.015 Source A Regression 2 229.4 26.8 0.0002 0.856 Residual 9 8.6 Constant 106.8 4.121 25.91 <0.0001 Diet Ca, % 4.069 0.614 6.63 0.0001 In vitro solubility 0.221 0.071 3.11 0.0127 Source B Regression 2 92.5 3.44 0.0777 0.433 Residual 9 26.9 Constant 99.6 8.326 11.96 <0.0001 Diet Ca, % 2.486 1.088 2.29 0.0481 In vitro solubility 0.171 0.132 1.29 0.2302 of the limestone). Thus, without increasing a hen s ability for in vivo solubilization, which might have an upper limit, the retained limestone would not exceed the capacity of the gizzard for the large limestone particles. The layers fed Source A limestone (less soluble in vitro) retained more limestone in the gizzard than layers fed Source B. The increased retention of Source A limestone by layers only occurred when limestone particle size was larger than a screen size of 27. The layers retained only a small amount of limestone when the smallest particle size was fed from both sources and at all dietary Ca levels. The low retention in the gizzard of smallparticle limestone was caused by the high passage rate from the gizzard because of physical size limitations and not the high in vitro solubility because the corresponding in vivo solubility was not improved (Table 2). The results agree with the observation made by Rao and Roland (1992) that the retention time in the gizzard for small (diameter less than 0.84 mm) particle limestone is relatively short. The in vivo solubility was significantly (P < 0.01) decreased as dietary Ca intake was increased for both limestone sources (Table 2). The in vivo solubility significantly (P < 0.05) decreased as the size of limestone was decreased. The in vivo solubility of the limestone particles tended to be negatively correlated with the in vitro solubility, however only the hens fed Source A limestone at a 3.72% Ca level showed a significant (P < 0.05) negative relationship. The highest in vivo solubility was consistently found for hens fed the second largest particle size of Source B, whereas the largest particle size of Source A produced the highest in vivo solubility (Table 2). The data showed the effect of in vitro solubility on in vivo solubility was dependent on limestone source or relative solubility (Source A generally less soluble than Source B). Although less limestone was retained for hens fed Source A when the dietary Ca level was greater than 3.72%, a corresponding in vivo solubility increase was not observed. This result indicates that the excessive supply of limestone from a less soluble source decreases limestone in vivo solubility by increasing the excretion of undissolved limestone by the layers. The gizzard may have an upper limit for the amount of ingested limestone particles that can be retained. Layers fed diets with large-particle Ca supplements or less-soluble Ca particles may reach a saturation state of limestone particles in the gizzard and a continuing intake of same diets may cause an increased output of undissolved limestone. At the lowest dietary Ca level (1.95%), higher in vivo solubility was obtained when the two large particles from Source A were fed than when the same sizes from Source B were fed (93.9, 92.3 vs 84.1 and 90.7%, Table 2). The low Ca feed level did not produce significant in vivo solubility differences when the smallest particle limestone (0.5 to 0.8 mm) were fed. Rao and Roland (1990) intubated layers with two Ca sources with different in vitro solubility and did not find a significant influence of in vitro solubility on in vivo Ca solubilization. The inability of the researchers to detect a correlation of in vitro and in vivo solubility may have been caused by the researchers using only small-particle (0.5 to 0.8 mm) limestone in the experiment. Several possible reasons exist that help explain why the in vivo solubility data from the research reported herein was dependent on both the Ca source and in vitro solubility. First, the rate of Ca2+ uptake may have been limited by the hen s intrinsic capacity for Ca absorption from the digestive
1706 tract. The available Ca from the less-soluble source may be absorbed more completely due to its slower Ca release than Ca from sources with greater solubility. Second, large-particle Ca may go through the tract more slowly because of greater retention in the gizzard, which allows the limestone to stay in an acidic environment for a longer period of time. The acidity would increase the opportunity to dissociate the CaCO 3 into ionic Ca, hence producing more available Ca for absorption. Hens require ionic Ca for intestinal absorption (Scott et al., 1982). Although smaller particles have higher in vitro solubility, the particles stay in the in vivo acidic environment a reduced amount of time. The relatively higher in vivo Ca release rate of small particles from the gizzard may exceed the Ca absorption capacity of the intestine so that the utilization of the dissolved Ca may be reduced. Third, feeding high levels of large Ca particles from a less soluble source will create conditions for Ca particles to dissolve more slowly in the gizzard. The Ca particles will accumulate in the gizzard when the amount ingested surpasses the hen s ability to solubilize the Ca particles. The accumulated Ca would eventually be forced out by the following ingested Ca so that the apparent in vivo solubility would be negatively affected. It should be pointed out that the in vivo solubility is not a direct measure of Ca retention. Generally, the in vivo solubilities obtained in the present study (Table 2) were higher than the normal 40 to 60% Ca retention for laying hens. Released Ca from limestone may not be completely absorbed by hens. The lower Ca retention may also be attributed to the loss of the absorbed Ca from the kidney. There are conflicting results in the literature on the beneficial effect of larger-particle calcium or with a lower in vitro solubility on eggshell and bone status. Reviews (Scott et al., 1971; Roland, 1986) have shown a positive effect of Ca with a coarse particle size on eggshell quality in half of the reported studies, whereas the other half of the studies showed no effect on the shell quality. The large scope of Ca source and size makes comparison of these results difficult. In summary, the results showed that solubility in vivo is reversibly related to solubility in vitro. A larger particle limestone (>0.8 mm) with a lower in vitro solubility (within a range of 30 50%) accumulates in the gizzard (reverse relationship to in vitro solubility) and produces a high in vivo solubility (94% maximum LSV depending mainly on dietary Ca level and in vitro solubility). Thus, it would potentially lead to increased Ca retention by layers fed a larger particle limestone. Long-term studies (Cheng and Coon, 1990a; Zhang and Coon, 1992) showed that low in vitro solubility for limestone is superior for eggshell quality and bone status compared to high in vitro solubility values for limestone. The low in vitro solubility of Ca supplements allows for the increased gizzard retention and in vivo solubility that is required for utilization. The maximum ZHANG AND COON weight of limestone particles that accumulated in the gizzard of hens fed 3.72% Ca (NRC, 1994; 3.25%) from large-particle dietary limestone (> 2.0 mm) of the lower in vitro soluble Source A (29.8 45.8%) ranged between 11 to 15 g. The weight of limestone particles retained by the gizzard in the feeding studies was dependent upon dietary level of Ca, particle size, and solubility. ACKNOWLEDGMENTS The authors are indebted to the Broiler and Egg Association of Minnesota and Iowa Limestone Company for partial financial support of the research. 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