IMPROVED IN VITRO METHODS OYSTER SHELL SOLUBILITY~

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1 01997 Applii Poultry Science, Inc IMPROVED IN VITRO METHODS FOR DETERMINING LIMESTONE AND OYSTER SHELL SOLUBILITY~ BINGFAN ZHANG and CRAIG N. COON2 Depment ofanimal Science, University of Minnesota, St. Paul, MN Phone: (612) FB: (612) Primary Audience: Feed Mill Managers, Limestone Processors, Nutritionists DESCRIPTION OF PROBLEM Research has demonstrated the beneficial effect of feeding layers a Ca source containing larger coarse particles or a source assayed to have a low in vitro solubility on layer egg shell quality and bone ash concentration [l, 2,3,4]. Solubility of Ca sources in vitro is highly correlated with solubility in vivo (reverse relationship) and Ca availability in laying hens [q. Since both egg shell quality and skeletal maintenance are economically important to the layer industry, there is an increasing justification to formulate calcium requirements based on in vitro Ca solubility. Use of different methods for determining Ca solubilityhave created difficulties for comparison and application of results from different studies. The percentage weight loss method (WLM) and the quicker ph change method (PHM) [6] have gained fairly wide acceptance because the testing conditions have been standardized. The main limitation of the ph change and weight loss methods is 1 Published as Paper Number , the Scientific Journal Series, Minnesota Agricultural Experiment Station. 2 To whom correspondence should be addressed

2 Research Report ZHANG and COON 95 that the amount of available H+ may become limiting when a highly soluble limestone is tested. The amount of H+ contained in 100 ml of 0.1N HC1 solution as recommended in the methods will be completely used to produce C02 by dissolving 1.0 g CaC03, which is equivalent to a maximum of about 50% solubility. The limitation of the methods in the amount of H+ can likely be overcome by increasing the normality of HCl, volume of the solution, or decreasing sample size and/or the time of reaction so that the sensitivity of the methods can be increased. Scott [I developed a procedure (SWLM) which can be considered a modified WLM [6]. This method differs from the WLM because of the smaller amount of sample used (the sample size was reduced to 0.5 g from 2.0 g as recommended in the WLM). This modification helps alleviate the problem of possible H+ deficiency with the WLM, but increases the risk of inaccurate evaluation of test samples containing a high percentage of large particles. We performed this study to find a more reliable in vitro method of measuring solubility of calcium sources. MATERIALS AND METHODS The experiment used limestone from two sources, oyster shell (OS) and limestone from commercial sources (LS). Limestone was screened to six different sizes (average United States screen numbers 5,6,8.5,14,19, and 50) for OS and seven sizes (six as screened for OS, plus an 80 screen size) for LS. Average United States standard screen number is defined as described by Cheng and Coon [6]. Three different concentrations (O.lN, 0.2N, and 0.3N) of HCl were combined with two different volumes (100 ml and 200 ml) to produce six different treatments. An additional treatment of 100 ml of 0.3N HCl used a 5-min reaction time (as opposed to 10 min for the other treatments) but otherwise followed the procedure described below. Three replicates of each sample were measured for each treatment. The procedure was performed as follows: beakers of 400 ml capacity containing the assigned volume and normality of HCl were warmed in a 42 C water bath oscillating at 80 Hz for 15 min, or until the temperature of the solution in the beakers was 42 C. The bath water surface was kept at least 1.0 cm higher than that of HCl solution in the beaker. A test sample of 2.0 g was placed in each of the 400-mL beakers except the one to be used as a blank (the blank was used to establish new ph change method). After allowing the sample to react with HCl in the bath for 10 min, the supernatant (about 5/6 of total) was transferred into a 250-mL beaker without losing undissolved sample (the same was done for the blank). About 200 ml of deionized water was added to each of the 400-mL beakers to stop the reaction, and the undissolved sample was later filtered on a pre-weighed and dried filter paper [8] with excess deionized water. The weight was determined after drying in a 70 C oven for 10 hr or until constant weight was reached for the determination of the actual weight loss. The transferred blank and the supernatant in the 250-mL beakers were agitated until they became homogeneous. Their ph levels were then immediately measured with a ph meter and electrode [9]. The ph change was calculated (the reading from the supernatant minus the reading of the blank). Because following the PHM [6] led to less than satisfactory stability of ph readings, the procedure was modified as described above in an attempt to obtain more stable readings. The solubility of two oyster shell and three limestone samples from commercial sources was determined by the 200 ml of 0.2N HC1 weight loss method (new WLM) as described herein, the WLM [6], and the SWLM [7l. n o of the threelimestone samples (samples A and B) were purposely made by mixing proportional various-sized limestones screened from two commercial sources to create a larger difference in size distribution between the two limestone samples. The solubility of six replicates of each sample was determined using each of the methods. To describe treatments sensitivity in distinguishing samples, we used Range divided by Least Signifcant Difference critical value for comparison, where range is defined as the difference between the highest and lowest solubility among the samples produced by a treatment (RDL). Data were analyzed using the regression procedure of the SAS statistical package [lo, 111.

3 96 LIMESTONE SOLUBILITY ASSAY RESULTS AND DISCUSSION METHOD DEVELOPMENT The two treatments, 200 ml of 0.2N HCI and 200 ml of 0.3N HCl, produced a larger solubility range for the samples tested than did the other treatments. The mean solubility values ranged from 20.%% to 78S% for 200 ml 0.2N HCl, and from 35.99% to 97.59% for 200 ml 0.3N HCl treatment ("hble 1). Although the 200 ml of 0.3N HCl treatment produced a slightly wider range of solubility values than 200 ml of 0.2NHCI treatment, the latter is considered better because it provides more space for separating more soluble Ca sources than those used in the present study. Furthermore, 200 ml 0.2N HCl produced the highest RDL values for both OS and LS (Table l), which indicated that this treatment was the most sensitive in distinguishing among samples. Although 100 ml 0.1N HCl yielded the lowest variability, this reduction in variabfity was not enough to compensate for the reduced solubility range; this treatment had a lower RDL than 200 ml 0.2N HCI. Comparing other treatments (100 ml 0.2N HC1 of OS and LS; 200 ml O.IN HCI of LS) to 200 ml 0.2N HCl yields similar results. 'hble 2 summarizes regression models to predict the absolute amount of limestone dissolved in HCI generated from different treatments by using h(ph change) as predictor. The models derived in the present study were satisfactory in terms of predicting power (R2 ranges from.9303 to 959) and simplicity. The PHM [6] predicts solubility directly from ln(ph change) with a cubic relationship between the solubility and ln(ph change). In the present study, only the linear and/or quadratic term of h(ph change) was significant in improving the models to predict the absolute weight loss instead of solubility (percentage basis) directly. The modification was made because the direct solubility prediction introduces potential systematic error due to the difficulty in weighing an exact amount (e.g., 2.0 g) of sample, especially in the case of a w

4 Research Report ZHANG and COON 97 TABLE 2. Re ression models predicting absolute amount of limestone dissolved in HCI (g) with In(pH change) as predicto? I Io51334 I I co.0001 I <O.o %e model is in the form of Y = a + BlX + Bfi where X = In H change). Data were the pooled measurements of both oyster shell (OS) and limestone from commercial sources(ps). BMSE = Mean square of error from the table of ANOVA analysis. large-particle sample. The same sample may thus yield different ph readings, and thus different solubilities, because of over- or underweighing of test samples. The inaccuracy in weighing samples mainly affects the absolute solubilized amount; it may not affect, or may affect only to a lesser degree, the solubilized proportion. The prediction equations derived from the present study should be more reliable than previous equations based on the PHM [6]. The solubility of the calcium sources can be easily calculated from the predicted weight loss. The accuracy of the PHM [6] relies on the accuracy of ph readings. The procedure of Cheng and Coon [6] sometimes resulted in unstable ph readings in the present study. Possible reasons for this instability include: 1) the solution was not homogenous when the reading was taken after the reaction; 2) the water bath temperature sometimes fluctuated, and 3) readings depended on the placement of the electrode (top, middle, or bottom portion of the solution), so that heterogeneity of the solution may have generated variant readings. The procedure developed in the present study rarely produced unstable readings. COMPARISON OF METHODS The newly developed method utilizing 200 ml of 0.2N HCl for 2-g samples produced the most satisfactory range of solubility values and was more sensitive in distinguishing samples (higher RDL values) in the present study. The newly developed method (new WLM) was compared with the SWLM [7] and the WLM [6] using two oyster shell and three limestone samples that contained varying percentages of various particle sizes ('Ihble 3). The new WLM produced higher values and a larger solubility range than the SWLM and the WLM (Table 4). The WLM [6] gave the lowest and smallest solubility range. The SWLM produced a slightly higher and wider range of numbers than the WLM, but these two methods showed no significant difference in ability to distinguish test samples (Table 4). The SWLM consistently gave higher variations in determined solubilities than the other two methods. The smaller sample size (0.5 g) may be responsible for the higher variation associated with the SWLM. Relatively small and similar standard deviations were obtained for the three limestone samples using the new WLM and the WLM [6], but the new method showed a higher variation than the WLM in testing the two oyster shell samples. Although the new method may produce a larger variation in the results for some samples (e.g., oyster shell samples) than the WLM, the larger solubility range achieved with the larger volume of 0.2N HC1 acid solution

5 98 JAPR LIMESTONE SOLUBILITY ASSAY provides an advantage for distinguishing among samples (Table 4). Literature on the determination of solubility usually employs a 0.1N HC1 solution in an attempt to simulate the acidic environment in the gizzard of hens. The in vitro condition, however, may change as affected by either intrinsic or extrinsic factors. It would be extremely difficult to find a satisfactory in vitro condition to simulate the con&- tion in vivo. Thus, it seems more reasonable to judge the in vitro solubility determination methods by their ability to distinguish among Ca sources than by their in simulation ofin vivo conditions. Whether the difference as determined by an in vitro method has significance invivo should be tested by in vivo studies. Results from the 200 ml of 0.2N HC1 solution utilized for the in vitro solubility procedure in the present study correlate significantly (in reverse: R2 ranged from.36 to.90 depending on dietary calcium level and source) to layer in vivo limestone solubility [12]. METHOD LSA LSB LSC OS A OSB POOLED SE Mean+SD 0.2N of 200 ml e 4157rt1.33b 4358-c153a Md ' HCI (new) SWLM [2] ' 15.72&1.32ab 17.23rt2.12a bc 13.33k2.67bc WLM ' 13.27k1.35' 13.61k1.26' ll.o1+o.6ob 11.75k0.67b LSD

6 Research Report ZHANG and COON 99 CONCLUSIONS AND APPLICATIONS 1. The newly developedmethod (200 ml of 0.2N HClwith2.0 g sample size) produces a larger solubility range than the WLM, PHM [l], or SWLM [7J 2. The sample size of 0.5 g as recommended in the SWLM [A increases standard error in the determination of solubility because it is not large enough to accommodate large-particle samples. 3. In the newly developed ph change method, the weight loss (WL) can be predicted by: WL (g) = x h(ph change) (adjusted R2=.%1), and the solubility is calculated as: Solubility (%) = WL (g) x loo/initial sample weight (g). 1. Chew T.K. and C.N. Coon, Effect of calcium source, particle size, limestone solubility in and calcium intake level on layer bone status and performance. Poultry Sci Scott, M.L, SJ. Hull, and P.A. MullenhoN, The calcium re uirements of laying hens and effects of dietar yter &ell upon eggshell quality. Poultry sci Brister, RD., Jr., S.S. Linton, and C.R Cregtr, Effect of dietary calcium sources and article size on laying hen performance. Poultry Sci. 60:2& Keshavq K. and C.C. McCodck, Effect of sodium aluminosilicate, oyster shell, and their combinations on acid-base balance and eggshell quality. Poultry Sci. 7Q Rao, K.S. and DARoland, Influence of dietary calcium level and particle size of calcium source on in y~ calcium solubiluation by commercial Leghorns. Poultry Sci % Chew T.K. and C.N. Coon, Comparison of various methods for the determination of limestone solubility. Poultry Sci Scott, M., How can calcium be supplied to high-producing hens? Feedstuffs 63(39):1& Whatman Inc., 6 Just Road, Fairfield, NJ (Whatman ashless filter paper No. 41). REFERENCES AND NOTES 9. Orion Research, Inc.,The Schrafft Center,529 Main Street, Boston, MA (Orion digital ionalyzer/501: Electrode model 8155). 10. Statistics involved a general regression using the amount of limestone dissolved in HCl as dependent variable, In@H chan ) and (In@H chan e))2 as predictors. Data from both & and LS were pm%d for the analysis. Predictorswere dropped from a modelwhen corresponding coefficients were not different from zero (P >.OS). Means were separated usin the LSD (Least Significant Difference) method of the &M (General Linear Model) procedure. Computations followed the Statistical Analysis Systems Institute, SAS/STAT User's Guide [ll]. 11. SAS Instltule, SAS/STAT User's Guide. SAS Institute, Inc., Cary, NC. 12. Zhang, Blngfan, 199S.The relationship of calcium intake, source, particle size, solubilityhyitrpand retention in gizzard of limestone, and calcium retention in layin4 hens. Chapter 3 in: Master of Science Thesis, Universityof Minnesota, St. Paul, MN. ACKNOWLEDGEMENT The authors are indebted to the Broiler and Egg Association of Minnesota and Iowa Limestone Company for partial financial support of the research.