
==== Front
Poult Sci
Poult Sci
Poultry Science
0032-5791
1525-3171
Elsevier

S0032-5791(24)00805-8
10.1016/j.psj.2024.104226
104226
METABOLISM AND NUTRITION
Determination of calcium digestibility and bioavailability in 5 limestone sources using commercial broiler and crossbred chickens
Drysdale R.L. *
Utterback P.L. *
Parsons B.W. †
Parsons C.M. poultry@illinois.edu
*1
⁎ Department of Animal Sciences, University of Illinois at Urbana-Champaign, IL 61801, USA
† Department of Poultry Science, University of Arkansas at Fayetteville, AR 72701, USA
1 Corresponding author: poultry@illinois.edu
22 8 2024
11 2024
22 8 2024
103 11 10422611 6 2024
12 8 2024
© 2024 The Authors
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Three experiments were conducted to determine effect of feeding 5 commercial limestones varying in solubility (88–97%), mean particle size (500–700 microns), and geographical origin on Ca bioavailability and digestibility in commercial broiler and crossbred chickens. In Experiment 1, both crossbred and commercial chickens were used to measure the effect of dietary Ca level on tibia bone ash to develop a slope-ratio Ca bioavailability assay. Chickens were fed diets that contained Ca levels ranging from 0.20% to 0.95% from 9 to 22 d-of-age. Regressions of bone ash (mg/tibia and %) on supplemental Ca intake yielded large linear responses in both types of chickens in Experiment 1. In Experiment 2, relative bioavailability of Ca in 5 limestones using bone ash as primary response criterion was determined. Thirteen diets were fed to commercial Ross 308 male broilers which were either a Ca-deficient diet (0.30% Ca) or that diet supplemented 0.15 or 0.30% Ca from either reagent grade calcium carbonate (RCaCO3) or 1 of the 5 commercial limestones from 9 to 22 d-of-age. Bioavailability of Ca in limestones relative to RCaCO3 was determined using multiple linear regression of bone ash (mg/tibia and %) on supplemental Ca intake, which yielded slope-ratio relative Ca bioavailability values ranging from 90% to 106% in Experiment 2. In Experiment 3, apparent ileal digestibility (AID) and apparent total tract retention (TTR) of Ca in broiler chickens was measured for the 5 limestones using corn-based diets. The AID and TTR of Ca at 21 d-of-age were low and variable with a range of 20 to 34% for AID of Ca and 12 to 31% for TTR of Ca. Results from these studies indicate that a slope-ratio bone ash assay with either crossbred or modern commercial chickens can be used to measure relative bioavailability of Ca in limestones and there were few consistent differences in relative Ca bioavailability, AID of Ca, and TTR of Ca among 5 commercial limestones evaluated herein.

Key words

calcium
bioavailability
digestibility
limestone
chicken
==== Body
pmcINTRODUCTION

Calcium is the most abundant macro mineral in the body and it is primarily found in the skeleton (Veum, 2010). Calcium has multiple functions within the body, such as maintenance of osmotic acid-base balance, muscle contraction, enzyme activation, and bone formation and maintenance (Proszkowiec-Weglarz and Angel, 2013). When there is a Ca deficiency, impairments in bone mineralization, growth, and feed efficiency can be observed. In contrast to Ca deficiencies, it is also possible to have excess Ca in poultry diets. Excess Ca can negatively affect feed efficiency, reduce phytate hydrolysis thereby reducing the availability of nutrients, and exacerbate infections (Paiva et al., 2013; Walk and Rao, 2020; Parsons and Rochell, 2024). The NRC (1994) recommends that broiler diets contain approximately 1% total Ca from 0 to 3 wk-of-age, with the Ca level in the diet decreasing as the age of the bird increases. Since broilers are selected for rapid growth, they require higher dietary Ca levels to support skeletal development and energy metabolism during the early stages of life compared with later feeding phases (Fleming, 2008). Current diet formulation focuses on meeting a total dietary Ca requirement; however, in order to improve the precision by which the Ca requirement of chickens is met, it is important to account for factors that affect the ability of birds to digest and absorb Ca in different feedstuffs.

There is increased interest in formulating poultry diets based on digestible Ca rather than total Ca. Walk et al. (2021b) stated that the primary challenge with a digestible Ca formulation system in broiler chickens is not the absence of digestibility values for raw materials, but rather highly variable results among experiments due ingredient variability, variations in feedstuff particle size, and differences in methodology such as dietary Ca to P ratios, the presence or absence of phytate, and the age of the birds used. Calcium digestibility can be expressed as apparent, standardized, or true digestibility. Apparent digestibility does not account for endogenous Ca losses, while standardized and true digestibility account for basal and total endogenous Ca losses, respectively (David et al., 2019). Aside from ileal Ca digestibility, total tract Ca retention can be measured by collecting excreta, and relative Ca bioavailability based on bone ash can be calculated using the slope-ratio bioassay (Dilworth et al., 1964; Ward et al., 1984; Littell et al., 1995; Henry and Pesti, 2002). Bone status, such as bone ash, weight, volume, breaking strength, and densitometry is often utilized as an indicator of mineral adequacy and to calculate relative mineral bioavailability because many minerals are important in the production of the matrix of bone and are required for maintaining bone structure (Reichmann and Connor, 1977; Rao et al., 1993; Khanal et al., 2020; Walk et al., 2021a).

Limestone is often the largest contributor of Ca in poultry diets, accounting for upwards of 50% of the total analyzable Ca in a broiler diet; thus, the digestibility and bioavailability of Ca in inorganic sources, such as limestone, has a substantial impact on the overall availability of Ca in the diet (Adeola and Zhang, 2018). It has been shown, however, that variations among limestone sources, such as particle size, rate of solubility, and geological origin can affect Ca digestibility and bioavailability in the feedstuff (Kim et al., 2019; David et al., 2021; Walk et al., 2021b). Therefore, the objective of this study was to: 1) identify the dietary Ca levels required to obtain a linear response in tibia bone ash for relative Ca bioavailability studies with both crossbred and modern commercial broiler chickens, 2) compare Ca availability estimates for limestones obtained from 2 bioassays, and 3) determine the Ca availability in 5 different commercial limestones, which varied in solubility (88 to 97%), particle size, and geographical origin in chickens.

MATERIALS AND METHODS

The protocol for this study was reviewed and approved by the Institutional Animal Care and Use Committee at the University of Illinois (number 19090).

Ingredients and Analyses

Five limestone sources varying in origin were obtained from Danisco Animal Nutrition & Health / IFF (1000 41st Ave Dr SW, Cedar Rapids, IA, 52404), and were analyzed to determine particle size (ANSI/ASAE method S319.4, 2008) and solubility at 30 min using pH 3 HCl solution buffered with 3 M glycine using the procedure of Kim et al. (2019). Titanium concentrations in experimental diets, ileal digesta, and excreta were measured using UV spectroscopy (Meyers et al., 2004). The Ca and P analyses were performed via inductively coupled plasma-mass spectrometry (method 985.01 A, B, and C; AOAC International, 2007). All analyses except particle size and solubility were conducted at University of Missouri-Columbia Experimental Station Chemical Laboratory, Columbia, MO. Particle size and solubility were determined by Danisco Animal Nutrition and Health/IFF.

Diets and Design

Experiment 1 was conducted to evaluate bone ash response to different dietary Ca levels in both male and female crossbred and male commercial broiler chickens in an attempt to develop a slope-ratio bioassay for determining relative Ca bioavailability in limestone. The latter was done to determine if Ca responses would be similar in both types of chickens. Crossbred chickens have been used previously for this type of bioassay by these authors (Boling-Frankenbach et al., 2001) because this particular cross provides birds that are more uniform than commercial broiler chickens, are less prone to developing leg issues when fed mineral-deficient diets, and more readily consume mineral deficient-diets, particularly semi-purified ones. Commercial broiler chickens, however, are more widely available compared with the crossbred line used herein. Crossbred (New Hampshire x Columbian) chickens used in this experiment were hatched at the University of Illinois at Urbana-Champaign poultry research field laboratory and commercial Ross 308 chickens were obtained from a commercial hatchery.

Crossbred and commercial broiler chickens were provided a nutritionally complete corn-soybean meal starter diet until 9 d-of-age. All chickens were housed in Petersime batteries with raised wire floors in an environmentally controlled room. Prior to the start of the experimental phase, birds were fasted overnight. At 9 d-of-age, birds were weighed, wing banded, and allotted to 1 of 6 dietary treatments, ensuring mean BW was equalized across treatments. A completely randomized design was used and there was a total of 6 diets that were fed to both crossbred and commercial broiler chickens. There were 4 replicate pens per treatment with 5 chickens per replicate, for a total of 240 chickens (120 crossbred, 120 commercial broilers). Initial mean weight of the broilers was 121.6 g / chicken, and initial mean weight for crossbred chickens was 94.3 g / chicken.

The 6 dietary treatments were corn-soybean meal-based diets (Table 1). Diet 1 was a Ca-deficient diet which contained 0.20% Ca, while diets 2 through 6 contained increased increments of 0.15% Ca from limestone. Thus, diet 2 contained 0.35% Ca, diet 3 contained 0.50% Ca, diet 4 contained 0.65% Ca, diet 5 contained 0.80% Ca, and diet 6 contained 0.95% Ca. Limestone was added in place of Solka Floc (powdered cellulose, International Fiber Corporation, Urbana, OH 43078). All diets contained a calculated nonphytate P level of 0.45% and a calculated CP level of 22%. At the end of the experiment, BW gain, feed consumption, and feed efficiency were calculated for each replicate. When birds were 22 d-of-age, they were euthanized using CO2 gas and the right tibia was autoclaved, cleaned of any adhering tissues, oven-dried at 100°C for 24 h, and ashed at 600°C in a muffle furnace for 24 h. The regressions were conducted as described by Finney (1964) and Littell et al. (1995) using the model below.LinearRegression:Y=a0+bx

QuadraticRegression:Y=a0+bx+cx2

Table 1 Ingredient composition of diets in Experiment 1 (%, as-fed basis).

Table 1	Dietary treatment	
Ingredient	1	2	3	4	5	6	
Corn	57.44	57.44	57.44	57.44	57.44	57.44	
Soybean meal	36.00	36.00	36.00	36.00	36.00	36.00	
Soybean oil	1.60	1.60	1.60	1.60	1.60	1.60	
Limestone	-	0.39	0.79	1.18	1.58	1.97	
Dicalcium phosphate	0.42	0.42	0.42	0.42	0.42	0.42	
Monosodium phosphate	1.11	1.11	1.11	1.11	1.11	1.11	
Potassium chloride	0.25	0.25	0.25	0.25	0.25	0.25	
Solka Floc1	1.97	1.58	1.18	0.79	0.39	-	
L-Lys HCl	0.28	0.28	0.28	0.28	0.28	0.28	
Vitamin mix2	0.20	0.20	0.20	0.20	0.20	0.20	
Mineral mix3	0.15	0.15	0.15	0.15	0.15	0.15	
DL-Met	0.37	0.37	0.37	0.37	0.37	0.37	
Choline chloride (60%)	0.08	0.08	0.08	0.08	0.08	0.08	
L-Thr	0.13	0.13	0.13	0.13	0.13	0.13	
Calculated values4							
CP	22.0	22.0	22.0	22.0	22.0	22.0	
Ca	0.20	0.35	0.50	0.65	0.80	0.95	
Nonphytate P	0.45	0.45	0.45	0.45	0.45	0.45	
1 Powdered cellulose from International Corporation, Urbana, OH 43078. Limestone was added in place of Solka Floc.

2 Provided per kilogram of diet: retinyl acetate, 4,400 IU; cholecalciferol, 25 µg; DL-α-tocopherol, 11 IU; vitamin B12, 0.01 mg; riboflavin; 4.41 mg; D-pantothenic, 10 mg; niacin, 22 mg; menadione sodium bisulfate, 2.33 mg.

3 Provided as milligrams per kilogram of diet: manganese, 75 from MnSO4·H2O; iron, 75 from FeSO4·H2O; 75 mg from ZnO; copper, 5 mg from CuSO4·5H2O; iodine, 75 from ethylene diamine dihydroidide; selenium, 0.1 from NaSeO3.

4 Diet 1 was analyzed to contain 21.4% CP, 0.22% Ca, and 0.65% total P.

Where Υ was tibia ash content (mg/tibia) or concentration (%), a0 was the y-intercept, and x was supplemental Ca intake.

Experiment 2 was conducted to determine bioavailability of Ca in 5 limestones relative to Ca in reagent grade CaCO3 using bone ash as the primary response criterion. Commercial Ross 308 males were housed in Petersime batteries with raised wire floors in an environmentally controlled room. Chickens were fed a standard, nutritionally complete corn-soybean meal diet for 8 d and had ad libitum access to feed and water. On d 8, chickens were fasted overnight. On d 9, chickens were weighed, wing banded, and allotted to 1 of 13 dietary treatments, ensuring BW was equalized across treatments in a completely randomized design. The mean starting weight was 139.4 g / chicken and there were 6 replicates per treatment with 5 chickens per replicate for a total of 390 birds. Experimental diets were provided ad libitum from d 9 to 22. Diet 1 was a Ca-deficient diet calculated to contain 0.30% Ca (Table 2). Diets 2 and 3 contained 0.15 and 0.30% supplemental Ca from reagent grade CaCO3. Diets 4 and 5, 6 and 7, 8 and 9, 10 and 11, and 12 and 13 contained 0.15 or 0.30% supplemental Ca, respectively, from the 5 commercial limestones (L1–L5). All diets contained 0.45% nonphytate phosphorus and 22% CP, and limestones were added in place of Solka Floc. Chickens were euthanized on the last day of the experiment using CO2 gas and the right leg was collected from each bird for subsequent tibia ash analysis. The right tibia was collected and processed as described previously in Experiment 1. A multiple linear regression was then conducted according to Finney (1964) and Littell et al. (1995) using the model below.Y=a0+b1x1+b2x2+b3x3+b4x4+b5x5+b6x6

Table 2 Ingredient composition of diets in Experiment 21,2 (%, as-fed basis).

Table 2	Dietary treatment	
Ingredient	1	2	3	4	5	6	7	8	9	10	11	12	13	
Corn	58.72	58.72	58.72	57.44	57.44	57.44	57.44	57.44	57.44	57.44	57.44	57.44	57.44	
Soybean meal	36.00	36.00	36.00	36.00	36.00	36.00	36.00	36.00	36.00	36.00	36.00	36.00	36.00	
Soybean oil	1.60	1.60	1.60	1.60	1.60	1.60	1.60	1.60	1.60	1.60	1.60	1.60	1.60	
R-CaCO3	-	0.40	0.80	-	-	-	-	-	-	-	-	-	-	
Limestone 1	-	-	-	0.40	0.80	-	-	-	-	-	-	-	-	
Limestone 2	-	-	-	-	-	0.40	0.80	-	-	-	-	-	-	
Limestone 3	-	-	-	-	-	-	-	0.40	0.80	-	-	-	-	
Limestone 4	-	-	-	-	-	-	-	-	-	0.40	0.80	-	-	
Limestone 5	-	-	-	-	-	-	-	-	-	-	-	0.40	0.80	
Dical	0.87	0.87	0.87	0.87	0.87	0.87	0.87	0.87	0.87	0.87	0.87	0.87	0.87	
MSP	0.63	0.63	0.63	0.63	0.63	0.63	0.63	0.63	0.63	0.63	0.63	0.63	0.63	
KCl	0.02	0.02	0.02	0.02	0.02	0.02	0.02	0.02	0.02	0.02	0.02	0.02	0.02	
NaCl	0.15	0.15	0.15	0.15	0.15	0.15	0.15	0.15	0.15	0.15	0.15	0.15	0.15	
Solka Floc3	0.80	0.40	-	0.40	-	0.40	-	0.40	-	0.40	-	0.40	-	
L-Lys HCl	0.28	0.28	0.28	0.28	0.28	0.28	0.28	0.28	0.28	0.28	0.28	0.28	0.28	
Vitamin mix4	0.20	0.20	0.20	0.20	0.20	0.20	0.20	0.20	0.20	0.20	0.20	0.20	0.20	
Mineral mix5	0.15	0.15	0.15	0.15	0.15	0.15	0.15	0.15	0.15	0.15	0.15	0.15	0.15	
DL-Met	0.37	0.37	0.37	0.37	0.37	0.37	0.37	0.37	0.37	0.37	0.37	0.37	0.37	
Choline Cl	0.08	0.08	0.08	0.08	0.08	0.08	0.08	0.08	0.08	0.08	0.08	0.08	0.08	
L-Thr	0.13	0.13	0.13	0.13	0.13	0.13	0.13	0.13	0.13	0.13	0.13	0.13	0.13	
Calculated values6														
CP	22.0	22.0	22.0	22.0	22.0	22.0	22.0	22.0	22.0	22.0	22.0	22.0	22.0	
Ca	0.30	0.45	0.60	0.45	0.60	0.45	0.60	0.45	0.60	0.45	0.60	0.45	0.60	
Nonphytate P	0.45	0.45	0.45	0.45	0.45	0.45	0.45	0.45	0.45	0.45	0.45	0.45	0.45	
1 Abbreviations: R-CaCO3 = reagent grade calcium carbonate; Dical = dicalcium phosphate; MSP = monosodium phosphate.

2 Limestone in diets was added in place of Solka Floc.

3 Powdered Cellulose; International Fiber Corporation, Urbana, OH 43078.

4 Provided per kilogram of diet: retinyl acetate, 4,400 IU; cholecalciferol, 25 µg; DL-α-tocopherol, 11 IU; vitamin B12, 0.01 mg; riboflavin; 4.41 mg; D-pantothenic, 10 mg; niacin, 22 mg; menadione sodium bisulfate, 2.33 mg.

5 Provided as milligrams per kilogram of diet: manganese, 75 from MnSO4·H2O; iron, 75 from FeSO4·H2O; 75 mg from ZnO; copper, 5 mg from CuSO4·5H2O; iodine, 75 from ethylene diamine dihydroidide; selenium, 0.1 from NaSeO3.

6 Diet 1 was analyzed to contain 21.7% CP, 0.29% Ca, and 0.63% total P.

Where: Υ = tibia ash content (mg/tibia) or concentration (%); a0 = y-intercept; b1 = slope for reagent grade CaCO3; x1 = Ca intake (g) from reagent grade CaCO3; b2 = slope for limestone 1; x2 = Ca intake (g) from limestone 1; b3 = slope for limestone 2; x3 = Ca intake (g) from limestone 2; b4 = slope for limestone 3; x4 = Ca intake (g) from limestone 3; b5 = slope for limestone 4; x5 = Ca intake (g) from limestone 4; b6 = slope for limestone 5; x6 = Ca intake (g) from limestone 5.

Experiment 3 was conducted to determine apparent ileal digestibility (AID) and apparent total tract retention (TTR) of Ca for the 5 test limestones. Commercial Ross 308 males were placed in Petersime batteries with raised wire floors in an environmentally controlled room. Chickens were provided a nutritionally complete corn-soybean meal starter diet for 17 d and had ad libitum access to feed and water. On d 17, birds were fasted overnight. On d 18, chickens were weighed and allotted to 1 of 5 dietary treatments, equalizing BW across treatment in a completely randomized design. The mean initial starting weight for chickens was 536.6 g and there were 8 replicates per treatment with 5 chickens per replicate, resulting in a total of 200 chickens. Experimental diets were provided ad libitum from d 18 to 21. All diets were corn-based (93.4% corn) with each diet containing 2.05% of 1 of the 5 test limestones (Table 3). Each diet had a calculated Ca level of 0.80%, a calculated CP level of 7.9%, and a calculated nonphytate phosphorus level of 0.45%. Titanium dioxide was added at 0.5% of the diet as an indigestible marker. Chickens were euthanized on the last day of the experiment (21 d-of-age) using CO2 gas and ileal digesta were collected from Meckel's diverticulum to ileocecal junction. Digesta were collected from the entire ileum due to the limited amount of digesta that were present. Excreta were also collected for each replicate. Diets and freeze-dried ileal digesta and excreta were analyzed for Ca and titanium (Meyers et al., 2004) at the University of Missouri. The AID and TTR of Ca were calculated as described below.AIDofCa(%)=[(Cadiet−Cailealdigesta)/Cadiet]×100

Where Ca diet = Ca in diet (%); Ca ileal digesta = Ca in ileal digesta (%) × titanium in diet (%) / titanium in ileal digesta (%).TTRofCa(%)=[(Cadiet−Caexcreta)/Cadiet]×100

Where Ca diet = Ca in diet (%); Ca excreta = Ca in excreta (%) × titanium in diet (%) / titanium in excreta (%).Table 3 Ingredient composition of diets in Experiment 3 (%, as-fed basis).

Table 3	Dietary treatment	
Ingredient	1	2	3	4	5	
Corn	93.40	93.40	93.40	93.40	93.40	
Soybean oil	2.00	2.00	2.00	2.00	2.00	
Limestone 1	2.05	-	-	-	-	
Limestone 2	-	2.05	-	-	-	
Limestone 3	-	-	2.05	-	-	
Limestone 4	-	-	-	2.05	-	
Limestone 5	-	-	-	-	2.05	
Monosodium phosphate	1.50	1.50	1.50	1.50	1.50	
Vitamin mix1	0.20	0.20	0.20	0.20	0.20	
Mineral mix2	0.15	0.15	0.15	0.15	0.15	
Sodium chloride	0.20	0.20	0.20	0.20	0.20	
Titanium dioxide	0.50	0.50	0.50	0.50	0.50	
Composition values:						
CP, (calculated)	7.9	7.9	7.9	7.9	7.9	
Ca, (analyzed)	0.94	0.88	0.91	0.92	0.91	
Nonphytate P, % (calculated)	0.45	0.45	0.45	0.45	0.45	
1 Provided per kilogram of diet: retinyl acetate, 4,400 IU; cholecalciferol, 25 µg; DL-α-tocopherol, 11 IU; vitamin B12, 0.01 mg; riboflavin; 4.41 mg; D-pantothenic, 10 mg; niacin, 22 mg; menadione sodium bisulfate, 2.33 mg.

2 Provided as milligrams per kilogram of diet: manganese, 75 from MnSO4·H2O; iron, 75 from FeSO4·H2O; 75 mg from ZnO; copper, 5 mg from CuSO4·5H2O; iodine, 75 from ethylene diamine dihydroidide; selenium, 0.1 from NaSeO3.

Statistical Analysis

SAS software (SAS Institute INC, 2010) was used for all statistical analyses. Data from Experiment 1 were analyzed using a 2-way ANOVA with chicken type (crossbred vs. commercial) and dietary Ca level as main effects. Linear and quadratic effects were then evaluated using PROC GLM in SAS by regressing performance, tibia ash content (mg/tibia), and tibia ash concentration (%) on supplemental Ca intake from reagent grade CaCO3. Data from Experiments 2 and 3 were analyzed using a 1-way ANOVA for a completely randomized design. Multiple linear regression analysis was then conducted using data from Experiment 2 to determine relative Ca bioavailability. For this analysis, bone ash content (mg/tibia) and concentration (%) were regressed on supplemental Ca intake from reagent grade CaCO3 or test limestone sources (Finney, 1964; Littell et al., 1995). The Ca bioavailability value for reagent grade CaCO3 was set at 100% and the regression coefficient (or slope) for each test limestone was divided by the regression coefficient (or slope) for reagent grade CaCO3 to yield relative Ca bioavailability values. Experiments 1 through 3 contained 4, 6, and 8 replicate pens of 5 chickens per pen, respectively, which served as the experimental unit. For Experiments 1 and 2, regressions were performed using each pen as an observation rather than the treatment mean to due to the limited number of dietary treatments per test ingredient and to ensure the model accurately explained the response of each pen of chickens. Differences among means for Experiments 1 through 3 were evaluated using Fisher's least significant difference test (Carmer and Walker, 1985) and slope contrasts were used to assess difference among relative Ca bioavailability values. The significance value for all analyses was P < 0.05.

RESULTS AND DISCUSSION

Nutrient Composition

The analyzed Ca content of test limestones was similar to the value reported by the NRC (1994) of 38%. The 5 limestones (L1 through L5) had Ca concentrations of 39.3, 36.7, 38.2, 38.7, and 37.9%, respectively. Mean particle size was 516, 590, 652, 507, and 714 microns, and limestone solubility was 97, 88, 95, 97, and 93% for L1 through L5, respectively.

Experiment 1

Growth performance and tibia ash results from Experiment 1 are presented in Table 4. As expected, BW gain, feed intake, and feed efficiency were higher (P < 0.05) for commercial broiler chickens compared with crossbred chickens. There was a quadratic response (P < 0.05) to increasing dietary Ca level, where BW gain, feed intake, and feed efficiency increased (P < 0.05) as dietary Ca increased at lower Ca supplemental levels of 0.15 and 0.30%, but there was no further increase at higher supplemental Ca levels. There was no interaction between dietary Ca level and chicken type (P > 0.05) for growth performance. For bone ash content (mg/tibia) and concentration (%), there was no interaction (P > 0.05) between dietary Ca level and chicken type. Further, there was a quadratic effect (P < 0.05) to increasing dietary Ca level for both crossbred and commercial broiler chickens for bone ash content (mg/tibia) and concentration (%).Table 4 Growth performance and tibia ash for crossbred and commercial broiler chickens in Experiment 1.1

Table 4Dietary treatment	BW gain2 (g/chick)	Feed intake2 (g/chick)	Gain:feed2 (g/kg)	Bone ash2 (mg/tibia)	Bone ash2 (%)	
Crossbred chickens						
1. Ca deficient diet - 0.20% Ca	204d	370c	551c	272c	32.3f	
2. As 1 + 0.15% Ca from limestone	235c	417b	563c	279c	34.8e	
3. As 1 + 0.30% Ca from limestone	295a	456a	647a	366b	37.3d	
4. As 1 + 0.45% Ca from limestone	270b	446ab	606b	392b	39.2c	
5. As 1 + 0.60% Ca from limestone	283ab	466a	608b	443a	41.3b	
6. As 1 + 0.75% Ca from limestone	287ab	468a	613b	478a	42.5a	
Pooled SEM	6.0	9.9	9.3	12.2	0.35	
Linear effect, P-value	<0.001	<0.001	0.007	<0.001	<0.001	
R2 for linear effect	0.60	0.66	0.29	0.94	0.96	
Quadratic effect, P-value	<0.001	0.005	0.005	0.012	0.005	
R2 for quadratic effect	0.78	0.76	0.52	0.96	0.97	
Commercial broiler chickens						
1. Ca deficient diet - 0.20% Ca	425c	604c	702c	383f	35.8e	
2. As 1 + 0.15% Ca from limestone	503b	696b	721bc	481e	39.0d	
3. As 1 + 0.30% Ca from limestone	612a	795a	770ab	655d	42.3c	
4. As 1 + 0.45% Ca from limestone	628a	804a	783a	732c	44.6b	
5. As 1 + 0.60% Ca from limestone	640a	822a	778a	795b	46.6a	
6. As 1 + 0.75% Ca from limestone	645a	811a	796a	850a	47.5a	
Pooled SEM	20.5	16.3	18.0	23.0	0.64	
Linear effect, P-value	<0.001	<0.001	<0.001	<0.001	<0.001	
R2 for linear effect	0.69	0.69	0.47	0.94	0.91	
Quadratic effect, P-value	<0.001	<0.001	0.119	<0.001	0.003	
R2 for quadratic effect	0.84	0.87	0.53	0.97	0.94	
a-f Means within a column with no common superscript differ (P < 0.05).

1 Values are means of 4 pens of 5 chickens; average initial body weight was 94.3 g for crossbred chickens and 121.6 g for commercial broilers. Diets were fed from 9 to 22 d-of-age.

2 For the 2-way combined ANOVA for both types of chickens, there was a significant main effect of chicken type (crossbred vs. commercial) and dietary Ca level (P < 0.05). There was no significant interaction (P > 0.05) between chicken type and dietary Ca level.

The NRC (1994) recommends a total dietary Ca level of 1.00% for broiler chickens from 0 to 21 d-of-age. Current total dietary Ca recommendations for starter and grower phases for Ross 308 broiler chickens are 0.95 and 0.75%, respectively (Aviagen, 2022). Bai et al. (2022) fed diets ranging from 0.60 to 1.20% Ca to broiler chickens from 0 to 21 d-of-age and found that there was a linear increase in tibia bone mineral content and a quadratic increase in tibia ash %, bone mineral density, and breaking strength as dietary Ca increased. In that study, a plateau occurred at 0.88% total dietary Ca for tibia ash %. Walk et al. (2022) reported similar results, where there was a quadratic increase in tibia ash % as the total dietary Ca content increased from 0.37 to 1.03% and a plateau occurred at approximately 0.95% total Ca. Results from the present study are in good agreement with Bai et al (2022) and Walk et al. (2022), where a quadratic response (P < 0.05) was observed for tibia ash content (mg/tibia) and concentration (%) as the dietary Ca content increased (Table 4). In contrast to those previous studies, however, no plateau had been reached in tibia ash content and concentration even at the highest Ca level of 0.95% in Experiment 1. Upon further evaluation, there was a strong positive linear response (P < 0.05; R2 = 0.91 to 0.96) to dietary Ca level with the absence of a quadratic effect (P > 0.05) when total dietary Ca level was titrated between 0.20% to 0.65% Ca for both bone ash content (mg/tibia) and concentration (%). These results indicate that large linear increases in bone ash can be obtained in both crossbred and commercial chickens by supplementing a corn-soybean meal diet with increasing levels of Ca. Thus, this dietary regimen or type of experiment can potentially be used for determining relative bioavailability of different dietary Ca sources.

Experiment 2

Growth performance and tibia ash for chickens in Experiment 2 are shown in Table 5. Weight gain, feed intake, gain to feed ratio, bone ash content (mg/tibia), and bone ash concentration (%) increased (P < 0.05) with increasing inclusion of reagent grade CaCO3 and the test limestones compared with the Ca-deficient diet 1. There was generally no difference (P > 0.05) among individual limestones within dietary Ca level except for higher BW gain for chickens fed 0.30% supplemental Ca from reagent grade CaCO3 compared with the same supplemental Ca level from L1, L2, or L4. In addition, gain to feed ratio for chickens fed 0.30% supplemental Ca from reagent grade CaCO3 was higher (P < 0.05) compared with chickens fed diets containing the test limestones L1 through L4. There was no consistent individual limestone effect within supplemental Ca level on the bone ash expressed as mg/tibia or as a %. The multiple regression equations for bone ash regressed on supplemental Ca intake from the limestone are shown in footnotes 3 and 4 of Table 5. There was a strong positive linear effect for all limestones for tibia bone ash content and concentration regressed on supplemental Ca intake (P < 0.05; R2 = 0.78-0.79).Table 5 Growth performance and tibia ash for commercial broiler chickens in Experiment 2.1,2

Table 5Dietary treatment	BW gain (g/chick)	Feed intake (g/chick)	Gain:feed (g/kg)	Bone ash3 (mg/tibia)	Bone ash4 (%)	
1. Ca deficient diet - 0.30% Ca	566e	784d	723c	521c	37.2f	
2. As 1 + 0.15% Ca from RCaCO3	686bc	896ab	766b	715b	41.7cd	
3. As 1 + 0.30% Ca from RCaCO3	758a	910ab	834a	836a	44.3ab	
4. As 1 + 0.15% Ca from limestone 1	676bcd	877abc	771b	690b	41.2d	
5. As 1 + 0.30% Ca from limestone 1	685bc	868abc	790bc	807a	43.6ab	
6. As 1 + 0.15% Ca from limestone 2	661cd	866abc	763b	704b	40.8de	
7. As 1 + 0.30% Ca from limestone 2	687bc	875abc	785b	788a	43.0bc	
8. As 1 + 0.15% Ca from limestone 3	654cd	858bc	762b	652b	39.8e	
9. As 1 + 0.30% Ca from limestone 3	715ab	905ab	791b	846a	44.8a	
10. As 1 + 0.15% Ca from limestone 4	634d	829dc	763b	667b	41.0de	
11. As 1 + 0.30% Ca from limestone 4	692bc	884abc	783b	802a	43.5ab	
12. As 1 + 0.15% Ca from limestone 5	686bc	885abc	776b	707b	41.4d	
13. As 1 + 0.30% Ca from limestone 5	724ab	919a	788ab	856a	44.0ab	
Pooled SEM	19.4	20.8	16.6	23.0	0.46	
a-f Means within a column with no common superscript differ (P < 0.05).

1 Abbreviations: RCaCO3 = reagent grade calcium carbonate.

2 Values are means of 6 pens of 5 chickens; average initial body weight was 139.4 g. Diets were fed from 9 to 22 d-of-age.

3 Multiple regression of tibia ash (Y; mg) on supplemental Ca intake (g) from reagent grade CaCO3 (X1), limestone 1 (X2), limestone 2 (X3), limestone 3 (X4), limestone 4 (X5),and limestone 5 (X6) yielded the equation: Y = 542.0 + 107.3 ± 8.61 X1 + 100.1 ± 8.98 X2 + 103.3 ± 9.57 X3 + 106.0 ± 8.95 X4 + 96.4 ± 9.04 X5 + 114.0 ± 9.25 X6 (R2 = 0.79). The (±) values are standard errors of the regression coefficients.

4 Multiple regression of tibia ash (%) on supplemental Ca intake (g) from reagent grade CaCO3 (X1), limestone 1 (X2), limestone 2 (X3), limestone 3 (X4), limestone 4 (X5), limestone 5 (X6) yielded the equation: Y = 37.8 + 2.38 ± 0.193 X1 + 2.20 ± 0.201 X2 + 2.10 ± 0.214 X3 + 2.35 ± 0.200 X4 + 2.13 ± 0.202 X5 + 2.30 ± 0.207 X6 (R2 = 0.78). The (±) values are standard errors of the regression coefficient.

The bioavailability values of Ca in test limestones relative to reagent grade CaCO3 from multiple linear regression analysis of bone tibia ash content (mg/tibia) and concentration (%) on supplemental Ca intake are presented in Table 6. Slope-ratio values ranged from 89.8 to 106.2% for tibia ash content (mg/tibia) and 88.2 to 98.7 for tibia ash concentration (%). Based on the regression equations, standard errors of regression coefficients, and slope contrasts (Table 5 footnotes 3 and 4), there were no differences (P > 0.05) among relative bioavailability values for Ca in limestones L1 through L5. Bioavailable Ca concentrations for the 5 limestones are also shown in Table 6; these did not differ greatly from the analyzed Ca level since relative bioavailability Ca values for the limestones were high.Table 6 Relative Ca bioavailability in the test limestone sources in Experiment 2.1

Table 6	Total Ca in limestone (%)	Bioavailability values2 (%)	Bioavailable content3 (%)	
Limestone source		Tibia ash (mg/tibia)	Tibia ash (%)	Tibia ash (mg/tibia)	Tibia ash (%)	
RCaCO3	-	100	100	-	-	
1	39.3	93.3	92.4	36.7	36.3	
2	36.7	96.3	88.2	35.3	32.4	
3	38.2	98.8	98.7	37.7	37.7	
4	38.7	89.8	89.5	34.8	34.6	
5	38.0	106.2	96.6	40.4	36.7	
1 Abbreviations: RCaCO3 = reagent grade calcium carbonate.

2 Calculated by the slope-ratio method using the regression equation in footnotes 3 and 4 in Table 5. Bioavailability values are relative to the Ca in reagent grade CaCO3 which was set at 100%. There were no significant differences (P > 0.05) from 100 for limestones 1-5 within columns.

3 Bioavailable content = (Total Ca x bioavailability value)/100. Values are presented on as-fed basis.

Experiment 3

Growth performance, AID of Ca, and TTR of Ca in Experiment 3 are presented in Table 7. Weight gain and gain to feed ratio did not differ (P > 0.05) among chickens fed diets containing the 5 limestones. The AID of Ca ranged from 21.5 to 34.3%, but there were no significant differences (P > 0.05) among limestones. In contrast to AID, TTR Ca did differ (P < 0.05) among limestones with TTR ranging from 15.6 to 38.6%. Limestone 3 and 4 had the highest TTR of Ca which was greater (P < 0.05) compared with TTR of Ca from L1, L2, and L5. The AID of Ca herein was generally higher than TTR. David et al. (2021) observed similar results with the values being higher for ileal digestibility compared with TTR. The TTR of Ca may be lower compared with AID of Ca due to urinary excretion of Ca, in which higher plasma Ca concentrations caused by absorbed dietary Ca may have induced Ca excretion in the urine herein.Table 7 Growth performance, apparent ileal calcium digestibility, and apparent total tract calcium retention values for ad libitum-fed chickens in Experiment 3.1,2

Table 7Limestone source3	BW gain (g/chick)	Feed intake (g/chick)	Gain:feed (g/kg)	AID of Ca (%)	TTR of Ca (%)	
1	67.8	243.9ab	278.0	20.4	19.2b	
2	67.9	258.3ab	262.8	21.5	15.6b	
3	67.1	240.5b	279.4	29.3	38.6a	
4	70.3	261.0a	270.0	33.5	30.9a	
5	67.8	249.0ab	272.6	34.3	20.4b	
Pooled SEM	2.5	6.6	8.1	4.94	3.23	
a-b Means within a column with no common superscript differ (P < 0.05).

1 Abbreviations: AID = apparent ileal digestibility; TTR = apparent total tract retention.

2 Values are means of 8 pens of 5 chickens at 22 d of age.

3 Corn-based diets calculated to contain 0.8% Ca from the limestone sources (Table 3).

In contrast to the present study, Kim et al. (2019) found that there were large differences in AID of Ca in diets containing different limestones. They also noted that limestones with smaller particle sizes had lower AID of Ca compared with limestones with larger particle sizes; however, no differences (P < 0.05) in AID of Ca among the 5 limestones varying in particle size were observed herein. Limestone with small particle sizes that solubilize more rapidly in the gastrointestinal tract may have reduced digestibility as the Ca can alter the pH in the gastrointestinal tract to reduce phytase activity and can bind to phytate to reduce its solubility prior to its degradation by phytases (Kim et al., 2018). The lack of differences among limestones varying in particle size and Ca solubility reported herein may be due to the fact that Ca availability in feedstuffs can be affected by multiple factors. For instance, Kim et al. (2019) reported that particle size explained less than 40% of the variation in Ca digestibility among limestones; therefore, those authors concluded that differences in AID of Ca among limestone samples may also be affected by the mine of origin, type of rock, and physical and chemical characteristics of the rock.

Walk et al. (2021b) conducted a review in which data reported for 55 samples of limestone were evaluated. The mean AID of Ca reported by those authors for limestone was 53%, which was higher than the present study (Table 7). It is important to note, however, that Walk et al. (2021b) reported that there was a large variation of 20 to 77% in Ca digestibility among the 55 limestones. Thus, the AID of Ca reported in the current study is within the overall range of values reported previously. One potential cause for the lower mean AID of Ca for limestone reported in the presented study compared with the study by Walk et al. (2021b) is differences in dietary adaptation length. An adaptation time of 72 h was utilized in the present study. The length of adaptation has been found to affect the digestibility of Ca. For example, David et al. (2019) reported large reductions in digestibility of Ca in limestones from 65 to 36% as dietary feeding or adaptation length increased from 24 to 168 h. It is, therefore, possible that the utilization of shorter adaptation times than what was utilized herein may result in greater AID of Ca in limestone.

When making a comparison of relative Ca bioavailability values with AID of Ca, it is interesting to note that L5 had both the highest relative bioavailability and AID of Ca of the 5 limestones. When comparing bioavailability values for the other 4 limestones to L5 (based on tibia ash content in mg/tibia), percentage values are 88, 91, 93, and 85 for L1, L2, L3, and L4, respectively. Likewise, when the same comparison is made for AID of Ca, percentage values are 65, 63, 85, and 98, respectively. Thus, variability among the 5 limestones was numerically greater for ileal digestibility compared with relative bioavailability and the ranking was inconsistent between the 2. It is important to note, however, that there were no differences (P > 0.05) in relative bioavailability and AID of Ca; albeit, L3 and 4 had greater (P < 0.05) TTR compared with other limestones. Further research is needed to compare relative Ca bioavailability with relative Ca digestibility and total tract retention for samples that are known to differ in Ca availability in order to better assess the similarities and differences among these bioassays.

In summary, a slope-ratio bioassay based on bone ash response was conducted using modern commercial broiler chickens to determine relative bioavailability of Ca in feed ingredients such as limestone. Bioavailability of Ca in 5 commercial limestones was high relative to Ca in reagent grade CaCO3, whereas the AID and TTR of Ca in the 5 limestones were much lower and highly variable.

DISCLOSURES

There is no conflict of interest on manuscript.
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