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This is unedited, preprint, author version of the article: 1

Swiatkiewicz S., Arczewska-Wlosek A., Jozefiak D. Bone quality, 2

selected blood variables and mineral retention in laying hens fed with 3

different dietary concentration and source of calcium. LIVESTOCK 4

SCIENCE, DOI: 10.1016/j.livsci.2015.09.011 5

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Bone quality, selected blood variables and mineral retention in laying hens fed with 10

different dietary concentration and source of calcium 11

S. Swiatkiewicz a,*, A. Arczewska-Wlosek a, D. Jozefiak b 12

a National Research Institute of Animal Production, 32-083 Balice, Poland 13

b Department of Animal Nutrition and Feed Management, Poznań University of Life Sciences, 14

60-637 Poznań, Poland 15

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* Corresponding author. Tel.: +48 66 6081343; fax: +48 12 2856733. 17

E-mail adress: sylwester.swiatkiewicz@izoo.krakow.pl (S. Swiatkiewicz). 18

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A B S T R A C T 20

The objective of this study was to determine the influence of limestone particle size in the diets 21

with different contents of Ca on the biomechanical and geometrical measurements of tibia and 22

femur bones, digestibility of nutrients, and selected biochemical blood variables. The experiment 23

was conducted with 108 laying hens, which were allocated to 9 treatments with 6 replicate cages 24

and 2 laying hens in each cage. A 3 × 3 factorial arrangement of treatments with 3 dietary 25

concentrations of Ca (3.20, 3.70, and 4.20%) and 3 dietary substitutions (0, 25, and 50%) of fine 26

particles of limestone (diameter, 0.2 to 0.6 mm) with large particles of limestone (diameter, 1.0 27

to 1.4 mm), was used. The hens were fed with experimental diets from 25 to 70 wk of age. At wk 28

45, a balance trial was conducted, and after termination of the experiment, i.e., at wk 70, tibia 29

and femur bones and blood samples were collected for analysis. Neither dietary Ca concentration 30

nor limestone particle size had an effect on dry matter, organic matter, ether extract, N-free 31

extracts, crude fiber, and crude ash digestibility, and P retention and excretion; however, Ca 32

excretion increased linearly and Ca relative retention decreased linearly with increasing dietary 33

Ca concentration (P < 0.05). No effect of limestone particle size on tibia and femur 34

biomechanical and geometrical measurements, tibia and femur mineralization, serum alkaline 35

phosphatase activity, or serum Ca and P concentrations was observed. Increased dietary Ca 36

concentration enhanced linearly tibia and femur bone breaking strength, yielding load, stiffness, 37

and Ca concentration (P < 0.05). Serum alkaline phosphatase activity decreased linearly with 38

increasing dietary Ca concentration (P < 0.05). In conclusion, the results of this study 39

demonstrated that a content of 3.20 to 3.70% Ca in a layer’s diet is not sufficient through the 40

entire laying cycle to maintain optimal bone quality; however, partial substitution of fine- with 41

large-particle limestone does not improve Ca and P retention and bones quality variables. 42

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Keywords: Laying hens, Dietary calcium, Limestone particle size, Mineral retention, Bone 43

quality 44

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1. Introduction 46

Skeletal disorders and poor bone quality, related mainly to osteoporosis, are widespread in 47

modern laying hens. Osteoporosis is a severe decrease in mineralised structural bone, in which 48

Ca is mobilised from the bone to be involved in eggshell formation (Whitehead, 2004; 49

Whitehead and Fleming, 2000), and frequently results in loss of bone strength, enhanced 50

brittleness, and high fracture incidence. Not only performance and economical losses for the egg 51

industry, but also important welfare problems because of acute and chronic pain and distress to 52

the birds, are the results of osteoporotic changes (Lay et al., 2011; Webster, 2004). It was 53

reported that almost 30% of hens experienced bones fractures during the end phase of laying 54

(Gregory and Wilkins, 1989). Several authors indicated that hens kept in conventional cages are 55

especially vulnerable to osteoporosis, exhibiting lower bone mineral density, bone mass, bone 56

cross-sectional bone area, and bone breaking strength than laying hens kept in furnished colony 57

cages, cages modified with nest boxes and perches, or in floor pens (Jendral et al., 2008; 58

Silversides et al., 2012). A study by McCoy et al. (1996) attributed 35% of mortality in high-59

producing caged laying hens to osteoporosis. Because of osteoclastic resorption and decline of 60

structural bone content during the laying period (Fleming et al., 1998; Whitehead and Fleming, 61

2000), the symptoms of osteoporosis are more frequently observed in older hens. 62

Nutritional management can serve as one of the tools for prevention or alleviation of 63

osteoporotic changes and skeletal defects in highly performing laying hens. Because of the fact 64

that calcification of eggshell uses extremely high amounts of Ca (approximately 2.2 g per egg), 65

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Ca nutrition, i.e., supply of the optimal amount and form of Ca, is the most crucial for proper 66

mineralization of eggshell and bones in high-producing laying hens. As the skeleton acts as the 67

Ca source for shell mineralization in the dark period of the day, inadequate Ca intake by hens not 68

only leads to a considerable decrease in eggshell quality, but can increase bones brittleness and 69

their susceptibility to fractures. For this reason several researchers (Bar et al., 2002; Castillo et 70

al., 2004; Costa et al., 2008; Lichovnikova, 2007; Safaa et al., 2008) have suggested that the 71

need of laying hens for dietary Ca can be higher than the value (3.25% Ca in the diet) included in 72

NRC recommendations (NRC, 1994). On the other hand, however, it is known that too high 73

dietary Ca concentration can negatively affect retention of some other essential minerals (Pastore 74

et al., 2012) or efficacy of phytase supplementation to the layer’s diet (Englmaierova et al., 75

2014). 76

Results of some experiments showed that replacing fine limestone with coarse limestone, 77

as a source of Ca for hens, which is dissolved more slowly, thus providing the hen continuously 78

with Ca (assuring maintenance of an adequate Ca blood concentration overnight, when the 79

process of shell calcification is intensive and laying hens do not have access to feed), may 80

beneficially affect selected bone quality traits (Cufadar et al., 2011; Fleming et al., 1998; 81

Guinotte and Nys, 1991; Koreleski and Swiatkiewicz, 2004; Saunders-Blades et al., 2009). Rao 82

et al. (1992) found that that 1.0 mm is the minimum limestone particle size required to improve 83

its selective retention in the gizzard of laying hens (Rao et al., 1992). However, the experimental 84

data on the interactive effect of limestone particle size and dietary Ca concentration on bone 85

quality in modern high-producing laying hens are limited. Therefore, this experiment was 86

conducted to study the effect of different dietary Ca concentrations and particle size of the 87

dietary Ca source, i.e., the level of substitution of fine-particle with large-particle limestone, on 88

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the biomechanical and geometrical measurements of tibia and femur bones, digestibility of 89

nutrients, and selected biochemical blood variables. 90

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2. Material and methods 92

2.1. Birds and experimental diets 93

A total of 108 seventeen-weeks-old hens (ISA Brown, Hendrix Genetics, Boxmeer, the 94

Netherlands) obtained from a commercial source were placed in a poultry house in cages (2 birds 95

per cage) on a wire-mesh floor under controlled climate conditions. The cage dimensions were 96

30 × 120 × 50 cm. During the pre-experimental period, i.e., from 17 to 24 wk of age, the hens 97

were offered a commercial diet (170 g/kg of crude protein, 11.6 MJ/kg of metabolizable energy 98

(ME), 37.0 g/kg of Ca. and 3.8 g/kg of available P) ad libitum. The Local Cracow Ethics 99

Committee for Experiments with Animals approved all experimental procedures relating to the 100

use of live animals. 101

At wk 25, the hens were randomly assigned to 1 of 9 treatments with 6 replicate cages and 102

2 hens per cage, and experimental diets were fed until wk 70. During the experiment, laying hens 103

were provided feed and water ad libitum, and were exposed to a 14:10 h light:dark cycle with a 104

light intensity of 10 lux. A 3 × 3 factorial arrangement of treatments with 3 dietary 105

concentrations of Ca (3.20, 3.70, and 4.20%) and 3 dietary substitutions (0, 25, and 50%) of fine 106

particles of limestone (FPL; diameter, 0.2 to 0.6 mm) with large particles of limestone (LPL; 107

diameter, 1.0 to 1.4 mm) as a Ca source, was used. The nutrient content of the diets was 108

calculated on the basis of the chemical composition of raw feedstuffs, and ME value was 109

calculated based on equations in the European Tables (Janssen, 1989). The chemical composition 110

other feed materials was determined by AOAC (2000) methods for moisture (930.15), crude 111

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protein (984.13), crude fat (920.39), fiber (978.10), and ash (942.05). Amino acids were 112

analyzed in acid hydrolysates after initial peroxidation of sulphur amino acids by color reaction 113

with the ninhydrin reagent (Beckman-System Gold 126 AA Automatic Analyzer; Beckman 114

Coulter, Inc., Pasadena, CA; Method 982.30; AOAC, 2000). The Ca content was determined by 115

flame atomic absorption spectrophotometry (Method 968.08; AOAC, 2000) and total P content 116

was determined by colorimetry using the molybdo-vanadate method (Method 965.17; AOAC, 117

2000). The composition of the experimental cereal-soybean diets is given in Table 1. 118

2.2. Measurements 119

At 45 wk of age, digestibility was determined by the total collection method. The total 120

collection of excreta was conducted over 5 d, and the feed consumption for each cage was 121

recorded. Excreta was stored in plastic bags at -20ºC for 2 wk and, after thawing, was dried in an 122

oven at 50ºC to a constant weight, weighed and finely ground. The content of nutrients in the 123

diets and excreta was estimated using the same methods as it was previously described for the 124

feed materials. Apparent total tract digestibility coefficient of dry matter was calculated as dry 125

matter intake – dry matter excretion/dry matter intake. Similarly, digestibility of organic matter, 126

crude fat, N-free extracts, crude fibre, and ash was calculated. Calcium or P retention (mg) was 127

calculated as: Ca or P intake – Ca or P excretion. Calcium or P relative retention (as a % of Ca or 128

P intake) was calculated as: Ca or P intake – (Ca or P intake – Ca or P excretion)/Ca or P intake 129

x 100. 130

At the end of the experiment (70 wk of age), all of the hens were sacrificed through 131

cervical dislocation. Blood samples were collected from the jugular vein of each hen before 132

slaughter, centrifuged at 3,000 × g for 15 min at 4ºC, and frozen stored (-20ºC) until analysis. 133

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Serum Ca and P concentration and alkaline phosphatase (ALP) activity were determined by 134

colorimetric assay using commercial kits (Pointe Scientific, Warsaw, Poland). 135

The tibia and femur from both legs were collected, cleaned of soft tissues, weighed and 136

frozen (-20ºC) until analysis. For determination of ash, the left tibias and toes were dried for 24 h 137

at 105ºC, weighed, and dry-ashed in a muffle furnace at 600ºC. A mass of 0.2 g of bone ash was 138

dissolved in 10 mL of 6 M hydrochloric acid. Calcium concentration in tibia bones was then 139

analysed by flame atomic absorption spectrophotometry (AOAC, 2000). 140

For measurements of the biomechanical and geometrical properties of bones, the right 141

tibias were used. Biomechanical properties were determined by means of the 3-point bending test 142

(Instron 5542; Instron, Norwood, MA, US). Bone breaking strength and yielding load were 143

measured as a graphical record from post-deformation curves. Stiffness in elastic conditions was 144

calculated as a yielding load/elastic deformation ratio. Tibia length, cortex thickness, and 145

external and internal diameters (for cross-section area calculations) were measured at the 146

breaking location, using an electronic slide caliper. The crosssection area was calculated from 147

the equation: 3.14 (HB – hb)/4, where H = external vertical diameter; B = external horizontal 148

diameter; h = internal vertical diameter; and b = internal horizontal diameter. 149

2.3. Statistical analysis 150

The data were subjected to statistical analysis as a completely randomised design using the 151

GLM procedure (Statistica 5.0; StatSoft, Inc., Tulsa, OK, US). Analysis for linear and quadratic 152

response for increasing dietary Ca concentration and increasing substitution rate of FPL with 153

LPL were calculated using orthogonal polynomial contrasts. The statistical significance was 154

considered to be P ≤ 0.05. 155

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3. Results and discussion 157

In this experiment, the dietary concentration of Ca or physical form of limestone did not 158

affect laying performance and eggshell quality, however, as it was presented in our previous 159

paper, substitutions of FPL with LPL improved eggshell quality in older laying hens 160

(Swiatkiewicz et al., 2015). The results of the measurements of the biomechanical and 161

geometrical properties of the bones and their mineralization are presented in Tables 2 and 3. The 162

particle size of limestone in the diet had no effect on the bones traits, and increasing level of 163

substitutions of FPL with LPL did not affect bone breaking strength, yielding load, stiffness, 164

cortex thickness and cross section area, weight, and length, as well as crude ash and Ca 165

concentrations in the bones. Some measurements of the tibia and femur bones were affected by 166

dietary Ca concentration, and breaking strength, breaking strength/cross section area ratio, 167

yielding load, stiffness, and bone Ca concentration increased linearly with increasing dietary Ca 168

content (P < 0.05). There were no interactions for measured bones measurements between 169

experimental factors (dietary Ca content and limestone particle size). Similar results were found 170

recently by Nascimento et al. (2014) who observed a beneficial effect of increasing dietary Ca 171

content (2.85 to 5.25%) on tibia breaking strength in hens in the second laying cycle. Likewise, 172

Cheng and Coon (1990) indicated that bone ash concentration and breaking strength were 173

linearly related to Ca intake (2.0 to 4.5 g/d). Our results might indicate that the requirement of 174

laying hens to maintain a high resistance of long bones to fractures is greater than for optimal 175

eggshell quality and greater than the recommendations of NRC (1994). Contradictory results 176

were reported by Safaa et al. (2008) who reported that the increase of dietary Ca concentration 177

(from 4.08 to 4.0%) beneficially affected eggshell quality, but had no effect on tibia 178

characteristics. 179

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The lack of the effect of substitutions of FPL with LPL on bone quality, as observed in our 180

experiment, is inconsistent with the results of the majority of studies, where particulate limestone 181

was evaluated as a Ca source for laying hens. For instance, Saunders-Blade et al. (2009) showed 182

that partial replacement of fine with particulate Ca source positively affected the weight, density, 183

mineralization, and breaking strength of tibia bones. In one earlier work by Fleming et al. (1998), 184

the use of particulate limestone as a source of Ca in the diet alleviated some of the symptoms of 185

osteoporosis in aged laying hens, i.e., increased tibia radiographic density and breaking strength 186

at 50 and 70 wk of age. As was indicated by the authors, this effect could be linked to the 187

extended time of Ca absorption from particulate limestone that resulted in a greater availability 188

of Ca for shell and bone formation and in a sparing effect on cancellous bone resorption. 189

Guinotte and Nys (1991) reported increased tibia strength and mineralization and enhanced Ca 190

and P plasma concentrations in laying hens fed a diet with particulate limestone. Cufadar et al. 191

(2011) found that large (2-5 mm) or very large (> 5 mm) limestone particle size had a beneficial 192

effect on tibia mechanical properties and Ca concentrations in the bones of moulted hens (76 to 193

88 wk of age). Simultaneously, they observed higher tibia force shear in hens fed a diet with an 194

increased dietary Ca concentration (4.20%), however, surprisingly Ca bone concentration was 195

decreased in these birds (Cufadar et al., 2011). More recently, Tunc and Cufadar (2015) reported 196

increased mineral (Ca, P, and Mg) concentration in tibia bones when hens were fed a diet 197

supplemented with large particle size source of Ca. Oliveira et al. (2013) found that partial 198

replacement of fine with medium sized limestone (0.60 mm diameter) beneficially affected tibia 199

bone breaking strength without an effect on tibia mineralization. De Witt et al. (2009) showed 200

that large particles of limestone (> 1.0 mm) had a beneficial effect on the mechanical properties 201

of tibia bones of older laying hens (at 70 wk of age). In the recent study by Wang et al. (2014) 202

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the positive effect of the large particle size of the dietary Ca source (0.85-2.00 mm) on tibia Ca 203

concentration and breaking strength was observed in laying ducks. The mechanism of such a 204

beneficial effect of LPL on bone quality can be connected with the possibility that the slower 205

solubility of LPL and its prolonged retention time in the gizzard also makes Ca available at 206

night, when the hens have no access to feed, but when the shell calcification process is occurring 207

intensively (Roland and Harms, 1973). This would prevent the mobilisation of bone Ca and P 208

reserves, a process that could affect eggshell quality (Farmer et al., 1986). Findings similar to the 209

results of our study, i.e., lack of an effect of limestone particle size on bone quality variables, 210

were only reported by Safaa et al. (2008), who reported that substitution of part of FPL in the 211

diet with coarse limestone did not affect the tibia characteristics of laying hens. It is difficult to 212

clarify the inconsistency of results of our experiment with the results of the majority of studies in 213

respect to the effect of the source of dietary Ca (limestone particle size) on bone characteristics, 214

however, we think that it might be partially explained by the differences in the solubility and 215

particle sizes of the coarse limestone used, as well as by the different duration of the experiments 216

(time of LPL supplied to hens), different hens’ age at the termination of the experiment (bones 217

collected for analysis) and the different genetic lines of the hens used in the experiments. 218

The effect of dietary treatments on the digestibility of nutrients is presented in Table 4. In 219

our study, neither the Ca content nor the limestone particle size had an effect on the digestibility 220

of dry matter, organic matter, ether extract, N-free extracts, crude fiber, and crude ash. We did 221

not find any information in the literature comparing the influence of different Ca concentrations 222

or limestone particle size on the digestibility of these nutrients. Substitution of FPL with LPL did 223

not affect the Ca and P balance, i.e., excretion and retention of these macroelements (Table 4). 224

Total amount of excreted and retained Ca increased linearly (P < 0.05) with an increasing dietary 225

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content of Ca, while the efficacy of Ca retention, i.e., relative retention of Ca (as a % of Ca 226

intake), decreased with increasing dietary Ca concentration. Excretion and retention of P were 227

not affected by the Ca content in the diet. No interaction for the digestibility of nutrients and the 228

balance of Ca and P between experimental factors was observed. Our results, i.e., linearly 229

decreasing relative Ca retention with increasing dietary Ca, clearly confirm that, at low dietary 230

Ca concentrations, laying hens are able to maximize utilization of Ca from the feed, reducing 231

excretion of this macroelement. Results similar to our findings were reported by Pelicia et al. 232

(2009) who found that an increased content of Ca in the diet (3.0 to 4.5%) increased excreta Ca 233

concentration without any effect on blood Ca, however there was no influence of Ca particle size 234

(fine vs coarse) on excreta Ca. Several other authors also reported that that excretion of Ca 235

increased and relative Ca retention decreased with increasing dietary Ca concentration 236

(Chowdhury and Smith, 2002; Jadhao and Sinha, 1998; Vieira et al., 2011). The number of 237

studies on the effect of the physical form of dietary limestone on the balance of Ca and P is 238

limited, however Scheideler (1998) observed no effect of partial (50%) substitution of fine with 239

large particle sized limestone on Ca digestibility in 33-wk-old laying hens, but LPL improved Ca 240

digestibility in older hens (116 wk of age). Similarly, Araujo et al. (2011) did not find any 241

differences in Ca and P digestibility between laying hens fed a diet containing fine or coarse 242

(1.00 mm diameter) limestone. Lichovnikova (2007) reported, however, increased Ca retention 243

in laying hens when 50% of FPL was substituted with LPL. 244

Partial substitution of FPL with LPL had no effect on serum Ca and P concentration and 245

ALP activity (Table 4). There was no effect of dietary Ca content on serum Ca and P 246

concentration, whereas serum ALP activity decreased linearly with increasing dietary Ca 247

concentration (P < 0.05). In general, the values of serum Ca and P concentration, and ALP 248

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activity, observed in our study were in the range of reference values for laying hens 249

(Mazurkiewicz, 2005). Similar Ca and P concentrations in the blood serum of laying hens were 250

observed, among others, in the experiments by Cerolini et al. (1990), Dobrzański et al (2011), 251

Neijat et al. (2014), and Suchy et al. (2001). ALP plays an important role in the process of 252

mineralization of eggshell and bone, and the results of several studies have shown an inverse 253

relationship between dietary Ca and blood ALP, thus ALP activity was increased in laying hens 254

fed the diets deficient in Ca (Hurwitz and Griminger, 1961; Rao et al., 2003; Reichman and 255

Connor, 1977). Accordingly, our findings, i.e. increased ALP activity in hens fed diet with a 256

reduced dietary Ca concentration, can indicate that 3.20% of Ca is too low to meet the 257

requirement of high-producing laying hens. 258

259

4. Conclusions 260

We therefore conclude, based on the results of this study, that the requirement of laying 261

hens for dietary Ca to maintain an optimal quality of bones and their high resistance to fractures 262

can be higher than for optimal eggshell quality and greater than the values of the 1994 NRC 263

recommendations. On the other hand, inconsistently with majority of previous studies, a partial 264

substitution of fine with large particle limestone does not have a beneficial influence on the 265

biomechanical and geometrical measurements of the tibia and femur bones and on the utilization 266

of nutrients, including Ca retention, in laying hens. 267

268

Conflict of interest statement 269

The authors confirm that this work has no conflict of interest. 270

271

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Acknowledgments 272

This work was supported from the National Research Institute of Animal Production 273

Statutory Activity (Research Project No. 05-008.1; Balice, Poland). 274

275

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Table 1 393 Composition of experimental diets (g/kg; as-fed). 394

Item Dietary Ca

Reduced Standard Increaesd

Ingredient (g/kg):

Corn 417.1 423.1 456.1

Wheat 240.0 210.0 150.0

Soybean meal 230.0 236.0 244.0

Rapeseed oil 13.0 19.0 26.0

Limestone 78.0 90.0 102.0

Monocalcium phosphate 12.5 12.5 12.5

NaCl 3.0 3.0 3.0

DL-Met 1.4 1.4 1.4

Vitamin-mineral premixa 5.0 5.0 5.0

Analyzed chemical composition:

ME (MJ/kg)b 11.60 11.60 11.60

Crude protein (g/kg) 170.0 170.0 170.0

Lys (g/kg) 8.35 8.35 8.35

Met (g/kg) 4.10 4.10 4.10

Ca (g/kg) 32.0 37.0 42.0

Total P (g/kg) 3.15 3.15 3.15

Available P (g/kg) 3.90 3.90 3.90 a Provided per kilogram of diet: vitamin A, 10,000; vitamin D3, 3,000 IU; vitamin E, 50 IU; vitamin K3, 2 mg; 395

vitamin B1, 1; vitamin B2, 4 mg; vitamin B6, 1.5; vitamin B12, 0.01 mg; Ca-pantotenate, 8 mg; niacine, 25 mg; folic 396 acid, 0.5 mg; choline chloride, 250 mg; manganese, 100 mg; zinc, 50 mg; iron, 50 mg; copper, 8 mg; iodine, 0.8 mg; 397 selenium, 0.2 mg; and cobalt, 0.2 mg. 398

b ME = metabolizable energy; calculated according to European Table (Janssen, 1989) as a sum of the ME 399 content of components. 400

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Table 2 401 Effects of dietary Ca and limestone particle size (Part) on tibia bones measurements a. 402

Item Dietary Ca (%) Large Part (%)b SEM P-value

3.2 3.7 4.2 0 25 50 Ca Part Ca x Part

Ln Qd Ln Qd

Bone breaking strength (N) 169 171 189 171 180 179 3 0.007 0.161 0.251 0.422 0.767

Cross section area (mm2) 17.8 18.1 17.0 17.6 17.2 18.1 0.3 0.179 0.217 0.433 0.264 0.902

Bone breaking strength/cross

section area ratio (N/mm2)

9.58 9.61 11.23 9.86 10.61 9.96 0.21 0.001 0.062 0.840 0.098 0.901

Yielding load (N) 108 107 118 110 109 114 2 0.013 0.081 0.236 0.443 0.540

Stiffness (N/mm) 132 135 144 132 137 141 2 0.023 0.524 0.096 0.893 0.627

Cortex thickness (mm) 0.913 0.915 0.916 0.892 0.902 0.949 0.013 0.928 0.996 0.092 0.544 0.741

Tibia weight (g) 9.21 9.30 9.87 9.47 9.50 9.41 0.10 0.008 0.259 0.407 0.784 0.605

Relative tibia weight (g/100 g of

body weight)

0.504 0.502 0.519 0.516 0.506 0.502 0.006 0.298 0.436 0.348 0.817 0.320

Tibia length (mm) 120 119 119 119 119 119 1 0.364 0.462 0.797 0.882 0.949

Crude ash content (g/kg) 405.0 412.6 422.4 412.7 418.6 417.4 4.2 0.101 0.404 0.654 0.689 0.414

Ca content (g/kg) 151 157 157 155 156 154 1 0.035 0.250 0.895 0.443 0.167 a SEM = standard error of the mean; Ln = linear; and Qd = quadratic. 403 b Substitution rate of fine with large particle limestone in the diet, i.e., % of large particle limestone (diameter, 1.0 to 1.4 mm). 404

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Table 3 405 Effects of dietary Ca and limestone particle size (Part) on femur measurements a. 406

Item Dietary Ca (%) Large Part (%)b SEM P-value

3.2 3.7 4.2 0 25 50 Ca Part Ca x Part

Ln Qd Ln Qd

Bone breaking strength (N) 173 182 192 180 184 182 3 0.003 0.973 0.742 0.559 0.786

Cross section area (mm2) 19.7 19.8 19.9 19.7 19.5 20.1 0.2 0.730 0.894 0.424 0.372 0.881

Bone breaking strength/cross

section area ratio (N/mm2)

8.85 9.24 9.69 9.19 9.51 9.08 0.12 0.019 0.935 0.751 0.224 0.823

Yielding load (N) 109 116 117 112 113 116 1 0.013 0.273 0.219 0.631 0.528

Stiffness (N/mm) 139 149 150 144 145 149 2 0.025 0.223 0.272 0.727 0.651

Cortex thickness (mm) 0.912 0.936 0.955 0.932 0.923 0.949 0.011 0.111 0.922 0.511 0.452 0.807

Femur weight (g) 7.16 7.35 7.48 7.15 7.42 7.41 0.07 0.079 0.864 0.149 0.355 0.241

Relative femur weight (g/100 g of

body weight)

0.392 0.396 0.393 0.390 0.395 0.395 0.004 0.930 0.655 0.560 0.755 0.135

Femur length (mm) 85.1 84.3 84.3 83.9 85.3 84.5 0.3 0.194 0.408 0.362 0.083 0.213

Crude ash content (g/kg) 430 455 460 444 449 453 6 0.065 0.453 0.533 0.959 0.263

Ca content (g/kg) 161 171 178 166 172 172 2 0.002 0.713 0.271 0.490 0.265 a SEM = standard error of the mean; Ln = linear; and Qd = quadratic. 407 b Substitution rate of fine with large particle limestone in the diet, i.e., % of large particle limestone (diameter, 1.0 to 1.4 mm). 408

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Table 4 409 Effects dietary Ca and limestone particle size (Part) on apparent digestibility of nutrients (%), balance of Ca and P, alkaline phosphatase (ALP) 410

activity in blood, and Ca and P concentration in blood a. 411

a SEM = standard error of the mean; Ln = linear; and Qd = quadratic. 412 b Substitution rate of fine with large particle limestone in the diet, i.e., % of large particle limestone (diameter, 1.0 to 1.4 mm). 413

Item Dietary Ca (%) Large Part (%)b SEM P-value

3.2 3.7 4.2 0 25 50 Ca Part Ca x Part

Ln Qd Ln Qd

Dry matter digestibility 74.8 73.9 74.2 74.2 74.3 74.4 0.2 0.284 0.205 0.701 0.936 0.996

Organic matter digestibility 78.2 77.4 78.1 77.8 77.9 78.0 0.2 0.947 0.112 0.671 0.954 0.975

Ether extract digestibility 71.6 70.8 72.0 77.8 77.9 78.0 0.3 0.571 0.111 0.439 0.512 0.195

N-free extracts digestibility 91.6 91.4 91.7 91.6 91.5 91.6 0.2 0.923 0.619 0.956 0.881 0.952

Crude fiber digestibility 4.43 3.56 5.66 4.03 4.71 4.91 0.62 0.458 0.302 0.597 0.867 0.974

Crude ash digestibility 55.4 53.7 53.0 53.8 54.2 54.0 0.4 0.069 0.602 0.937 0.806 0.882

Ca excretion (mg/hen per

day)

1,623 1,921 2,268 1,967 1,925 1,920 45 <0.001 0.629 0.427 0.718 0.935

Ca retention (mg/hen per day) 2,266 2,465 2,818 2,472 2,533 2,539 41 <0.001 0.121 0.202 0.601 0.346

Ca retained (% of Ca intake) 58.3 56.2 55.4 55.8 57.0 57.1 0.5 0.017 0.525 0.262 0.606 0.908

P excretion (mg/hen per day) 571 564 580 573 569 574 4 0.339 0.174 0.964 0.591 0.235

P retention (mg/hen per day) 201 192 213 199 205 201 3 0.229 0.093 0.878 0.531 0.824

P retained (% of P intake) 26.0 25.4 26.8 25.8 26.5 35.9 0.4 0.499 0.317 0.905 0.496 0.825

ALP activity in blood serum

(IU/l)

385 326 283 349 343 303 13 <0.001 0.734 0.111 0.496 0.161

Ca content in blood serum

(mg/dl)

28.2 28.7 28.6 29.3 27.9 28.1 0.5 0.798 0.801 0.355 0.463 0.542

P content in blood serum

(mg/dl)

6.93 6.87 6.86 7.13 6.51 7.03 0.21 0.898 0.960 0.836 0.206 0.653