Influence of particle size and calcium source on production performance, egg quality, and bone parameters in laying ducks

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1 Influence of particle size and calcium source on production performance, egg quality, and bone parameters in laying ducks S. Wang, W. Chen, H. X. Zhang, D. Ruan, and Y. C. Lin1 Institute of Animal Science, Guangdong Academy of Agricultural Sciences, State Key Laboratory of Livestock and Poultry Breeding, Key Laboratory of Animal Nutrition and Feed Science in South China, Ministry of Agriculture Guangdong Public Laboratory of Animal Breeding and Nutrition Guangdong, Key Laboratory of Animal Breeding and Nutrition Guangzhou , China ABSTRACT The influence of calcium source (limestone and oyster shell) and particle size (<0.1 mm; 0.85 to 2 mm) on laying performance, egg quality, and bone properties were examined in laying ducks by a 2 2 factorial arrangement of treatments. Longyan females (288) with similar BW at 24 wk of age were randomly allotted into 4 treatments, each with 6 replicates of 12 individually caged birds and studied over the following 12 wk. Particle size affected egg weight and feed conversion (P < 0.05). Large particle size increased shell breaking strength, albumen height, Haugh unit, and shell content of phosphorus and magnesium (P < INTRODUCTION Calcium is a critical factor for egg production, but little is known about the optimal way of providing this for laying ducks. Many studies with hens have shown that calcium sources with large particle size prolong the residence time of the particles in the stomach of poultry, favoring solubilization so that calcium ions are made available in sufficient quantities for absorption. Zhang and Coon (1997a) noted that large particle size limestone (>0.8 mm) with lower solubility in vitro (30 to 50%) was retained in the gizzard for a longer time, thereby increasing its solubility in vivo to as much as 94%. Rao and Roland (1989) showed that an increased proportion of calcium was solubilized from larger particles of limestone in the digestive system during a 25-h period, providing greater availability of calcium ions for absorption. Additional sources of calcium have been introduced and are in practical use with birds, such as egg shell, oyster shell, and others. Gongruttananun (2011) indicated that ground, sterilized eggshell could 0.05), but had no effect on egg shape, yolk color, shell thickness, or the weight proportion of shell. There were no effects of particle size on tibial properties: dry defatted weight, calcium content, or breaking strength. Limestone increased albumen height, shell content of calcium and phosphorus, and the breaking strength of tibia (P < 0.05). It is concluded that limestone with a large particle size provided for superior productive performance, egg quality, and bone characteristics and is more suitable than oyster shell for practical applications. Key words: calcium source, particle size, productive performance, egg quality 2014 Poultry Science 93 : Poultry Science Association Inc. Received February 10, Accepted June 25, Corresponding author: lyc0123@tom.com be used as a complete calcium source for laying hens and Safaa et al. (2008) showed that 40% oyster shell, substituted for limestone, did not affect the laying performance of brown-shell laying hens. There are few studies on the use of 100% oyster shell as the calcium source and none examining different of calcium sources and particle sizes for laying ducks. A large number of experiments has been carried out in hens, but the literature for ducks is very limited. The crop of ducks is just a distensible gullet, whereas that of hens is a storage organ. The ovipository cycle and laying time also differ between ducks and hens. These differences may prevent simple extrapolation of findings on calcium sources and different particle size from hens to ducks. The present study has investigated the effect on laying performance, egg quality, and tibial indices of oyster shell or limestone, each of 2 particle sizes as calcium sources for laying ducks. MATERIALS AND METHODS Experimental Design, Birds, and Housing This study was approved by the Animal Care and Use Committee of Guangdong Academy of Agriculture Science. Longyan ducks (288), of the same genetic 2560

2 PARTICLE SIZE AND CALCIUM SOURCE FOR DUCKS 2561 Table 1. Composition and nutrient levels of the diets (g/kg, as fed basis) Small particles (SP) Large particles (LP) Item Limestone Oyster shell Limestone Oyster shell Ingredient Corn Soybean meal Wheat bran Calcium hydrogen phosphate Limestone (LP 34.23%) 1 94 Oyster shell (LP 34.85%) Limestone (SP 35.65%) Oyster shell (SP 34.85%) dl-methionine l-lysine HCl Sodium chloride Premix Total 1,000 1,000 1,000 1,000 Calculated analysis 3 AME (kcal/kg) CP (%) Total Lys (%) Total Met (%) Ca (%) Total P (%) Available P (%) Measured values of calcium content. 2 Provided the following (per kg of diet): vitamin A, 12,000 IU; vitamin D 3, 2,000 IU; vitamin E, 38.7 IU; vitamin K 3, 1.0 mg; vitamin B 1, 3.0 mg; vitamin B 2, 9.6 mg; vitamin B 6, 6.0 mg; vitamin B 12, 0.03 mg; choline chloride, 1,000 mg; d-pantothenic acid, 28.5 mg; folic acid, 0.6 mg; biotin, 0.15 mg; Fe, 50 mg; Cu, 10 mg; Mn, 90 mg; Zn, 90 mg; I, 0.50 mg; Se, 0.40 mg. 3 The CP, Ca, and total P are measured values in the mixed feeds. background and of comparable BW at 24 wk of age, were assigned at random to 4 experimental groups, each containing 6 replicates of 12 individually caged ( cm 3 ) birds and studied for 12 wk. Each replicate consisted of 2 banks of 6 adjacent cages and each bank shared a deep feed trough enabling individual feeding of the 6 birds without spillage at 0700 and 1500 h twice daily. Birds were fed the same, thoroughly mixed corn-soybean-based diet, formulated for laying ducks (Table 1), but with 4 different calcium components (see below), each providing 3.6% calcium and exceeding the NRC recommendation (NRC, 1994). The daily allowance of feed (average 144 g) was the maximum for individuals without their leaving refusals. A 2 2 factorial arrangement was used to compare the calcium sources, ground limestone or oyster shell, each provided at 2 particle sizes. Each calcium source was purchased commercially and ground coarsely and finely. Large particles (LP) were between 1.18 and 2 mm (>84%), as determined by sieve analysis using a vibratory shaker and small particles (SP) were almost all less than 0.1 mm (Table 2). The ducks were all housed in the same room, with incandescent lighting of 10 lx, providing 15L:9D. After 12 wk of feeding, 2 birds of average BW for the treatment group were selected from each replicate and heparinized blood was collected from the wing vein at 1000 h, 7 to 9 h after the previous ovipositioning, when the next ovum is in the shell gland. Those birds were then stunned and exsanguinated, and the tibias were collected. Productive Performance The birds were fed the 4 diets for a 7-d adaptation period after which feed intake was recorded daily on a per replicate basis (2 troughs). Likewise, the number of total, broken, and shell-less eggs were recorded daily by replicate. All eggs produced were individually weighed and graded daily (European Economic Community, 1989). Egg production, egg weight, egg yield (average egg weight per day per duck), ADFI, and feed conversion ratio (FCR, feed/eggs) were calculated daily from Table 2. Particle size distribution of limestone and oyster shell Small particles (%) Large particles (%) Size (mm) Limestone Oyster shell Size (mm) Limestone Oyster shell > > to to to to < <

3 2562 Wang et al. these data but are presented as the averages for the complete 12-wk period. Egg Quality Egg quality was measured on 8 eggs collected at random from each replicate once weekly during wk 9 to 12, and the average of 32 eggs from each replicate was calculated. Yolk color, albumen height, and Haugh units were measured on the day of collection using an Egg Analyzer (model EA-01, ORKA Food Technology, Ramat HaSharon, Israel). The strength of the shell was determined on the vertical axis using an Egg Force Reader (model EFR-01, ORKA Food Technology). Egg length, egg width, and eggshell thickness were measured using a digital micrometer, the distance between the blunt and pointed ends for length, and the diameter at mid-length for the width. The egg shape index was calculated as egg length divided by width. The shells with membranes were weighed, washed under a gentle flow of water to remove adhering albumen, then ashed at 700 C. Shell (1 g) was heated with 15 ml of nitric acid and 2 ml of perchloric acid until the appearance of white fumes then quantitatively diluted to 100 ml with water. Calcium was determined by titration with EDTA and phosphorus by ammonium molybdenate and UV spectrophotometry. Magnesium was determined by atomic absorption spectrometry (Hitachi/Z-2000, Hitachi Limited, Tokyo, Japan), as described by Alfrey et al. (1974). Plasma Analysis Freshly collected blood (2 samples from each replicate) was centrifuged (1,200 g) at 4 C for 10 min and plasma was held at 20 C until used. Plasma calcium, phosphorus, and alkaline phosphatase contents were determined for all samples by an automatic biochemical analyzer (Synchron cx5, Beckman Coulter Inc., Los Angeles, CA), with reagents from BioSino Bio-Technology and Plasma Science Inc. (Beijing, China). Tibial Characteristics Two pairs of tibias were collected from each replicate for analyses. The left tibias were ashed for 24 h, and the content of tibial ash was determined on a dry defatted weight basis (Lin and Shen, 1979). Calcium and phosphate in bone ash were determined, as described above for shell. Right tibias were cleaned of all tissue and bone mineral density of the right tibia was measured at the Guangzhou Overseas Chinese Hospital with a x- ray osteodensitometer (Lunar Prodigy, General Electric Company, Fairfield, CT) and the breaking strength of the middle of the bone was determined with a materials tester (Instron 4411, Instron Corporation, Grove City, PA) using software version 8.09, a standard 50-kg load cell, and a modified shear plate (8 cm in length and 1 mm in width), as described by Riczu et al. (2004). Scanning Electron Microscopy The morphology of freshly fractured, nondecalcified eggshell fragments from the limestone diets with SP and LP was evaluated. For each treatment, 12 shell fragments from the blunt end were air-dried, mounted with conductive carbon cement, scanned, and imaged with an environmental scanning electron microscope (XL-30-ESEM, FEI, Eindhoven, the Netherlands) at an accelerating voltage of 20 kv; stubs were oriented to provide a transverse view of the eggshell (Figure 1). Images of transverse sections of eggshell from ducks fed limestone with LP and SP are shown at 400. The crystal layers are shown at higher magnifications (6,400 ). Statistical Analysis Replicate (n = 6) was taken as the experimental unit. Except where noted otherwise, 2 sampled birds per replicate were used. The effects of source and particle size were analyzed by the 2-way ANOVA procedure of SAS (SAS Institute Inc., 2003) with P < 0.05 indicating significance. Analyses used the following model: Y ijk = μ ijk + S i + PS j + (S PS) ij + ε ijk, where Y ijk is the replicate value of a given variable, μ ijk is the mean, S i is the effect of calcium source (i = 1 or 2, limestone or oyster shell), PS j is the effect of particle size (j = 1 or 2, LP or SP), (S PS) ij is the effect of interaction between the 2 main effects, and ε ijk is the random error. No significant interactions were detected, so the model was reduced to just the 2 main effects. For some variables, pooling or averaging within replicates over time was used as there was no experimental interest in possible temporal changes. RESULTS Productive Performance There was no statistical difference between the slightly higher egg production of ducks on the LP than the SP diets (Table 3). Increasing the particle size from SP to LP increased egg yield and decreased FCR (both P < 0.05). The source of calcium had no effect on these indices of productive performance over the whole 12-wk period. There was no mortality during the experiment. Egg Quality As shown in Table 4, LP increased the breaking strength of the shell (P < 0.05), and improved the appearance of eggs based on increased albumen height and Haugh units (P < 0.01). Particle size did not affect egg shape index, yolk color, eggshell thickness, or weight. The LP diets resulted in higher phosphorus and lower magnesium contents of eggshells (P < 0.01) than did SP diets, but calcium content was unaffected. The

4 2563 PARTICLE SIZE AND CALCIUM SOURCE FOR DUCKS Table 3. Effect of calcium source and particle size on productive performance in laying ducks Size1 Item Egg production (%) Egg yield (g/d) Feed conversion4 (g/g) P-value3 SP LP Limestone Oyster shell SEM2 Size <0.05 < Smaller particles (SP) and larger particles (LP). from ANOVA error mean square for n = 6 replicates. 3No interactions were significant. 4All ducks were given 144 g/d of feed. 2Derived Plasma Variables and Tibial Characteristics Particle size and sources of Ca did not significantly affect plasma calcium, phosphorus, or alkaline phosphatase activity (Table 5). The larger particle size of the calcium sources increased (P < 0.05) the breaking strength of fresh tibia, dry weight, and calcium content of defatted tibia of the ducks (Table 6). The slightly greater density of the defatted tibia and increased tibial ash and phosphorus in the diets with LP were not significant. Inclusion of limestone rather than oyster shell did increase the breaking strength of the tibias (P < 0.05), but not the other tibial characteristics. Overall, the best tibial indices were obtained from use of limestone of large particle size. Scanning Electron Microscopy Typical images of scanned transverse sections of eggshells from ducks fed the limestone diets, LP and Figure 1. Environmental scanning electron microscope images of transverse sections of eggshell from ducks fed limestone with larger particles (LP) is shown at 400 in panel a, and from ducks fed limestone with smaller particles (SP) in panel b. Higher magnifications, showing the crystal layer (6,400 ), indicate greater regularity with the LP diet (panel c) than the ragged, loose organization from the SP diet (panel d). Arrows in panel a indicate the shell membrane (SM) and mammillary layer (ML). source of calcium affected the albumen height, shell calcium content, and shell phosphorus content (P < 0.05), both being higher with the limestone diet. There were no differences between limestone and oyster shell on the remaining indices of egg quality.

5 2564 Wang et al. Table 4. Effect of particle size and source of calcium on egg quality in laying ducks Size 1 P-value 3 Variable SP LP Limestone Oyster shell SEM 2 Size Shape index Breaking strength (kg) < Albumen height (mm) <0.01 <0.05 Yolk color Haugh units < Shell thickness (mm) Shell weight (g) Shell calcium (% DM) <0.05 Shell phosphorus (% DM) <0.01 <0.01 Shell magnesium (% DM) < Smaller particles (SP) and larger particles (LP). 2 Derived from ANOVA error mean square for n = 6 replicates. 3 No interactions were significant. SP, are shown in Figure 1. The overall structure of the eggshell from the LP diet (Figure 1a) appeared to be dense and pyknotic and showed close arrangement, tightly connected into a mass with fuzzy boundaries. Eggshell from the SP diet was less substantial with a loose arrangement of clearly discernible mammillae (Figure 1b). Connection was poorer, with obvious gaps, between the mammillary layer and shell membranes. The crystal layer of the LP eggshell was more regular (Figure 1c), whereas that of the SP was ragged and loose (Figure 1d). DISCUSSION Effect of Particle Size on Productive Performance, Egg Quality, Tibial Characteristics, and Scanning Electron Microscopy of Eggshell The present study demonstrated a positive effect on most productive traits, including egg yield and FCR, in laying ducks with intake of larger particle size of the calcium source. There is little information in support of this observation in the available literature, and nothing for ducks. Skřivan et al. (2010) showed that feeding younger and older hens a calcium source with large particle size (0.8 to 2 mm) increased egg production, egg weight, and FCR. Koreleski and Swiatkiewicz (2004) observed a nonsignificant increase in hen-day egg production (from 90.1 to 91.8%) when one-half of the fine limestone (<0.4 mm) was replaced with particulate limestone grit (2.0 to 4.0 mm). Consistent with Koreleski and Swiatkiewicz (2004), who found that the limestone grit increased eggshell breaking strength, the present results using ducks demonstrate greater breaking strength and enhanced egg appearance. A similar effect on albumen height and Haugh unit was shown in chickens fed limestone of large particle size (Skřivan et al., 2010). Little is known about factors influencing the mineral composition of the shell so the reasons why shell %P was increased by LP and by limestone, whereas %Mg decreased with LP are not obvious. There are reports (Guinotte and Nys, 1991; Pizzolante et al., 2009; Araujo et al., 2011) indicating no significant effect of limestone particle size on productive performance and egg quality. In the ducks studied here, particle size of the calcium source influenced the quality of bone, indicated by breaking strength, dry defatted weight, and calcium content of the tibia, but not that of calcium, in eggshells. This outcome was consistent with particulate limestone increasing accumulation of medullary bone in the proximal tarsometatarsus, improved breaking strength of the tibia and radiographic densities of tibia and keel (Fleming et al., 1998). Similarly, Saunders- Blades et al. (2009) showed that a mixed particle size calcium source (1/3 particulate) improved tibial weight, density, mineral content, and breaking strength. Somewhat different results have also been obtained with Table 5. Effect of particle size and source of calcium on plasma variables in laying ducks Size 2 P-value 4 Variable 1 SP LP Limestone Oyster shell SEM 3 Size Ca (mm) P (mm) AKP (U/L) Alkaline phosphatase (AKP). 2 Smaller particles (SP) and larger particles (LP). 3 Derived from ANOVA error mean square for n = 6 replicates. 4 No interactions were significant.

6 PARTICLE SIZE AND CALCIUM SOURCE FOR DUCKS 2565 Table 6. Effect of calcium source and particle size on tibial characteristics in laying ducks Size 1 P-value 3 Variable SP LP Limestone Oyster shell SEM 2 Size Breaking strength (N) <0.05 <0.05 Density (g/cm 2 ) Dry defatted weight (g) < Tibia ash (%) Tibia P (%) Tibia Ca (%) < Smaller particles (SP) and larger particles (LP). 2 Derived from ANOVA error mean square for n = 6 replicates. 3 No interactions were significant. hens, such as increased shell calcium (as well as phosphorus) from large particle limestone (Skřivan et al., 2010) and a conditional effect of particle size on tibial strength (Koreleski and Swiatkiewicz (2004). 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 (Rao and Roland, 1989; Zhang and Coon, 1997a). The longer retention time increases the duration of providing Ca to the intestine, allowing a more consistent supply of calcium during the night when the calcification takes place. Although this could account for the beneficial effects of LP found here in ducks, it did not result in any change in plasma calcium or phosphorus measured at 1000 h, but eucalcemia is tightly regulated. Net enteric uptake of calcium was not measured here, but the presumed greater availability from the LP sources could account for the increase observed in tibial calcium. Bain (1992) found that the fracture toughness of the eggshell was a measure of its resistance to unstable crack growth, and in this respect there appeared to be a link between the latter and the structural organization of the mammillary layer. Larger particle size clearly improved the mammillary layer of the duck s eggs, which could explain the significant increase in their mechanical strength found here. Effect of Calcium on Productive Performance, Egg Quality, and Tibial Characteristics The source of calcium, limestone, or oyster shell did not generally affect productive performance, egg quality, or tibial characteristics with the exception of albumen height, shell content of calcium and phosphorus, and breaking strength of the tibia, all of which were higher with limestone. Most published research with hens has the same findings. For example, Richter et al. (1999) found that various calcium sources (limestone, limestone together with oyster shells, marine shells, and shells treated with phosphoric acid) did not affect feed intake, laying performance, egg weight, feed conversion, or mortality. There were minimal influences on stability variables of eggshells such as deformation, ultimate strength, weight/100 g of eggs, shell weight/cm 2, cracks, and eggs without shells. Similarly, Safaa et al. (2008) showed that 40% substitution of limestone with oyster shell did not affect productive performance or egg quality of brown-shell laying hens. In contrast to the present findings, Çatlı et al. (2012) indicated that partial substitution of limestone with oyster shell meal in laying hens resulted in significant increases in egg production (numbers and mass) and greater feed intake. Earlier studies concerning oyster shell meal supplementation showed beneficial effects on eggshell breaking strength (Brister et al., 1981; Makled and Charles, 1987; Proudfoot and Hulan, 1987; Guinotte and Nys, 1991; Grizzle et al., 1992; Ahmad and Balander, 2004). In the ducks studied here, oyster shell decreased albumen height and marginally (P = 0.06) reduced Haugh unit, suggesting that it compromised freshness of the eggs. Because the oyster shells sometimes contained meat residues, there is the possibility of microbial contamination in shells and most research indicates that oyster shell is less soluble than limestone (Zhang and Coon, 1997b; Lichovnikova, 2007). Limestone and oyster shell of the same particle size would be expected to experience similar retention in the digestive tract, so digestibility of calcium and phosphorus must differ. It is assumed that reduced net absorption of calcium and phosphorus in ducks fed oyster shell resulted in decreased shell contents of these elements and breaking strength of the tibia compared with ducks given limestone. In conclusion, limestone with large particle size was shown to increase productive performance, and improve the quality of eggs and bone. Calcium sources with large particle size, rather than finely ground, significantly improved productive performance, egg appearance, egg quality, and the quality of the tibia in ducks in heavy production. There is no specific information regarding particle size and calcium source in NRC (1994). Oyster shell could be used as a substitute for limestone with a small decrease in egg quality and tibial characteristics. On the basis of all variables considered here, limestone of larger particle size was the better calcium source for laying ducks.

7 2566 Wang et al. ACKNOWLEDGMENTS The authors sincerely thank W. Bruce Currie (emeritus professor, Cornell University, Ithaca, NY) for his help in presentation of this manuscript. This work was supported by the Earmarked Fund for Modern Agroindustry Technology Research System (CARS-43-13), National Natural Science Foundation of China (Grant No ), and Technology Research of Animal Feed of the Guangdong Academy of Agricultural Sciences (2011A ). REFERENCES Ahmad, H. A., and R. J. Balander Physiological response of layers to alternative feeding regimen of calcium source and phosphorus level. Int. J. Poult. Sci. 3: Alfrey, A. C., N. L. Miller, and R. Trow Effect of age and magnesium depletion on bone magnesium pools in rats. J. Clin. Invest. 54: Araujo, J. A. d., J. H. V. d. Silva, F. G. P. Costa, J. M. B. d. Sousa, P. E. N. Givisiez, and N. K. Sakomura Effect of the levels of calcium and particle size of limestone on laying hens. R. Bras. Zootec. 40: Bain, M. M Eggshell strength a relationship between the mechanism of failure and ultrastructure organization of the mammillary layer. Br. Poult. Sci. 33: Brister, R. D. Jr., S. S. Linton, and C. R. Creger Effects of dietary calcium sources and particle size on laying hen performance. Poult. Sci. 60: Çatlı, A., M. Bozkurt, K. Küçükyılmaz, M. Çınar, E. Bintas, F. Çöven, and H. Atik Performance and egg quality of aged laying hens fed diets supplemented with meat and bone meal or oyster shell meal. S. Afr. J. Anim. Sci. 42: European Economic Community Hen eggs and egg products. Council Directive 89/437/EEC. Off. J. L 212: Fleming, R., H. McCormack, and C. Whitehead Bone structure and strength at different ages in laying hens and effects of dietary particulate limestone, vitamin K and ascorbic acid. Br. Poult. Sci. 39: Gongruttananun, N Effects of eggshell calcium on productive performance, plasma calcium, bone mineralization, and gonadal characteristics in laying hens. Poult. Sci. 90: Grizzle, J., M. Iheanacho, A. Saxton, and J. Broaden Nutritional and environmental factors involved in egg shell quality of laying hens. Br. Poult. Sci. 33: Guinotte, F., and Y. Nys Effects of particle size and origin of calcium sources on eggshell quality and bone mineralization in egg laying hens. Poult. Sci. 70: Koreleski, J., and S. Swiatkiewicz Calcium from limestone meal and grit in laying hen diets-effect on performance, eggshell and bone quality. J. Anim. Feed Sci. 13: Lichovnikova, M The effect of dietary calcium source, concentration and particle size on calcium retention, eggshell quality and overall calcium requirement in laying hens. Br. Poult. Sci. 48: Lin, I., and T. Shen Studies on duck nutrition. II. Calcium and phosphorus requirements of mule ducklings. Poult. Sci. 58: Makled, M., and O. Charles Eggshell quality as influenced by sodium bicarbonate, calcium source, and photoperiod. Poult. Sci. 66: NRC Nutrient Requirements of Poultry. 9th rev. ed. Natl. Acad. Press, Washington, DC. Pizzolante, C., E. Saldanha, C. Laganá, S. Kakimoto, and C. Togashi Effects of calcium levels and limestone particle size on the egg quality of semi-heavy layers in their second production cycle. Braz. J. Poult. Sci. 11: Proudfoot, F., and H. Hulan Effect on shell strength of feeding supplemental sources of calcium to adult laying hens given insoluble grit during the rearing period. Br. Poult. Sci. 28: Rao, K., and D. Roland Influence of dietary calcium level and particle size of calcium source on in vivo calcium solubilization by commercial Leghorns. Poult. Sci. 68: Richter, G., G. Kiessling, W. Ochrimenko, and H. Luedke Influence of particle size and calcium source on limestone solubility in vitro, performance and eggshell quality in laying hens. Arch. Geflugelkd. 63: Riczu, C. M., J. L. Saunders-Blades, A. K. Yngvesson, F. E. Robinson, and D. R. Korver End-of-cycle bone quality in white and brown-egg laying hens. Poult. Sci. 83: Safaa, H. M., M. P. Serrano, D. G. Valencia, M. Frikha, E. Jimenez- Moreno, and G. G. Mateos Productive performance and egg quality of brown egg-laying hens in the late phase of production as influenced by level and source of calcium in the diet. Poult. Sci. 87: SAS Institute Inc SAS User s Guide: Statistics. Version 9.0. SAS Institute Inc., Cary, NC. Saunders-Blades, J., J. MacIsaac, D. Korver, and D. Anderson The effect of calcium source and particle size on the production performance and bone quality of laying hens. Poult. Sci. 88: Skřivan, M., M. Marounek, I. Bubancova, and M. Podsedníček Influence of limestone particle size on performance and egg quality in laying hens aged weeks and weeks. Anim. Feed Sci. Technol. 158: Zhang, B., and C. N. Coon. 1997a. The relationship of calcium intake, source, size, solubility in vitro and in vivo, and gizzard limestone retention in laying hens. Poult. Sci. 76: Zhang, B., and C. N. Coon. 1997b. Improved in vitro methods for determining limestone and oyster shell solubility. J. Appl. Poult. Res. 6:94 99.