Calcium deposition in egg due to substitution of limestone by eggshell flour in feed of laying hens

The aim of this research was to evaluate calcium deposition in egg using eggshell flour as a limestone substitute in feed. Two hundreds laying hen of Isa Brown strain of 25 weeks were used in this study. Treatments were diet with 7.5% limestone as control (T0), 2.5% limestone is substituted with eggshell flour (T1), 5% limestone is substituted with eggshell flour (T2) and limestone is substituted with eggshell flour (T3). A completly randomized design were used to allocated the treatments with 5 replications of each. Each experimental unit consists of 10 laying hens. Parameters measured were egg weight, yolk weight, albumen weight, eggshell weight, calcium of egg (yolk, albumen and eggshell), length, weight and Ca of tibia bone. The results showed that substitution of limestone with eggshell flour had significantly effect (P<0.05) on eggshell weight, Ca deposition on yolk, albumen, and Ca of tibia bone but non significantly effect on egg weight, weight and percentage of yolk, weight and percentage Calcium Deposition in Egg by Eggshell Flour in Laying Hens (S. Kismiati et al.) 257 of albumen, percentage and Ca of eggshell, length and weight of tibia bone. In conclusion, calcium deposition in yolk was the highest in the use of 7.5% eggshell flour to substitute limestone but obtained the lowest Ca of bone , while calcium deposition in albumen was the highest in the use of 2.5% eggshell flour.


INTRODUCTION
Poultry industry development may give positive and negative impacts.The positive impact was nutrition sufficiency and job opportunities, the negative impact was waste product of excreta and eggshell.Excreta can be used as organic fertilizer and source of energy, while eggshell if not handled properly will effect on environmental pollution.
Egg production of Indonesia was 1401079.2tons/year (Direktorat Jenderal Peternakan dan Kesehatan Hewan, 2016), it is estimated that there are 140107.92tons/year eggshell waste and not utilized.The eggshell is covered with cuticles.The cuticle has a compositional with proteins, sulfated polysaccharides and phosphates (Rodríguez-Navarro et al., 2013).The eggshell membranes consists of highly collagens, glycoproteins and cysteine-rich, eggshell membrane proteins as used in the development of the embryo and protect it against pathogen invasion (Cordeiro and Hincke, 2016).
Calcium is one of minerals essential for poultry.Requirement of Ca of poultry for laying period was around 4 -5 times compared with brooding and growing period.Ca content of the feed will affected the eggshell and bone quality of laying hens (An et al., 2016).Neijat et al. (2011) stated that Ca consumption was deposite on the bone, meat, eggshell, albumen, yolk and some other will excrete through excreta.Medullary bone acts as Ca storage for eggs formation especially eggshell (Kim et al., 2012) and reserve of eggshell formation when Ca deficiency in feed (Olgun and Aygun, 2016).Ca consumption of laying hen was 4000 mg and deposit to the egg 3100 mg, faeces 500 mg and urine 400 mg (Leeson dan Summers, 2005).Ca of egg is needed for embryo development (Uni et al., 2012, Tahara andObara, 2014;Scanes, 2015).
Ca source is widely used as poultry feed material is limestone.Limestone contains many minerals but no protein, while eggshell flour contains mineral and a little protein.Noviyanti et al. (2015) stated that limestone composed by Ca, K, Na, Al, dan Si.Limestone was composed by macro mineral Ca, P, Mg, Na, K, Cl, S and micromineral Mn, Zn, Fe, Cu, Co, and Se (Khalil and Anwar, 2009).Limestone was composed 35.7 -36.4% of Ca (Pelicia et al., 2009).
Alu et al (2013) stated that eggshell flour may used for substitution of bone flour on broiler diet without blood hematology and nutrient digestibility changing.Olgun et al. (2015) stated that differences of Ca source showed significance effected on egg production and eggshell quality.Research of Cufadar et al. (2014) showed that used of eggshell flour, limestone, oyster shell, mixture of limestone with eggshell, limestone with oyster shell and eggshell with oyster shell showed non significance effect on the egg weight and eggshell weight.Research of Scheideler (1998) showed that Ca of eggshell has higher digestibility then of limestone.According to Dolinska et al. (2016), calcium carbonat (CaCO 3 ) of eggshell has a higher solubility than of synthetic CaCO 3 .Szeleszczuk et al. (2015) stated that the in vitro solubility Ca of eggshell is higher than CaCO 3 .The aim of this research was to evaluate a substitution of limestone with eggshell flour on the Ca deposition in egg of laying hens.

MATERIALS AND METHODS
Two hundred laying hens (Isa Brown) were used in this study.The experimental period was 12 weeks.The laying hens were distributed in a Completely Randomized Design with 4 treatments and 5 replications.The treatment were limestone of 7.5% in feed (T0), substitution 2.5% lime limestone with eggshell flour (T1), substitution of 5% limestone with eggshell flour (T2) and the replacement of limestone with eggshell flour (T3).The composition of feedstuff and nutrient content of dietary treatment is presented in Table 1.Before use, the eggshell was immersed in water temperature 80 °C for 15 minutes, drained and then immersed into 5% phosphoric acid, dried by the sun for 3 days and grounded with 3 mm particle size.
Parameter measured were weight of egg, weight and percentage of yolk, weight and percentage of albumen, weight and percentage of eggshell, Ca of yolk, Ca of albumen, Ca of eggshell and length, weight and Ca content of tibia bone.Analysis of calcium yolk, calcium albumen and calcium eggshell using the AOAC method (Horwitz and Latimer, 2005).Data was analyzed by Analysis of Variance (ANOVA) and continued with Duncan's Multiple Range Test (DMRT) when there was significantly difference.

Calcium Deposition on Egg
Calcium deposition on egg with feed suplementation eggshell flour as a substitution of limestones is presented in Table 2. Used of eggshell flour for substitution of limestone showed an effect (P<0.05) on eggshell weight, Ca of yolk, Ca of albumen but there was no effect on egg weight, weight and percentage of yolk, weight and percentage of albumen, percentage of eggshell weight and Ca of eggshell .

Egg Weight
Results of this research showed that used of eggshell flour up to 7.5% as substitution of limestone did not affect on egg weight.Eggshell flour contains Ca almost the same as limestone, so that the use of eggshell flour as a substitute for limestone had no effect on egg weight.Gongruttananun (2011) stated that Ca content of eggshell is 34.89%, while Ca content of limestone is 36.4-35.7% (Pelicia et al., 2009)

Weight and Percentage of Yolk
The used of eggshell flour as substitution of limestone did not affect the yolk weight and percentage of yolk.The results of the study was in agree to the results of previous studies.Saldanha et al. (2009) stated that percentage of yolk was not affected by source of Ca.The research of Safaa et al. (2008) showed that the percentage of yolk was not different between used limestone or oyster shell powder as a source of Ca in the feed.Gongruttananun (2011) stated that substitution of limestone with eggshell flour did not effect the yolk percentage.Pelicia et al. (2009) reported that Ca in the diet was not affected on percentage of yolk.Factor affected the percentage of yolk is genetic (Hanusová et al. , 2015), protein level of the feed (Gunawardana et al., 2008), eggs storage (Scott dan Silversides, 2000) and age of laying hens (Silversides and Budgell, 2004).Longer time of storage will increase weight of yolk (Khan et al., 2014).Yolk percentage was positive correlated to weight of egg and age of laying hens (Ulmer-Franco et al., 2012).

Weight and Prcentage of Albumen
Result of the research showed that used of eggshell flour as substitution of limestone did not affect the weight and percentage of albumen.Saldanha et al. (2009) and Safaa et al. (2009) stated that Ca source of feed did not affect percentage of albumen.The results of the study were in agree with the research of Gongruttananun ( 2011) that the limestone substitution with eggshell flour in feed of Rhode Island Red strain did not significantly affect the percentage of albumen.Pelicia et al. (2007) state that difference source of Ca (limestone or marine calcium) did not affect the weight of albumen.Ca level in the feed did not affect the albumen percentage (Pelicia et al. , 2009).The factor influencing albumen weight was genetic (Suk and Park, 2001;Hanusová et al., 2015).Silversides and Budge (2004)

Weight and Percentage of Eggshell
Table 2 showed that substitution of 5% and 7.5% limestone with eggshell flour will increase weight of eggshell (P<0.05).Increasing of eggshell weight was affected by protein content and minerals (Fe, Mg, Zn, Cu, and Se) of eggshell flour.Panheleux et al. (2000), Mine at al. (2003), Rodríguez-Navarro et al. (2015), Marie et al. (2015) and Zang et al. (2016) stated that eggshell contains many minerals and very low of protein.Schaafsma et al. (2000) stated that eggshell contained high Ca and other minerals such as P, Mg, Fe, Zn, Se, Cr, Na, F, Cr, V and slightly protein.Khalil and Anwar (2009) and Noviyanti et al. (2015) stated that limestone contains minerals but without protein.Supplementation of Mn, Zn, and Cu increased eggshell quality (Stefanello et al. 2014).
Eggshell percentage showed not different.Khalil and Anwar (2009) reported that eggshell persentage was not affected by different of Ca source of feed.Research of Gongruttananun (2011) showed that used of eggshell flour as substitution of limestone showed non significance effect the eggshell percentage.Olgun et al. (2015) stated that used of eggshell flour as substitution of limestone (66.7%) did not affect the percentage of eggshell.

Ca of Yolk
Substitution of limestone by eggshell flour affected (P<0.05) the Ca deposition in yolk.Ca deposition on yolk increased along with used of eggshell flour as substitution for limestone in feed.The result showed that Ca utilization from eggshell flour was higher compared to limestone.This result was supported by statement of Scheideler (1998) in which digestibility of eggshell flour was higher than limestone and Szeleszczuk et al. (2015) stated that bioavailability Ca of eggshell was high.Leeson dan Summers (2005) stated that calcium of diet was storage in bone, deposited in eggs and excreted on the excreta.Differences of Ca source was not affected the Ca of yolk (Vargas-Rodríguez et al., 2016).

Ca of Albumen
There was significantly effect (P<0.05) of substitution of limestone with eggshell flour on Ca deposition in albumen.Substitution of limestone with eggshell flour would increase Ca albumen.The highest of Ca deposition was found on T1 (2.5% or 33.33% eggshell flour).Kiczorowska et al. (2015) stated that Ca of yolk positively correlates with Ca albumen, but the results of this study Ca of albumen would increase on T1 and followed by `decreasing in T2 and T3, but T2 and T3 were still higher than T0.Calcium of albumen dereased along with increased calcium of yolk.This result was different from the result of Pastore et al. (2012) and Vargas-Rodríguez et al. (2016) that Ca of the feed did not affect the deposition Ca in albumen.

Ca of Eggshell
Ca deposition in eggshell was not significantly different, whereas Ca deposition in yolk and albumen was higher (P<0.05) on the use of eggshell flour than limestone (Table 2).Tunç and Cufadar (2015) stated that different Ca sources of feed had no significantly effect on eggshell weight, but significant on Ca deposition in eggshell.The research of Olgun et al. (2015) showed that the different calcium sources had significantly effect on eggshell weight and eggshell calcium content.

Length, Weight and Calcium (Ca) Content of Tibia Bone
Result of this study showed that substitution of limestone by eggshell flour significantly effect on Ca content of tibia bone but no significantly effect on the length and weight of tibia bone (Table 3).

Length and Weight of Tibia Bone
The length and weight of tibia bone were not significantly different.The results of this study were in accordance with the study of Gongruttananun (2011) that the use of eggshell flour had no significant effect on the length and weight of tibia bone.

Calcium Content of Tibia Bone
Table 3 showed that the replacement of 5% limestone with eggshell flour increased the calcium percentage and calcium content of tibia bone, but substitution of 7.5% limestone with eggshell flour decreased the percentage and weight calcium of bone.This is due to increase Ca deposition in yolks (Table 2).The results showed that the use of 7.5% eggshell flour has occurred Ca mobilization of tibia bone for egg formation, especially egg yolks.Tunç et al. (2015) stated that the Ca content tibia bone is affected by the calcium source, but the results of this study show not significant effect.
The results of this study was different from research of Saunders-Blades et al. (2009) in which the weight and calcium of tibia were not affected by calcium sources.Tůmová et al. (2016) and Swiatkiewicz et al. (2015) suggested that tibia bone Ca is affected by Ca levels of feed.

CONCLUSION
Calcium deposition in yolk was the highest in the use of 7.5% eggshell flour to substitute limestone, while calcium deposition in albumen was the highest in the use of 2.5% eggshell flour.The use of 7.5% eggshell flour as a substitute for limestone resulted in the mobilization of Ca from the tibia bone so that the Ca content of the tibia bone decreased.

Table 1 .
. The use of eggshell flour as a substitute of limestone in diet Composition of Feed Stuff and Nutrient Content of Dietary Treatment Adeyemo et al. (2012)005) by Eggshell Flour in Laying Hens (S.Kismiati et al.)259 of laying hen was not effect on the egg weight with the egg weight is 47.47 -47-81 g(Gongruttananun, 2011).Result of the research ofCufadar (2014)showed that different source of Ca was not affected on the egg weight.According toSultana et al. (2007)andSafaa et al. (2008)differences of Ca source and Ca content in diet did not effect on egg weight.An et al. (2016)confirmed that Ca was not the factor affecting to the egg weight.Hanusová et al. (2015)stated that egg weight was affected by genetic factor.Leeson and Summers (2005)and King'ori (2012) stated that factor affecting the egg weight were age, body weight and nutrition consumption.According toAdeyemo et al. (2012), protein content in diet was effect on the egg weight.

Table 2 .
stated that factors affecting the Calcium Deposition in Egg on using Eggshell Flour as Substitution of Limestone in the Feed of Laying Hens

Table 3 .
Length, Weight and Ca Content of the Tibia Bone on Using Eggshell Flour as Substitution of Limestone in the Feed of Laying Hens