Use of extruded eggshell as a calcium source substituting limestone or oyster shell in the diet of laying hens

Abstract The nutritional values of limestone, oyster shell and extruded eggshells were evaluated using different methods. In total, 120 ready‐to‐lay pullets, 18‐week‐old ISA Brown were distributed into six dietary groups, namely D1 (4% limestone), D2 (8% limestone), D3 (4% oyster shell), D4 (8% oyster shell), D5 (4% eggshell) and D6 (8% eggshell), to assess the effect of calcium sources on egg production, egg quality, dry matter and cholesterol content of the egg. Kitchen‐extruded eggshell contained 98.52, 4.24, 29.75 and 14.82% DM, CP, Ca and P, whereas hatchery‐extruded eggshell contained 99.20, 13.80, 25.53 and 13.87% DM, CP, Ca and P, respectively. Limestone and Oyster shells contained 99.60 and 99.51% DM, and 37.12 and 35.20% Ca, respectively. Body weight, egg, hen day and egg mass production, and FCR did not differ among diets (p > .05). Egg production tended to increase with the increase of hen‐housed egg production (p < .001) in D6, followed by D2, D5, D3, D4 and D1, respectively. The lowest production cost and the highest net profit were observed in D6, followed by D2, D4, D5, D1 and D3, respectively. Diet with 8% Ca sources performed better than the diets with 4% Ca sources in terms of egg quality and dry matter content, where D6 was comparable to D2 or D4. The weight of egg, albumen, yolk, eggshell, dry yolk and yolk–albumen ratio increased, while dry albumen and eggshell weight, eggshell strength and thickness, Haugh unit, yolk index and egg‐specific gravity decreased with the increase in bird's age. The cholesterol content of yolk was statistically similar among diets. Therefore, no adverse effect of calcium sources on the production of laying hen was observed. Of these, extruded eggshell especially the 8% extruded eggshell may be beneficial to use in the diet of laying hen for producing a quality, safe and profitable egg.

Calcium carbonate from oyster shells contains lead vestige among the other potential toxic elements such as aluminium, cadmium and mercury. In this case, eggshell has a great advantage for not containing toxic elements.
Laying hens need Ca to lay and have strong eggshells. If hens do not get enough Ca in diet, their bodies pull Ca from their bones. As birds have no sweat gland, they rely on the panting system to dissipate excessive heat from the body. This causes excessive loss of carbon dioxide (CO 2 ) which forms calcium carbonate (CaCO 3 ) in the presence of Ca in the uterus. The net result is lower eggshell quality with soft or thin-shelled eggs. This is why more calcium is needed to provide in layer ration. Eggshell is a good source of Ca, and the hens prefer this Ca very much even more than crust oyster shell or limestone. Large amounts of extruded eggshells in restaurants, hatchery, kitchen and egg product factories are thrown out as wastes every day. Extruded eggshell with membrane can be used as a source of Ca and a small amount of protein in layer diets without any adverse effects on egg production and egg quality (Gongruttananun, 2011;Liehovnikova, 2007). Sheideler (1998) has reported that extruded eggshell with a large particle size of limestone or oyster shell improved the eggshell quality. Therefore, the present study was aimed at determining the effect of extruded eggshell substituting limestone or oyster shell on egg production performance, egg and eggshell quality of laying hen for producing a quality, safe and cost-effective egg.

| Collection and process of eggshell, limestone and oyster shell
Kitchen and hatchery-extruded eggshell were collected from the student Hall of BSMRAU, restaurant and the hatchery, washed with fresh water and allowed to boil at a temperature of 100°C for 3-4 min, as a result all pathogenic bacteria, viruses and parasites were destroyed. In addition, the eggshells were dried under the sun, ground using a grinder and then stored. Limestone and oyster shells were collected from the local market.

| Nutrient analysis of extruded eggshell, limestone and oyster shell
The nutrient content of kitchen and hatchery-extruded eggshell, limestone or oyster shells were determined by a proximate analysis and spectrophotometric method (Ca & P) (AOAC, 2011) at the laboratory of the Department of Livestock Services, Dhaka, Bangladesh.

| Feeding trial
A total of 120 ready-to-lay pullet, 18-week-old ISA Brown were collected from CP Bangladesh Company Ltd. and distributed into six dietary groups, namely D 1 (diet with 4% limestone), D 2 (Diet with 8% limestone), D 3 (diet with 4% oyster shell), D 4 (Diet with 8% oyster shell), D 5 (Diet with 4% eggshell) and D 6 (Diet with 8% eggshell) having 20 pullets in each dietary group. The birds were reared in an individual cage management system providing standard management practices as per standard given by the breeder during the experimental period (Table 1).

| Data recording
The nutrient content of eggshell, limestone and oyster shell was evaluated. Body weight, feed intake and egg and egg mass production were recorded fortnightly, mortality was recorded when occurred during the laying period. Hen day and hen-housed egg production were calculated during the experimental period.
Egg quality and dry matter content of egg at 28, 36, 48, 56 and 68 weeks of age of the bird were recorded. Egg yolk cholesterol at 32, 44 and 56 weeks of age of the bird was recorded.
FCR (Feed/dozen eggs and Feed/kg eggs), production cost and net profit (Tk/dozen eggs) were calculated during the experimental period.

| Statistical analysis
The collected data were analysed using the Statistix10 computer package program (Statistix 10, 1985). Mortality of birds and cholesterol content of egg yolk were subjected to chi-squared test and t test, respectively.
Statistical model: The following statistical model was used for the analysis of egg production performance data.
where Y ij is the observation of the jth number of individuals in the ith dietary group; µ is the overall mean; D i is the fixed effect of the ith dietary group (i = 1, 2 ………6); and e ij is the random error.
The following statistical model was used for the analysis of data of egg quality and dry matter content of the egg.

ISLAM And nISHIBORI
where Y ijk is the observation on the kth number of individuals in the ith dietary group and jth age group; µ is the overall mean; D i is the fixed effect of the ith dietary group (i = 1, 2 ………6); A j is the effect of the jth age group (j = 1, 2 ………5); and e ijk is the random error.

| Egg production performance of laying hen-fed diet with extruded eggshell, limestone and oyster shell for 365 days of laying period
Dietary groups were significantly different for feed intake, henhoused egg production, production cost (Tk/dozen or kg eggs) and net profit (p < .001), but not significantly different for body weight, egg production, hen day egg production, egg mass production, mortality and FCR (Feed/kg egg) (p > .05) ( Table 3). Body weight of hen at the end of the experiment was almost similar among diets (p > .05).
But the highest feed intake was observed in D 1 , followed by D 5 , D 6 , D 4 and D 2 , respectively (p < .001). Evidently, but not significantly, the highest egg production, hen day egg production and egg mass production were observed in D 6 , followed by D 2 , D 5 , D 3 , D 4 and D 1 , respectively. Feed conversion ratio (FCR) tended to decrease in D 6 , followed by D 2 , D 3 , D 5 , D 1 and D 4 , respectively. The lowest production cost and the highest net profit were observed in D 6 , followed by D 2 , D 4 , D 5 , D 1 and D 3 , respectively. Therefore, the diets with 8% Ca sources performed better than the diets with 4% Ca sources.
Of these, D 6 (8% eggshell) performed the best among the dietary groups in terms of egg production, production cost and net profit.  Table 4). The highest egg weight was observed in D 1 , followed by D 3 , D 6 , D 4 , D 5 and D 2 , respectively. The highest eggshell strength was measured in D 4 , followed by D 6 , D 2 , D 3 , D 5 and D 1 , respectively. Therefore, the eggshell was stronger in diets with 8% Ca sources than in diets with 4% Ca sources. The diet D 6 showed the highest yolk colour followed by D 5 , D 4 , D 2 , D 3 and D 1 , respectively. Albumen and yolk width were almost similar among the dietary groups (p > .05). However, the highest albumen height was in D 1 , followed by D 2 , D 3 , D 5 , D 6 and D4, respectively. The highest albumen weight was in D 3 and D 1 , and the lowest in D 2 , D 4 , D 6 and D 5 . Yolk weight was almost similar among dietary groups (p > .05), but the lowest eggshell with membrane weight was in D 3 and D 5 , moderate in D 6 and D 4 and the highest in D 2 and D 1 . The higher eggshell with membrane or eggshell thickness was observed in diets with 8% Ca sources compared to the diets with 4% Ca sources. Of these, the diet with 8% extruded eggshell was comparable to the diet with 8% limestone or oyster shell in terms of the eggshell thickness or eggshell strength. The highest Haugh unit was observed in D 2 , followed by D 1 , D 5 , D 3 , D 6 and D 4 , respectively. The highest yolk index was measured in D 3 , D 6 , D 5 , D 2 and the lowest in D 1 and D 4 . The highest specific gravity was measured in D 2 , followed by D 4 , D 1 , D 6 , D 5 and D 3 , respectively. The yolk-albumen ratio was almost similar among dietary groups (p > .05).
Age affected the egg quality traits (p < .001). Egg weight, albumen and yolk width, yolk height, albumen weight, yolk weight, eggshell with membrane weight and yolk-albumen ratio increased with the increase in bird's age. However, eggshell strength, yolk colour, albumen height, eggshell with membrane thickness, eggshell thickness, membrane thickness, Haugh unit, yolk index and the specific gravity of egg decreased with the increase in bird's age.
Interaction between diet and age was observed for yolk colour, albumen width and height, eggshell with membrane thickness, eggshell thickness, membrane thickness and Haugh unit (p < .001) but no interaction of diet and age was found for egg weight, eggshell strength, yolk width, yolk height, albumen weight, yolk weight,

| Dry matter content of egg of laying hen-fed diet with extruded eggshell, limestone and oyster shell for 365 days of laying period
There was no significant difference among dietary groups for dry matter content of egg of laying hen (p > .05) except the eggshell and dry eggshell weight and dry yolk weight (p < .05) ( Table 5). The highest eggshell weight was observed in D 2 , followed by D 4 , D 1 , D 3 , D 6 and D 5 , respectively. The diet D 2 showed the highest dry yolk weight, followed by D 1 , D 4 , D 6 , D 5 and D 3 , respectively. But the highest dry eggshell weight was observed in D 4 , followed by D 2 , D 6 , D 5 , D 3 and D 1 , respectively. The other dry matter traits of egg were tended to increase in diets with 8% Ca sources than in diets with 4% Ca sources (p > .05). Thereof, the diet with 8% extruded eggshell was comparable to the diet with 8% limestone or 8% oyster shell in terms of the dry matter content of the egg.
Age influenced the dry matter traits of the egg (p < .01) except the dry egg weight, moisture content and albumen weight (p > .05). Egg weight, yolk weight and dry yolk weight increased with the increase in bird's age, but the dry albumen, eggshell and dry eggshell weight decreased with the increase in bird's age. No interaction effect of diet x age was observed on dry matter content of egg (p > .05).

| Nutritive value of extruded eggshell, limestone and oyster shell
Extruded eggshell not only contains Ca and P but also contains crude protein (CP), because of the presence of eggshell membrane which contains crude protein. The estimated crude protein in the present study was higher than that reported by Lertchunhakiat et al. (2016).
They found 2.14% CP in eggshell. However, the present study supported them for containing DM and Ca. They reported 99.40% DM and 29.87% Ca, a similar amount was measured in the present study.
Hatchery-extruded eggshell contained CP% which was higher than that of kitchen-extruded eggshell. However, hatchery-extruded eggshell contained Ca and P which was lower than kitchen-extruded eggshell because of taking Ca and P by embryo from hatching egg

| Egg production performance of laying hen-fed diet with extruded eggshell, limestone and oyster shell for 365 days of laying period
Body weight, egg, hen day egg, egg mass egg production and FCR were almost similar among the dietary groups. Evidently, but not significantly, the highest egg and hen day egg production was observed in D 6 , followed by D 2 , D 5 , D 3 , D 4 and D 1 , respectively. Similarly, FCR tended to be the lowest in D 6 , followed by D 2 , D 3 , D 5 , D 1 and D 4 , respectively. Therefore, the present findings indicated that Ca sources had no adverse effect on egg production performances. The diet D 6 showed the highest hen-housed egg production, followed by D 2 , D 5 , D 3 , D 4 and D 1 , respectively. These findings were supported by Gongruttananun (2011), Frontng, & Bergquist (2015. Olgun et al. (2015) partially supported these findings. They reported the significant effect of Ca sources on egg production, feed intake but not on body weight and FCR. Elsayed et al. (2014) reported that eggshell may be used as a Ca source in laying hen diet without any adverse effect on body weight, egg production and feed consumption. Saleh, Ahmed, et al. (2019) and Saleh, Kirrella, et al. (2019) reported the improved hen day egg production, egg weight, egg mass and FCR when they included the flaxseed and/ or fenugreek seed and cumin oil in the diet of laying hen that supports the present findings. The lowest production cost and the highest amount of net profit were observed in D 6 , followed by D 2 , D 4 , D 5 , D 1 and D 3 , respectively. Thereof, it was found that the diets with 8% Ca sources performed better than the diets with 4% Ca sources in terms of egg production performances.

LSD value and Level of Significance
However, the diet with 8% extruded eggshell (D 6 ) performed the best in terms of egg production, production cost and net profit.

| Egg quality traits of laying hen-fed diet with extruded eggshell, limestone and oyster shell for 365 days of laying period
Albumen and yolk width, yolk weight, membrane thickness and yolkalbumen ratio were statistically similar among dietary groups but the other egg quality traits, namely egg weight, albumen weight, eggshell strength, eggshell weight and thickness, albumen and yolk height, yolk colour, Haugh unit, yolk index and specific gravity of egg differed significantly among dietary groups. For these traits, diets with 8% Ca sources performed better than the diets with 4% Ca sources. Of these, the diet D 6 (8% extruded eggshell) was comparable to the diet with 8% limestone or 8% oyster shell in terms of egg quality traits. The present findings were consistent with the findings of Olgun et al. (2015). Therefore, 8% extruded eggshell in the diet of laying hen might be beneficial to improve eggshell as well as egg quality.
As an effect of age, egg, albumen and eggshell weight, albumen and yolk width, yolk height and yolk-albumen ratio increased, while eggshell strength and thickness, yolk colour, albumen height, Haugh unit, yolk index and the specific gravity of egg decreased with the increase in the bird's age, which was supported by Mitrovic et al. (2010) and Tumova, and Ledvinka (2009

| Cholesterol content of egg of laying hen-fed diet with extruded eggshell, limestone and oyster shell for 365 days of laying period
No previous work was found on the cholesterol content of laying hen eggs that influenced by Ca sources. In the present study, no significant difference was observed among dietary groups for the cholesterol content of egg yolk of laying hen. Therefore, the cholesterol content of egg yolk was almost similar among dietary groups.
Even the amount of cholesterol content of the commercial farming egg measured in the present study was also similar to the egg of test diets. However, Saleh, Ahmed, et al. (2019) and Saleh, Kirrella, et al. (2019) found the reduced cholesterol in blood plasma and egg yolk when they added flaxseed and/ or fenugreek seed and cumin oil in the diet of laying hen.

| CON CLUS IONS
The present study reveals that kitchen-extruded eggshell contains a higher amount of Ca and P but a lower amount of crude protein than the hatchery-extruded eggshell. Limestone or oyster shell contains | 11

ISLAM And nISHIBORI
Ca only. The diet with 8% extruded eggshell performed the best among the dietary groups in terms of egg production, FCR and net profit. Egg quality traits and dry matter content of egg were better in diets with 8% Ca sources than in diets with 4% Ca sources. However, the diet with 8% extruded eggshell was comparable to the diet with 8% limestone or 8% oyster shell in terms of egg quality and dry matter content of the egg. The cholesterol content of the egg was almost similar among the dietary groups. Egg quality traits and dry matter content of egg except for egg, albumen, eggshell and yolk weight decreased with the increase in bird's age. Therefore, the extruded eggshell especially the 8% extruded eggshell may be beneficial to use in the diet of laying hen for producing a safe, quality and profitable egg.

ACK N OWLED G EM ENTS
The authors are grateful to the Ministry of Education, Government of the People's Republic of Bangladesh for providing a fund to carry out the research and prepare the scientific article.

CO N FLI C T O F I NTE R E S T
The authors declare that there is no conflict of interest regarding the publication of this article.

E TH I C A L S TATEM ENT
The authors confirm that the ethical policies of the journal, as noted on the journal's author guidelines page, have been adhered to.

PE E R R E V I E W
The peer review history for this article is available at https://publo ns.com/publo n/10.1002/vms3.544.