The Influence of Hen Aging on Eggshell Ultrastructure and Shell Mineral Components

  • Park, Ji-Ae (Department of Animal Science and Biotechnology, Gyeongnam National University of Science and Technology) ;
  • Sohn, Sea-Hwan (Department of Animal Science and Biotechnology, Gyeongnam National University of Science and Technology)
  • Received : 2018.08.17
  • Accepted : 2018.09.10
  • Published : 2018.10.31


The eggshell, which is a complex and highly ordered structure, is very important factor for food safety and egg marketing. This study investigated the changes in eggshell structure and shell components in relationship to hen age. For this study, we examined the histological change of the endometrium of the 30-, 60-, and 72-wk-old commercial layers, and analyzed the ultrastructure and ionic composition of their eggshells. The results showed that histological deformation, fibrosis, atrophy and elimination of micro-villi in the uterus endometrium were found through microscopic observation that was associated with increasing hen age. Concentration of blood-ion components such as $Ca^{2+}$, $Na^+$, $K^+$, and $Cl^-$ ions did not change with age. Along with the results from the ultrastructure analysis of the eggshell, the palisade layer ratio and the density of mammillary knobs were significantly decreased in older hens. In addition, the type B mammillary knobs were frequently observed with increasing hen age. In the mineral element assay from the eggshell, $Ca^{2+}$, $S^{2-}$, and $Co^{2+}$ significantly decreased with increasing hen age, whereas $Na^+$, $K^+$, and $V^{2+}$ significantly increased. Therefore, the damages of endometrial tissue inhibit the processes of ion transmission and the crystallization of eggshell formation, resulting in a large and non-uniform mammillary knob formation. This means the conditions of endometrial cells affect the formation of the eggshell structure. In conclusion, hen aging causes the weakness of the eggshell and degrades the eggshell quality.


Supported by : Gyeongnam National University of Science and Technology


  1. Abe H. 1996. The mammalian oviductal epithelium: Regional variations in cytological and functional aspects of the oviductal secretory cells. Histol Histopathol 11:743-768.
  2. Ahmed AM, Rodriguez-Navarro AB, Vidal ML, Gautron J, Garcia-Ruiz J, Nys Y. 2005. Changes in eggshell mechanical properties, crystallographic texture and in matrix proteins induced by moult in hens. Br Poult Sci 46:268-279.
  3. An SH, Kim DW, An BK. 2016. Effects of dietary calcium levels on productive performance, eggshell quality and overall calcium status in aged laying hens. Asian-Australas J Anim Sci 29:1477-1482.
  4. Arias JL, Fernandez MS, Dennis JE, Caplan AI. 1991. Collagens of the chicken eggshell membranes. Connect Tissue Res 26:37-45.
  5. Athanasiadou D, Jiang W, Goldbaum D, Saleem A, Basu K, Pacella MS, Bohm CF, Chromik RR, Hincke MT, Rodriguez-Navarro AB, Vali H, Wolf SE, Gray JJ, Bui KH, McKee MD. 2018. Nanostructure, osteopontin, and mechanical properties of calcitic avian eggshell. Sci Adv 4:3219.
  6. Bahr JM, Palmer SS. 1989. The influence of aging on ovarian function. Crit Rev Poult Biol 2:103-110.
  7. Bar A. 2008. Calcium homeostasis and vitamin D metabolism and expression in strongly calcifying laying birds. Comp Biochem Phys A Mol Integr Physiol 151:477-490.
  8. Birgani ZT, Gharraee N, Malhotra A, van Blitterswijk CA, Habibovic P. 2016. Combinatorial incorporation of fluoride and cobalt ions into calcium phosphates to stimulate osteogenesis and angiogenesis. Biomed Mater 11:015020.
  9. Carrino DA, Dennis JE, Wu TM, Arias J, Ferandez MS, Rodriguez JP, Fink DA, Heauer AH, Caplan AI. 1996. The avian eggshell extracellular matrix as a model for biomineralization. Connect Tissue Res 35:325-329.
  10. Chen W, Zhao F, Tian ZM, Zhang HX, Ruan D, Li Y, Wang S, Zheng CT, Lin YC. 2015. Dietary calcium deficiency in laying ducks impairs eggshell quality by suppressing shell biomineralization. J Exp Biol 218:3336-3343.
  11. Cusack M, Fraser AC, Stachel T. 2003. Magnesium and phosphorus distribution in the avian eggshell. Comp Biochem Physiol B Biochem Mol Biol 134:63-69.
  12. Dunn IC, Joseph NT, Bain M, Edmond A, Wilson PW, Milona P, Nys Y, Gautron J, Schmutz M, Preisinger R, Waddington D. 2009. Polymorphisms in eggshell organic matrix genes are associated with eggshell quality measurements in pedigree Rhode Island Red hens. Anim Genet 40:110-114.
  13. Dunn IC, Rodriguez-Navarro AB, Mcdade K, Schmutz M, Preisinger R, Waddington D, Wilson PW, Bain M. 2012. Genetic variation in eggshell crystal size and orientation is large and these traits are correlated with shell thickness and are associated with eggshell matrix protein markers. Anim Genet 43:410-418.
  14. Fathi MM, El-Dein AZ, El-Safty, Radwan LM. 2007. Using scanning electron microscopy to detect the ultrastructural variations in eggshell quality of Fayoumi and Dandarawi chicken breeds. Int J Poult Sci 6:236-241.
  15. Fathi MM, El-Dlebshany AE, El-Deen MB, Radwan LM, Rayan GN. 2016. Effect of long-term selection for egg production on eggshell quality of Japanese quail (Coturnix japonica). Poult Sci 95:2570-2575.
  16. Fernandes JIM, Murakami AE, Sakamoto MI, Souza LMG, Malaguido A, Martins EN. 2008. Effects of organic mineral dietary supplementation on production performance and egg quality of white layers. Braz J Poult Sci 10:59-65.
  17. Fernandez MS, Araya M, Arias JL. 1997. Eggshells are shaped by a precise spatio-temporal arrangement of sequentially deposited macromolecules. Matrix Biol 16:13-20.
  18. Gantois I, Ducatelle R, Pasmans F, Haesebrouck F, Gast R, Humphrey TJ, Van Immerseel F. 2009. Mechanisms of egg contamination by Salmonella Enteritidis: Review article. FEMS Microbiol Rev 33:718-738.
  19. Gongruttananun N. 2011. Effects of eggshell calcium on productive performance, plasma calcium, bone mineralization, and gonadal characteristics in laying hens. Poult Sci 90:524-529.
  20. Hincke MT, Nys Y, Gautron J, Mann K, Rodriguez-Navarro AB, McKee MD. 2012. The eggshell: Structure, composition and mineralization. Front Biosci 17:1266-1280.
  21. Huntley DM, Holder DP. 1978. Ultrastructure of shell gland tissue from hens producing good and poor eggshells. Poult Sci 57:1365-1368.
  22. Jiang S, Cui L, Shi C, Ke X, Luo J, Hou J. 2013. Effects of dietary energy and calcium levels on performance, egg shell quality and bone metabolism in hens. Vet J 198:252-258.
  23. Jonchere V, Brionne A, Gautron J, Nys Y. 2012. Identification of uterine ion transporters for mineralisation precursors of the avian eggshell. BMC Physio 12:10.
  24. Kulshreshtha G, Rodriguez-Navarro A, Sanchez-Rodriguez E, Diep T, Hincke MT. 2018. Cuticle and pore plug properties in the table egg. Poult Sci 97:1382-1390.
  25. Lee MH, Cho EJ, Choi ES, Bang MH, Sohn SH. 2016a. The effect of hen age on egg quality in commercial layer. Korean J Poult Sci 43:253-261.
  26. Lee MH, Cho EJ, Choi ES, Sohn SH. 2016b. The effect of storage period and temperature on egg quality in commercial eggs. Korean J Poult Sci 43:31-38.
  27. Liao B, Qaio HG, Zhao XY, Bao M, Liu L, Zheng CW, Li CF, Ning ZH. 2013. Influence of eggshell ultrastructural organization on hatchability. Poult Sci 92:2236-2239.
  28. Mohammadpour A, Zamanimoghadam A, Heidari M. 2012. Comparative histomorphometrical study of genital tract in adult laying hen and duck. Vet Res Forum 3:27-30.
  29. Molnar A, Maerten L, Ampe B, Buyse J, Kempen I, Zoons J, Delezie E. 2016. Change in egg quality traits during the last phase of production: Is there potential for an extended laying cycle? Br Poult Sci 57:842-847.
  30. Nys Y, Gautron J. 2007. Structure and formation of the eggshell. In Bioactive egg compounds. Huopalahti R, Lopez-Fandino R, Anton M, Schade R (ed). Springer-Verlag, Berlin, German. pp 99-102.
  31. Nys Y, Hincke MT, Arias JL, Garcia-Ruiz JM, Solomon SE. 1999. Avian eggshell mineralization. Poult Avian Biol Rev 10:143-166.
  32. Panchal SK, Wanyonyi S, Brown L. 2017. Selenium, vanadium, and chromium as micronutrients to improve metabolic syndrome. Curr Hypertens Rep 19:10.
  33. Park JA, Cho EJ, Park JY, Sohn SH. 2017. Histological change of uterus endometrium and expression of the eggshell-related genes according to hen age. Korean J Poult Sci 44:19-28.
  34. Park JA. 2018. Study on the eggshell ultrastructure, shell composition and eggshell-related gene expression according to hen age. Ph.D. dissertation, Gyeongnam National Univ. of Science and Technology, Korea.
  35. Parsons AH. 1982. Structure of the eggshell. Poult Sci 61:2013-2021.
  36. Radwan LM. 2016. Eggshell quality: A comparison between Fayoumi, Gimieizah and Brown Hy-Line strains for mechanical properties and ultrastructure of their eggshells. Anim Prod Sci 56:908-912.
  37. Rayan GN, Galal A, Fathi MM, El-Attar AH. 2010. Impact of layer breeder flock age and strain on mechanical and ultrastructural properties of eggshell in chicken. Int J Poult Sci 9:139-147.
  38. Richard JD, Zhao J, Harrel RJ, Atwell CA, Dibner JJ. 2010. Trace mineral nutrition in poultry and swine. Asian-Australas J Anim Sci 23:1527-1534.
  39. Roberts JR, Chousalkar K, Samiullah S. 2013. Egg quality and age of laying hens: Implications for product safety. Anim Prod Sci 53:1291-1297.
  40. Rodriguez-Navarro A, Kalin O, Nys Y, Garcia-Ruiz JM. 2002. Influence of the microstructure on the shell strength of eggs laid by hen of different ages. Br Poult Sci 43:395-403.
  41. Rodriguez-Navarro AB, Dominguez-Gasca N, Munoz A, Ortega-Huertas M. 2013. Change in the chicken eggshell cuticle with hen age and egg freshness. Poult Sci 92:3026-3035.
  42. Rose-Martel M, Hincke MT. 2015. The eggshell as a barrier to contamination. In Handbook of eggs in human function. Wageningen Academic Publishers, Wageningen, Netherlands. pp 172-177.
  43. Sanchez-Gonzalez C, Moreno L, Lopez-Chaves C, Nebot E, Pietschmann P, Rodriguez-Nogales A, Galvez J, Montes-Bayon M, Sanz-Medel A, Llopis J. 2017. Effect of vanadium on calcium homeostasis, osteopontin mRNA expression, and bone microarchitecture in diabetic rats. Metallomics 9:258-267.
  44. Scibior A. 2016. Vanadium (V) and magnesium (Mg)- In vivo interactions: A review. Chem-Biol Interct 258:214-233.
  45. Solomom SE. 1991. Egg and eggshell quality. Wolf Publishing Ltd., London.
  46. Stefanello C, Santos TC, Murekami AE, Martins EN, Carnerio TC. 2014. Productive performance, eggshell quality, and eggshell ultrastructure of laying hens fed diets supplemented with organic trace minerals. Poult Sci 93:104-113.
  47. Tumova E, Gous RM. 2012. Interaction of hen production type, age, and temperature on laying pattern and egg quality. Poult Sci 91:1269-1275.
  48. van Toledo B, Parsons AH, Combs CF. 1982. Role of ultrastructure in determining eggshell strength. Poult Sci 61:569-572.
  49. Vetter AE, O'Grady SM. 2004. Sodium and anion transport across the avian uterine (shell gland) epithelium. J Exp Biol 208:479-486.
  50. Wilson PB. 2017. Recent advances in avian egg science: A review. Poult Sci 96:3747-3754.
  51. Woudstra T, Thomson AB. 2002. Nutrient absorption and intestinal adaptation with ageing. Best Pract Res Clin Gastroenterol 16:1-15.