Food Structure

Volume 5 Number 1 Article 13

1986

The Microstructure of the Hen's Egg Shell - A Short Review

R. M. G. Hamilton

Follow this and additional works at: https://digitalcommons.usu.edu/foodmicrostructure

Part of the Food Science Commons

Recommended Citation Hamilton, R. M. G. (1986) "The Microstructure of the Hen's Egg Shell - A Short Review," Food Structure: Vol. 5 : No. 1 , Article 13. Available at: https://digitalcommons.usu.edu/foodmicrostructure/vol5/iss1/13

This Article is brought to you for free and open access by the Western Dairy Center at DigitalCommons@USU. It has been accepted for inclusion in Food Structure by an authorized administrator of DigitalCommons@USU. For more information, please contact [email protected]. FOOD ~l!CROSTRUCTURE, Vol. 5 (1986). pp. 99-110 0730-S4 19/86$ 1. 00•.0S SEM. Inc .• AMF O'Hare (Chicago), IL 60666- 0507 U.S.A.

THE MICROSTRUCTUR E OF THE HEN'S EGG SHELL - A SHORT REVIEWl

R. M.G. Hamilton

Animal Research Centre, Agriculture Canada, Ottawa, Ontario, Ca nada KlA OC6

Introduction

The structure of the hen's egg shell can The hen's egg shell surrounds the biological be d iv1 ded 1nto five separate 1ayers . The 1 nner­ material essential to the reproduction of the most layer consists of two distinct membranes species (Fig. lA). Besides providing mechanical known as the inner and outer shell membranes. These membranes are compos ed of networks of protein/polysaccharide fibres and are =70 ~m thick. Attached to the outer fibres of the outer membrane are polycrystals of calcite (calcium carbonate) which extend outward in an inverse conical manner until the cones from several sites of crystal i nitiation fuse together. The fibre/ crystal attachment sites, known as basal caps, and the cones form the mammillary knobs layer which is :: 100- 110 llm thick. After the cones fuse with each other, continuing calcite deposi­ tion produces columnar crystals 10 - 30 "'m in diameter and :::2 00 llm in length. These crys­ tals form the palisade layer and are intermingled with a protein/polysaccharide matrix that differs in composition From the shell membranes. Over the columnar crystals is a thin layer (::S -8 "'m thick), known as the vertical crystal layer, of small calcite crystals that are orientated perpendicular to the shell's surface. The cuti­ cle is the outermost layer of the shell; it is =10 "'m thick and contains predominantly pro­ tein. Passing vertically through the palisade layer of the shell from "valleys" between the marrmi llary knobs to the surface of the vertical crystal layer are funnel - shaped, unbranched pores, These pores are capped by the cuticle whi ch i s crac ked and thus allows the diffusion of gases between the contents of the egg and its environment. The geometrical configuration of the cones in the mammillary knobs layer is related to the thickness of shell. Specific ~ Schematic diagrams of the principal amino acids in the membrane fibres of the basal components of the hen's egg (A) and of caps influence shell strength. the layers that constitute the shell ( B). These diagrams are not drawn to KEY WORDS: Egg shell, microstructure, shell scale. Figure B based on diagrams membranes. marrmillary knobs, palisade/matrix, from Schmidt (1962), S1mk1ss (1968), cuticle. Tyler (l969a) and S1mons (197lb).

Initial paper received Ma rch 10 1986 protection to the egg' s contents or the develop­ Manuscript received May 21 1986 ing embryo, the shell is a barrier to microorgan­ Direct inquiries to R. M.G. Hamilton isms, a reservoir of calcium for the embryo, a Telephone number: 613 993 6002 x 261 permeable membrane that facilitates the exchange of respiratory gases between the egg and its

99 R.M.G. Hamilton environment without excessive dehydration and is, ovum is mature, it is released from th•e ovary for wild birds, a camouflage (Board, 1982). (ovulated) and begins its descent of the hen's Interest in the egg shell dates back to at reproductive tract where the albumen and s.he ll least the time of Aristotle (384-322 8C). According to Tyler (1969a), Purkinje published one of the first detailed studies of the egg shell in 1825. In a series of over 30 11 classi­ cal11 papers from work done between 1821 and 1899 as a hobby, the industrialist von Nathusius demonstrated clearly that the avian egg shell is a complex, high ly ordered structure composed of calcified matrix superimposed on fibrous membranes. Even though von Nathusius had only a simple microscope, his drawings rival in detail photomicrographs obtained with modern light and electron microscopic techniques. The original papers of von Nathusius have been translated into English and edited by Tyler (1964) and include detailed line drawings of the shell structure of eggs from about 130 species, including the domestic hen. More recently, Simons (1971a,b) published the results of a comprehensive study of the hen's egg shell and membranes i nvo 1vi ng 1 i ght, and transmission and scanning electron microscopy. This monograph also contains an extensive review of previously published data. Reviews on the morphological aspects of the hen's egg shells have been published by Stewart (1935), Burmester (1940), Simkiss (1968), Tyler ( 1969a) and Parsons ( 1982). Microscopic studies of egg shell formation have been reported by, among others, Simons (197la), Hoffer (1971), Draper et al. (1972), Creger et al. (1976), Sternberger et al. (1977), Pooley (1979) and Makita (1981). In addition, reviews have been published on shell ca l cification (Wilbur and Simkiss, 1968; Eastin and Spaziani, 1978a,b), organic components of the shell (Krampitz and Witt, 1978; Krampitz, 1982; Leach, 1982) and the relationship between shell structure and egg shell breakage (Parsons, 1982; Washburn, 1982). It is now generally accepted that the shell of the hen's egg (Fig. 2) contains at least five distinct layers as indicated schematically in Figure lB. These layers are 1) inner and outer shell membranes, 2) mammillary knobs, 3) pali­ sade/shell matrix, 4) vertical crystals, and 5) the cuticle (Simons, 197la). Because this pre­ sentation is a tutorial, the discussion will be limited to the major structural and biochemical ~ Radially fractured surface of the shell aspects of these layers. Little evidence wi 11 from a white-shell egg showing the be presented to confirm these aspects since, in shell membranes (SM), marm1illary knobs some cases, the data is extensive. (MK), palisade and vertical crystals layer (P-VC) and cuticle (C T). Shell ~hell Formation section was sputter coated with gold and examined with a scanning electron The following brief description of egg and microscope (EMR lOOA; 10 kV). Bar = shell formation is given to indicate the specific 100 "m. area of the hen's reproductive tract and the time ~ Enlargement of the rectangular section required for the synthesis of these layers. The in Fig. 2 showing the shell membranes size of the yolk (Fig. lA) present in the ovary and mammillary knobs. Bar= 10 J.Jffi. of the hen increases rapidly over a period of ~ Outer fibres of the outer shell membrane 10-14 days before ovulation. During this time viewed towards the mammillary knobs in its weight increases from about 200 mg to 15-18 the background. The overlying membrane g through the incorporation of proteins and material was stripped manually. Section lipids that are synthesized mainly in the liver was prepared and examined as described (Redshaw and Follett, 1972). After the yolk or in Fig. 2. Bar 10 J.Jin.

100 Egg Shell Microstructure

are forned. The average time between successive Table 2. Means and Standard Deviations of eggs is 24-25 hours for high producing birds. Egg and Shell Characteristics Th e reproductive tract consists of at least for White- and Brown-Shell Eggs five di itinct regions, namely: 1) infundibulum, (30 Eggs per Sample) l 2) magnJm , 3) isthmus, 4) uterus or shell gland, and 5) va gina. The time taken and the component Mean (Standard Deviation) added to the egg during passage through the Characteristics White- Shell Brown -Shell reproductive tract of the hen are surrvnarized in Table 1 Shell color pigments are incorporated Egg weight, g 65.2 4.4) 65.0 5.6) Length, nm 60.6 2 .2) 59.5 2. 5) Table l Site of Formation of Egg and Shell Diameter, rrrn 43.6 l.O) 44 .l l. 4) Components 1 Shell weight, 5. 91 0.4) 5 . 36 0. 5) Shell Section of Time Developing Components thickness2 ,JJm 334 ( 21. 7) 324 ( 20. 7) Reproductive Egg Spends in Produced or Compression fracture System Section (hrl Added strength3 N 29.7 4.2) 29.5 ( 5. 2) Deformation4, J.Lm 63.4 7 .4) 75.2 (15. 9) Ovary Yolk Infundi bu lum 0.25- 0.5 (Site of fertilization) lAdapted from Thompson et al., 1981. Magnum 3 Albumen as a gel 2Measured at the equator (diameter) of the eggs. Isthmus 1. 25 Water and miner­ 3compression fracture force applied at 20.0 als that dilute mm/min to the equator of the egg. N = Newtons. the albumen; and 4For an applied load of 1.0 kg. shel 1 membranes Uterus 20 - 21 Thin albumen and shel 1 characteristics for white- and brown­ (shell gland) and minerals, shell eggs laid by mature hens (Thompson et al., shell, shell 19Bl}. Characteristics such as shell thickness, pigments and compression fracture strength and deformation cuticle. vary among eggs at a particular site (Table 2) Vagina Passage to ex ­ and also among sites on a single egg. For ex­ Cloaca terior for the ample, the shell is thickest at the equator and deve 1oped egg thinnest at the narrow pole; thickness at the blunt end is intermediate between the narrow pole and equator (Tyler, 1961; Tyler and Geake, 1965). lAdapted from Geiger et al., 1974. She 11 Membranes within the last 5 hours of shell deposition (Warren and Conrad, 1942). The brown pigment Even though the membranes located on the that colors the eggs from some hens is porphyrin inner surface of the egg shell appear to be a synthesized by the shell gland f rom 6- amino single layer (Fig. 3), they can be divided, by levulinic acid (Polin, 1957). Further informa­ careful manipulation, into two distinct layers tion ma y be obtained from Robinson (1972) and of fibrous material. They adhere closely to Gilbert (1971) for albumen synthesis and Simkiss each other except at the blunt end of the egg and Taylor (1971), Creger et al. (1976) and Reed where they are separated by the air cell (Fig. and Yurkiewicz (1982) for shell formation. 18). One layer surrounds the albumen while the other is attached to the "tips" of the calcified Gross Composition and material of the shell (Fig. 3); these layers are Physical Characteristics of Eggs known as the inner and outer shell membranes, respectively. The hen • s egg contains about 58% a 1 bumen, Microscopic examination of these layers 32% yolk and 10% shell by weight, with the first reveals an interwoven meshwork of fibres (Fig. two components containing approximately 76 and 4) arranged in a random manner (Stewart, 1935; 24% water, respectively (Gilbert, 1971). About Moran and Hale , 1936; Kaplan and Siegesmund, 96% of the material in the shell is inorganic 1973; Creger et al., 1976; Wong et al., 1964). and 4% organic (Geiger et al. , 1974); the latter The fibres of the inner membrane are about one is composed of a number of discrete proteins, half as thick as those in the outer membrane (22 glycoproteins and glycosaminoglycans. Calcium vs 48 lJm; Simons, 197la). The thickness of carbonate constitutes 98% of the inorganic mate­ the shel 1 membranes vary within an egg and among rial while magnesium carbonate and calcium phos­ eggs (Balch and Tyler, 1964). Electron micro­ phate contribute equally to the remainder; trace scopy studies of Masshoff and Stolpmann (1901) amounts of other inorganic ions are present. showed that each fibre of the membranes had a Numerous factors influence egg size (weight) central core that was fine and fibrillar in such as the genotype of the hen, age of hen, structure and this core was surrounded by a position of the egg in a sequence, rate of lay, sheath. Delicate strands of material spanned environmental temperature, diet and disease the gaps present between the core and the sheath. status (Gilbert, 1971). The data presented in Fibres from the inner and outer membranes were Table 2 show the means and variability in egg similar in structure, except the former were

101 R.M .G. Hamilton thinner. Draper et al. (1972) described the (1970) also found small quantities of glucos; amine material in the core as 11 electron dense 11 and that and galactosamine, but due to the absenc e of in the sheath as 11 1ess dense" substance. Simons uronic acid they proposed that the galactos; amine and Wiertz (1963) and Hoffer (1971) reported was not associated with chondroitin sulphate. similar observations for membranes from hen 1 s Abatangelo et al. (1978), however, reported the eggs and Japanese quail eggs, respectively. presence of 0.5% uronic acid in the shell mem­ Frequently the mantle of adjacent fibres co­ branes. alesce, giving the appearance of extensive Lipids have been isolated in small quanti­ branching of the fibres (Masshoff and Stolpmann, ties from the shell membranes by Hasiak et al. 1961; Fig. 4). Also , evident on these fibres (l970a,b) and Britton (1977) . These included are small protuberances (see Creger et al., 1976; mono, di and triglycerides, free fatty acids , Leach , 1982 ; Fig. 4), which can be useful when cholesterol and its esters, lecithin, lysoleci­ studying the relationship of composition to thin, cephalin and sphingomyelin (Hasiak et al., shell structure such as occurs with dietary 1970a , b). Trace amounts of sodium, potas sium. copper and manganese deficiencies (Baumgartner manganese, zinc, copper, boron and aluminum were et al., 1978; leach and Gross, 1983; found to be present in both the inner and outer respectively) . shell membranes by Wedral et al . (1974). Calcium Chemical analyses indicate that the shell which was also observed was thought to have a membranes in their native state contain about structural role . 20% water, 75-76% proteinaceous material and 4-5% carbohydrate (Gilbert , 1971). The nature Marrrni llary Knob Layer of the proteinaceous material of the shell mem­ branes remains to be resolved. Some researchers In the marrrnillary knob layer, the calcified have concluded that these membranes contain ker­ portion of the egg shell connects with the f ibres atin or ovokeratin (Simkiss and Taylor, 1971) of the outer portion of the outer shell membrane whereas others consider the proteins as col lagen­ (Fig. lB and 3) . The part of the marrvnillary knob like due to the presence of hydroxyproline layer that is embedded in the outer membrane (Balch and Cooke, 1970) and hydroxylysine fibres is termed the basal caps while the portion (Candlish and Scougall, 1969). An elastin-like above and between these membrane fibres and the protei n has been suggested also to occur in tne palisade layer is known as the cone layer (Tyler, shell membranes (Simkiss and Tyler, 1957; Harris 1965). The membrane fibres penetrate at least et al., 1980). The keratinous nature of the mem­ 70 lJm into the palisade layer (Simons, 197la). brane proteins has been questioned because of Removal of the membrane, by sodium hydroxide (5% differences in structure, amino acid content, w/v) or protease digestion revealed microscopic and protein solubility of soft and hard keratins tracts as evidence of attachment of the marrrnil­ and the proteinaceous material from shell mem­ lary knobs in the fibres (Kaplan and Siegesmund, branes (Wedral et al., 1974; Vadehra et al. , 1973; Stevenson , 1980; respectively) . The 1971). Similarly, results from amino acid ana l­ appearance of the marrrnillary knobs after the ysis, radioimmunoassay and enzymatic hydrolys i s removal of the membranous fibres is sometimes 11 11 led to the conclusion that elastin is not present described as rose-bud • About one-third of the in the shell membranes (Starcher and King, 19BO; shell thickness is accounted for by the marrrnil­ Leach et al., 1981; Crombie et al., 1981) even lary knob layer (Burmester, 1940). though desmosine and isodesmosine, lysine-derived The calcified portion of the s hell is com­ molecular crosslinks found in elastin, occur in posed of small individual crystallites (Simons, the hydrolysates of the membrane s (Baumgartner et 197la) of calcium carbonate in the form of al . , 197B; Harris et a1., 1980; leach et al., calcite (Terepka, 1963a). These individual 1981). Wong et al. (1984) demonstrated the crystallites radiate in all directions from the presence of both membranes of two forms of centre of the mallYlli llary core where they attach collagen- like proteins, similar to collagen I to the membrane fibre. According to Schmidt and V, in the ratio of approximately 100:1 . (1965), growth of individual crystallites, Immunofluorescence microscopy indicated that sometimes known as spherulites (Wilbur and within each membrane, collagen I was associated Simkiss, 196B), inwards to the outer membrane is predominately with the large, coarse fibres impeded by the resistance of the outer membrane (::::2.5 ~m diameter) and the collagen V with fibres. Their growth is further decreased by a the delicate, narrow fibres (::::0.6 \Jm reduced flow of calcifying material from the diameter). At the electron microscopic level, uterine tissue due to more rapid growth of the normal 67-nm banding usually seen with type crystals illYllediately adjacent to that tissu.e. I collagen was not present in either the large, As the crystals grow outward from the basal coarse, or the delicate, narrow fibres . caps, they form irregular polygonal cones and The simple sugars represent 70 - 80% of the eventually fuse with those from adjacent forma­ carbohydrate present in the shell membranes tions to complete the cones of the mammillary (Balch and Cooke, 1970). Glucose, galactose, knob layer. The c rystallites that grow into and mannose were reported by both Balch and Cooke membranes are also known as eiospherites and (1970) and Wedral et al. (1974). However, the those that grow outward from the membranes as former also reported the presence of fructose and exopherites (Tyler and Fowler, 1978). the latter xylose; the reason for the discrepancy Electron microscopic studies indicate that is not evident. Both research groups found the structure and spatial configuration of t he sialic acid in small amounts. Balch and Coo ke mammillary knob layer is an important determi-

102 Egg She ll Microstructure

nant of s hell strength. El - Boushy et al. (1968), 1% (w/v) sodium hydroxide so lution , and of the Rob i nson and King (1970), King and Rob i nson cuticle layer with 5% (w/v) ethylenediaminetetra­ (1972) and Bunk and Balloon (1978) observed that acetic acid (EDTA , disodium sa lt at pH 7.5- 8) , the marrmi llary layer was frequentl y d isorganized produces the "true" shell as defined by Tyler in shells of low s trength compared to those of (1969a). The residue that remains after the high strength. The number of marrmillary knobs "true" shell has been decalcified with 20% EDlA per square centimeter wa s higher for shells of (pH 7.5-8) is known as t he • true" shell matrix low breaking strength than those of high (Tyler, 1969a). strength, the former shells were thinner than Detailed studies of the crystalline struc­ the latter (307 vs 356 ~m ) (van Toledo et al. ture of the egg shell have shown that the 1982). These res ults s upport the findings of palisade layer contains individua l columnar Tyler and Fowler (1978) that the s hell thickness crystals of calcium carbonate a s rhombohedral of eggs from wild birds was highly correlated calcite (Schmidt, 1962; Terepka, 1963a). Each (r = 0 . 94) to the "nearest neighbour" di stance co lumn of calcite consists of crystallites with between manvntllary cone s. Lea ch and Gros s reported diameters of 10- 15 1-1m (Terepka, 1963a) (1983) , however, found that although the shell t o 20- 30 "m (Perrott et al., 1981 ). Both impact strength of eggs from manganese defi c ient Terepka ( 1963a) and Schmidt ( 1964) cone luded hens was lower than for those from non- deficient from mi c roscopic studies with polarized light hens, the former shells had fewer and wider that the orientation of £-axis of the he xagonal manvnillary knob s per unit area than the latter. unit cell of the crystallites in the pali sade Simki s s and Tyler (195 7) showed, by hi s to­ layer wa s perpendicular to the s urface of the chemical techniques , that the concentration of egg s hell. On the other hand, Cain and Heyn organic materia 1 was higher i n the manvni llary (1964) reported that the f. - axi s of the calcite cones than elsewhere in the manrnillary knob crysta l s in the pa li sade layer wa s inclined 28° layer. Subsequently, Robinson and King (1968) ± 16° from the perpendicular to the shell demonstrated that the organic portion o f the surface in X-ray diffraction s tudies. From a manvn11lary cone s contained mainly neutral muco­ limited X- ray diffraction study, Favejee et al. polysaccharides. The results of Cooke and Bal ch (1965} observed that the £-axis of ca l cite in (1970a) confirm the histochemical observations egg s hell was randomly arranged and on the aver­ of Robinson and King (1968) and provide quanti­ age was inclined 45° to the s hell's surface. tative data that about one half of the carbohy­ Sharp and Silyn-Roberts (1984) point out that drate material present in the manvnillary cones £.-axis angles reported by Cain and Heyn (1964) was glucosamine. are improbable because of the manner in which The chemi cal composition of the fibre s in t hey were determined . In contrast, Perrott et the manvntllary cones also affect shell strength. al. (1981) found that, while the £ - axes of the The ami no acid content of the outer portion of crystallites were inc l ined at angles between 12° the outer membrane influences the s hell strength and 48° from the pe rpendicular to the s urface of eggs from young and ol d hens (Britton and within a crystal column, there was no evidence Hale 1977; Blake et a l. 1985). Methionine, from chemica ll y thinned specimens of a preferred seri ne, phenyl alanine and a spa rti c acid along ori entation among the columns i n the palisade with lysine (Britton and Hale , 1977) and proline layer as a whole. However , when specimens were (Blake et al., 1985) were found to influence t hinned with a n ion beam some preferred orienta­ s hell deformati on and egg specific gravity; tion within a single crysta l was observed, such de formation and specific gravity are two methods that the £-axis lay within a range of 30 ± 18° convnonly used t o estimate egg shel l streng t h from the perpendicular to the surfa ce. Sha r p (see Hami lton, 1982). On the other hand, Frank and Silyn-Robert s (1984 ) showed by X- ray diffrac ­ et al. (1965) reported t hat there was l ittle t ome try that a preferred orientat ion developed relati onship between t he ratio of t he membra ne s graduall y through ou t the shell of hen' s eggs, to total s hell and s hell breaking strength. beginning at the shell membranes and reac hing a maximum at the exterior surfa ce. Two of 20 Palisade/Shell Matri x a nd Vertical Crystal layers s hell s examined exhibited a si ngle preferred orientation in which the pole of the (001) plane The palisade layer begins at the point wa s perpend i c ular to the s hell' s s urface; where the individual co ne s from adjacent fo r ma ­ wherea s , a second preferred orientation, (104) tions fu se with eac h other in the manvni llary pl ane , occurred simultaneously with the (001) knob layer, and extends to within a bout 8 JJm p lane in the remain ing eggs. This second orien­ of the shell 's surface (Fig. lB ; Simons, 1971a). tation occurred only in the outer half of the These layers account for about two ~thi rds of the shell where the (104) pole was perpendi cular to overall thickness of the egg shell (Stewart , the surface. The data reported by Favejee et 1935; Burmes ter, 1940). Interwoven among and al. ( 1965 ) was inter preted by Sharp and Silyn­ through the crystals of the pali sade layer is a Roberts (1984) a lso to indicate the presence of matrix of organi c ma terial (Simons, 197 la ,b; c rystal s in two preferred orientations. In Pooley, 19 79) wh ic h Terepka (1963b) desc r i bed as another study by Si l yn- Roberts and Sharp (1985a) hav i ng a herr ing - bone appearance. The vertical found that the ( 001) preferred orientation was crystal, also called t he s urface c rys tal, layer characteristic of the s he l l s of eggs from ratite is deposited on top of the palisade layer; this and ttnamou s pecies of bi rds and the (104) orien­ layer varies in thi ck ne ss between 3 and 8 ~m . t ation appeared in the s he l l s of only a sma ll Remo val of the s hell membranes, by boiling in a proportion of the species and then only a s a sec-

103 R. M.G. Hamilton ondary development. This phenomenon was found same region Sharp and S i1 yn- Robe rt s ( 1984) found also to occur in the shells of reptilian eggs by the second preferred orientation in the crystal­ S i l yn - Robert s and Sharp ( l985b) . line structure started to develop in the majority Simon s (l91la,b) and Pooley (1919) observed of the shells they examined. Simons (197lb) that the calcite crystals in the vertical crystal observed , by electron microscopy, that fractures layer were small with their greatest dimension in the egg shell follow the layers of organic at approximately right angles to the s hell's material in the palisade layer; consequently, surface. Perrott et al. (1981) also found that the presence of both radial and tangential frac­ the preferred orientation of the crystals in the tures in his photomicrographs may be due to the vertical crystal layer was parallel to the influence of the organic matrix material on the surface. preferred orientation of crystals in the palisade Holes with diameters of about 0.4 ~m are layer. Both Simkiss and Tyler (1958) and Simons present in the decalcified preparations of the (197la) postulate that the deposition of organic true shell. These holes, known as vesticular material precedes that of the inorganic material. holes (Simons, 197la,b), are apparently more Frank. et al. (1965) found little evidence that numerous in the mammillary knobs than in the the amino acid content of the shell matrix influ­ palisade layer. Sometimes minute crystals lie ences she 11 break. i ng strength. close to the holes (Simons, 197la; Heyn, 1903) Terepka (1903b) suggested an inverse rela­ which are air filled spaces (Pooley, 1979). tionship between the concentration of shell The organic material that constitutes the matrix material and mineralization. Si mk.iss and matrix of the calcified portion of the shell Tyler (1958) hypothesiz~d that the shell matrix consists of a protein/polysaccharide complex acts as a chelating agent probably due to the (Simkiss and Tyler. 1958; Saker and Balch, 1962; mucoitin sulphuric acid, or hyaluronic acid as Cooke and Balch, 1970a,b; Heaney and Robinson, it is presently known. Abatangelo et al. (1978), 1976). This complex contains at least 70% pro­ however, demonstrated that the egg shell matrix tein and 11% polysaccharide along with a sma11 could bind calcium ions only if carboxylic side amount of fat (Baker and Balch, 1962). The chain groups were available. Subsequently, matrix protein is characterized by the absence Cortivo et al . (1982) isolated a peptide that of hydroxyproline (Baker and Balch, 1962; Frank was resistant to alkaline hydrolysis. About 50% et al., 1965). a low content of aromatic and of the amino acid residues in this peptide were sulphur amino acids and a ratio of about 2- to-1 aspartic and glutamic acids which were found to for dicarboxylic amino acids to basic amino acids be important in the binding of calcium ions. (Baker and Balch, 1962). frank et al. (1965) These researchers found no y - carboxyglutamic reported a moderate correlation (r ; 0.88) be ­ acid present in the shell matrix contrary to the tween the amino acid composition of the shell results of Krampitz et al. (1980). Blake and matrix and that of non -collagenous protein iso­ Kling (1984) demonstrated that iil vivo decarbox­ lated from porcine hyaline cartilage. Chondro­ ylation of y- carboxyglutamic acid had no signi­ itin sulphates A and B. and hyaluronic acid that ficant effect on shell strength when measured by contained equal molar amounts of glucosamine and specific gravity. galactosamine, were identified in the s hell Histochemical staining of the s hell matrix matrix by Baker and Balch (1902). Cooke and indicates the presence of the enzyme carbonic Bal ch (1970b) and Heaney and Robinson (1976), anhydrase (Robinson, 1970) which ha s subsequently respectively. Smal l amounts of sialic acid were been isolated (Krampitz, 1982). This enzyme reported by Frank et al. (1905) and Cooke and catalyzes the reversible reaction: Balch (1970b), but when the shell matrix material was analyzed directly, no measurable (l) sialic acid was found in the water soluble residue obtained from the matrix (Abatangelo et (Robinson and King, 1963) and is considered to al., 1978; and Cortivo et al., 1982). Other be involved in shell calcification of the hen's carbohydrates reported to be present in the egg {Krampitz and Witt, 1978). shell matrix include mannose, glucose, fucose In addition to calcium carbonate as calcite and xylose (Cooke and Balch , 1970b). in the true shell , phosphate and magnesium occur Cooke and Balch (1970b) demonstrated an in the outer part of the shell ( Itoh and Hatano, uneven distribution of the organic matrix mate­ 1964; Si mons, 1971a). Small amounts of potas­ rial within and among the crystalline material sium, sodium, iron, copper, manganese, sulphur of the shell. They found its concentration in­ and zinc are present in the shell but their creased to maximum in a region about two - thirds location and purpose is not known (Simons, of the distance from the inner membrane and then 197la). The magnesium content in the shell has rapidly decreased towards the shell's surface. been demonstrated to increase from the membranes These results do not support the postulate of to the shell's surface (Brooke and Hale, 1955; Tyler and Geake (1958) or the conclusion of Simons, 197la). However, Board and Love (1983) Carter (1909) that the organic content of the found, from electron probe microanalysis across "true shell" or •incremental shell", respec­ the radial axis of the egg shell, the occurrence tively, is constant. of a narrow band of magnesium- rich shell at· the It is interesting to note the decrease in marrrnillary knobs layer and, after an initi al the acid solubility of the calcified material in decrease, a progressive increase in magnesium the region of the true shell observed by Cooke concentration to a maximum at the outer surface and Balch (1970b) occurred in approximately the of the shell. According to the results of Smith

104 Egg Shell Microstructure

et al. (1954) most of the phosphate(s) present Balch, 1970a; Wedral et al., 1974), fucose (Baker in the shell was also located in the outer part. and Balch, 19&1), xylose (Wedral et al., 1974), and sialic acid (Baker and Balch, 1962; Wedral et al., 1974). Although galactosamine has been detected in cuticular material by Cooke and Balch Cuticle is the term used to identify the (1970a), the absence of uronic acid was consid­ layer of organic material that is deposited over ered to indicate that acid mucopolysaccharides the vertical crystal layer of the egg shell were not present. In addition to the presence (Simons, 197la,b); it is sometimes called the of sulphur and phosphorus (=2 and 0.1 mg/g, "bloom". The thickness of the cuticle was found respectively), Wedral et al. (1974) reported by Simons (197la) to vary between 0 .5 and 12.8 that the cuticle contains aluminium, boron, l-Im among eggs and at different sites on the copper, iron, magnesium, manganese, potassium, same egg. Many star-shaped crack. systems are sodium and zinc (0.01 - 4.0 ~g/g) and calcium apparent with scanning electron microscopy (• 15 "g/g). (Simons, 197la,b); these cracks in the cuticle Samet i mes the cut i c 1es of eggs from young contain vesicles up to about 1 JJm. Large broiler- type hens may be covered with a layer crack. systems tend to be oval in shape and have (:::- 45 ~m thick) of calcified material espe ­ clearly defined edges (Simons, 197la) ; they are cially if oviposition ha s been delayed due to considered to represent the surfaces of oval the presence of two eggs in the shell gland at pore plaques. Small crack. systems also occur in the same time (Simons, 1971a). The chemical the cuticular material on the surface of the composition of the "cover" material is similar egg's shell. The microscopic appearance of the to that in the palisade layer. cuticular material changes with time due possibly to drying and oxidation of sulphydryl ( - SH) and disulphide (-S-S-) groups present in the pro­ tein(s} of the cuticle. At the time the egg is Passing vertically through the palisade laid, the cuticle may be easily disrupted mechan­ layer of the shell from the "valleys" between ically, especially if it comes into contact with the ma11111illary knobs (Board et al ., 1977) to the the wire floor of a cage, or the c laws or beak. surface of the vertical crystal layer are of the hen (Denison, 1967; Tyler and Standen, funnel - shaped pores (Fig. lB; Tyler, 1956). 1969; Talbot and Tyler, 1974). The areas where These pores in the s hells of eggs from the cuticle has been disrupted are translucent domesticated hens are unbranched and capped by in appearance, contain more water and are weaker the organic material of the cuticle (Board et than the surrounding shell material (Tyler and al., 1977). Each pore vari'es in width from 15 Geak.e , 1964). The presence of translucent areas to 65 JJm at the surface to between 6 to 23 on the egg shell is known as mottling and is JJm at the inner level s of the shell (Tyler, more evident in eggs that have been stored 1956}. Scanning electron microscopy indi cated possibly because of shrinkage of the cuticle that the wall s of the pores are rough but without over time. a definite ultrastructure and contain no fibrous The cuticle of the shell is considered to be or crystalline material in their lumen (Tullett important in the waterproofing of the avian egg et al., 1975). Simkiss (19&1) found that the (Board and Halls, 1973). However, these re­ pores of eggs removed directly from the oviduct searchers found that of 453 brown shelled eggs were filled with sulphur- containing protein. examined, 3 . 5% had no demonstrable cuticle and The opening at the shell surface is plugged with another 8% had no cuticle at either the pointed cuticular material that contains cracks which or broad end of the egg. Belyavin and Boorman allows the diffusion of respi ratory gases. (1980) found that removal of the cuticle from The di s tribution of pores in the egg she ll the shell with EDTA significantly reduced their of the domestic hen is not random (Tyler, 1965; thickness , specific gravity and nondestructive 1969b). It is estimated that there are between deformation whi ch are factors associated 100 and 300 pores/cm2 of shell surface indirectly with shell strength. {Gilbert, 1971). There is a s trong positive Chemical analysis indicates that the cuticle linear correlation (r "' 0.92) between the number contains 85-90% protein (Wedral et al . • 1974; of pores/cm2 and mammillary knobs/0.25 rrrn2 Baker and Balch, 1962) most of which is insol ­ which Tullett (1975) speculated may be due to uble in water or potassium chloride solution (1% the distribution of cells i n the uterus that (w/v); Wedral et al., 1974), 4-5% carbohydrate secrete the sites on which calc ification is (Wedral et al. , 1974; Cooke and Balch, 1970a), subsequently initiated . Tyler and Fowler (1978) 1.5- 3.5% lipid (Wedral et al., 1974) and 3- 3.5% postulated that when three or more cones having ash (Wedral et al., 1974; Baker and Balch, 19&2). approximately circular cross sections meet at Based on paper or column chromatographic pro­ the beginning of the pali sade layer a "channel" cedures, Baker and Balch ( 1962) and Wedral et will be formed between them and this channel al. (1974), respectively, showed that aspartic might, under certain circumstances, continue and glutamic acids, glycine and arginine were through the palisade layer to form a pore. In the major amino acids in ttle proteinaceous mate­ addition, Tyler (1956) speculates that liquid rial of the cuticle. The major carbohydrate might pass through these "channels" of the moieties reported present in the shell cuticle forming s hell into the egg, but gradually some include galactose and mannose (Baker and Balch, of the channels become blocked by the developing 19&1; Wedral et al. 1974), glucose (Cooke and crystals until shell deposition is completed.

105 R.M .G. Hamilton

As a result, only a relatively small proportion Blake JP, Kling LJ. (1984). Examination of of the intitial channels would remain as pores the potential role of y- carboxyglutamic acid when the egg is 1aid. in eggshell quality. Poultry Sci. g, 981-986.

Future Research Blake JP, Kling LJ, Halteman WA. (1985). The relationship of the amino acid composition of a Even though the hen's egg shell is less than portion of the outer eggshell membrane to 400 ~m in thickness, it has a complex micro- eggshell quality. Poultry Sci.§!, 176-1B2. structure that consists of a 11 Composite• materi al. Further research is needed to estab- Board RG . (1982). Properties of avian egg lish the interrelationships among the inorganic shells and their adaptive value. Biol. Rev. and organic constituents that form this composite 11.. 1-28. material, the preferred orientation of the crys­ tals of the she ll and the strength of an egg's Board RG, Halls NA . (1973). The cuticle : shell, especially at the microstructure level. barrier to liquid and particle penetration of Composition of the organic matrh material the shell of the hen's egg. Brit . Poultry Sci . present in the palisade and vertical c rystal ll. 69-97. layers should be reinvestigated with modern analytical techniques. The mechanism involved Board RG, Love G. (1983). Magnesium distri- when the egg shell fractures due to the appli ­ bution in avian egg shells with particular cation of forces slowly or rapidly also requires reference to those of wild fowl (Anatidae). elucidation. Additional studies are required to Comp. Biochem. Physiol . 75A, lll - ll6. increase our understanding of the "biology of egg shell formations" so that it can be explained Board, RG, Tullett, SG, Perrott HR. (1977) . in terms of biochemical and physiological pro­ An arbitrary classification of the pore systems cesses. In order to resolve this problem, the in avian egg shells . J. Zool. (Lond.) ]J!£, expertise of such groups as electon microscop­ 251-265. ists, biochemists, material science engineers and poultry scientists are needed . Britton WM. (1977). Shell membranes of eggs differing in shell qua11ty from young and old Acknowl edqements hens. Poultry Sc i . i§., 647 - 653.

The author thanks A.F . Yang, Electron Britton WM, Hale Jr KK. (1977). Amino acid Mi croscopy Section, Chemistry and Biology analysis of shell membranes of eggs from young Research Institute, Agriculture Canada, Ottawa and old hens varying in ·shell quality . Poultry for the electron photomicrographs of egg Sci . ~. 865 -87 1. shells. The figures were prepared by Re search Program Services, Research Branch, Agri culture Brooke J, Hale HP . (1955). Strength of the Canada, Ottawa. shell of the hen's egg. Nature (Lond.) ill. B48-849.

Bunk MJ , Balloun SL. (1978). Ultrastructure Abatangelo G, Oaga-Gordini 0, Castellani I. of the marrmillary region of low puncture strength Cortivo R. (1978). Some observations on the avian eggshells. Poultry Sci. 21.· 639 - 647. calcium ion binding to the eggshell matrix. CalcH. Tiss. Res.£§., 247-252. Burmester BR. (1940). A study of the physical and chemica 1 chang es of the egg during its Baker JR, Balch DA. (1962). A study of the passage through the isthmus and uterus of the organic material of hen's egg shell. Biochem. hen's oviduct. J. £xp. Zool. ~. 445 -500. J . g, 352-361. Cain CJ, Heyn ANJ . (1964). X- ray diffraction Balch DA, Cooke A. (1970). A study of the studies of the crystalline structure of the composition of hen's eggshell membranes. Ann. avian egg shell. Biophys. J. 1_, 23-39. Biol. Anim. Biochim. Biophys. J.Q, 13- 25. Candlish JK, Scougall RK. (1969). L- 5- hydrox- Balch DA, Tyler C. (1964). Variation in some ylysine as a constituent or the shell membranes shell memb rane characteristics over different of the hen's egg. Int. J. Protein Res. parts of the same shell. Brit. Poultry Sci. l. 299-302. _j_, 201 - 215. Carter TC. (1969). The hen's egg: Relation­ Baumgartner S, Brown OJ, Salevshy Jr E. Leach ship between shell thickness and the amount of Jr RM. (197B) . Copper deficiency in the laying organic matter in the shell. Brit . Poultry Sci. hen . J. Nutr. lOB, 804-811. J.Q, 165-174.

Belyavin CG, Boorman KN . (1980). The Cooke AS, Balch DA. (1970a) . Stud ies of influence of the cuticle on egg- shell strength . membrane, manrnillary core and cuticle of the hen Br it. Poultry Sci. li. 295-298. egg shell. Brit . Poultry Sci. J.l, 345- 352.

106 Egg Shel 1 Microstructure

Cooke AS, Balch OA. (l970b). The distribution Harris 0, Blount JE, Leac h Jr M. (1980). and carbohydrate composition of the organic Localization of lysyl oxidase ln hen oviduct: matrix i n hen egg shell. Brit. Poultry Sci. Implications in egg shell membrane formation and ll. 353-365. compo sition. Science 208, 55-56.

Cortivo R, Castellani I. Martelli M, Ha si ak RJ, Vadehra DV, Baker RC. (1970a). Michelotto G, Abatangelo G. (1982). Chemical Fatty acid composition of the egg exterior characterization of the hen eggs he 11 matrix: structures of Gallus ill..!.!!i- Camp. Biochem. Isolation of an alkali-resistant peptide. J. Physiol. ;)2, 751-755. Chromatography ill. 127-135. Hasiak RJ, Vadehra DV, Baker RC. (l970b). Creger CR, Phillips H, Scott JT. (1976). Lipid composition of th(' egg exteriors of the Formation of an egg shell. Poultry Sci. chick (Gallus 9A..l.!.Y.i). Comp. Biochem. Physiol. _j2, 1111 - 1123. ;u.. 429-435.

Crombie G, Snider R, Faris B, Franzblau C. Heaney RK, Robinsofl OS. (1976). The isolation (1981). Lysine-derived cross-links in the egg and characterization of hyaluronic acid in egg shell membrane. Biochim. Biophys. Acta shell. Biochim. Biophys . Acta ffi, 133-142. 640. 365-36 7. Heyn ANJ. (1963). The crystalline structure Deni son JW. (19&7). The effect of mechanical of calcium c arbonate in the avian egg shel l . An disturbance of the egg cuticle in shell electron microscope study . J. Ultrastructure mottling. Poultry Sc i. i§., 771 - 772 . Res. ~. 176-188.

Ora per MH, Davidson MF, Wyburn GM, Johnston Hoffer AP. (1911). The ultrastructure and HW. (1972). The f ine structure of the isthmus cytochemistry of the shell membrane - secreting of the oviduct of Gallus domesticus. Quart. J. region of the Japanese quail oviduct. Amer. J. Exp. Physiol. _li, 297- 309. ---- Anat. ffi, 253-288.

Ea stin Jr WC, Spaziani E. (1978a). On the ltoh H. Hatano T. (1964). Variation of control of calcium secretion in the avian s hell magnesium and phosphorus deposition rates during gland (uterus). Biol. Reprod. li. 493-504. egg shell formation. Poultry Sc i. iJ, 77-BO.

Eastin Jr WC, Spaziani E. (1978b). On the Kaplan S, Siegesmund KA. (1973). The mechanism of calci um secretion in the avian s tructure of the chicken egg shell and shell shell gland (uterus). Biol. Reprod. li. 505 - 518. membranes as studied with the scanning electron microscope and energy dispersive X- ray El - Boushy AR, Simons PCM, Wiertz G. (1968). microanalysis. Poultry Sci. g, 1798-1801. Structure and ultra - s t r ucture of the hen's egg shell as influenced by environmental temperature, King NR, Robinson OS. (1972). The use of the humidity and vitamin C additions. Poultry Sci. scanning electron microscope for comparing the !I. 456- 467. structure of weak and strong egg s hells. J. Mi croscopy li. 437-443. Favejee JChl, van der Plas L, Schoorl R. (1965). X- ray diffraction of the crystalline Krampitz GP. (1982). Structure of the organic structure of the avian egg shell: Some critical matrix in mollusc shells and avian eggshells . remarks. Biophys. J. ~. 359 - 361. In: "Biological Mi nera lization and Oemineraliza­ tion11 Ed. by GH Nancollas. Springer- Verlag, NY, Frank FR, Burger RE, Swanson MH. (1965). The 219-232. relationships among shell membrane, selected chemical properties, and the resistance to s hell Krampitz G, Witt W. (1978). Biochemical failure of Gallus domesticus eggs. Poultry Sci. aspects of biomineralization. In: "Topi cs in 11.. 63 - 69. Current Chemistry" Ed. by MJ S Dewar, K Hafner, E Heilbronner, S Ito, JM Lehn, K Niedenzu, SW Geiger GS, Maclaury OW, Quigley GO, Rollins Rees, K Schafer, G Wittig. Springer- Verlag, NY, FO, Sc hano EA, Talmadge OW. (1974). Science 57-144. studies in poultry biology. Poultry Sci. g, 455-503. Krampitz G, Heisel H, Witt -Krause W. (1980). Identification of y-carboxyglutamic acid in Gilbert AB. (1971). The egg: Its physical and ovocalcium. Naturwissenschaften g, 38-39. chemical aspects. In: "Physiology and Biochemistry of the Fowl M Ed. by OJ Be 11, BM Leach Jr RM. {1982). Biochemistry of the Freeman. Academic Press, NY, vol 3, 1379- 1399. organic matrix of the eggshell. Poultry Sci . 61 • 2040- 204 7. Hamil ton RMG. (1982). Methods and factors that affect the measurement of egg shell Leach Jr RM, Gross JR. (1983). The effect of quality. Poultry Sci. §.1_, 2022-2039. manganese deficiency upon the ultrastructure of the eggshell. Poultry Sci. §..?., 499 -504.

107 R.M.G. Hamilton

Leach Jr RM, Rucker RB, Van Dyke GP. (1981). Schmidt WJ. (1962). Liegt der Eischalenkalk Egg shell membrane protein: A nonelastin der Vogel als submikroskopische Kristallite desmos i ne/i sodesmos i ne~c ontain i ng protein. vor? Z. Zellorsch. mikrosk.. Anat. .2.1.. 848- 880. Arch. Biochem. Biophys. f.!Jl. 353- 359. Schmidt WJ. (1964). Uber die Struktur einiger Makita T. (1981). X ~ ray microanalysis of the abnormer Voge l - Ei scha 1 en nebs t Bemerk.ungen zu avian shell gland and eggshell. Scanning neueren Auffassungen betreffend Bau und Bi ldung Electron Mi croscopy 1981; II: 473 ~ 480. der kalkschale. Z. Morph. Olcal. Tiere 21. 3ll-361. Masshoff W, Stolpmann HJ. (1961). licht ~ und elektronenmikropische Untersuchungen an der Schmidt WJ. (1965). Die Eisosphariten (Basal Schalenhaut und Kalkschale des Huhnereies. Z. k.alotten) der Schwanen-Eischale. Z. Zel1forsch. Zellforsch. mikrosk. Anat. i2_, 818 ~ 832. mikrosk. Anat. §l, 151-164.

Moran T, Hale HP. (1936). Physics of the Sha rp RM, Silyn-Roberts H. (1984). hen's egg. 1 . Membranes in the egg. J. Exp. Development of preferred orientation in the Biol. }]_, 35-40. eggshell of the domestic fowl. Biophys. J. ~. 175- 180. Parsons AH. (1982). Structure of the eggshell. Poultry Sci. Ql, 2013-2021. Silyn-Roberts H, Sharp RM. (1985a). Preferred orientation of calcite in the ratite Perrott HR, Scott VO, Board RG. (1981). and timarou eggshells. J. Zool. (Lond.) Crystal orientation in the shell of the domestic 205. 39 - 52. fowl: An electron diffraction study. Calcif. Tissue Int. Jl, 119- 124. Silyn-Roberts H, Sharp RM. (1985b). Preferred orientation of calcite and aragonite Polin 0. (1957). Formation of porphyrin from in the reptilian eggshells. Proc. Roy. Soc. delta -amino levulenic acid by uterine and liver Lond. 8 ££2, 445- 455. tissue from laying hens. Proc. Soc. Exp. Biol. Med. 21. 276-279. Simkiss K. (1961). Calcium metabolism and avian reproduction. Biol. Rev.~· 321 - 367. Pooley AS. (1979). Ultrastructural relationships of mineral and organic matter in Simkiss K. (1968). The structure and avian eggshells. Scanning Electron Microscopy formation of the shell and shell membranes. In: 1979; II: 475-482. ''Egg Quality: A Study of the Hen's Egg" Ed. by TC Carter. Oliver and Boyd, Edinburgh. Redshaw MR, Follett BK. (1972). The physiology of egg yolk production in the hen. Simkiss K, Taylor TG. (1971). Shell In: "Egg Formation and Production" Ed. by BM formation. In: "Physiology and Biochemistry of Freeman, PE Lake. British Poultry Science Ltd, the Domestic Fowl" Ed. by OJ Bell, BM Freeman. Edinburgh, 35 - 49. Academic Press, NY, vol 3, 1331 - 1343.

Reed JM, Yurkiewicz WJ. (1982). Avian egg Simk.iss K, Tyler C. (1957). A histochemical shell physiology: A review. Proc. Pens. Acad. study of the organic matrix of hen egg- shells. Sci. ~. 55-60. Quart. J. Microscop. Sci.~. 19-28.

Robinson OS. (1970). The structure of the Simkiss K, Tyler C. (1958). Reactions between mammillary layer of the domestic hens' egg- shell matrix and metallic cations. Quart. eggshell. Ann. Biol. Anim. Biochim. Biophys. J. Microscop. Sci. _21, 5-13. l_Q(Suppl. 1). 27 - 35. Simons PCM. (197la). Ultrastructure of the Robinson OS. (1972). Egg white glycoproteins hen eggs he 11 and its phys i o 1 ogi ca 1 ; nterpreta­ and physical properties of egg white. In: 11 Egg tion. Centre for Agricultural Publishing and Formation and Production" Ed. by BM Freeman, PE Documentation, Wageningen, The Netherlands . 90pp . Lake. British Poultry Science Ltd, Edinburgh, 65-86. Simons PCM. (l971b). Ultrastructure of the hen eggshell and its physiological Robinson OS, King NR. (1963). Carbonic interpretation. Figures. Centre for anhydrase and formation of the hen's egg shell Agricultural Publishing and Documentation, Nature ( lond.) ill. 497-499 . Wageningen, The Netherlands. 46 pp.

Robinson OS, King NR. (1968). Simons PCM, Wiertz G. (1963). Notes on the Mu copolysaccharides of an avian egg shell structure of membranes and shell in the hen 1 s membrane. J. Roy. Micros. Soc.§..!!, 13-22. egg. An electron microscopical study. Z. Zellforsc h. mik.rosk.. Anat. 2.2_, 555-567. Robinson OS , King NR. (1970). The structure of the organic mammillary cores in some weak egg shelh. Brit. PouHry Sci. 11. 39-44.

108 Egg Shell Microstructure

Smith AH, Wing et CM, Blackard JR. (1954) . The Tyler C. (1969b). The distribution of pores transfer of phosphorus to the hen's egg, under in the eggshells of the domestic fowl: A further controlled environment,as traced with radiophos ­ study. Brit. Poultry Sci. lQ, 375-3BO . phorus (p32). Poultry Sc i. li. 90B-919. Tyler C, Fowler S. (1978). The distribution Starcher BC, Ki ng GS. (19BO). The presence of of organic cores, cones, cone junctions and desmos i ne and i sodesmos i ne in eggs he 11 membrane pores in the egg shells of wild birds. J. Zool. protein. Connective Tissue Res. !!_, 53 - 55. (Lond.) JJ!§., 1-14.

Sternberger H, Mueller J, Leach Jr M. (1977) . Tyler C, Geake FH. (195B). Studies on egg Microscopic study of the initial stages of egg shells. IX. The influence of individuality, shell calcification. Poultry Sci. 2Q, 537-543. breed and season on certain characteristics of egg s hells from pullets. J. Sci. Food Agric. Stevenson IL . (1980). The removal of egg ~. 473-4B3. shell membranes by enzymatic treatment to facilitate the study of shell microstructure. Tyler C, Geake FH. (1964). The effect of Poultry Sci. 2i. 1959- 1960. water of egg shell strength including a study of the translucent areas of the shell. Brit. Stewart GF. (1935). The s tructure of the Po ultry Sc i .?_, 277 - 2B4 . hen's eggshell . Poultry Sci. l.i. 24-32. Tyler C, Geake FH. (1965). Egg shell Talbot CJ, Tyler C. (1974). A study of the thickness patterns as shown by i ndividual fundamenta 1 cause of art ific ia 1 trans 1ucent domestic hens. Brit . Poultry Sc i . §., 235-243. areas in egg shells. Brit. Poultry Sci. u.. 205 - 215. Tyler C, Standen N. (1969). The artificial production of translucent streaks on egg shells Terepka AR. (1963a). Structure and and various factors influencing their calc1fication in avian egg shell. Expt. Cell. development. Brit. Poultry Sci. lQ, 359 - 369. Res . ~. 171 - 1B2. Vadehra OV, Nath KP, Baker RC. (1971). Terepka AR. (1963b). Organic - inorganic Physi coc hemical properties of the proteins of interrelationships in avian egg shells. Ex pt. egg shell membranes. Poultry Sc i. iQ, 1638 Cell. Res.~. 1B3 - 192 . (Abstract).

Thompson BK, Hamilton RMG, Voisey PW. (19Bl). van Toledo 8, Parsons AH , Combs Jr Gf. Relationships among various egg traits related (1982). Role of ultrastructure in determining to shel1 strength among and within five avian eggshell strength. Poultry Sci. g , 569-572. species. Poultry Sci. QQ., 2388-2394. Warren DC, Conrad RM. ( 1942). Time of pigment Tullett SG . ( 1975). Regulation of avian deposition in brown shelled hen eggs and in eggshell porosity. J. Zoo 1. ( Lond . ) turkey eggs . Poultry Sci. n_ , 515-520. m. 339- 34B. Washburn KW. (1982) . Incidence, cause and Tullett SG, lutz PL, Board RG. (1975). The prevention of egg shell breakage in corrmercial fine structure of the pores in the s hell of the production. Poultry Sci. g, 2005 - 2012. hen's egg. Brit. Poultry Sci. 1.§., 93-95. Wedral EM, Vadehra DV, Baker RC. (1974). Tyler C. (1956). Studies on egg shells. VII. Chemical composition of the cuticle, and the Some aspects of structure as shown by plastic inner and outer shell membranes from eggs of models . J. Sci. Food Agric. 1.. 4B3 - 493. Gallus Yill..!:!.i- Comp. Biochem. Physiol. 11.!!_. 631 - 640. Tyler C. (1961). Studies on egg shells. XVI. Variation in shell thickness over different Wilbur KM, Simkiss K. (196B). Calcified parts of the same shell. J. Sci. Food Agric. shells. In: "Comprehensive Biochemistry., Ed. by J.l, 459 -470 . M Florkin, EH Stotz . Elsevier Publishing Co., NY, vol 26, ptA, 229-295. Tyler C. (1964). Wilhelm von Nathusius 1821 - 1899 on Avian Egg shells. The Berkshire Wong M, Hendrix MJC, von der Mark K, Little C, Printing Co . , Ltd, Reading. 104 pp. Stern R. (19B4). Collagen in the egg shell membranes of the hen . Develop . Biol. lQi, 28 - 36. Tyler C. (1965) . A s tudy of the egg s hells of t he Sphenisciformes. J. Zool. (Lond.) liL 1- 19. Discussion with Reviewers

Tyler C. (1969a). Avian egg shells: Their A.S. Pooley: Does the structure of the eggshell structure and characteristics. Int. Rev. Gen. have any i nfluence on the egg as a food material? Exp. Zool. 1. Bl - 130. Author: No, eggshell structure has no direct influence on the egg's co ntents. However, if

109 R. M. G. Hamilton thickness, a structural property of the shell, is considered, then shell structure may be considered to have an indirect effect because thin shelled eggs wi 11 be more susceptible to moisture loss and/or microbial contamination of their content during storage.

A.S. Pooley: Is anything that has been learned about eggshells in the past 30 years likely to lead to breeding selection that w11 1 provide increased resistance to breakage of eggs? Author: Yes, research on the measurement of eggshell strength has provided techniques that are routinely used in selection programs by the primary breeder to produce the corrrnercial stocks used for egg production. In addition, informa ­ tion from this research, such as cool eggshells are stronger than warm ones, has been discussed w1th egg producers for use in their operations.

11 0