- Email: [email protected]
Ultrastructure of The Mammillary Region of Low Puncture Strength Avian Eggshells1
Ultrastructure of The Mammillary Region of Low Puncture Strength Avian Eggshells1 MICHAEL J. BUNK2 and S. L. BALLOUN Department of Animal Science, Iowa State University, Ames, Iowa 50011 (Received for publication July 20, 1977)
INTRODUCTION T h e t e r m "egg shell q u a l i t y " has been expressed by a n u m b e r of p a r a m e t e r s in r e c e n t years. Loss in dollars per year t o t h e p o u l t r y industry has been estimated, and physical measurements such as shell thickness, specific gravity, and breaking or p u n c t u r e strengths have been the m o s t c o m m o n l y r e p o r t e d techniques. Although general egg shell m o r p h o l o g y has been studied for m a n y years, t h e precise developmental stages of shell f o r m a t i o n , along with a t t e m p t s t o correlate t h e u l t r a s t r u c t u r e of t h e egg shell with practical p r o d u c t i o n parameters, have escaped detailed research. Because calcified structures are i n c o m p a t i b l e with advanced microscopic techniques developed for softer biological tissues.
1 Journal Paper No. J-8842 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa. Project 2240. 2 Department of Poultry Science and Division of Nutritonal Sciences, Cornell University, Ithaca, New York 14853. 3 Quasi-static loading technique for Instron Instrument.
1978 Poultry Sci 57:639-647
General egg shell ultrastructure has been reviewed b y Simons ( 1 9 7 1 ) . Research concerning egg shell quality, however, has primarily been concerned with t h e rate of deposition of calcium salts o n t o shell m e m b r a n e fibers t o form a thick high-quality shell. An alternative approach is t o p o s t u l a t e t h a t shell strength is affected b y t h e organized formation of crystal (palisade c o l u m n ) lattices as t h e shell is calcified. According t o this h y p o t h e s i s , t w o egg shells of equal thickness and calcium c o n t e n t , varying only in structural organization, w o u l d be e x p e c t e d t o display different levels of intrinsic strength and different shell quality. Preliminary observations (King and R o b i n son, 1 9 7 2 ; Bunk, 1977) have indicated t h a t t h e basal m a m m i l l a r y region of t h e shell is freq u e n t l y altered in low-quality shells. Meyer et al. ( 1 9 7 3 ) , however, concluded t h a t m o s t of t h e changes responsible for differences in shell breaking (Instron m e t h o d 3 ) strength seemed t o take place in t h e palisade layer, while t h e mammillary layer appeared unaffected. ElBoushey et al. ( 1 9 6 8 ) observed irregular, loose and basally r o u n d e d mammillae in egg shells p r o d u c e d b y hens subjected t o e n v i r o n m e n t a l physical stress. McFarland et al. ( 1 9 7 1 ) later
639
Downloaded from http://ps.oxfordjournals.org/ at University of Pennsylvania Library on March 4, 2015
ABSTRACT The mammillary region of poor quality avian egg shells displays a number of aberrant structural alterations not commonly observed in high quality egg shells. Three general categories of mammillary alterations were observed in radial sections of extremely low quality shells: 1) Shells with a proliferation of discrete rounded calcified bodies situated upon the shell membrane fibers; 2) shells with an extremely disorganized, multinucleate mammillary region without numerous calcified bodies; and 3) mammillary knobs possessing cavernous tips without mammillary core formations. Adhesion discs (fracture surfaces between adjacent calcified structures) were observed and classified into two types, small-rounded (Type I) adhesion discs between adjacent calcified bodies, or between calcified bodies and larger mammillary formations; and large oval (Type II) adhesion discs between adjacent mammillary knob formations. The nucleation zone or external surface of the outer shell membrane fibers, upon which calcium salts initially precipitate, was also observed to vary in low-quality shells. Waves or bulges in the nucleation zone were observed directly beneath the multinucleate unorganized mammillary knob formations. These findings suggest that the initial process of shell mineralization (mammillary knob formation) is responsible for the base upon which the palisade (main shell) layer is deposited. Disorganization of the basal calcified region may give rise to poorly indigitating calcite (palisade) columns and decreased intrinsic shell strength.
640
BUNK AND BALLOUN
reported t h a t C o t u r n i x quail dosed with D D T laid eggs with alterations in mineral deposition and increased porosity of t h e mammillary layer. These latter observations suggest t h a t factors o t h e r t h a n the density and thickness of t h e palisade may also be d e t e r m i n a n t s of shell strength. MATERIALS AND METHODS
'Sputter coated (Hummer II, Techniques, Inc.). 5 JOEI HSM-35 (13 KvSOMA).
Each egg was placed o n t o t h e scale prongs, blunt end directed t o w a r d s the right of the scale, and highest elevation of t h e shell e q u a t o r directly u n d e r the needle tip (Fig. 2). The average value of four separate p u n c t u r e s , conducted along t h e e q u a t o r of each eggshell, was recorded. Regions of shell i n t e n d e d for microscopic e x a m i n a t i o n were r e p u n c t u r e d at 1 cm intervals along t h e e q u a t o r , and areas b e t w e e n t w o identical p u n c t u r e scores were marked for examination. Shells then were fractured through m a r k e d areas, c o n t e n t s were rinsed away with distilled water, and shells were processed for microscopic analysis. Microscopic Preparation. Sections of rinsed egg shell (.5 cm X .5 cm) with m e m b r a n e s attached were separated from the e q u a t o r of t h e shell half at r o o m t e m p e r a t u r e , air-dried, cemented to brass microscope stubs, gold coated 4 , stored in a dessicator, and examined with a scanning electron microscope .
FIG. 1. Operation of the shell-puncture meter to quantitate intrinsic eggshell strength. FIG. 2. Eggshell punctured in two locations and marked for microscopic analysis. FIG. 3. Ultrastructural evaluation of a shell puncture location. A crater-like surface depression was observed with subsurface fracture planes extending obliquely toward the shell membranes. The mammillary knob formations beneath the needle impact point, however, did not appear extensively damaged and rested upon the intact shell membranes. (75X).
Downloaded from http://ps.oxfordjournals.org/ at University of Pennsylvania Library on March 4, 2015
Shell Quality Determinations. G r o u p s of 1 0 0 eggs were collected at midday within several h o u r s of oviposition from Single C o m b Leghorn (SCWL) hens housed in wire layer pens, 2 birds per cage, with water and standard corn-soybean laying ration available ad lib. Shell strength was measured by a calibrated shell p u n c t u r e device according to t h e m e t h o d of M u n r o ( 1 9 6 6 ) (Fig.
1).
641
EGG SHELL ULTRASTRUCTURE Freeze fracture surfaces were prepared according to the method of Bunk and Balloun (1977). RESULTS
6 Charging is the loss of SEM final image quality due to accumulation of loose unconducting material.
Puncture strength 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31
No. 2 1 1 3 3 11 13 14 17 14 3 7 2 2 2 4 1
crete, rounded bodies situated upon membrane fibers of the shells was the first type of alteration observed (Fig. 4—7). Various sizes and degrees of unification were observed in membrane bodies of poor quality egg shells. The term "intermammillary adhesion disc" (Fig. 5) has been employed to describe round or oval adhesion surfaces between adjacent membrane bodies or between membrane bodies and larger shell formations. Adhesion discs seemed to be areas where the membrane bodies enlarged and coalesced into a single structure. Several discs (AD) are shown in Figs. 5 to 7. The membrane body in Fig. 7 seemed to have been attached to the larger shell formation via the adhesion disc but separated from the adjacent surface when the shell was fractured for analysis. The membrane body was more firmly attached to the membrane fibers than to the adjacent calcified structure because it retained the membrane connection after the fracture occurred. Two types of adhesion discs were noted. The first type (Type I), already mentioned, occurred between membrane bodies close to the shell membranes. A second type (Type II) of adhesion disc (arrow, Fig. 4) occurred higher, more externally, upon the radial surface. These discs were larger, more oval, and seemed to be surfaces where adjacent mammillary knobs united during the initial stages of palisade layer
Downloaded from http://ps.oxfordjournals.org/ at University of Pennsylvania Library on March 4, 2015
Shell Quality Determination. A radial shell section gauged for shell quality is shown in Fig. 3. The shell was fractured directly through a puncture depression, and the arrow (right side of micrograph) points towards the cuticular (other) shell surface. Effect upon the shell was noted as a round crater-like surface depression with subsurface fracture planes extending laterally from the point of impact. The mammillary region and internal portion of the palisade layer, beneath the puncture depression, rested upon the shell membranes and were separated from the outer regions of the shell resulting in an incomplete puncture of the shell. Mammillary knobs and membrane fibers beneath the point of impact did not appear extensively damaged. Severe pulverization of calcite from the point of needle impact was observed as white=charged areas in the outer regions of the palisade layer. The distribution of puncture scores for 100 randomly selected egg shells is shown in Table 1. Similar distributions were obtained in repeated trials with approximately 7 to 12% of the shells per trial displaying considerably reduced shell puncture strengths. For this study, the shells were arbitrarily classified as low (14 to 20 oz), average (21 to 25 oz) and above-average quality (25 to 32 oz). Ultrastructural Evaluation. Shells with average and above average quality measurements resembled specimens shown by previous authors (Simons, 1971; McFarland et ai, 1971). Egg shells with extremely low puncture scores, however, appeared grossly inferior with shell checks and/or surface irregularities. Microscopically, extreme disorganization and conformational alterations were noted in the mammillary region. Sections of these shells possessed few uniform conical mammillary knobs as described in previous reports (Romanoff and Romanoff, 1949). The structural alterations observed in mammillary regions of poor-quality shells were classified into three general categories. Membrane bodies, a proliferation of small, dis-
TABLE 1 .—Puncture strength in ounces of 100 randomly selected Single Comb White Leghorn egg shells
642
BUNK AND BALLOUN
;
•-.'..
'
f j j w
3:
FIG. 4. Mammillary region of poor-quality eggshell. The shell membrane layers, while thin, retained a physical distinction between inner and outer membrane fiber groups. Areas of these shells did not possess well formed, rounded mammillary formations, but instead showed numerous nucleation sites and incompletely calcified structures. The term membrane body has been employed to describe small round incompletely calcified shell components attached to the outer shell membrane fibers. Areas of the palisade region (right side of micrograph) were observed without direct membrane connections, as seen in higher quality eggshells. (250X). FIG. 5. Membrane bodies and type 1 adhesion discs of poor-quality eggshells. An enlargement of Fig. 4 (arrow) shows a membrane body (Mb) attached to outer shell membrane fibers (osm) and several adhesion discs (Ad) where membrane bodies were formerly attached. Larger type II adhesion discs are shown at the upper left of arrow in Figure 4. (1000X ). FIG. 6. Radial shell surface of poor-quality eggshell showing larger, more irregular membrane bodies. Two layers of membrane bodies are observed beneath the palisade layer. The surface of the primary (lower) membrane bodies served as the nucleation site for secondary layer of membrane bodies which then enlarged into the base of the palisade region. (250X). FIG. 7. Enlargement of base of "more normal appearing" mammillary knob formation of poor quality eggshell. At low magnification, adjacent areas appeared to have large, broad mammillary formations similar to regions observed in average and higher quality shells. However, upon closer examination, these mammillary knobs appeared to be composed of membrane bodies that had fused "more completely" into a single structure. This micrograph shows a more complete fusion of membrane bodies with a fusion line (Fl) between adjacent nucleation sites, and a singular membrane body with adhesion (fracture) surface to the larger calcified region. Note that the membrane body (Mb) was more firmly attached to the outer shell membrane fibers than to adjacent calcified structures, and separated upon fracture. (1160X).
formation. Large mammillary k n o b formations of lowquality shells (Fig. 7) seemed to be t h e result of i n c o m p l e t e fusion of several m e m b r a n e bodies. T h e fusion line ( F L , Fig. 7) was considered to be a d e m a r c a t i o n b e t w e e n two adjacent m e m b r a n e bodies. As such, t h e first t y p e of struc-
tural alteration was considered t o b e d u e to small r o u n d e d discrete bodies t h a t originated uniformly across t h e outer shell m e m b r a n e surface and resulted in an unorganized base for the mammillary and palisade layers. T h e second general t y p e of alteration observed in poor-quality shells was t h a t of an
Downloaded from http://ps.oxfordjournals.org/ at University of Pennsylvania Library on March 4, 2015
'
EGG SHELL ULTRASTRUCTURE
show various states of multinucleation, and Figures 9 and 11 also s h o w a minimal k n o b to m e m b r a n e relationship n o t observed in higher quality shells. Freeze-fractured surfaces revealed no mammillary core, central channel, or core space described in previous reports (Bunk a n d B a l l o u n , 1977). A third general t y p e of deformation observed was caverns or cavities at t h e tip of large, more uniform mammillary k n o b formations (Figures 12—15). No singular site of crystal initiation was obvious, and the internal (lumenal) surfaces of tip cavities (Fig. 13) seemed similar in crystalline nature to the exterior k n o b surface. Also, the e x t e n t to which m e m brane fibers penetrated the k n o b s was decreased from that in average and above average quality shells. Freeze-fractured surfaces (Figs. 14—15) showed no mammillary core s t r u c t u r e .
FIG. 8. Large multinucleated mammillary region of low-quality eggshell. A wave or bulge in the nucleation zone (plane) was observed, and mammillary formations (knobs) were composed of crystal columns originating from numerous nucleation sites. (390X). FIG. 9. Enlargement of multinucleated mammillary formation from Fig. 8. A minimal shell membrane-calcite relationship was observed. (970X). FIG. 10. Extensive multinucleated mammillary formation. (460X). FIG. 11. Mammillary formation originating from two nucleation sites with a minimal calcite-shell membrane fiber relationship. (540X).
Downloaded from http://ps.oxfordjournals.org/ at University of Pennsylvania Library on March 4, 2015
extremely disorganized, multinucleated m a m millary region (Figs. 8—11). Calcified m e m brane bodies as described previously were n o t observed in these regions, b u t w h a t could be considered a single mammillary k n o b of an average quality shell seemed to be the result of several i n d e p e n d e n t , partly fused calcite colu m n s originating from several locations and projecting in several directions. In addition, a wave or bulge in the nucleation z o n e existed such t h a t all t h e mammillary k n o b s did n o t originate from a single uniform plane or surface. For clarification, the nucleation zone refers to the outer surface of the outer shell m e m b r a n e layer. On the right side of F'ig. 8, a r o u n d e d structure (b) was observed t h a t had n o direct m e m b r a n e c o n n e c t i o n b u t enlarged to form a base of t h e palisade layer. Figures 10 and 11
643
BUNK AND BALLOUN
644
•-
wL
"• 2jS^B
f . . i--.
-^
M
.m .
DISCUSSION An i m p o r t a n t biological consideration lies in the c o n c e p t t h a t the cell is a basic u n i t of s t r u c t u r e and function. In an analogous phrase, t h e mammillary k n o b and palisade c o l u m n t h a t originate from t h a t k n o b are the basic u n i t of s t r u c t u r e and function of the avian eggshell. Eggshell research, however, has tended to ignore any idea of a cellular or unitary approach to shell strength, and has focused primarily u p o n the idea t h a t a firm thick shell results solely from a d e q u a t e calcium deposition during t h e formation of the palisade layer. T h e d e f o r m a t i o n s categorized here have s h o w n t h a t the strength of the p o o r quality shell lies also in t h e organization of t h e primary calcified structures (mammillary knobs) rather t h a n
strictly in t h e thickness of t h e palisade region. T h e area of t h e shell to m e m b r a n e relationship especially seems critical, and a large (7 to 10%) portion of p r o d u c t i o n losses could actually be due to small changes in the c o m p o s i t i o n of t h e outer shell m e m b r a n e fibers or alterations in factors causing t h e initial precipitation of calcium salts, rather than only to limitation of substrate (calcite) during palisade f o r m a t i o n . It has been observed t h a t a d e q u a t e calcium, palisade thickness, is present in low quality shells (Figs. 4 and 6) b u t t h a t its c o n f o r m a t i o n is unorganized and in need of further study. T h e areas of unorganized, multinucleated m a m millary k n o b s indicate that the shell m e m b r a n e fibers initially b e c o m e over-precipitated with calcium salts and t h a t t h e n u m e r o u s m e m b r a n e
Downloaded from http://ps.oxfordjournals.org/ at University of Pennsylvania Library on March 4, 2015
MHHHHHBSNK 1
FIG. 12. Cavernous tip mammillary knob. Membrane fiber penetration of knob tip is decreased, and a fusion line extends from the right side of the cavern opening. (1 590X ). FIG. 13. Interior or lumen of cavern shown in Fig. 12. A lack of sharp discrete crystal edge surfaces is observed, while no organized primary mammillary radial crystals are noted. (1 590X ). FIG. 14. Fractured cavernous mammillary knob. Note the fusion line that originates at the mid-dorsal cavern surface and extends toward the upper left of the micrograph. The adhesion surfaces between this mammillae and adjacent structures lacks definition, and occurs in spots rather than unifrom surfaces. (1380X). FIG. 15. Enlargement of the mammillary tip shown in Fig. 14. Note the vesiculated heterogeneous nature of the membrane fiber-knob tip interface. The lumen of the cavern appears to possess a definite "organic coating" and a fusion line originates from the dorsal aspect of die cavern (2770X).
EGG SHELL ULTRASTRUCTURE bodies d o n o t enlarge with enough breadth t o form a wide s t u r d y base for t h e palisade columns. Palisade columns of p o o r quality shells arising from u n e q u a l and (or) unparallel m a m millary k n o b formations then give rise to poorly interlocking calcite columns. It has been shown in Figure 7 t h a t the a t t a c h m e n t of the m e m b r a n e (mammillary core) is stronger t h a n the a t t a c h m e n t between adjacent calcified structures.
decreased degree of interdigitation and less intrinsic strength (see Fig. 19). Although t h e thickness of the palisade c o l u m n has been q u a n t i t a t e d , t h e degree of column interdigitation can n o w only be speculated. In s u p p o r t of an idea or t h e o r y of interdigitation are Figures 16 to 18. Here multinucleated mammillary k n o b s were observed (Fig. 16 shows b o t h a cavernous tip and m e m b r a n e b o d y ) and a striking fact was t h e observable cracks or micro-checks seen at t h e ultrastructural level. In Figures 17 and 18, t h e multinucleated k n o b itself displays intrinsic fissures or micro-checks. A fracture line was observed to start at the m e m b r a n e level between individual nucleation sites and e x t e n d externally t o w a r d s t h e palisade layer. A second fissure was also observed b e t w e e n this k n o b and the adjacent mammillary f o r m a t i o n (right side
FIG. 16. Intermammillary fissure; note membrane body (mb) and cavernous tip (ct) in adjacent mammillary regions. The fissure or microcheck extended peripherally towards the palisade region. (760X). FIG. 17, 18. Intramammillary fissure (a) and intermammillary fissure (b) are observed in a multinucleated mammillary knob. Note the adjacent mammillary knob with apparent cavernous tip (Fig. 17). The intramammillary fissure (a, Fig. 18) originated at the outer membrane fiber level and extended peripherally towards the palisade layer. The fracture line appeared to result from decreased interdigitation or cohesive strength of primary radical crystal growth of the mammillary knob. (Fig. 17, 880X ; Fig. 18, 1630X).
Downloaded from http://ps.oxfordjournals.org/ at University of Pennsylvania Library on March 4, 2015
Very little consideration has been directed t o w a r d s t h e exact relationship of adjacent palisade columns, and the degree t o which individual calcite c o l u m n s interlock (interdigitation) m a y be t h e intrinsic weakness to the physical c o n c e p t of eggshell strength. T w o adjacent columns originating from different levels (waves in the nucleation plane) unparallel t o the m e m b r a n e could be e x p e c t e d to show a
645
646
BUNK AND BALLOUN
of micrograph) and also extended toward the palisade. One could speculate that these areas are the actual weak spots of an eggshell, and responsible for the cracks and (or) checks observed after processing. These areas seem to be the result of altered mammillary knob synthesis, and would require the least force or pressure to expand, and serve as the initiation of complete cracks through the palisade layer. Correlated to the extent of interdigitation is the degree and distribution of shell organic matrix. Tightly fused crystal columns would possess a reduced quantity of trapped organic material chiefly from the oviducal fluids. Conversely, shells enlarging with disorganized adjacent crystal columns would entrap a greater amount of oviducal fluids which would then precipitate as the shell dehydrated after oviposition. Simons (1971) observed a 60% increase in the number of vesicular (pit-like) holes per unit of surface area in poor quality shells of equal thickness but widely different breaking strengths. It was also noted (Simons, 1971) that the structure of the decalcified palisade layers of weak shells resembled the outer part of the cone (mammillary) layer of normal shells rather than that of the normal palisade layer. This suggests that the initial process of mammillary knob formation plays a greater role in shell strength than previously acknowledged. The mammillary region has been reported to contain a greater
Additional investigations lie chiefly in developing specific techniques that result in a direct correlation between eggshell ultrastructure and quantitative shell quality measurements. This investigation has considered a single method of gauging shell quality (needle puncture) for a number of reasons. First, shell and membrane properties are noted to vary over regions of a shell and over portions of the shell equator (Romanoff and Romanoff, 1949; Balch and Tyler, 1964). Adjacent points as close as .5 cm could be independently gauged for shell strength and directly identified with the microscope. Methods that result in one numerical value per egg (Instron, Bomber, or specificgravity) were considered to be at a definite disadvantage, since only crude approximations of ultrastructure of a quadrant of shell circumference could be estimated. The needle puncture method also was considered because it eliminated soaking and major crushing distortions created during specific gravity or Instron determination.
Downloaded from http://ps.oxfordjournals.org/ at University of Pennsylvania Library on March 4, 2015
FIG. 19. Diagramatic representation of the concept of interdigitation of crystal columns in avian eggshells. A represents waves or irregularities in the nucleation zone (plane), and numerous non-uniform nucleation sites on the outer shell membranes. These then give rise to extremely disorganized, poorly interdigitating cystal columns in the palisade layer, and increased quantities of organic material (oviducal fluids and cellular debris) become entrapped between adjacent columns. While shell thickness and total organic content may appear similar, the even uniform distribution and increased interlocking arrangement in B would be indicative of an eggshell with higher intrinsic strength and better shell quality.
quantity of organic material (Simkiss, 1968), and the observation of an increased organic content of palisade region of weak eggshells indicates an alteration in the growth of palisade crystal columns allowing a greater degree of fluid entrapment. The initial crystals of the mammillae appear somewhat disorganized in freeze-fracture surfaces of normal shell surfaces (Bunk and Balloun, 1977), and organic shell material may simply be a function of space filling or packing between adjacent crystal surfaces rather than a supportive framework in shell formation. To help in determining the underlying reasons for malformed and disorganized mammillary regions, factors such as the amino acid patterns or trace element relationships of the outer shell membrane fibers are in need of further study in more precise detail than previously accomplished. In unpublished studies, we have observed a flattening and (or) increased "knottiness" of the outer shell membrane fibers of poor quality shells. Although their results are unexplained at present, Masshoff and Stolpmann (1961) observed membrane fibers to be consisting of a core of keratin fibrils embedded within a matrix, surrounded by a less electron dense mantle layer. If calcium salts precipitate onto the outer mantle surface of the membrane fibers, then the surface structure or composition of this mantle layer becomes an important biological interface.
EGG SHELL ULTRASTRUCTURE ACKNOWLEDGMENT Deep appreciation is expressed t o Dr. H. T. Horner, Jr., I.S.U. D e p a r t m e n t of B o t a n y a n d Plant Pathology, for m a n y hours of personal instruction to t h e senior a u t h o r , and for use of microscope facilities. Also, we wish t o a c k n o w ledge m e m b e r s of t h e I.S.U. P o u l t r y Section (Drs. Wm. J. Owings, A. Nordskog, R. J. Hasiak, a n d Prof. L. Z. Eggleton) for help a n d consultation during t h e senior a u t h o r ' s Master's program while at Iowa S t a t e .
Balch, D. A., and C. Tyler, 1964. Variation in some shell membrane characteristics over different parts of the same shell. Br. Poultry Sci. 5:201-215. Bunk, M. J., 1977. An ultrastructural evaluation of avian eggshell quality. M.S. Thesis, Iowa State University, Ames, Iowa. Bunk, M. J., and S. L. Balloun, 1977. Structure and relationship of the mammillary core to membrane fibers and initial calcification of the avian eggshell. Br. Poultry Sci. 1 8 : 6 1 7 - 6 2 1 . El-Boushy, A. R., P. C. M. Simons, and G. Wiertz, 1968. Structure and ultrastructure of the hen's egg shell as influenced by environmental temperature, humidity, and vitamin C additions. Poult. Sci. 47:456-467. King, N. R., and D. S. Robinson, 1972. The use of the
scanning electron microscope for comparing the structure of weak and strong egg shells. J. Microscop. 9 5 : 4 3 7 - 4 4 3 . McFarland, L. Z., R. L. Garrett, and J. A. Nowell, 1971. Normal eggshells and thin eggshells caused by organochlorine insecticides viewed by the scanning electron microscopy. Scanning Electron Microscopy. Proc. 4th Ann. Scanning Electron Microscope Symp. (I.I.T. Research Institute, Chicago, IL 60616, USA) 4:377-384. Masshoff, W., and H. J. Stolpmann, 1961. Licht- und elektronemiktroskopische Untersuchungen an der Schalenhaut und Kalkschale des Huhnereis. Z. Zellforsch. Mikrosk. Anat. 55:818-832. Meyer, R., R. C. Baker, and M. L. Scott, 1973. Effects of hen egg shell and other calcium sources upon egg shell strength and ultrastructure. Poultry Sci. 52:949-955. Munro, S. S., 1966. 1. Studies on needle puncture as a method of measuring shell strength. 1. Development of the method, effect of needle shape, variation between areas of the shell, and differences between operators. Hy-Line Res. Rep. II (III-A-I): 1 5 - 2 3 . Romanoff, A. L., and A. J. Romanoff, 1949. The avian egg. Wiley, New York. Simkiss, K., 1968. The structure and formation of the shell and shell membranes. Vol. 4. In Egg,quality: A study of the hen's egg. T. C. Carter, ed., Oliver and Boyd, Edinburgh, pp. 3—25. Simons, P. C. M., 1971. Ultrastructure of the hen eggshell and its physiological interpretation. Commun. No. 175 Cent. Inst. Poultry Res. "Het Spelderholt" Beekbergen, The Netherlands.
Downloaded from http://ps.oxfordjournals.org/ at University of Pennsylvania Library on March 4, 2015
REFERENCES
647
INTRODUCTION T h e t e r m "egg shell q u a l i t y " has been expressed by a n u m b e r of p a r a m e t e r s in r e c e n t years. Loss in dollars per year t o t h e p o u l t r y industry has been estimated, and physical measurements such as shell thickness, specific gravity, and breaking or p u n c t u r e strengths have been the m o s t c o m m o n l y r e p o r t e d techniques. Although general egg shell m o r p h o l o g y has been studied for m a n y years, t h e precise developmental stages of shell f o r m a t i o n , along with a t t e m p t s t o correlate t h e u l t r a s t r u c t u r e of t h e egg shell with practical p r o d u c t i o n parameters, have escaped detailed research. Because calcified structures are i n c o m p a t i b l e with advanced microscopic techniques developed for softer biological tissues.
1 Journal Paper No. J-8842 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa. Project 2240. 2 Department of Poultry Science and Division of Nutritonal Sciences, Cornell University, Ithaca, New York 14853. 3 Quasi-static loading technique for Instron Instrument.
1978 Poultry Sci 57:639-647
General egg shell ultrastructure has been reviewed b y Simons ( 1 9 7 1 ) . Research concerning egg shell quality, however, has primarily been concerned with t h e rate of deposition of calcium salts o n t o shell m e m b r a n e fibers t o form a thick high-quality shell. An alternative approach is t o p o s t u l a t e t h a t shell strength is affected b y t h e organized formation of crystal (palisade c o l u m n ) lattices as t h e shell is calcified. According t o this h y p o t h e s i s , t w o egg shells of equal thickness and calcium c o n t e n t , varying only in structural organization, w o u l d be e x p e c t e d t o display different levels of intrinsic strength and different shell quality. Preliminary observations (King and R o b i n son, 1 9 7 2 ; Bunk, 1977) have indicated t h a t t h e basal m a m m i l l a r y region of t h e shell is freq u e n t l y altered in low-quality shells. Meyer et al. ( 1 9 7 3 ) , however, concluded t h a t m o s t of t h e changes responsible for differences in shell breaking (Instron m e t h o d 3 ) strength seemed t o take place in t h e palisade layer, while t h e mammillary layer appeared unaffected. ElBoushey et al. ( 1 9 6 8 ) observed irregular, loose and basally r o u n d e d mammillae in egg shells p r o d u c e d b y hens subjected t o e n v i r o n m e n t a l physical stress. McFarland et al. ( 1 9 7 1 ) later
639
Downloaded from http://ps.oxfordjournals.org/ at University of Pennsylvania Library on March 4, 2015
ABSTRACT The mammillary region of poor quality avian egg shells displays a number of aberrant structural alterations not commonly observed in high quality egg shells. Three general categories of mammillary alterations were observed in radial sections of extremely low quality shells: 1) Shells with a proliferation of discrete rounded calcified bodies situated upon the shell membrane fibers; 2) shells with an extremely disorganized, multinucleate mammillary region without numerous calcified bodies; and 3) mammillary knobs possessing cavernous tips without mammillary core formations. Adhesion discs (fracture surfaces between adjacent calcified structures) were observed and classified into two types, small-rounded (Type I) adhesion discs between adjacent calcified bodies, or between calcified bodies and larger mammillary formations; and large oval (Type II) adhesion discs between adjacent mammillary knob formations. The nucleation zone or external surface of the outer shell membrane fibers, upon which calcium salts initially precipitate, was also observed to vary in low-quality shells. Waves or bulges in the nucleation zone were observed directly beneath the multinucleate unorganized mammillary knob formations. These findings suggest that the initial process of shell mineralization (mammillary knob formation) is responsible for the base upon which the palisade (main shell) layer is deposited. Disorganization of the basal calcified region may give rise to poorly indigitating calcite (palisade) columns and decreased intrinsic shell strength.
640
BUNK AND BALLOUN
reported t h a t C o t u r n i x quail dosed with D D T laid eggs with alterations in mineral deposition and increased porosity of t h e mammillary layer. These latter observations suggest t h a t factors o t h e r t h a n the density and thickness of t h e palisade may also be d e t e r m i n a n t s of shell strength. MATERIALS AND METHODS
'Sputter coated (Hummer II, Techniques, Inc.). 5 JOEI HSM-35 (13 KvSOMA).
Each egg was placed o n t o t h e scale prongs, blunt end directed t o w a r d s the right of the scale, and highest elevation of t h e shell e q u a t o r directly u n d e r the needle tip (Fig. 2). The average value of four separate p u n c t u r e s , conducted along t h e e q u a t o r of each eggshell, was recorded. Regions of shell i n t e n d e d for microscopic e x a m i n a t i o n were r e p u n c t u r e d at 1 cm intervals along t h e e q u a t o r , and areas b e t w e e n t w o identical p u n c t u r e scores were marked for examination. Shells then were fractured through m a r k e d areas, c o n t e n t s were rinsed away with distilled water, and shells were processed for microscopic analysis. Microscopic Preparation. Sections of rinsed egg shell (.5 cm X .5 cm) with m e m b r a n e s attached were separated from the e q u a t o r of t h e shell half at r o o m t e m p e r a t u r e , air-dried, cemented to brass microscope stubs, gold coated 4 , stored in a dessicator, and examined with a scanning electron microscope .
FIG. 1. Operation of the shell-puncture meter to quantitate intrinsic eggshell strength. FIG. 2. Eggshell punctured in two locations and marked for microscopic analysis. FIG. 3. Ultrastructural evaluation of a shell puncture location. A crater-like surface depression was observed with subsurface fracture planes extending obliquely toward the shell membranes. The mammillary knob formations beneath the needle impact point, however, did not appear extensively damaged and rested upon the intact shell membranes. (75X).
Downloaded from http://ps.oxfordjournals.org/ at University of Pennsylvania Library on March 4, 2015
Shell Quality Determinations. G r o u p s of 1 0 0 eggs were collected at midday within several h o u r s of oviposition from Single C o m b Leghorn (SCWL) hens housed in wire layer pens, 2 birds per cage, with water and standard corn-soybean laying ration available ad lib. Shell strength was measured by a calibrated shell p u n c t u r e device according to t h e m e t h o d of M u n r o ( 1 9 6 6 ) (Fig.
1).
641
EGG SHELL ULTRASTRUCTURE Freeze fracture surfaces were prepared according to the method of Bunk and Balloun (1977). RESULTS
6 Charging is the loss of SEM final image quality due to accumulation of loose unconducting material.
Puncture strength 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31
No. 2 1 1 3 3 11 13 14 17 14 3 7 2 2 2 4 1
crete, rounded bodies situated upon membrane fibers of the shells was the first type of alteration observed (Fig. 4—7). Various sizes and degrees of unification were observed in membrane bodies of poor quality egg shells. The term "intermammillary adhesion disc" (Fig. 5) has been employed to describe round or oval adhesion surfaces between adjacent membrane bodies or between membrane bodies and larger shell formations. Adhesion discs seemed to be areas where the membrane bodies enlarged and coalesced into a single structure. Several discs (AD) are shown in Figs. 5 to 7. The membrane body in Fig. 7 seemed to have been attached to the larger shell formation via the adhesion disc but separated from the adjacent surface when the shell was fractured for analysis. The membrane body was more firmly attached to the membrane fibers than to the adjacent calcified structure because it retained the membrane connection after the fracture occurred. Two types of adhesion discs were noted. The first type (Type I), already mentioned, occurred between membrane bodies close to the shell membranes. A second type (Type II) of adhesion disc (arrow, Fig. 4) occurred higher, more externally, upon the radial surface. These discs were larger, more oval, and seemed to be surfaces where adjacent mammillary knobs united during the initial stages of palisade layer
Downloaded from http://ps.oxfordjournals.org/ at University of Pennsylvania Library on March 4, 2015
Shell Quality Determination. A radial shell section gauged for shell quality is shown in Fig. 3. The shell was fractured directly through a puncture depression, and the arrow (right side of micrograph) points towards the cuticular (other) shell surface. Effect upon the shell was noted as a round crater-like surface depression with subsurface fracture planes extending laterally from the point of impact. The mammillary region and internal portion of the palisade layer, beneath the puncture depression, rested upon the shell membranes and were separated from the outer regions of the shell resulting in an incomplete puncture of the shell. Mammillary knobs and membrane fibers beneath the point of impact did not appear extensively damaged. Severe pulverization of calcite from the point of needle impact was observed as white=charged areas in the outer regions of the palisade layer. The distribution of puncture scores for 100 randomly selected egg shells is shown in Table 1. Similar distributions were obtained in repeated trials with approximately 7 to 12% of the shells per trial displaying considerably reduced shell puncture strengths. For this study, the shells were arbitrarily classified as low (14 to 20 oz), average (21 to 25 oz) and above-average quality (25 to 32 oz). Ultrastructural Evaluation. Shells with average and above average quality measurements resembled specimens shown by previous authors (Simons, 1971; McFarland et ai, 1971). Egg shells with extremely low puncture scores, however, appeared grossly inferior with shell checks and/or surface irregularities. Microscopically, extreme disorganization and conformational alterations were noted in the mammillary region. Sections of these shells possessed few uniform conical mammillary knobs as described in previous reports (Romanoff and Romanoff, 1949). The structural alterations observed in mammillary regions of poor-quality shells were classified into three general categories. Membrane bodies, a proliferation of small, dis-
TABLE 1 .—Puncture strength in ounces of 100 randomly selected Single Comb White Leghorn egg shells
642
BUNK AND BALLOUN
;
•-.'..
'
f j j w
3:
FIG. 4. Mammillary region of poor-quality eggshell. The shell membrane layers, while thin, retained a physical distinction between inner and outer membrane fiber groups. Areas of these shells did not possess well formed, rounded mammillary formations, but instead showed numerous nucleation sites and incompletely calcified structures. The term membrane body has been employed to describe small round incompletely calcified shell components attached to the outer shell membrane fibers. Areas of the palisade region (right side of micrograph) were observed without direct membrane connections, as seen in higher quality eggshells. (250X). FIG. 5. Membrane bodies and type 1 adhesion discs of poor-quality eggshells. An enlargement of Fig. 4 (arrow) shows a membrane body (Mb) attached to outer shell membrane fibers (osm) and several adhesion discs (Ad) where membrane bodies were formerly attached. Larger type II adhesion discs are shown at the upper left of arrow in Figure 4. (1000X ). FIG. 6. Radial shell surface of poor-quality eggshell showing larger, more irregular membrane bodies. Two layers of membrane bodies are observed beneath the palisade layer. The surface of the primary (lower) membrane bodies served as the nucleation site for secondary layer of membrane bodies which then enlarged into the base of the palisade region. (250X). FIG. 7. Enlargement of base of "more normal appearing" mammillary knob formation of poor quality eggshell. At low magnification, adjacent areas appeared to have large, broad mammillary formations similar to regions observed in average and higher quality shells. However, upon closer examination, these mammillary knobs appeared to be composed of membrane bodies that had fused "more completely" into a single structure. This micrograph shows a more complete fusion of membrane bodies with a fusion line (Fl) between adjacent nucleation sites, and a singular membrane body with adhesion (fracture) surface to the larger calcified region. Note that the membrane body (Mb) was more firmly attached to the outer shell membrane fibers than to adjacent calcified structures, and separated upon fracture. (1160X).
formation. Large mammillary k n o b formations of lowquality shells (Fig. 7) seemed to be t h e result of i n c o m p l e t e fusion of several m e m b r a n e bodies. T h e fusion line ( F L , Fig. 7) was considered to be a d e m a r c a t i o n b e t w e e n two adjacent m e m b r a n e bodies. As such, t h e first t y p e of struc-
tural alteration was considered t o b e d u e to small r o u n d e d discrete bodies t h a t originated uniformly across t h e outer shell m e m b r a n e surface and resulted in an unorganized base for the mammillary and palisade layers. T h e second general t y p e of alteration observed in poor-quality shells was t h a t of an
Downloaded from http://ps.oxfordjournals.org/ at University of Pennsylvania Library on March 4, 2015
'
EGG SHELL ULTRASTRUCTURE
show various states of multinucleation, and Figures 9 and 11 also s h o w a minimal k n o b to m e m b r a n e relationship n o t observed in higher quality shells. Freeze-fractured surfaces revealed no mammillary core, central channel, or core space described in previous reports (Bunk a n d B a l l o u n , 1977). A third general t y p e of deformation observed was caverns or cavities at t h e tip of large, more uniform mammillary k n o b formations (Figures 12—15). No singular site of crystal initiation was obvious, and the internal (lumenal) surfaces of tip cavities (Fig. 13) seemed similar in crystalline nature to the exterior k n o b surface. Also, the e x t e n t to which m e m brane fibers penetrated the k n o b s was decreased from that in average and above average quality shells. Freeze-fractured surfaces (Figs. 14—15) showed no mammillary core s t r u c t u r e .
FIG. 8. Large multinucleated mammillary region of low-quality eggshell. A wave or bulge in the nucleation zone (plane) was observed, and mammillary formations (knobs) were composed of crystal columns originating from numerous nucleation sites. (390X). FIG. 9. Enlargement of multinucleated mammillary formation from Fig. 8. A minimal shell membrane-calcite relationship was observed. (970X). FIG. 10. Extensive multinucleated mammillary formation. (460X). FIG. 11. Mammillary formation originating from two nucleation sites with a minimal calcite-shell membrane fiber relationship. (540X).
Downloaded from http://ps.oxfordjournals.org/ at University of Pennsylvania Library on March 4, 2015
extremely disorganized, multinucleated m a m millary region (Figs. 8—11). Calcified m e m brane bodies as described previously were n o t observed in these regions, b u t w h a t could be considered a single mammillary k n o b of an average quality shell seemed to be the result of several i n d e p e n d e n t , partly fused calcite colu m n s originating from several locations and projecting in several directions. In addition, a wave or bulge in the nucleation z o n e existed such t h a t all t h e mammillary k n o b s did n o t originate from a single uniform plane or surface. For clarification, the nucleation zone refers to the outer surface of the outer shell m e m b r a n e layer. On the right side of F'ig. 8, a r o u n d e d structure (b) was observed t h a t had n o direct m e m b r a n e c o n n e c t i o n b u t enlarged to form a base of t h e palisade layer. Figures 10 and 11
643
BUNK AND BALLOUN
644
•-
wL
"• 2jS^B
f . . i--.
-^
M
.m .
DISCUSSION An i m p o r t a n t biological consideration lies in the c o n c e p t t h a t the cell is a basic u n i t of s t r u c t u r e and function. In an analogous phrase, t h e mammillary k n o b and palisade c o l u m n t h a t originate from t h a t k n o b are the basic u n i t of s t r u c t u r e and function of the avian eggshell. Eggshell research, however, has tended to ignore any idea of a cellular or unitary approach to shell strength, and has focused primarily u p o n the idea t h a t a firm thick shell results solely from a d e q u a t e calcium deposition during t h e formation of the palisade layer. T h e d e f o r m a t i o n s categorized here have s h o w n t h a t the strength of the p o o r quality shell lies also in t h e organization of t h e primary calcified structures (mammillary knobs) rather t h a n
strictly in t h e thickness of t h e palisade region. T h e area of t h e shell to m e m b r a n e relationship especially seems critical, and a large (7 to 10%) portion of p r o d u c t i o n losses could actually be due to small changes in the c o m p o s i t i o n of t h e outer shell m e m b r a n e fibers or alterations in factors causing t h e initial precipitation of calcium salts, rather than only to limitation of substrate (calcite) during palisade f o r m a t i o n . It has been observed t h a t a d e q u a t e calcium, palisade thickness, is present in low quality shells (Figs. 4 and 6) b u t t h a t its c o n f o r m a t i o n is unorganized and in need of further study. T h e areas of unorganized, multinucleated m a m millary k n o b s indicate that the shell m e m b r a n e fibers initially b e c o m e over-precipitated with calcium salts and t h a t t h e n u m e r o u s m e m b r a n e
Downloaded from http://ps.oxfordjournals.org/ at University of Pennsylvania Library on March 4, 2015
MHHHHHBSNK 1
FIG. 12. Cavernous tip mammillary knob. Membrane fiber penetration of knob tip is decreased, and a fusion line extends from the right side of the cavern opening. (1 590X ). FIG. 13. Interior or lumen of cavern shown in Fig. 12. A lack of sharp discrete crystal edge surfaces is observed, while no organized primary mammillary radial crystals are noted. (1 590X ). FIG. 14. Fractured cavernous mammillary knob. Note the fusion line that originates at the mid-dorsal cavern surface and extends toward the upper left of the micrograph. The adhesion surfaces between this mammillae and adjacent structures lacks definition, and occurs in spots rather than unifrom surfaces. (1380X). FIG. 15. Enlargement of the mammillary tip shown in Fig. 14. Note the vesiculated heterogeneous nature of the membrane fiber-knob tip interface. The lumen of the cavern appears to possess a definite "organic coating" and a fusion line originates from the dorsal aspect of die cavern (2770X).
EGG SHELL ULTRASTRUCTURE bodies d o n o t enlarge with enough breadth t o form a wide s t u r d y base for t h e palisade columns. Palisade columns of p o o r quality shells arising from u n e q u a l and (or) unparallel m a m millary k n o b formations then give rise to poorly interlocking calcite columns. It has been shown in Figure 7 t h a t the a t t a c h m e n t of the m e m b r a n e (mammillary core) is stronger t h a n the a t t a c h m e n t between adjacent calcified structures.
decreased degree of interdigitation and less intrinsic strength (see Fig. 19). Although t h e thickness of the palisade c o l u m n has been q u a n t i t a t e d , t h e degree of column interdigitation can n o w only be speculated. In s u p p o r t of an idea or t h e o r y of interdigitation are Figures 16 to 18. Here multinucleated mammillary k n o b s were observed (Fig. 16 shows b o t h a cavernous tip and m e m b r a n e b o d y ) and a striking fact was t h e observable cracks or micro-checks seen at t h e ultrastructural level. In Figures 17 and 18, t h e multinucleated k n o b itself displays intrinsic fissures or micro-checks. A fracture line was observed to start at the m e m b r a n e level between individual nucleation sites and e x t e n d externally t o w a r d s t h e palisade layer. A second fissure was also observed b e t w e e n this k n o b and the adjacent mammillary f o r m a t i o n (right side
FIG. 16. Intermammillary fissure; note membrane body (mb) and cavernous tip (ct) in adjacent mammillary regions. The fissure or microcheck extended peripherally towards the palisade region. (760X). FIG. 17, 18. Intramammillary fissure (a) and intermammillary fissure (b) are observed in a multinucleated mammillary knob. Note the adjacent mammillary knob with apparent cavernous tip (Fig. 17). The intramammillary fissure (a, Fig. 18) originated at the outer membrane fiber level and extended peripherally towards the palisade layer. The fracture line appeared to result from decreased interdigitation or cohesive strength of primary radical crystal growth of the mammillary knob. (Fig. 17, 880X ; Fig. 18, 1630X).
Downloaded from http://ps.oxfordjournals.org/ at University of Pennsylvania Library on March 4, 2015
Very little consideration has been directed t o w a r d s t h e exact relationship of adjacent palisade columns, and the degree t o which individual calcite c o l u m n s interlock (interdigitation) m a y be t h e intrinsic weakness to the physical c o n c e p t of eggshell strength. T w o adjacent columns originating from different levels (waves in the nucleation plane) unparallel t o the m e m b r a n e could be e x p e c t e d to show a
645
646
BUNK AND BALLOUN
of micrograph) and also extended toward the palisade. One could speculate that these areas are the actual weak spots of an eggshell, and responsible for the cracks and (or) checks observed after processing. These areas seem to be the result of altered mammillary knob synthesis, and would require the least force or pressure to expand, and serve as the initiation of complete cracks through the palisade layer. Correlated to the extent of interdigitation is the degree and distribution of shell organic matrix. Tightly fused crystal columns would possess a reduced quantity of trapped organic material chiefly from the oviducal fluids. Conversely, shells enlarging with disorganized adjacent crystal columns would entrap a greater amount of oviducal fluids which would then precipitate as the shell dehydrated after oviposition. Simons (1971) observed a 60% increase in the number of vesicular (pit-like) holes per unit of surface area in poor quality shells of equal thickness but widely different breaking strengths. It was also noted (Simons, 1971) that the structure of the decalcified palisade layers of weak shells resembled the outer part of the cone (mammillary) layer of normal shells rather than that of the normal palisade layer. This suggests that the initial process of mammillary knob formation plays a greater role in shell strength than previously acknowledged. The mammillary region has been reported to contain a greater
Additional investigations lie chiefly in developing specific techniques that result in a direct correlation between eggshell ultrastructure and quantitative shell quality measurements. This investigation has considered a single method of gauging shell quality (needle puncture) for a number of reasons. First, shell and membrane properties are noted to vary over regions of a shell and over portions of the shell equator (Romanoff and Romanoff, 1949; Balch and Tyler, 1964). Adjacent points as close as .5 cm could be independently gauged for shell strength and directly identified with the microscope. Methods that result in one numerical value per egg (Instron, Bomber, or specificgravity) were considered to be at a definite disadvantage, since only crude approximations of ultrastructure of a quadrant of shell circumference could be estimated. The needle puncture method also was considered because it eliminated soaking and major crushing distortions created during specific gravity or Instron determination.
Downloaded from http://ps.oxfordjournals.org/ at University of Pennsylvania Library on March 4, 2015
FIG. 19. Diagramatic representation of the concept of interdigitation of crystal columns in avian eggshells. A represents waves or irregularities in the nucleation zone (plane), and numerous non-uniform nucleation sites on the outer shell membranes. These then give rise to extremely disorganized, poorly interdigitating cystal columns in the palisade layer, and increased quantities of organic material (oviducal fluids and cellular debris) become entrapped between adjacent columns. While shell thickness and total organic content may appear similar, the even uniform distribution and increased interlocking arrangement in B would be indicative of an eggshell with higher intrinsic strength and better shell quality.
quantity of organic material (Simkiss, 1968), and the observation of an increased organic content of palisade region of weak eggshells indicates an alteration in the growth of palisade crystal columns allowing a greater degree of fluid entrapment. The initial crystals of the mammillae appear somewhat disorganized in freeze-fracture surfaces of normal shell surfaces (Bunk and Balloun, 1977), and organic shell material may simply be a function of space filling or packing between adjacent crystal surfaces rather than a supportive framework in shell formation. To help in determining the underlying reasons for malformed and disorganized mammillary regions, factors such as the amino acid patterns or trace element relationships of the outer shell membrane fibers are in need of further study in more precise detail than previously accomplished. In unpublished studies, we have observed a flattening and (or) increased "knottiness" of the outer shell membrane fibers of poor quality shells. Although their results are unexplained at present, Masshoff and Stolpmann (1961) observed membrane fibers to be consisting of a core of keratin fibrils embedded within a matrix, surrounded by a less electron dense mantle layer. If calcium salts precipitate onto the outer mantle surface of the membrane fibers, then the surface structure or composition of this mantle layer becomes an important biological interface.
EGG SHELL ULTRASTRUCTURE ACKNOWLEDGMENT Deep appreciation is expressed t o Dr. H. T. Horner, Jr., I.S.U. D e p a r t m e n t of B o t a n y a n d Plant Pathology, for m a n y hours of personal instruction to t h e senior a u t h o r , and for use of microscope facilities. Also, we wish t o a c k n o w ledge m e m b e r s of t h e I.S.U. P o u l t r y Section (Drs. Wm. J. Owings, A. Nordskog, R. J. Hasiak, a n d Prof. L. Z. Eggleton) for help a n d consultation during t h e senior a u t h o r ' s Master's program while at Iowa S t a t e .
Balch, D. A., and C. Tyler, 1964. Variation in some shell membrane characteristics over different parts of the same shell. Br. Poultry Sci. 5:201-215. Bunk, M. J., 1977. An ultrastructural evaluation of avian eggshell quality. M.S. Thesis, Iowa State University, Ames, Iowa. Bunk, M. J., and S. L. Balloun, 1977. Structure and relationship of the mammillary core to membrane fibers and initial calcification of the avian eggshell. Br. Poultry Sci. 1 8 : 6 1 7 - 6 2 1 . El-Boushy, A. R., P. C. M. Simons, and G. Wiertz, 1968. Structure and ultrastructure of the hen's egg shell as influenced by environmental temperature, humidity, and vitamin C additions. Poult. Sci. 47:456-467. King, N. R., and D. S. Robinson, 1972. The use of the
scanning electron microscope for comparing the structure of weak and strong egg shells. J. Microscop. 9 5 : 4 3 7 - 4 4 3 . McFarland, L. Z., R. L. Garrett, and J. A. Nowell, 1971. Normal eggshells and thin eggshells caused by organochlorine insecticides viewed by the scanning electron microscopy. Scanning Electron Microscopy. Proc. 4th Ann. Scanning Electron Microscope Symp. (I.I.T. Research Institute, Chicago, IL 60616, USA) 4:377-384. Masshoff, W., and H. J. Stolpmann, 1961. Licht- und elektronemiktroskopische Untersuchungen an der Schalenhaut und Kalkschale des Huhnereis. Z. Zellforsch. Mikrosk. Anat. 55:818-832. Meyer, R., R. C. Baker, and M. L. Scott, 1973. Effects of hen egg shell and other calcium sources upon egg shell strength and ultrastructure. Poultry Sci. 52:949-955. Munro, S. S., 1966. 1. Studies on needle puncture as a method of measuring shell strength. 1. Development of the method, effect of needle shape, variation between areas of the shell, and differences between operators. Hy-Line Res. Rep. II (III-A-I): 1 5 - 2 3 . Romanoff, A. L., and A. J. Romanoff, 1949. The avian egg. Wiley, New York. Simkiss, K., 1968. The structure and formation of the shell and shell membranes. Vol. 4. In Egg,quality: A study of the hen's egg. T. C. Carter, ed., Oliver and Boyd, Edinburgh, pp. 3—25. Simons, P. C. M., 1971. Ultrastructure of the hen eggshell and its physiological interpretation. Commun. No. 175 Cent. Inst. Poultry Res. "Het Spelderholt" Beekbergen, The Netherlands.
Downloaded from http://ps.oxfordjournals.org/ at University of Pennsylvania Library on March 4, 2015
REFERENCES
647