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Observations of the Effects of Eggshell Pimpling on Shell Ultrastructure
Observations of the Effects of Eggshell Pimpling on Shell Ultrastructure D. COSTELLO, S. M. MEOLA,1 and T. W. ODOM2 Department of Poultry Science, Texas Agricultural Experiment Station, Texas A&M University, College Station, Texas 77843 (Received for publication March 8, 1984)
INTRODUCTION Rough or pimpled eggshells produced by the occurrence of calcareous deposits protruding from the surface of the eggshell have been a subject of interest in the determination of shell quality. A reduction in the marketability of pimpled eggs due to appearance, losses due to breakage, and the formation of leakers are the major effects of eggshell pimpling on the egg industry. It is generally agreed that normal eggshell formation follows a sequential pattern, and that this pattern is reflected in the relationships of shell ultrastructural components. The incidence of shell pimples represents an observable anomaly in these relationships. Thus, the scrutiny of these anomalies may provide insight as to the mechanisms of the developmental process itself. Two types of eggshell pimpling were described by Roland et al. (1975). Type I pimples were identified as superficial deposits of calcareous material attached only to the surface of the shell. Type II pimples, the most predominate type, were defined as those in which the pimple was attached at any point between the shell membranes and the exterior surface. Formation of the shell seemed to take place over and around a mass of intrusive material.
1 USDA, SEA-ARS. Veterinary Toxicology and Entomological Laboratory, College Station, TX 77841. 2 To whom correspondence should be addressed.
Type I pimples are thought to produce minimal effects on the shell ultrastructure; consequently, only pimples of the second type were considered in this study. Significant variations in the eggshell pimpling scores of hens have been shown to be extremely difficult to produce by manipulation of dietary and environmental conditions (Farmer and Roland, 1980; Goto et al, 1982). Direct manipulation of the uterine environment by the introduction of calcium carbonate or dilute hydrochloric acid has also produced little variation in the extent of eggshell pimples (Roland et al, 1975). This difficulty suggests that the origins of eggshell pimples are widely distributed in the process of shell development and are relatively unaffected by the determinants of the mineral constituents of the shell. The objective of the present investigation was to determine the location of pimple intrusive material with respect to other structural components of the shell and to compare the appearance of the intrusive material itself with that of other shell constituents. MATERIALS AND METHODS Eggs with outstanding occurrences of Type II pimples were obtained from both the Texas Agricultural Experiment Station research farm and a local commercial layer operation. After breaking out and washing the eggs, a small area of the shell surrounding each predominate pimple was carefully excised from the shell. Pimples and their underlying membranes were observed under a light microscope in calcified and decalcified whole mounts. Several speci-
1484
Downloaded from http://ps.oxfordjournals.org/ at University of New Orleans on June 14, 2015
ABSTRACT In the present study eggshell pimples were observed under a scanning electron microscope to determine the relationships of shell structural constituents to the intrusive mass. Structural observations suggest that the intrusions occur at various stages in shell formation and that the time at which the intrusion took place determined its effects on subsequent shell ultrastructure. (Key words: eggshell pimpling, eggshell ultrastructure) 1985 Poultry Science 64:1484-1487
STRUCTURE OF EGGSHELL PIMPLES
mens were stained with either methyl violet or alcian blue. Twenty samples were selected at random for observation under the scanning electron microscope. These were broken across the pimple, mounted on stubs, and sputter coated with a 200 A layer of gold. The samples were observed with a Cambridge Model 25 scanning electron microscope.
RESULTS AND DISCUSSION
apices of mammillary knobs immediately above an intrusion that had occurred during mammillary deposition (Figure IE). These spherites seem to have been observed by several researchers in the early stages of shell development. They appear to be mamillary precursors of organic composition. This observation suggests that the presence of an outer shell membrane may not be required for the deposition of mammillae or the formation of mammillary knobs. However, the presence of mammillae in some form seems to be required for the subsequent development and organization of shell structure. Pimple intrusions occurring after the mammillary stage of formation produced much greater effects on subsequent shell development. The production of relatively wide or nonexistant pallisades and large fissures between the pimples and the surrounding shell were generally observed when intrusions were made at later stages of development. The presence of such fissures would contribute greatly to decreased structural integrity of the eggshell. Examination of the pimple material itself suggested that pimple intrusions consisted of various substances. Although a material with a glassey fracture similar to that of dried egg albumen was observed in some samples, others contained material with a planar fracture. It was thought that the air drying of samples may have produced an artifact in the appearance in the intrusive material. For this reason a few samples were critical point dried. Figure I F shows the appearance of one such sample. Globular substances as well as fine microfibrils (about , 1 /urn diameter) can be seen. It appears that pimple intrusions may consist of many different materials. Clearly the biochemical identification of these materials should be undertaken. The fact that pimple intrusions occur at any point in eggshell development may raise questions concerning the distinction between Type I and Type II pimples. Type I pimples scraped from the shell and decalcified in 5% ethylenediaminetetraacetic acid appeared to have a nonmineral core that behaved histologically as Type II intrusive material. The negative correlation between pimpling score and shell thickness reported by Goto et al. (1982) may be because an increase in eggshell thickness may smooth over the appearance of pimple intrusions at the shell surface.
Downloaded from http://ps.oxfordjournals.org/ at University of New Orleans on June 14, 2015
Examinations of pimples by light microscopy generally supported the observations made by Ball et al. (1973). No disturbances of the inner shell membrane lying directly beneath the pimples were detected, nor was there any histological evidence of pimple intrusive material in the inner membrane. The outer layer of the outer shell membrane appeared matted and seemed to have a lower density of attached mammillae when compared to the areas surrounding the pimple intrusion. The presence of mammillae below the majority of pimple intrusions suggested that the most noticeable intrusions occur during or immediately subsequent to the deposition of mammillae on the outer membrane. Under the scanning electron microscope, pimple intrusions occurred at many points in normal shell development. Intrusions were observed anywhere from beneath the outer membrane to the surface of the shell. (See Figure 1, A and B.) The location of pimple intrusions appears to be independent of extent of shell formation. At whichever point intrusions occurred, shell development was neatly interrupted beneath. Figure 1,. C and D show "craters" in the shell surface from which large pimples were removed. The development of the mammillary structures beneath strongly resemble electron micrographs taken by Creger et al. (1976) of developing eggshells at 4.5 and 5.0 hr postoviposition. Above the intrusive material the process of shell formation proceeds. Intrusions occurring prior to or during the development of mammillary knobs produced little disorganization of subsequent shell ultrastructure. It can be assumed that if structures such as mammilae are necessary for organization, such structures must then be present above pimple intrusions. Indeed, in one case, small spherites (about 10 /im diameter) were observed attached to the
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STRUCTURE OF EGGSHELL PIMPLES
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FIG. 1. A. Eggshell pimple occurring during mammillary deposition. Note the abnormally large pallisades (P), the presence of mammillae (M) attached to the outer shell membrane, and the pimple intrusion (I). X58. B. A pimple intrusion occurring beneath the outer shell membranes. Note that the development of the mineralization portion of the shell has proceeded normally. X61. C. A "crater" in the shell surface revealed by the removal of an overlying shell pimple. The mammillary development has halted at a point strongly resembling photomicrographs taken at 4.5 hr postoviposition (Creger et al, 1976). X56. D. Another pimple "crater." Mammillary development has reached a stage comparable to photomicrographs taken by Creger et al. (1975) 5 hr postoviposition. X23. E. Mammillary knob development above a pimple intrusion which occurred during mammillary deposition. Note the presence of nonmineralized spheroids (S) at the pieces of mammillary knob (MK). X 1038. F. Critical point dried pimple intrusive material seen at high magnification. The material appears to be composed of microfibrils (F) as well as mineralized globular structures (G). X5600.
Ball, R. F., R. J. Macken, J. F. Hill, and A. J. Wyatt, 1973. The nature and probable cause of rough egg shells laid by two lines of white leghorns. Poultry Sci. 52:500-506. Creger, C. R., Hall Phillips, and J. T. Scott, 1976. Formation of an egg shell. Poultry Sci. 55:1717— 1723. Farmer, M., and D. A. Roland, 1982. The influence of dietary ingredients, photoperiod, and cage
density on egg shell pimpling. Poultry Sci. 6 1 : 495-502. Goto, K., G. C. Harris, and P. W. Waldroup, 1982. Relationship between pimpling of egg shells, environmental temperature, and carbonic anhydrase activity of certain body tissues. Poultry Sci. 61:364-366. Roland, D. A., J. B. Thompson, R. A. Voitle, and R. H. Harms, 1975. Studies on the cause, prevention and artificial creation of pimpled egg shells. Poultry Sci. 54:1485-1491.
Downloaded from http://ps.oxfordjournals.org/ at University of New Orleans on June 14, 2015
REFERENCES
INTRODUCTION Rough or pimpled eggshells produced by the occurrence of calcareous deposits protruding from the surface of the eggshell have been a subject of interest in the determination of shell quality. A reduction in the marketability of pimpled eggs due to appearance, losses due to breakage, and the formation of leakers are the major effects of eggshell pimpling on the egg industry. It is generally agreed that normal eggshell formation follows a sequential pattern, and that this pattern is reflected in the relationships of shell ultrastructural components. The incidence of shell pimples represents an observable anomaly in these relationships. Thus, the scrutiny of these anomalies may provide insight as to the mechanisms of the developmental process itself. Two types of eggshell pimpling were described by Roland et al. (1975). Type I pimples were identified as superficial deposits of calcareous material attached only to the surface of the shell. Type II pimples, the most predominate type, were defined as those in which the pimple was attached at any point between the shell membranes and the exterior surface. Formation of the shell seemed to take place over and around a mass of intrusive material.
1 USDA, SEA-ARS. Veterinary Toxicology and Entomological Laboratory, College Station, TX 77841. 2 To whom correspondence should be addressed.
Type I pimples are thought to produce minimal effects on the shell ultrastructure; consequently, only pimples of the second type were considered in this study. Significant variations in the eggshell pimpling scores of hens have been shown to be extremely difficult to produce by manipulation of dietary and environmental conditions (Farmer and Roland, 1980; Goto et al, 1982). Direct manipulation of the uterine environment by the introduction of calcium carbonate or dilute hydrochloric acid has also produced little variation in the extent of eggshell pimples (Roland et al, 1975). This difficulty suggests that the origins of eggshell pimples are widely distributed in the process of shell development and are relatively unaffected by the determinants of the mineral constituents of the shell. The objective of the present investigation was to determine the location of pimple intrusive material with respect to other structural components of the shell and to compare the appearance of the intrusive material itself with that of other shell constituents. MATERIALS AND METHODS Eggs with outstanding occurrences of Type II pimples were obtained from both the Texas Agricultural Experiment Station research farm and a local commercial layer operation. After breaking out and washing the eggs, a small area of the shell surrounding each predominate pimple was carefully excised from the shell. Pimples and their underlying membranes were observed under a light microscope in calcified and decalcified whole mounts. Several speci-
1484
Downloaded from http://ps.oxfordjournals.org/ at University of New Orleans on June 14, 2015
ABSTRACT In the present study eggshell pimples were observed under a scanning electron microscope to determine the relationships of shell structural constituents to the intrusive mass. Structural observations suggest that the intrusions occur at various stages in shell formation and that the time at which the intrusion took place determined its effects on subsequent shell ultrastructure. (Key words: eggshell pimpling, eggshell ultrastructure) 1985 Poultry Science 64:1484-1487
STRUCTURE OF EGGSHELL PIMPLES
mens were stained with either methyl violet or alcian blue. Twenty samples were selected at random for observation under the scanning electron microscope. These were broken across the pimple, mounted on stubs, and sputter coated with a 200 A layer of gold. The samples were observed with a Cambridge Model 25 scanning electron microscope.
RESULTS AND DISCUSSION
apices of mammillary knobs immediately above an intrusion that had occurred during mammillary deposition (Figure IE). These spherites seem to have been observed by several researchers in the early stages of shell development. They appear to be mamillary precursors of organic composition. This observation suggests that the presence of an outer shell membrane may not be required for the deposition of mammillae or the formation of mammillary knobs. However, the presence of mammillae in some form seems to be required for the subsequent development and organization of shell structure. Pimple intrusions occurring after the mammillary stage of formation produced much greater effects on subsequent shell development. The production of relatively wide or nonexistant pallisades and large fissures between the pimples and the surrounding shell were generally observed when intrusions were made at later stages of development. The presence of such fissures would contribute greatly to decreased structural integrity of the eggshell. Examination of the pimple material itself suggested that pimple intrusions consisted of various substances. Although a material with a glassey fracture similar to that of dried egg albumen was observed in some samples, others contained material with a planar fracture. It was thought that the air drying of samples may have produced an artifact in the appearance in the intrusive material. For this reason a few samples were critical point dried. Figure I F shows the appearance of one such sample. Globular substances as well as fine microfibrils (about , 1 /urn diameter) can be seen. It appears that pimple intrusions may consist of many different materials. Clearly the biochemical identification of these materials should be undertaken. The fact that pimple intrusions occur at any point in eggshell development may raise questions concerning the distinction between Type I and Type II pimples. Type I pimples scraped from the shell and decalcified in 5% ethylenediaminetetraacetic acid appeared to have a nonmineral core that behaved histologically as Type II intrusive material. The negative correlation between pimpling score and shell thickness reported by Goto et al. (1982) may be because an increase in eggshell thickness may smooth over the appearance of pimple intrusions at the shell surface.
Downloaded from http://ps.oxfordjournals.org/ at University of New Orleans on June 14, 2015
Examinations of pimples by light microscopy generally supported the observations made by Ball et al. (1973). No disturbances of the inner shell membrane lying directly beneath the pimples were detected, nor was there any histological evidence of pimple intrusive material in the inner membrane. The outer layer of the outer shell membrane appeared matted and seemed to have a lower density of attached mammillae when compared to the areas surrounding the pimple intrusion. The presence of mammillae below the majority of pimple intrusions suggested that the most noticeable intrusions occur during or immediately subsequent to the deposition of mammillae on the outer membrane. Under the scanning electron microscope, pimple intrusions occurred at many points in normal shell development. Intrusions were observed anywhere from beneath the outer membrane to the surface of the shell. (See Figure 1, A and B.) The location of pimple intrusions appears to be independent of extent of shell formation. At whichever point intrusions occurred, shell development was neatly interrupted beneath. Figure 1,. C and D show "craters" in the shell surface from which large pimples were removed. The development of the mammillary structures beneath strongly resemble electron micrographs taken by Creger et al. (1976) of developing eggshells at 4.5 and 5.0 hr postoviposition. Above the intrusive material the process of shell formation proceeds. Intrusions occurring prior to or during the development of mammillary knobs produced little disorganization of subsequent shell ultrastructure. It can be assumed that if structures such as mammilae are necessary for organization, such structures must then be present above pimple intrusions. Indeed, in one case, small spherites (about 10 /im diameter) were observed attached to the
1485
COSTELLO ET AL.
1486
IBkJt »'*$?&* ^J^%
It/ ^M wPk/N^
!»'*• ^ 2 ^ * % i
Downloaded from http://ps.oxfordjournals.org/ at University of New Orleans on June 14, 2015
C
STRUCTURE OF EGGSHELL PIMPLES
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FIG. 1. A. Eggshell pimple occurring during mammillary deposition. Note the abnormally large pallisades (P), the presence of mammillae (M) attached to the outer shell membrane, and the pimple intrusion (I). X58. B. A pimple intrusion occurring beneath the outer shell membranes. Note that the development of the mineralization portion of the shell has proceeded normally. X61. C. A "crater" in the shell surface revealed by the removal of an overlying shell pimple. The mammillary development has halted at a point strongly resembling photomicrographs taken at 4.5 hr postoviposition (Creger et al, 1976). X56. D. Another pimple "crater." Mammillary development has reached a stage comparable to photomicrographs taken by Creger et al. (1975) 5 hr postoviposition. X23. E. Mammillary knob development above a pimple intrusion which occurred during mammillary deposition. Note the presence of nonmineralized spheroids (S) at the pieces of mammillary knob (MK). X 1038. F. Critical point dried pimple intrusive material seen at high magnification. The material appears to be composed of microfibrils (F) as well as mineralized globular structures (G). X5600.
Ball, R. F., R. J. Macken, J. F. Hill, and A. J. Wyatt, 1973. The nature and probable cause of rough egg shells laid by two lines of white leghorns. Poultry Sci. 52:500-506. Creger, C. R., Hall Phillips, and J. T. Scott, 1976. Formation of an egg shell. Poultry Sci. 55:1717— 1723. Farmer, M., and D. A. Roland, 1982. The influence of dietary ingredients, photoperiod, and cage
density on egg shell pimpling. Poultry Sci. 6 1 : 495-502. Goto, K., G. C. Harris, and P. W. Waldroup, 1982. Relationship between pimpling of egg shells, environmental temperature, and carbonic anhydrase activity of certain body tissues. Poultry Sci. 61:364-366. Roland, D. A., J. B. Thompson, R. A. Voitle, and R. H. Harms, 1975. Studies on the cause, prevention and artificial creation of pimpled egg shells. Poultry Sci. 54:1485-1491.
Downloaded from http://ps.oxfordjournals.org/ at University of New Orleans on June 14, 2015
REFERENCES