- Email: [email protected]
Changes in the Gross Morphological Appearance of Chicken and Turkey Blastoderms During Preincubation Storage1,2
Changes in the Gross Morphological Appearance of Chicken and Turkey Blastoderms During Preincubation Storage1'2 K. L. ARORA3 AND I. L. KOSIN Department of Animal Sciences, Washington State University, Pullman (Received for publication January 10, 1966)
(1872) reported the O ELLACHER presence of vacuoles in the germinal
1
Scientific Paper No. 2747, College of Agriculture, Pullman. Project No. 1255. 2 This investigation was supported in part by research grant 5544 from the Division of General Medical Sciences, U.S. Public Health Service. 3 Present address: Drake University, Des Moines, Iowa.
819
Downloaded from http://ps.oxfordjournals.org/ at :: on January 22, 2015
area of incubated and unincubated infertile chicken eggs, observing that such vacuoles were absent in fertile eggs. According to Lecaillon (1910) segmentation in the infertile unincubated avian germ disc is restricted to its central portion and does not progress as far as in the fertile, unincubated egg. Furthermore, he found that incubating infertile eggs, or keeping them at room temperature for a time results in a rapid breakdown of the segmentation area. The vacuoles are characteristic not only of the germinal area of chicken eggs, but also those of the duck (Chappelier, 1912) and turkey (Kosin, 1951). Kosin (1944) observed that the presence of a varying number of large and small vacuoles both along the periphery and in the center of the blastodisc of unincubated chicken eggs in extreme cases gives its surface a reticular appearance. The normal blastoderm in a recently-laid unincubated egg is characterized by distinct area pellucida and area opaca. A few small vacuoles may be present along the periphery of such blastoderms, although the area pellucida in freshly-laid chicken eggs has always been found to be devoid of them. Kosin (1944) sug-
gested that blastoderms with one or more large vacuoles on the periphery might be cases of early moribundity. This was demonstrated to be so by Gowe (1950) for the chicken, and by Kosin (1951) for the turkey on the basis of macro- and microscopic analysis of germ discs from eggs subjected to preincubation holding. Kosin (1951) observed that turkey blastoderms with numerous vacuoles around the periphery developed abnormally when incubated. In most cases, development ceased within the first three days of incubation. Additional support on this point came from the study on chickens by Krizenecky et al. (1955) who reported that high temperatures during preincubation storage increased the incidence of vacuoles in the unincubated germ discs. Kosin (1948) demonstrated that in addition to vacuolation, nuclear pycnosis is characteristic of moribund turkey blastoderms. The observations of Zelnik (1961) are in close agreement with those of the above-cited investigators. On the other hand, Weisbroth (1960) reported that vacuoles appear independently of storage; i.e. extended storage per se does not appear to induce vacuolation in normal blastoderms. He further found that peripheral vacuoles do not interfere with development of the embryo, at least during the first 40 hours of incubation. The study to be reported below had two objectives: (1) to observe the progressive changes in the gross morphological appearance of the chicken and the turkey blastoderms during the preincubation storage,
820
K. L. ARORA AND I. L. KOSIN
and (2) to explore relationships between the morphological characteristics of the blastoderms during storage and the subsequent early embryogenesis. MATERIALS AND METHODS
Downloaded from http://ps.oxfordjournals.org/ at :: on January 22, 2015
The experimental material was provided by eggs from mass-mated White Leghorn pullets and Broad Breasted Bronze turkeys. The eggs were collected between 8 a.m. and 12:30 p.m. following which the eggs were brought to a laboratory for the preliminary determination of the fertility status. The fertility of the eggs was checked through the window, measuring 1 cm.2, which was cut in the shell in the large end of the egg. The freehand drawing of each blastoderm was then made depicting such "landmarks" as presence or absence of vacuoles, their distribution, as well as shape of the two major surface areas: area pellucida and area opaca. To be selected for this study, the blastoderms had to be devoid of vacuoles except, perhaps, for a few minute ones along the periphery of the periblast. Following this classification, the selected eggs were sealed and transferred to storage at 13°C. and 85% relative humidity. During storage, the windows were opened at least four times and the morphological appearance of each blastoderm was noted and compared with the original drawing. If any observable difference occurred between the successive examinations, additional drawings were made to record them. Storage and the examination procedure continued until the blastoderm showed no further changes in its gross morphology. The egg was then incubated for 24-36 hours at 37.5°C. and 65% relative humidity, or order to correlate the macroscopic appearance of the blastoderm with subsequent development. Appropriate representative specimens were saved from many blastoderms for further histological and cytological studies.
RESULTS
The normal chicken or turkey blastoderm after prolonged storage at 13.0°C. was found to pass, sequentially, through a series of recognizable morphological changes which finally led to the condition of moribundity and death. This included the following stages, all of which, except stage A, were sooner or later observed in eggs subjected to storage: Stage A—Normal blastoderm: characterized by several, regularly outlined bands (periblastic and polar rings) and a more solid band of the area opaca (Figure 1); the degree of "solidity" varied among specimens, however. The semi-translucent area pellucida frequently showed centrally located Nucleus of Pander. This stage was found to be devoid of vacuoles in the area pellucida, although small vacuoles were sometimes present either along the periphery of the area opaca or in the periblast (Figure 2). Stage B—Initial fluxation: with few exceptions, all blastoderms following storage showed some decrease from the original dimensions. Invariably, the area pellucida was devoid of the vacuoles. Stage C—Conspicuous Nucleus of Pander: The centrally localized white yolk became conspicuous, filling almost completely the central area of the blastoderm. In many specimens, vacuoles became both larger and more numerous in the periblast area. The latter often merged into the area opaca (Figure 3). Some blastoderms, especially in turkey eggs, showed this condition even at oviposition. Stage D—Initial vacuolation: A few scattered vacuoles, small to medium in size, appeared in the area pellucida (Figure 4). The area opaca became "flattened"; sometimes it lacked vacuoles. The latter, however, were always present in the periblast. This stage was also observed in some specimens of blastoderms in turkey eggs at the
821 APPEARANCE OF BLASTODERMS
Downloaded from http://ps.oxfordjournals.org/ at :: on January 22, 2015
822
K. L. AEORA AND I. L. KOSIN
basis of gross morphological changes the chicken blastoderms were found to be more resistant to the detrimental effects of 7-10 day storage than the turkey blastoderms. However, the frequency of blastoderms showing "degenerative changes" (changes as described under D, E, and F stages above) increased progressively as the storage was extended to 14 days or beyond. Some turkey blastoderms, on the other hand, showed signs of moribund conditions, corresponding to stage E, within 2-3 days of initiation of storage. In fact, turkey hens have been observed to lay eggs that were invariably judged moribund after 3-4 days' storage at 13°C. Both chicken and turkey stage A, B and C blastoderms on incubation for 24-36 hours always developed normally. The decrease in diameter (influxation) associated with blastoderms subjected to cool storage environment (13°C.) did not interfere, on the whole, with normal embryogenesis, although a few embryos from stage C blastoderms showed delay in development. Some of the blastoderms in stage D, when incubated, developed abnormally or died. These blastoderms, at the time of the initiation of incubation, exhibited extensive vacuolation in the area opaca, with small to medium size vacuoles being present in the
Illustrations FIGS. 1-5. Diagrammatic drawings of turkey blastoderms. The horizontal bars in the lower right corner of Figures 1 and 5 indicate the actual diameter of an average blastoderm prior to and after 28 days' storage. FIGS. 1 and 2. Normal blastoderms at or shortly after oviposition. In Figure 1, the dark band represents the area opaca which surrounds the semi-translucent area pellucida. Beyond the area opaca is the periblast. In Figure 2, the Nucleus of Pander can be barely seen in the middle of the area pellucida. The area opaca is less prominent than in Figure 1. A number of vacuoles are visible along the periphery of the periblast. FIG. 3. The Nucleus of Pander is prominent. Some dislocation may occur between the relative positions of the areas pellucida and opaca. The vacuoles are numerous in the periblast. FIG. 4. The area opaca frequently "expands" at the expense of the periblast. A few vacuoles appear in the area pellucida. FIG. S. Vacuoles cover the entire area of the blastoderm. Boundaries between the various blastodermal regions are all but completely obliterated.
Downloaded from http://ps.oxfordjournals.org/ at :: on January 22, 2015
time of oviposition (Figure 6). This is a critical stage because such a blastoderm can be erroneously classified as either being a case of "preoviposital" mortality, or as "infertile." Stage E—Initial moribundity: Numerous vacuoles of increased size appear in the area pellucida as well as in the area opaca. The diameter of the blastoderm grew markedly smaller than it was originally, though all of its major "landmarks" still remained intact. The originally dense, broad area opaca sometimes became less distinctly outlined. The outer and inner periblastic rings were still visible, although they were becoming increasingly indistinct. Stage F—Terminal moribundity: Practically all of the structural details of the normal blastoderm, except polar and periblastic rings disappeared by now. The outlines of the area opaca and area pellucida were almost completely obliterated by the presence of large numbers of vacuoles of various sizes and shapes over the entire surface of the blastoderm. The blastoderm was reduced in overall dimensions. This stage resembled the appearance of different variants of preoviposital mortality or even biological (true) infertility (Figures S and 7)Despite wide individual variation, on the
823
APPEARANCE OF BLASTODERMS
* '
Fie. 6. An unincubated chicken blastoderm in which numerous vacuoles have started to make their appearance in the periblast after a 14-day storage. These vacuoles were not present before storage (Stage D). FIG. 7. An unincubated turkey blastoderm after 28 days' storage. Note the presence of numerous vacuoles and a somewhat reduced size in the germ disc. FIG. 8. A section from unincubated blastoderm in a chicken egg stored 14 days. Note vacuoles and surface discontinuity (gaps) in the epiblast of the blastoderm (Stage E ) . (X650)
area pellucida. The Nucleus of Pander appeared as a prominent vacuolated white spot. The compactness of the area pellucida was completely lost. Following were the chief developmental abnormalities observed in such blastoderms: Marked retardation in development, incomplete axial system or sometimes total absence of the system and disorganization or duplication of the growth centers. Occasionally, the cellular proliferation in the central and proximal areas continued without expansion of the blastoderm over the yolk. Such blastoderms showed extensive cellular moribundity or
necrosis at the zone of junction. Histological examination of the blastoderms showing vacuolation of the area pellucida indicated by the presence of the vacuoles of varying sizes in the blastoderms, and separation of area pellucida into scattered masses of cells (Figure 8). Upon incubation, the blastoderms in stages E and F resulted either in outright mortality (i.e. total failure to initiate development) or in disintegration of the blastoderm after brief initial development. These blastoderms had an appearance similar to that generally associated with
Downloaded from http://ps.oxfordjournals.org/ at :: on January 22, 2015
*
824
K. L. ARORA AND I. L. KOSIN
preoviposital mortality or biological (true) infertility. Numerous vacuoles were present over the entire surface of the blastoderm; the dimensions of these vacuoles were accentuated by incubation. DISCUSSION
SUMMARY
A sequential series of regressive changes in the gross morphology of blastoderms were observed in chicken and turkey eggs during preincubation storage at 13°C. This included the gradual loss of structural detail normally associated with the area pellucida and area opaca and the appearance, in increasing numbers, of vacuoles within these regions. The presence of such vacuoles, particularly in the area pellucida, was indicative of growing blastodermal moribundity. Blastoderms with large vacuoles in the area pellucida were incapable of further development. Some turkey hens consistently laid eggs of a type just described, i.e. their blastoderms were either moribund or "dead" at oviposition. REFERENCES Chappellier, A., 1912. La cicatricule de l'oeuf dans le croisement: canard de Rouen (Anas boschas var. domestica (L.) x canard de Barbarie (Cairina moschata (L.)) et les especes parentes. Comp. Rend. Assoc, franc. Avance. Sci. 40: 541-544. Gowe, R. S., 1950. Techniques for identifying fertile hens' eggs. Poultry Sci. 29: 409-413. Kosin, I. L., 1944. Macro- and microscopic methods of detecting fertility in unincubated hens' eggs. Poultry Sci. 23 : 266-269. Kosin, I. L., 1948. Parthenogenesis and abortive embryonic development in the domestic fowl. Proc. 8th World's Poultry Congress 1: 345351. Kosin, I. L., 1951. A study of the morphology of
Downloaded from http://ps.oxfordjournals.org/ at :: on January 22, 2015
Chicken and turkey blastoderms, even when kept under what is considered to be "optimal" conditions (13°C. and 85% relative humidity), do not remain static during storage, but continue to undergo regressive structural changes. In spite of rather wide individual variation in susceptibility to detrimental effect of storage, each "stored" blastoderm passes through a series of well-recognized regressive changes which first lead to the condition of moribundity and, finally, death. Blastoderms in the latter stage were characterized by the extreme vacuolation of the germinal disc area and loss of structural details. Extensive vacuolation at periphery of the disc or, more so, the presence of numerous vacuoles in the area opaca and, certainly, in the area pellucida were always associated with moribundity of the blastoderm: upon incubation, such blastoderms resulted in either outright mortality, or, at best, in abnormal embryogenesis. This supports the earlier view that vacuolation of the area pellucida is associated with moribundity (Kosin, 1951). In fact, results of the present study clearly show that the presence of large vacuoles within the area pellucida is indicative that the blastoderm is completely incapable of further development. To all intents and purposes such blastoderm is dead. The vacuoles probably result from a progressive disintegration and contraction of the protoplasmic mass in the germ disc, ultimately leading to its discontinuity or the formation of gaps in the blastoderm. Because the morphological changes that were observed in the aging turkey and chicken blastoderm in the present study
were consistent, both in their sequence of appearance, as well as their association with the capacity of the blastoderm to continue early development upon incubation at 37.5°C, the recent suggestion by Weisbroth and Kosin (1966) that the morphological appearance of the unincubated blastoderm can be used as a guide in determining its potential for subsequent normal embryogenesis, has received additional corroboration.
APPEARANCE OF BLASTODERMS moribund turkey blastoderms. Proc. 9th World's Poultry Congress 3 : 75-87. Krizenecky, J., J. Sajner, V. Orel and F. Musil, 1955. Detection of fertilized, infertile and nonfertilized eggs in biological control of incubation and in testing of commercial eggs. Ministerstvo Potravinarskeho Promyslu, Prague, pp. 112. Lecaillon, A., 1910. Le parthenogenese chez les oiseaux. Segmentation et degenerescence de l'oeuf non feconde. Arch, d'anat. micros. 12: 511-638. Oellacher, J., 1872. Die Ver'anderungen des unbefruchteten Keimes des Hiihnereies im Eileiter
825
und bei Bebrutungsversuchen. Zeit. f. wiss. Zool. 22: 181-234. Weisbroth, S. H., 1960. The morphological response of gastrulae from three genetic lines of Broad Breasted Bronze turkeys to preincubation storage. M.S. Thesis. Washington State University, Pullman, pp. 62. Weisbroth, S. H., and I. L. Kosin, 1966. Gross spatial changes in the turkey blastoderm following extended preincubation storage. Proc. Soc. Exper. Biol. Med. 121: 795-800. Zelnik, J., 1961. Morfologia zardocnych diskov morky. Pol'nohospodarstvo 8: 211-216.
S. S. RIZK, J. C. AYRES AND A. A. KRAFT Department of Dairy and Food Industry, Iowa State University, Ames, Iowa (Received for publication January 10, 1966)
HE contents of the egg as laid by the normal healthy hen are sterile and it is possible that the shell may not be contaminated during the act of laying (Stuart and McNally, 1943). However, the ovaries may occasionally become infected with SalmoneUae which may be transferred to the egg contents (Scott, 1930). Consequently, the incidence of Salmonellae on or in shell eggs depends upon three factors:
T
"Journal Paper No. J-5291 of the Iowa Agricultural and Home Economics Experiment Station, Ames. Project No. 1544; Center for Agricultural and Economic Development cooperating. A report of work done under contract with the U. S. Department of Agriculture and authorized by the Research and Marketing Act of 1946. The contract is being supervised by the Western Utilization Research and Development Division of the Agricultural Research Service. Reference to a company or product name does not imply approval or recommendation of the product by the U. S. Department of Agriculture to the exclusion of others that may be suitable.
the environmental condition of the eggs, handling and storage, and the physical structure of the shell. Studies have been made on the ability of egg spoilage bacteria to utilize the material of the shell membranes for growth (Stokes and Osborne, 1956; Elliott and Brant, 1957; Garibaldi and Stokes, 1958; Board et al., 1964). Lancaster and Crabb (1953) demonstrated that although bacteria penetrated the shell, only rarely did organisms pass through the shell membrane into the white of the eggs. Stokes and Osborne (1956) believed that the egg shell membrane delayed the penetration of Salmonellae to egg contents by functioning as a mechanical rather than as a bactericidal barrier. Yet Salmonellae have been isolated from spray dried whole egg powder manufactured in the United States (Gibbons and Moore, 1944; Schneider, 1946). The source of contamination of spray dried whole egg with Salmonellae was investigated by Solowey et
Downloaded from http://ps.oxfordjournals.org/ at :: on January 22, 2015
Effect of Holding Condition on the Development of Salmonellae in Artificially Inoculated Hens' Eggs 1
(1872) reported the O ELLACHER presence of vacuoles in the germinal
1
Scientific Paper No. 2747, College of Agriculture, Pullman. Project No. 1255. 2 This investigation was supported in part by research grant 5544 from the Division of General Medical Sciences, U.S. Public Health Service. 3 Present address: Drake University, Des Moines, Iowa.
819
Downloaded from http://ps.oxfordjournals.org/ at :: on January 22, 2015
area of incubated and unincubated infertile chicken eggs, observing that such vacuoles were absent in fertile eggs. According to Lecaillon (1910) segmentation in the infertile unincubated avian germ disc is restricted to its central portion and does not progress as far as in the fertile, unincubated egg. Furthermore, he found that incubating infertile eggs, or keeping them at room temperature for a time results in a rapid breakdown of the segmentation area. The vacuoles are characteristic not only of the germinal area of chicken eggs, but also those of the duck (Chappelier, 1912) and turkey (Kosin, 1951). Kosin (1944) observed that the presence of a varying number of large and small vacuoles both along the periphery and in the center of the blastodisc of unincubated chicken eggs in extreme cases gives its surface a reticular appearance. The normal blastoderm in a recently-laid unincubated egg is characterized by distinct area pellucida and area opaca. A few small vacuoles may be present along the periphery of such blastoderms, although the area pellucida in freshly-laid chicken eggs has always been found to be devoid of them. Kosin (1944) sug-
gested that blastoderms with one or more large vacuoles on the periphery might be cases of early moribundity. This was demonstrated to be so by Gowe (1950) for the chicken, and by Kosin (1951) for the turkey on the basis of macro- and microscopic analysis of germ discs from eggs subjected to preincubation holding. Kosin (1951) observed that turkey blastoderms with numerous vacuoles around the periphery developed abnormally when incubated. In most cases, development ceased within the first three days of incubation. Additional support on this point came from the study on chickens by Krizenecky et al. (1955) who reported that high temperatures during preincubation storage increased the incidence of vacuoles in the unincubated germ discs. Kosin (1948) demonstrated that in addition to vacuolation, nuclear pycnosis is characteristic of moribund turkey blastoderms. The observations of Zelnik (1961) are in close agreement with those of the above-cited investigators. On the other hand, Weisbroth (1960) reported that vacuoles appear independently of storage; i.e. extended storage per se does not appear to induce vacuolation in normal blastoderms. He further found that peripheral vacuoles do not interfere with development of the embryo, at least during the first 40 hours of incubation. The study to be reported below had two objectives: (1) to observe the progressive changes in the gross morphological appearance of the chicken and the turkey blastoderms during the preincubation storage,
820
K. L. ARORA AND I. L. KOSIN
and (2) to explore relationships between the morphological characteristics of the blastoderms during storage and the subsequent early embryogenesis. MATERIALS AND METHODS
Downloaded from http://ps.oxfordjournals.org/ at :: on January 22, 2015
The experimental material was provided by eggs from mass-mated White Leghorn pullets and Broad Breasted Bronze turkeys. The eggs were collected between 8 a.m. and 12:30 p.m. following which the eggs were brought to a laboratory for the preliminary determination of the fertility status. The fertility of the eggs was checked through the window, measuring 1 cm.2, which was cut in the shell in the large end of the egg. The freehand drawing of each blastoderm was then made depicting such "landmarks" as presence or absence of vacuoles, their distribution, as well as shape of the two major surface areas: area pellucida and area opaca. To be selected for this study, the blastoderms had to be devoid of vacuoles except, perhaps, for a few minute ones along the periphery of the periblast. Following this classification, the selected eggs were sealed and transferred to storage at 13°C. and 85% relative humidity. During storage, the windows were opened at least four times and the morphological appearance of each blastoderm was noted and compared with the original drawing. If any observable difference occurred between the successive examinations, additional drawings were made to record them. Storage and the examination procedure continued until the blastoderm showed no further changes in its gross morphology. The egg was then incubated for 24-36 hours at 37.5°C. and 65% relative humidity, or order to correlate the macroscopic appearance of the blastoderm with subsequent development. Appropriate representative specimens were saved from many blastoderms for further histological and cytological studies.
RESULTS
The normal chicken or turkey blastoderm after prolonged storage at 13.0°C. was found to pass, sequentially, through a series of recognizable morphological changes which finally led to the condition of moribundity and death. This included the following stages, all of which, except stage A, were sooner or later observed in eggs subjected to storage: Stage A—Normal blastoderm: characterized by several, regularly outlined bands (periblastic and polar rings) and a more solid band of the area opaca (Figure 1); the degree of "solidity" varied among specimens, however. The semi-translucent area pellucida frequently showed centrally located Nucleus of Pander. This stage was found to be devoid of vacuoles in the area pellucida, although small vacuoles were sometimes present either along the periphery of the area opaca or in the periblast (Figure 2). Stage B—Initial fluxation: with few exceptions, all blastoderms following storage showed some decrease from the original dimensions. Invariably, the area pellucida was devoid of the vacuoles. Stage C—Conspicuous Nucleus of Pander: The centrally localized white yolk became conspicuous, filling almost completely the central area of the blastoderm. In many specimens, vacuoles became both larger and more numerous in the periblast area. The latter often merged into the area opaca (Figure 3). Some blastoderms, especially in turkey eggs, showed this condition even at oviposition. Stage D—Initial vacuolation: A few scattered vacuoles, small to medium in size, appeared in the area pellucida (Figure 4). The area opaca became "flattened"; sometimes it lacked vacuoles. The latter, however, were always present in the periblast. This stage was also observed in some specimens of blastoderms in turkey eggs at the
821 APPEARANCE OF BLASTODERMS
Downloaded from http://ps.oxfordjournals.org/ at :: on January 22, 2015
822
K. L. AEORA AND I. L. KOSIN
basis of gross morphological changes the chicken blastoderms were found to be more resistant to the detrimental effects of 7-10 day storage than the turkey blastoderms. However, the frequency of blastoderms showing "degenerative changes" (changes as described under D, E, and F stages above) increased progressively as the storage was extended to 14 days or beyond. Some turkey blastoderms, on the other hand, showed signs of moribund conditions, corresponding to stage E, within 2-3 days of initiation of storage. In fact, turkey hens have been observed to lay eggs that were invariably judged moribund after 3-4 days' storage at 13°C. Both chicken and turkey stage A, B and C blastoderms on incubation for 24-36 hours always developed normally. The decrease in diameter (influxation) associated with blastoderms subjected to cool storage environment (13°C.) did not interfere, on the whole, with normal embryogenesis, although a few embryos from stage C blastoderms showed delay in development. Some of the blastoderms in stage D, when incubated, developed abnormally or died. These blastoderms, at the time of the initiation of incubation, exhibited extensive vacuolation in the area opaca, with small to medium size vacuoles being present in the
Illustrations FIGS. 1-5. Diagrammatic drawings of turkey blastoderms. The horizontal bars in the lower right corner of Figures 1 and 5 indicate the actual diameter of an average blastoderm prior to and after 28 days' storage. FIGS. 1 and 2. Normal blastoderms at or shortly after oviposition. In Figure 1, the dark band represents the area opaca which surrounds the semi-translucent area pellucida. Beyond the area opaca is the periblast. In Figure 2, the Nucleus of Pander can be barely seen in the middle of the area pellucida. The area opaca is less prominent than in Figure 1. A number of vacuoles are visible along the periphery of the periblast. FIG. 3. The Nucleus of Pander is prominent. Some dislocation may occur between the relative positions of the areas pellucida and opaca. The vacuoles are numerous in the periblast. FIG. 4. The area opaca frequently "expands" at the expense of the periblast. A few vacuoles appear in the area pellucida. FIG. S. Vacuoles cover the entire area of the blastoderm. Boundaries between the various blastodermal regions are all but completely obliterated.
Downloaded from http://ps.oxfordjournals.org/ at :: on January 22, 2015
time of oviposition (Figure 6). This is a critical stage because such a blastoderm can be erroneously classified as either being a case of "preoviposital" mortality, or as "infertile." Stage E—Initial moribundity: Numerous vacuoles of increased size appear in the area pellucida as well as in the area opaca. The diameter of the blastoderm grew markedly smaller than it was originally, though all of its major "landmarks" still remained intact. The originally dense, broad area opaca sometimes became less distinctly outlined. The outer and inner periblastic rings were still visible, although they were becoming increasingly indistinct. Stage F—Terminal moribundity: Practically all of the structural details of the normal blastoderm, except polar and periblastic rings disappeared by now. The outlines of the area opaca and area pellucida were almost completely obliterated by the presence of large numbers of vacuoles of various sizes and shapes over the entire surface of the blastoderm. The blastoderm was reduced in overall dimensions. This stage resembled the appearance of different variants of preoviposital mortality or even biological (true) infertility (Figures S and 7)Despite wide individual variation, on the
823
APPEARANCE OF BLASTODERMS
* '
Fie. 6. An unincubated chicken blastoderm in which numerous vacuoles have started to make their appearance in the periblast after a 14-day storage. These vacuoles were not present before storage (Stage D). FIG. 7. An unincubated turkey blastoderm after 28 days' storage. Note the presence of numerous vacuoles and a somewhat reduced size in the germ disc. FIG. 8. A section from unincubated blastoderm in a chicken egg stored 14 days. Note vacuoles and surface discontinuity (gaps) in the epiblast of the blastoderm (Stage E ) . (X650)
area pellucida. The Nucleus of Pander appeared as a prominent vacuolated white spot. The compactness of the area pellucida was completely lost. Following were the chief developmental abnormalities observed in such blastoderms: Marked retardation in development, incomplete axial system or sometimes total absence of the system and disorganization or duplication of the growth centers. Occasionally, the cellular proliferation in the central and proximal areas continued without expansion of the blastoderm over the yolk. Such blastoderms showed extensive cellular moribundity or
necrosis at the zone of junction. Histological examination of the blastoderms showing vacuolation of the area pellucida indicated by the presence of the vacuoles of varying sizes in the blastoderms, and separation of area pellucida into scattered masses of cells (Figure 8). Upon incubation, the blastoderms in stages E and F resulted either in outright mortality (i.e. total failure to initiate development) or in disintegration of the blastoderm after brief initial development. These blastoderms had an appearance similar to that generally associated with
Downloaded from http://ps.oxfordjournals.org/ at :: on January 22, 2015
*
824
K. L. ARORA AND I. L. KOSIN
preoviposital mortality or biological (true) infertility. Numerous vacuoles were present over the entire surface of the blastoderm; the dimensions of these vacuoles were accentuated by incubation. DISCUSSION
SUMMARY
A sequential series of regressive changes in the gross morphology of blastoderms were observed in chicken and turkey eggs during preincubation storage at 13°C. This included the gradual loss of structural detail normally associated with the area pellucida and area opaca and the appearance, in increasing numbers, of vacuoles within these regions. The presence of such vacuoles, particularly in the area pellucida, was indicative of growing blastodermal moribundity. Blastoderms with large vacuoles in the area pellucida were incapable of further development. Some turkey hens consistently laid eggs of a type just described, i.e. their blastoderms were either moribund or "dead" at oviposition. REFERENCES Chappellier, A., 1912. La cicatricule de l'oeuf dans le croisement: canard de Rouen (Anas boschas var. domestica (L.) x canard de Barbarie (Cairina moschata (L.)) et les especes parentes. Comp. Rend. Assoc, franc. Avance. Sci. 40: 541-544. Gowe, R. S., 1950. Techniques for identifying fertile hens' eggs. Poultry Sci. 29: 409-413. Kosin, I. L., 1944. Macro- and microscopic methods of detecting fertility in unincubated hens' eggs. Poultry Sci. 23 : 266-269. Kosin, I. L., 1948. Parthenogenesis and abortive embryonic development in the domestic fowl. Proc. 8th World's Poultry Congress 1: 345351. Kosin, I. L., 1951. A study of the morphology of
Downloaded from http://ps.oxfordjournals.org/ at :: on January 22, 2015
Chicken and turkey blastoderms, even when kept under what is considered to be "optimal" conditions (13°C. and 85% relative humidity), do not remain static during storage, but continue to undergo regressive structural changes. In spite of rather wide individual variation in susceptibility to detrimental effect of storage, each "stored" blastoderm passes through a series of well-recognized regressive changes which first lead to the condition of moribundity and, finally, death. Blastoderms in the latter stage were characterized by the extreme vacuolation of the germinal disc area and loss of structural details. Extensive vacuolation at periphery of the disc or, more so, the presence of numerous vacuoles in the area opaca and, certainly, in the area pellucida were always associated with moribundity of the blastoderm: upon incubation, such blastoderms resulted in either outright mortality, or, at best, in abnormal embryogenesis. This supports the earlier view that vacuolation of the area pellucida is associated with moribundity (Kosin, 1951). In fact, results of the present study clearly show that the presence of large vacuoles within the area pellucida is indicative that the blastoderm is completely incapable of further development. To all intents and purposes such blastoderm is dead. The vacuoles probably result from a progressive disintegration and contraction of the protoplasmic mass in the germ disc, ultimately leading to its discontinuity or the formation of gaps in the blastoderm. Because the morphological changes that were observed in the aging turkey and chicken blastoderm in the present study
were consistent, both in their sequence of appearance, as well as their association with the capacity of the blastoderm to continue early development upon incubation at 37.5°C, the recent suggestion by Weisbroth and Kosin (1966) that the morphological appearance of the unincubated blastoderm can be used as a guide in determining its potential for subsequent normal embryogenesis, has received additional corroboration.
APPEARANCE OF BLASTODERMS moribund turkey blastoderms. Proc. 9th World's Poultry Congress 3 : 75-87. Krizenecky, J., J. Sajner, V. Orel and F. Musil, 1955. Detection of fertilized, infertile and nonfertilized eggs in biological control of incubation and in testing of commercial eggs. Ministerstvo Potravinarskeho Promyslu, Prague, pp. 112. Lecaillon, A., 1910. Le parthenogenese chez les oiseaux. Segmentation et degenerescence de l'oeuf non feconde. Arch, d'anat. micros. 12: 511-638. Oellacher, J., 1872. Die Ver'anderungen des unbefruchteten Keimes des Hiihnereies im Eileiter
825
und bei Bebrutungsversuchen. Zeit. f. wiss. Zool. 22: 181-234. Weisbroth, S. H., 1960. The morphological response of gastrulae from three genetic lines of Broad Breasted Bronze turkeys to preincubation storage. M.S. Thesis. Washington State University, Pullman, pp. 62. Weisbroth, S. H., and I. L. Kosin, 1966. Gross spatial changes in the turkey blastoderm following extended preincubation storage. Proc. Soc. Exper. Biol. Med. 121: 795-800. Zelnik, J., 1961. Morfologia zardocnych diskov morky. Pol'nohospodarstvo 8: 211-216.
S. S. RIZK, J. C. AYRES AND A. A. KRAFT Department of Dairy and Food Industry, Iowa State University, Ames, Iowa (Received for publication January 10, 1966)
HE contents of the egg as laid by the normal healthy hen are sterile and it is possible that the shell may not be contaminated during the act of laying (Stuart and McNally, 1943). However, the ovaries may occasionally become infected with SalmoneUae which may be transferred to the egg contents (Scott, 1930). Consequently, the incidence of Salmonellae on or in shell eggs depends upon three factors:
T
"Journal Paper No. J-5291 of the Iowa Agricultural and Home Economics Experiment Station, Ames. Project No. 1544; Center for Agricultural and Economic Development cooperating. A report of work done under contract with the U. S. Department of Agriculture and authorized by the Research and Marketing Act of 1946. The contract is being supervised by the Western Utilization Research and Development Division of the Agricultural Research Service. Reference to a company or product name does not imply approval or recommendation of the product by the U. S. Department of Agriculture to the exclusion of others that may be suitable.
the environmental condition of the eggs, handling and storage, and the physical structure of the shell. Studies have been made on the ability of egg spoilage bacteria to utilize the material of the shell membranes for growth (Stokes and Osborne, 1956; Elliott and Brant, 1957; Garibaldi and Stokes, 1958; Board et al., 1964). Lancaster and Crabb (1953) demonstrated that although bacteria penetrated the shell, only rarely did organisms pass through the shell membrane into the white of the eggs. Stokes and Osborne (1956) believed that the egg shell membrane delayed the penetration of Salmonellae to egg contents by functioning as a mechanical rather than as a bactericidal barrier. Yet Salmonellae have been isolated from spray dried whole egg powder manufactured in the United States (Gibbons and Moore, 1944; Schneider, 1946). The source of contamination of spray dried whole egg with Salmonellae was investigated by Solowey et
Downloaded from http://ps.oxfordjournals.org/ at :: on January 22, 2015
Effect of Holding Condition on the Development of Salmonellae in Artificially Inoculated Hens' Eggs 1