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A Heritable Difference in the Resistance of Turkey Blastoderms to Prolonged Preincubation Storage—Cytological Evidence1,2
A Heritable Difference in the Resistance of Turkey Blastoderms to Prolonged Preincubation Storage— Cytological Evidence1'2 K. L. ARORA3 AND I. L. KOSIN Department of Animal Sciences, Washington State University, Pullman, Washington 99163 (Received for publication May IS, 1968)
OSIN and Arora (1966) have reported that following a period of extended storage, eggs from a Broad Breasted Bronze (B.B.B.) line selected for large body size at 24 weeks of age (line 2) produced, in the first three days of incubation, more dead and abnormal embryos than those from a B.B.B. line selected for high hatchability (line 4). Furthermore, the surviving normal embryos in the latter were consistently superior in their rate of early development. The purpose of the present paper is (a) to augment our earlier observations on the gross appearance of the affected blastoderms and (b) to report the results of a cytological study of the blastoderms, from the same two genetically isolated lines (cf. Kosin and Arora, 1966) subjected in-ovo, first, to extended preincubation storage and, then, to brief incubation. The latter treatment was introduced to test the capacity of cells in "aged" blastoderms to recuperate during incubation, as measured by their mitotic activity at a time when it can be expected to be at a high level. MATERIALS AND METHODS
The experimental material was provided by the same two B.B.B. lines, line 2 and 'Scientific Paper No. 3125, College of Agriculture, Washington State University, Pullman. Project No. 12SS. 2 This investigation was supported in part by Research Grant 5544 from the Division of General Medical Sciences, U.S.P.H. Service. 'Present Address: College of animal Sciences, Agricultural University, Hissar, Haryana, India.
line 4, mentioned above. In general, the procedure for gathering the necessary data followed the plan already described in the earlier papers of this series (Kosin and Arora, 1966; Arora and Kosin, 1966). The eggs were collected daily at hourly intervals between 9 a.m. and 4:30 p.m., and transferred to an egg storage room kept at 13°C. and 18% relative humidity. Series 1. Following an overnight storage, the eggs were brought into the laboratory where the ambient temperature was about 22°C, and held on their sides for 2-3 hours. This was to float the blastoderm to the top. Later, a small window (1 cm.2) was cut in the shell of the upper surface of the egg. The cut piece and its shell membranes were then deflected, to permit the measuring of the diameter of the blastoderm (cf. Weisbroth and Kosin, 1966) and, where necessary, its further morphological examination. The latter was done according to the method developed in this laboratory (Arora and Kosin, 1966). After this, the cut shell and membranes were replaced and sealed with paraffin, and the eggs were then stored at 13°C. and 80% relative humidity. The first examination was carried out after 7 days' storage. At that time, the blastoderms were again measured and their morphogenetic status evaluated. The eggs containing blastoderms which were judged to be morphologically "normal" were resealed and stored for additional 7 days. The latter treatment was followed by the final examination for any evidence of degenerative changes.
2000
Downloaded from http://ps.oxfordjournals.org/ at Michigan State University on January 18, 2015
K
2001
BLASTODERM RESISTANCE TO STORAGE
RESULTS AND DISCUSSION
The data summarized in Table 1 show that irrespective of genetic line, the frequency of blastoderms with gross morphoTABLE 1.—Frequency of turkey blastoderms showing degenerative changes following 7 and 14 days of storage. Series 1
line
Total number of blastoderms examined
2
64
4
76
Days of storage 7
14
39.1" (25) 21.1 (16)
60.9 (39) 51.3 (39)
• Blastoderms, expressed as percent of the original total number of specimens, which showed degenerative changes after a given storage period; figures in parentheses, on the lower line, represent the actual number of degenerative blastoderms involved in each classification.
TABLE 2.—Decrease in the blastoderm diameter after 7-day preincubation storage. Series 1 Mean diameter (mm.) T . u n
2 4
Total number of blastoderms 51 48
Before storage
After storage
3.74 3.70
3.60 3.54
logical changes rose as the storage length was extended from 7 to 14 days. On the basis of our earlier experience (Arora and. Kosin, 1966), we adjudged these changes to be degenerative: there was loss in structural detail and increase in the number of vacuoles which appeared in the area pellucida and area opaca. In addition, these data suggest that the blastoderms from line 4 suffered less in this respect than their counterparts from line 2: 25 of the 64 line 2 blastoderms (39.1%) showed degenerative changes after one week of storage and all of the remaining 39 (or 60.9% of the total sample) were so affected. The corresponding percentages for line 4 blastoderms were 21.1 and 51.3. The Chi-square test showed these differences to be statistically significant (P < 0.05). Influxation (i.e., decrease) in the mean blastoderm diameter followed a 7-day preincubation storage in both lines (Table 2). The interline difference in influxation was not statistically significant, however. The cytological examination of the sections of blastoderms incubated for 5 hours after preincubation storage revealed an increased frequency of fragmented and of necrotic nuclei (Table 3). Increases in the latter value for the 14-day and 21-day groups were greater in line 2 blastoderms (P < 0.05). The same was true for the fragmented nuclei classification on the 14-day group. The cytological analysis cited above has provided a clear-cut corroboration, now on cellular basis, of the already reported heri-
Downloaded from http://ps.oxfordjournals.org/ at Michigan State University on January 18, 2015
Series 2. The eggs were assigned at random to one of the following storage periods: 1, 7, 14 and 21 days at 13°C. and 80% relative humidity. After this, the eggs were incubated for 5 hours at 37°C. and 65% relative humidity. The next step was to excise the blastoderms and fix them for 4 hours in a mixture of absolute ethanol and acetic acid (19:1) at - 6 ° C . to - 8 ° C . (cf. Wolman and Behar, 1951). The blastoderms, 10-12 per line—storage classification, were then dehydrated overnight at 22°C. in absolute ethanol. Finally, the material was cleared, imbedded in paraffin and sectioned at 7 [x,. The staining was done according either to the procedure developed by Conn et al. (1960), involving the use of Delafield's haematoxylin and eosin, or the method for the Feulgen-Fast Green reaction described by Humason (1962). The frequency of necrotic and fragmented nuclei in the sections, expressed in percent, was determined on the basis of at least 500 nuclei from 30 randomly selected unit areas in several sections of each blastoderm.
2002
K. L. ARORA AND I. L. KOSIN
TABLE 3.—Frequency (%) of fragmented and necrotic nuclei in line 2 and line 4 blastoderms after 5 hours of incubation of stored eggs. Series 2 Type of nuclei
Storage length (days) 7
14
21
Dead Line 2 Line 4
3.66 3.28
3.52 3.14
4.86 3.79
7.80 5.70
Fragmented Line 2 Line 4
1.12 1.31
2.00 2.22
4.38 2.50
2.08 2.34
table difference in the resistance of turkeyblastoderms to the deleterious effect of prolonged preincubation storage (cf. Kosin and Arora, 1966). Again, the blastoderms of a genetic line known for its low hatchability (and, consistently, for large mature body size) suffered more from the storage treatment than the blastoderms from a line characterized by relatively high hatchability (and, incidentally, small body size): the superiority of the latter (line 4) was evident both in terms of a lower incidence of necrotic cells and of nuclear fragmentation. The demonstrated existence of this differential in the incidence of necrotic cells between the two genetic lines is another step in providing a biological explanation for the difference in the livability of their embryos and, hence, their hatchability. SUMMARY
Eggs from two genetically isolated lines of Broad Breasted Bronze turkeys were involved in the present study: "High-hatch-
REFERENCES Arora, K. L., and I. L. Kosin, 1966. Changes in the gross morphological appearance of chicken and turkey blastoderms during preincubation storage. Poultry Sci. 4S: 819-825. Conn, H. J., M. A. Darrow and V. M. Emmel, 1960. Staining Procedures Used by the Biological Stain Commission. The Williams and Wilkins Company, Baltimore, pp. 289. Humason, G. L., 1962. Animal Tissue Techniques. W. H. Freeman and Company, San Francisco, pp. 468. Kosin, I. L., and K. L. Arora, 1966. The pattern of early embryonic development in two genetically isolated lines of Broad Breasted Bronze turkeys. Poultry Sci. 45: 622-629. Weisbroth, S. H., and I. L. Kosin, 1966. Gross spatial changes in the turkey blastoderm following extended pre-incubation storage. Proc. Soc. Exper. Biol. Med. 121: 795-800. Wolman, M., and A. Behar, 1951. Methods of fixation for enzyme-cytochemistry and cytology. Exper. Cell Res. 3:619-621.
NEWS AND NOTES (continued from page 1999) U.S.D.A. NOTES Dr. W. P. Flatt was recently designated as Assistant Director of the Animal Husbandry Re-
search Division, Agricultural Research Service, U.S. Department of Agriculture. He was the Project Leader of the Energy Metabolism of the Dairy
(continued on page 2012)
Downloaded from http://ps.oxfordjournals.org/ at Michigan State University on January 18, 2015
1
ability" line 4 and "Low-hatchability" line 2. The eggs had been subjected, before analysis, to storage up to 21 days' duration at 13°C. and 80% relative humidity. Bulk of the observations was based on unincubated blastoderms from these eggs, although one series involved storage eggs subsequently incubated for 5 hours at 37°C. and 65% relative humidity. The results indicate that when gross morphology and gross cytology of the affected blastoderms were used as criteria, line 4 specimens proved to be relatively more resistant to storage stress than those from line 2: even after 21 days of storage, the former blastoderms showed fewer gross abnormalities and lower frequency of necrotic and fragmented cells.
OSIN and Arora (1966) have reported that following a period of extended storage, eggs from a Broad Breasted Bronze (B.B.B.) line selected for large body size at 24 weeks of age (line 2) produced, in the first three days of incubation, more dead and abnormal embryos than those from a B.B.B. line selected for high hatchability (line 4). Furthermore, the surviving normal embryos in the latter were consistently superior in their rate of early development. The purpose of the present paper is (a) to augment our earlier observations on the gross appearance of the affected blastoderms and (b) to report the results of a cytological study of the blastoderms, from the same two genetically isolated lines (cf. Kosin and Arora, 1966) subjected in-ovo, first, to extended preincubation storage and, then, to brief incubation. The latter treatment was introduced to test the capacity of cells in "aged" blastoderms to recuperate during incubation, as measured by their mitotic activity at a time when it can be expected to be at a high level. MATERIALS AND METHODS
The experimental material was provided by the same two B.B.B. lines, line 2 and 'Scientific Paper No. 3125, College of Agriculture, Washington State University, Pullman. Project No. 12SS. 2 This investigation was supported in part by Research Grant 5544 from the Division of General Medical Sciences, U.S.P.H. Service. 'Present Address: College of animal Sciences, Agricultural University, Hissar, Haryana, India.
line 4, mentioned above. In general, the procedure for gathering the necessary data followed the plan already described in the earlier papers of this series (Kosin and Arora, 1966; Arora and Kosin, 1966). The eggs were collected daily at hourly intervals between 9 a.m. and 4:30 p.m., and transferred to an egg storage room kept at 13°C. and 18% relative humidity. Series 1. Following an overnight storage, the eggs were brought into the laboratory where the ambient temperature was about 22°C, and held on their sides for 2-3 hours. This was to float the blastoderm to the top. Later, a small window (1 cm.2) was cut in the shell of the upper surface of the egg. The cut piece and its shell membranes were then deflected, to permit the measuring of the diameter of the blastoderm (cf. Weisbroth and Kosin, 1966) and, where necessary, its further morphological examination. The latter was done according to the method developed in this laboratory (Arora and Kosin, 1966). After this, the cut shell and membranes were replaced and sealed with paraffin, and the eggs were then stored at 13°C. and 80% relative humidity. The first examination was carried out after 7 days' storage. At that time, the blastoderms were again measured and their morphogenetic status evaluated. The eggs containing blastoderms which were judged to be morphologically "normal" were resealed and stored for additional 7 days. The latter treatment was followed by the final examination for any evidence of degenerative changes.
2000
Downloaded from http://ps.oxfordjournals.org/ at Michigan State University on January 18, 2015
K
2001
BLASTODERM RESISTANCE TO STORAGE
RESULTS AND DISCUSSION
The data summarized in Table 1 show that irrespective of genetic line, the frequency of blastoderms with gross morphoTABLE 1.—Frequency of turkey blastoderms showing degenerative changes following 7 and 14 days of storage. Series 1
line
Total number of blastoderms examined
2
64
4
76
Days of storage 7
14
39.1" (25) 21.1 (16)
60.9 (39) 51.3 (39)
• Blastoderms, expressed as percent of the original total number of specimens, which showed degenerative changes after a given storage period; figures in parentheses, on the lower line, represent the actual number of degenerative blastoderms involved in each classification.
TABLE 2.—Decrease in the blastoderm diameter after 7-day preincubation storage. Series 1 Mean diameter (mm.) T . u n
2 4
Total number of blastoderms 51 48
Before storage
After storage
3.74 3.70
3.60 3.54
logical changes rose as the storage length was extended from 7 to 14 days. On the basis of our earlier experience (Arora and. Kosin, 1966), we adjudged these changes to be degenerative: there was loss in structural detail and increase in the number of vacuoles which appeared in the area pellucida and area opaca. In addition, these data suggest that the blastoderms from line 4 suffered less in this respect than their counterparts from line 2: 25 of the 64 line 2 blastoderms (39.1%) showed degenerative changes after one week of storage and all of the remaining 39 (or 60.9% of the total sample) were so affected. The corresponding percentages for line 4 blastoderms were 21.1 and 51.3. The Chi-square test showed these differences to be statistically significant (P < 0.05). Influxation (i.e., decrease) in the mean blastoderm diameter followed a 7-day preincubation storage in both lines (Table 2). The interline difference in influxation was not statistically significant, however. The cytological examination of the sections of blastoderms incubated for 5 hours after preincubation storage revealed an increased frequency of fragmented and of necrotic nuclei (Table 3). Increases in the latter value for the 14-day and 21-day groups were greater in line 2 blastoderms (P < 0.05). The same was true for the fragmented nuclei classification on the 14-day group. The cytological analysis cited above has provided a clear-cut corroboration, now on cellular basis, of the already reported heri-
Downloaded from http://ps.oxfordjournals.org/ at Michigan State University on January 18, 2015
Series 2. The eggs were assigned at random to one of the following storage periods: 1, 7, 14 and 21 days at 13°C. and 80% relative humidity. After this, the eggs were incubated for 5 hours at 37°C. and 65% relative humidity. The next step was to excise the blastoderms and fix them for 4 hours in a mixture of absolute ethanol and acetic acid (19:1) at - 6 ° C . to - 8 ° C . (cf. Wolman and Behar, 1951). The blastoderms, 10-12 per line—storage classification, were then dehydrated overnight at 22°C. in absolute ethanol. Finally, the material was cleared, imbedded in paraffin and sectioned at 7 [x,. The staining was done according either to the procedure developed by Conn et al. (1960), involving the use of Delafield's haematoxylin and eosin, or the method for the Feulgen-Fast Green reaction described by Humason (1962). The frequency of necrotic and fragmented nuclei in the sections, expressed in percent, was determined on the basis of at least 500 nuclei from 30 randomly selected unit areas in several sections of each blastoderm.
2002
K. L. ARORA AND I. L. KOSIN
TABLE 3.—Frequency (%) of fragmented and necrotic nuclei in line 2 and line 4 blastoderms after 5 hours of incubation of stored eggs. Series 2 Type of nuclei
Storage length (days) 7
14
21
Dead Line 2 Line 4
3.66 3.28
3.52 3.14
4.86 3.79
7.80 5.70
Fragmented Line 2 Line 4
1.12 1.31
2.00 2.22
4.38 2.50
2.08 2.34
table difference in the resistance of turkeyblastoderms to the deleterious effect of prolonged preincubation storage (cf. Kosin and Arora, 1966). Again, the blastoderms of a genetic line known for its low hatchability (and, consistently, for large mature body size) suffered more from the storage treatment than the blastoderms from a line characterized by relatively high hatchability (and, incidentally, small body size): the superiority of the latter (line 4) was evident both in terms of a lower incidence of necrotic cells and of nuclear fragmentation. The demonstrated existence of this differential in the incidence of necrotic cells between the two genetic lines is another step in providing a biological explanation for the difference in the livability of their embryos and, hence, their hatchability. SUMMARY
Eggs from two genetically isolated lines of Broad Breasted Bronze turkeys were involved in the present study: "High-hatch-
REFERENCES Arora, K. L., and I. L. Kosin, 1966. Changes in the gross morphological appearance of chicken and turkey blastoderms during preincubation storage. Poultry Sci. 4S: 819-825. Conn, H. J., M. A. Darrow and V. M. Emmel, 1960. Staining Procedures Used by the Biological Stain Commission. The Williams and Wilkins Company, Baltimore, pp. 289. Humason, G. L., 1962. Animal Tissue Techniques. W. H. Freeman and Company, San Francisco, pp. 468. Kosin, I. L., and K. L. Arora, 1966. The pattern of early embryonic development in two genetically isolated lines of Broad Breasted Bronze turkeys. Poultry Sci. 45: 622-629. Weisbroth, S. H., and I. L. Kosin, 1966. Gross spatial changes in the turkey blastoderm following extended pre-incubation storage. Proc. Soc. Exper. Biol. Med. 121: 795-800. Wolman, M., and A. Behar, 1951. Methods of fixation for enzyme-cytochemistry and cytology. Exper. Cell Res. 3:619-621.
NEWS AND NOTES (continued from page 1999) U.S.D.A. NOTES Dr. W. P. Flatt was recently designated as Assistant Director of the Animal Husbandry Re-
search Division, Agricultural Research Service, U.S. Department of Agriculture. He was the Project Leader of the Energy Metabolism of the Dairy
(continued on page 2012)
Downloaded from http://ps.oxfordjournals.org/ at Michigan State University on January 18, 2015
1
ability" line 4 and "Low-hatchability" line 2. The eggs had been subjected, before analysis, to storage up to 21 days' duration at 13°C. and 80% relative humidity. Bulk of the observations was based on unincubated blastoderms from these eggs, although one series involved storage eggs subsequently incubated for 5 hours at 37°C. and 65% relative humidity. The results indicate that when gross morphology and gross cytology of the affected blastoderms were used as criteria, line 4 specimens proved to be relatively more resistant to storage stress than those from line 2: even after 21 days of storage, the former blastoderms showed fewer gross abnormalities and lower frequency of necrotic and fragmented cells.