Growth of the Chick Embryo In Vitro B . E . D U N N AND M. A. BOONE
Department of Poultry Science, Clemson University, Clemson, South Carolina 29631 (Received for publication September 15, 1975)
POULTRY SCIENCE 55: 1067-1071, 1976
INTRODUCTION
L
ONG-TERM cultivation of the chick embryo in vitro facilitates chorioallantoic grafting by eliminating the need to locate a graft through a small hole in the egg shell. Boone et al. (1964) used an in vitro technique to study the effects of thalidomide on developing chick embryos. Recently, applications have involved studies of tumor-induced angiogenesis (Auerbach et al., 1975) and culture of central nervous tissue (Corner and Richter, 1973). Cultivation of the chick embryo also promises to be useful in behavioral, teratological, and physiological studies of development. The available techniques (Ramsey, 1970; Corner and Richter, 1973; Dunn, 1974; Auerbach et al, 1974; Dunn and Boone, 1975) are subject to improvement, however. Ramsey (1970) cultured three-day embryos through a maximum of nine and one-half days of total incubation in glass beakers but did not indicate the final developmental stage of cultured embryos. Corner and Richter (1973) reported 50% livability of two-day embryos in specially-designed ceramic containers through Hamburger-Hamilton stage 30 (seven
Published with permission of the Director of the S.C. Agricultural Experiment Station as technical contribution number 1296.
days), with the best embryos developing to stage 41 (15 days). Dunn (1974) using threeday embryos suspended in plastic wrap in 5 cm. diameter chambers, reported 70-80% survival to 15 days of total incubation with a maximum development to stage 41. Auerbach et al. (1974) cultured three- to four-day embryos in standard petri dishes and obtained approximately 40% livability through 14 days of total incubation with maximum development to stage 44 (18 days). Dunn and Boone (1975), using three-day embryos, reported 54% livability to 13 days of total incubation and maximum development to stage 39 (13 days). The present study includes, for the first time, a direct comparison of wet weight, dry weight, and right third toe length between cultured and control embryos based on the Hamburger-Hamilton series of developmental stages (Hamilton, 1952).
MATERIALS AND METHODS The present improved technique involves use of simple chambers constructed of plastic pipe in which embryos are suspended in plastic wrap. The cumbersome saturation humidity chambers described previously (Dunn, 1974) have been eliminated thereby increasing the incubator capacity from 24 chambers to approximately 100. Chamber
1067
Downloaded from http://ps.oxfordjournals.org/ at University of Pennsylvania Library on January 22, 2015
ABSTRACT An improved technique is described for in vitro culture of the chick embryo from three days through 21 days of total incubation (three days in shell plus 18 days in culture). Mean survival time for cultured embryos is 18 days of total incubation, and mean morphological stage is Hamburger-Hamilton stage 41 (15 days). Mean measurements of wet weight, dry weight, and right third toe length for stage 45 (partial or complete abdominal yolk sac retraction, 19-20 days) cultured embryos are comparable with corresponding means for stage 42-43 (16-17 days) embryos from control intact eggs. Possible causes of growth retardation in vitro are briefly discussed.
1068
B. E. DUNN AND M. A. BOONE
surface area is enlarged from 5.0 cm. (Dunn, 1974) to 7.8 cm. in accordance with the reports of Corner and Richter (1973) and Auerbach et al. (1974) who used surface diameters of 8 cm. and 10 cm., respectively. Fertile eggs were selected for uniformity of color, size, and weight (53.35 ± 3.37 g.) from two flocks of Single Comb White Leghorns. Eggs to be cultured were incubated in a Robbins 131 incubator at 37.5° C , 60% relative humidity, and were turned hourly for 72 ± 1 hours before use. The remaining control intact eggs were incubated for 10 to 21 days to allow normal embryonic growth and development. Methods of removal of egg contents from shells were similar to those previously described (Dunn, 1974) except that egg contents suspended in Kitchen Kraft™ plastic wrap were supported in 5.0 cm. tall, 7.8 cm. diameter tripods constructed of plastic pipe (Figure 1). Tripods were covered with the top of a 100 x 15 mm. plastic disposable petri dish allowing clearance for gas exchange. Embryos were cultured at 37° C. in a humidified atmosphere with 1-2% CO, (Auerbach et al., 1974) in a Forma C 0 2 incubator. No media or antibiotics were added to the cultured egg contents. Embry-
Four trials, utilizing a total of 111 cultured three-day embryos, were conducted. Survival time and morphological stage only were recorded in the first two trials consisting of a total of 31 embryos. In the third and fourth trials, consisting of 48 and 32 embryos, respectively, measurements also included wet weight, dry weight, and right third toe length. The same parameters were measured in two series of 65 and 61 embryos from control intact eggs incubated for 10 to 21 days under standard conditions of incubation. Analysis of variance for all data was performed using the methods of Steel and Torrie (1960).
RESULTS AND DISCUSSION The combined data for survival time and morphological stage for all cultured embryos are presented in Figure 2, which illustrates that 75% of the embryos survived through 17 days, 50% survived through 18.5 days,
Downloaded from http://ps.oxfordjournals.org/ at University of Pennsylvania Library on January 22, 2015
FIG. 1. View of a three-day embryo and egg contents supported in plastic wrap in a 5.0 cm. tall, 7.8 cm. diameter plastic tripod. The petri dish cover has been removed to facilitate photography.
onic mortality was monitored at least twice daily. Embryos were considered dead when they failed to exhibit gross movement, and there were no signs of circulation in the chorioallantoic vascular system. Survival time and morphological stage of each embryo were estimated to nearest one-half day and nearest whole Hamburger-Hamilton stage, respectively. Staging criteria used were general body conformity, development of the down, right third toe length, and yolk sac retraction. Dead cultured embryos and embryos from control intact eggs were dissected from adherent membranes. Yolk sacs were removed from stage 45-46 control and cultured embryos. Embryos were rinsed thoroughly to remove adherent egg contents, then blotted on a damp towel to remove excess water. Wet weight was determined to the nearest 0.01 g. and length of the third toe of the right foot measured to the nearest 0.1 mm. Dry weight was determined to the nearest 0.1 mg. after drying at 100° C. for 24 hours.
EMBRYO GROWTH IN VITRO 100
L
—1_
— 1 _
90. 80.
—
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~l_
DAYS OF TOTAL INCUBATION
i
70.
1
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l_
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< >
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1 I 1
.
40. 30.
I_
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31
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i i i i 1 ! 14 15 16 12 9 10 II DAYS OF TOTAL INCUBATION
•
'
i
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18
19
42 43 36 37 38 39 4 0 41 HAMBURGER-HAMILTON STAGE
44
i
i 35
i
IF
1
i
T
i
i 45
i _ *-^ 20 21 i 46
FIG. 2. Survival to various incubation ages and Hamburger-Hamilton stages of development of 111 cultured chick embryos.
and 3% survived to 21 days. Figure 2 further indicates that morphological development of cultured embryos lags behind the corresponding total incubation age by two to three days, such that an embryo which survives for 15 days of total incubation may possess the morphology of a control 13 day embryo (Hamburger-Hamilton stage 39), while an embryo which survives for 19 days may have the morphology of a control 16 day embryo (Hamburger-Hamilton stage 42). The maximum morphological stage we have observed in vitro is stage 45 (Figure 3), based on partial or complete abdominal retraction of the yolk sac, which normally occurs on day 19-20 (Hamilton, 1952). Mean survival time and mean morphological stage for all cultured embryos were 18 days of total incubation (three days in shell plus 15 days in culture) and HamburgerHamilton stage 41 (15 days), respectively. No significant differences (P > .05) occurred in mean survival time or in mean morphologi-
FIG. 3. Three cultured stage 45 embryos (b, c, d) exhibiting complete abdominal yolk sac retraction compared with a control stage 45 embryo (a).
Downloaded from http://ps.oxfordjournals.org/ at University of Pennsylvania Library on January 22, 2015
Z> CO
1 1
1070
B. E. DUNN AND M. A. BOONE
TABLE 1.—A comparison of mean wet weight, dry weight, and toe length between control and cultured embryos with respect to Hamburger-Hamilton (H-H) stage Wet weight (g.)
H-H stage
Age (days)
Control
29 36 37 38 39 40 41 42 43 44 45 46
6 10 11 12 13 14 15 16 17 18 19-20 21
2.64 3.95 5.43 7.84 10.85 14.33 16.77 21.11 23.70 30.49 39.27
Dry weight (g.)
Culture
Control
4.12 5.94 7.85 9.59 11.41** 13.15** 15.63** 16.56** 15.79**
.1674 .2580 .4249 .6930 1.2449 1.8857 2.7200 3.6020 4.2855 5.5471 7.0132
Culture
Toe length (mm.) Culture
No. embryos
Control
Control
8.4 10.1 12.1 14.5 16.3 19.2 20.8 22.8 24.6
12 12 12 11 11 11 11 11 11 20 4 126
Culture 1
8.2 10.7 12.0 14.0 14.7** 16.9** 17.0** 16.4**
1 3 6 13 11 22 12 1 10 80
*(P < .05). **(P < .01).
cal stage among the four trials. Mean wet weight, mean dry weight, and mean right third toe length data for the two series of control and cultured embryos are presented in Table 1. Table 1 indicates that no significant differences (P > .05) between control and cultured embryos occurred with respect to wet weight prior to stage 41, with respect to dry weight prior to stage 40, or with respect to toe length prior to stage 42. Comparison between cultured and control embryos indicates that the reduction in wet weight of cultured embryos was highly significant (P < .01) at stage 41 and at subsequent stages and the reduction in dry weight of cultured embryos was significant (P < .05) at stage 40 and highly significant (P < .01) at subsequent stages. Toe length reduction in cultured embryos compared with controls was highly significant (P < .01) at stage 42 and at subsequent stages. The difficulty of accurately determining the stage of older cultured embryos must be noted. While our data indicate that cultured stage 43-44 embryos approximate the mean wet weight, dry weight, and toe length of control stage 42 embryos (Table 1), the general body conformity and down development of the cultured embryos is more advanced
than that of control stage 42 embryos. Our data further indicate that cultured embryos exhibiting partial or complete yolk sac retraction (stage 45) have the average wet and dry weights of control stage 41-42 embryos and the average toe length of control stage 42 embryos. Retracted yolk sacs frequently are reduced in size due to leakage of their contents during culture. Retardation of growth in vitro may be due to one or a combination of factors. Temperature and/or humidity requirements for cultured embryos may be different from those for intact eggs. Gas exchange in vitro may be restricted as cultured egg contents have an average of 38 cm. 2 of surface area unobstructed by contact with the supporting plastic wrap, which is about one-half the surface area of a standard egg (Wangensteen et at, 1970/71). It is suggested that gas exchange through the supporting plastic wrap may enhance embryonic development in these chambers. The increase and decrease, respectively, in air cell P C 0 2 and P ^ thought to stimulate pulmonary respiration during the latter half of incubation (Freeman and Vince, 1974), do not occur in vitro. Perhaps simulation of the air cell gas environment would enhance embryonic growth and development.
Downloaded from http://ps.oxfordjournals.org/ at University of Pennsylvania Library on January 22, 2015
.2517 .4188 .6669 1.0073* 1.5011** 1.8122** 2.1850** 2.7826** 2.4037**
EMBRYO GROWTH IN VITRO
Leakage of yolk sac contents in vitro probably decreases the availability of yolk nutrients to the developing embryo. Transfer of albumen through the sero-amniotic connection is delayed in unturned intact eggs resulting in inhibited albumen uptake and greatly decreased hatchability (Randies and Romanoff, 1950). The combination of abnormal egg geometry and lack of turning may restrict albumen assimilation in vitro. Finally, of the calcium in the newly-hatched chick (Romanoff, 1967; Simkiss, 1967), is unavailable for skeletal calcification in vitro. ACKNOWLEDGMENTS The authors wish to thank Dr. W. E. Johnston of the Experimental Statistics Unit for statistical analysis of the data.
REFERENCES Auerbach, R., R. Arensman, L. Kubai and J. Folkman, • 1975. Tumor-induced angiogenesis: lack of inhibition by irradiation. Int. J. Cancer, 15: 241-245. Auerbach, R., L. Kubai, D. Knighton and J. Folkman, 1974. A simple procedure for long-term cultivation of chicken embryos. Devel. Biol. 41: 391-394. Boone, M. A., M. Hammond and B. D. Barnett, 1964.
The effect of thalidomide on chickens and embryos. Poultry Sci. 43: 506-507. Corner, M. A., and A. P. J. Richter, 1973. Extended survival of the chick embryo in vitro. Experientia, 29: 467-468. Dunn, B. E., 1974. Technique for shell-less culture of the 72-hour avian embryo. Poultry Sci. 53: 409412. Dunn, B. E., and M. A. Boone, 1975. Shell-less culture of the avian embryo. Poultry Sci. 54: 1336. Freeman, B. M., and M. A. Vince, 1974. Development of the Avian Embryo. John Wiley and Sons, New York, New York. Hamilton, H. L., 1952. Lillie's Development of the Chick. Holt, Rinehart, and Winston, New York, New York. Ramsey, J. B., 1970. The effects of normal atmosphere and increased C 0 2 on chick embryos incubated in opened shells and in vitro. M.S. Thesis, Clemson University. Randies, C. A., and A. L. Romanoff, 1950. Some physical aspects of the amnion and allantois of the developing chick embryo. J. Exp. Zool. 114: 87-101. Romanoff, A. L., 1967. Biochemistry of the Avian Embryo. John Wiley and Sons, New York, New York. Simkiss, K., 1967. Calcium in Reproductive Physiology. Chapman and Hall, London, England. Steel, R. G. D., and J. H. Torrie, 1960. Principles and Procedures of Statistics. McGraw-Hill, New York, New York. Wangensteen,0. D., D. Wilsonand H. Rahn, 1970/71. Diffusion of gases across the shell of the hen's egg. Resp. Physiol. 11: 16-30.
Downloaded from http://ps.oxfordjournals.org/ at University of Pennsylvania Library on January 22, 2015
egg shell calcium, which contributes 75-80%
1071
Department of Poultry Science, Clemson University, Clemson, South Carolina 29631 (Received for publication September 15, 1975)
POULTRY SCIENCE 55: 1067-1071, 1976
INTRODUCTION
L
ONG-TERM cultivation of the chick embryo in vitro facilitates chorioallantoic grafting by eliminating the need to locate a graft through a small hole in the egg shell. Boone et al. (1964) used an in vitro technique to study the effects of thalidomide on developing chick embryos. Recently, applications have involved studies of tumor-induced angiogenesis (Auerbach et al., 1975) and culture of central nervous tissue (Corner and Richter, 1973). Cultivation of the chick embryo also promises to be useful in behavioral, teratological, and physiological studies of development. The available techniques (Ramsey, 1970; Corner and Richter, 1973; Dunn, 1974; Auerbach et al, 1974; Dunn and Boone, 1975) are subject to improvement, however. Ramsey (1970) cultured three-day embryos through a maximum of nine and one-half days of total incubation in glass beakers but did not indicate the final developmental stage of cultured embryos. Corner and Richter (1973) reported 50% livability of two-day embryos in specially-designed ceramic containers through Hamburger-Hamilton stage 30 (seven
Published with permission of the Director of the S.C. Agricultural Experiment Station as technical contribution number 1296.
days), with the best embryos developing to stage 41 (15 days). Dunn (1974) using threeday embryos suspended in plastic wrap in 5 cm. diameter chambers, reported 70-80% survival to 15 days of total incubation with a maximum development to stage 41. Auerbach et al. (1974) cultured three- to four-day embryos in standard petri dishes and obtained approximately 40% livability through 14 days of total incubation with maximum development to stage 44 (18 days). Dunn and Boone (1975), using three-day embryos, reported 54% livability to 13 days of total incubation and maximum development to stage 39 (13 days). The present study includes, for the first time, a direct comparison of wet weight, dry weight, and right third toe length between cultured and control embryos based on the Hamburger-Hamilton series of developmental stages (Hamilton, 1952).
MATERIALS AND METHODS The present improved technique involves use of simple chambers constructed of plastic pipe in which embryos are suspended in plastic wrap. The cumbersome saturation humidity chambers described previously (Dunn, 1974) have been eliminated thereby increasing the incubator capacity from 24 chambers to approximately 100. Chamber
1067
Downloaded from http://ps.oxfordjournals.org/ at University of Pennsylvania Library on January 22, 2015
ABSTRACT An improved technique is described for in vitro culture of the chick embryo from three days through 21 days of total incubation (three days in shell plus 18 days in culture). Mean survival time for cultured embryos is 18 days of total incubation, and mean morphological stage is Hamburger-Hamilton stage 41 (15 days). Mean measurements of wet weight, dry weight, and right third toe length for stage 45 (partial or complete abdominal yolk sac retraction, 19-20 days) cultured embryos are comparable with corresponding means for stage 42-43 (16-17 days) embryos from control intact eggs. Possible causes of growth retardation in vitro are briefly discussed.
1068
B. E. DUNN AND M. A. BOONE
surface area is enlarged from 5.0 cm. (Dunn, 1974) to 7.8 cm. in accordance with the reports of Corner and Richter (1973) and Auerbach et al. (1974) who used surface diameters of 8 cm. and 10 cm., respectively. Fertile eggs were selected for uniformity of color, size, and weight (53.35 ± 3.37 g.) from two flocks of Single Comb White Leghorns. Eggs to be cultured were incubated in a Robbins 131 incubator at 37.5° C , 60% relative humidity, and were turned hourly for 72 ± 1 hours before use. The remaining control intact eggs were incubated for 10 to 21 days to allow normal embryonic growth and development. Methods of removal of egg contents from shells were similar to those previously described (Dunn, 1974) except that egg contents suspended in Kitchen Kraft™ plastic wrap were supported in 5.0 cm. tall, 7.8 cm. diameter tripods constructed of plastic pipe (Figure 1). Tripods were covered with the top of a 100 x 15 mm. plastic disposable petri dish allowing clearance for gas exchange. Embryos were cultured at 37° C. in a humidified atmosphere with 1-2% CO, (Auerbach et al., 1974) in a Forma C 0 2 incubator. No media or antibiotics were added to the cultured egg contents. Embry-
Four trials, utilizing a total of 111 cultured three-day embryos, were conducted. Survival time and morphological stage only were recorded in the first two trials consisting of a total of 31 embryos. In the third and fourth trials, consisting of 48 and 32 embryos, respectively, measurements also included wet weight, dry weight, and right third toe length. The same parameters were measured in two series of 65 and 61 embryos from control intact eggs incubated for 10 to 21 days under standard conditions of incubation. Analysis of variance for all data was performed using the methods of Steel and Torrie (1960).
RESULTS AND DISCUSSION The combined data for survival time and morphological stage for all cultured embryos are presented in Figure 2, which illustrates that 75% of the embryos survived through 17 days, 50% survived through 18.5 days,
Downloaded from http://ps.oxfordjournals.org/ at University of Pennsylvania Library on January 22, 2015
FIG. 1. View of a three-day embryo and egg contents supported in plastic wrap in a 5.0 cm. tall, 7.8 cm. diameter plastic tripod. The petri dish cover has been removed to facilitate photography.
onic mortality was monitored at least twice daily. Embryos were considered dead when they failed to exhibit gross movement, and there were no signs of circulation in the chorioallantoic vascular system. Survival time and morphological stage of each embryo were estimated to nearest one-half day and nearest whole Hamburger-Hamilton stage, respectively. Staging criteria used were general body conformity, development of the down, right third toe length, and yolk sac retraction. Dead cultured embryos and embryos from control intact eggs were dissected from adherent membranes. Yolk sacs were removed from stage 45-46 control and cultured embryos. Embryos were rinsed thoroughly to remove adherent egg contents, then blotted on a damp towel to remove excess water. Wet weight was determined to the nearest 0.01 g. and length of the third toe of the right foot measured to the nearest 0.1 mm. Dry weight was determined to the nearest 0.1 mg. after drying at 100° C. for 24 hours.
EMBRYO GROWTH IN VITRO 100
L
—1_
— 1 _
90. 80.
—
~»_.
~l_
DAYS OF TOTAL INCUBATION
i
70.
1
HAMBURGER -HAMILTON STAGE
l_
60.
< >
> or
50.
1 I 1
.
40. 30.
I_
i 1 I 1
20. 10. 0
-S
L
1
4
5
i
i
6
7
29
31
i
i 8 i
34
i i i i 1 ! 14 15 16 12 9 10 II DAYS OF TOTAL INCUBATION
•
'
i
17
18
19
42 43 36 37 38 39 4 0 41 HAMBURGER-HAMILTON STAGE
44
i
i 35
i
IF
1
i
T
i
i 45
i _ *-^ 20 21 i 46
FIG. 2. Survival to various incubation ages and Hamburger-Hamilton stages of development of 111 cultured chick embryos.
and 3% survived to 21 days. Figure 2 further indicates that morphological development of cultured embryos lags behind the corresponding total incubation age by two to three days, such that an embryo which survives for 15 days of total incubation may possess the morphology of a control 13 day embryo (Hamburger-Hamilton stage 39), while an embryo which survives for 19 days may have the morphology of a control 16 day embryo (Hamburger-Hamilton stage 42). The maximum morphological stage we have observed in vitro is stage 45 (Figure 3), based on partial or complete abdominal retraction of the yolk sac, which normally occurs on day 19-20 (Hamilton, 1952). Mean survival time and mean morphological stage for all cultured embryos were 18 days of total incubation (three days in shell plus 15 days in culture) and HamburgerHamilton stage 41 (15 days), respectively. No significant differences (P > .05) occurred in mean survival time or in mean morphologi-
FIG. 3. Three cultured stage 45 embryos (b, c, d) exhibiting complete abdominal yolk sac retraction compared with a control stage 45 embryo (a).
Downloaded from http://ps.oxfordjournals.org/ at University of Pennsylvania Library on January 22, 2015
Z> CO
1 1
1070
B. E. DUNN AND M. A. BOONE
TABLE 1.—A comparison of mean wet weight, dry weight, and toe length between control and cultured embryos with respect to Hamburger-Hamilton (H-H) stage Wet weight (g.)
H-H stage
Age (days)
Control
29 36 37 38 39 40 41 42 43 44 45 46
6 10 11 12 13 14 15 16 17 18 19-20 21
2.64 3.95 5.43 7.84 10.85 14.33 16.77 21.11 23.70 30.49 39.27
Dry weight (g.)
Culture
Control
4.12 5.94 7.85 9.59 11.41** 13.15** 15.63** 16.56** 15.79**
.1674 .2580 .4249 .6930 1.2449 1.8857 2.7200 3.6020 4.2855 5.5471 7.0132
Culture
Toe length (mm.) Culture
No. embryos
Control
Control
8.4 10.1 12.1 14.5 16.3 19.2 20.8 22.8 24.6
12 12 12 11 11 11 11 11 11 20 4 126
Culture 1
8.2 10.7 12.0 14.0 14.7** 16.9** 17.0** 16.4**
1 3 6 13 11 22 12 1 10 80
*(P < .05). **(P < .01).
cal stage among the four trials. Mean wet weight, mean dry weight, and mean right third toe length data for the two series of control and cultured embryos are presented in Table 1. Table 1 indicates that no significant differences (P > .05) between control and cultured embryos occurred with respect to wet weight prior to stage 41, with respect to dry weight prior to stage 40, or with respect to toe length prior to stage 42. Comparison between cultured and control embryos indicates that the reduction in wet weight of cultured embryos was highly significant (P < .01) at stage 41 and at subsequent stages and the reduction in dry weight of cultured embryos was significant (P < .05) at stage 40 and highly significant (P < .01) at subsequent stages. Toe length reduction in cultured embryos compared with controls was highly significant (P < .01) at stage 42 and at subsequent stages. The difficulty of accurately determining the stage of older cultured embryos must be noted. While our data indicate that cultured stage 43-44 embryos approximate the mean wet weight, dry weight, and toe length of control stage 42 embryos (Table 1), the general body conformity and down development of the cultured embryos is more advanced
than that of control stage 42 embryos. Our data further indicate that cultured embryos exhibiting partial or complete yolk sac retraction (stage 45) have the average wet and dry weights of control stage 41-42 embryos and the average toe length of control stage 42 embryos. Retracted yolk sacs frequently are reduced in size due to leakage of their contents during culture. Retardation of growth in vitro may be due to one or a combination of factors. Temperature and/or humidity requirements for cultured embryos may be different from those for intact eggs. Gas exchange in vitro may be restricted as cultured egg contents have an average of 38 cm. 2 of surface area unobstructed by contact with the supporting plastic wrap, which is about one-half the surface area of a standard egg (Wangensteen et at, 1970/71). It is suggested that gas exchange through the supporting plastic wrap may enhance embryonic development in these chambers. The increase and decrease, respectively, in air cell P C 0 2 and P ^ thought to stimulate pulmonary respiration during the latter half of incubation (Freeman and Vince, 1974), do not occur in vitro. Perhaps simulation of the air cell gas environment would enhance embryonic growth and development.
Downloaded from http://ps.oxfordjournals.org/ at University of Pennsylvania Library on January 22, 2015
.2517 .4188 .6669 1.0073* 1.5011** 1.8122** 2.1850** 2.7826** 2.4037**
EMBRYO GROWTH IN VITRO
Leakage of yolk sac contents in vitro probably decreases the availability of yolk nutrients to the developing embryo. Transfer of albumen through the sero-amniotic connection is delayed in unturned intact eggs resulting in inhibited albumen uptake and greatly decreased hatchability (Randies and Romanoff, 1950). The combination of abnormal egg geometry and lack of turning may restrict albumen assimilation in vitro. Finally, of the calcium in the newly-hatched chick (Romanoff, 1967; Simkiss, 1967), is unavailable for skeletal calcification in vitro. ACKNOWLEDGMENTS The authors wish to thank Dr. W. E. Johnston of the Experimental Statistics Unit for statistical analysis of the data.
REFERENCES Auerbach, R., R. Arensman, L. Kubai and J. Folkman, • 1975. Tumor-induced angiogenesis: lack of inhibition by irradiation. Int. J. Cancer, 15: 241-245. Auerbach, R., L. Kubai, D. Knighton and J. Folkman, 1974. A simple procedure for long-term cultivation of chicken embryos. Devel. Biol. 41: 391-394. Boone, M. A., M. Hammond and B. D. Barnett, 1964.
The effect of thalidomide on chickens and embryos. Poultry Sci. 43: 506-507. Corner, M. A., and A. P. J. Richter, 1973. Extended survival of the chick embryo in vitro. Experientia, 29: 467-468. Dunn, B. E., 1974. Technique for shell-less culture of the 72-hour avian embryo. Poultry Sci. 53: 409412. Dunn, B. E., and M. A. Boone, 1975. Shell-less culture of the avian embryo. Poultry Sci. 54: 1336. Freeman, B. M., and M. A. Vince, 1974. Development of the Avian Embryo. John Wiley and Sons, New York, New York. Hamilton, H. L., 1952. Lillie's Development of the Chick. Holt, Rinehart, and Winston, New York, New York. Ramsey, J. B., 1970. The effects of normal atmosphere and increased C 0 2 on chick embryos incubated in opened shells and in vitro. M.S. Thesis, Clemson University. Randies, C. A., and A. L. Romanoff, 1950. Some physical aspects of the amnion and allantois of the developing chick embryo. J. Exp. Zool. 114: 87-101. Romanoff, A. L., 1967. Biochemistry of the Avian Embryo. John Wiley and Sons, New York, New York. Simkiss, K., 1967. Calcium in Reproductive Physiology. Chapman and Hall, London, England. Steel, R. G. D., and J. H. Torrie, 1960. Principles and Procedures of Statistics. McGraw-Hill, New York, New York. Wangensteen,0. D., D. Wilsonand H. Rahn, 1970/71. Diffusion of gases across the shell of the hen's egg. Resp. Physiol. 11: 16-30.
Downloaded from http://ps.oxfordjournals.org/ at University of Pennsylvania Library on January 22, 2015
egg shell calcium, which contributes 75-80%
1071
