/ . Embryol. exp. Morph., Vol. 14, Part 3, pp. 273-280, December 1965
Printed in Great Britain
The effect of high temperature incubation upon
the myocardial glycogen in the chick embryo
by JOHN M. DELPHIA1 and JOHN ELLIOTT2
From the College of Medicine, Ohio State University
INTRODUCTION
NUMEROUS studies have shown that high temperature incubation for the chick
embryo will accelerate growth. Ancel & Lallemand (1941) reported that, with
incubation temperatures of 39 • 0 to 42 • 0° C. during the 3rd through the 6th day of
incubation, 49 • 2 per cent, to 62 • 7 per cent, of the specimens expressed Coelosomia.
This malformation is characterized by the eventration of the viscera through a
body cleft. Delphia & Eveleth (1961) found that continuous incubation of chick
embryos at 40-0°C. resulted in Coelosomia of the heart, liver, ventriculus and
small intestines in 63 per cent, of the specimens. Further studies by Delphia (1963)
indicated that, with a continuous incubation temperature of 40 • 0° C , the embryos
were advanced in growth stages (Hamburger-Hamilton, 1951) by one stage and
were 1 • 3 times heavier than normally incubated specimens at 8 days incubation.
At this time (8 days incubation) coelosomia is demonstrated clearly. The
coelosomic specimens incubated at 40-0°C. for 12 days incubation were less
advanced in stage of growth and smaller than normal incubation specimens.
Rott (1957a) reported a general increase in the embryo mortality with an
elevation of the incubation temperature. Rott (1957b) also demonstrated that
elevated specimens incubated at 38 -5° C. were larger than specimens incubated
at normal temperature; further, they obtained the increased weight because of
accelerated rate of growth during the first half of incubation. Romanoff (1960)
and others have demonstrated that elevated incubation temperature accelerates
the rate of the heart beat in the chick embryo. Shelley (1961) states that a decrease
in the glycogen concentration in the myocardium of the foetus is associated with
a period of rapid growth during which the cardiac muscle fibre increases in size.
Lee (1951) has shown that the per cent, glycogen in the myocardium of the chick
embryo is much greater than in the other glycogen storage areas during the first
half of incubation and the per cent, glycogen in the myocardium decreases
progressively toward hatching.
1
Author's address: Department of Anatomy, College of Medicine, Ohio State University,
Columbus 10, Ohio, U.S.A.
2
Author's address: College of Medicine III, Ohio State University, Columbus 10, Ohio,
U.S.A.
274
j . M. D E L P H I A and J. ELLIOTT
The present study is concerned with glycogen accumulation in the myocardium
of the chick embryo during abnormally high incubation temperature. Since
elevated temperature is associated with increased heart rate and increased in
size and acceleration of the rate of growth during the first half of incubation and,
further, since glycogen concentration in the myocardium of the foetus decreases
during the growth of the cardiac muscle fibre, it is of interest to know whether
glycogen accumulation will be altered during the establishment of hyperthermically induced coelosomia.
MATERIALS AND METHODS
All control specimens were incubated at 37-5°C. The high temperature
incubation was done in two ways. Specimens incubated at 40-0°C. during the
3rd through the 6th day of incubation, following the procedure of Ancel & Lallemand (1941), were designated Experimental Group I. Specimens incubated
continuously at 40-0°C, following the procedure of Delphia & Eveleth (1961),
were designated Experimental Group II. Only coelosomic specimens were considered from Experimental Groups I and II. All embryos used in the study of
myocardial glycogen were alive and staged according to the developmental
stages of Hamburger-Hamilton (1951) at the time of removal from the egg. The
heart was removed from the egg and weighed within 1 min. from the removal of
the embryo from the egg. After weighing, the heart was placed in a dry ice chamber until it was used in glycogen analysis.
The Anthrone method of Lee (1951) was used for the determination of glycogen
in the myocardial tissue with the exception that the acid hydrolysis step was
omitted. The carbohydrate value obtained in this procedure, as suggested by
Carroll et al. (1956), includes a small amount of material other than glycogen.
Since the other carbohydrates involved are negligible in amount, the total carbohydrate values in this study will be interpreted as pure glycogen. The weight of the
embryonic heart and the amount of glycogen determined by the above modification of the Anthrone method of colorimetric determination of glycogen were
used to determine the per cent, glycogen in the myocardium of the embryo's heart.
This figure, expressed in grams glycogen/100 g. of myocardial tissue, was obtained
for all specimens.
Three to thirteen specimens from the Control Group and each Experimental
Group were collected for the 6th, 8th, 10th, 1 lth and 12th days of incubation. The
study was terminated at 12 days incubation because the expression of hyperthermic coelosomia and its related excessive enlargement of the visceral organs
has been completed prior to this time.
OBSERVATIONS
Table 1 shows the number of specimens, the mean-stage of development
according to the Hamburger-Hamilton (1951) stages of development, the mean
heart weight, the mean per cent, glycogen, the standard error of the mean per cent.
Effect of high temperature on myocardial glycogen
275
glycogen and the standard deviation from the mean per cent, glycogen for the
Control Group and Experimental Groups I and II for the 6th, 8th, 10th, 1 lth and
12th day of incubation. The embryo mortality increased progressively from
15 per cent, at 6 days incubation to 80 per cent, at 12 days incubation for Experimental Group I and from 20 per cent at 6 days incubation to 80 per cent at 12 days
incubation for Experimental Group II.
TABLE 1
Mean per cent, glycogen for myocardium of chick embryo during normal
incubation temperature and elevated incubation temperature
Days
incubation
Group
Mean
HamburgerNumber of Hamilton
specimens
stage
Mean
Mean %
heart
Glycogen
weight
in Mg. (grams/100 g.)
Standard
deviation
6
Control
Exp. I
Exp. II
10
10
11
29
30
31
510
6-25
613
0-1319±
0 0934±
0 1629±
00182
00176
0 0390
0 0574
00557
01233
8
Control
Exp. I
Exp. II
11
10
9
34
34
35
1208
11-40
16-90
0-4379±
0-4656±
0-2415±
00614
0 0662
0-0573
0-2047
0-2205
0 1908
10
Control
Exp. I
Exp. II
10
10
13
35
36
36
22-18
2601
26-90
0-5461 + 0-0614
0-5657± 00375
0-2922± 0 0442
0-1952
0-1285
01396
11
Control
Exp. I
Exp. II
9
3
9
37
36
38
33-80
38-40
38-08
0-9718± 0 0425
0-3821 + 0 0664
0-1341± 0 0440
01274
01150
01319
12
Control
Exp. I
Exp. II
10
7
3
38
38
39
60-62
53-60
41-70
0-5169±
0 1700±
OO363±
01835
0-1272
00251
00581
0 0481
0 0144
Examination of the data in Table 1 suggests that the continuous high temperature specimens remain advanced in stage of development by at least one stage
over those undergoing normal incubation. It should be pointed out that each
individual was classified to stage according to the way it met the majority of the
external morphological characteristics of a specific stage in the HamburgerHamilton (1951) stages.
It is of interest to note that Rott (1957a, b) found that there was a great increase
in body weight and heart weight at about the 12th day of normal temperature
incubation. This is the time when the mean heart weight for Experimental Group
II is decidely lower than the Control Group but the stage of growth is not different.
Although the per cent, glycogen for the myocardial tissue of the Control Group is
276
j . M. D E L P H I A and J. ELLIOTT
lower for each day than noted by Lee (1951), the pattern of daily increase is
comparable with that found by the latter worker.
The same technic used in the present study of the heart tissue was applied by
the authors to liver tissue in the chick embryo. The readings were lower than Lee's
(1951) recordings but in the same proportion as was found for the heart tissue.
The significance found in the statistical studies used in the present report substantiate the use of the data.
Superficial survey of the per cent, mean glycogen (Table 1) suggests a considerable decrease in the amount of glycogen in the myocardium of the specimens
undergoing continuous high temperature incubation. The mean per cent.
TABLE 2
' / ' Test for Comparison of Groups
Day of
incubation
6
8
10
11
12
Experimental
group I versus
control group
1-44
0-28
-0-25
-7-07$
4-31$
Experimental
Group II versus
control group
- 0-69
2-23*
3-47$
13-66J
-4-40*
Experimental group I
versus experimental
group II
-1-56
2-09f
4-61J
1-56
1-27
Note: The data necessary for the calculation of the't' values in the Comparison of Groups are
found in Table 1.
1. Control Group = continuous incubation at 37 • 5° C.
2. Experimental Group I=raised incubation temperatures (40 0°C) during 3rd through
6th days of incubation.
3. Experimental Group II = continuous incubation at 40 • 0° C.
* 5 % level of significance
t Near 5 % level of significance
J 1 % level of significance
glycogen in the myocardium of the Control Group appears to be almost double
that of Experimental Group II at 8 and 10 days incubation and several times
larger than that of Experimental Group II at 11 and 12 days incubation. The mean
per cent, glycogen in the myocardium of the Control Group was approximately
three times as large as that found in Experimental Group I at 11 and 12 days
incubation.
In order to study the effects of high temperature incubation upon the glycogen
deposition and/or storage in the myocardial tissue, each Experimental Group was
compared statistically with the Control Group for each day of incubation. The
information for the comparison of each Experimental Group with the Control
Group and for comparison of the Experimental Groups with each other is found
in Table 1. The resulting' t' test values are shown in Table 2. Comparison of the
mean per cent, glycogen of the Control Group with Experimental Group I
Effect of high temperature on myocardial glycogen
277
showed significant difference at the 1 per cent, level of significance for the 1 lth and
12th day of incubation. These ' t ' values were found to be - 7 - 0 7 and 4-31 for
the 1 lth and 12th day of incubation. Comparison of the mean per cent, glycogen
of the Control Group with that of Experimental Group II showed a significant
difference at the 5 per cent, level of significance at eight days incubation and a
significant difference at the 1 per cent level of significance for the 10th, 1 lth and
12th days of incubation. The' t' values were 2 • 23,3 • 47,13 • 66 and - 4 • 40 for the
8th, 10th, 11th, and 12th days of incubation respectively. The difference between
the standard deviations in the Control Group and Experimental Group II appears
large. Nevertheless, the mean 1 per cent, glycogen values in the two groups are so
different that the ' t ' test for these groups is acceptable.
A ' t' value was determined for the comparison of Experimental Group I with
Experimental Group II for each day of incubation studied. A significant difference in the myocardial glycogen of these two groups was found for the 10th day of
incubation; this' t' value, 4-612, was significant at the 1 per cent level. A ' t' value
of 2 -089 for the comparison of the two experimental groups at 8 days incubation
was near the 5 per cent level of significance. Thus, the Experimental Groups I and
II behaved similarly before the 8th day and after the 10th day of incubation: both
groups (table 2) show significant decreases in myocardial glycogen in comparison
with the Control Group during the 11th and 12th days of incubation.
DISCUSSION
The data in Table 1 and 2 indicate that there is no significant difference in the
amount of glycogen in the myocardium of the Control Group and both experimental groups at 6 days incubation. The comparison of the mean per cent,
glycogen for the Control Group and Experimental Group I shows that the use of
elevated high temperature during the 3rd through the 6th day of incubation
results in a significant decrease (1 per cent, level of significance) in the glycogen
accumulation in the myocardial tissue after ten days' incubation. The comparison
of the mean per cent glycogen for the Control Group and Experimental Group II
indicates that, beyond the eighth day of incubation, there is a significant decrease
(per cent, level) in the accumulation of myocardial glycogen as a result of continuous high temperature incubation. The decreasing effect on glycogen accumulation occurs later in the Experimental Group I. In other words, the two high
temperature procedures eventually produce similar results.
One might ask why specimens incubated at 40° C. either continuously or
according to the procedure of Ancel & Lallemand (1941) demonstrate, eventually,
a significant decrease in myocardial glycogen. It is to be recalled that elevated
incubation temperature accelerates the rate of the heartbeat (Romanoff, 1960).
Rott (1957a, b) and Delphia (1963) have shown that high and abnormally high
temperatures, respectively, increase the rate of growth and size of the embryo in
the first half of incubation. It has also been stated (Shelley, 1961) that myocardial
19
278
J. M. D E L P H I A and J. ELLIOTT
glycogen is decreased during periods of rapid growth of the muscle tissue.
Accelerated growth of the myocardium and accelerated rate of the heartbeat
possibly account for the decreased myocardial glycogen that occurred during the
two abnormally high incubation temperature procedures followed in the present
study.
It appears to the writers that there is a similarity between the extremely
decreased myocardial glycogen in both Experimental Groups and the decreased
myocardial glycogen obtained by Shelley (1961) in experimental foetal anoxia.
It is possible that abnormally high incubation temperature creates anoxic conditions. Further, this could have a bearing on the increased mortality rate in
abnormally high incubation of the chick embryo.
Perhaps the extremely decreased per cent, glycogen in the Experimental Groups
I and II at 11 and 12 days incubation coincides with the time when these embryos
have begun to experience homiothermy. The embryos would then be reacting to
the stress of temperature elevation. This would tend to deplete the glycogen
accumulation and/or storage in the myocardial tissues even more than in the
earlier stages.
SUMMARY
1. Glycogen accumulation in the myocardium during incubation at 40-0° C.
has been investigated. Normal temperature incubation (37 • 5° C.) was used for the
Control Group. Specimens in Experimental Group I were incubated at 40 -0°C.
during the 3rd through the 6th day of incubation while the specimens in Experimental Group II were incubated continuously at a temperature of 40-0° C.
2. The Anthrone method of glycogen determination was employed. The
glycogen from the heart was studied in three to thirteen embryos from the Control
Group and each Experimental Group for each of the following days of incubation:
6,8, 10, 11 and 12.
3. The mean per cent, glycogen, standard error and standard deviation from
the mean per cent glycogen was determined for each group studied on the days of
incubation indicated above. The ' t ' test for the comparison of two groups was
applied. The following results were obtained:
(a) There was no significant difference in the per cent, myocardial glycogen in
the embryos from the groups at 6 days incubation.
(b) The Experimental Group I showed a significant decrease (1 per cent, level
of significance) in myocardial glycogen from the Control Group at 11 and
12 days incubation.
(c) The Experimental Group II showed a significant decrease (5 per cent, level
of significance) in myocardial glycogen from the Control Group at 8 days
incubation. The myocardial glycogen in Experimental Group II is
decreased significantly (1 per cent level) at 10, 11 and 12 days incubation.
Effect of high temperature on myocardial glycogen
279
(d) The myocardial glycogen in Experimental Group II was significantly
lower (1 per cent, level of significance) than that of Experimental Group I
at 10 days incubation.
4. The application of elevated temperature (40 • 0° C.) sufficient to cause visceral
enlargement and coelosomia, whether it is applied during the 3rd through the
6th day of incubation or continuously in the incubation of the chick embryo,
results in a significant decrease in the accumulation of myocardial glycogen.
RESUME
Effets d'une temperature d'incubation elevee sur le glycogene du myocarde de
Vembryon de poulet.
1. On a examine l'accumulation de glycogene dans le myocarde au cours d'une
incubation a 40° C. Une temperature normale d'incubation (37-5°C.) a ete
utilisee pour le groupe de temoins. Les embryons du groupe experimental I ont
ete incubes a 40° C. du 3e au 6e jour d'incubation, tandis que ceux du groupe experimental II ont ete continuellement incubes a une temperature de 40° C.
2. On a utilise la methode a l'anthrone pour la determination du glycogene.
On a etudie le glycogene cardiaque dans trois a treize embryons du groupe de
temoins et de chaque groupe experimental pour chacun des jours d'incubation
suivants: sixieme, huitieme, dixieme, onzieme et douzieme.
3. Pour chaque groupe etudie aux jours indiques ci-dessus, on a determine le
pour cent, moyen du glycogene, I'erreur standard et la deviation standard a partir
du pourcentage moyen. On a applique le test' t' pour comparer les deux groupes.
Les resultats suivants ont ete obtenus:
(a) II n'y a pas eu de difference significative dans le pour cent, du glycogene du
myocarde chez les embryons des groupes etudies le 6e jour de l'incubation.
(b) Par comparaison avec le groupe temoin, le glycogene du myocarde du
groupe experimental I presentait une diminution significative (niveau
significatif= 1 pour cent.), les l l e et 12e jours d'incubation.
(c) Par comparaison avec le groupe temoin, le glycogene du groupe experimental II presentait une diminution significative (niveau significatif = 5 per
cent.), le 8C jour de l'incubation. Le glycogene cardiaque du groupe II
diminue de maniere significative (niveau = 1 pour cent), les 10e, l l e et 12e
jours d'incubation.
(d) Le glycogene myocardiaque du groupe experimental II est moins abondant
(niveau significatif = 1 pour cent.) que celui du groupe I au 10e jour de
l'incubation.
4. L'application d'une temperature elevee (40° C ) , suffisante pour provoquer
une dilatation des visceres et dela coelosomie, si elle intervient du 3e au 6e
jour d'incubation, ou de maniere continue, provoque une diminution significative de l'accumulation du glycogene du myocarde.
280
J. M. DELPHIA and J. ELLIOTT
ACKNOWLEDGEMENTS
The authors wish to acknowledge the support for this study given by the Central Ohio
Heart Association, Columbus, Ohio, and the Bremer Foundation of the College of Medicine,
Ohio State University, Columbus, Ohio.
REFERENCES
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CARROLL, N. V., LONG LEY, R. W. & ROE, J. H. (1956). The determination of glycogen in liver
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ANCEL,
(Manuscript received 11th May 1965)