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Development of the egg of Gallus domesticus in vitro.

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DEVELOPMENT O F THE EGG O F GALLUS
DOMESTICUS I N VITRO
a. P. M. VOGELAAR AND J. B. VAN DEN BOOGERT
Histological Department of t h e Anatomical Laboratory, University of Leyden
ONE PLATE
( N I N E FIGURES)
INTRODUCTION
It is a curious fact that only a few investigators have
studied the development of the bird's egg in vitro. Of these
the first to be mentioned is Preyer ( '85), who demonstrated,
in his well-known work on the physiology of the embryo, the
possibility of studying the development of. the chick outside
its natural environment. Preyer removed the fertilized egg
from the shell and placed it in a little glass dish which had
already been treated with thymol to prevent infection of the
white of the egg. Development could then be followed till
the end of the second day. BBguelin (1757), whose work
pregeded that of Preyer's by a quarter of a century, studied
the living germinal disc, at intervals, through a hole in the
shell at the thick end of the egg. When the embryo was not
under observation, the opening was covered with a piece of
shell from another egg. Obviously, in this experiment of
BBguelin, there is no mention of a development in vitro, and
under such conditions his observations could not be very
exact. Among the investigators who followed Preyer were
FQr6 ( 'Oa), Loisel ( 'OO), and Paton ('11). These authors
adopted the method of placing the contents of the egg shell
in glass dishes in an incubator and were able to follow
developmental processes to a limited degree. I n 1912
McWhorter and Whipple ('12) removed the blastoderm of the
chick and studied it in vitro, but did not attempt to transplant the whole egg.
385
386
J. P. M. VOGELAAR A N D J . B. V A N D E N BOOGERT
The principal difficulties which must be overcome in such
experiments are infection of the white of the egg and drying
up of the egg, it being the tendency of the yolk to rise above
the surface of the liquid in which it is enveloped. The latter
can be prevented by adding to the white of the egg that of
another egg o r a physiological salt solution. I n repeating
the experiments of the above-mentioned investigators, w e
modified them in order to get a method by which the development of the embryo could be followed for a long period without interruption. For no matter how excellent the technique
developed by Gerlach ('84) may be, by which one can study
the embryo through a window in the egg shell, there are certain objections to it. I n the first place, one can only study the
embryo for a short period at a time, as otherwise there is
a danger of suffocation, since no air can penetrate t o the
vessels of the vascular area through the window. That this
method, in the hands of a good experimentist, can give excellent results is apparent from the experiments of Graper
( 'll), and in certain cases one cannot say that the method
of Gerlach should not b eemployed. But it is never possible
in using his method to leave the embryo in perfect rest during the experiment. To insure this, we have taken up the
study of the development of the bird's egg in vitro, a short
summary of which is given below.
MATERIAL AND TECHNIQUE
For the experiments fresh fertilized eggs of Qallus domesticus are used. Before removing the shell it is necessary
to sterilize it thoroughly. This can best be done by following Gerlach's method of placing the egg in a solution of 3
per cent carbolic acid. Experience has taught us that a snbmergence in this solution for two and a half minutes k sufficient. To avoid contamination in handling, the drying of
the egg shell can best be done with the aid of a warm air
current (Fohn apparatus). The egg should then be held
between the thumb and the index-finger of the left hand so
that the equatorial zone of the egg may not be touched. I t
DEVELOPMENT O F EGG O F GALLUS DOMESTICUS
387
is even better to place the egg on three upright glass rods,
so that it is then supported in three places. Within a few
minutes the surface of the egg is dried by the air current.
I n some cases a little drop of fluid hangs stubbornly on the
under surface of the egg. This can be displaced by a small
piece of cotton wool, kept in a solution of phenol, which has
been cut out beforehand. After the surface of the egg has
been entirely dried, the egg should be cut open with a pair
of small curved scissors, the initial opening being made with
a needle. The cut must be in the equatorial zone. On the
top side of the egg a small piece of the shell is left intact.
I n cutting the egg open it is best to hold it in the left hand
following the directions given above. Care should be taken
not to rotate the egg during these manipulations, in order
to keep the germinal disc on the upper side of the yolk. After
the cut has been made, the two parts of the shell are withdrawn in opposite directions, thereby permitting the contents of the egg shell to fall into a sterile glass dish. Halfglobe dishes with an internal diameter of about 8 em. were
used in the experiments. The quantity of the albumen of
a single egg is not sufficient to cover the yolk. It is therefore necessary to add the albumen of one or more eggs or to
use a physiological salt solution. Whenever a part of the
yolk is uncovered, and it is of course always that part which
bears the germinal disc, a further development is impossible.
The additian of the albumen of another egg does not hinder
the development of the embryo.
Since, at certain times during development, the specific
gravity of the yolk mass is lighter than that of the albumen,
it is impossible to prevent the yolk from floating by adding
more albumen, and therefore a liquid of a lighter specific
gravity must be used. For this purpose a salt solution of
the following formula was used: 1000 cc. water, 3 grams of
potassium chloride, 3.59 grams of sodium chloride, 0.31 gram
of calcium chloride. The added salt solution covers the yolk
and the albumen with a thin perfectly transparent layer. As
the egg is practically covered only by the salt solution, it is
388
J. P. M. VOGELAAR AND J. B. VAN DEN BOOGERT
possible to see the finest details in development. I n order to
prevent too great an evaporation of the fluid and an infection of the culture medium it is necessary to cover the dish
with a glass plate. But when the covered dish is placed in
the incubator, large fluid drops collect within a short time
on the under side of the plate, making continued observation
impossible. I n order to avoid this difficulty, an incubator
was chosen which opened from above. I n the lid of the incubator a circular opening was then cut, of such a size as to
permit the dish to be lowered through the opening and yet
be supported by the edge of this opening. The glass plate
which covered the dish, was then wired with a resistance
coil and connected with an electric current. By keeping the
temperature of the glass at a slightly higher temperature
than that of the contents of the dish, the dimness of the glass
caused by evaporation was overcome. The wires were so
attached as not t o interfere with the exact observation of
the development of the egg. While the photographs were
being made, the heating wires were put aside and afterward
replaced on the lid.
I n taking photographs allowance had to be made for the
upward movement of the egg following progressive changes
in the specific gravity of the yolk. This was accomplished
by attaching the camera to the tube of a microscope and varying its position above the egg by means of the adjustment
screws of the microscope. By this means it was possible
t o study and photograph the egg without moving or jarring
the preparation. This is of special importance in solving the
problems which are met with in studying the development
of the egg in vitro.
RESULTS
The most important change in the egg to be seen in the
first twenty-four hours of life is a definite increase in size of
the germinal disc, which gradually continues for the first,
few days, so that at last the blastoderm covers a great part of
the surface of the egg. Figure 1 shows an egg which has
DEVELOPMENT O F EGG O F GALLUS DOMESTICUS
389
been developing for twenty-two hours and ten minutes in
vitro. Figure 2 represents the same preparation twentysix hours later. I n the first photograph the attention is
specially drawn to the strongly developed germinal disc.
Further, both chalazae can be clearly seen and also the
albuminous zone which is around the egg. Surrounding this
is the salt solution which is perfectly transparent. Besides
the great transformation of the germinal disc, attention is
specially drawn to an increase in size of the egg which can
be particularly remarked by a visible tension of the yolk
membrane. While this shows a number of small wrinkles in
the newly opened egg, these disappear after several hours.
One has the impression that the yolk membrane is greatly
stretched. This is particularly well shown by a number of
small folds radiating from the point of attachment of both
chalazae over a small portion of the surface of the yolk
membrane. It was not possible for us to determine precisely
the exact increase in volume of the egg. It was too slight
to be made visible on the photograph.
Figure 2 shows the special case of a blastoderm growing
out in ring form without the development of an embryo. Such
deviation from the normal development can also be found in
other circumstances and has already been seen by others.
The ring-form germinal discs are formed, according to
Schimkewitsch, by an unequal growth of the central and
peripheral parts of the blastoderm while the anlage of the
embryo is degenerating. From the researches of Schimkewitsch ('02) it is clear that the germinal disc is much more
resistant to harmful influences than the embryo itself. This
is also true of the development in vitro. One can repeatedly
see the development of a large germinal disc, while the embryo
is hardly being developed at all. It is very probable that
the development of the embryo was disturbed by elevation
and drying of that part of the surface of the egg which contained the embryo. It was not possible for us always to
avoid this.
390
J. P. M. VOGELAAR A N D J . B. V A N D E N BO0GER.T
Figure 2 also shows another remarkable regular feature
of these preparations, namely, a sort of cleavage of the yolk
mass. The rents in the preparation show a radiating course.
These rents are undoubtedly formed by the regular increase
in volume of the egg by the absorption of liquid from the
surrounding media. The absorption probably takes place
over the whole surface of the egg. It follows, therefore,
that the yolk mass cannot fill the whole place within the yolk
membrane, so that a sinking of the heavier yolk elements
takes place. Looking at the egg from the side, one can see
clearly a separation of the contents into two layers. Underneath, the yellow, non-transparent yolk mass and above, a
broad layer of an opalescent liquid. The constitution of the
latter is being studied further. The rents in the yolk mass
appear for the first time after absorption of liquid from the
surrounding fluid. It is the first sign of the sinking of the
yolk substance.
That the development of an embryo in vitro is possible
can be seen from figures 3 and 4, which are taken from the
same preparation. These were made before we made use of
the photographic apparatus described above. That is the
reason why both representations of the egg are not in the
same position. I n figure 3 the large embryo is clearly visible.
One sees both ca-ebral vesicles and the extremities. This
embryo, photographed at the end of the fifth day, showed
typical spontaneous movements, especially a stretching of the
body from time to time.
Very remarkable was the fact that great changes in temperature in the surroundings were not harmful. Thus, some
hours before the photograph was taken, some liquid of a
temperature of 18.3"C. was added to the preparation which
had a temperature of 38°C. in order to prevent the drying up
of the egg, a part of which was lying above the surface of the
liquid. As a matter of course, the temperature of the perparation became much lower, with the result that the frequency of the heart beat was very slight. It sometimes
seemed that the heart was not beating any more. After about
DEVELOPMENT OF EGG O F GALLUS DOMESTICUS
391
half an hour it had recovered its normal movements and
beat as frequently as before.
I n examining figure 4 attention should be drawn in particular to the peculiar shape of the yolk, which is constricted
into two halves, the embryo lying on the smaller portion.
The greater part of the vascular area is also lying here. This
can be more clearly seen in figure 3, where the smaller lobe
is subdivided into three sacculations, the embryo lying on
the boundary between these and the rest of the egg. The
cause of these sacculations is the great increase in tension
of the yolk membrane accompanying the increase in size of
the egg. At a certain moment the tension becomes so great
that the yolk membrane cannot resist it any longer and ruptures. Probably in our case several ruptures were formed,
thus creating the complex hernia shown in figure 3. By
the further increase in volume of the egg the remaining pieces
of the yolk membrane which separated the hernias from each
other would be ruptured, thus giving rise to the large hernia
in figure 4. This rupture of the yolk membrane was a regular
occurrence in our experiments, and suggests that the formation of a hernia has a direct connection with the osmotic
pressure of the culture medium. This will be investigated
further.
The photographs in figures 5, 6, and 7 are all taken from
the same preparation and show very clearly the formation
of a hernia. This process has special significance, as it exercises a harmful influence on the development of the embryo,
as does the great increase in volume which the egg undergoes within a short time after the first appearance of the
hernia. Figure 5 shows the preparation after ninety-two
hours’ life in vitro; figure 6, twenty-nine hours later; figure
7, still tbirteen hours later. One can clearly see the enormous increase in volume of the egg.
One of the most remarkable appearances that can be seen
in the early formation of a hernia is the abnormal development of the vascular area. The blood islands, as it were,
are pulled away from each other. No typical vessels are
392
J. P. M. VOGELAAR AND J. B. VAN DEN BOOGERT
formed. The embryo, which is clearly visible on the photographs 6 and 7, has developed in quite an abnormal way.
I n oraer to be certain that the development of a hernia
is the direct result of a rupture in the yolk membrane, we
have made experiments with eggs that had developed for
some days under perfect normal circumstances in an incubator. By careful opening of the egg shell after this had
been bored on the wide end by a needle, it could be clearly
seen that the formation of a hernia had also taken place here
(fig. 8). The embryo with the whole vascular area lies here
on the hernia. One sees clearly the curled edge of the ruptured yolk membrane. The following figure (9) shows
another egg which exhibited no sign of hernia when the shell
was opened. But upon pricking it with a fine needle it was
possible to produce a rent in the yolk membrane. Within a
few minutes a large hernia was formed similar t o that in
figure 6. The position of the vascular area in both cases is
the same. It is not possible to explain why the hernia is
formed much faster in the latter experiment than during the
development of the egg in vitro. Probably, in the latter
case, the rent is not formed so quickly.
SUMMARY
According to the technique described above, it is possible
to develop the fertilized eggs of Gallus domesticus repeatedly in vitro to a point where a large vascular area and an
embryo can be seen. The longest time in which it was possible to maintain development was about five days. The
embryo thus produced showed typical spontaneous movements. But in-vitro development, thus far, has always resulted in rupture of the yolk membrane-a disadvantageous
circumstance due to an increase in volume of the egg. This
hernia formation can also occur under normal conditions
of development.
D E V E L O P M E N T O F EGG O F G A L L U S D O M E S T I C U S
393
LITERATURE CITED
B~GUELIN
1757 Abhandlung von der Kunst geoffnete Eier beim Lampenfeuer
auszubriiten. Translated from the French by J. G. Kriinitz. Hamburgisch. Magaz. Hamburg.
1900 Remarques sur l’incubation des oeufs de poule prives de leur
coquille. C. R. SOC.Biol., T. 52, p. 601.
GERLACH,
L. 1884 Uber neuere Methoden auf dem Gebiete der experimentellen
Embryologie. Anat. Anz., Bd. 2, 1884, S. 583.
GRAPER,L. 1911 Beobachtung von Wachstumsvorgangen a n Reihenaufnahmen
lebender Hiihnerembryonen nebst Bemerkungen iiber vitale Farbung.
Arch. Entw. Mech., Bd. 33, S. 303.
LOISEL,G. 1900 Incubation d’oeufs de poule retires de leur coquille. C. R. SOC.
Biol., T. 52, p. 582.
1900 DBveloppement d ’ovules de poule incubes dans de l’albumen
de canard. C. R. Soc. Biol., T. 52, P. 757.
MCWHORTERAND WHIPPLE 1912 The development of the blastoderm of the
chick in vitro. Anat. Rec., vol. 6, p. 121.
PATON,
S. 1911 Experiments on developing chicken’s eggs. Jour. Exp. Zool.,
vol. k l , p. 469.
PREYER,
W. 1885 Specielle Physiologie des Embryo. Leipzig : Th. Grieben ’s
Verlag (L. Fernau).
SCHIMKEWITSCH,
W. 1902 Ueber die Entwickelung des Hiihnchens unter kunstlichen Bedingungen. Anat. Anz., Bd. 20, p. 507.
F d ~ d ,CH.
PLATE 1
EXPLANATION OF FIGURES
1 A hen’s egg, twenty-two hours in vitro. Note germinal disc, yellow yolk,
albumen (with two chalazae), and outermost zone of salt solution containing air
bubbles.
2 The same egg, forty-eight hours in vitro. Note ring-shaped blastoderm,
rapidly increasing in size, and formation of rents in yolk mass.
3 A chick embryo, four days in vitro. Note constriction of yolk into two
parts, the upper half of which (carrying the embryo) is again subdivided into
three sacculations.
4 The same embryo, five days in vitro. The yolk mass is now constricted into
two portions through coalescence of the three sacculations in the upper pole.
Note differentiation of cerebral vesicles a n d limb buds. This embryo showed
spontaneous movements.
Figures 3 a n d 4 were made with Hauff-Diapositiv plates, the others with
Gevaert-Filtered-Orthochrom plates. That is the reason why the yolk in figures
3 and 4 takes black in these photographs and white in the others.
5 to 7 An embryo 92, 121, and 134 hours in vitro. Note formation of
large hernia through rupture of yolk membrane. As the blastoderm is stretched,
the blood islands are separated, so t h a t no blood vessels a r e formed.
8 Normally incubated egg, showing a hernia of the yolk. The embryo with
the whole vascular area lies on the hernia. Note curled edge of ruptured yolk
membrane.
9 Normally incubated egg in which a hernia of the yolk has been produced
by tearing the yolk membrane with a needle.
394
DEVELOPMENT OF EGG O F GALLUS DOMESTICUS
J. P. M. VOQELAAR AND J. B . VAK LIES BOOGERT
395
PLATE 1
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