the circular sac, and, in the course of mutation into eyeball, lines its
posterior part with a layer which becomes the _retina_. The transparent
layer covering the forepart of that sac and the inclosed lens is formed
into the _cornea_. Other layers of the sac are formed into the
_choroid_, the _ciliary processes_, the _iris_, and the _pecten_.
Of the appendages of the eye the membrana nictitans is the first to
develop. Then develop the lower lid and, last, the upper lid.
After the development of the essential organs of sense, the skin is
developed. Modifications of the skin form the outer ear and the eyelids.
Then are formed the maxillary arch, the hyoidean arch, and the scapular
arch.
The vessels which return the blood from the vitellicle are the
transverse and the longitudinal _vitelline veins_. The first are so
called because these trunks pass to the embryo at right angles to its
axis. They are the largest returning canals. The _longitudinal veins_
extend parallel with the axis of the embryo; they are of smaller size.
The right anterior longitudinal vein becomes the right precaval and
receives the remains of the right transverse vitelline vein, as the
right vena azygos. The left anterior longitudinal vitelline vein is also
persistent as the left precaval, and enters in the mature bird, as in
the embryo, at the posterior or the lower part of the auricle. The left
transverse vitelline vein is also subsequently reduced, by receiving
only the vertebral veins of that side, to the condition of a so-called
azygos vein. The main trunk of the post-caval is the result of the
returning vessels from the abdominal viscera and the posterior limbs at
a later stage of development. There is but one principal posterior
longitudinal vitelline vein, and this anastomoses with the left
transverse vein as it enters the embryo.
The _auricle_, which, by its dilatation of the left side, appears to be
double, receives the venous blood at its right division. The left one,
subsequently receives the veins from the lungs, is ultimately separated
from the left precaval and the right auricle to which that vein is
conducted and restricted.
The _ventricular part of the heart_, at the second day of incubation, is
in the form of a bent tube, curving from behind downward, forward, to
the right and upward, continuing insensibly into the part representing
the aortic bulb, in which the septum first appears, and ultimately
dividing the ventricle into two.
At this stage the piers of the _maxillary arch_ appear as buds from
beneath the eyeballs. The naso-premaxillary process is above their
interspace. The piers of the mandibular arch and those of the hyoidean
arch follow in close succession. The blastemal base of the scapular arch
projects slightly at the sides of the fovea cardiaca; the piers, now
separate, ultimately meet in front of the heart, and accompany it in its
retrograde course. The mesencephalon is the largest of the segments of
the brain which are connected with the eyeballs.
When the heart has assumed its form as such, distinct from the great
trunks rising from it, the two _arteries_ from the base of the
ventricles appear. The artery to the right bifurcates, one division
supplying the head and the wings, the other winding over the right
bronchus. That to the left also bifurcates. Its left division arching
over the left bronchus and anastomosing with the right arch a little
below and behind the apex of the heart. Its right division arches over
the back of the heart, bending to the right and anastomosing with the
right aortic arch just above the outer ductus arteriosus. Each of these
divisions of the left primary arterial trunks sends off a branch to its
corresponding lung. As the lung expands, and especially at the beginning
of the act of expansion toward the close of the period of incubation,
the blood is diverted into the pulmonary vessels, and the channels below
them shrink and disappear. The left primary artery is retained as the
trunk of the pulmonaries, and, through the changes in the interior of
the ventricle, this artery comes to discharge exclusively the ventricle
corresponding to the right in mammals. The retained aorta rises from the
left ventricle.
The _air-sacs_ begin at the lower point of the lung, appearing like
small hydatids, and extend further and further into the abdomen, in
front of the kidneys. They are at first full of fluid. Soon after the
development of the abdominal air-sacs others are developed.
The _lungs_ are at first free, but afterward begin to be attached to the
ribs and to the spine.
In the female embryo we first observe two _oviducts_, one on each side
of the basis, or stroma of the ovarium, which appears in a relation to
the primordial kidneys similar to that of the testes in the male. At the
period when the permanent kidneys have sent their ureters to the cloaca,
the oviducts have been developed as prolongations from that part, and,
up to a certain point of development, they are of equal size and length.
Subsequently the left oviduct alone continues to grow; the right
remaining stationary or shrivels; occasionally it may be discerned as a
rudimentary in the mature bird, but usually all trace of it has
disappeared before hatching. The left oviduct expands above or at its
free end into the infundibular orifice, where its parietes are very
thin. As it descends, these increase in thickness, and the efferent tube
gradually acquires the texture and form of an intestine. It is attached
and supported by a duplicature of the peritoneum.
[Illustration:
FIG. 82.—Embryological studies.
_A._ 1, The chorion and allantois. 2, The allantoic cavity. 3, The
amnion. 4, The yolk sac. 5, A small quantity of remaining albumin.
_B._ Brain tube of a chick 25½ hours old showing partly closed brain
tube with eleven folds of neuromeres.
_C._ The development of the alimentary tract. 1, Trachea. 2, Lung. 3,
Esophagus. 4, Stomach. 5, Pancreas. 6, Bile duct. 7, V-shaped loop
of midgut. 8, Cloaca. 9, Vitello-intestinal duct.
_D._ 1 to 6 inclusive, same as C. 7, Cæca. 8, Cloaca.
_E._ Kidneys, Wolffian bodies, and testes of an embryo chick. 1, The
adrenals. 2, The genital. 3, Primitive oviduct. 4, Permanent kidney.
5, Ureters. 6, Duct of primitive kidney which conducts the excretion
into the cloaca.
_F._ A transverse section of a chicks’ head 48 hours of incubation. 1,
Forebrain. 2, Pigmented layer of retina. 3, Ectoderm. 4, Nervous
part of retina. 5, Optic stalk. 6, Invagination of ectoderm to form
the lens rudiment.
]
At first, the right and the left ovaria are similar in size, but the
symmetry is soon disturbed by concentration of development in the left
ovary. The right ovary remains stationary and ultimately, in most birds,
completely disappears by the time the chick is ready to emerge from the
shell.
Three fetal membranes are developed, the _chorion_, the _amnion_, and
the _allantois_. There is also developed the _yolk sac_ (Figs. 82 and
83). The amnion is connected to the body wall at the umbilicus. The
amniotic fluid is found in this sac. The chorion is at first surrounded
by the albumen, but as the albumen is absorbed the chorion comes in
contact with the inner shell membrane. It is probable that the chorion
consists of ectoderm on the outside and of mesoblast on the inside. The
amnion, on the other hand, is formed of mesoblast on the outside and
ectoderm on the inner, or embryonal, side. The allantois springs from
the embryo soon after the fourth day, and develops from the ventral wall
of the primitive gut. By some embryologists, the yolk sac is included in
the embryonic membranes. It commences as the splanchno-pleure
surrounding the mass of yolk. It becomes smaller as the yolk is
absorbed. At first its outline is round but later its walls become
folded in. The yolk is dissolved and absorbed by the entodermic lining
of the sac, and is carried to the embryo by veins called the vitelline
veins (Fig. 83, No. _B_, 4), which ramify on the walls of the sac.
Some time after the fourteenth day, the chick assumes a position
lengthwise within the egg shell so that the head is near the broad end
of the egg. The head is bent upon the chest and the beak is usually
tucked under the wing. Later the head assumes a position, by a double
curve of the neck, so that the beak is in contact with the air cell.
About the fifteenth day, the coils of intestine, which heretofore have
been outside the abdominal cavity, are withdrawn into the abdominal
cavity, as is also the abdominal yolk sac. As the chick pips out of the
shell, the umbilicus becomes occluded.
The outer upper part of the tip of the beak is provided with a short,
stout, spike-like arrangement, called the _egg tooth_. On the twentieth
day this part of the beak is forced against the wall of the egg and
gradually breaks through the egg shell. The breathing by the lungs
commences some time before hatching; this is evidenced by the chick
within the shell giving chirping sounds.
[Illustration:
FIG. 83.
_A._ Longitudinal section of chick, 4 days incubation. 1, Cephalic
fold. 2, Caudal fold. 3, True amniotic cavity. 4, Epiblast. 5,
Somatic mesoblast. 6, Visceral mesoblast. 7, Hypoblast. 8, Future
anua still closed. 9. The allantoic vessicle. 10, The mesentery. 11,
Intestine. 12 and 13, Yolk sac. 14, Cavity of true amnion. 15, The
mouth. 16, Pleuroperitoneal cavity. 17, Fore gut.
_B._ Membranes of the chick at the third day of incubation. 1,
Membrane surrounding albumin. 2, Amnion. 3, Allantois. 4,
Vitellicle.
_C._ The fore part of an embryo chick at the second day. 1,
Mesencephalon. 2, The eye. 3, Olfactory organ. 4, Mandibular arch.
5, Maxillary arch. 6, Maxillary arch. 7, Hyoidean arch. 8, Scapular
arch. 9, Depression organ of hearing. 11, Process from encephalon
for union with nerve of hearing.
_D._ Primitive blood-vessels at second day of incubation (Owen). 1,
Mesocephalon. 2, The right anterior longitudinal vein. 3, The eye.
4, The mandibular arch. 5, The hyoidean arch. 6, Scapular arch. 7,
The same. 8, The ventricular portion of the heart. 9, The right
transverse vitelline vein. 10, Posterior longitudinal vitelline
vein. 11, Vertebral vein. 12, Tributaries of same. 14, The auricle.
16, Aortic bulb.
_E._, A transverse section of chick embryo, 29 hours incubation. 1,
Neural canal. 2, Neural crest. 3, Somatopleure. 4, Splanchnopleure.
5, Omphalomesenteric vein. 6, Aorta. 7, Notocord. 8,
Pleuroperitoneal cavity.
_F._ Chick embryo at the ninth day of incubation. 1, Allantois. 2,
Amnion. 3, Air cell at large end of the egg. 4, Egg shell. 5, Outer
shell membrane. 6, Inner shell membrane. 7, Yolk sac. 8, Albumin
(Bradley).
]
The _circulation of the blood_ is somewhat like the circulation in the
fetus of quadrupeds. The primitive tubular heart (Fig. 83, No. _D_, 8)
is bent in S-shape; the two ends are connected with blood-vessels; and
later, by the development of the septa, the cavities of the adult heart
are defined. The foramen ovale is found in the median septum. This
opening brings the right and the left side into communication. The septa
between the cavities of the heart are completed by the end of the sixth
day. The two vitelline, or omphalo-mesenteric, veins carry the blood
containing the nutrients from the yolk sac to the liver, where it is
mixed with the blood drained from the intestines by the portal vein, and
this blood is finally carried by the posterior vena cava into the right
auricle of the heart. From here it passes through the foramen ovale to
the left auricle; from the left auricle it enters the left ventricle,
from which it is forced into the aorta, and then out into the systemic
circulation. Practically all, if not all, of the blood of the pulmonary
artery is sent into the aorta through a connection with this vessel
called the ductus arteriosus.
_Down_ appears about the thirteenth day of incubation. There are two
kinds of down on the chick, one long, which comes first, about two or
three days before hatching; a second, or fine, down forms at the roots
of the other. As the embryo develops the air cell, at the large end of
the egg which is developed between the two shell membranes, or _membranæ
putaminæ_, gradually enlarges.
OUTLINE FOR LABORATORY STUDY OF THE CHICK
The objects of laboratory study of the embryos are as follows:
1. To study the living embryo.
2. To study the entire embryo:
With the dissecting microscope, as an opaque object.
With the compound microscope after killing, hardening,
clarifying, and mounting.
3. To study embryos by dissection, in later stages.
4. To study the serial sections with the compound microscope.
THE LIVING EMBRYO
The egg is opened under warm physiological salt solution: 0.8 per cent.
sodium chlorid in distilled water heated to a temperature of 38°C.
Gradually pick away the shell at the large end. Note that there are two
membranes and an air cell. Strip off the membranes. When sufficient
shell and membranes have been removed from the large end, invert the
open end of the egg in the salt solution and allow the contents to flow
out. Care must be taken not to break the yolk. The embryo, or
blastoderm, lies upon the surface of the yolk, which is usually turned
with this body uppermost. Separate the blastoderm by cutting around the
outside of the area vasculosa. In doing this a small pair of slightly
curved scissors is needed. After the embryo, or blastoderm, has been
separated, gently float it into a watch crystal with the flat bottom
submerged in the salt solution. The watch crystal with its contents may
now be gently lifted out. Next remove the vitelline membrane. The
vitelline membrane is the delicate transparent membrane covering the
blastoderm, The embryo is now ready for study.
THE PREPARATION FOR STUDY OF ENTIRE EMBRYOS AND SECTIONS
The following processes may be used in killing embryos up to four days,
or ninety-six hours, of age. After removing the embryo as described
above, spread the blastoderm out in the watch crystal and pipette off
the salt solution. Allow it to stand till the edge of the tissue begin
slightly to adhere. Then slowly add the killing fluid by aid of a
pipette, dropping it on the center of the embryo. The pipette must be
held low or the mechanical interference will dislocate the parts.
Older embryos are submerged with their membranes intact into the killing
fluid. The quantity of fluid should be several times the bulk of the
specimen. Kleinenberg’s picrosulphuric acid may be used as a killing
fluid. This fluid is a saturated solution of picric acid plus 2 per
cent. sulphuric acid, to which is added twice its volume of water.
Chick embryos from one to two days old should be left in this fluid from
one and one-half to six hours. Embryos from two to four days old two and
one-half to six hours. Remove the specimen from the killing fluid, and
place it in 70 per cent. alcohol. Change the alcohol every twenty-four
hours until the color ceases to come out of the embryo. Preserve in 80
per cent. alcohol.
If the specimen is to be mounted whole, transfer it from 80 per cent.,
then to 50 per cent., then to 35 per cent., and, finally to water. Small
embryos should remain in each fluid thirty minutes and large ones sixty
minutes.
The following method may be used for _staining embryos_: Dilute
Delafield’s hematoxylin with four times its volume of water. To every 6
cubic centimeters of this diluted hematoxylin, add one drop of
Kleinenberg’s undiluted picrosulphuric acid, and leave specimen in the
fluid thus prepared until it is stained through. This will require from
one to three hours. Now pass up through the series of alcohols to 70 per
cent. Next extract the excessive stain with 1 per cent. hydrochloric
acid in 70 per cent. alcohol. Wash repeatedly with 70 per cent. alcohol
to free from the acid; then transfer the specimen to 80 per cent.
alcohol and leave in this for several hours for complete removal of the
acid; then transfer to 95 per cent. alcohol for thirty minutes. Allow
the specimen to remain in absolute alcohol for one hour. Introduce a
layer of oil of cloves or xylol beneath the alcohol. This may be done by
gradually allowing the fluid to run down the side of the bottle. After
the embryos have sunk into the oil and begun to appear transparent,
remove the fluid and add fresh oil. After the specimen is sufficiently
transparent, mount in balsam, supporting the cover slip so that it will
not rest on the embryo.
In staining for section, place the embryo in borax carmine from the 50
per cent. alcohol and leave twelve hours. Then wash in 50 per cent.
alcohol; after which transfer to 70 per cent. alcohol for six hours.
Clarify in oil of cedar or oil of cloves, and place in melted paraffin
for two hours. Imbed in paraffin, and section.
Embryos may be hardened, imbedded, and sectioned after the usual
methods, using either paraffin or celloidin, and the sections stained
with hematoxylin and eosin after sectioning.
POINTS TO BE OBSERVED IN THE STUDY
1. THE EMBRYO TWENTY-NINE TO THIRTY-FOUR HOURS OLD WITH FROM TEN TO
FOURTEEN SOMITES.
A study of the egg.
In opening the egg, observe that the shell membranes are double.
Observe that the shell is porous.
Note the air cell at the large end of the egg and note that the space or
cell lies between the outer and the inner shell membrane.
After the egg contents have dropped into the salt solution, note
extending from the ends of the yolk the twisted denser cords of albumen.
These are the chalazæ, which act as stays to the yolk. Note that the
yolk is surrounded by a delicate membrane. This is the vitelline
membrane. The yolk is the true ovum and serves as food for the
developing embryo.
A study of the living embryo.
Note the amount of yolk that is covered by the blastoderm. Note the
slipper-shaped, transparent center of the blastoderm. This is the area
pellucida. In the center of this there is a narrow white streak. The
area opaca is the area lying external to the area pellucida. In this
there is the area vasculosa.
A study of the embryo entire, including the vascular area.
The blood islands show as irregular deeply stained masses in the
vascular area. At this stage they are inclosed in wide anastomosing
tubes, the extra-embryonic blood-vessels, which open peripherally into
the bounding sinus terminalis.
The following structures may be identified:
The neural tube forming the axis of the embryo. In the anterior region
may be noted the forebrain, the optic vesicles, the midbrain. The
hindbrain is subdivided into the neuromeres. The cord, or myelon, of the
neural tube back of the hindbrain is closed in front but is open behind.
The head projects above the blastoderm. The fold which unites the
ventral surface of the head with the blastoderm is called the head fold.
The diverging folds of the myelon encloses the primitive streak.
On each side of the neural tube are the mesoblastic somites. The series
is continued behind by the undivided segmental plate.
The heart is beneath the hindbrain. The portion of the body cavity in
which it lies, is bounded in front by the head fold and behind by the
diverging limbs of the splanchno-pleure. Its posterior or venous end
receives the vitelline veins from the vascular area. The anterior or
arterial end is prolonged into the ventral aorta.
Note that the axis is somewhat bent.
The head fold of the amnion is noted to extend over the anterior end of
the head.
A study of the transverse sections.
The sections about 20 micro-millimeters thick should be cut serially,
and so mounted. Knowing how many micro-millimeters the embryo is in
length will enable one to make a diagram of the fetal structure in the
study of the series of transverse sections. The sections should be drawn
in the order studied so as to obtain relative structural ideas.
With the microscope study the following regions:
1. Optic vesicle.
2. Midbrain.
3. Posterior half of the heart.
4. Myelon in the closed region.
5. Myelon in the open region.
6. Through the primitive streak.
2. THE EMBRYO TWENTY TO TWENTY-FOUR HOURS OLD WITH FROM TWO TO SIX
SOMITES.
Compare the parts in this embryo with those of the embryos from
twenty-nine to thirty-four hours old. Note and describe the relations of
the embryo, the area pellucida, and the area opaca. Note how much of the
yolk the blastoderm covers.
In studying the entire embryos, note the condition of the medullary
plate. Observe if the tube is formed in any part; how far back the head
plate extends, if the heart can be seen; and if the primitive streak is
longer or shorter than in 1.
Study relations of structures and make drawings through the point of
divergence of the walls of the fore gut; also through the somatic
region, and through the primitive streak.
Study all parts, as the head fold, the heart, and the fore gut.
3. THE EMBRYO FORTY-FOUR TO FORTY-EIGHT HOURS OLD WITH TWENTY-FOUR TO
TWENTY-NINE SOMITES.
Remove the embryo with the entire area, and preserve it.
Note and carefully describe the changes visible to the naked eye since
the thirty-fourth hour.
In making a study of the entire embryo we note there has been a rapid
growth of the dorsal surface of the head, which has become more bent.
This bend, in the region of the midbrain is called the cephalic flexure.
The forebrain and part of the midbrain form almost a right angle with
the rest of the head. The head is compressed laterally and free from the
blastoderm. The dorsal side of the trunk is turned up, and there is a
twisting of the axis of the embryo just back of the heart. The tail fold
begins at about this time and may or may not be visible. The optic
vesicles are relatively smaller in relation to the brain than in 1. Note
the part of the forebrain to which they are attached. Observe the inner
and outer layers of the retina, the lens, the choroid fissure, and the
cavity of the vitreous humor.
Note the auditory vesicles and whether or not they are closed sacs.
Note that the heart has grown in length and has become doubled on
itself. The two ends are fixed. Note the relation of the heart to the
afferent and efferent blood-vessels. Note that two, and possibly three,
visceral pounches are visible. Note whether or not they are ventral to
the midbrain. The first, the hyomandibular pouch, is bounded in front by
the first visceral, or mandibular, and behind by the second visceral,
arch. The second pouch is bound in front by the hyoid, and behind by the
third visceral arch. The third pouch is bounded in front by the third
visceral arch, and behind by the fourth. Note the number of mesoblastic
somites and the condition of the mesoblastic segmental plates.
Note how far back the foregut is closed. Locate the head fold of the
amnion, and note how far back it is closed. Note changes that have taken
place in the vascular area. In studying the sections it will be found
that a section cut transversely to the trunk will pass horizontally
through the forebrain and through the midbrain.
Study a section through the trunk a short distance behind the heart.
Observe the elevation of the axis of the body. Note the way in which the
lateral folds, or the lateral limiting sulci, in the somatopleure,
delimit the embryonic from the extra-embryonic area.
Note the appearance of the mesenchyme, the approximation of the two
dorsal aortæ, the appearance of the amniotic folds. Observe in the
mesoderm the posterior cardiac veins, and the myotomes, or muscle
plates. The sclerotome is made up of the mass of mesenchyme between the
myotome, on the one hand, and the neural tube and the notochord, on the
other. Note the folding of the splanchno-pleure, and note if there is
present the Wolffian duct, or the nephrotome.
Study sections through the optic vesicles. Note if there is the
beginning of the lens. Do you note the diverticula of the pharynx? Can
you identify the closed amnion and the chorion?
Study sections through the auditory pit. Note fusion of the gill pouches
with the ectoderm. Note the blood-vessels. Study sections through the
region of the heart, through the roots of the vitelline veins, and
through the primitive streak, if it is still present.
For this study it will appear that the anterior end has developed in
advance of the posterior end. The tail fold has probably just begun.
Write a description of the pharynx, and of the circulation at this
stage.