Chapter XII: Part II: 1, The os occipitale (1, dorsal; 2, two lateral; 3, ventral (11)

The _spurs_ are conical with a flat base. The basal part rests upon an enlargement of the shank bone. Soft structure is found between the horny spur and the bone. The upper cells, like those of the nails and of the skin, are constantly being worn or cast off, and new cells push up from the lower layers of cells. These newly formed flattened cells soon become cornified. The oldest formation is found at the tip and the youngest at the base.

EMBRYOLOGY OF THE CHICK

That a new individual may be brought into existence, there must be accomplished the union of the male element, or _spermatozoon_, with the female element, the _ovum_. This union is called _fertilization_. In the fowl this fertilization is accomplished at the anterior portion of the oviduct, after the calyx has ruptured and discharged its yolk, and before the albumen has been formed around it. The blastoderm is found on the surface of the yolk. One spermatozoon is all that is required; in fact, only one can be used in this union.

=Spermatogenesis.=—The spermatozoa are formed by the seminiferous tubules of the testis. From these cells, called the _spermatogonia_, are formed other cells called _spermatocytes_, which in turn form the _spermatids_, the immediate forerunners of the spermatozoa. During the period of multiplication the spermatogonia divide repeatedly by mitosis. Numbers of small cells are thus produced, each containing in its nucleus the number of chromosomes typical of the somatic cell of that fowl. In the second period the cells become larger and spermatocytes of the first order are formed. Then comes the period of maturation, during which two succeeding divisions rapidly occur. The first division results in the formation of two cells exactly alike. These are spermatocytes of the second order. They differ from the somatic cells in that they contain only one-half the typical number of chromosomes. The second division produces two similar spermatids from one spermatocyte of the second order. Therefore four spermatids exactly alike may be formed from one spermatocyte of the first order. From these spermatids the spermatozoa are formed (Fig. 55). The heads of the spermatozoa contain the nuclei derived from the spermatids; the necks contain the centrosomes; and the tail, consisting of three parts is probably formed from the protoplasm. Three parts of the tail are as follows: first, the pars conjunctionis, which unites the tail to the neck; second, the pars principalis, which constitutes the main length of the tail; and, third, the pars terminalis, which consists of an axial filament which transverses the entire tail and is surrounded by a protoplasmic sheath.

=Oögenesis.=—The ovum during its formation passes through three stages.

FIG. 81.—Section of ovum in a hen. 1. Nucleolus. 2. Nucleus. 3. Liquor
folliculi. 4. Stratum granulosum. 5. Follicular cells. 6. Theca
folliculi. 7. Peripheral stroma.
]

The first stage, that of _division_, takes place before the chick is hatched, and, according to Bradley, comes to an end about the time of hatching. This stage consists of the rapid formation of ova in the female chick. In the second stage, which begins about the time of hatching, there is an increase in the size of the units of the ovary, accompanied by _yolk formation_. At this time each ovum is in its own follicle (Fig. 81), and is surrounded by a layer of cuboidal cells and a _theca_. The theca is formed from the adjacent fibrous stroma. The third stage, that of _maturation_, commences during the development of the yolk and is complete after it has escaped to the oviduct. Maturation consists of each cell’s dividing into two unequal parts. In each division the cell is split into a small cell known as the _polar body_, which is cast off and disappears, and a larger cell, which is the ovum proper. In this process half of the original chromosomes are cast off.

=Fertilization.=—The sperm travels rapidly; experiments have shown eggs to be fertile laid twenty-four hours after service by a male. When the ovum is discharged into the ovarian pocket it is surrounded by spermatozoa.

After the male pronucleus has united with the female pronucleus in the single-celled ovum, there is a cleavage of the cell in the long axis of the egg, making two cells; and then a cleavage at right angles, which progressively continues, makes the mulberry-like mass. The remaining content of the egg consists of food for the development of the embryo. From this mass of cells before the egg is laid the _blastoderm_ is formed. The cells of the blastoderm are differentiated into two layers. The superficial layer is the _ectoderm_ and the lower layer is the _entoderm_. In the newly laid egg the blastoderm may be observed. It is about 4 millimeters in diameter. It has a transparent central area, the zona pellucida, which is located over the subgerminal region. There is a peripheral, less transparent area called the zona opaca.

In the fertile egg, as soon as it is subjected to the proper temperature, cell multiplication in the blastoderm begins. The first signs of such change are noted in the pellucid area of the blastoderm where embryonal traces appear in the form of the parallel lines called the _plicæ primitivæ_, which diverge to form the cephalic dilatation. At about this time takes place the formation of the _myelencephalous columns_, in which the blood lakes expand in the surrounding halones and in the tracts along which pass colorless blood particles. These tracts extend from below the cephalic expansion to the peripheral sinuses, as the _proto-vertebræ_, which begin to appear at the sides of the myelon. The red color is acquired by the blood, and the heart by its movements, is made more manifest as the _punctum saliens_. A distinct membrane, the serous layer, is formed upon the germ and the blastoderm. The cephalic end of the embryo rises from the surface of the blastoderm, and then, curving down, sinks into it, forming for itself a kind of hood of the serous layer. This hood gradually extends from the margin of the fossa over the body, and, meeting a similar fold formed by the projecting and incurved tail, closes over the germ on the upper side, making a circumscribed cavity, which is the _amnion_. The progress of differentiation of layers of the blastoderm has, meantime, gone on beneath. The serous layer is in part reflected from the vascular and from the mucous layer. The mucous layer is concerned in the formation of the intestinal canal; and beyond this part, which is at first an open groove, the mucous layer expands over the yolk, which it ultimately incloses, the margins of the _vitellicle_ so formed contracting and uniting at the side opposite the embryo at a sort of cicatrix, to which the last part of the abdominal yolk adheres. The vitellicle is richly vascular, and the surface next to the yolk is augmented by rugæ.

The fowl’s egg, at about the fortieth hour, shows the buds from which the limbs are developed. A vesicle is seen to protrude near the anal end of the intestine, which, rapidly expanding, spreads over the embryo, acquiring a close adhesion to the amnion, but remaining distinct from the vitellicle, over which it spreads. It finally encloses the albumen and interposes itself between the latter and the lining membrane of the shell. Umbilical vessels are associated with this membrane. Hunter called this membrane the _allantois_ from its containing urine, and Owen states that the sac which surrounds the albumen acts as the chorion or _placenta_; for it is most probable that from this surface the albumen is absorbed and the chick supported on its developmental food. The external part of the sac apparently acts as lungs as it comes into contact with the shell of the egg through pores of which there is an exchange of air. Oxygen is consumed and carbon dioxide is given off. The blood in the vessels of this membrane is in color more like arterial blood and that in the interior more like venous blood.

The embryonic mass of the incubating egg always floats to the top side. As the embryo grows it turns upon its left side, exhibiting a profile view; it then indents the yolk, and finally almost divides it into two parts.

The peripheral layers of cells rise from the margin of the germ mass, and extend and contract toward the opposite pole. This tract of germ substance is the _primitive streak_. Along the median line it next forms a furrow, which stops short of the ends of the streak. This streak terminates opposite the point from which the germ begins, and swells into the head. The median furrow expands upon it. The cephalic borders are next united by a thin layer of epithelial cells above the furrow, converting it into a cavity, or ventricle. The myelonal furrow is similarly covered by a layer, uniting the lateral columns. The embryonal trace becomes longer, narrower, and bends round the vitellus. A layer of epithelial cells forms a network over the whole dorsal surface of the embryo. Oblique striæ appear in the broadening germ mass radiating from the primitive streak. These indicate divisional segments. These beginnings of aponeurotic septa probably accompany and support nervous productions from the myelon columns.

Two transverse constrictions begin to divide the cephalic enlargements into three lobes, the second and the third of which expand into vesicles. An accumulation of cells at the side of the middle expansion appears to add greatly to its breadth. This forms the basis of the eyes.

The differentiation and the confluence of the cell constituents of the primitive streak have led to the formation of a pair of albuminous cords along the sides of the median furrow, forming the _myelon_ proper. The cells exterior to and above them are converted into muscle and fibrous septa; and beneath the column is a jelly-filled cylinder, with a transversely striated sheath, pointed at both ends, forming the _notocord_. Its anterior point passes a little in advance of the acoustic vesicle. Beneath the notocord and surrounding the blastema is stretched the vegetative, or mucous, layer of cells, in contact with the yolk. Both the head and the tail of the now cylindrical embryo are liberated from the surface of the yolk. A fold of the blastema, reflected from the under part of the head, sinks like a pouch into the yolk, and soon includes the rudiment of the heart, like a bent cord, which begins to oscillate about the seventh day. From the midline of the inferior surface of the embryo, or its mucous layer, two longitudinal plates descend, diverging into the yolk-substance, and form the _primitive intestinal_ groove.

The _ophthalmic vesicle_ elongates and curves outward until the two ends almost come into contact. Between these two ends and beneath the delicate tegumentary layer connecting them the _crystalline lens_ is formed. About the same time, the _otoliths_ appear in the acoustic vesicles, which have now acquired a cartilaginous case. The _cerebral lobes_ begin to be formed by a small fold, rising laterally and overlapping the forepart of the second enlargement, which has expanded to greater breadth. The _olfactory cavities_ appear as small cutaneous follicles.

The two myelonal columns, expanding between the ear sacs and receding so as to show the notocord beneath, bend upward and inward, and unite, to be continued into the posterior of the _optic lobes_, thus commencing the cerebellar bridge across the epencephalic ventricle. The encephalic vacuities have begun to be filled by the granular basis of the cerebellar substance.

The _intestinal groove_ begins to be converted into a canal at its two ends. Beneath the anterior end, and behind the heart, there gradually accumulates the cellular basis of the liver.

The commencement of the development of the _organ of hearing_ is by a superficial depression of the cephalic blastema to meet the process from the encephalon, which forms the acoustic nerve. The lining of the depression becomes, on closure of the slit, the proper tunic of the labyrinth.

The vesicle of the labyrinth swells into four dilatations, of which three are ampullar and the fourth cochlear. The _ampullar dilatations_ extend into very slender canals, at first almost in the same plane, by which they are brought into mutual communication. As the canals expand and elongate, they assume their characteristic relative positions as external, superior, and posterior, the posterior end of the external canal being extended beneath the posterior canal. The cochlear dilatation curves as it elongates. An inner layer becomes distinct from the common membrane and forms the _acoustic lamina_.

As in the development of the ear, so in the development of the eye, the production of the nerve process from the cerebral center is the first step; the infolding of the superficial blastema to meet the nerve is the next. The so-called _cutaneous follicle_ becomes a circumscribed sac or vesicle, in which the changes and the development next proceed, converting the vesicle into an acoustic labyrinth or into an eyeball. In each case neural elements of two vertebræ become modified to lodge and to protect the sense organs, forming respectively the recess called _otocrane_ and that called the _orbit_. The one is located between the occipital and the parietal vertebræ, and the other between the frontal and the nasal vertebræ. The part of the outer blastemal layer of the head which sinks to meet the process from the mesencephalic dilatation, rapidly changes its follicular into a vesicular state. The vesicle thus formed elongates, bending around the cell mass in which the _crystalline lens_ is formed, and the meeting of the two ends results in forming the choroid fissure at the lower part of the eyeball.

The _mesencephalic process_, or optic nerve, expands at the posterior of 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.

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.

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.

4. THE EMBRYO SIXTY-EIGHT TO SEVENTY-TWO HOURS OLD WITH CERVICAL FLEXURES FORMED.

In a study of the living embryo note the changes visible to the naked eye since forty-eight hours old. Note the difference in the blood-vessels of the vascular area. Name the arteries and veins. Note the beating of the heart.

In a study of the entire mount note that a second, the cervical flexure, has appeared in the head. Note that the tail fold is well formed. Note the position of the embryo on the blastoderm. Determine if the amnion is completely closed. Note the olfactory pits on the ventral surface of the head, a short distance in front of the optic stalks. Note the telencephalon, a rudiment of the cerebral hemispheres and an extension of the primary forebrain. It is bilobed anteriorly. The optic stalks are attached to the floor near the anterior end of the thalamencephalon. Note the infundibular region, which is the depressed region behind the optic stalks. In the roof of the thalamencephalon there is a short diverticulum, the epiphysis. The mesencephalon, or midbrain, forms the apex of the cranial flexure, and is united to the hindbrain by a narrow isthmus. The metencephalon, or rudimentary cerebellum, appears as a thick portion on the most anterior division of the hindbrain. The rest of the hindbrain is provided with a transparent roof and constitutes the myelencephalon, or the rudimentary medulla oblongata. Observe the inner and the outer wall of the optic cup, the lens, the choroid fissure, and the posterior, or vitreous, chamber.

Note the form of the otocyst, or auditory sac. Note above which visceral arch it lies. Note the number of visceral clefts. The visceral arches are formed by the thickening of the walls of the bounding clefts. The visceral arches are as follows: the first is the mandibular, or hyomandibular, arch, which is in front of the first cleft. From this there is developed the lower jaw. Note if there is a maxillary process arising from the dorsal angle of the arch. The second arch is the hyoid arch, which is located behind the first cleft. Then follow in order the third, the fourth, and the fifth visceral arches. Note above the mandibular arch the rudimentary trigeminal ganglion, and above the hyoid arch the rudimentary acoustico-facialis. The latter is in contact with the anterior walls of the auditory sac. The rudimentary glosso-pharyngeal ganglion is noted above the third visceral arch. The vagus, or pneumogastric, ganglion is located above the fourth and the fifth. Note the form and the position of the heart. Note the anterior and the posterior limb rudiment in the trunk.

In a study of the sections it is found that cuts transverse to the trunk pass about horizontally through the forebrain. At this age the following sections of the embryo should be studied:

First, through the hindbrain, at which level will be noted the auditory sacs, the neuromeres, the trigeminal, the acoustico-facialis, and the glosso-pharyngeal and the vagus ganglion.

Second, through the upper part of the pharynx, at which level will be observed the midbrain, the hindbrain, the visceral pouches, the nerves, and the blood-vessels.

Third, through the choroid fissure of the optic cups. Note the parts of the eye, and, on the other side of the section, the heart.

Fourth, a study of a section through the olfactory pits.

Fifth, a study of a section through the pancreatic and the hepatic diverticula.

Sixth, a study at the beginning of the allantois through the hind-gut.

At this age it is of interest to study the systems of organs. Observe the manner in which the splanchno-pleure folds to form the walls of the intestine. Note the commencement of the mesentery. Note that the foremost part of the alimentary tract is formed from the stomodeal invagination of the ectoderm. The hypophysis is formed from a dorsal outgrowth of this. Note its relation to the brain.

The following structures are formed from outgrowths of the ectoderm at this stage:

First, the visceral pouches.

Second, the median rudiment of the thyroid. This is an outgrowth from the pharynx between the two hyoid arches.

Third, the rudimentary lungs, which develop in a pair from a median ventral diverticulum of the alimentary tract, just behind the last visceral pouch. The esophagus is just posterior to this. The esophagus, very short at this stage, is continuous with a slightly wider part that develops into the stomach.

Fourth, the first liver diverticulum, and, at a short distance posterior to this, the second liver diverticulum.

Fifth, the pancreas is at a point where the intestine opens ventrally. It first appears as a slight thickening of the dorsal angle of the intestine.

Sixth, the ventral wall of the hind-gut forms a wide evagination.

Seventh, the beginning of the allantois.

After the whole series of transverse sections have been studied and drawn, construct a longitudinal section of the fetus, including a reconstruction of the alimentary tract.

At this stage the =heart= is a simple tube. The following divisions are distinctly visible: the auricular portion, the ventricular portion, the sinus venosus, and the bulbus arteriosus. The union of the two ductus Cuvieri and the ductus venosus form the sinus venosus. The two ductus Cuvieri are formed by the union of the anterior and the posterior cardinal veins. The ductus venosus is formed by the union of the small right and the large left vitelline vein. These latter veins return the blood from the yolk sac. The sinus venosus empties into the single auricle above which it is located. The single auricle is later divided into two chambers, the right and the left auricle. At this stage it is widest in the lateral direction. The auricle empties directly into the ventricle. The ventricle lies ventrally and behind the auricle. This location is due to the bending of the heart at this stage of development. Its hindmost portion forms the future apex of the heart. If the series of sections be studied from the posterior forward, the ventricle will be first to appear in the sections. Just beneath the auricular portion of the heart there is the bulbus arteriosus. The bulbus arteriosus soon divides into a number of aortic arches. There is an ascending pair in each of the visceral arches. The dorsal aorta is formed by the union of the aortic arches above the visceral arches. The aortic arches are first continued a short distance forward as the carotid arteries. The dorsal, or posterior, aorta passes backward under the notochord. The dorsal aorta divides into two parallel aortæ which give off on each side the vitelline arteries. Note other branches of this aorta.

The veins at this stage consist of the anterior and posterior cardinal, the ductus venosus, the ductus Cuvieri, and the vitelline veins.

Make drawing of the circulatory system after a completion of the study of the series of sections.

Make a study of the =nervous system= according to hints already given.

The dorsal and the ventral roots of the spinal nerves are given off separately, and secondarily unite. From the neuroblasts of the cord there are at regular intervals outgrowths representing the ventral roots. From the neural crest there develop segmental collections of neuroblasts which form the spinal ganglia, from which the dorsal spinal nerve roots develop. In fact this developmental stage can be observed in embryos only forty-eight hours old, first appearing as a line of cells springing on each side from the angle between the neural canal tube and the external epiblast. In the section from the embryo seventy-two hours old there are observed the rudiments of the development of these spinal nerves. Four primary ganglia develop in the neural crest of the head. These ganglia are as follows: the acoustico-facialis ganglia, which is located over the hyoid arch; the ganglia of the trigeminus, which is located over the mandibular arch; the ganglia of the glosso-pharyngeus, which is located over the third visceral arch; and the ganglia of the vagus, which is located over the third and the fourth visceral cleft.

In this stage of development the trigeminal and the acoustico-facialis are clearly visible.

On each side and dorsal to the aorta is noted the Wolffian body, or mesonephros. The Wolffian body (Fig. 82, _E_) consists of a series of tubules imbedded in the mesenchyme. The openings into the Wolffian duct lie just beneath the cardinal vein.

In a study of the =Wolffian duct= determine just how far anteriorly and how far posteriorly it extends. Note whether it empties into the cloaca.

Each tube beginning in a blind extremity is later dilated. It has a thin wall, and is situated near the median portion of the Wolffian body. The tubule proper passing transversely, opens into the duct. The upper wall of the thin walled part is invaginated by a mass of mesenchyme that receives a small vessel from the dorsal aorta. The Malpighian corpuscle, consisting of a glomerule and Bowman’s capsule, is thus established. In the study of the four days old chick note the further development of these parts.

In a study of a chick four days, or ninety-six hours old, note to what extent the yolk is covered by the blastoderm. It will be noted that the embryo lies in the extra-embryonic cavity. This cavity is bounded above by the chorion and below by the splanchno-pleure. In removing the amnion from the embryo, note the relation to the somatic umbilicus. Note the relation of the splanchnic umbilicus to the splanchno-pleure. What relation has the allantois to the above?

In examining the head, locate the cerebral hemispheres, and note their development. Locate the pineal gland. Note changes in the olfactory pit and the eye. Locate the lens and the choroid fissure. Note that the maxillary process of the mandibular arch lies beneath the eye and behind the olfactory pits. Note the otocyst and the relations of the other arches to the above structures.

In a study of the =trunk=, note the tail, the allantois, Wolffian ridges, the heart, and the condition and the position of the rudimentary limbs.

Make drawing of embryo from the side view. Carefully cut off the head immediately behind the last visceral arch, and study and draw the structures observed on the ventral side. Note the maxillary processes, the mandibular and the hyoid arch, the nasal pits, and the fronto-nasal process, which is just beginning its development.

In a study of transverse sections observe from your drawing at what level the section is made. Study and draw a section made through the region of the anterior limbs. Note the spinal ganglion, the muscle plate, or myotome, the condensation of mesenchyme around the notochord, the pancreas, the liver, and the intestine; and note the distribution of the mesenchyme. Note the ventral roots of the spinal nerves, and the neuroblasts in the spinal cord.

In sectioning the embryo from before backward, the first sections will pass horizontally through the hindbrain and the midbrain region. Note the parts, including the auditory vesicle. In the first series of sections also locate the ganglia of the pneumogastric, or vagus, the acoustico-facialis, the trigeminus, and the glosso-pharyngeal nerves. Note the notochord and the cardinal veins. After the disappearance of the ear, we observe the midbrain, which, is located at one end of the section, and the cord at the other end. The region between lies just above the pharynx. In the following series study the visceral arches. Note the third pair of cranial, or motor ocular, nerves. This latter nerve springs from the floor of the midbrain. From the ventral prolongation of the floor of the thalamencephalon there arises the infundibulum. The hypophysis is located just beneath the infundibulum. At this stage the hypophysis appears as a tube to empty into the mouth. This is an ingrowth of the oral epithelium.

In a study of a section through the center of the eye we should observe the lens and optic stalk. This is in the region of the optic chiasm. Note the choroid fissure and the pineal gland, the latter appearing just beyond the eyes. Just forward lies the telencephalon, or rudiments of the cerebral hemispheres.

In a study of the =alimentary tract= we note that the mouth is bounded by the mandibular arches. Note the maxillary processes, and the ventral surface of the head. A finger-like diverticulum, extending from the roof of the ruptured double membrane, formerly separating the pharynx from the arches, forms the hypophysis. The great development of the visceral pouches makes the pharynx rather complex. In studying sections horizontally through the pharyngeal region note the various visceral arches and pouches. Note the arteries of the thyroid diverticulum.

In the series note the changed development of the lung rudiments, the glottis, the esophagus, the trachea, and the bronchi. The bronchi appear in pairs. Note that the liver has assumed proportions, and that it surrounds the common trunk of the vitelline veins, which it divides into two parts. The sinus venosus lies close to the heart. The ductus venosus is also surrounded by the liver. Above the tip of the ventricle we note a dilatation which represents the stomach. The hepatic, or bile duct is located immediately behind the stomach. This duct is formed by the fusion of the right and the left duct. Locate, draw, and describe the pancreas; trace the intestine; locate the splanchnic umbilicus, or yolk stalk; locate the allantois stalk, and trace its connection with the hind-gut.

In the series, locate and study the Wolffian ducts, the beginning of the Müllerian duct, the embryonic kidney, or mesonephros, the permanent kidney, or metanephros. The urino-genital ridge is made up of all the above except the last named. The urino-genital ridge forms a rounded projection on each side of the mesentery into the dorsal angles of the body cavity.

The Wolffian ducts empty into the cloaca. There are two ducts which may be traced far forward, and which are found to extend backward along the lateral margin of the ridge to the cloaca. Along the greater part of their length we note tubules emptying into them.

Beginning near the anterior end of the urino-genital ridge, we note that the Müllerian ducts arise from a thickened line of epithelium. The greater part of the ridge is formed by the mesonephros. This is made up of a series of tubules in each of which we may distinguish two parts as follows: a tuft of capillaries from the aorta forming the glomerules, surrounded by a thin walled invaginated capsule, making up the Malpighian corpuscle; and the tubules proper. The tubules lead from the corpuscles, or glomerules, to the Wolffian duct.

The germinal epithelium constitutes the essential portions of the gonad, or ovary, or testis. The germinal epithelium arises from a thickening of the peritoneum of the median wall of the ridge. The gonad is found near the anterior end of the ridge. At about this age of the embryo there should appear the primitive ovary or testis. Near the posterior termination of the Wolffian duct and from the dorsal diverticulum there arises the ureter, or metanephros duct.