THE EMBRYOLOGY or THE couo SALMON,
oucoaumcuus msurcn (WALBAUM)

Thesis for the Degree of M. S.
MECHIGAN STATE UNIVERSITY
IBRAHEM H. ZEITOUN
1970

 

 

 

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ABSTRACT
THE EMBRYOLOGY OF THE cono SALMON,
ONCORHYNCHUS KISUTCH (WALBAUM).
By

Ibrahim H. Zeitoun

This survey was an attempt to complete our knowledge about
the life history of the coho salmon, Oncorhynchus kisutch
(Nalbaum). Its early development was typically teleostean. A
prolonged period of a discoidal-type cleavage followed ferti-
lization. By the fourth day of incubation the blastula stage
was completed and on the closure of the blastoporal lips most of
the primordia organ systems were established. The peculiar fea-
tures of teleosts were attained following the formation of the
yolk sac. Hatching occurred after about forty days under
continuously flowing water with a constant temperature of
10 1 1 C. Prior to hatching the twisting of the heart and the
formation of the branchial arches were observed, both of which
are characteristic of primitive vertebrates. The discoidal
cleavage, the development of the finfold, and the pharyn-
geal extension pushing the ventral mouth forward prove the

higher phylogenetic position of the coho among teleosts.

THE EMBRYOLOGY OF THE COHO SALMON,

ONCORHYNCHUS KISUTCH (WALBAUM).

By

Ibrahim H. Zeitoun

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Fisheries and Wildlife

1970

 

Géc/xo
3,—M'70

ACKNOWLEDGEMENTS

I wish to express my special thanks to my major professor
Dr. Peter Tack, to committee members Dr. Eugene Roelofs and
Dr. Wayne Porter, who have given me much advice and encourage-
ment during all stages of work. Thanks are also due to
Dr. Howard Johnson, who assisted in obtaining the eggs, and to
Dr. D. L. Haynes and Stuart Gage of the Department of Entomology,
for their help in taking the photographs.

I want the staff and graduate students of the Fisheries
and Wildlife Department to know that I have greatly appreciated
their friendship and support to me as a foreign student here

at Michigan State University.

 

ii

TABLE OF CONTENTS

Page
INTRODUCTION I O O O O O O O O O O O O O O O O O O O O 1

MATERIALS AND METHODS. . . . . . . . . . . . . . . . . 4

DEVELOPMENT. 0 O O O O O O C O O O O 0 0 O O O O 0 O O 7
Description of fertilized eggs and the sperm. . . 7
Early segmentation stages . . . . . . . . . . . . 10
Gastrulation. . . . . . . . . . . . . . . . . . . 14
The early embry0. o o o o o o o o o o o o o o o o 15
Stages of the late embryo . . . . . . . . . . . . 21

13 to 20 days of incubation. . . . . . . . . 21
21 to 27 days of incubation. . . . . . . . . 22
28 to 34 days of incubation. . . . . . . . . 23
35 days to hatching. . . . . . . . . . . . . 27

DISCUSSION 0 O O O O O O O O O O O O O O O I O O O O O 3L1,
SUMMARY 0 O O O O O O O O O O O O O O I O 0

LITERATURE CITED 0 O 0 O O O O O O O O I O O O O 0 O O 39

iii

LIST OF FIGURES

FIGURE Page
1. Diagrams of the sperm and the fertilized egg. . . 9
2. Early cleavage stages . . . . . . . . . . . . . . 13
3. Early embryonic stages. . . . . . . . . . . . . . 20
b. Stages of the late embryo . . . . . . . . . . . . 26
5. The development of the heart. . . . . . . . . . . 29
6

. The hatched embryo. . . . . . . . . . . . .

iv

INTRODUCTION

Oncorhynchus kisutch (Walbaum) is a member of the family
salmonidae. It has several common names: silver salmon, coho
salmon, or Jack salmon. It is an anadromous fish usually
found along the Pacific coast of North America extending from
the Coronado Islands, Mexico, north to Alaska and south on the
Asiatic side to the Japanese coast. It prefers to inhabit
cool, clear waters (Shapovalov and Taft, l95b). Recently fishery
biologists of the United States succeeded in introducing this
species in the Great Lakes.

The spawning season in the rivers and streams lasts from
September to November. Other cohos are reported to spawn in
January and some as late as March (Nalden, 196A). The adults
move upstream and the female constructs a nest or redd in the
gravel of shallow waters where she deposits her eggs which are
immediately fertilized by her male mate's milt. The parents
then die and are washed downstream.

The adults show a secondary sexual dimorphism prior to and
during the spawning season. In the sea they are silver in color
on both sides with small dark spots on their backs. In the spawn-
ing season, however, the female is dark in color while the male

becomes reddish and its Jaws are elongated, the lower Jaw

forming the kype. The smolts usually migrate to the sea
after one year and after two years in the sea they attain
sexual maturity and return to the river to spawn and die.
Some males, known as jacks or grilse, mature and return to
spawn after only one year in the ocean (Shapovalov and Taft,
1954). Jones (1966) reported that the average weight of the
coho, Q. kisutch, in southern waters ranges from five to ten
or twelve pounds and slightly higher in British Columbia.
Shapovalov and Taft (195h) stated that the males in the spawn-
ing season are slightly larger than the females and the mean
length of the male is 64.7 cm and that of the female is 63.8 cm.
There is little and scattered information about the embry-
ology of teleosts, although it is important to any fishery bio-
logist who deals with the life history or production of fish.
This information is necessary to the study of ontogenetic and
phylogenetic relationships. Balfour (1885), McEwen (1953), and
Balinsky (1966) described the embryology of vertebrates in
general, with a poor treatment of teleosts. Price (193ua, 193kb,
1935) made a thorough study of the Whitefish Coregonus clupea-
formis. Battle (19h0) worked on the early stages of the develop-
ment of goldfish, Carassius auratus, from the time of fertilization
to hatching. Armstrong and Child (1956) made extensive obser-
vations on the normal early development of the mummichog
Fundulus heteroclitgg. Recently Oris (1968) made a compre-
hensive survey of the embryology of the English sole,

Parophyrys vetulus, briefly treating the early development

of each organ, using a few photomicrographs.

In regard to the work which has been done on the
salmoninae, Biddle (1917) described the early stages of the
development of chinook salmon Oncorhynchus tschawytscha from
the time of fertilization to the gastrula stage. Wales (1941)
described the embryology of steelhead trout without dealing
with the early cell cleavage, which has been recently described
by Knight (1963). Battle (1944) made an extensive description
of the embryology of the Atlantic salmon, §gl§§‘§glgr, dealing
with many organs during early development. The present work
presents a survey on the embryology of the coho salmon, Q.

kisutch, in order to fill the gap in its life history.

MATERIALS AND METHODS

The eggs used in this survey were obtained from the
Platte River Station of the Michigan Department of Natural
Resources, Benzie County, Michigan. Approximately three
thousand eggs were collected on October 31, 1969 from two
females. The eggs were placed in a plastic bowl filled with
enough river water to cover them. The milt was then stripped
from three healthy males over the eggs. The contents were
stirred for three minutes to allow fertilization. The excess
milt was washed away by several changes of the natural water,
and the eggs were placed in big Jars filled to the neck with
stream water. The eggs were brought back to Michigan State
University where they were placed in a Heath Fish Incubation
Cabinet, Model lB-l6,* which was combined with a temperature
regulator tank and flowing water regulator. In this way, the
eggs were continuously subJected to well-aerated and constant
temperature flowing water. The constant temperature used in
this work was 10 3,1 C.

Two groups of the fertilized eggs were collected at

each sampling time, ten eggs in each group. The samples

 

*Available through Heath Teena Corporation, 19819 9hth Avenue
South, Kent, Washington.

Li

 

IE

28

were taken at one-hour intervals from the time of fertili-
zation to the Zh-hour stage, then one sample every six hours
until the third day of incubation, and one-day interval samples
through the whole period of incubation until hatching.

The first group of fertilized eggs was fixed in Bouin's
solution while the second group was fixed in Smith's solution.
The Smith solution was replaced by Kahle's fixative after
the eyed stage. The above mentioned killing and fixative
solutions are recommended by Guyer (1936), since they fix
satisfactorily and cause much less distortion and shrinkage to
the germ tissues. Two methods of dehydration were used, the
alcohol and xylol method was usually applied to the whole mount
and the transverse series. Also the aniline method, using
toluene as a clearing agent, was used before the infiltration
step of the embryo. Two series have been prepared in this
study, one of whole mounts and one of transverse sections at
different stages of development.

The embryos were embedded in Fisher Tissuemat 52.5 i 0.5 C,
and.sectioned at 5 p and 8‘p, the maJority being cut at 8,» and
mounted by the water-albumen method. The whole mount embryos
were stained with alum cochineal solution and because of the
large size of the coho salmon's eggs the embryos were taken
Off‘ the yolk mass. The transverse sections were stained with
‘Delxifield's hematoxylin using eosin as a counter stain. Canada
balsam was the mounting substance used in both sets.

Observations were made at 10X, 20X, MEX, and 125x. Some

photographs of the embryos were made with a Nikon AFM-M-35
camera and Olympus microscope: others were taken with a
Leitz-Wetzlar microscope equipped with a 35 mm Leitz camera.
The hatching occurred in approximately forty days at a
water temperature of 10 i l C. This period varies with the
temperature of the water (Embody, l93h). No serious fungous
infection appeared during the incubation, and the total
mortality rate of the eggs was approximately twelve percent.
The greatest rate of mortality occurred during the tender period
of development and hatching. The high percentage of dead eggs
may be due to the handling during the sensitive period and to

incomplete development of the hatched embryos.

DEVELOPMENT

Description of fertilized eggs and the sperm

The fertilized egg of the coho salmon, Oncorhynchus
kisutch, is spherical, light yellow in color, and has a dia-
meter ranging between 5-5.5 mm (Figure l,B). The yolk occupies
the largest part of the egg. It is semifluid when the egg is
fresh. At the animal pole the blastodisc is mounted over the
yolk sphere and is continuous with the plasma membrane (or
vitelline membrane). The chorion or zona radiata, which is a
protective membrane secreted in the female ovary by the folli-
cular cells, forms the outer membrane or shell of the egg. In
between the two egg membranes there is the perivitelline space,
which is filled with fluid. Many oil droplets are distributed
through the yolk, but there is a conspicuous concentration of
them at the animal pole. Riddle (1917) suggested that they are
nutrient materials for the developing embryo. The oil globules
regulate the specific gravity of the egg during development
(Richards, 1931).

The milt is a whitish, colloidal fluid containing a huge
number of spermatozoa. The sperm is minute, slender, and has

a length of approximately .01 mm (Figure 1,A).

Figure 1,-A, Diagram of the sperm of the coho salmon:
B, Diagram of the fertilized egg. B1,, blastodisc;
Ch., chorion; 3., head: M., middlepiece; 0., oil
globules Pv.s., perivitelline space; T., tail;
V.m., vitelline membrane: Y., yolk.

 

 

 

 

 

10

Early segmentation stages

The segmentation and development in the teleost's eggs
are restricted to the blastodisc: it is a discoidal-type of
cleavage. After fertilization the blastodisc is formed by
the streaming of the cytoplasm toward the animal pole, where
the egg nucleus rests, forming a patch of protoplasm over
the yolk sphere (Solberg, 1938). The regular rounded germ
disc is attained at the fourth-hour stage, at which it
bulges slightly over the yolk level.

The splitting of the one-cell germ disc to two cells
commenced at about the ninth hour. At this stage a slight
lengthening of the blastodisc can be noticed, and the first
cleavage plane is meridional on the blastodisc and does not
extend deeply through it, but a basal cytoplasmic sheet is
left. The splitting occurred on the short axis of the blasto-
disc, giving rise to two equal blastomeres (Figure 2,A).

Within two hours of the first cleavage the second furrow
of cleavage occurred at a right angle to the first along the
long axis of the blastodisc, and the result of cleavage was four
equal blastomeres (Figure 2,B).

Prior to the third cleavage the blastodisc showed more
lengthening along the second plane of cleavage. The third
cleavage occurred fourteen hours after fertilization (Figure 2.0).
It was a latitudinal division parallel to the second plane and
the resulting eight blastomeres are arranged in two rows, four

cells each. The furrows following the first cleavage are

ll

deeper and extend through the protoplasm, completely dividing
it.

The sixteen-cell stage appeared in eighteen hours. The
blastodisc in this stage retains the rounded pattern and starts
to raise from the yolk beneath it (Figure 2,D). Multiple seg-
mentation gives the two-layered 32-cell stage at 22 hours, and
the three-layered 6h-cell stage at 2h hours (Figure 2,F), with
more layers of cells added as development proceeds. In the
following stages the cells become smaller and it is difficult to
count them and to trace the cleavage planes, which start to be
irregular. The blastodisc in these stages is properly called
the blastoderm.

The periblast layer, which is continuous with the plasma
membrane and surrounds the blastoderm, began to be distinguish-
able at the 32-cell stage around the edge of the blastodisc, and
as development proceeded it spread inward beneath the germ disc,
forming the subblastodermic periblast. The periblast is res-
ponsible for the digestion of the yolk for the growing blasto-
derm (Balinsky, 1966). The external cells of the blastoderm
are closely attached while those of the internal layers are
loosely Joined together, creating intercellular spaces which
form the beginning of the segmentation cavity (Figure 2,F).

As division proceeds the uprising of the blastoderm increases
and the segmentation cavity lies with the blastodermal layers
.above and the periblast layer below. The latter stage is
called the blastula stage (36 hours, Figure 2,G). The blastula

Figure 2.-Early cleavage stages of the coho salmon.
A, The first cleavage. Two cells at nine hours,
tux; B, Four-cell stage at eleven hours, 40X;
C, Eight-cell stage at fourteen hours, tux.
D, Sixteen-cell stage at eighteen hours, uox.
E, 32-cell stage at 22 hours, uox; F, Transverse
section of the 32-cell stage, showing the beginning
of the segmentation cavity; G, Transverse section
of the early blastula, 36 hours: B, Transverse
section of the late blastula, showing the flatten-
ing of the blastoderm over the yolk. Seg.C.,
segmentation cavity; Ep.. epidermal layer.

12

 

 

 

14

stage of the coho salmon, Q. kisutch, lasts a relatively long
period, during which the blastoderm is flattened over the yolk
without any increase in the area it rests upon. The blastoderm
is composed of several layers of cells, usually called the

epiblast (Figure 2,B).

Gastrulation

At about the 8u-hour stage the germ ring is clearly
observed as a result of the accumulation of the blastoderm
cells around its edge (Figure 3,A). This cell accumulation is
presumably due to the more rapid divisions of the marginal cells
than those in the middle of the epiblast. The gastrulation
actually starts at the 96th hour of incubation. The cells at
one edge of the germ ring begin to involute and spread over the
yolk (Figure 3,B). The involuted layer is the hypoblast of the
embryo. The subgerminal cavity lies between the epiblast and
the hypoblast. It is known that the involution of the epiblast
occurs from the posterior margin of the germ ring which is also
the dorsal blastoporal lip (McEwen, 1953).

Along with the involution the epiboly begins and the
anterior and the lateral margins of the germ ring commence to
extend over the yolk sphere, attempting to close the blasto-
pore and forming the yolk sac. The dorsal lip of the blasto-
pore shows only a slight movement during epiboly.

On the sixth day of incubation the embryonic shield first
appeared by the convergence of the blastodermal cells on the

dorsal blastoporal lip. The embryonic shield coincides with

15

the long axis of the future embryo and the embryo axis

corresponds to the second plane of early cleavage (McEwen, 1953).
As development proceeds the embryonic shield increases in

size. The extra-embryonic ectoderm spreads around the yolk

and closes the blastopore after approximately eleven days of

incubation.

The early embryo

As the inflected cells move toward the anterior end of
the embryo, the extra-embryonic blastoderm moves around the
yolk to the opposite side and the embryonic shield increases
in size and thickness (Figure 3,C).

On the seventh day of incubation the primary organ rudi-
ments are distinguishable, the endoderm exists Just above the
periblast, the mesoderm which is derived partly from the
epiblast and partly from the hypoblast, occupies the mid-
region of the embryo and the uppermost layer is the ectoderm.
Along the midline of the embryo the ectoderm shows a distin-
guishable internal concentration of cells representing the
ectoderm neural cells, and Just beneath it the mesodermal axial
cells of the notochord can be observed (Figure 3,D).

A day later the extra-embryonic ectoderm covers one-
fourth of the yolk and at the same time the embryonic shield
increases in length. The medullary keel is observed, especially
at the anterior end of the embryonic shield, which will form
the brain. The thinner middle and posterior portions of

the neural keel represent the rudimentary spinal cord. The

l6

optic anlagen arise as lateral swellings from the presen-
cephalon and Just behind them four masses of cells arise on
both sides of the neural keel, but they do not distinguish the
mesencephalon from the rhombencephalon. The notochord becomes
visible and extends from the posterior margin of the brain
region to the anterior end of the undifferentiated tail knob.
In sections the notochord appears as a circular, hollow and
narrow axial structure beneath the neural ectoderm. The meso-
dermal tissues are condensed as lateral plates along the noto-
chord margins, and eleven pairs of mesodermal somites are
visible externally.

The ninth day the blastoderm spreads and covers approxi-
mately half of the yolk sphere: the embryo is better seen rising
over the yolk with twenty pairs of mesodermal somites. The
embryo increases in length particularly toward the caudal end,
which is thinner than the anterior end of the embryo. The
neural keel is enlarged and grows deeper than the preceding
stage and the eye stalk can be detected as a means of communi-
cation between the forebrain and the eye anlagen. The auditory
placodes, from which the internal ear vesicles develop, appear
as two solid masses of cells on the inner surface layer of the
epidermis on both sides of the hindbrain. The pectoral fin buds
can be observed as minute, external lateral proJections on both
sides of the embryo Just behind the ear placodes.

At the ten-day stage the extra-embryonic ectoderm spreads

over three-quarters of the yolk mass. The epiboly movement

17

is more active on the ventral blastoporal lip and it gradually
decreases along the ventral lips toward the dorsal lip where

it shows only a slow posterior movement. In this stage the
caudal knob changes to form a thick and definite round

structure and the finfold appears as a very narrow ectodermal
outgrowth, which starts dorsally from the posterior end of the
head region, moves around the caudal knob and advances ventrally
to about the middle of the trunk region (Figure 3,E). The
thickening of the ectodermal neural keel increases especially

in the brain region (Figure 3,F). The brain begins to differ-
entiate into its primary three vesicles: the prosencephalon or
forebrain, the mesencephalon or midbrain, and the rhombencephalon
or hindbrain. The olfactory placodes are distinguishable in the
form of two thickenings of the epidermis on either side of the
forebrain and in front of the eye vesicles which have been
invaginated inward and transformed into a double-walled, cuplike
structure (Figure 3,F). The eye lens anlagen are developed as
two thickenings from the inner epidermal walls which correspond
to each eye cup, and the optic cup is still in connection

with the prosencephalon through the optic stalk.

On the closure of the blastopore (11 days) the changes
become rapid. The rudimentary eye lens consists of a rounded
and undifferentiated mass of cells which is still in contact
with the inner wall of the epithelium. The retinal wall of
the optic cup is thickened and its inner cells can be seen as

fusiform in shape. The auditory vesicles are enlarged, forming

18

a small, hollow structure in the hindbrain region and still

are attached to the epidermal dorsal wall of the body. The
brain has increased in thickness and the hindbrain splits into
two divisions, the metencephalon or cerebellum, and the
myelencephalon or medulla oblongata. The medulla oblongata
shows a series of segmentations before its entrance in the
spinal cord. The midbrain remains the thinner part of the
brain, though its lateral and ventral floors undergo thickening.
The divisions of the forebrain are not clearly seen in the whole
mounts, but in the transverse sections the telencephalon or
cerebrum can be detected as well as the diencephalon. The
infundibulum arises as a slight depression from the diencephalon
floor. There are 36 pairs of mesodermal somites. The heart
primordium is marked clearly in this stage by the concentration
of the mesodermal cells along the midline of the ventral side

of the body Just behind the eye vesicles and above the peri-
blast. The notochord does not show a noticeable lengthening,
but it increases slightly in diameter and it is still hollow
with no signs of vacuolation. The finfold is prominent and

in the transverse sections it appears as a ridge of two
separated epithelial layers, each one is one cell in thickness.
The Kupffer's vesicle is distinguishable below the caudal region
as a small, round invagination of the yolk. McEwen (1953) has
concluded that it has a neural function. The pectoral fin buds
project slightly laterally Just behind the auditory anlagen.

In this stage the endoderm under the mesoderm, as well as the

Figure 3.-The early embryonic stages. A, The germ

ring and the early embryonic shield, aux. B,
Transverse section showing the beginning of
involution: C, The early embryo showing the
optic anlagen and the caudal knob after six days
of incubation, 20X: D, Transverse section of the
trunk region of the seven-day embryo, showing
the primary organ rudiments; E, The structure

of the embryo Just before the closure of the
blastopore at ten days, 20X; F, Head region of
the same embryo showing the ectodermal neural
keel and the sensory organ rudiments. end..
endoderm: Ep., epiblast: ep., epidermis: Hyp.,
hypoblast: mes., mesoderm; n.k., neural keel:
neur., neural cells; not., notochord.

19

 

 

21

notochord, forms a simple tube which is easily observed in
the transverse sections and represents the pharynx in the head

region 0

The late embryo

(1) Stages from 13 to 20 days of incubation

As development proceeds, the embryo increases in size
and the caudal region begins to curl Over the yolk sac. The
embryo lies over a slightly thickened part of the extra-
embryonic tissue. The head in these stages shows torsion
over the yolk. The notochord has increased in diameter and
extends to the caudal tip, and the vacuolations commence during
the end of this period. The brain is greatly increased in
size, particularly the hindbrain region, where the metenceph-
alon and the myelencephalon increase in width. The expansion
of the hindbrain (rhombecephalon) cavity gives rise to the
fourth ventricle, which is clearly seen from the dorsal view
(Figure h,C). The spinal cord at the end of this period has
started to appear in the caudal region as a separate cord lying
over the notochord. The olfactory placodes in the antero-
lateral region of the head have invaginated, thus forming the
olfactory sacs, and their naris opens exteriorly. Each eye cup
gives rise to a definite outer, thin iris and an inner retina
proper. The pigmentation of the eyes begins after this differ-
entiation by the invasion of the chromatophores within the
retinal wall. The eye lens is separated completely from the

corresponding ectodermal wall and lies within the eye cup

22

cavity. The lens shows further differentiation, its outer
margin transformed to the lens epithelium and the cells of the
inner bulk are elongated and transformed to the lens fibers
(Figure h,B). The gut can be seen from the ventral view of
the embryo as a simple tube extending to the posterior end of
the trunk region. In the pharyngeal region and Just behind
the auditory vesicles, the ectoderm walls form a series of
grooves, each one corresponding to one of the endodermal gill
pouches of the pharynx. The pectoral fin buds are enlarged
and were observed by the naked eye as small, lateral exten-
sions immediately behind the pharyngeal region of the embryo
(Figure 4,A). There are 6” pairs of mesodermal somites. The
coelomic mesoderm shows more differentiation. The endocardium
forms a single tube extending ventrally below the head region and
its venosus portion extends forward. The pronephric ducts and
tubules can be traced in the transverse sections as lateral
tubes from the dorsal portion of the coelomic mesoderm (Figure

4,D).

(ii) Stages from 21 to 27 days

The development of the gill arches is the important feature
in these stages: they are developed by the perforation of the
ectoderm wall of the gill grooves, a process described in the
preceding period. Later on the hyoid arch extends backward
from its external surface in order to cover the gill buds
behind. From the dorsal view, the mandibular arch is completely

hidden, and the hyoid arch is attached to it, forming the

23

hyomandibular arch, while the other gill arches are exposed.

The dorsal aorta is distinguishable and can be traced along

the body to the caudal region, below which the caudal vein runs.
The vitelline vein is evident and is directly connected to the
heart. It is filled with oval blood cells containing an
apparent nucleus. The cells located on the notochord rim

become thicker and form the notochord sheath and their proto-
plasmic strands form a netlike structure, filling the notochord
center. The notochord vacuolation is now complete in the entire
notochord. The eyes are heavily pigmented but the retinal wall
undergoes more differentiation with the appearance of the sclera.
The head is darker in color, due to the small, scattered spots
of melanophores on its dorsal region (Figure 4,E). The myotomes
are enlarged, the myosepta are definite, and the myofibrils

are observed in both the whole and the transverse specimens
((Figure 4,F). The gut rests ventrally and extends along the
midline of the body cavity, and in these stages is observed to
be lifted off the yolk. Below the intestine the subintestinal
vein is seen in the transverse sections. The tail through these
stages has increased in both length and in width since it is
completely flattened over the yolk and the dorsal and the ventral
margins of the finfold lie on both sides of the body.

(111) Stages from 28 to 34 days

The gradual increase in the embryo's size is accompanied
by a slight decrease in the yolk sphere. The opercle, which

started to grow posteriorly in the previous period. covers the

24

first gill arch by the 28th day and the second gill arch by
the 34th day. The notochord increases in diameter and extends
from its anterior end to the midbrain region and posteriorly
to the tip of the tail where it and the neural cord curve up
(Figure 4,D). The brain shows enlargement, especially in its
middle vesicle, which starts to overlap all the other parts

of the brain. The sense organs, the eyes, the auditory vesi-
cles, and the nostrils have enlarged and show more differen-
tiation. The cells of the optic cup have been arranged in
several layers representing the choroid, and the retinal and
ganglionic layers of the retina proper; the optic stalk is
transformed into the optic nerve and enters the floor of the
diencephalon. The lens fibers which occupy the inner portion
of the eye lens sphere are more condensed. The olfactory pits
move dorsally and their epithelium is surrounded by mesenchyme
cells, which also surround the auditory vesicles, later giving
rise to cartilage and producing the olfactory and auditory cap-
sules, respectively. The transverse sections show that a sheet
of cartilage beneath the floor of the brain has developed as a
part of the chondrocranium. The eye cups undergo a similar
change, forming the eye capsules. The liver makes its first
appearance during this period as a ventral pouch coming from
the gut in the body cavity and the intestine ends at approxim-
ately the posterior end of the trunk, marking the location of
the anus. The ventricle of the heart is well-defined and the

atrium is demarcated by the vitelline vein which enters the

Figure 4.-Stages of the late embryo. A, Whole mount of
the twenty-day embryo, showing the pectoral fin
buds and the beginning of pigmentation of the
eyes. 20X; B, Transverse section of the eye region
of the same embryo showing the olfactory pits
and the eyes; C, Transverse section in the hind-
brain region showing the hollow notochord and the
heart: D, Transverse section of the anterior trunk
region: E, 24-day embryo showing the position of
the vitelline vein, 6.5x. F, Transverse section
in the caudal region of the same embryo; G, The
undifferentiated caudal finfold, 125X. d.f.f..
dorsal finfold: e.c., eye cup; e.l., eye lens;
f.b., forebrain: H., heart; m.b., midbrain: n.k.,
neural keel: n.r.. nose rudiment: p.neph.. prone-
phric duct: v.f.f., ventral finfold.

25

 

2?

sinus venosus (Figure 5,E and F). The rudiment of the lower
Jaw is inferior to the forebrain and lies over the yolk. The
pigmentation spots are increased and the dorsum of the head

is mottled (Figure 5,D).

(iv) Stages from 35 days to hatching (40 days)

During this period the heart shows two important changes.
First the twisting, which may have begun during the final days
of the previous period, is now completed. The anterior or the
venous portion of the endocardial tube has inflected and grown
backwards. Its posterior or arterial end has grown upward
and forward and in the meantime, the heart has coiled to the
left as seen in diagrams A, B, and C of Figure 5. The heart
appears as an oval structure with a distinct ventricle and
atrium. Secondly, the heart moves back to lie Just behind the
gill arches. The heart and the blood vessels are filled with
erythrocytes. Semicircular canals of inner ear are distinct
and the ear capsules are formed. The eyes are prominent and
heavily pigmented so that they hide the eye lens (Figure 6,B).
The movement of the pupils of the eyes is observed, and the
anterior part of the head shows a slight extension, forming
the snout. The pharynx advances, pushing the mouth cavity
forward and the four pairs of gill arches are completely
covered with the opercle, forming the branchial chamber. By
focusing on the gill buds one can see the development of the
gill papillae on both surfaces of each gill bud, marking the

rudimentary gill rakers and filaments. The lower Jaw is well-

Figure 5.-The development of the heart. A,B,C, Diagrams
showing the twisting of the heart. the arrows indi-
cate the order of the looping; D, Thirty-day
embryo showing the darkly pigmented body, 6.5K; E,
Transverse section of the anterior trunk region of
the same embryo; F, Dorsal view of the looping
heart and the connection of the sinus venosus with
the vitelline vein, 125X; G, Transverse section of
the 35-day embryo showing the muscled ventricle of
the heart. A., atrium: C., conus arteriosuss 3..
heart: 3., sinus venosus: V., ventricle; v.v., vite-
lline vein.

28

 

 

 

 

30

developed but it is immobile and is supported by cartilagenous
elements (Figure 6.D). The somitic movements are distinguish-
able. The rudimentary dorsal, adipose, and anal fins can be
seen as thickening parts of the disintegrating finfold. The
pectorals are thin folds in which the supporting rays develop.
The embryo Just before hatching forms almost a complete circle
over the yolk, with a loosely attached head and detached tail.
The first signs of hatching occurred after approximately
36 days of incubation, but all the hatched larvae died during
or shortly after hatching. This may be due to some defect in
the hatching enzymes or to an earlier faulty development which
interrupted the normal processes of hatching (Battle, 1944).
But Riddle (1917) stated that the mortality of the hatching
chinook salmon, Q. tshawytscha, may be due to certain physio-
logical changes which did not occur in the chorion prior to
hatching. Bishai (1962) said that the yolk-sac disease is one
of the causes of the high mortality of the alevins. The yolk-
sac disease is caused by the rough handling of the eggs during
incubation or the malfunction of the thyroid gland. A period
of intensive hatching occurred in the 39th and 40th days.
Most of the fish emerged from the chorion by the tail while
the remaining ones emerged headfirst. Then the alevin under-
went an active lashing movement by which it freed itself from
the chorion. Riddle (1917) mentioned that the pectoral fins
move Just before hatching, piercing the chorion. After break-

ing away the alevin lies on its side for a period of rest, and

31

does not move except when disturbed. The Just-hatched alevin
is sixteen millimeters in length, scaleless, and shows a
coordinated movement of the opercle and lower Jaw as a part of
the respiratory process, which is mostly accomplished by the
vascular system covering the yolk (Figure 6,A). Rays in

the caudal fin are faintly seen with some pigment cells along
them (Figure 6,E). The abdomen is attached to the yolk sac,
the weight of which is responsible for the lashing movement
of the body, although the muscular system of the body is
well-developed. At the end of 28 days the alevin has almost
absorbed the yolk and enters the larval stage, being ready

to feed on external food.

"—

Figure 6.-The hatched embryo. A, The newly-hatched

embryo showing the lower Jaw and the vascular
system around the yolk sac, 6.5x. B, Transverse
section of the eye region showing the differen-
tiated retinal wall and the eye lens: C, Trans-
verse section of the trunk region Just behind the
anus; D, Transverse section of the oral region,
showing the lower and upper Jaws: E, The faint
caudal fin rays and the upcurved notochord, 125x.
F, Transverse section of the trunk region Just

in front of the anus. d.a., dorsal aortas e.l.,
eye lens; e.r., eye retinal wall; int.. intestine;
l.J., lower Jaw; my., myotome.

32

 

DISCUSSION

It is obvious that the early stages of development of the
coho salmon, Oncorhynchus kisutch, are typically teleostean.
Hatching occurred after forty days of incubation at a temper-
ature of 10 i l C, or after the embryo wassubJected to 720
thermal units. The thermal units required for the whitefish is
325 (Price, 1935), the Atlantic salmon requires 470 T.U. (Battle,
1944), and the steelhead trout needs 573.9 T.U. (Wales, 1941).
This indicates that the coho salmon embryo has a relatively
long period of incubation. According to Embody (1934), the
incubation period varies with the temperature. The similarity
of the coho's cleavage with that of other teleosts deviates with
the slight lengthening of the blastodisc, which was observed
in the first cleavage stage of the echo eggs. Also the swim-
ming bladder was not seen during the embryological stages of
the coho. Both results are unlike those observed in the deve-
lopment of other teleosts. This reveals that the development
of the swimming bladder may take place after hatching.

The concentration of oil globules at the animal pole and
around the blastodisc prior to the initial segmentation is of
physiological significance since the globules are composed of

phospholipids and lipoproteins and are absorbed by the embryo

34

35

from the beginning of development until the gastrular over-
growth (Smith, 1957). The periblast, which was seen beneath
the blastoderm and in contact with the yolk does not partici-
pate in the formation of the embryo but it may serve in
breaking down the yolk for the growing embryo (Balinsky, 1966).
It was found that at the closure of the blastoporal lips
most of the primordia of the organ systems had been established
and could be traced easily, such as the development of the
brain and the establishment of its three main vesicles, the
forebrain, the midbrain, and the hindbrain. The establishment
of the sensory organs was accompanied by the differentiation of
the retinal wall of the eye cups, the appearance of the nostrils,
and the development of the internal ears. The gut as an endo-
dermal organ, and its well-developed pharynx, was also found.
Following the disappearance of the yolk plug the embryo-
logical features peculiar to teleosts were apparent. The sub-
divisions of the brain and their nerve connections with the
sensory organs were easily observed. The differentiation of
both the cells of the retinal wall forming the four retinal
and ganglionic layers, and the eye lens forming the lens
epithelium and lens fibers were clearly seen. Another of
these features was the development of the infundibulum. The
gill arches, which are partly endodermal and partly ectodermal,
were distinguishable, as was the development of the operculum
as an external extension from the hyomandibular arch. Pectoral

fins appeared as lateral, fleshy extensions. The fish attained

36

the complete hatching number of somites which is 64 pairs at
the twenty-day stage and in the following stages the somites
underwent enlargement and the myofibrils and myosepta were
well-defined.

The establishment of the heart from the ventral portion
of the coelomic mesoderm, the pronephric ducts developing
from its dorsal portion, and the twisting of the heart and its
migration to the pharyngeal region are all characteristic of
primitive vertebrates. The discoidal cleavage of the blasto-
disc, the finfold as a primitive ectodermal outgrowth giving
rise to the unpaired fins of the alevin, and the pharyngeal
extension pushing the ventral mouth forward, prove the higher
phylogenetic position of this species among fishes, since these

structures can be found in the primitive teleosts.

SUMMARY

Approximately 3,000 coho salmon. Q. giggtgh, eggs were
obtained from the Platte River Station of the Michigan Depart-
ment of Natural Resources. The eggs were placed in a Heath Fish
Incubation Cabinet supplied with continuous flowing water with a
constant temperature of 10 1.1 C. The eggs were fixed in Bouin's
and Smith's solutions: the latter was replaced by Kahle's solu-
tion after the sensitive period of the embryo. Staining was done
with alum cochineal for the whole mounts and with Delafield's
hematoxylin and eosin for the transverse sections, which have
been cut at 5,u.and 8,u. Photographs were selected from both
series from the initial cleavage through hatching. The coho
salmon, Q. kisutch, embryology is typically teleostean. The
blastula stage was completely formed by the fourth day of
incubation and in the sixth day the gastrulation began by the
involution, epiboly, and convergence of the blastoderm cells.

At the end of the gastrulation, after eleven days, the yolk
sac was formed and most of the rudimentary organs were
distinguishable. Prior to hatching the organ systems showed
a high differentiation and more development. Hatching began
on the 36th day and intensive hatching occurred on the 39th

and 40th days of incubation. The yolk sac was completely

3?

38

absorbed 28 days after hatching and the alevin was then

ready to feed on external food.

LITERATURE C ITED

 

LITERATURE CITED

Armstrong, P.B. and J.S. Child. 1965. Stages in the normal
development of Fundulus heteroclitus. Biol. Bull.
128 (2) 3143-16 .

Balfour, F.M. 1885. A Treatise on Comparative Embryology.
Macmillan and Co.. London. Vol. 2. 792 p.

Balinsky, B.J. 1966. An Introduction to Embryology. W.B.
Saunders Co.. Philadelphia, 673 p.

Battle, H.J. 1940. The embryology of the goldfish.
Carassius auratus, from Lake Erie. Ohio Jour. Sci.
1:0 (2, 832-930

Battle, H.J. 1944. The embryology of the Atlantic salmon,
Salmo salar. Canad. Jour. Res. 22 (D,5) :105-125.

Bishai, J.M. 1962. Hydrocoele embryonalis in salmonids.
Salmon and Trout Magazine. 165:111-114.

Embody, G.C. 1934. Relation of temperature to the incuba-
tion period of eggs of four species of trout. Trans.
Amer. Fish Soc. 64:281-289.

Guyer, M.F. 1936. Animal Micrology. The University of Chicago
Press, Chicago, Illinois, 331 p.

Jones, J.W. 1961. The Salmon. Collins, St. James' Place,
London, 192 p.

Knight, A.E. 1963. The embryonic and larval development of
the rainbow trout. Trans. Amer. Fish Soc. 92 (4) :344-355.

McEwen, R.S. 1953. Vertebrate Embryology. Henry Hold and Co..
New York, 699 p.

Oris, J.J. 1968. The embryology of the English sole, Parophyrys
vetulus. Calif. Fish and Game. 54 (3) 3133-155.

Price, J.W. 1934(a). The embryology of the whitefish, Coregonus
clupeaformis. Ohio Jour. Sci. 34 (5) :287-305.

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41

Price, J.W. 1934(b). The embryology of the whitefish.

Coregonus clupeaformi . Ohio Jour. Sci. 34 (6) 3399-414.

Price, J.W. 1935. The embryology of the whitefish. Coregonus
clupeaformis. Ohio Jour. Sci. 35 (1) 340-53.

Richards, A. 1931. Outline of Comparative Embryology.
New York, John Wiley and Sons, Inc.. 432 p.

Riddle, M.C. 1917. Early development of the chinook salmon.
Publ. Puget Sound Marine Sta. 1 (28) 3319-339.

Shapovalov, L. and A.C. Taft. 1954. Life histories of the
steelhead rainbow trout, Salmo gairdenerii. and silver
salmon, Oncorhygchus kisutch. Ca ifornia Dept. of Fish
and Game Fish Bulletin, No. 98, 375 p.

Smith, S. 1957. Early development and hatching. In: The
Physiology of Fishes. Vol. 1. (M.E. Brown, Ed.).
Academic Press, Inc.. New York, 447 p.

Solberg, A.N. 1938. The development of a bony fish. Prog.
Fish Cult. 4031-19.

Walden, H.T. 1964. Familiar Fresh Water Fishes of America.
Harper and Row Publ., New York, 324 p.

Wales, J.H. 1941. Development of steelhead trout eggs.
Calif. Fish and Game. 27 (4) 3250-260.

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