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THE DEVELOPMENTAL PATTERN OF THE COLORADO RIVER
TOAD (BUFO ALVARIUS GIRARD) FROM STAGE
ONE THROUGH TWENTY
A Thesis
Presented in
Partial Fulfillment of the Requirements
for the Degree of
Master of Science in Biology
by
Willie James Hill
Arizona State University
Tempe, Arizona
1961
Approved
Gerald A. Cole
Graduate Adviser
TABLE OF CONTENTS
CHAPTER
I. INTRODUCTION
II. TAXONOMIC POSITION, DISTRIBUTION AND ECOLOGY
III. METHODS
The Induction of Gamete Formation; Method and Results
Methods Employed in Sectioning Embryos
IV. DISCUSSION OF DEVELOPMENTAL STAGES 1-20
Rates of Development
External Morphology
Internal Morphology
V. SUMMARY AND CONCLUSIONS
BIBLIOGRAPHY
PAGE
1
3
8
8
10
11
11
15
24
27
29
LIST OF TABLES
TABLE
1. Rates of Development of Two Species of Rana (at 65° F) and B. alvarius (at 72.5° F)
2. Rates of Development of R. Pipiens at 25°C as Compared to B. alvarius at 25.5°C
PAGE
13
14
LIST OF FIGURES
FIGURES
1. Map showing Geographical Distribution of B. alvarius
PAGE
7
LIST OF PLATES
PLATES
1. External Morphology of the Unfertilized Egg and Developing Embryo of B. alvarius
from Stage One Through Nine
2. External Morphology of the Developing Embryos of B. alvarius from Stage
Ten Through Fifteen
3. External Morphology of the Developing Embryos of B. alvarius from Stage
Sixteen Through Eighteen
4. External Morphology of the Developing Embryos of B. alvarius from Stage
Nineteen Through Twenty
5. Semi-diagrammitic Cross Sections of Embryos of B. alvarius at hatching
Stage Twenty
PAGE
21
21
23
23
26
CHAPTER I
INTRODUCTION
The developmental patterns of several North American anurans have been described in detail.
These include Rana pipiens Schreber, Bufo americanus Holbrook, and Rana sylvatica LeConte.
Since the beginning of modern embryology, extensive research has been done on the grass frog,
Rana pipiens, and its developmental history has been worked out in the most detailed manner.
Fertilized eggs have been studied under natural conditions as well as in the laboratory. Induced
ovulation by means of pituitary injection has been of great value in such research.
As early as 1897, T.H. Morgan reported on the development of the frog's egg. In 1931, A. E.
Adams reported on artificial induction of ovulation in frogs and toads. Additional embryologists who
have investigated frogs and toads include Shumway, (1927), Weiss (1939), Rugh (1934, 1935),
Pollister (1937), and several others.
Although there are many published accounts of the embryological development of amphibians,
including several anuran species, no such description of the ontogeny of the Colorado River Toad
(Bufo alvarius Girard) is available. It is the purpose of this dissertation to report on the early
embryology patterns of this unusual amphibian.
Development following fertilization has been classified according to a series of stages, to
accentuate certain embryonic structures or patterns. These stages normally range from one through
twenty-five. Stages one through twenty (the time of hatching) mark the most dramatic stages of
embryological development. For this reason, I have concentrated my study on these stages in B.
Alvarius.
Specimens for use in this study were collected near the Thunderbird Academy, Scottsdale,
Arizona; the mouth of the Verde River, Payson, Arizona; and near irrigated fields in the vicinity of
Glendale, Arizona. Others were borrowed from the Poisonous Animal Research Laboratory, Arizona
State University, Tempe, Arizona.
CHAPTER II
TAXONOMY, DISTRIBUTION, AND ECOLOGY
The rare toad, B. alvarius, is a member of the family Bufonidae. The characteristics of the family
are: generally short limbed, chunky, broad-waisted, warty anurans with conspicuous parotid glands;
usually with varied development of ridges (cranial crest) between and behind the eyes; pectoral girdle
arciferous; procaelous vertebrae; no ribs; urostyle attached to double condyle (simple in pelahotids on
sacrum); anterior end of centrum of sacral vertebra concave; sacral diapaphyses more or less dilated; no
teeth; tongue elliptical or pear shaped and free posteriorly; pupils horizontal; eardrums distinct,
obscure, or absent; two metatarsal tubercles, inner one larger.
Most members of the Bufonidae are terrestrial during most of the year, entering water only to
breed. They commonly deposit their eggs in two gelatinous strings (Stebbins, 1951).
B. alvarius is the largest of the western toads. Adults may attain a snout-vent length of six or
seven inches. The skin is generally smooth, but small scattered warts appear on the dorsal and lateral
surfaces, and many small tubercles are present. Warts are light-colored and set in dark areas in
juveniles, but the dark zone is obscure or absent in adult individuals; the parotid glands are somewhat
bean shaped, divergent posteriorly, two to three times as long as wide and smooth. The area between
the glands (dorsally) is three to four times the width of the gland. Cranial crests are prominent and
crescent-shaped. The eardrums are conspicuous. Several prominent rounded warts usually arranged in
a row extend backward from the angle of the jaw. There are several dorsal femoral warts, one usually
elongate, and much enlarged, that stand out from the surrounding smooth or minutely warty skin.
There is a membranous fold of skin on the inner side of the tarsal region. The color of B. alvarius
ranges from dark brown to brownish green, to olive or grayish with some warts often pale orange, or
orange brown. The vertical surface is light colored and usually unmarked. At the anterior corner of
each eye appears a whitish area. The irides are rust colored.
The sexes can be distinguished by the dark nuptial pads on the thumbs and inner fingers of the
male; the thumb is larger than that of the female. Another sexual difference is the presence of reddish
colored warts in straight lines on the dorsal surface of the female; these are absent or only slightly
pigmented in the male.
As is true of most toads, B. alvarius is largely nocturnal. Although summer rains seem to start
seasonal activity, wet weather is not always responsible for its appearance, for it has been known to
emerge in the absence of rain. Though an inhabitant of arid regions, it is dependent on the presence of
streams or ponds for breeding and frequently breeds in temporary pools and irrigation ditches.
Toads assigned to the genus Bufo are distributed throughout the world except for New Guinea,
Polynesia, Australia, and Madagascar. In the Americas, species of Bufo occur from the Hudson Bay
region, Labrador, possibly the Aleutian Islands, along the southern coast of central Alaska southward
through North and Central America well into South America. It has been established that there are
approximately 250 species belonging to this genus (Stebbins, 1951)
B. alvarius occurs only in the lower and upper Sonoran life-zones, and in Arizona it occurs
principally in the drainage of the Gila River. (Fig. 1)
Fig. 1. Map showing the distribution
of B. alvarius. The darkened area
represents its total geographical
distribution as far as known.
CHAPTER III
METHODS
I. INDUCTION OF GAMETE FORMATION AND RESULTS
Several males and females of B. alvarius were collected and kept in the laboratory for the purpose
of obtaining fertilized eggs. These toads were collected in late September, 1960, and kept under
temperature-controlled conditions. Two males and two females were kept at 65° F, and two males and
two females were maintained between 70° F and 80° F. Thew were observed and tested daily for two
weeks, but no sign of sperm production or ovulation could be detected. Then they were placed in a
refrigerated room, at 55° F, where they remained dormant for two weeks.
The temperature of 55° F seemed to be low enough to induce dormancy, and was selected
because temperatures of 40° F to 45° F had detrimental effects upon the toads. On numerous occasions
toads were placed in a refrigerated room at 40° F and within one week they had perished. If they were
removed periodically (after twelve hours at 40° F) and allowed two or three hours to warm up, they
survived in every case. Apparently these toads cannot tolerate even relatively short periods of
uninterrupted low temperatures. This may be a significant factor in the limited geographical range of
the species.
After two weeks at 55° F the toads were removed from the cold room and kept three hours at
room temperature. They were then injected with 2ml. of beef pituitary extract, dissolved in absolute
ethanol. The suspension was drawn into a 5 ml. luer-type syringe, and was injected with an 18 gauge
needle. The injections were made with the toad placed with the dorsal region facing the palm of the
hand, and the cloacal region pointing towards the finger tips. Holding the toad in the above manner
with the left hand, the injection was made in the lower left quadrant, about one-fourth inch into the
abdominal region. Care was taken to avoid injury to vital organs, especially blood vessels.
After these injections, the toads were observed for 60 hours to see if ovulation had been induced.
The results were negative in all instances. The same steps as outlined above were repeated on other
male and female toads, but instead of using absolute ethanol to prepare the suspension, a 65% saline
solution was used. Within 48 hours after injection, amplexus occurred. The eggs were laid in single
strings early in the morning. With such positive results observed it was desirable to see just how fast
the beef pituitary extract worked. Two males were injected and placed in separate beakers. Four hours
later the urine from each beaker was examined microscopically with the results that both samples
contained many spermatozoa. This simple technique proved to be a very useful and efficient method
for testing the effectiveness of the injected gonadotropic hormone.
Whole pituitaries excised from B. alvarius and injected into other males and females of the same
species worked extremely well. Also, pituitaries excised from Rana pipiens produced positive results
in B. alvarius.
Using beef pituitary powder, whole beef pituitary powder, both dissolved in 65% saline solution,
and whole pituitaries (from either B. alvarius or Rana pipiens) it was possible to obtain sperm and eggs
within 48 hours in almost every case.
It should be noted that certain precautionary methods were found necessary to ensure success of
the techniques described above. For example, it was found that extreme care had to be exercised in
injecting the toads, because their vital organs were easily damaged, resulting in death in some
instances. Furthermore, it was found necessary to keep syringes, needles, and glassware meticulously
clean. Also, the laboratory containers and water supply were kept clean and fresh. Filtered pond water
was used for culturing the eggs and was changed at regular intervals, care being taken to keep the
temperature the same as that at which the eggs were previously developing.
II. METHODS EMPLOYED IN SECTIONING EMBRYOS
Embryos o be section or to be prepared as whole mounts were fixed in Smith's modification of
Tellyesnicky's flud, and preserved in 10% formalin.
Selected embryos were taken from 10% formalin and washed for three hours in distilled water.
They were then stained for 24 hours in Semichon's Carmine, after which they were removed from the
stain, impregnated with celloidin, and embedded in paraffin. Single sections were then prepared by
splitting the embryos with a safety razor (flooded with cedarwood oil) in any plane desired. This
method proved to be very efficient where serial sections were not desired.
Serial cross sections were prepared by using celloidin-impregnated and paraffin-embedded
embryos sectioned with a microtome at a thickness of 25 microns.
CHAPTER IV
DISCUSSION OF DEVELOPMENTAL STAGES 1-20
The characteristic stages 1 through 20 could be recognized during the development of B.
alvarius. From stage 13 through 20, as the embryos increased in length, they appeared narrower, both
laterally and longitudinally, than embryos of B. americanus, Rana pipiens, or R. sylvatica. Except for
this slender profile, the external appearance did not appear atypical.
I. RATES OF DEVELOPMENT
Developing embryos were kept at 65° F, 70° F and 80° F. At 65° F development was slower
than at 70° F. An increase from 75° F to 80° F did not accelerate development niticably. Temperatures
below 60° F resulted in death of most of the embryos. Optimum developmental temperatures ranged
from 70° F to 75° F. An average temperature of 72.5° was maintained for the developing embryos
from stage 1 through 20.
Care was taken to protect the embryos from sudden temperature changes. Abrupt temperature
changes resulted in their death early in the study.
All embryos did not hatch simultaneously, as has been reported for other anurans. There was a
time lapse of ten minutes between the escape of the first and last embryo from the gelatinous envelope.
The time required for B. alvarius to reach the hatchling stage can be increased by lowering the
temperature of the pond-water medium. However, even with the temperature lowered to 65° F, or
slightly lower, the rate of development is relatively fast.
When compared to other frogs and toads, B. alvarius develops from zygote to hatchling at a
remarkably fast rate. Table 1 and 2 illustrates this rapid development compared to two species of Rana.
Data for the latter were taken from Rugh (1951) and Hamburger (1960).
Table 1
Rates of Development of Two Species of
Rana and B. alvarius (time in hours)
STAGE
R. PIPIENS
65°F
R. SYLVATICA
65°F
B. ALVARIUS
72.5°F
3 – Two Celled
3.5
2.5
1.5
4 – Four Celled
4.5
3.0
2.5
5 – Eight Celled
5.7
4.5
3.5
8 – Blastula
16.0
12.0
7.5
9 – Gastrula
21.0
16.0
11.0
10 – Mid-gastrula
20-24
16-20
13-15
11 – Late Gastrula
25.0
20-23
17.0
12 – Neural Plate
29-37-5
23-26
18-20
13 – Neural Fold
38-44
37.7
21-22
14 – Rotation
43.5-48
37.7
21-25
15 – Neural Tube
50.54.5
40.5
28-30
16 – Tail Bud
51-58
40-45
28-30
17 – Muscular Response
60-69
-------
39.0
18 – Gill Buds
70-84
50.0
43.0
19 – Heart Beat
85-96
66.0
58.0
20 - Hatching
95-103
72-87
68-76.5
TABLE 2
Rates of development of R. pipiens at 25°C as
compared to B. alvarius at 22.5°C. (time in hours)
STAGE
R. PIPIENS
B. ALVARIUS
1
0
0
2
½-1
½
3
2½
1.5
4
3½
2.5
5
4½
3.5
6
–
3.5
7
–
04/04/05
8
23
7.5
9
20
11.5
10
--
13-15
11
--
17.0
12
40
18-20
13
48
21-22
14
--
21-25
15
56
28-30
16
66
34
17
76
39.0
18
--
43.0
19
--
58.0
20
--
68-76.5
II. EXTERNAL MORPHOLOGY
The eggs of B. alvarius appear black upon superficial examination. A closer examination under
a dissecting microscope reveals that they range from dark brown to black. The characteristic dark
hemisphere (animal hemisphere) and the lighter hemisphere (vegetal hemisphere) prevail. These areas
of contrasting pigmentation are shown in plate 1, A.
The eggs are encased in a single, long tube of jelly. The envelope is somewhat loose, but quite
gelatinous material is clear, transparent, and seems not to be very adhesive. No partitions exist between
the individual eggs, whose arrangement varies from an almost perfect linear series of near spheres to a
zigzag patten of broadly wedge-shaped eggs. The number of eggs per inch averages about eighteen,
ranging from twelve to twenty-eight. Measurement of a number of eggs revealed a mean diameter of
2.2 mm.
The unfertilized egg is stage 1 in the development of B. alvarius. Stages 2 through 20 cover the
embryological development of B. alvarius from fertilizationthrough hatching (stage 20).
The fertilized egg (stage 2) showed the characteristic gray crescent area between te animal and
vege tal hemisphere. This area developed on the side of the egg opposite sperm entrance, (plate 1, B).
The second cleavage plane (stage 4) also began at the animal pole, crossing the first cleavage
furrow at right angles and dividng the embryo into four blastomeres. Careful inspection of the animal
hemisphere showed that these two furrows are often not exactly at right angles. Se Plate 1, D for
external morphology at this stage.
The third cleavage (stage 5) is marked by the formation of a latitudinal furrow slightly above
the equator of the blastomeres and lying in the animal hemisphere. This latitudinal furrow divides the
cleaving embryo into eight blastomeres. This furrow is not an exact circle but has irregularities where
it intersects the other cleavage planes, (plate 1, E).
The fifth cleavage division (stage 7) is marked by two furrows again being formed, one in the animal
hemisphere and one in the vegetal hemisphere. On the completion of the upper and lower furrows,
thirty-two blastomeres are formed. As is true in R. pipiens, R. sylvatica, and some other anurans, the
blastomeres in the animal hemisphere are smaller than those in the vegetal hemisphere. From this stage
on, increasing irregularity of the cleavage planes is apparent. This results in the plastomeres becoming
increasingly smaller in both hemispheres, but the difference between those in the animal hemisphere
(micromeres) and those in the vegetal hemisphere (macromeres) becomes more pronounced.
Stage eight (a mid-cleavage stage ) shows no unusual peculiarities.
By stage nine (Plate 1, I), the pigmented area has increased relatively greater than the
unpigmented area. The cells in the pigmented area are so small at this time that they can be seen only
with difficulty when examined with a hand lens, but can be seen clearly with the aid of a dissecting
microscope. The blastocoel was noted by sectioning embryos at this stage. This is also characteristic
of the embryos of other anurans of the same stage (i.e., R. pipiens, R. sylvatica). The roof of this cavity
is very thin; its floor, made up of cells of the vegetal hemisphere, is very thick.
After the formation of the blastula, the embryo undergoes further differentiation into a gastrula
and the three germ layers start to differentiate. Externally the process of gastrulation, or germ layer
formation is marked by the progressive development of the blastopore. The first indication of
gastrulation is the appearance of a somewhat crescent-shaped ridge on one side of the surface of the
late blastula at the junction of the micromeres and macromeres. Study of sagittal sections of embryos
at this stage showed that in this region, surface cells were involuting to form a slit-like cavity, the
gastrocoel. As the dorsal lip seemed to advance over the surface of the vegetal hemisphere its ends
elongated to form the lateral lips. As it advanced, the margin of the blastopore moved almost
completely over the vegetal hemisphere and the lateral lips united to form the ventral lip.
Stages ten, eleven, and twelve were studied by makindg preparations of the dorsal lip, and
ventral lip (yolk-plug) stages of gastrulation. In this wal the position o all three germ layers can be
noted. The short impregnating and sectioning method described previously, using celloidin and
cedarwood oil plus a safety razor was employed to study these stages. Position of germ layers seemed
to be typical as described by Adamstoned and Shumway (1954) for R. pipiens.
Stage thirteen is marked by early differentiation of a nervous layer in the dorsal region. The
thickening of this layer produces a longitudinal flattening on the dorsal surface of the embryo and is
called the neural plate. At this stage the blastopore is nearly closed and is merely a dorso-ventral slit.
Stage fourteen marks the neural fold stage. The embryo at this time is increasing in length. The
margins of the neural plate are elevated to form te neural folds. These folds bound a shallow groove,
the neural groove. At the anterior end of the embryo extending around the neural folds on either side is
a crescentric elevated area known as the sense plate. The blastopore is surrounded by the neural folds
on the right and left. Sections show that the neural folds are produced by a thickening of the nervous
layer in that region. The lateral portion of each neural fold is destined to give rise to the cells of the
neural crest. The notochord is separated from the mesoderm on each side, but it is in rather close
contact with the advancing sheets of entoderm, which will form the roof of the gut. Plate 2, E and F,
shows the neural folds and the neural groove.
At stage 15 the embryo has increased in length. The neural folds are in contact with each other
along the mid-dorsal line, but have not yet fused. Posterior to the sense plate, the gill plate can be seen
(slightly) on either side of the neural folds. Sections show that the neural crests are being drawn over
the developing neural tube, and the neural folds enclose a longitudinal canal, the neurocoel. The
mesoderm nearest the notochord is segmented into blocks, or somites, of which three or four pairs may
be distinguished. Sagittal sections were prepared of stage-15 embryos. The two halves were studied
with the aid of a dissecting microscope.
Stage sixteen is marked by the formation of the neural tube. The embryo of B. alvarius at this
stage is a little more than 4.5 mm. In length. From the dorsal aspect the embryo is somewhat pear
shaped, and, on each side, transverse grooves inclosing the gill plate can be seen. B. alvarius at this
stage seems to be much narrower than comparable embryos of R. pipiens or R. sylvatica. On the
ventral surface at the anterior end are two swellings, the primordia of the mucous glands. Between
them a depression can be seen which is the developing stomodeum. At the posterior end, the
blastopore is closed over by the fused neural folds. At the postero-ventral end of the fused blastopore
the beginning of the proctodeum can be seen. Six somites can be seen.
Stage seventeen is marked by the presence of a distinct tail bud. The mucous glands at this time
have enlarged considerably. A ventral view of the mucous glands shows a groove in each extending
from the posterior end forward giving each gland the appearance of an elongated U. Between them the
stomodeum may be identified. At the posterior end of the body, a short distance ventral and anterior to
the base of the tail bud, the proctodeum can be plainly seen now. In lateral view, a swelling in the
sense plate region indicates the outgrowth of the optic vesicle. Posterior to the gill plate, a series of
slight transverse grooves marks the presence of from eleven to thirteen somites.
Stage eighteen is marked by muscular response to mechanical stimuli. Embryos of B. alvarius
respond to prodding of a glass needle at this time.
Stage nineteen is marked by the first heart beats. These may be observed readily with a
dissecting microscope. Also, at this stage the gill buds are prominent. The mean length of the B.
alvarius embryos measured at this stage was 6 mm.
Plate No. 1.
A. Stage one, unfertilized egg.
B. Stage two, fertilized egg (gray crescent).
C. Stage three, two-celled.
D. Stage four, four-celled.
E. Stage five, eight-celled.
F. Stage six, sixteen-celled.
G. Stage seven, fifth cleavage.
H. Stage eight, mid-cleavage.
1. Stage nine, late cleavage (blastula).
Plate No 2.
A. Stage ten, early gastrula.
B. Stage eleven, mid-gastrula.
C. Stage twelve, late gastrula.
D. Stage thirteen, neural plate.
E. Stage fourteen, neural fold.
F. Cross section of neural fold stage.
G. Stage fifteen, late neural fold.
H. Cross section of late neural fold.
PLATES NO 1 & 2
Plate No. 3.
A. Stage sixteen, neural tube and early tail bud.
B. Lateral view of neural tube stage.
C. Stage seventeen, tail bud and some muscular response.
D. Lateral view of tail bud stage.
E. Stage eighteen, muscular response and gill buds.
F. Lateral view of muscular-response stage.
Plate No. 4.
A. Stage nineteen, heartbeat.
B. Lateral view of heartbeat stage.
C. Stage twenty, hatching.
1. Lateral view of hatching stage.
III. INTERNAL MORPHOLOGY
In order to study the internal development of B. alvarius, serial sections were prepared of
embryos 3 – 4 mm. Long and embryos at hatching stage.
In the 3 mm. Embryos, characteristic structures, including early organ systems could be
detected. The brain, liver, proctodeum, epidermis, and mid-gut could be detected.
The serial sections of the newly hatched embryos showed several deviations from the
comparable stages of other known anurans. A section through the anterior division of the brain showed
that the epiphysis is somewhat immature at this time (Plate 5, A). It is a hollow dorsal evagination
from the diencephalon. In comparable stages of development in other frogs and toads, the epiphysis is
relatively long, and solid by the time hatching occurs. Other sections through the head showed that the
lens vesicles were still attached to the head ectoderm, (Plate 5, B).
The liver is also relatively immature at hatching time, (Plate 5, D). In other anurans, at the
hatching stage, the liver has developed an anterior branch. This was not noted in my B. alvarius
preparations.
Another peculiarity was seen in development of the somites, (Plate 5, E, F). It appears that the
somites are quite advanced at hatching time, and have become conspicuously V-shaped. Comparable
sections of other frogs and toads show the somites to be similar, but they are not as well developed.
Although somewhat V-shaped, they are not bent so abruptly, and are less advanced than those of B.
alvarius.
Plate No. 5.
(semi-diagrammatic cross sections).
A. Position of the epiphysis.
B. Location of the lens vesicles.
C. Morphology of the hypophysis.
D. The appearance of the liver.
E. The appearance of the somites.
F. The appearance of the somites.
Plate No 5
CHAPTER V
SUMMARY AND CONCLUSIONS
During the development of B. alvarius the characteristic, described stages, one through twenty,
were recognized and studied. These stages showed similar patterns of development to those known for
other frogs and toads, although some minor differences were apparent.
The most striking difference was the rate at which B. alvarius developed from the time of
fertilization until hatching. The whole process was completed between 68 and 76.5 hours. This is
exceedingly faster than the development of other frogs and toads such as B. americanus, R. pipiens, R.
sylvatica, and R. catesbeiana.
Temperature is a very important factor in the development of B. alvarius. The optimum
temperature ranged from 70° to 75° F. Temperatures above 75° F did not appreciably accelerate
development. Abrupt temperature changes were detrimental. Lowering of the temperature to 65° F
retarded development somewhat, but development still progressed, and at a fast rate. Temperatures
below 60° F killed many of the embryos.
During the early stages of development the external morphology of the embryos seemed
normal. However, the general body profile appeared to become unusually narrow as the embryos
began to increase in length.
Through the study of serial cross sections some deviations in internal development were noted.
No differences from other anurans were detected in the serial sections of three to four mm. embryos,
but in the hatching-stage embryos, the lens vesicles, liver, and epiphysis seemed to be relatively underdeveloped. Another difference was noted in the development of the somites, which appear to be more
advanced than some known anurans at this same stage.
The results of this study indicated that ovulation can be induced in B. alvarius by injecting
whole pituitaries from its own species, from pituitaries of R. pipiens, or from injecting pituitary
powder, or whole beef pituitary powder (dissolved in 65% saline solution). When ethanol was used as
a solvent, negative results were obtained. It is believed that ethanol denatured protein substances in the
pituitary extract.
There remain many problems to be worked out concerning the rare toad, B. alvarius. Among
these are the nature of factors limiting in its geographical distribution, the effect of temperature upon its
late breeding habits, the nature of the poisonous glands within its skin, and its ontogeny from hatching
to adulthood.
BIBLIOGRAPHY
Adams, A. E. 1931. Induction of ovulation in frogs and toads. Proc. Soc. Exp. Biol. Med. 26:677.
_____, 1934. The gonad and thyroid stimulating potencies of Phycone. Hebin. Anat. Rec. 59:349
_____, 1941. Studies in Experimental Zoology. Ann Arbor: Edward Bros.