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LIBRARY
Michigan State
University .
THE EFFECT OF INJECTED THIOURACIL ON BODY WEIGHT AND
HATCHABILITY OF THE CHICK EMBRYO
BY
MADHESHWAR DHARI SINHA
AN ABSTRACT
Submitted to the College of Agriculture
Michigan State University of Agriculture and
Applied Science in partial fulfillment of
the requirements for the degree of
MASTER OF SC IENCE
Department of Poultry Science
1959
Approved:
MADHESHWAR DHARI SINHA 1
This study was done to determine the effect of thioura-
cil on early chick embryos. Most of the data published were
mainly after thiourea or thiouracil were injected.
First, different dosages of thiouracil were tried
to determine the toxic level of this drug. Five milligrams
of thiouracil were found to be the toxic level for the chick
embryos and 2.0 mg. was found to be the maximum level to be
used in any physiological work.
One-tenth, 0.2, 0.5 and 2.0 mg. of thiouracil was
injected into the small end of fertile eggs that had been
incubated for h8 hours.
Two groups of controls were kept. One group was
injected with Ringer's solution and one group received no
injection. The eggs were then incubated in forced draft
incubator at 37.50 C and a relative humidity of 66%. The
eggs were turned every two hours and were candled at LB hour
intervals after the treatment.
Between the 7th and the let day of incubation,
twelve eggs from each of the treatments were randomly selected
and sacrificed so embryos' body weights could be determined.
The average body weights of all thiouracil injected
embryos autOpsied on the 7th through the let day of incuba-
tion were lower than those of controls. Significant weight
differences between individuals receiving different levels
of thiouracil were noted after the 11th day of incubation.
MADHESHWAR DHARI SINHA 2
In each group, 12 fertile eggs were left in the incubator
after the let day of incubation until they hatched or died.
The experiment lasted for 6 more days and on the 27th day
all the remaining eggs were sacrificed. Embryos receiving
0.1, 0.2, 0.5 and 1.0 mg. hatched by the 25th day of incu-
bation. 0n the 27th day, some live embryos were found in
the eggs which had received an injection of 2.0 mg. of
thiouracil.
Both groups of controls hatched on the 20th and let
day of incubation. All of the control embryos showed re-
traction of the yolk sac at hatching time; whereas in many
of the treated embryos, yolk sacs were still outside the
body cavity at hatching time.
THE EFFECT OF INJECTED THIOURACIL ON BODY WEIGHT AND
HATCHABILITY OF THE CHICK EMBRYO
BY
MADHESHWAR DHARI SINHA
A THESIS
Submitted to the College of Agriculture
Michigan State University of Agriculture and
Applied Science in partial fulfillment of
the requirements for the degree of
MASTER OF SCIENCE
Department of Poultry Science
1959
ACKNOWLEDGEMENT
The author wishes to express his sincere appre-
ciation and thanks to Dr. Robert K. Ringer for his invaluable
assistance and guidance which enabled the completion of
this study.
He is indebted to Dr. Theo H. Coleman for his help
and discussion on the thesis.
The writer wishes to acknowledge his indebtedness to
Mr. Kenneth Rood and Mr. E. W. Speakmann for their assistance
in compiling and evaluation of the data.
The author also wishes to thank Dr. H. C. Zindel
for his c00peration in making facilities available for this
study.
11
TABLE OF CONTENTS
CHAPTER
I.
II.
III.
VI.
VII.
INTRODUCTION AND REVIEW OF LITERATURE
History . . . . . . . . . . . . . .
Mode of Action . . . . . . . . . .
OBJECTIVE O O O O O O O O O O O O O 0
To determine the Dosage Level of Thiouracil .
To determine the effect on the .aely Life
the ChiCk Enbryo O C O O O O O C
To determine the Effect on Hatchability .
METHODS AND PROCEDURE . . . . . . . .
EXperiment No. 1 . . . . . . . . .
EXperiment No. 2 . . . . . . . . .
RESULTS AND DISCUSSION . . . . . . .
The Influence of Different Dosages of
Thiouracil . . . . . . . . . . .
Effect of Thiouracil on Body Weight
Effect of Thiouracil on Harching and Yolk
Re traCtion e e o o o o o o o o o
SUWARY o o o o o o o o o o o o o o
BIBLIOGRAPHY . . . . O . O . . O O .
APPENDIX . .2.. . . . . . . . . . .
iii
PAGE
O‘O‘UIP
\ONNOO
11
11
ll
19
22
38
TABLE
I.
II.
III.
.v.
VI.
VII.
VIII.
IX.
LIST OF TABLES
Effects of different dosages of thiouracil on
chick embryos injected at us hours of
incubation. O O O O 0 O O O O O O O O O O 0
Average body weight in gms. of chick embryos
injected with thiouracil at 2h hours of
incubation compared with controls injected
with Ringer's solution or untreated . . . . .
Effect of Thiouracil on length of incubation
periOd O O O O O O O O O O O O O O O O O O 0
Body weight in gms. of 7-day old chick embryos
injected with thiouracil at #8 hours of
incubation compared with controls injected
with Ringer's solution or untreated . . . . .
Body weight in gms. of 8-day old chick embryos
injected with thiouracil at h8 hours of
incubation compared with controls injected
with Ringer's solution or untreated . . . . .
Body weight in gms. of 9-day old chick embryos
injected with thiouracil at h8 hours of
incubation compared with.controls injected
with Ringer's solution or untreated . . . . .
Body Weight in gms. of 10-day old chick embryos
injected with thiouracil at #8 hours of
incubation compared with controls injected
with.Ringer's solution or untreated . . . . .
Body weight in gms. of ll-day old chick embryos
injected with thiouracil at h8 hours of
incubation compared with controls injected
with.Ringer's solution or untreated . . . . .
Body weight in gms. of 12-day old chick embryos
injected with thiouracil at h8 hours of
incubation compared with controls injected
with Ringer's solution or untreated . . . . .
Body weight in gms. of 13-day old chick embryos
injected with thiouracil at R8 hours of
incubation compared with controls injected
with.Ringer's solution or untreated . . . . .
iv
PAGE
21
#5
he
in
ta
#9
50
TABLE
XI.
XII.
XIII.
IXV.
XVI.
XVII.
XVIII.
LIST OF TABLES
Body weight in gms. of lR-day old chick embryos
injected with thiouracil at R8 hours of
incubation compared with controls injected
with Ringer's solution or untreated . . . . .
Body weight in gms. of 15-day old chick embryos
injected with thiouracil at R8 hours of
incubation compared with controls injected
with Ringer's solution or untreated . . . . .
'Body weight in gms. of l6-day old chick embryos
injected with thiouracil at R8 hours of
incubation compared with controls injected
with Ringer's solution or untreated . . . . .
Body weight in gms. of 17-day old chick embryos
injected with thiouracil at R8 hours of
incubation compared with controls injected
with.Ringer's solution or untreated . . . . .
Body weight in gms. of 18-day old chick embryos
injected with thiouracil at R8 hours of
incubation compared with controls injected
with Ringer's solution or untreated . . . . .
Body weight in gms. of 19-day old chick embryos
injected with thiouracil at R8 hours of
incubation compared with controls injected
with Ringer's solution or untreated . . . . .
Body weight in gms. of 20-day old chick embryos
injected with thiouracil at R8 hours of
incubation compared with controls injected
with Ringer's solution or untreated . . . . .
Body weight in gms. of 31-day old chick embryos
injected with thiouracil at R8 hours of
incubation compared with controls injected
with Ringer's solution or untreated . . . . .
PAGE
51
52
53
51:
SS
56
57
58
LIST OF FIGURES
FIGURE PAGE
1. Average body weight in gms. of 7-day old chick
embryos injected with thiouracil at R8 hours
of incubation compared with controls injected
with Ringer's solution or untreated. . . . . . 23
2. Average body weight in gms. of 8-day old chick
embryos injected with thiouracil at R8 hours
of incubation compared with controls injected
with Ringer's solution or untreated . . . . . 2R
3. Average body weight in gms. of 9-day old chick
embryos injected with thiouracil at R8 hours
of incubation compared with controls injected
with Ringer's solution or untreated . . . . . 25
R. Average body weight of gms. of 10-day old chick
embryos injected with thiouracil at R8 hours
of incubation compared with controls injected
with Ringer's solution or untreated . . . . . 26
5. Average body weight in gms. of ll-day old chick
embryos injected with thiouracil at R8 hours
of incubation compared with controls injected
with.Ringer's solution or untreated . . . . . 27
6. Average body weight in gms. of 12-day old chick
embryos injected with thiouracil at R8 hours
of incubation compared with controls injected
with Ringer's solution or untreated . . . . . 28
7. Average body weight in gms. of 13-day old chick
embryos injected with thiouracil at R8 hours
of incubation compared with controls injected
with Ringer's solution or untreated . . . . . 29
8. Average body weight in gms. of lR-day old chick
embryos injected with thiouracil at R8 hours
of incubation compared with controls injected
with Ringer's solution or untreated . . . . . 30
9. Average body weight in gms. of 15-day old chick
embryos injected with thiouracil at R8 hours
of incubation compared with controls injected
twith Ringer's solution or untreated . . . . . 31
10. Average body weight in gms. of 16-day old chick
embryos injected with thiouracil at R8 hours
of incubation compared with controls injected
with Ringer's solution or untreated . . . . . 32
vi
FIGURE
11.
12.
13.
15.
LIST OF FIGURES
Average body weight in gms. of 17—day old chick
embryos injected with thiouracil at R8 hours
of incubation compared with controls injected
with Ringer's solution or untreated . . . .
Average body weight in gms. of 18-day old chick
embryos injected with thiouracil at R8 hours
of incubation compared with controls injected
with Ringer's solution or untreated . . . .
Average body weight in gms. of 19-day old chick
embryos injected with.thiouracil at R8 hours
of incubation compared with controls injected
with Ringer's solution or untreated . . . .
Average body weight in gms. of 20-day old chick
embryos injected with thiouracil at R8 hours
of incubation compared with controls injected
with.Ringer's solution or untreated . . . .
Average body weight in gms. of 21rday old chick
embryos injected with thiouracil at R8 hours
of incubation compared with controls injected
with Ringer's solution or untreated . . . .
vii
PAGE
31:
35
36
37
CHAPTER I
INTRODUCTION AND REVIEW OF LITERATURE
History. Certain drugs such as sulfocyanide, thiourea
and thiouracil, inhibit the formation and secretion of
thyroid hormone and thus are a powerful goitrogenic sub-
stance in many different animals and man (MacKenzie,
MacKenzie and McCollum, 19R1; Ritcher and Clisby, 19R1;
Kennedy, 19R2; Astwood, Sullivan, Bissell, and Tyslowitz,
19R3; Mackenzie and Mackenzie, 19R3). These workers further
reported that the characteristic morphological changes
produced in the thyroid gland by these drugs are a loss
of colloid, hyperemia, and enlargement (hypertrophy) and
multiplication of cells (hyperplasia) leading to an in-
creased size and weight of the thyroid.
Adams and Bull (19R9) injected thiourea and thiouracil
(Deracil) into yolk sac of developing chick embryos of
White Plymouth Rocks. Dosages used were 0.5 m1. of a
solution of 0.R% thiourea and 0.1% to 0.2% thiouracil on
the 8th, 1Rth, and 18th day of incubation. They reported
that the main effects were retardation of hatching in
those eggs incubated to the 21st through.2Rth day and
lack of retraction of the yolk, decreased body and limb
growth, enlargement of the thyroid gland with typical
hyperemia, hypertrophy and hyperplasia, a marked increase
in height of follicular epithelium and colloid depletion.
Grossowitez (19R6) found that chick embryos whose
yolk sacs were injected with thiourea between the 7th and
17th days of incubation were delayed in hatching and their
yolk sacs were not retracted. McCreight (1950) reported
similar reaction in another strain of chicks. Adams and
Buss (1952) working with White P1ymouth.Rock chick embryos
which were given a single injection of thiourea and :methyl
thiouracil found that cell proliferation occurred in the
thyroid‘gland.
Vidal (1952) reported that thiourea injected at the
7th day and 15th day of incubation caused generalized
retardation of growth with no specific abnormality but
high.mortality.
Romanoff and Lauffer (1956) reported the effect of
injected thiourea on the develOpment of some organs of the
chick embryi They confirmed some of the above findings
and showed that the administration of thiourea resulted in
prolongation of embryonic development and high.mortality.
In all cases the thyroid was markedly hypertrophied, and
increased in weight. ‘They also reported an increase in
the weight of the adrenal and testes and a considerable
decrease in liver and ovarian weights.
Many investigators have reported hypertrophy and
hyperplasia of the thyroid gland in fish, amphibians,
reptiles, birds and mammals from.treatment with anti-thyroid
drugs; with emphasis on the increased size of the follicular
3
cells and weight of the thyroid (Charipper and Gordan, 19R7;
Adams and Bull, 19R9; Adams and Craig, 19R9, 1950, 1951;
D'Angelo, Gordon and Charipper, 19R7; Domm and Beivaiss,
19R8; Goldsmith, 19R9; Lynn and Wachowski, 19Rl; McCreight,
1950).
Mitotic activity shortly after injection of anti-
thyroid drugs or goitrogenic compounds have been reported
(Paschkis, Cantarow, Rakoff, and Rothenberg, 19R5; Adams
and Bull, 19R9). Paschkis et a1. 19R5, reported that thioura-
cil given daily to rats in drinking water produced a hyper-
plasia as soon as 2R hours after treatment. Adams and Buss,
1952, determined the mitotic activity in chick embryos given
one injection of an anti-thyroid drug on the 1Rth day of
incubation and killed after 2R additional hours of incubation.
The 1Rth day was chosen for the single injection because
on the 13th day of incubation the follicles of the thyroid
have become distinct and contain colloid (Bradway, 1929).
The glandis completely developed by the 15th.day of incuba-
tion (Sem, 1932; Martindale, 19R1; Bull, 19R8). The results
from Adams and Buss (1952) using White P1ymouth.Rock chick
embryos showed that counts of mitosis in comparable areas
with cell proliferation began as early as the cellular
hypertroPhy and colloid loss in the thyroids of the treated
embryos. Twenty-four and forty-eight hours after injection
mitosis in the injected embryos exceeded that in the controls.
Thus they confirmed that the weight of the thyroid of chick
embryos treated with anti-thyroid drugs was not only due
to hypertrOphy of follicular cell but also due to a marked
increase in the number of cells by mitosis.
Working with day-old chicks, Astwood, Bissell and
Hughes (19RR) found that when 0.1% thiouracil was given in
the ration for 10 days the thyroid was enlarged 5 - 7 times
more than that in the controls. Turner and Schultze (19R5)
reported that the action of thiouracil on body weight is
relatively greater in young, growing birds. Glazener and
Jull (19R6) reported that thiouracil decreased the growth
and feed consumption of broilers.
Moreng and Shaffner (19R9); Shaffner (1951); indicated
that thiouracil depressed weight gains in chickens except
when fed at low levels (0.012 to 0.05 per cent).
Thiouracil fed to hens at the level of 0.1 per cent
for six months does not affect egg production or fertility
and hatchability in chickens but the higher level (0.3
per cent) reduced these traits (McCartney and Shaffner,
191:9).
Chicks hatched from thiouracil treated hens had
enlarged thyroids and hypoactive thyroids (Wheeler and
Hoffman, 19R8, 19h9, 1950; McCartney and Shaffer, 19R9;
Booker and Sturkie, 19R9). These latter workers injected
.thyroxine into these chicks hatched from the hypothyroid
hens and reported a reduction in the size of the thyroid.
Mellen (1957) working with metabolic rate (M.R.)
of chicks found that thiouracil depressed the M. R. as long
as the drug was given in feed, but lasted for only 18 hours
when the drug was withheld after which the M.R. averaged
the same as in the controls.
‘gggg 2: Action. The exact mode of action of anti-
thyroid compounds is not known, but evidence supports the
theory that the thiocarbamides among them prevent the
iodination of tyrosine. Pitt-Rivers (1950); Larson, Keating,
Peacock, and Rawson (19R5) found that thiouracil doesnot
decrease the uptake of inorganic iodine, but prevents the
gland from binding to protein or decreases the organic bond.
Dempsy (19RR) by histological technique, found that the
enzyme peroxidase is present in thyroid cells and disappears
after thiouracil administration. The hypertrophy and hyper-
plasia of the thyroid gland following treatment with anti-
thyroid drugs evidently results from excessive secretion
of thyrotrOphic hormone (T.S.H.) by the anterior pituitary
gland. Stimulation causing this excessive T.S.H. results
from.the decreased amount of thyroid hormone in the chulat-
ing blood.
CHAPTER II
OBJECTIVE
Thyroid inhibiting drugs have been used in an attempt
to measure the relative requirement for the thyroid hormone
in varying physiological conditions.
The objective of these experiments were:
1. To determine the dosage level of thiouracil for
further physiological and embryological exPeriments and to
determine the level of toxicity of this drug.
2. To determine the effect on subsequent liviability
and growth after injections of thiouracil into R8 hour embryos.
In.most reports in the literature, injections have been
made after the 7th day of incubation; therefore after the
thyroid has started forming. No previous workers have
determined the effects of injections of thiouracil into R8
hour embryos.
3. To determine the effect on hatchability, after
injections of thiouracil into R8 hour chick embryos.
CHAPTER III
METHODS AND PROCEDURE
Experiment No. 1. — To Determine the Toxicity Level of
—ThIouracilon the Chick Embryo.
Twelve dozen fertib eggs of White Leghorn hens
(Gallus gallus d.) were obtained from the Michigan State
University Poultry Research Farm. The eggs were selected
for good shell texture and uniformity of size and weight.
The eggs were incubated in a force-draft incubator at
37.5°C and a relative humidity of 60%. The maintenance of
temperature and humidity at this level is recommended by
the manufacturer of the incubator and also by Romanoff (1956)
who states that at this level the maximum hetchability is
obtained. The eggs were turned every two hours and were
candled at R8 hour intervals after treatment started.
After R8 hours of incubation the eggs were divided
into 7 groups, each group containing 12 eggs. The groups
were injected as follows: 0.1 mg. of thiouracil, 0.5 mg.
of thiouracil, 1.0 mg. of thiouracil, 2.0 mg. of thiouracil
and 5.0 mg. of thiouracil; and two controls--one with the
injection of Ringer's solution and one without any injection
at all. '
The mode of injection was via the Yushok methods
(1950). This constitutes an injection being made into
8
the albumin at the small end of the egg so as not to strike
the major allantoic blood vessel area.
The thiouracil was difuted in Ringer's solution
(sodium chloride, 9 gm., potassium chloride, 0.R2 gm.,
calcium chloride, 0.29 gm. and distilled water, 1000 m1.).
The solutions were sterilized by autoclaving for 30 minutes.
The small ends of the eggs were washed with 70%
ethyl alcohol and dried. The Operator's hands were also
scrubbed and washed with ethyl alcohol. The egg shell was
pierced by a needle which was flamed and inserted at the
small end. Then 0.5 ml. of sterile thiouracil was injected
for each of the different treatments and 0.5 ml. of Ringer's
solution was injected to one of the controls. Then the hole
made by the needle was sealed with liquid paraffin. All
eggs were then incubated. As a spot check to test for con-
tamination and whether or not the treatments had killed the
started embryos, eggs were candled 2R hours after injections
were made.
The eggs were left in the incubator up to the
21st day and then opened. Results of the above experiment
at let day of incubation are shown in Table I.
The embryos in Group 5 which contained 5 mg.
thiouracil, started to die R8 hours after injection and
all the embryos were dead on the 9th day of incubation.
TABLE I
Effects of different dosages of thiouracil on chick embryos
injected at R8 hours of incubation.
No. of No. of No. of
Dead Eggs LIve
95222 Dosages Embryos .Sgt Embryos
1 0.1 mg. of thiouracil ‘ 3 12 9
2 0.5 mg. of thiouracil 2 12 10
3 1.0 mg. of thiouracil R 12
R 2.0 mg. of thiouracil 7 12 5
5 5.0 mg. of thiouracil 12 12 0
6 Control with 0.5 ml. of Ringer's
solution» 1 12 ll
7 Control without injection 2 12 10
This demonstrated that 5.0 mg. of thiouracil was toxic
thus inhibited embryonic development and completely
suppressed body metabolism and the death of the embryos
resulted.
Experiment N9. g. - Effect of Thiouracil on Body Weight
and Hatchability.
As the 5.0 mg. was toxic to the embryos the dosage
level used in this experiment was: 0.1 mg. of thiouracil,
0.2 mg. of thiouracil, 0.5 mg. of thiouracil, 1.0 mg. of
thiouracil and 2.0 mg. thiouracil. Controls consisted of
uninjected eggs and eggs injected with Ringer's solution.
10
One hundred and twelve dozen fertile eggs were
obtained from Michigan State University Poultry Research
Farm. All the eggs were from White Leghorn hens (Gallus
gallus d.). They were selected for uniformity of size and
shape.
Random selection during the setting of the eggs in
the incubator trays was used. Each group was set with 16
dozen eggs in the forced draft incubator under the previously
stated condition of temperature and humidity. Eggs were
again turned every 2 hours.
Following the technique of Yushok (1950) as previously
described, injections were made after 2R hours of incubation.
After 2R hours of incubation the eggs were candled by
an ordinary egg candler to check fertility.
On each day from the 7th through the 21st day of
incubation, twelve eggs from each treatment were randomly
selected and sacrificed at which time the embryo body weights
were taken. In addition to body weight, the number of live
embryos was recorded so as to determine fertility. After
the let day one dozen of the eggs in each group was allowed
to remain in the incubator until they hatched or died. This
procedure was used to determine the effect of an anti»
thyroid drug (thiouracil) on the delay of hatching as
reported for thiourea (Grossowicz, 19R6; Adams, 19R9; Adams
and Buss, 1952; Vidal, 1952).
11
CHAPTER IV
RESULTS AND DISCUSSION
A. The Influence of Different Dosages of Thiouracil:-
Before starting the final experiment, the toxic
level of thiouracil was determined as discussed in Chapter
III. .
The experiment showed that 5.0 mg. of the thiouracil
when injected into chick embryos resulted in 100% mortality
after 9the day of incubation. 2.0 mg. was less toxic to
the embryos, thus it was included in the experiments. (Table
I) *'
Romanoff and Lauffer (1956) injected 2, 3, 5 and
10 mg. of thiourea and obtained 100% mortality in the 5
and 10 mg. of treated embryos. Similar results were obtained
by Vidal (1952) with 5.0 mg. of thiourea.
There are no data available regarding the toxic
level of thiouracil.
B. Effect of Thiouracil on Body Weight:-
From an examination of Table II it is evident that
in general the average body weight of chicks injected with
, thiouracil was less than that of the controls injected
with.Ringer's solution or left untreated. The embryo
weights of the eggs treated with thiouracil were less than
12
the controls, until the 11th day of incubation at which
time the thyroid had'become functional (Hansbrough and Khan,
1951). Before the 11th day there is no significant differ-
ence between the thiouracil treatments (except the 0.1 mg.
1evel--the low level) but all the treated embryos with thiour-
acil are significantly different than the controls. Thus
it indicates that the earlier injection, i.e., injection
of different doses of thiouracil, does inhibit the body
weight before the thyroid is completely formed. This
suggests that thiouracil inhibits body development even
though the thyroid per Se is not functional and indicates
that colloid, which is present at the seventh day, may be
involved in develoPment.
There are no published data to show the effect on
embryos of injections of.thiouracil before the deve10pment
of the thyroid except Vidal (1952) who injected high levels
of thiourea to determine any abnormality in development
and mortality. Vidal (1952) mentioned that injecting
thiourea at O or R8 hours of incubation caused no abnormality
in develOpment although it did cause high mortality: however,
he did not study the effect on body weight and hatchability.
His primary object was to determine the effect of the
injections on subsequent livibility of embryos. With the
exception of the controls, most of the embryos died before
the 11th day of incubation.
13
As shown in Table II there is a change in signifi-
cant differences with individual treatments after the 11th
day of incubation, the period when the thyroid is completely
formed (Khan, 1951). Reduction of body weight caused by
2.0 mg. of thiouracil was significantly greater than the
reduction caused by 1.0 mg. of thiouracil. But there is
no significant difference between treatments of 0.1,
0.2 and 0.5 mg. of thiouracil when injected into chick
embryos. The greatest difference is between the controls
and treated embryos.
The average body weights of all embryos aut0psied
on the 7th through the let days of incubation were lower
than those of controls. From the 17th day of incubation
through the 2lst, those of the embryos injected with
thiouracil were consistently lower than those of the con-
trols. The difference between the higher concentration
injected (2.0 mg. of thiouracil) and controls are approxi-
mately 13 gms. on the let day, 10 gms. on the 20th, 8
gms. on 19th, 6 gms. on the 18th and 5 gms. on the 17th
day of incubation. There was a pronounced difference in
weight between individuals receiving different treatments
of thiouracil. There is an approximate difference of 6 to
7 gms. between 2.0 mg. and 0.1 mg. of thiouracil injected
chick embryos on the 2lst day of incubation. There is a
difference in body weight of approximately 3 -'5gns. between
the 2.0 mg. and 0.1 mg. treatments from the 17th to 2lst day.
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moc.m mom.m‘ www.mv oao.n sem.m ms».m mem.m eHo ease HH
mmmed wmmqmu. mom.m Hmm.m omm.m Ham.a omm.H. see same OH
mam.m maa.w, mme.H Hme.a NJ:.H wom.a emm.a eao ease a
sem.ai ~:0.m wmm.a meH.H wfla.a mda.a ‘wmo.a oao name a
mmm.o pem.o mme.o Hoe.o mme.o mem.o om:.o eHo name A
.Ghuaoo Hohpzoo
. assessoasa no meson eseaeneao seat moasnsm scene no names;
.cepeoapss ho Goausaon n.nownfim spas mopoonna maonpsoo spat veneqaoo soapenwoaa
ho manon w: as HHOeHBOHmp_£pHB beacons“ nomanae Means Mo .eaw a“ pnmfieh huon owwaopm
HH mgmda
15
Ho>oa mm as ugeoamadwfimuwoq memos mafia eaaom n
soapsHOm m.nowsam go .HE mo. oepoonnH u N Hohpnoo
cepsompnb n H donunoo
«Newmi ee.owq om.mm ee.mm eH.Hm mm.ma merma
mm.mm me.mm mm.:m mmeww, w ms.am md.om . se.ma
mm.smV . me.~m Ho.:m em.mm m:.am mm.om inasmw
m. m. .ma.a.o .ms m.o .mm w.o .ms o.H .ms o.m
Hoaunoo Hoapaoo
assessoasa to demon cassettes seat moaaosm scene so named;
Apessatsoov HH memes
eHo ease Hm
eHo mace om
eao mace ma
owaasm.mm ems
16
The difference is approximately the same in all the
treatments from 13th to the let day of incubation.
The retardation of growth with exposure of chick
embryos to anti—thyroid compound, was first studied by
Andrews and Schnetzler (19R5) who raised chicks from eggs
laid by hens given thiouracil. They demonstrated the
presence of the drug in the yolk of such eggs, but they did
not describe any modification in development of the embryos.
The second paper, that of Grossowicz (19R6) already men-
tioned, emphasized retardation of hatching and lack of
retraction of yolk sac. Adams and Bull (19R9) injected 0.R%
of thiourea on the 1Rth day of incubation and embryos killed
daily thereafter showed a statistically significant differ-
ence. They also found that when a series of chick embryos
were injected with thiourea on the 8th, or 8th and 1Rth, or
8th, 1Rth and 18th days of incubation, the body weights at
autopsy on the let day were consistently less than those
of the controls. Decreased body weight in New Hempshire
chicks after doses of thiourea and thiouracil have been
reported (Macright, 1950). These chicks were injected on
the 6th, 12th and 18th days of incubation and killed at
daily intervals thereafter. Some papers, however, include
no data on body weights or growth of chicks injected with
thiourea or hatched from eggs laid by hens fed thiouracil.
Romanoff and Lauffer (1956) injected a single dose
of 5.0 mg. of thiourea into different groups on successive
17
days from 0 to 18 days of incubation. All the embryos died
except for eight, which hatched on the twenty-first day of
incubation from eggs injected on the 18th day. The body
weights of dead embryos were less than controls. This in-
dicated that 5.0 mg. of thiourea was toxic to chick embryos.
The above results are in agreement with data presented in
Table II.
There are several reports on the administration of
goitrogenic drugs to very young chicks from one day to a
week old, that have a bearing on the possible influence of
such compounds on early growth. In these experiments, the
anti-thyroid drug, thiourea, or thiouracil was included in
the food or drinking water for varying periods of time and
in varying quantities (Astwood, Bissell and Hughes, 19RR;
Mixner, Reineke and Thrner, 19RR; Schultze and Turner, 19R9;
Briggs and Lillie, 19R6; D'Angelo, Gordon and Charipper,
19R7). One day old chicks of several breeds usually responded
to two weeks of age on 0.1% thiouracil in their food by
slight losses in body weights (Mixner, et. al., 19RR).
Thiouracil (0.1% to 0.8%) included in food or White Leghorn
chicks for two weeks from the day of hatching lowered the
body weights approximately 10 gms. compared with the con-
trols (Sultze and Turner, 19R5). Weak solutions of thiourea
(0.05% or 0.07%) caused only negligible reduction in the
weight of White Rock chicks that were fed the drug for 3
18
weeks from hatching. However, thiourea of 0.1% strength
reduced the weight of female chicks R8.8 gms. (37.2%) and
of males 3R.8 gms. (28.2%) below those of their controls
(Schultze and Turner, (19R5). There was a reduction of
27.2% in chicks of a New Hampshire cross given 0.5% thiouracil
in their food for 5 weeks (Briggs and Lillie, 19R6). _Body
weights lower than those of their controls were also re-
ported in chicks of a Barred Rock X New Hampshire cross
fed 0.1% thiourea in the ration for 20 weeks (D'Angelo,
Gordon and Charipper, 19R7). '
In these experiments, the general effects upon the
body weight of newly hatched chicks of treatment with
moderately strong solution of anti-thyroid drugs were: (1)
long treatment of 3 to 20 weeks usually reduced it. (2) short
ones sometimes reduced it and sometimes did not; breeds
differed in their responses; and thiourea was more effective
than thiouracil.
In mammals many instances of retarded growth after
administration of anti-thyroid drugs have been reported
(Astwood, et. al., 19R3; Christensen, 19R5; Dempsy and
Astwood, 19R3; Fitzhugh and Nelson, 19R7; Goldsmith, Gordon
and Charipper, 19R5; Higgins, 19R5; Hughes, 19RR; William
Weinelass, Bissell and Peter, 19RR).' Only dogs and monkeys
seem to be refractory to the treatment with these drugs
(Aranow, Engle and Sperry, 19R6; Donowski, Man and Winkler,
19R6; Mayer, 19R7). These drugs inhibit metamorphosis in
19
anuran and urodele tadpole (Gordon, Goldsmith and Charipper,
19R3), but in some instances body growth in the anurans has
continued although limb growth has not (Adams and Craig,
19R9a).
C. Effect of Thiouracil on Hatching and Yolk Sac Retraction
In each group, 12 fertile eggs were left in the
incubator after the 21st day of incubation until they hatched
or died.
Chick embryos receiving the different dosages of
thiouracil were delayed in hatching if the eggs containing
them.were incubated to hatching dates or beyond. However,
no chicks injected with thiouracil hatched on or before the
2lst day of incubation with the exception of 3 chicks out of
12 fertile eggs of Group 1 receiving 0.1 mg. of thiouracil
and R chicks in Group 2 receiving 0.2 mg. of thiouracil and
2 chicks from Group '3 receiving .5 mg. of thiouracil.
The results of this phase of the experiments are shown
in Table III. The results show that chicks receiving 2.0
mg. of thiouracil had not hatched at the 27th day of incuba-
tion. At this time they were sacrificed and embryos were
removed. Four embryos were alive and eight were dead.
In Groups I to IV all the chicks hatched by the 25th
day of incubation.
The controls receiving Ringer's solution and without
' any treatment hatched on the 20th and let days of incubation;
20
and the yolk.sacs were entirely within the body.
This effect was similar to that reported by Grossowicz
(19R6) but less striking since the eggs were only allowed to
go six days beyond normal hatching, compared with ten days in
his experiment. He reported a correlation between the time
of injection (the 7th to the 17th day) and dosages administered
(0.3 to 3.0 mg. of thiourea) and the degree of retardation.
Adams and Bull (19R9) used 2.0, R.0 and 6.0 mg. of thiourea
and found no differences between treatments. Beginning treat-
ment on the 8th or 1Rth day made no apparent difference. In
further studies by Adams and Buss (1952), they found no chicks
injected with thiourea or propyl thiouracil hatched on or
before the let day of incubation.
Romanoff and Lauffer (1956) compared the effect of
different doses when eggs were injected with 2, 3, 5 and 10
mg. of thiourea on the eleventh day of incubation (a state of
incubation at which the thyroid is known to be functional).
Observation showed that the administration at this time re-
sulted in prolongation of embryonic development by 3 to 10
days beyond the normal period of 21 days and increased pre-
natal mortality up to 100% in some cases, as compared to
25% mortality among the controls.
' Similar results were obtained in the present experiment
when the chicks received thiouracil before the formation of
thyroid. There is no information in the literature on the
action of anti-thyroid drugs administered before thyroid
21
formation, i.e. before the 11th day of incubation.
The present experiment shows that thiouracil, when
injected into R8 hour embryos will prolong the incubation
period and will delay yolk sac retraction.
TABLE III
Effect of Thiouracil on Length of Incubation
Period
Days 0.1 28° 0.2 gg. 0.5 gg. 1.0 mg. 2.0 gg. Control
21st 3 R 2 - - 10
22nd 1 O l - -
23rd R 6 5 6 .-
25th 3 l 2 R -
27th - - - - l2 (sacrificed)
22
CHAPTER V
SUMMARY
Thiouracil, in amounts varying from 0.1 to 2.0 mg.,
was injected into the albumen of chicken (gallus gallus d.)
eggs after 2R hours of incubation. The embryos were sacri-
ficed or were permitted to deve10p until they hatched or
died. The controls received Ringer's solution or were not
injected. '
Observation showed that administration of 5.0 mg.
of thiouracil after R8 hours of incubation caused 100%
mortality by the ninth day of incubation. '
There was a highly significant loss of body weight of
all the treated chick embryos compared to controls. Signifi-
cant differences between individual treatments were noted
after the 11th day of incubation.
Experiments showed that the administration of thiouracil
resulted in prolongation of embryonic development beyond
the normal period of 21 days and lack of retraction of the
yolk sac.
Body FIGURE 1 23
Weight
_. .. 1,0
L 1 l 1
1 1 T 1 '
A
1
Control Control 0.1 mg. 0.2 mg. 0.5 mg. 1.0 mg. 2.0 mg.
# l # 2 Dosage Thiouracil injected
Average body weight in gms. of 7 day old chick embryos in-
jected with Thiouracil at R8 hours of incubation compared
with controls injected with Ringer's solution or untreated.
FIGURE 2
Body
Weight
db 2.5
\
A A
I T
l
I
I 1 l
I I w
Control Control 0.1 mg. 0.2 mg. 0.5 mg. 1.0 mg. 2.0 mg
#1 # 2 Dosage Thiouracil injected
Average body weight in gms. of 8 day old chick embryos in-
jected with Thiouracil at R8 hours of incubation compared
with controls injected with.Ringer's solution and untreated.
FIGURE 3
Body
Weight
L3.5
L 1 1 J a L
Central Control 0.1 mg. 0.2 mg. 0.5 mg. 1.0 mg. 2.0 mg.
# l # 2 Dosage Thiouracil injected
Average body weight in gms. of 9 day old chick embryos in-
jected with Thiouracil at R8 hours of incubation compared
with controls injected with Ringer's solution and untreated.
Body
Weight 26
FIGURE
L 21.5 1‘
1
A A
t
C ntrol Control 0.1ng. 0.2ng. 0.5 $3. 1.0 mg. 2.0 mg.
# 1 # 2 Dosage Thiouracil injected
L
Average body weight in gms. of 10 day old chick embryos in-
jected with Thiouracil at RB hours of incubation compared
with controls injected with Ringer‘s solution and untreated.
27
FIGURE 5
Body
Weight
it 6.0
.. 3.0
at 2.5. \
Control Control 0.1 mg. 0.2 mg. 0.5 mg. 1.0 mg. 2.0 mg.
# 1 # 2 ' Dosage Thiouracil injected
Average body weight in gms of 11 day old chick embryos in-
jected with Thiouracil at i118 hours of incubation compared
with controls injected with Ringer's solution and untreated.
28
FIGURE 6
Body
Weight
l l
V I
l
P
I
Control Control 0.1 mg. 0.2 mg. 0.5 mg. 1.0 mg. 2.0 mg.
# l # 2 Dosage Thiouracil injected
Average body weight in gms. of 12 day old chick embryos in-
jected with Thiouracil at R8 hours of incubation compared
with controls injected with Ringer's solution and untreated.
29
FIGURE 7
Body
Weight
"r 11.0
1- 9.0
it 7.0
J A L .1 I
U ' j ' v w
Control Control 0.1 mg. 0.2 mg. 0.5 mg. 1.0 mg. 2.0 mg.
# l # 2 Dosage Thiouracil injected
Average body weight in gms. of 13 day old chick embryos in-
jected with thiouracil at RC hours of incubation compared
with controls injected with Ringer's solution or untreated.
3O
t 12.0
*- 10.0
A l l a l 1
CU w ' w t ' ‘—
L
U
Control Control 0.1 mg. 0.2 mg. 0.5 mg. 1.0 mg. 2.0 mg.
# l # 2 Dosage Thiouracil injected
Average body weight in gms. of 1R day old chick embryos in-
jected with Thiouracil at R8 hours of incubation compared
with controls injected with Ringer's solution or untreated.
31
Body
Weight FIGURE 9
- 17.0
1
C ntrol Control 0.1'nlg. 0.2 mg. 0.5 mg. 1.0 mg. 2.0 mg.
# l # 2 Doses of Thiouracil injected
Average body weight in gms. of 15 day old chick embryos in-
jected with Thiouracil at R8 hours of incubation compared
with controls injected with Ringer's solution or untreated.
32
FIGURE 10
Body
Weight
«L 19.0
«1- 17.0
L
" ISeo
. 13.0 \
l
l a l L J
Control Control 0.1 mg. 0.2 mg. 0.5 mg. 1.0 mg. 2.0 mg.
# l # 2 Dosage Thiouracil injected
Average body weight in pms. of 16 day old chick embryos in-
jected with Thiouracil at R8 hours of incubation compared
with controls injected with Ringer's solution or untreated.
33
FIGURE 11
Body
Weight
.L21.0
'Control Control 0.1Img. 0.2 mg. 0.5 kg. 1.0 kg. 2.0 mg.
# 1 # 2 Dosage Thiouracil injected
Average body weight in gms. of 17 day old chick embryos in-
jected with Thiouracil at R8 hours of incubation compared
with controls injected with Ringer's solution or untreated.
_ 3h
FIGURE 12
Body
Weight
25.0
.. 211.0
m 23.0
t 22.0
4t- 21.0
¢ 20.0
+.19.0
L L J A
—v
c ntrol Control 0.1Ing. 0.2r-g. 0.5 mg. 1.0'ng. 2.0 ;g.
# 1 # 2 Dosage Thiouracil injected
Average body weight in gms. of 18 day old chick embryos in-
jected with Thiouracil at RR hours of incubation compared
with controls injected with.Ringer's solution or untreated.
35
FIGURE 13
Body
Weight
it 27
l l #L_
s : i . . a
Control Control 0.1 mg. 0.2 mg. 0.5 mg. 1.0 mg. 2.0 mg.
# l # 2 Dosage Thiouracil injected
Average body weight in gms. of 19 day old chick embryos in-
jected with Thiouracil at;RB hours of incubation compared
with controls injected with Ringer's solution or untreated.
Body 36
w
°13ht FIGURE 1R
'1' 30e0
a a l A l
1
fl.
Control Control 0.1 mg. 0.2 mg. 0.5 mg. 1.0;g. 2.0 mg.
# l # 2 Dosage Thiouracil injected
Average body weight in gms. of 20 day old chick embryos in-
jected with Thiouracil at R8 hours of incubation compared
with controls injected with.Ringer's solution or untreated.
37
FIGURE 15
Body
,Weight
+- 33
w- 30
w 27
1 2R
1'. 21
i i 2 4 is %
Control Control 0.1 mg. 0.2 mg. 0.5 mg. 1.0 mg. 2.0 mg.
# l # 2 Dosage Thiouracil injected
Average body weight in gms. of 21 day old chick embryos in-
jected with Thiouracil at R8 hours of incubation compared
with controls injected with Ringer's solution or untreated.
we. was
38
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13fi1ated embryonic tissue." Jour. Expt. Zool., 93:1.
19 3. .
Sadhu. "Physiological mechanism of experimental goitro-
gensis." gm. g. Physiol., 1952:150. l9h8.
Schultze and Turner. "The determination of rate of thyroxine
secretion of certain domestic animals." Mg, Ag . Expt.
Sta. Res. Bull. 392. 19h5. - .
Sun, T. P. "Histo-physiogenesis of the gland of internal
secretion thyroid-adrenal, parathyroid and thynius
of the chick embryos." Physiol. Zool., 5:384. 1932.
. “fax f},
A3
Turner, C. W. "Effect of rape seed on the th roid of the
chick." Poult. Sci., 25:188-187. 19h a. ,
. "Comparison of the effect of feeding thiobarbital
and thiouracil on the thyroid gland of the chick."
Poultry Science, 25:186-187. 19h6b. ,
VanderLaan, W. P. and A. Bissell. "The influence of selected
goitrogenic compound on the th oid gland of chicks."
Endocrinology, 38:308-3lh. 19 6a. .
’ ‘ffiififl'
. "Effects of propyl thiouracil and of potassium .51
thiocynate on the uptake of iodine by thyroid gland Q ‘
of the rat." Endocrinology, 39:157-160. 19h6b. r ‘
Vidal, A. "Influence de la thiourie sur le developpement
del embryon de poulet." Ann. Endocrinol. 13(6): {»
982-990. 1952. - g
Willer, B. H. "The endocrine glands and development of the
chick." Am.‘g. Anat., 33:67-103. 192h.
Williams, R. H., A. R. Weinglass, G. W. Bissell and J. B.
Peters. "Anatomical effects of thiouracil." Endocrin-
ologl. 3u=317-328. lath.
Woodside, G. L. "Experimentally induced h per-thyroidition in
the chick embryo." Anat. Rec., 67: 3-h30. 1937.
x Yushok, W. D. "Effect of anti-thyroid drugs on chick embryo."
Thesis C. V., 1950. _
APPENDIX
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