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€95?

DETERMINATION OF THYROJI) SECRETION RATES
IN RAINBCN TROUT, §ALIQ GARDNER]: .

USING RADIOACTIVE IODINE

By
JACK RUSSELL HOFFERT

AN ABSTRACT

Suhnitted to the College of Science and Arts
Michigan State University of Agriculture and
Applied Science in partial fulfillment of

the requiremnts for the degree of

MASTER OF SCIENCE
Department of Physiology and Pharmacology

1959

D C E !
Approved by i ‘K I'M/W»

Jack R. Hoffert

ABSTRACT

The control and regulation of metabolic activity by hormonal factors
has been established in warm blooded vertebrates. The existence of sim-
ilar hormonal systems in poikilothermic vertebrates and invertebrates
has also been established. The functional and anatomical characteristics
of these hormonal systems may vary from those found in higher warm-blooded
vertebrates.

Thyroid tissue in the teleost is known to produce iodinated amino
acids identical to those of mammals. The utilization of thyroidal
compounds by the teleost does not follow the same pattern as in higher
vertebrates since it has been shown that thyroidectomy does not change
oxygen consumption in the teleost.

Estimation of glandular activity will aid in understanding the
function of the thyroid hormone intseleosts. This may in turn streng-
then our understanding of the thyroidal systems in other organisms.

The following thesis presents findings on the determinations of
thyroid activity of rainbow trout. §§lfl9 gairdneri . The secretion rate
determinations were based on changes produced on the 1-131 output rate
of thyroid tissue by administration of exogenous thyroxine. The assay
for thyroid secretion rates has been satisfactorily applied to several
species of warmpblooded vertebrates. Determination of thyroid activity
by this method. has to the author’s knowledge, never been applied to
poikilothermic vertebrates.

Thyroid secretion rates for §glmg neri were found to range
from 0.190 to 0.415 pgm. l-thyroxine/loo gms. body wt./ day. No signifi-
cant difference in the mean thyroid secretion rates were found for any

ii

Jack R. Hoffert

of the experimental groups. Determinations run on two-year old and
one-year old trout yielded mean secretion rates of 0.302 and 0.2U3 pgms.
l-thyroxineIiOO gms body wt. /day respectively. One-year old fish at

13°C. and 3°C. gave rates of 0.2h3 and 0.139 pgm./1£K)tmu/an resyectivcly.

Radioactive uptake and output studies indicated that the peak accu-
mulation of approximately 20% of the injected dose occurred within 2h
hours after a plerupoeritoneal injection of I—131. Ln zigg counting
under the geometry described in the thesis. indicated a two component
output curve. The first component (t% 3 9 days) is followed by a much
slower output rate (t% = :F? o days). The first component of this out-
put curve may possibly be the result of the high accumulation of non-
thyroidal I-l3l activity occurring immediately after the injection of
1-131. The site of maximal accumulation of non-thyroidal I-ljl activity
was shown to gradually shift anteriorly for a days following injection
of I-131 until it reached the thyroidal area. After 4 days there was no
further change in the position of maximal activity.

The mean values of oxygen consumption at 13°C. and 3°C. were found
to be 0.087 and 0.040 ml. /gm./hr. respectively. Operculum rates at 13°C.
and 3°C. had values of 117 and 61 movements per minute respectively.

Q10 values indicate that the oxygen consumption of trout follows the
pattern of a normal thermochemical type of reaction (Q10 8 2.0) for the
temperature range of 3°C. to 13°C. Oxygen.consumptions and operculum
rates yielded the following Q10 values: oxygen consumption = 2.17; oper-
culum rates I 1.92. The Q10 value of the mean thyroid secretion rates

for this temperature range was 1.72.

iii

DETERMINATION OF THYROID SECRETION F ATES

IN RAINBOW TROUT..§ADEQ GAlEDflEfiII,
USING RADIOACTIVE 101mm

By
JACK Russsu. HOFFERT

ATHESIS

Submitted to the College of Science and Arts
Michigan State University of Agriculture and
Applied Science in partial fulfillment of
the requirements for the degree of

MASTER (1" SCIENCE

Department of Physiology and Pharmacology

1959

ACKNG'I WTS

The author acknowledges his indebtedness to all who have done
pioneering work in laying a foundation for the study of comparative
endocrinology of the thyroid gland. The following persons deserve
special thanks for contributions which have enhanced the value of this
work; Dr. P. O. From. and Dr. E. P. Reineke of the Department of

Physiology and Pharmacology, Michigan State Univers' ty.

TABLE CF CONTENTS

PAG

ACKNOWLEDGEMENTS......... .............. .... ............ ..... v

LIST OF TABLES .... .................... . ........ ............viii

LIST OF FIGURES ..... ....... . ............. . ..... ....... . .. ix
CHAPTER

I. nwmmmmnm..n.. ..... ”.u.u.. .............. u. 1

General Remarks ...... ........ .................. 1

Literature Review .............................. 1

Anatomy and Embryological Development of
the Teleost Thyroid .................... 1
Histology of the Teleost Thyroid .......... 2
Blood Supply to the Thyroid Tissue ........ 3
Iodine Metabolism in Mammals .............. 3
Iodine Metabolism in Pisces ............... 4
Control of Iodine Metabolism in the Teleost-
Hypophysectomy and Its Effect .......... 6
deletion of Thyroid to General Metabolism.. 7
The Effects of Anti-Thyroid Drugs ......... 5
Migration and the Thyroid ................. 9
Estimation of Glandular Activity .......... 10
Statement of the PrOblem ............. ........ .. 13
II. GENERAL METHODS AND MATERIALS
Experimental Animals .......... ....... .......... 14
Radioactive Iodine ............................. 15

Thyroptropin (TSH) .... ...................... ... 15

vi

CHAPTER

IV.

V.

L I I L .11. .n.‘ 'J 1

APPENDIX

APPENDIX

Thyroxine ......................................
Radiation Standards and Injected Doses of I-131.
QMCounting Method ..
Gross Radioactive Mapping ...... ..... ...........
Physical Decay of I-131 Standards ..............
Body Background Correction .....................
EXPERDIEN'I‘AL RESULTS
Uptake and Output Rates of Body Background and
Thyroid .....................................
Gross Distribution of Injected I-131 ...........
Effects of TSH on Ianggg_Thyroid Counts .......
Thyroid Secretion Rates ........................
Effects of Temperature on Oxygen Consumption

am oerUImn Rates ......OOOOOOOOOOOOOOO....

DI$U$ION ....OOOOOOOOOOOOOOOIOOOOOOOOOOOOOOOOOOOO
SUMMARY AND CONCLUSIONS .......... ...... ...........
cu CITED coo. ooooooooo o. oooooooooooooooooo e ccccccc on

Io Statistical Treatment Of Data 0.000000000000000.
II. Thyroid Secretion Rate Data for Groups I, II,

III, IV, and V of Table V. ....................

vii

PAGE

15

16
19
22

23

27
34

34

54

58

TABLE
I.
II.

III.

LIST OF TABLES

Decay of I-l3l Standard ............................
Body Background Correction .........................
Mean Activity of Thyroid and of Tissues Counted for
Body Background Following Injection of 1.131 . . . .
Effect of TSH on In 11.19 Thyroid Activity . . . . . . . . . .
Data for Thyroid Secretion Rate Determimtions . . . . .
Mean Values and Standard Deviation of 02 Consumptions

and Operculum Rates of 1 Year Old Trout at 3°C.

am13oc. ......OOOOOOOOOOO.......OOOOOOCCOOOOOOO

viii

PAGE
2 3

26

27

34

39

4O

FIGURE

2.

3.

9.

LIST OF FIGURES

In Vivo Counting Apparatus ........................
The Anatomy of the Trout, the Injection Site and
Counting Area for Thyroid and Background Acti-
vity ...........................................
(A) A Semi-log Plot of the Standards Activity .....
(B) A semi—log plot of the Measured Activity with
line Fitted by Method of mast Squares .....
(A) Output Curve of Thyroid Activity ..............
(B) ,‘t Injected Dose of Animal Background ..........
Reproductions of Gross Distribution Maps of

Injected I—l3l for Times: Zero, One Hour and

one Day ......OOCOOOOOOOOOOOOOOOOOOOQCOOOOOOOQO.

Reproductions of Gross Distribution Maps of Injected

I-lBl for Times: Four Days, Seven Days and Twenty

One Days cocoon.oeeeeooeeoepoeoeeeeeeeeeee000.00

Output Curve of Average In Vivo Thyroid Activity
With TSH Given on the Sixth Day After L131
Injection °--°.°---~'-----0~-----°-------a---°°-

[é Injected Dose of Animal Background with TSH Given
on the Sixth Day After 1-131 Injection .........

Statistical Method Used in Estimating the Thyroid
Secretion Rate of Eleven l-year Old Trout at

1300. 000......0..0.0.0.0........IOOOOOOOOOO....

PAGE

18

21

24

24

28

29

31

33

36

37

41

IICTRLU JCT lOl.’

 

General Remarks

 

Animal physiology, a science of function, attempts to explain how
an animal or its organs, tissues and cells perform their varied functions.
The study of animal physiology has laid the foundations for our present
understanding of the physiology of the higher forms of life, including
man. Scientists dealing with more advanced living organisms have often
found it necessary and helpful to turn to the study of either related
or more primitive forms of life for a more complete understanding of a
complicated biologiual phenomena.

Endocrinology, a field of physiology, has advanced rapidly in recent
years. As with many fileds of science dealing in some way with medicine,
endocrinolOgy got its start through clinical medicine. That the control or
regulation of cellular structures of an organism by internal secretions was
a very early phylogenetic dovelOpmcnt has been shown through the study of
various invertebrate phyla.

Investigations of animals, including vertebrates and protochordates, show
that tiyro d tissue exists in all vertebrates and in some of the protochordates
(Lynn and uachowski, 1951). Gudernatsch (1911) discovered that amphibian
metamorphism.was controlled by the thyroid gland. This opened wide the field
of investigation dealing with thyroid tissue in lower vertebrates.

Literature Review

____A

Anatomy and EmbryOIOgic§l_Develorment of the Teleost Thyroid.

 

 

The thyroid of fishes should not be regarded as a gland since in most
cases thyroid follicles enclosed in a connective tissue capsule do

not occue. A few discrete thyroid glands in fish have been described.

In the teleost $3,211” W (swordfish) a compact well circmnscribed
mass of thyroid tissue is found near the cephalic end of the ventral
aorta (Addison and Richter. 1932). This gland is very vascular and
deep red in color. Matthews and Smith (1948) found a compact thyroid
gland in the parrot fish. §pagi§gma.

Thyroid tissue develops early'in teleosts. Stratified epithelium
enlarges to form an unpaired structure on the ventral side of the phar-
ynx. between the let. and 2nd. gill pockets. At this time the thyroid
tissue is located near the tubular heart but a subsequent shift in posi-
tion of the heart and ventral aorta causes a removal of the thyroidal
tissue from its site of origin to its scattered location in mature fish.
In adult fish thyroidal tissue is generally most dense in the location
of the ventral aorta and/or branchial arteries. The thyroid tissue is
brownish yellow in color. however. in most cases it is impossible to
make a definite identification in teleosts without microscopic examin-
ation (Gudernatsch, lQll).

Through the use of radioautographs employing radioiodine Chavin
(1956) showed that functional thyroidal material occurred in the throat
and also in the lymphoidal pronephric remnants (the head kidney) of gold-
fish (Cagassius m 13.). Under thyroid hyperplasia follicles may
appear in tissues adjacent to the throat regions and even in the kidney.
Ninety percent of normal goldfish have thyroid follicles in the head
kidney (Pickfordand Atz, 195'!) and this is not a path010gical condition.
M221 22‘. tasteless: name

There is a great variability in the histology and cytology of the
thyroid of different individuals taken from the same environment. In

warmpblooded vertebrates there is much less variation. Because of the

great variance among individuals, one must exercise caution with regard
to conclusions concerning the functional state of the thyroid which are
based on histological evidence alone.

The structural form of the tm'roid follicle changes as the fish
ages. The thyroid follicle cell of teleosts has a very prominent nuclei
with scanty cytoplasm. and the cell membranes are usually hard to see.
The shape of the nuclei vary with the corflition of the gland. The
thyroid follicle is supported by loose connective tissue (Hoar. 1939).
3.19221; £92231 to. 13m MT?! 01 1111559.

Gudernatsch (1911) reports that the thyroid artery arises from the
dorsal branch of the united right and left fourth commissural arteries.
This vessel is believed to serve the main mass of follicleslocated in
this area. while follicles more widely scattered may, and probably do
receive capillaries from vessels other than the thyroid artery. Blood
is removed from the thyroid follicles by the thyroid vein. a vessel which
also drains the musculature below the ventral aorta. The thyroid vein
enters directly into the sinus venosus. The lymphatic system may also
play an important role in drainage of the thyroid tissue.

Mil-g Metabolism in {annals

The general scheme of iodine metabolism leading to the formation of
thyroxine in the mammal is as follows: Host ingested iodine is reduced
to iodide during digestion and absorption and it is in this form that
most of the element is found in the blood. Iodide is comentrated in
the tlvroid gland where it is enzymatically oxidized to iodine by the
thyroid cells. The iodine replaces two hydrogens of the benzene nuclei
of tyrosine forming 3,5 diiodotyrosine. The 3.5 diiodotyrosine next

undergoes oxidation and condenses to form thyroxine. with extrusion of

aminOpropionic acid. A peroxidase system having sufficient potential
to promote oxidation of iodide has been found in thyroid tissue (Werner,
1955).

The use of I-131 in studies of thyroxine formation has confirmed
the classic scheme of thyroid hormone formation. But these studies.
along with chromatcr‘aphic separation techniques. have demonstrated the
presence of very appreciable quantities of monoiodotyrosine and tri-
iodothyronine in the normal gland. The synthesis of thyroxine is a
rapid process and is believed to take place on the protein molecules of
the colloid. Diiodotyrosine and thyroxine cannot be readily removed by
dialysis of homogenates of thyroid tissue.

The intermediate biochemistry of monoiodtyrosine and triiodothyronine
production has not been worked out. These substances could be formed by
iodination or by deiodination. Triiodothyronine has been demonstrated
in thyroid extracts and in certain tissues such as the liver and kidney
of the higher vertebrates (Werner. 1955). Triiodcthyronine is more
potent in producing a metabolic response than either diiodotyrosine or
thyroxine. It has been suggested that triiodothyronine is the active
cellular form of the thyroid hormone, however, its site of formation is
not definitely known. Thyroxine is the major thyroidal hormone component
found in the blood.
lsaizafletamliaainziasea

Gorbman and associates have done much work on the biochemistry of
the thyroid hormones found in fish. Berg and Gorbman (195“) found a
peak thyroidal accumulation of 3 per cent of the injected dose of 1-131
in goldfish. Carassius auratus. Injections of thyrotropic hormone (TSH)

prior to I-131 injections increased the uptake of 1-131 to 9 per cent

of the injected dose and keeping goldfish in water of low iodine content
also caused an increased I-lBl uptake. A relatively slow rate of thyro-
xine synthesis was indicated by the fact that one week after injection
of radioiodine most of the 1.131 was in the form of monoiodotyrosine and
diiodotyrosine. with little. if any as thyroxine. TSH caused the thy;
roid tissue of the goldfish to produce small amounts of thyroxine as
early as 24 hours after injection of 1-131.

Again Gorbman at al.. (1952) working with §gzligghings ganigglg
(shark) showed that the gross uptake of injected 1-131 was irregular but
rapid. with the peak uptake of 20 to 3“ per cent of the injected dose
in 6 to 17 hours. The output half-life was said to be rapid. Mono-
iodotyrosine and diiodotyrosine were woduced first. and smll but
significant amounts of labelled thyroxine were formed within 17 hours
after injections of the I-131. No appreciable amounts of inorganic
iodine were found after 2“ hours. Radioautographs showed the I-l3l to
be localized in the colloid one hour after injections with no significant
amount of radioiodine in the epithelial cells at this time.

By use of chromatograms Gorbman and Berg (1955) were able to find mono-
idotyrosine. diiodotyrosine and thyroxine in MALE; Mange, and E.
.hetergglitgs. He found no triiodOthyronine. The iodine compounds found.
their order of appearance in the chromatograms. and their relative prop-
ortions did not differ in any significant way from the thyroxineogenic
cycle of mammals but the rate of syntheses of the thyroxine is not known.

In various species of mammals including man. and as far as is known
also in other classes of vertebrates. the iodinated amino acids derived
from hydrolysis of colloid are the sane (Pickford and eta, 1957). She states

that the available data thus provides no grounds for believing that the

teleostean thyroid releases a special hormone different from those
secreted in higher vertebrates.

mmeigamwmb iaiie_la__Te os -§zaaahzs_aaicmaadlia
§££222

Chavin (1956) stated that the basic endocrine mechanism.for control
of thyroid function in goldfish is similar to that of mammals. His
conclusions were based on the fbllowing results:

(1) The thyroidal I—131 uptake of intact goldfish was 8-103 of
the injected dose. TSH increased the uptake some 23“ per
cent. Injected Hal-131 was rapidly excreted for within 24
hours after injection 653 of the radioactivity was found in
the aquarium water.

(2) Hypophysectomy cut the uptake of I-131 to less than 1% and
hypophysectomized fish showed a 3.000% increase in I-lBl
uptake when treated with TSH.

(3) Thyroxine. cortisone. thiouracil and the stress of saline
immersion decreased thyroid activity in intact goldfish but
hypophysectomized fish were unaffected.

Using E, diaphagus maintained in fresh running water Gorbman-ferg(1€55)
showed that seven days after injection the fish were still accumulating
1-131 in the thyroid. There was a slight plateau at 22% of the injected
dose. Since no recycling of excreted I—131 could occur he believed that
the tracer iodine must have come from depots located in the peripheral
tissue. It has never been shown in higher vertebrates that the peripheral
tissues could retain iodine in storage form for periods longer than a
week so that it could be fed into the blood stream.

Pickford (1953b)found that in hypophysectomized E. hetergglitgg the

thyroid follicle cells were relatively flat with no cells over 12 microns
in height. Intact fish fed an iodine deficient diet exhibited thyroid
hyperplasia but in hypophysectomized fish iodine deficiency had no effect.
She has summarized the effects of hypophysectomy on the male killifish
as follows:

(1) Did not grow in length. Weight changes are irregular.

(2) Increased liver size

(3) Testes undergo complete regression.

(a) Fish may develop renal calculi and urinary duct obstruction.

(5) Loss of osmoregulatory capacity.

(6) Thyroid glands are inactive.

(7) Fish become anemic.

(8) No effect on pancreatic islets, stannius cornuscles. or adrenal

(Giacomini) tissue in the area of the posterior cardinal vein.

Relation g_f. Tm _t_o_ Genera; ktabolism

Reports prior to 1956 (see Fromm and Reineke. 1956) generally agree
that the piscine thyroid has no influence on the oxygen consumption. The
three papers discussed below present additional data on this problem.

Matty (1957) surgically thyroidectomized parrot fish (Pseudosgazgs
guacamaia) and he measured the oxygen consumption of individual fish be-
fore and after thyroidectomy. No thyroidectomized fish showed changes
in oxygen consumption for periods up to 124 days after thyroidectomy.
Intraperitoneal injections of dried parrot fish thyroid. l-thyroxine
and 0.7 per cent saline had no effect on the oxygen consumption. hov-
ever. extracts of parrot fish thyroid was shown to elevate markedly the
oxygen consumption of rats. The possibility that teleosts may respond

metabolically only to thyroid extracts of teleost origin was not demon-

strated in this work.

From and Reineke (1956) used radiothyroidectonv to destroy the
thyroid tissue in rainbow trout (m W. The radiothyroid-
ectony was accomplished by a single injection of 250 no. of 1-131 as
well as by higher doses. It was shown that radiothyroidectomy did not
decrease the oxygen consumption of trout fingerlings below that of the
control animals.

Hoar (1958) has presented data showing that both thyroxine and
gonadal steroids affect the metabolism of goldfish. Only the steroids
affect oxygen consumption while both steroids and thyroxine increase
nitrogen excretion. Hoar stated "0n the basis of these and of a great
may in gm studies it has been suggested that the primary effect of
this hormone (thyroxine) is one of accelerating the splitting of protein
and that the products so produced lead to an increased oxygen consump-
tion." He also suggests that the hormone acts in maintaining an equil-
ibrium between protein anabolism and protein catabolism.

It has been shown that the metabolism of poikilotherms varies
directly with environmental temperature. Bullock (1955) points out that
trout (m; w removed from a given temperature and placed in one
10°C. higher will have an increased oxygen consumption. The mechanism
of this increased oxygen consumption is not known.

11}; Effects of Anti-Thyroid Drugs

mm W and W galley; immersed in solu-
tions of thiourea showed marked inhibition of growth. Histological
examination of the thyroid tissue of the thiourea treated animals showed
a definite hyperplasia when compared to normal controls (Goldsmith.

1949). Thiourea produces hypothyroidism through inhibition of synthesis

of thyroid hormone.

Many reports state that during prolonged treatment with anti-
thyroid drugs a gradual restoration of thyroid activity may occur. Pick-
fordand Atz (1957) stated that antithyroid drugs at high doses can kill an
animal by general inhibition of cell oxidation. It has also been pointed
out that the direct action of antithyroid drugs on other organs cannot
be wholly ignored. Pickford found that high dosage levels. needed for
complete inhibition of the thyroid produced toxic effects not seen.after
surgical thyroidectomy.

Some investigators have found that antithyroid materials such as
thiourea decreases the demand for oxygen in teleosts. Since thiourea
is a strong antioxident the reduced oxygen consumption might be due to
the antioxidant property of thiourea rather than the decreased amount
of thyroid hormone.

Mjggatiog.agd,thg Thyroid

After about two years. young rainbow trout of Michigan waters under-
go a color transformation following which the silver trout or "steelhead"
migrates to the Great Lakes. These changes are believed to be analogous
to the changes from parr to smolt in salmon. Hoar (1953) found seasonal
changes in the histological picture of the thyroid of salmon parr. His
interp etation was that the thyroid was more active in the spring than
in the summer or fall. Robertson (1948) found similar evidence in rain,
bow trout. Examination of the thyroid glands of the rainbow trout smolt
revealed markedly increased "functional activity" when compared with the
parr thyroids.

Migrating trout taken from streams leading to Lake Michigan and Lake

Superior showed marked hyperplasia of the thyroid in the sexually ripe

10

or spent fish in contrast to the relatively quiescent state found in

immature trout (RObBrtSOD.§§iE&3, 1953). The hyperplasia of sexually maturing
Lake Michigan trout is believed due to the low iodine content of the

water causing hypofunction during the time of increased demand for the
thyroid hormone. The egg mass of the mature trout was shown to contain

more iodine than the combined total of all the other tissues. including

the thyroid. Iodine must be of great importance in the development of

the embryo.

In studying the effects of thyroid preparations on Salmogidae
LaRoche and Leblond (1952) report that when M 1.13;; in fry. parr and
smolt stages were given thyroid preparations the following changes took
place:

(1) Thickening of the integument (slight in fry and parr) intense

in the smolt.

(2) Broadening of the head produced in Salvelinus fontinalig (Brook
trout) at parr stage. Believed to be due to hyperplasia of
interorbital connective tissue.

(3) Administration of iodide to salmon parr produced a similar.
though less pronounced. pallor than that due to thyroid extract.

Estimation _o_§ Glandgar M

There have been many varied means by which‘workers have tried to
measure thyroid activity. Much of the early work was done by histolog-
ical examination of thyroid follicles. Active follicles are described
as having increased colloid material. more prominent cells with rounded
nuclei. increased cytoplasm and large dark nucledli; the lack of these
characteristics would indicate an inactive gland. Neither the size nor

the histological appearance of a gland is necessarily correlated with

11

the amount of secretion into the circulation (Swift. 1953). In the case
of an animal on an iodine deficient diet. the TSH mechanism of the ante-
rior pituitary will respond to the decreased thyroxine blood level
causing an increased growth (simple goiter) of the thyroid but there
would be no ultimate increase in the rate of hormone secretion.

It has been shown by Nadler and Leblond (1955\ that differences
in colloid diameter when statistically examined could be shown to be
identical to cross sections taken at different planes through the
spherical follicle. Changes in the histological appearance of a given
gland will show whether or not the tissue is being stimulated but cannot
be used as a means of indication of thyroid activity in terms of the
amount of hormone being used by the organiSM.

One of the major means of studying thyroid function has been to
measure the basal metabolic rate (BER) of the organism. This method
cannot be used with fish because thyroid hormone has not been shown to
be involved in the regulation of the oxidative metabolism.

There are many indications of increased or decreased thyroidal
activity as shown by gross changes in the organism. Changes from parr
to smolt stages, migration. sexual.neturation, fin regeneration, thin-
ning of the integument and exopthalmos have been correlated with changes
in the histological appearance of the thyroid in fish.

‘With the availability of radioactive iodine more investigators have
been using output and uptake half-times as a means of detecting changes
in thyroid activity. The rate and amount of I-131 uptake by the thyroid
does not indicate the rate at which thyroxine is being produced. The
same holds true for measurements of the output rate of I—l3l. However.

these measurements may be used to indicate a relative functional activity

of thyroid tissue.

Chemically the assay for total iodine in the gland has been used
as an indication of secretion rate. It has been shown that hypophy-
sectomy fails to affect the iodine content of the thyroid gland. It
could be that this is due to a decrease in both the rate of secretion
and the ability of the thyroid to concentrate iodine (Wolff. 1951). He
injected I-131 into rats and after a short period of time measured the
rate at which the 1-131 was leaving the gland (biological half-time).
Hypophysectomy had the same effect as an addition of thyroxine. that is.
it prolonged the biological half—time. Perry (1951) believed that this
decline in 1-131 removal from the thyroid gland is an indication of a
decreased secretion rate of the hormone. If the above were true then
any known factors that would increase thyroid secretion rate should also
increase the rate of 1-131 output from the gland. The major controlling
factor of thyroid secretion is the thyrotropin (TSH) from the anterior
pituitary. He found an increased output of 1-131 following injection
of TSH. This supported his basic plan for a method to measure thyroid
secretion rates.

Perry. using groups of rats injected with different levels of thy-
roxine, showed that the inhibition of thyroidal 1-131 output was propor-
tional to the dosage of thyroxine given. Reineke and Singh (1955)
studying the effects of increased dosage of thyroxine to individual
animals, also found that thyroidal I-l3l output is proportional to the
dosage of thyroxine administered. Based on these results a method for
the estimation of thyroid secretion rates was proposed by these investi-

gators.

S tenant-4: the Problem

At the present time our knowledge of thyroid function in the tele-
osts is slight. The major block to further understanding in this area
has been the inability to estimate the true rate of thyroxine utilization
by the animal. With the development of a satisfactory method of detec-
ting thyroid secretion rates many of the still unanswered problems of
teleost thyroid function may be answered.

The actual rate of thyroid hormone formation.ixxpoikilothermic
vertebrates has, to the author's knowledge, never been Hwasured- Inves-
tigators working with teleosts indicated that on the bases of "histolog-
ical evidence" such factors as age and environmental temperature may
effect thyroid activity. A preliminary examination into these factors
has been attempted.

Through secretion rate determinations future workers will be able
to clarify existing data and investigate further the problem of thyroid

function in the teleosts and other vertebrates.

GENERAL METHODS AEQ,MATERIKLS
EXQgrimentgl Anima 3

Rainbow trout. §almgpgaiggneri1. were obtained from the‘wolf Lake
Hatchery. which is operated by the State of Michigan. Department of
Conservation. The average weight of the fish used was lb.0 gms. with
a standard deviation of‘; 3.4 gms.

At the laboratory the trout were held in 26»gallon glass aquaria
in a constant temperature room. They were kept at 13°30.5°C. under
constant illumination. Each tank was aerated and equipped with an air
lift filter which contained gravel. glass wool and activated charcoal.
Every third day the fish were transferred to a clean holding tank of
aged water. Fish were fed dried trout pellets currently used by the
Michigan Department of Conservation. The pellets contained 2% iodized
NaCl.

On arrival all fish were dipped in a fresh 15 p.p.m. solution of
malachite green for 15-30 seconds. The dip prevented contamination
of the stock tanks with rather common pathological conditions of trout
such as tailrot. Ichty. and other parasitic infections.

Fish were anesthetized by immersion for approximately 15 seconds
in a 0.033% solution of MS-222 (Sandoz Chemical Co.). A syringe connec-
ted to a microburet and fitted with a 27 gauge needle was used for all
injections. The needle was inserted into the pleuroperitoneal cavity
approximately 2.0 cm. anterior to the pelvic fins. All injections were
kept to a volume less than 0.2cc. to minimize internal damage caused by
osmotic changes or increased pleuroneritoneal pressure.

One of the most convenient ways of marking fish is to clip the fins.

In this study usually only one fin was removed and when two fine were

14

15

removed one of them was the adipose fin which is not used in swimming.
About 2# hours after the clipping, the fish shoWed signs of tail-rot.
and subsequently 2/3 of the eXperimental group died. Noting that only
operated fish developed tail—rot, a strict aseptic operative procedure
was next used. The i‘ish were dipped in fresh malachite green (1:1500)
for 15 seconds before and after the operation. The fin was removed and
the cut surface dried. An antibiotic powder. Ureka Sulmide Powder
(Jensen Salsbery Laboratories). was applied to the wound. which was in
turn coated with "collodion". The fish showed no tail-rot after three
weeks of post operative experimentation.
Radioggtgve ggdigg

Carrier free radiodine as NaI-l31 was obtained from the Oak Ridge
National Laboratory. The I-l31 solution was diluted with distilled
water so that 0.1 m1. of solution contained the desired activity.
Thzzgtronin.12§§l

The hormone used was obtained from the Armour Laboratories (Veter-
inary Standard - Lot. No. R377158) in the form of a sterile powder of
purified thyrotronic principle of bovine anterior pituitary glands.
Thyroxigg

Crystalline l—thyroxine. supplied by Glaxo Laboratories. Greenford
Middlesex. England. was purified by Dr. E. P. Reineke. Ten mgs. of the
crystallized thyroxine were dissolved in distilled water made slightly
alkaline with NaCH. H01 was then added to make the solution slightly
cloudy and at this point the monosodium salt of thyroxine was formed.
This solution was then.diluted to contain 100 ’8. of l-thyroxine per
ml. of stock solution. Dilutions for injection were made from this

stock solution at the start of each experiment.

16

flagigtigg Standards egg Injected Dgses 9; 1-151

Each fishr'eceived 12.xc.of 1-131. and this activity was contained
in 0.1 ml. Twenty per cent (by volume) of the injected dose was placed
in a small porcelain cup. six drops of a solution containing casein.

Hal and NaHSO3 were added and the mixture evaporated to dryness. This
solution produces a more stable mixture minimizing loss of I—131 by
chemical or physical means. Standards were counted by placing them in
the holding tube (figure 1) and centering them over the scintillation
detector. In the holding tube the porcelain cups remained at the same
level as the lower jaw of the average trout. Thus the standard was
counted at the same geometry as the 1p_yiyg counts of the trout. Correc-
tions for physical decay and changes in the counting apparatus were made
by expressing the activity of the fish as a per cent of the injected
dose. Using the activity of the standard. the activity of the total
injected dose was calculated.

Ig4Vivo nggti g Hethgi

The apparatus used to maintain a fish in a constant position for
the purpose of measuring the gamma radiation from the 1.131 is shown
diagramatically'in figure 1. The refrigerated water (13 3 2O 0.). at a
constant pressure and free of air bubbles. passes into the anterior end
of the holding tube. leaves the tube by the posterior drain. and is
returned to the holding tank.

The holding tube is placed on top of a lead block collimator and is
centered over the 2.6 cm. hole. The lead chamber encloses a Nuclear-
Chicago Mbdel D55 Scintillation Detector with a 3/h x 3/” inch sodium
iodide crystal. Counts were recorded using a Nuclear-Chicago Model 1620

Analytical Count Rate Meter. An Esterline Angus Graphic Instrument was

FIGURE 1

In4Vivo Counting Apparatus - A. refrigerator; B,
holding tank; C, numb; D, p'essure control; E. bub—
ble trap; F, holding tube; 0. scintillation tube; H,
lead chamber; I, 1620 Count Rate Meter; J. Esterline

Angus Graphic Instrument.

18

wmaol

 

19

used to make a permanent record. The equilibrium time is the time taken
by the count rate meter tor'each a final reading within the probable
error of the true average. The per cent probable error of a single
reading of data obtained varied between 0.7% and 5.2%.

The holding tube. containing fiSh.was centered over the collimator.
The activity in the thyroid area was counted by adjusting the position
of the fish until the highest count was recorded. A second igbyiyg
count was made of each fish. This is the animal background and includes
the activity of the stomach. intestines. kidney and gonads. No thyroid
tissue is included in this count. The activity in this area is due to
I-131 in the blood. intestine. pleurcperitenal cavity; and general body
cells. either as free iodine. or as radioactive iodinated amino acids
produced by the thyroid or somatic metabolism. The count was continued
until a stable horizontal line was recorded on the Esterline Angus
Recorder. By this time the "Equilibrium Timeé had been reached and the
per cent probable counting error was as noted above. Figure 2 shows the
gross anatomy of the tPOUtwith the injection site and counting area for
thyroid and background activity.
Q32§§,flagioactive Mappigg

The Model C-lOO Actigraph Strip Feeder (Nuclear-Chicago) was used
with a shielded scintillation tube. 1620 Count Rate Meter and Esterline
Angus Recorder. The strip feeder is attached to the Esterline-Angus
Recorder by means of a flexible coupling cable. As the recording chart
is fed out of the Esterline-Angus the 6-100 Actigraph feeds an aluminum
strip table. at constant geometry. over the scintillation tube at a
speed of 3.h inches per minute. Suitable collimation was gained by using

a lead shield with a slit running perpendicular to the direction of the

FIGURE 2

The Anatomy of the Trout showing the Injection Site
and Counting Area for Thyroid and Background Activity -
A. pyloric caeca; B. liver; C, stomach. D, heart; E,
large intestine; F. pleuroperitoneal cavity; 0, fat;

H. transverse septum (false diaphragm); I. pericardial
cavity; J. operculum. The area above "thyroid" and
"background" is included in the in vivo measurements

of each.

20

N mmDGE

_0230m0v_0<m

 

 

 

 

 

 

 

 

 

22

aluminum strip table. The fish. after being killed. was placed on the
aluminum strip table and fed over the slit collimator while a record
of the counting rate was made.

game; pear 9.2 L131 mamas:

To correct for physical decay of the I-l3l all measured activities
were eXpressed as per cent of the total injected dose as calculated
from the decaying I-l3l standard. The 1-131 standard should decay at a
rate similar to the decay constant for 1-131. Investigating this assump—
tion two standards of 2.0.nc. I-l31 prepared as noted above were counted
daily for several days. The daily determinations as shown in Table I
are the average cpm. of four determinations of the two standards correc-
ted for room background. At time zero (A0) the standards had an average
count of 2.950 cpm. This value when corrected for physical decay by
use of a table based on t% = 8.06 days gives At (physical) values as
shown in Table I. Column At (measured) shows the activity of the decaying
standard as measured with the counting setup.

The line drawn to points A0. At (physical) and At (measured) are
shown in Figure 3A. Figure BB shows At (measured) values with the lire
fitted by method of least squares. The t% based on values of At (meas-
ured) taken from the fitted line gave:

A0 3 2.950 cpm.

At " 2.077 cpm.
t h days
ti

7.898 days
The variance between the "t%" of the standard's count and the decay "t%"

constant was probably caused by changes in the counting sensitivity of
the instruments coupled with a slight amount of physical or chemical

loss from.the prepared standard. Making decay corrections of ip,v;vo

thyroid measurements by expressing the activity as a per cent of the
injected dose allows for compensation of the changes in the counting
instruments sensitivity. The L131 standard did show an overall "ti"

slightly less than the "ti." value taken from physical decay tables.

TABLE I

DECAY 0F I-131 STANDARD

 

 

Days of Decay At (physical? At measured)"
0 2950 cpm. 2950 cpm.
1 2707 cpm. 2700 cm.
2 2h81+ cm. 2460 cm.
3 2279 cpm. 2197 cm.
5 1905 cpm. 1795 cpm.
6 1761 cpm. 1716 cpm.
7 1617 cpm. 1591 cpm.

* Counts per minute (cpm.) of injected dose corrected for
room background.

gm Backggggpd Cgrrectign

Because of the inability of the counting apparatus to count oily
thyroid I-l3l activity a certain per cent of the animal background was
included in the total activity of tie in 1117.0. thyroid measurements.
The problem of correcting for body background was approached as follows:
Nine fish were injected with I-l3l. The in mg activity of the thyroidal
area and body background of three fish was determined 1 day after injec-
tion. After these measurements were made the fish were sacrificed and
a portion of the lower jaw was removed. The activity of these lower
jaws was determined at the same geometry as the in 1125‘. counts. Similar
procedures were carried out on 3 fish for two succeeding days.

In Table II the values under "T" refer to the activity in the iso—

COUNTS PE R MIN.

COU NTS PER MIN.

if

4000

   
 
 

3¢W0

2000‘

 

 

I oooL

Fig. 3A.

Fig. 3B.

24

HGURE 3A

P HYSICAL CORRECTION

MEASURED CORRECTION

A I L I A l A #1

l 2 3 4 s c 7
TIME IN DAYS

HGURE 3B

 

A ‘ ‘ A J A A

I 2 a 4 s 6
TIME IN DAYS

A semi-log plot of the standards activity corrected by
physical decay tables based on the zero time activity.
Measured correction is a plot of the measured activity of
the standard.

A semi-log plot of the measured activity with line fitted by
method of least squares. "t%" = 7.89 days.

25

lated lower jaws of the fish. The values in column "A" are the igflgiyg
counts 0’ the thyroidal area minus total body background. The "B"
values are the i_.yiyg counts of the thyroidal area minus one—half the
total body background. All values are in terms of per cent of total
injected dose. The fact that values for "T" were in all cases smaller
than those for either "A" or "B" may indicate that all of the thyroidal
tissue was not contained in the excised low jaws or that complete
correction for body background was not being made.

Ra and Rb are ratios of A/T and B/T respectively. These ratios
are less on days 2 and 3 than on day 1 indicating that both "A" and "B"
values are coming closer to the "T" value. Ar and Br are values for
the per cent change in Ha or Rb from day 1 to day 3. A value of 100
for either A1. or Br would inaicate that we have reached a true estimate
of the thyroid activity based on the ”T" values. Since the Br value
falls closer to 100 than the Ar value it was concluded that correction
of thyroid activity count for body background was most accurately accom-
plished by subtraction of one-half the total body background.

Animal background shortly after injection of I-131 is quite high.
This activity decreases rapidly during the following 3 to # days. Thus
the importance of making corrections for body background becomes
relatively less as the interval between time of injection and time of
measurement increases.

Since data for the determination of thyroid secretion rates were
obtained starting u days after injections of I-l3l. corrections of these
values for body background is less important than oarrections needed in

the determination of uptake and early output rates.

26

TABLE II

BODY BACKGROUND CCRRECTION

“goo“-uo

 

 

 

Time" T ' A' B' Ra Rb Ar Br
1 8.2245 16.363 23 .51” 1.985 20852 ------- ‘-----‘-
2 6.87h 7.u76 9.611 1.088 1.398 5h.811 49.018
3 6.891 8.751 9.970 .270 l.h46 116.728 103.h33

 

 

“-.-.~ —— - -..—.-

* Days after injection of 1-131
' Per cent injected dose (average of three animals)
Note: See above text for explanation of values found in this table.

EKEERTMENTAL RESULTS
MMWMQMWaflW
A group of eight fish were each injected with 12‘pq of I-131 and
counts of the thyroid and animal background activity made over a period
of 8 days. The average activity of the thyroid and of the tissues counted

for the animal background determinations are presented in Table III.

TABLE III
MEAN ACTIVITY OF THYROID AND OF TISSUES COUNTED FOR BODY

BACKGROUND FOLLOWING INJECTION OF I-131

Days after Number of

 

injection Observations Thyroid* Body Background
1 7 2302 3, 1109*I 809 j; 702**
2 8 23.1 12.8 706 2.7
3 ‘3 20A 11.6 u.8 1.5
1+ 8 19.9 11.5 z+.6 2.5
6 8 20.3 12.1 3.9 0.8
7 8 18.0 11.5 4.“ 1.9
a 8 18.5 12. u.6 1.2

* Activity as per cent of injected dose
’* Standard deviation

The corrected values for thyroid activity plotted in Figure AA
indicate that during the first 4 days following injection there is a
rapid loss of 1-131 from the thyroid area (t% = Six) days). This is
followed by a slower rate of loss which has a half-time of :5? iays.

Figure hB shows the animal background activity which again appears
to be divided into two components. There occurs initially a rapid loss
of activity (ti = 2:5 tays) followed by an increase in activity. (t% =
13 days uptake). All lines drawn in Figures 1m arr! as were fitted

by visual methods.

27

INJECTED DOSE

'56

30

DO
C3

l0

1'

 

  
 
   

28

/T I/2 - 9 DAYS

T IIZ I 37 DAYS

/

Fig. 4A - Output Curve of Thyroid notivity.
Activity expressed as per cent in-
jected dose corrected for animal
background, vs. time in days after
I-lSl injection.

36 INJECTED DOSE

0)

(fl

 

  
  

29

’T ll2 ' 2.5 DAYS

T V2 1' l3 DAYS

 

Fig. 4B - Fer Cent Injected nose of Animal
Background. Days after 1-131 in-
jection shown on "x" axis. Lach

point is the average of 12 fish.

FIGURE 5

o - 3K1o - 3/u

Gross Radioactive Map of Dead
Fish Injected with 1-131

1 - 3x10 - 3/4 : Gross Radioactive Map of Fish One
Hour After I-lBl Injection

.0

Gross Radioactive Map of Fish One
Day After I-l3l Injection

10 - 3x10 - 3/u

 

 

 

 

 

 

 

 

 

FIGURE 5

31

hD - 3K10 — 3/4:
inn - 3K10 - 3/u:

219 - 3KlO - 3/u:

FIGURE 6

Gross Radioactive Map Made
Four Days After Injection of
I-l3l.

Gross Radioactive Map Made
114 Days After Injection of
I-131.

Gross Radioactive Map Made
21 Days After Injection of
I—131.

 

 

 

 

 

 

 

 

 

FIGURE 6

33

34

.Qzeas.Qiaizibaiian.afflInjaaied.IaLIL

For this study one fish was sacrificed and then injected with I-131.
Five other fish were injected with I.131 and then sacrificed at varying
time intervals. A "gross radioactive map" was made of each fish as
previously described. Reproductions of these maps including an outline
of the fish drawn to scale are shown in Figures 5 and 6.

Some of the I-131 injected into the dead fish showed a slight migra-
tion both posteriorly and antericrly from.the site of injection. After
1 hour the greatest accumulation of 1-131 was in an area anterior to the
injection site but posterior to the transverse septum. Activity was also
present in the thyroidal area at this time. Between 1 and h days after
injection the greatest accumulation of I-l31 shifted to the thyroid area.
There was no further change in the position of maximal activity.

Effects __f; _T_S__ fl _1__ 17.119 Thus id Mtg

A group of 12 fish were injected with 1-131 and counts takencavery
24 hours thereafter. 0n the sixth day after I-131 injections each fish
received one mg. of TSH, and counts were continued as before. Data are
presented in Table IV and shown graphically in Figures 7 and 8.

TABLE IV

~“—--.m

 

Days after I-131 3 Injected Dose 3 Injected Dose
Injection Thyroid and S.D.‘ Background
1 19.1 9.1" 12.3
2 13.1 7.7 5.3
3 11.7 8.0 2.9
U 11.1 7.3 2.7
5 12.6 6.2 2.8
6 TSH given 10.8 7 .311 3.0
7 9.2 5.2 2.7
8 8.7 h.9 3.2
9 9.1 6.2 3.1

10 11.0 6.2 3.2

* Standard Deviation (9.)

Injections of TSH caused an increase in the rate of release of I-131
from the thyroid which lasted for approximately 3 days. This increased
output rate caused an increase in the animal background as shown in
Figure 8.

Thyroid Segretion Bates

Determination of thyroid secretion rates were carried out on
severalggnoups of animals under different eXperimental conditions.

Table V (page 39) gives a summary of the data collected for each deter-
mination.

The first in 2129 count was taken three days after the I—131 injec-

tion, and continued every two days thereafter. Starting five days after
the injection of I-131 a series of increasing daily thyroxine injections
were given. The dose (in J1g.,’ 100 gm. body wt.) was increased on the day
of counting. Figure 9 shows the response of eleven 1-year old trout
kept at 13°C. Data for the other experimental groups were treated in a
similar fashion. The solid line used to estimate 100% previous count
and the secretion rates was fitted by the "method of least squares".
The dotted lines are plots of the standard deviations about the fitted
line. An eXplanation of the statistical methods used is given in
Appendix one.

- Secretion rates were made on both one and two year old fish kept
at 13°C. Rates were also determined for one—year old fish which had
been cooled from 13°C. to 3°C. in 2h hours and maintained for one week
at 3°C. before the secretion rates were determined.

The thyroid secretion rates as determined in both 1 and 2 year old
fish at 13°C. show a range from 0.190 to O.u15.ng. l-thyroxine/loo gm. body

“W:./day. Due to the large variation no significant differences occurred

3% INJ ECTED DOSE

no
9

OD

' 5

U

 

O)

TSH

DAYS

Fig. 7 - Output curve of average in vivo
thyroid activity. Six day—5 a‘f‘t‘er
1-131 injecticn each of the 12
fish were injected with 1 mg. T83.

56

INJEC TED DOSE

‘1

N
4°.

23

OD

O)

4?

 

TSH

Fig. 8 - Yer Cent of Injected ~ose of Animal
Background. Each point is average
of 12 fish. On the sixth day after
I-lSl injections each fish received
1 mg. TSH.

37

38

between these groups (I through IV). For fish kept at 3°C. the thyroid
secretion rate was 0.139.ug. l-thyroxineflfmlgme body wt./day. No difference
was found between this group and others. The t—test of secretion rate
showed no signifiCant differences at the 5% level.

Effect pf Temperature on ngren Consgmption gnd_0gercglgm Rates

Oxygen consumptions were determined by the analysis for dissolved

 

oxygen using a modified "Microninkl r Method" and a constant flow
apparatus as described by Fromm (1958). Fish (1 year old) were main-
tained for at least one week at experimental temperatures before they
were used for oxygen consumption determinations. Operculum rates were
determined while each fish was in the holding tube during the oxygen

consumption experiment. The results are shown in Table VI.

39

TABLE V

DATA FOR THYROID SHIRETION RATE DETERMINATIONS

 

.. I II III IV v

Date 8/1u/58 8/23/58 9/1/58 10/1/58 10/3/58
Temp 13°C. 13°C. 13°C. 13°C. 3°C.
Age 2 2 1 1 1
N 27 27 21 33 30
Ex 3.375 6.750 2.625 9.125 1.880
sxz 0.711 2.817 0.553 0.869 0.200
Exy 3A1.292 675.118 2u7.576 392.96u 178.041
Ey 2.607.060 2.516.45u 1.882.5h1 2.907.8u7 2.629.274

Eyz 260.507.915 239.011.531 170.383.996 262.u05.275 238.660.052

a 89.895 83.019 82.830 77.98“ 77.523
b 53.300 40.731 5h.516 83.436 161.u7u
s. R. 0.190 0.u15 0.223 0.26» 0.139
ax; 17.833 10.200 50.330 9.592 1u.730
5x: 0.335 0.250 0.130 0.115 0.091
SE 0.066 0.0u9 0.029 0.020 0.017
oy2 9.323.4u7 8.686.613 8.036.226 7,76n.607 7.681.295
:2 0.966 0.6u7 0.u11 0.232 0.260
r - sig. 1% level 1% level 15 level 1% level 13 level

++ See Appendix one.

40

TABLE VI
MEAN VALUI'B AND STANDARD DEVIATICN OF 02 mNSUMPTIONS

AND OPERCULUM RATES or 1 YEAR OLD TROUT AT 3°C. and 13°C.

 

 

 

Temp. (00.) No. of 02 Cons. Operculum Rate
‘ Observations (ml-lgm/hr.) (number/minute)
13°C. 16 0.087 t 0.032 117 £7.28

30C. 8 0.040 $0.032 61 314.1%“

 

41

.mop\.uts .3600. .wEw OOCeCHXOMLEPIH 5qu I m .wflh

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

'iunog snotnead queg Jed

DISCUSSION

 

The thyroid secretion rate determinations made on rainbow trout
ranged from 0.1 to 0.4 ug. l-thyroxine/lOO gm./day. No comparable data for
cold-blooded vertebrates have been found in the literature. A few
values for thyroid secretion rates have been reported for warm-blooded
anim:ls. Flamboc (1958) reported a value of 0.278 mg. l-thyroxine/100
lbs./day (S.d. - 0.036) for non-lactating goats. Amin (1956) working
with several strains of mice resorted values of the order of 2.5 ug. l-
thyroxine/IOO gm/day. In work by Reineke and Singh (1955) values for rats
averaged around 2.0 ug. l-thyroxine/lOO gm./day. All of these data were ob-
tained using the similar techniques as those used with rainbow trout. The
thyroid secretion rate of trout appears to be significantly below that
of warmrblooded vertebrates. 0n the basis of histological appearance of
the trout thyroid one would expect this to be the case. histological sec-
tions of the trout thyroid (not included in this thesis) show the follicles
to have an extremely low epithelium. lhe over-all picture was very similar
to sections of rat thyroid which had undergone atrobhy as the reslut of
hypothysectomyx

Secondly the extremely slow output of I-131 from the thyroid of
trout (ti - 37 days) when compared with output values (t3 - 6 days)
for mice (Amin, 1356) would indicate a slow rate of release of iodinated
amino acids from the gland.

Irrespective of the large amount of research that has been done on
thyroid function in teleosts no definite continous function of thyroxine

has been established. There may be periods during which the thyroid is

42

43

quite inactive. Hoar (1953). for example, has described seasonal varia-
tions in the activity of the teleost thyroid based on differences in the
histology of thyroidal tissue.

The data obtained from the radioactive mapping procedure and uptake-
output studies with the geometry described herein can be used to explain
some of the variation in uptake-output data reported in the literature.

The measurement of thyroidal accumulation and output of I-131 in
Fish made by injecting large numbers of fish. sacrificing at appropriate
time intervals, and determining the activity in excised thyroid tissue
introduces the factor of experimental variability between animals. ‘When
.LQIEiyg counting methods are used each animal serves as its own control
and individual animal variance is reduced, giving more valid results.

The possibility of not surgically removing all thyroid tissue is elimin-
ated by the in yiyg counting procedure.

lilngg,estimations of the I-131 content of thyroidal tissue may be
erroneous due to the accumulation of injected I—lBl in the anterior part
of the pleuroperitoneal cavity. This nOn-thyroidal activity amounts to
a significant per cent of the injected dose of I-l3l for three days follmp-
ing injection but is relatively unimportant thereafter. The great diff-
erences in per cent uptake as well as the uptake and output rates of
I-131 appearing in the literature prdbably reflect differences in the
amount of animal background (non-thyroidal above) included in the thyroid
counts. The previous literature dealing with the metabolism.of I-131
by the teleost thyroid show no corrections for animal background.

As an example of the influence of animal background on igwgixg
counts was shown by Fromm and Reineke (1956). After radiothyroidectomy

considerable amounts of tracer doses of 1-131 accumulated in the head

region of trout. This non-thyroidal activity was shown to leave the
area rapidly having a half life of 1.8 days. This value compares favor-
ably with the rapid decrease in animal background found in the present
study.

The rapid decrease in animal background is probably the result of
an accelerated rate of excretion of excess I-lBl. During the period of
accelerated excretion of I-l3l some 65} of the total injected dose was
excreted into the aquarium water. Much of the excreted 1-131 was removed
from the water by the charcoal filter and changes of the aquariumxwater
every 3 days prevented most recycling of the excreted I-lBl.

For an accurate estimation of thyroid secretion rates it is important
that the in 1:19 thyroid activity be a measure of the true activity of
the thyroid gland. The interference of animal background in these studies
can be minimized in three general ways: (1) By injecting larger doses
of 1-131 the determination of secretion rates could be started at a
much later time. This increased interval between the injection and the
beginning of secretion rate determinations would result in a low body
background while the thyroid would still have sufficient activity to
give a relatively low counting error. The effects of the beta and gamma
radiation must be taken into account when increasing the dosage. (2) Suit-
able collimation could be developed which would enable counts to be taken
with minimal animal background activity. Because of the penetrating
power of gamma radiation from I—131 appropriate collimation is very
important. (3} If the rate of output of the animal background is known.
along with values for igflyitrg activities. a mathematical expression could
be derived to correct ig‘yiyg thyroid activity for animal background. In

this first attempt to estimate the thyroid secretion rate of rainbow

45

trout all of the above methods were used to some degree.

on the basis of the following statistical approach no significant
differences were found between the thyroid secretion rates for each
experimental group. A single standard deviation (5y) (see Fig. 9. page
41 ) was plotted as parallel lines to the fitted line. At the intercept
of the 3y lines with the 100% previous count line perpendiculars were
dropped from the "100% line" to the x axis. The distance from these
intercepts with the "x" axis (ZSx) is equivalent to two standard devia-
tion of the estimated thyroid secretion rate. Therefore the standard
deviation of the secretion rate is given by Sx. "3x" will be valid if
Sy is a measure of the true variance about the fitted line. Since two-
thirds of the observations fall within one standard deviation of the
fitted line the assumption that Sy is a valid measure of the standard
deviation is strengthened. From the standard deviation (Sx) the standard
error of the thyroid secretion rate was calculated. Using these data.
tests for significant differences between experimental groups were made
using the t-test.

It is well known that one of the main functions of thyroid secre-
tions in homoiotherms is the control or regulation of oxidative metabo-
lism. It is also known that exposure of warm-blooded animals to lowered
environmental temperatures causes increased thyroidal.activity. It has
tmwmlreported that when fish are exposed to low temperatures thyroid
function is maintained or slightly enhanced as shown by histological
examination of thyroidal material (Pickfordamd LtZ. 1957)-

The rate of reactions in the organism will increase as the temper-
ature increases. For meaningful comparisons of the effects of tempera-

ture on the rates of various biologic processes, the rates of reaction

46

may be compared for some given temperature interval. The ratio of the
rate of activity at one temperature to the rate at a temperature 10°C.
lower is called the ”temperature coefficient" (Q10). Thermochemical
types of reactions have 010 values of about 2.00 (Siese. 1957). Appen-
dix 1 gives the equation for obtaining Q10 values.

Trout at 13°C. and 3°C. have oxygen consumptions and operculum
rates that yield the following Q10 values: oxygen consumption = 2.17;
operculum rates = 1.92. The Q10 values indicate that the respiratory
metabolism decreases by about one-half per 10°C. drop in temperature.
The Q10 value of the mean thyroid secretion rates at the two temperatures
was found to be 1.72, indicating that thyroidal activity had neither
remained the same nor increased as previously postulated but that it had
followed a proportional iecrease in metabolic activity similar to that
of the general oxidative metabolism. in the face of the above data it
seems rather unlikely that thyroxine plays an essential role in the con-

trol of oxidative metabolism of the teleost at low temperatures.

1.

2.

30

SUMMARY.A CONCLUSIONS

A method for the measurement of thyroid secretion rates of fish was
developed. Thyroid secretion rates for a poikilothermic vertebrate.
Salmg ggigdgeri , was found to range from 0.190 to 0.#15‘pgm. l-

thyroxine per 100 grams body weight per day.

No significant differences in the thyroid secretion rate was found
between fish kept at 3°C. and 13°C. For this temperature interval
the thyroid secretion rates had a Q10 value (1.72) characteristic

of a thermochemical type of reaction.

One-year old and two-year old trout showed no significant difference

in their mean thyroid secretion rates.

Under the counting geometry and corrections described the peak up-
take of 1-131 by the thyroid tissue occurred within 2% hours after
pleuroperitoneal injection of 1-131. A two component output curve
was found. The first component (rapid output rate) may possibly be
the result of incomplete animal background corrections. Approxi-
mately 20% of the injected dose was accumulated in the thyroidal

area within the first 2“ hours.

The site of maximal accumulation of non-thyroidal I-lBl activity
was shown to gradually shift anteriorly for 4 days following injec-
tion of 1-131 until it reached the thyroidal area. After b days

there was no further change in the position of maximal activity.

010 values indicate that the oxygen consumption of trout follows

the pattern of a normal thermochemical type reaction (010 I 2.0)

47

48

for the temperature range of 3°C. to 13°C. Oxygen consumptions
and operculum rates yielded the following 010 values: oxygen consump-

tion = 2.17; operculum.rates = 1.92.

It is suggested that complete or standardized animal background
corrections are needed before igL I g counting data from different

workers may be compared.

LITERnlURb ClTnD

 

 

Addison, W. H. F., and M. N. Richter, 1932. u Note on the Thyroid Gland
of the Swordfish (Xiphias gladias, 1?). Biol. Bull., 63:472-476.

 

Amin, Abolghassem. 1956. Differences in Thyroid Activity of Several
Strains of Inbred and F1 Hybrid Rice. lhesis. Mich. State Univ.

Armour Laboratories. 1956. Retail Reference Catalog. Chicago, Ill.
Berg, 0., and A. Gorbman, 1954. Normal and “ltered Thyroidal runction

in Domesticated Goldfish, Carassius auratus. Proc. Soc. Exp.
8101. and Lied. 83:155-159.

 

Bullock, T.H. 1955. Compensation for Temperature in Metabolism and
Activity of Poikilotherms. Biol. Rev. 30:311-342.

Chavin, M. 1956a. Thyroid uistribution and Function in the fToldfish,
Carassius auratus L. as detenmined by the Uptake of Tracer Doses
of Radioiodingjfl—Raper presented at Conference on Radioactive Iso-
topes in Agriculture held at M.S.U. Jan. 12-14, 1956.

Chavin, M. 1956b. Thyroid Function in Goldfish. J. Exp. 2001. 133:254-
275.

Flamboe, E. 1958. A study of Thyroid Activity in Dairy Goats Relating
to Age, Lactation, Pregnancy and Season of the Year. PhD. Thesis.
Hich. State Univ.

Fromm, P. 0. 1958. A Hethod for Keasuring the Oxygen Consumption of
FiSh. Prop. fish-Cult. 20(5) 157—159.

Fromm, P. 0., and E.P. Reineke. 1956. Some Aspects of Thyroid Physi-
ology In Rainbow Trout. J. Cell and Comp. Physiol. 48:398-404.

Giese, A. C. 1957. Temperature and Life. Cell Physiology. Philadelphia:
W. V. Saunders Company. p. 52-64.

Goldsmith, E. U. 1949. Phylogeny of the Thyroid Descriptive and Pxper-
imcntal. in. New York Acad. Sc. 50:283-316.

worbman, A., andO. Berg. 1955. Thyroid Function in the fishes fund-
ulus heteroclitus, £5 majalis. and £3 digphanus. Endocrinology
56 3 £6.92 0

Gorbman, A., Lissitzky, S., Midiel, R., and J. Roche. 1952. Thyroidal
metabolism of Iodine in the Shark Scyjiorhinus (scyllium) canicula.
Endocrinology 51:311-521.

Gudernatsch, J. F. 1911. The Thyroid Uland of Teleosts. J. Horph. 21:
709-722 0

49

(fl
0

Hoar, W. S. 1939. The Thyroid Gland of the Atlantic Salmon. J. Morph.
65:257-295.

Hoar, W. S. 1953. Control and Timing of Fish Migration. Riol. Rev.
28:437-452.

Roar, R. S. 1958. Effects of Synthetic Thyroxine and Conadal Steroids
of the Metaoolism of Goldfish. Can. J. Zool. 136:113;121.

LeRoche, 6., and C. 7. Leblond. 1952. Effect of Thyroid Preparations
and Iodide on Calmonidae gilles. LndocrinolOgy 51:524-545.

 

Lynn, G. W., and H. E. Wachoqski. 1951. The Thyroid Gland and Its
Function in Cold-Blooded Vertebrates. Quart. Rev. Piol. 26:123-168.

Matthews, S. A., and U. C. Smith, 1948. Concentration of Radioactive
Iodine hy the Thyroid Gland of the Parrot Tish, Sparisoma 32.
Physiol. 152-153z222-225.

Matty, S. J. 1957. Thyroidectcmy and its Effect Upon nygen Consump-
tion of a Teleost Fidi, Pseudoscarus guacamaia. EndocrinoloEy.
1531-80

 

Nadler, N. R. and C. P. Leblond. 1955. Rate of Thyroid Hormone Form-
ation. Upton, N.Y.: U.S. Brookhaven Rational Laboratory, p.40-60.

Perry, W. F. 1951. A Method for Measuring Thyroid Hormone Secretion
in the Rat with its Application to the Bioassay of Thyroid Extracts.
Endocrinology 48:643.

Pickford, G. E. 1953a. The Fish in Endocrinological h’esearch. Trans.
N. Y. Road. Sci. 15:186-189.

Pickford, G. b. 1953b. A Study of the Hypophysectomized Kale Killifish,
Fundulus heteroclitus(Linn.) Bulletin of the Bingham Oceanographic
Collections. ‘Pish Endocrinology, 14:5-41.

 

E’ickford, “. 3., and T. W. Atz. 1957. The Thyroid and Thyroptropin.
The PhySiOIOby of the Pituiiary Gland of Fishes. New York: New
York Zoolobical Society.

Reineke, R. 3., and O. P. Singh. 1955. Estimation of Thyroid Hormone
Secretion Rate of Intact Rats. Proc. Soc. Exper. Biol. and Ned.
{83203-2070

Robertson, 0. H. 1948. The Occurrence of Increased Activity of Thyroid
Gland in Rainbow Trout at Time of Transformation from Parr to
Silvery Smelt. Physiol. 2001. 21:282-295.

Robertson, 0. R., and n. L. Chenory, 1953. Thyroid Hyperplasia in
Spawnina Rainbow Trout. Physiol. 2001., 26:328-334. '

51

Swift, D. R. 1355. Seasonal Variations it the Jrowth Rate, Thyroid
'land pctivity an; :ood Reserves of brown Trout (Salmo trutta Linn)
J. xpt. Biol., 32:751-764

 

Werner, D. C. 1955. (Editor) lhe Thyroid. Row York: Hoeber-Harper.
pp. 789.

Kolff, J. 1951. done tasters that Influence the Release of Iodine from
the Thyroid Vland. EndocrinolOEy 48:284-297.

PERTlNLfiI RLFEKENCLS NOT CIIED

Albert, A., 1951. The In Vivo Determination of the Biological Decay
of Thyroidel dadioiodine. Endocrinology. 4t:334-340.

Arkin, U., and R. Dalton. 1957. Tables for Statist'cians. bollege
Outline Series. hew York City N. Y.: hornes and Loble Inc. P.152.

Berg, 0., and A. Corbman, 1953. Utilizat on of Iodine by the Thyroid
or Platyfish, Xiphophorus (9}atypoecilus) meculatus. Proc. Soc. for
’xp. Viol. and Red. 83:751-756.

 

 

”rown, M. E. (Editor) 1957. The PhysiOIOgy of :ishes Volume I. Acad-
emic Press Inc.

Chambers, H. A. 1953. Toxic Effects of Thiourea on the Liver of the
Adult Gene Yillifish,(Fundulus heteroclitus Linn.) Bulletin of
the flingham OceanOgraphic Bollections. Fish Endocrinology 14:69-93.

 

Dixon, W., and i. Massey Jr. 1957. Introduction to Statistical Anal-
ysis. New York: McGraw-Hill 800k company Inc. op. 48b.

Fleischmann, Walter. 1951. Compnrvtive Physiology of the Thyroid and
Parathyroid blends. Fpringfiled Ill; Bannerstone House. pp. 78.

Langrord, H. G. 1957. Production of Exophthalmos in Fundulus heter-
oclitus (Var. Bermudas) by Triiodothyronine and Uesiccated Thyroid .
EndocrinOIOgy 60:390-392.

 

 

Hatthews, S. A., and D. C. Smith. 1947. Thehffect of Thiourea on the
Oxygen Consumption of iundulus. Physiol. 2001. 20:161-164.

Nigrelli, R. F. 1953. The Iisn in Biological Reasenrch. Trans.
1‘;- Yo Acad. SC]... 15:183-186.

Pickford, G.E. 1953. The Response of Kypophysectorizod Kale Killifish

to Trolonged ;rostmcrt with Surll toses of Thyoptropiz. Endocrin-
ology, 55:589-592.

52

Prosser, C. L., Bishop, D., Brown, F., Jahn, T., and f. fiolff. 1952.
Conparative Lnim l Rhysiology. Ihiladelphia and London: J. B.
Saunders Company, pp. 888.

Scheer, h. T. 1954. C mparative Ihysiology. Vertebrate New York:
John Wiley and Sons Inc. pp. 359-531.

Standard Hethode for the Examination of Water, Sewage, and Industrial
Wastes. 1955. Publication Office American 1 blic Health Associa-
tion Inc. flew York 19, 1.Y. Tenth Eiditon. op. 522.
Turner, C. D. 1955. The Thyroid and the ituitarv wland. fienerel Endocr-
’ inolcgy. Vhiledslphis: U. i. Saunders Conpany. p 17-64 and

3ng-443 0

APPENDDIES

APPENDIX I

STATISTICAL TREATMENT OF DATA

54

0‘.
CW

_S_TATISTICAL TREATMEEI; g; DAIA

mag ..e.<;__.5 retina 3:22
1. Metth 9;; ngst Smart:

number of observations

Sum of thyroxine dose X(N)

Sum of thyroxine dose 5 uared (N)

Sum of thyroxine dose ( preceding count)
Sum of i3 preceding cnmt

Sum of i preceding count (5 preceding count)
:5 preceding count

thyroxine dose in mg./gm. body weight/day.

miefi’i’urz

y=a+b(x)

E(y) N (a) + b (Mar)
E(xy) E(x)(a) + b ( )

solve for (a) and (b) where a = intercept arri b =
slope of line.

2. WWQQEEZQIQQJM “mm: -
Sy2= E12 -a§§x) - b13351)

N-Z
Sy = 3 Standard deviation of least square line.

Variance of the estimated secretion rate on the x axis is
defined as Sx and calculated by the following:

3x = r. SY/b
The M error of the stimated secretion rate is found by:

ss : _._ Sx/ N-l
3. 09931;;th of Correlation "r".
0Y2 (Ear/N)2

2 - b - N crz
r new: 5J- "(9:12.“ > L4

"r" values found in Barnes and Noble (1957) page 1%.

LP- wmmwm

To determine the existence of significant differences between

the mean secretion rates of any two groups the following calcul-

ations were made:

'1‘ = d/SEd
where T = test statistic (Barnes and Noble (1957))
d = differences between secretion rate "1" and secretion
rate "2"
SEd = standard error of the difference.

SEd = (SE1)2 + (532)2
degrees of freedom (df) =N1 + N2 - 2
MQIMMMLQEWQIEM
Exlyl = E(xy) -_&c_§§z}_
Exf = m2-ggfi

N
b = Ex1171

a?

slepe of line passing through the mean of the x and y axis.
time axis

activity axis

sum of time (N)

sum of activity

sum of activity (time)

sum of (thue)2(N)°

sum of (activity)‘w

total observation

Method 9; Detemimtggg Stmard Deviation 9; "ti," fitted line
.2 s a standard deviation of activity

32 = Ev? - (Ev)§i/ N
N-l

Methgg Used Lg.Calcg;gte 't%"

-o.693(t)/t%
At/AC = e

activity at time zero
activity at time "t"
time

half-life

p.
6'
"III

N
ll

Calculations 2; gm Values
10 t - t
Q10 = (Kz/Ki) / 2 1

where k1 = rate at temperature "1"
k2 2 rate at temperature "2"
t2 = temperature 2 (°c. + 273)
t1 I temperature 1 (0C. 4- 273)

Q10 ratio of activity per 10 degree change in temperature

APPENDIX II

THYROID SECRETION RATE DATA FOR GROUPS
I-II—III-IV—V

OF TABLE V

58

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