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In vitro measurement of circulating thyroid hormone levels in beef cattle

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Title:
In vitro measurement of circulating thyroid hormone levels in beef cattle
Creator:
Cowley, Jerry Jennings, 1940-
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English
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51 leaves : illustrations ; 28 cm

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Subjects / Keywords:
Analysis of variance ( jstor )
Blood proteins ( jstor )
Cattle ( jstor )
Chromatography ( jstor )
Heifers ( jstor )
Iodides ( jstor )
Pastures ( jstor )
Secretion ( jstor )
Thyroid gland ( jstor )
Thyroid hormones ( jstor )
Beef cattle ( fast )
Cattle -- Physiology ( fast )
Thyroid gland ( fast )
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bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

Notes

Bibliography:
Includes bibliographical references (leaves 46-49).
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Jerry Jennings Cowley.

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University of Florida
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IN VITRO MEASUREMENT OF CIRCULATING

THYROID HORMONE LEVELS IN BEEF CATTLE









By

JERRY JENNINGS COWLEY











A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY










UNIVERSITY OF FLORIDA 1968














ACKNOWLEDGMENTS


The author wishes to express his sincere appreciation to

Dr. Alvin Warnick, chairman of his supervisory committee, for his counsel and assistance throughout this study.

Appreciation is also expressed to Dr. Marvin Koger, Dr. Ray

Shirley.and Dr. G. T. Edds who served as members of the supervisory committee and to Dr. J. P. Feaster for guidance in the laboratory.

The author wishes to thank Mr. Dean Pogue and Mr. Hernando Gutierrez for their assistance in the gathering of data and care of experimental animals.

The author extends his deepest gratitude to his wife, Marilyn, for her constant encouragement and untiring efforts which were invaluable in achieving this goal.

























ii
















TABLE OF CONTENTS


Page

ACKNOWLEDGMENTS ............................................. ii

LIST OF TABLES .. ..................... ....................... iv

LIST OF FIGURES ........................................... vi

INTRODUCTION .......................... .................... 1

REVIEW OF LITERATURE....................................... 3
General Thyroid Physiology............................ 3
Reproduction and Lactation............................ 6
Temperature and Thyroid Activity....................... 8
Sephadex Chromatography .......................................... 10

EXPERIMENTAL PROCEDURE..... .................................. 13

RESULTS............. ............ .... ...................... 15
Effect of Different Columns on Percent Uptake
of Duplicate Samples............................. 15
Repeatability of Observations on Subsequent
Days for Estimating Thyroid Status in
Beef Cattle...................................... 17
Effect of Age of Isotope on Percent Uptake of
1-131 Labelled T3 by Cattle Serum Proteins ....... 18
- Effect of Temperature, Breed, and Sex on Thyroid
Secretion Rate of Beef Cattle............... .. 22

GENERAL SUMMARY AND CONCLUSIONS ............................. 43

BIBLIOGRAPHY .. .................. . ....... ......... ... . 46















LIST OF TABLES

Page

TABLE

I. PERCENTAGE UPTAKE OF 1-131 LABELLED T BY
DUPLICATE SAMPLES OF CATTLE SERUM THROUGH
DIFFERENT COLUMNS .............................. 16

II. PERCENT UPTAKE OF 1-131 LABELLED T3 BY CATTLE
SERUM FOR 5 DAYS AS MEASURED BY SEPHADEX
CHROMATOGRAPHY................................ 19

III. PERCENT UPTAKE OF OLD AND NEW 1-131 LABELLED T3 BY CATTLE SERUM PROTEINS ................... 21

IV. MEAN PERCENT UPTAKE OF 1-131 LABELLED T3 FOR
TEN MONTH PERIOD ............................. 24

V. ANALYSIS OF VARIANCE OF MEANS OF PERCENT UPTAKE
FOR TEN MONTH PERIOD .......................... 25

VI. ANALYSIS OF VARIANCE OF PERCENT UPTAKE OF 1-131
LABELLED T3 FOR MARCH .......................... 28

VII. ANALYSIS OF VARIANCE OF PERCENT UPTAKE OF 1-131 LABELLED T3 FOR APRIL ......................... 29

VIII. ANALYSIS OF VARIANCE OF PERCENT UPTAKE OF 1-131
LABELLED T FOR MAY...... ................... 30

IX. ANALYSIS OF VARIANCE OF PERCENT UPTAKE OF 1-131
LABELLED T3 FOR JUNE .......................... 31

X. ANALYSIS OF VARIANCE OF PERCENT UPTAKE OF 1-131
LABELLED T3 FOR JULY .......................... 32

XI. ANALYSIS OF VARIANCE OF PERCENT UPTAKE OF 1-131
LABELLED T3 FOR AUGUST ........................ 33

XII. ANALYSIS OF VARIANCE OF PERCENT UPTAKE OF 1-131 LABELLED T3 FOR SEPTEMBER ...................... 34

XIII. ANALYSIS OF VARIANCE OF PERCENT UPTAKE OF 1-131
LABELLED T3 FOR OCTOBER....................... 35


iv










LIST OF TABLES (continued) Page
TABLE

XIV. ANALYSIS OF VARIANCE OF PERCENT UPTAKE OF 1-131 LABELLED T3 FOR NOVEMBER ........................ 36

XV. ANALYSIS OF VARIANCE OF PERCENT UPTAKE OF 1-131
LABELLED T3 FOR DECEMBER ....................... 37













































v
















LIST OF FIGURES

Page

Figure

1. Monthly percent uptake of 1-131 labelled T3 for
treatment classification............... 26

2. Monthly percent uptake of 1-131 labelled T3 for
breed classification............................ 39

3. Monthly percent uptake of 1-131 labelled T3 for
sex classification......... ... ................. 41







































vi
vi














INTRODUCTION


In reviewing the role of the thyroid hormones in the reproductive physiology of the female, Reineke and Soliman (1953) pointed out that although over half a century of investigation indicates that the thyroid hormones are implicated in some manner, their precise role has not been determined. There is also ample evidence that the thyroid gland plays an important role in growth and development. Many factors are associated with thyroid hormone secretion rate including breed, age, sex, season of year, pregnancy, lactation, temperature and humidity, plane of nutrition, and a variety of diseases.

When nuclear reactor-produced 1-131 became available in 1946, the study of the thyroid gland received new impetus. Most of the subsequent research was in clinical diagnosis and therapy.

The introduction of an in vitro technique in 1962 has made it possible to study and compare circulating thyroid hormone levels without exposing the animals to radioactive material or endogenous thyroid hormones.

Earlier work has suggested that one of the factors influencing

adaptability is thyroid activity. Thyroid activity has been suggested as one of the factors involved in the difference in adaptability between European (Bos taurus) and Asiatic (Bos indicus) breeds of beef cattle.

This project was'designed to study the feasibility of an in vitro


1








technique to measure thyroid status in beef cattle and to measure differences in thyroid activity between Herford and Brahman cattle maintained in climate control chambers at 320 C and 210 C and on pasture for 12 months.















REVIEW OF LITERATURE


General Thyroid Physiology


A fundamental knowledge of the formation and release of the

thyroid hormones is essential in any study employing thyroid physiology. A single layer of epithelial cells, the follicular epithelium, encompassing a central mass of protein, the colloid, is the fundamental parenchymal unit of the gland. The small sacs or follicles of which the adult thyroid is composed may be considered from both the structural and functional point of view to be the secretory unit of the thyroid gland. Within the follicle and filling its lumen is the homogenous, thin, clear substance, the colloid. The synthesis of the thyroid hormones has been reported by Nadler (1962) to take place within the colloids of the follicles. Heidelberger and Pedersen (1935) found that the colloid consists largely of thyroglobulin, a large protein having a molecular weight of approximately 700,000.

Iodide is the only chemical form in which the thyroid gland can

assimilate iodine for formation of the thyroid hormones. Halmi (1964) stated that active transport is by far the most important mechanism whereby the gland can accumulate iodine. The thyroid gland, in addition to concentrating iodide, concentrates a series of other anions of no physiological use to the gland. Lewitus et al. (1961) explained



3







4

the "trapping mechanism" on the basis of valency and ionic size stating that the "trapping mechanism" in the thyroid gland involves a certain specially adapted protein in the cell membrane which will incorporate all monovalent ions which fulfill certain requirements of size.

Iodide that is transported into the gland from the circulation undergoes enzymatic oxidation. The subsequent iodination of tyrosine in the thyroglobulin to form the thyroid hormones occurs in the colloids of the follicular cells. Pitt-Rivers and Cavalieri (1964) gave the following sequence of events that lead to the formation of the thyroid hormones:

1. Iodide is transported into the gland by the blood.

2. Iodide is converted by some oxidative enzyme to monoiodotyrosine (T1) and diiodotyrosine (T2).

3. Thyroxine (T4) is formed by the coupling of 2 molecules

of T2 and the loss of 1 alanine side chain.

4. Triiodothyronine (T3) is formed by the coupling of 1

molecule each of T and T2 and the loss of 1 alanine

side chain.

Reactions 2 through 4 occur within the thyroglobulin molecule located in the follicles.

The thyroid hormones either enter the blood or are secreted into the lumen of the follicle and stored in the colloid bound to thyroglobulin. Because of its large molecular size, thyroglobulin is degraded to its constituent amino acids before the thyroid hormones are secreted into the blood stream. Pitt-Rivers and Tata (1959) state that this step is under the influence of thyroid stimulating hormone






5

(TSH) and is inhibited by excess iodide. The exact mechanism by which the hormones pass from the thyroid tissue into the blood is not known, but Tata (1964) suggests that passive diffusion across membranes is the most likely process. Taurog et al. (1956) found that both T4 and T3 were secreted into the circulatory system but neither T2 or T1 left the thyroid gland even when the gland contained 70 percent of its 1-131 in these forms.

It has been known for many years that for proper functioning,

the thyroid gland is dependent upon the anterior pituitary secretion, thyroid stimulating hormone (TSH). Also, the thyroid hormones themselves are known to influence the secretion of TSH. This reciprocal relationship provides a homeostatic mechanism which insures adequate synthesis and secretion of thyroid hormones.

Upon release from the gland, the hormones enter the circulation and are distributed throughout the body. The interaction between thyroid hormones and serum proteins, which bind the hormone more firmly than any tissue component, is the key to the present-day understanding of hormone transport. Deiss et al. (1953) reported both T3 and T4 bound to a specific alpha globulin just ahead of alpha-2-globulin. Frienkel et al. (1955) also reported that most 1-131 labelled T4 localized with a protein intermediate in mobility between alpha-l and alpha-2-globulin. They listed the globulins, albumins, and erythrocytes as the primary, secondary and tertiary carriers, respectively of T3 and T4.

Pitt-Rivers and Tata (1959) listed three principal types of

pathways at the organ level for the distribution of thyroid hormones after the administration of radioactive T3 and T4. These included:








(1) liver, kidney and posterior pituitary which show a high turnover rate; (2) skeletal muscle and intestine which exhibit a slow turnover rate; and (3) brain, spleen and gonads which accumulate only very low levels. Albert and Keating (1952) have shown the liver to be the most active tissue in concentrating and metabolizing thyroid hormones. The intense enterohepatic circulation of thyroid hormones is primarily responsible for rapid thyroid hormone catabolism and excretion.

Robbins and Rall (1955) stated that the bilary-fecal pathway by which most of the iodine is excreted as unchanged hormone in the feces is the predominant pathway in rats. Albright and Larson (1959) have shown urine to be the predominant excretory pathway in man.


Reproduction and Lactation


Brody and Frankenbuch (1942) were among the first to show that thyroidectomized cows fail to manifest normal physical signs of heat, and that administration of thyroid material restores normal estrual behavior in some thyroidectomized cows. Blaxter (1945) reported that thyroid therapy shortened the post-partum breeding interval.

Brunstad and Fowler (1958) found that crown-rump length of normal embryos carried by thyroprotein-treated and control gilts were significantly larger than those of thiouracil-treated gilts. Lucas et al. (1958) reported pigs born to thyroprotein-treated gilts to be heavier than those born to control and thiouracil-treated-gilts. They reported increased embyronic mortality from the 25th day of pregnancy to






7

parturition in the thiouracil-treated group and reduced embryonic mortality in the thyroprotein-treated group.

Spielmann et al. (1945) noted that the main effect of thyroidectomy on mature cows was a failure to manifest the physical signs of estrus. Normal ova were produced as evidenced by the birth of 3 calves from 3 cows thyroidectomized 161, 242, and 453 days before breeding.

Henneman et al. (1955) reported lactation to create increased demand on the thyroid gland, and lactating ewes showed a significantly higher thyroid secretion rate than either nonpregnant or pregnant ewes.

Spielmann et al. (1944) found that cows thyroidectomized prior to gestation, during pregnancy, or during lactation would cease lactation in about 180 days. It was also observed that incomplete removal of the thyroid gland produced a temporary decline in milk production, followed by a gradual return to the former levels.

Hart (1960) reported a 9.5 percent increase in lambs born from ewes treated with L-thyroxine pellets 10 to 11 days before mating. Ryle (1961) found that ewes exposed to an experimentally hot environment and ewes under normal conditions which received thyroxine had significantly more-embryos than those ewes that received no thyroxine. In 1963, Ryle proposed that dwarfing of fetuses exposed to high ambient temperatures, as reported by Yeates (1958) may be prevented by thyroxine therapy.

It has been repeatedly shown that milk yield in cattle can be increased by thyroid therapy. Graham (1934) and Folley and White









(1936) were among the first to demonstrate the beneficial effects of thyroxine on lactation in the bovine. Reineke (1946) stated that by giving repeated small doses of thyroxine, or by adding iodinated protein to the feed, it was possible to maintain increased milk yield for many months without causing any apparent harm to the cow. The main effect of the treatment, according to Myant (1964), is upon yield of fat rather than upon yield of total solids in the milk. Although thyroid hormones may be secreted in the milk, Robertson (1945) stated that the amount present in the milk of cows given iodinated proteins was insufficient to affect the metabolism of humans. This technique is not widly practiced for it is not economically feasible.


Temperature and Thyroid Activity


A series of experiments conducted at the Missouri Agricultural Experiment Station -have established a relationship between temperature and thyroid activity. The thyroid uptake of 1-131 and the conversion ratio (ratio of blood plasma thyroxine-like 1-131 to total 1-131) were used as parameters of thyroid activity. Blincoe and Brody (1955) maintained Holstein, Jersey, Brown Swiss and Brahman cows in a "comfort zone" (4-210 C). Increasing ambient temperature to 350 C decreased the thyroid activity 30 to 65 percent with Holstein cows showing the greatest decrease and Brahman the least. Decreasing ambient temperature to -8o C increased the thyroid activity in Jersey and Brahman 60 to 100 percent but did not increase thyroid activity in Holstein and Brown Swiss cows. Air velocities less than 10 m.p.h. had no measurable effect on thyroid






9

activity but the addition of radiant energy depressed the thyroid activity. Thus, increasing the thermal stress on cows either by increased ambient temperature or by increased radiation reduced the thyroid activity. As heat stress increased, the rate of clearance of plasma iodide by the thyroid decreased and the excretion rate increased.

Blincoe and Brody (1955) studied the influence of diurnally

variable temperatures on the thyroid activity of Jersey and Holstein cows. A daily temperature cycle of -10 to 40 C increased thyroid activity by 20 percent over the value of a "comfort zone" cycle. A temperature cycle of 21 to 380 C'decreased the thyroid activity by about 30 percent below its value in the "comfort zone" cycle. These data roughly paralleled the heat production data for the same animals.

Blincoe (1958) studied the influence of constant ambient

temperature on the thyroid activity of Shorthorn, Santa Gertrudis and Brahman calves. The 3 heifers of each breed were maintained at 100 C, 270 C, and in an open shed. The rate constant for hormone release of the Shorthorn heifers was decreased 45 percent at 270 C. The Brahman heifers showed no decrease and the Santa Gertrudis were slightly decreased.

The Shorthorn calves raised at 100 C were most affected by high temperature. At 38' C their thyroid secretory activity was reduced 60 percent below the reading at 100 C. The Santa Gertrudis were less affected and the Brahmans practically unaffected.

Swanson et al. (1957) studied the 1-131 uptake of 8 Jersey and Guernsey cows for 18 months. They found effects which could be






10

attributed to seasonal temperatures, but the average differences found between seasonal measurements were relatively unimportant. They concluded that "measuring thyroid activity of cows by this method on an uncontrolled survey basis could not be expected to provide reliable comparable data."

Howes (1964) measured the 1-131 uptake on 24 nonpregnant

Herefcrd and Brahman heifers. He found that the thyroid glands of

the Hereford concentrated 1-131 faster than the Brahmans and the thyroid glands of the Herefords retained a significantly greater percentage of the injected 1-131 at comparable time intervals than did the Brahman heifers.


Sephadex Chromatography


Gel filtration first became an established laboratory technique with the introduction of Sephadex in 1959. The use of a chromatographic method employing Sephadex gel for estimating thyroid status was reported by Shapiro and Rabinowitz (1962).

Sephadex is a modified dextran. The dextran macromolocules are cross linked to give a three-dimensional network of polysaccharide chains. Because of its high content of hydroxyl groups, Sephadex is strongly hydrophilic and the Sephadex beads swell considerably in water and electrolyte solutions giving a porus gel. Only molecules below a certain size (below 5,000 molecular weight with Sephadex G-25) can enter the interstices. Heavier molecules are excluded from the pores and pass through the bed in the liquid phase outside the particles, passing through the column more rapidly than the smaller molecules entering the pores. Molecules are therefore eluted from a Sephadex bed in the order of decreasing molecular size.








The mechanism of Sephadex binding is not well known, but it is known to bind compounds with aromatic rings. Thus, the Sephadex removes the unbound T3 but apparently cannot compete with the stronger T3 binding of blood serum, and this protein-bound T3 passes through the column. Shapiro and Rabinowitz (1962), Cuaron and Fucugauchi (1964) and Cowley (1965) have shown that a chromatographic column packed with Sephadex G-25 gel can be used to separate 1-131, unbound 1-131 labelled T3 and protein-bound 1-131 labelled T3. They found that protein-bound 1-131 labelled T3 would be eluted first, followed by free 1-131 and the unbound I-131 labelled T3 would remain in the gel. This unbound T3 could then be removed by the addition of blood serum. Jacobsson and Widstrom (1962) reported that by thorough washing, the columns could be used over 100 times with no adverse effects.

Shapiro and Rabinowitz (1962) compared Sephadex chromatography with the red blood cell uptake technique of Hamolsky et al. (1957) for the estimation of thyroid status. They reported that since the Sephadex technique was rapid, uses serum instead of whole blood and allows the user to correct for free iodide in the reagent, it overcomes the major objections to the red blood cell uptake technique. These objections were: (1) Lengthy multiple washings of red blood cells; (2) No correction for free iodide in the reagent; (3) Hemolysis and (4) A correction for hematocrit. Cowley (1965) also compared the two techniques and found Sephadex chromatography superior in determining hyperthyroid, hypothyroid and euthyroid status in sheep.

Cuaron and Fucugauchi (1964) found diagnosis of thyroid status by Se'phadex chromatography to agree with the clinical diagnosis in 95.9






12

percent of the 141 patients studied. They stated that it should be used as a screening test for thyroid function since it is sufficiently reliable, does not need internal administration of radioactive material and is simple and quick to perform. Jones andSchultz (1966) reported that gel filtration using Sephadex G-25 offers a simple, reliable and highly reproducible means of separating iodide and the thyroidal iodoamino acids.















EXPERIMENTAL PROCEDURE


Sephadex column chromatography was used to ascertain thyroid

status of beef cattle. The assay employed the technique of Shapiro and Rabinowitz (1962) as modified by Cowley (1965). The complete procedure is as follows:

1. Swell 3.0 grams Sephadex G-25 medium grade in 0.2 normal

phosphate buffer for 6 hours at room temperature. Pour

the gel into a Sephadex K-9 laboratory column fitted with

a luer ending outlet port.

2. Add 0.1 milliliter of 1-131 labelled T3 diluted to 1,000

counts per minute in buffer solution to 3 milliliters of

blood serum.

3. Incubate sample for 15 minutes in a water bath at 370 C.

4. Remove 1 milliliter of serum and assay for activity in a

scintillation counter equipped with a sodium iodide crystal.

This serves as the standard.

5. Place 1 milliliter of sample on top of gel bed taking care

not to disturb the gel surface.

6. After the serum enters the bed, add 2 separate 1 milliliter

aliquots of buffer solution.

7. When the buffer solution completely enters the gel bed, fill

the column with buffer solution and start collection of 1

milliliter elutes until 12 eluates are obtained.

8. Wash the column with 20 milliliters of blood serum diluted 10 13






14

times in buffer, and discard this serum.

9. After the diluted serum passes through the column, wash

with buffer until the column is again white.

10. Assay each of the 12 eluates for radioactivity as was

done for standards in step 4.

11. Determine percent uptake of protein-bound 1-131 labelled

T3 by the following calculation procedure:

a. deduct background from all counts.

b. sum up counts in those fractions containing proteinbound T3 (eluates 1-4).

c. sum up counts in those fractions containing free 1-131

(eluates 5-12).

d. subtract count obtained in c. above from net counts in

serum (standard obtained in step 4) to obtain net counts

1-131 labelled T3 in incubated serum.

e. percent uptake equals 100 X b or percent uptake equals

net counts protein-bound T3 fractions (b) divided by

net counts 1-131 labelled T3 in standard (d) multiplied

by 100.

This technique was used to determine thyroid status in all subsequent experiments.














RESULTS


Effect .of Different Columns on
Percent Uptake of Duplicate Samples


Sisson (1965) stated that an undesirable characteristic of a glass chromatographic column was the retention of radioactivity. Release of this radioactivity from the column could inject a source of error into the experiment. Cowley (1965) studied the repeatability of percent uptake of 1-131 labelled T3 of duplicate samples of sheep serum in different glass chromatographic columns packed with Sephadex G-25. He found no significant differences in uptake due to the column used with a correlation between uptake by the two columns of

0.99.

The objective of this study was to determine the repeatibility of 1-131 labelled T3 uptake by cattle serum proteins using duplicate samples in 2 different plastic chromatographic columns. Procedure

Ten cows were used as blood donors. Twenty-five mililiters of

blood was collected from the jugular vein of each and allowed to clot. Five mililiters of serum was removed and treated with 0.2 mililiter 1-131 labelled T3 diluted to 1,000 counts per minute. The sample was then placed on separate columns. The procedure described on page 13 for application, collection and calculation was followed. Results

The data obtained from this study, presented in Table I, were 15





TABLE I

PERCENTAGE UPTAKE OF 1-131 LABELLED T BY DUPLICATE
SAMPLES OF CATTLE SERUM THROUGH
DIFFERENT COLUMNS




Cow Column 1 Column 2 1 76.30 77.10 2 79.52 78.94 3 78.69 79.42 4 72.94 73.77 5 75.53 76.10 6 63.88 64.90 7 64.53 64.35 8 66.23 67.03 9 67.47 68.41

10 66.22 65.60 Means 71.13 71.56






17

analyzed by the paired t test using the hypothesis that ul = u2 and the correlation coefficient using the columns as variables (Li, 1961). The calculated t value of 2.14 was not significant (t.05 = 2.26). A highly significant correlation coefficient of 0.99 was found between the columns. These data indicate that there was no significant column effect on percent uptake of 1-131 labelled T3 by cattle blood serum, allowing the worker to use either column to rerun a particular sample without having to determine a correction factor, or reuse the same column.


Repeatability of Observations on Subsequent Days for Estimating Thyroid
Status in Beef Cattle


To be of significant value in any study of thyroid activity, the technique used for estimating thyroid status must be highly repeatable. Hocman (1966) stated that Sephadex chromatography is standardized to the extent that it yields similar results in similar experiments within the range of normal deviation. Shapiro and Rabinowitz (1964) reported that on 769 patients Sephadex chromatography determinations had a repeatability of 92 percent for estimating thyroid status when compared with results of other laboratory diagnoses.

The objective of this study was to determine if samples collected on different days from the same animal would give similar percent uptake as determined by Sephadex chromatography. Procedure

Five cattle were bled each day for five days. The blood was allowed to clot and then 3 mililiters of serum removed. The procedure given on page 13 was used to determine percent uptake of 1-131 labelled T3 by serum proteins.






18

Results

The data from this study, presented in Table II, were analyzed by the correlation coefficient using days as variables and by an analysis of repeatability (R). Highly significant correlation coefficients above 0.92 were found for each comparison. The R value was 0.921. These data indicate very good repeatability on a day to day basis. On a long term study, differences in percent uptake could be assumed to be due to thyroid hormone secretion rate and not to variation in the technique employed.


Effect of Age of Isotope on Percent Uptake
of 1-131 Labelled T3 by Cattle Serum Proteins


In routine laboratory procedure, 1-31 is usually discarded after one half-life. Half-life of an isotope is defined as the length of time required for one-half of the radioactive atoms in an isotope to undergo decay. Chase and Rabinowitz (1964) state that the halflife of 1-131 is 8.05 days. During the decay of 1-131 labelled T3 there is a disassociation of the 1-131 molecule from the triiodothyronine molecule resulting in free 1-131 in the reagent as well as a decrease in the total amount of radioactivity.

Shapiro and Rabinowitz (1962) reported that there is no problem with free 1-131 in the Sephadex chromatography technique because of a correction factor employed. This correction should provide for longer utilization of a particular sample of 1-131 labelled T3. Cowley (1965) reported that 1-131 labelled T3 that had been through

8 half-lives did not yield significantly different percent uptake than did the isotope in its first half-life.

The purpose of this study was to determine if 1-131 labelled T3







TABLE II

PERCENT UPTAKE OF 1-131 LABELLED T3 BY CATTLE SERUM FOR 5 DAYS AS MEASURED BY SEPHADEX CHROMATOGRAPHY



Cow Day 1 Day 2 Day 3 Day 4 Day 5 Mean Variance

1 66.39 65.92 66.89 66.51 65.22 66.19 0.349 2. 60.70 60.30 61.40 61.90 62.00 61.26 0.440 3 61.00 61.10 60.20 61.10 61.50 60.98 0.540 4 59.90 60.80 61.00 61.20 60.90 60.76 0.202 5 59.50 61.00 59.70 61.80 61.00 60.60 0.756 Means 61.50 61.82 61.84 62.50 62.12 61.96






20
could be used with cattle blood serum for more than one half-life without significantly affecting the results. This would allow for better utilization of the isotope and reduce the cost of estimating

thyroid status.

Procedure

Twenty milliliters of blood were collected. from the jugular vein of each animal and allowed to clot. The serum was decanted and a 4 milliliter aliquot placed in each of 2 flasks. Each sample was treated with 0.1 milliliter of 1-131 labelled T3 diluted to 1,000 counts per minute. One sample received 1-131 labelled T3 which had passed through 4 half-lives. The other sample received 1-131 labelled T3 in the first half-life. For determination of percent uptake by serum proteins, and amount of free 1-131 in the reagent, the technique described on page 13 was employed. Results

Data from this study (Table III) were analyzed using the

paired t test. The calculated t value was 0.676 with 7 degrees of freedom (t.05 = 2.365). The percent of free I-131 in the reagent increased with time as expected. The percent free 1-131 in the 1-131 labelled T3 increased from 1.89 percent on the date received to 4.68 at the end of one half-life. The maximum free 1-131 after 4 half-lives was 6.84 percent. The failure to find a significant difference in percent uptake between old and new isotope indicates that age of isotope has no effect. This appears to be an unusual characteristic of this technique and further work with aged 1-131 labelled T3 is warranted to explain it.






TABLE III

PERCENT UPTAKE OF OLD AND NEW 1-131 LABELLED T BY CATTLE SERUM PROTEINS



Cow 4 Half-lives First Half-life

1 64.54 . 66.52 2. 65.65 68.10 3 64.63 63.10 4 65.47 66.38 5 59.48 57.68

6 65.85 67.17 7 58.88 59.81 8 61.45 60.35 Mean 63.24 63.64






22

Effect of Temperature, Breed, and Sex
on Thyroid Secretion Rate of Beef Cattle


It has been well documented that the thyroid hormones play an important role in an animal's ability to withstand heat stress.

It has been proposed that thyroid activity may be a factor in the difference in ability of Bos taurus and Bos indicus cattle to withstand heat stress. Sex differences in heat tolerance have been noted by Myant (1964).

The purpose of this experiment was to compare both sexes of

Hereford and Brahman cattle under different environmental conditions. Procedure

Thirty six cattle approximately one year old were used in this study. Nine Brahman heifers, 9 Brahman bulls, 9 Hereford heifers, and 9 Hereford bulls were allotted to 3 treatments: pasture, hot chamber and comfort zone chamber. Twelve animals,

3 of each breed and sex, were placed on pasture exposed to normal environmental variation. Twelve of the remaining 24 animals, 3 of each breed and sex, were placed at random in an environmental chamber maintained at a constant temperature of 320 C and approximately 95 percent relative humidity. The third group of 12 animals was maintained in the comfort zone at 210 C and approximately 65 percent relative humidity.. The animals were maintained in the chambers for 12 months.

The cattle in the two chambers received the same diet, with

the amount of concentrate being allocated upon the basis of National Research Council requirements for body weight for growing breeding animals. Roughage was placed before the animals ad libitum. The 12 cattle on pasture received the same amount of concentrate but






23

had access to grass during the experiment. Treatment was confounded with phenotype of the test animals because the 12 pasture animals had been selected as breeding stock and were superior to the chamber animals. However, these pasture cattle were of value in indicating the response of good quality cattle under good nutritional conditions.

During the twelve-month experimental period, a 25 milliliter blood sample was collected from each animal every 28 days. The blood sample was allowed to clot and the serum withdrawn. The blood serum was analyzed for thyroid hormones employing the Sephadex chromatography procedure given on page 13. Results

Due to a malfunction of the scintillation counter used to assay for radioactivity, the first 2 months data were lost. Therefore, data is only available for 10 consecutive months. Treatment effects

The mean percent uptake for the 10 month period for treatment effects is presented in Table IV. The data for all animals on treatment shows a higher thyroid secretion rate, or lower percent uptake, for cattle on pasture (61.67 percent uptake) than for the cattle in either chamber. The cattle in the 320 C chamber had the highest percent uptake, 64.67 percent, with the cattle in the 210 C chamber being intermediate at 63.25 percent.

The analysis of variance of the 10 month means is presented in Table V. A highly significant difference between treatments indicates that there were different responses to the treatments.

A graph of the means for treatment effect is presented in

Figure 1. A definite diurnal rhythm was noted in all three groups.








TABLE IV

MEAN PERCENT UPTAKE OF 1-131 LABELLED T3 FOR TEN-MONTH PERIOD



All Hereford Brahman All All
Treatment Animals All Heifers Bulls All Heifers Bulls Bulls Heifers 320 C
Chamber 64.67 66.67 63.85 69.49 62.28 61.24 63.84 67.23 62.55 210 C
Chamber 63.25 63.47 61.25 65.70 63.03 60.79 65.26 65.48 61.02 Pasture 61.67 62.37 61.38 63.35 60.97 59.24 62.71 63.03 60.31






TABLE V

ANALYSIS OF VARIANCE OF MEANS OF PERCENT UPTAKE FOR TEN MONTH PERIOD




Source of Sum of Mean Variance D.F. Squares Squares

Total 35 261.71 Breed 1 34.44 34.44** Sex 1 126.35 126.35** Treatment 2 50.56 25.28** Breed X Sex 1 1.77 1.77 Breed X Treatment 2 23.08 11.54** Sex X Treatment 2 6.35 3.18** Breed X Sex X Treatment 2 6.87 3.44** Error 23 12.29 0.534


** Probability Less Than 0.01.






Lt-n
























- Pasture
- 210 C .. 320 C





Apts NaY Jn July Aug. Sept. Oclt. Nov. Dec.


=Ponthly pelrcnt upakeof 1=131 lbelled TS for treatment classification.






27

This would be expected for animals exposed to normal environmental variation but its appearance in the chamber groups indicates an overriding of the constant temperature effect by the animal's nervous system. The cause for this rhythm is not known but the availability of natural light through windows in the chambers may partially explain the rhythm in the chamber animals. The difference between the 320' C and 210 C chambers tended to disappear in the later months (September through December) while the pasture group maintained a lower percent uptake.

The analysis of variance for monthly collections is given in Tables VI through XV. The treatment effect remained highly significant throughout the experimental period even though the two chamber groups had approximately the same percent uptake during the last four months. This is explained by the pasture group being significantly different from the chamber groups during this period.

The higher percent uptake by the cattle in the 320 C chamber

indicates that heat stress affected thyroid function. The increased number of binding sites in the blood serum available for tying up 1-131 labelled T3 indicates a reduction in the thyroid hormone secretion rate and a reduction in the amount of circulating thyroid hormones.

Breed effects

The mean percent uptake for the 10 month period for breeds is presented in Table IV. The Hereford cattle had a higher percent uptake on all treatments than the Brahman cattle. The Herefords exhibited the same ranking by treatment as the All Animal classification.







TABLE VI

ANALYSIS OF VARIANCE OF PERCENT UPTAKE OF 1-131 LABELLED T3 FOR MARCH



Source of Sums of Mean Variance D.F. Squares Squares

Total 35 520.72 Breed .1 24.68 24.68** Sex 1 253.88 253.88** Treatment 2 116.74 58.37** Breed X Sex 1 12.23 12.23** Breed X Treatment 2 27.69 13.85** Sex X Treatment 2 36.06 18.03** Breed X Sex X Treatment 2 35.74 17.87** Error 23 13.70 0.595


** Probability Less Than .01.








TABLE VII

ANALYSIS OF VARIANCE OF PERCENT UPTAKE OF 1-131 LABELLED T3 FOR APRIL




Source of Sums of Mean Variance D.F. Squares Squares

Total 35 492.56 Breed 1 30.53 30.53** Sex 1 298.42 298.42** Treatment 2 94.67 47.34** Breed X Sex 1 1.41 1.41 Breed X Treatment 2 10.77 5.39* Sex X Treatment 2 30.20 15.10** Breed X Sex X Treatment 2 4.69 2.35 Error 23 21.87 0.951


* Probability Less Than 0.05.

** Probability Less Than 0.01.







TABLE VIII

ANALYSIS OF VARIANCE OF PERCENT UPTAKE OF 1-131 LABELLED T3 FOR MAY




Source of Sums of Mean Variance D.F. Squares Squares

Total 35 492.54 Breed 1 19.95 19.95** Sex 1 295.84 295.84** Treatment 2 59.15 29.56** Breed X Sex 1 0.01 0.01 Breed X Treatment 2 55.02 27.51** Sex X Treatment 2 11.90 5.95** Breed X Sex X Treatment 2 28.42 14.21** Error 23 22.25 0.967


** Probability Less Than 0.01









TABLE IX

ANALYSIS OF VARIANCE OF PERCENT UPTAKE OF 1-131 LABELLED T3 FOR JUNE



Source of Sums of Mean Variance D.F. Squares Squares

Total 35 527.22 Breed 1 14.83 14.83** Sex 1 308.01 308.01** Treatment 2 52.90 26.45** Breed X Sex 1 0.36 0.36 Breed X Treatment 2 58.08 29.04** Sex X Treatment 2 2.35 1.18 Breed X Sex X Treatment 2 47.30 23.65** Error 23 43.39 1.887


** Probability Less Than 0.01.








TABLE X

ANALYSIS OF VARIANCE OF PERCENT UPTAKE OF 1-131 LABELLED T3 FOR JULY



Source of Sums of Mean Variance D.F. Squares Squares

Total 35 496.68 Breed 1 87.73 87.73** Sex 1 119.53 119.53** Treatment 2 66.91 33.45** Breed X Sex 1 0.33 0.33 Breed X Treatment 2 13.91 6.95 Sex X Treatment 2 80.14 40.07** Breed X Sex X Treatment 2 42.26 21.13** Error 23 85.87 3.733


** Probability Less Than 0.01.







TABLE XI

ANALYSIS OF VARIANCE OF PERCENT UPTAKE OF 1-131 LABELLED T3 FOR AUGUST



Source of Sums of Mean Variance D.F. Squares Squares

Total 35 406.41 Breed 1 140.43 140.43** Sex 1 116.29 116.29** Treatment 2 54.96 27.48** Breed X Sex 1 1.64 1.64 Breed X Treatment 2 19.70 9.85* Sex X Treatment 2 14.78 7.39 Breed X Sex X Treatment 2 0.41 0.205 Error 23 58.61 2.548


* Probability Less Than 0.05.

** Probability Less Than 0.01.







TABLE XII

ANALYSIS OF VARIANCE OF PERCENT UPTAKE OF 1-131 LABELLED T3 FOR SEPTEMBER



Source of Sums of Mean Variance D.F. Squares Squares

Total 35 311.59 Breed 1 40.11 40.11** Sex 1 169.87 169.87** Treatment 2 26.61 13.30** Breed X Sex 1 2.56 2.56 Breed X Treatment 2 3.89 1.95 Sex X Treatment 2 30.09 15.04** Breed X Sex X Treatment 2 1.83 0.915 Error 23 36.63 1.593


** Probability Less Than 0.01.









TABLE XIII

ANALYSIS OF VARIANCE OF PERCENT UPTAKE OF 1-131 LABELLED T3 FOR OCTOBER




Source of Sums of Mean Variance D.F. Squares Squares

Total 35 322.43 Breed 1 24.51 24.51** Sex 1 100.33 100.33** Treatment 2 59.08 29.54** Breed X Sex 1 4.07 4.07 Breed X Treatment 2 14.31 7.15 Sex X Treatment 2 59.65 29.82** Breed X Sex X Treatment 2 6.88 3.44 Error 23 53.60 2.33


** Probability Less Than 0.01.



t-Il








TABLE XIV

ANALYSIS OF VARIANCE OF PERCENT UPTAKE OF 1-131 LABELLED T3 FOR NOVEMBER



Source of Sums of Mean Variance D.F. Squares Squares

Total 35 313.65 Breed 1 59.55 59.55** Sex 1 19.81 19.81** Treatment 2 20.61 10.30** Breed X Sex 1 18.34 18.34** Breed X Treatment 2 68.47 34.24** Sex X Treatment 2 19.48 9.74* Breed X Sex X Treatment 2 48.50 24.25** Error 23 58.59 2.547


* Probability Less Than 0.05.

** Probability Less Than 0.01.







TABLE XV

ANALYSIS OF VARIANCE OF PERCENT UPTAKE OF 1-131 LABELLED T3 FOR DECEMBER




Source of Sums of Mean Variance D.F. Squares Squares

Total 35 330.82 Breed 1 0.59 0.59 Sex 1 18.20 18.20 Treatment 2 116.79 58.39** Breed X Sex 1 1.26 1.26 Breed X Treatment 2 58.31 29.15** Sex X Treatment 2 32.26 .16.13 Breed X Sex X Treatment 2 5.04 2.52 Error 23 116.57 5.068


** Probability Less Than 0.01.






38

The higher percent uptake by Herefords in the 320 C chamber indicates that high ambient temperatures decreased the amount of circulating thyroid hormones.

The Brahman cattle did not follow the same ranking as the All Animals classification. In this breed there was a reversal between the 320 C chamber at 62.28 percent, and the 210 C chamber at 63.03 percent. The pasture group still had the lowest percent uptake at 60.97 percent. These data indicate that the Brahman cattle did not respond to heat stress by lowering the amount of circulating thyroid hormones but were able to maintain their secretion rate at the same level as in the 210 C chamber. This reversal in ranking is evident in the highly significant interaction between breed and treatment shown in Table V. Tables VI through XV show that this interaction did not remain significant for all months. Therefore, for three months in the experimental period the animals did not respond the same. There may be a seasonal effect involved as the three nonsignificant months were July, September and October with the August collection showing a significant breed by treatment interaction but not a highly significant difference

as in other months.

A graph of the means for each breed by months is presented in Figure 2. This graph shows a clear distinction between the breeds in every month but December. The same diurnal rhythm as noted for treatment effects is evident for breeds as expected since the data for treatment effects and breed effects are from the same animals.

Statistical analysis of the means in Table V shows a highly












50



5



60







70
- Brahman

- Hereford
5

Mar. Apr. May June July Aug. Sept. Oct. Nov. Dec. Figure 2. -- Monthly percent uptake of 1-131 labelled T3 for breed classification.





40

significant difference between breeds. There was no significant difference between breeds in the December collection (Table XV). This can also be seen in Figure 2.by the two lines nearly meeting in December. These data tend to reinforce the assumption that thyroid secretion rate is one of the factors accounting for the difference in adaptability of Hereford and Brahman cattle to heat stress., Since the Hereford cattle had a higher percent uptake on all treatments than did the Brahman cattle, it is assumed that the Brahman cattle had a higher amount of circulating thyroid hormone than the Hereford cattle in this experiment. Since the percent uptake in the 320 C chamber was 66.67 for Herefords and 62.28 for Brahmans, it may be assumed that the Brahman cattle in this experiment were better equipped to maintain thyroid secretion rate under heat stress than were the Hereford cattle. Sex effects

The mean percent uptake for the 10 month period for sex

effect is presented in Table IV under All Bulls and All Heifers. The bulls had a higher percent uptake on all treatments than did the heifers. The All Bulls and All Heifers classifications exhibited the same ranking by treatments as did the All Animals classification.

A highly significant difference due to sex is shown in Table V.

There was a larger mean square for sex effect than for any other source of variation. Analysis of monthly collections, Tables VI through XV, showed this highly significant sex effect in all but the last month (Table XV). The graph by months, Figure 3, shows this difference between sexes and a reversal in the last month that resulted in no significant difference due to sex in December.









50



5



60



5



70
All Heifers

- All Bulls
5

Mar. Apr. May June July Aug. Sept. Oct. Nov. Dec. Figure 3. -- Monthly percent uptake of 1-131 labelled T3 for sex classification.






42

There was no significant interaction between breed and sex, Table V, indicating that the sexes respond the same to the treatments in both breeds. Analysis by monthly collection showed two highly significant breed by sex interactions. These occurred in March (Table VI) and November (Table XIV). The sex by treatment interaction in Table V was highly significant. Analysis by months did not show this significant interaction in June (Table IX), August (Table XI), and December (Table XV).. A part of these interactions can be seen in the data on Table IV. There was a reversal of ranking in response to treatment by the Brahman bulls and heifers. The percent uptake was 63.84 in the 320 C chamber and 65.26 in the 210 C chamber for bulls and 61,24 and 60.79 respectively for heifers. The Hereford heifers exhibited no difference between the 210 C chamber at 61.25 percent and the pasture group at 61.38 percent. The animals with the highest uptake of all classes were the Hereford bulls in the 320 C chamber with 69.49 percent. The animals with the lowest uptake were the Brahman heifers on pasture with 59.24 percent.

The fact that percent uptake for heifers was consistently lower than the percent uptake for bulls indicates that heifers have a higher thyroid secretion rate than bulls and are better able to maintain their thyroid secretion levels under heat stress.















GENERAL SUMMARY AND CONCLUSIONS


An in vitro technique employing Sephadex column chromatography was used to estimate circulating thyroid hormone levels in beef cattle. Preliminary work with this technique was conducted to assertain the applicability of Sephadex chromatography to the evaluation of thyroid status in this species. The factors studied were: (1) the effect of different columns on percent uptake of duplicate samples; (2) repeatability of observations on subsequent days for estimating thyroid status; and (3) effect of age of isotope on percent uptake of 1-131 labelled T3 by cattle serum proteins.

It was found that there were no statistical differences

between duplicate samples placed on different columns. A highly significant correlation was found between the two columns. It was concluded that there were no differences between columns and replicate determinations of a particular sample did not necessitate the utilization of the same column. Since any residual radioactivity released by a column caused no significant differences in the results, there was no need for a correction factor for retention of radioactivity by the columns.

Daily blood samples obtained from an individual animal over

a 5-day period were highly correlated with a repeatability coefficient of 0.921. Therefore, it was concluded that this technique could be used to study an animal's thyroid secretion rate over an extended 43






44

period as any significant differences noted in the percent uptake by a particular animal would be due to a change in the amount of circulating thyroid hormone and not to the technique employed.

Length of storage of 1-131 labelled T3 caused no significant difference in percent uptake by serum proteins when old (4 halflives) and new (first half-life) reagents were compared. The percent of free 1-131 in the reagent increased and the total activity decreased with age. It was concluded that adjustment of all dosages to 1,000 counts per minute and correcting for free 1-131 in the reagent allowed for the complete utilization of each shipment of reagent disregarding half-lives.

The second phase of this study was directed towards comparing the effect of temperature, breed, and sex on circulating thyroid .hormone levels in Hereford and Brahman beef cattle. Three animals of each breed and sex were maintained at 320 C, 210 C and on pasture for 12 months.

Statistical analysis revealed that the three main effects studied; treatment, breed, and sex, were all highly significant. A difficulty in interpretation of these significant effects arose from the fact that the breed X treatment, sex X treatment and breed X sex X treatment interactions were also significant.

The data from this study showed that the pasture cattle had a

lower percent uptake or higher thyroid secretion rate than the cattle in either chamber.- The cattle in the 320 C chamber had the highest percent uptake with the cattle in the 210 C chamber being intermediate between the other two groups. All three groups exhibited a diurnal rhythm in a graph of percent uptake by months. These data indicated






45

that temperature variations are important in thyroid secretion rates as even the animals maintained in the "comfort zone" at a constant 210 C had a lower thyroid output than the animals on pasture. However, no conclusion could be reached on this point due to the difference in quality between the pasture and chamber animals. It was concluded from these data that the animals were stressed at 320 C and responded with a decreased thyroid hormone secretion rate.

On all three treatments the Hereford cattle had a higher percent uptake than the Brahman cattle. The Hereford cattle responded to the 320 C treatment by lowering thyroid hormone secretion rate while the Brahman cattle were able to maintain approximately the same thyroid secretion levels in the 320 C and 210 C chambers. From these data, it was concluded that Brahman cattle are better equipped to withstand heat stress than Hereford cattle by maintaining circulating thyroid hormone levels.

The bulls had a higher percent uptake on all treatments than did the heifers. The Hereford bulls and heifers responded to the 320 stress by lowering thyroid hormone secretion rates while the Brahman bulls and heifers did not. Due to the lower percent uptake by heifers on all treatments, it was concluded that the heifers had a higher thyroid secretion rate than the bulls and were better equipped to maintain this higher secretion rate in response to heat stress.

The animals with the lowest percent uptake were the Brahman

heifers on pasture with a mean uptake of 59.24 percent. The animals with the highest percent uptake were the Hereford bulls in the 320 C chamber with a mean uptake of 69.49 percent. The range in percent uptake for all animals on treatment was from 55.2 to 74.3 percent.















BIBLIOGRAPHY


Albert, A. and F. R, Keating. 1952. The role of the gastro-intestinal
tract, including the liver, in the metabolism of radiothyroxine.
Endocr. 51:427.

Albright, E. C. and F. C. Larson. 1959. Metabolism of L-thyroxine
by human tissue slices. J. Clin. Invest. 38:1899.

Blaxter, K. L. 1945. The effect of iodinated protein feeding on the
lactating cow. J. Endocr. 4:237.

Blincoe, C. 1958. The influence of constant ambient temperature on
the thyroid activity and iodide metabolism of Shorthorn, Santa Gertrudis and Brahman calves during growth. Mo. Agr. Exp. Sta.
Res. Bul. 649.

Blincoe, C., and S. Brody. 1955. The influence of ambient temperature,
air velocity, radiation intensity and starvation on thyroid
activity and iodide metabolism in cattle. Mo. Agr. Exp. Sta.
Res. Bul. 576.

Blincoe, C. and S. Brody. 1955. The influence of diurnally variable
temperatures on the thyroid activity and iodide metabolism of
Jersey and Holstein cows. Mo. Agr. Exp. Sta. Res. Bul. 579.

Brody, S. and R. F. Frankenbuch. 1942. Age changes in size, energy
metabolism and cardiorespiratory activities of thyroidectomized
cattle. Mo. Agr. Exp. Sta. Res. Bul. 349.

Brunstad, G. E. and S. H. Fowler. 1959. Thyroid status and embryonic
mortality in swine. Am. J. Physiol. 196:287.

Chase, G. D. and J. L. Rabinowitz. 1964. Principles of Radioisotope Methodology. 2nd Edition. Burgess Publishing Co., Minneapolis, Minn.

Cowley, J. J. 1965. A Comparison of Two in vitro Radioisotope
Procedures for Estimating Thyroid Status in Sheep. M. S. Thesis,
Louisiana State University, Baton Rouge, La.

Cuaron, A. and M. E. Fucugauchi. 1964. The binding of I-131-triiodothyronine by serum proteins as an in vitro test of thyroid
function. Acta. Encocrinologica 46:161.



46






47

BIBLIOGRAPHY (continued)

Deiss, W. P., E. C. Albright and F. C. Larson. 1953. Comparison of
in vitro serum protein binding of thyroxine and triiodothyronine.
Proc. Soc. Exp. Biol. Med. 84:513.

Folley, S. J. and P. White. 1936. The effect of thyroxine on milk
secretion and on the phosphotase of the blood and milk of the
lactating cow. Proc. Roy Soc. B. 120:346.

Frienkel, N., J. J. Dowling and S. H. Ingbar. 1955. The interaction
of thyroxine with plasma protein; localization of thyroxinebinding proteins in Cohn Fraction of plasma. J. Clin. Invest. 34:
1698.

Graham, W. R. 1934. The action of thyroxine on the milk and milk-fat
production of cows. Biochem. J. 28:1368.

Halmi, N. S. 1964. The accumulation and recirculation of iodide by
the thyroid. The Thyroid Gland. I. R. Pitt-Rivers and
W. R. Trotter, (eds.). Butterworths, Inc., Washington, D.C.

Hamolsky, M. W., M. Stein and A. S. Freedberg. 1957. The thyroid
hormone plasma protein complex in man. II. A new in vitro
method for study of "uptake" of labeled hormonal components of
human erythrocytes. J. Clin. Endocr. 17:33.

Hart, D. S. 1960. Fertility responses in ewes treated with thyroxine.
New Zealand J. Agr. Sci. 3:365.

Heidelberger, M. and K. O. Pedersen. 1935. The molecular weight and
isoelectric point of thyroglobulin. J. Gen. Physiol. 19:95.

Henneman, H. A., E. P. Reineke and S. A. Griffin. 1955. Thyroid
secretion rate of sheep as affected by season, age, breed,
pregnancy, and lactation. J. Animal Sci. 14:419.

Hocman, G. 1966. The use of Sephadex in the chromatography of thyroxine
containing compounds: a critique. J. Chromatog. 21:413.

Howes, J. J. 1964. The Comparative Physiology of Bos Taurus and Bos
Indicus cattle on Two Nutritional Levels. Ph.D. dissertation,
University of Florida, Gainesville, Florida.

Jacobsson, L. and G. Widstrom. 1962. Separation of Iodine compounds
in serum by gel filtration. Scand. J. Clin. Lab. Invest. 14:285.

Jones, J. E. and J. J. Schultz. 1966. Identification of thyroidal
iodoamino acids by gel filtration and automated 1-127 determination. J. Clin. Endocr. 26:975.

Lewitus, Z., M. Anbar and S. Guttman. 1961. A contribution to the
understanding of the "trapping mechanism" in the thyroid gland.
Advances in Thyroid Research. R. Pitt-Rivers (ed.). Pergamon
'Press, New York.






48

BIBLIOGRAPHY (continued)

Li, J. C. R. 1961. Introduction to Statistical Inference. Edwards
Brothers Inc. Ann Arbor, Mich.

Lucas, J. J., G. E. Brunstad and S. H. Fowler. 1958. The relationship
of altered thyroid activity to various reproductive phenomena
in gilts. J. Endocr. 17:54.

Myant, N. B. 1964. The thyroid and reproduction in mammals. The
Thyroid Gland. I. R. Pitt-Rivers and W. R. Trotter (eds.).
Butterworths Inc., Washington, D. C.

Nadler, N. J. 1962. Synthesis and release of thyroid hormones. Fed.
Proc. .21:628.

Pitt-Rivers, R. and J. R. Tata. 1959. The Thyroid Hormones. Pergamon
Press, New York.

Pitt-Rivers, R. and R. R. Cavalieri. 1964. Thyroid hormone biosynthesis.
The Thyroid Gland. I. R. Pitt-Rivers and W. R. Trotter (eds.).
Butterworths Inc.,-Washington, D. C.

Reineke, E. P. 1946. Thyroactive iodinated proteins. Vit. and Horm.
4:207.

Reineke, E. P. and F. A. Soliman. 1953. Role of the thyroid hormone
in reproductive physiology of the female. Iowa State Coll. J.
Sci. 28:67.

Robbins, J. and J. E. Rall. 1955. Thyroxine-binding capacity of
serum in normal man. J. Clin. Invest. 34:1324.

Robertson, J. D. 1945. The effect on basal metabolism of milk from
cows fed with iodinated protein. J. Endocr. 4:300.

Ryle, M. 1961. Early reproductive failure of ewes in a hot environment. I. J. Agr. Sci. 51:84.

Ryle, M. 1963. Early reproductive failure of ewes in a hot environment. III, IV, V. J. Agr. Sci. 60:95.

Shapiro, B. and J. L. Rabinowitz. 1962. A chromatographic method
utilizing Sephadex for the separation of free iodide, proteinbound and unbound triiodothyronine in sera. J. Nuclear Med.
3:417.

Sisson, J. C. 1965. Principles of, and pitfalls in, thyroid function
tests. J. Nuclear Med. 6:853.

Spielmann, A. A., W. E. Petersen, and J. B. Fitch. 1944. Effect of
thyroidectomy on lactation in the bovine. J. Dairy Sci. 27:441.






49

BIBLIOGRAPHY (continued)

Spielmann, A. A., W. E. Petersen, J. B. Fitch and B. S. Pomeroy.
1945. General appearance, growth and reproduction of the
thyroidectomized bovine. J. Dairy Sci. 28:329.

Swanson, E. W.,F. W. Lengemann and R. A. Monroe. 1957. Factors
affecting the thyroid uptake of 1-131 in dairy cows. J.Animal
Sci. 16:318.

Tata, J. R. 1964. Distribution and metabolism of thyroid hormones.
The Thyroid Gland. I. R. Pitt-Rivers and W. R. Trotter.(eds.).
Butterworths Inc., Washington, D. C.

Taurog, A., J. O. Wheat, and L. L. Chaikoff. 1956. Nature of the
iodine-131 compounds appearing in the thyroid vein after injection of iodine-131. Endocr. 58:121.

Yeates, N.T.M. 1958. Foetal dwarfism in sheep - an effect of high
atmospheric temperature during gestation. J. Agr. Sci. 51:84.















BIOGRAPHICAL SKETCH


Jerry Jennings Cowley, son of Pauline Cowley and the late Jack Cowley, was born July 14, 1940, in Marshall, Texas. He attended Marshall public schools and was graduated from Marshall Senior High School in 1958.

He received the degree of Bachelor of Science in Animal

Science from Texas A & M University in June, 1963. He then entered graduate school at Louisiana State University and received the degree of Master of Science in Animal Science in August, 1965. In September, 1965, he entered the University of Florida and is now a candidate for the degree of Doctor of Philosophy.

Mr. Cowley is married to the former Marilyn Jones. He is a member of Gamma Sigma Delta, national honorary agricultural society.









This dissertation was prepared under the direction of the chairman of the candidate's supervisory commitee and has been approved by all members of that committee. It was submitted to the Dean of the College .of Agriculture and to the Graduate Council, and was approved as partial fulfillment of the requirements for the degree Doctor of Philosophy.


December 1968





bean, Co~lege of Agriculture





Dean, Graduate School



Supervisory Committee:




Chairman




Full Text
TABLE VI
ANALYSIS OF VARIANCE OF PERCENT UPTAKE OF
1-131 LABELLED T3 FOR MARCH
Source of
Variance
D.F.
Sums of
Squares
Mean
Squares
Total
35
520.72
Breed
1
24.68
24.68**
Sex
1
253.88
253.88**
Treatment
2
116.74
58.37**
Breed X Sex
1
12.23
12.23**
Breed X Treatment
2
27.69
13.85**
Sex X Treatment
2
36.06
18.03**
Breed X Sex X Treatment
2
35.74
17.87**
Error
23
13.70
0.595
** Probability Less Than .01


This dissertation was prepared under the direction of the chair
man of the candidate's supervisory committee and has been approved by
all members of that committee. It was submitted to the Dean of the
College of Agriculture and to the Graduate Council, and was approved
as partial fulfillment of the requirements for the degree Doctor of
Philosophy.
December 1968
w>
Dean, Graduate School
Supervisory Committee:
Chairman


4
the "trapping mechanism" on the basis of valency and ionic size
stating that the "trapping mechanism" in the thyroid gland involves
a certain specially adapted protein in the cell membrane which will
incorporate all monovalent ions which fulfill certain requirements
of size.
Iodide that is transported into the gland from the circulation
undergoes enzymatic oxidation. The subsequent iodination of tyro
sine in the thyroglobulin to form the thyroid hormones occurs in the
colloids of the follicular cells. Pitt-Rivers and Cavalieri (1964)
gave the following sequence of events that lead to the formation of
the thyroid hormones:
1. Iodide is transported into the gland by the blood.
2. Iodide is converted by some oxidative enzyme to mono-
iodotyrosine (T^) and diiodotyrosine (T2).
3. Thyroxine (T4) is formed by the coupling of 2 molecules
of T2 and the loss of 1 alanine side chain.
4. Triiodothyronine (T^) is formed by the coupling of 1
molecule each of T and T and the loss of 1 alanine
1 2
side chain.
Reactions 2 through 4 occur within the thyroglobulin molecule
located in the follicles.
The thyroid hormones either enter the blood or are secreted
into the lumen of the follicle and stored in the colloid bound to
thyroglobulin. Because of its large molecular size, thyroglobulin
is degraded to its constituent amino acids before the thyroid hormones
are secreted into the blood stream. Pitt-Rivers and Tata (1959) state
that this step is under the influence of thyroid stimulating hormone


TABLE XII
ANALYSIS OF VARIANCE OF PERCENT UPTAKE OF
1-131 LABELLED T3 FOR SEPTEMBER
Source of
Variance
D.F.
Sums of
Squares
Mean
Squares
Total
35
311.59
Breed
1
40.11
40.11**
Sex
1
169.87
169.87**
Treatment
2
26.61
13.30**
Breed X Sex
1
2.56
2.56
Breed X Treatment
2
3.89
1.95
Sex X Treatment
2
30.09
15.04**
Breed X Sex X Treatment
2
1.83
0.915
Error
23
36.63
1.593
** Probability Less Than 0.01


LIST OF TABLES
Page
TABLE
I.PERCENTAGE UPTAKE OF 1-131 LABELLED T BY
DUPLICATE SAMPLES OF CATTLE SERUM ?HROUGH
DIFFERENT COLUMNS 16
II.PERCENT UPTAKE OF 1-131 LABELLED T-j BY CATTLE
SERUM FOR 5 DAYS AS MEASURED BY SEPHADEX
CHROMATOGRAPHY 19
III.PERCENT UPTAKE OF OLD AND NEW 1-131 LABELLED
t3 BY CATTLE SERUM PROTEINS 21
IV.MEAN PERCENT UPTAKE OF 1-131 LABELLED Tn FOR
TEN MONTH PERIOD 24
V.ANALYSIS OF VARIANCE OF MEANS OF PERCENT UPTAKE
FOR TEN MONTH PERIOD 25
VI.ANALYSIS OF VARIANCE OF PERCENT UPTAKE OF 1-131
LABELLED T3 FOR MARCH 28
VII.ANALYSIS OF VARIANCE OF PERCENT UPTAKE OF 1-131
LABELLED T3 FOR APRIL 29
VIII.ANALYSIS OF VARIANCE OF PERCENT UPTAKE OF 1-131
LABELLED T FOR MAY ; 30
3
IX.ANALYSIS OF VARIANCE OF PERCENT UPTAKE OF 1-131
LABELLED T3 FOR JUNE 31
X.ANALYSIS OF VARIANCE OF PERCENT UPTAKE OF 1-131
LABELLED T3 FOR JULY 32
XI.ANALYSIS OF VARIANCE OF PERCENT UPTAKE OF 1-131
LABELLED T3 FOR AUGUST 33
XII.ANALYSIS OF VARIANCE OF PERCENT UPTAKE OF 1-131
LABELLED T3 FOR SEPTEMBER 34
XIII.ANALYSIS OF VARIANCE OF PERCENT UPTAKE OF 1-131
LABELLED T3 FOR OCTOBER 35
iv


TABLE VIII
ANALYSIS OF VARIANCE OF PERCENT UPTAKE OF
1-131 LABELLED T3 FOR MAY
Source of
Variance
D.F.
Sums of
Squares
Mean
Squares
Total
35
492.54
Breed
1
19.95
19.95**
Sex
1
295.84
295.84**
Treatment
2
59.15
29.56**
Breed X Sex
1
0.01
0.01
Breed X Treatment
2
55.02
27.51**
Sex X Treatment
2
11.90
5.95**
Breed X Sex X Treatment
2
28.42
14.21**
Error
23
22.25
0.967
** Probability Less Than 0.01


REVIEW OF LITERATURE
General Thyroid Physiology
A fundamental knowledge of the formation and release of the
thyroid hormones is essential in any study employing thyroid physi
ology. A single layer of epithelial cells, the follicular epithelium,
encompassing a central mass of protein, the colloid, is the funda
mental parenchymal unit of the gland. The small sacs or follicles
of which the adult thyroid is composed may be considered from both
the structural and functional point of view to be the secretory
unit of the thyroid gland. Within the follicle and filling its
lumen is the homogenous, thin, clear substance, the colloid. The
synthesis of the thyroid hormones has been reported by Nadler
(1962) to take place within the colloids of the follicles. Heidel-
berger and Pedersen (1935) found that the colloid consists largely of
thyroglobulin, a large protein having a molecular weight of approxi
mately 700,000.
Iodide is the only chemical form in which the thyroid gland can
assimilate iodine for formation of the thyroid hormones. Halmi (1964)
stated that active transport is by far the most important mechanism
whereby the gland can accumulate iodine. The thyroid gland, in addi
tion to concentrating iodide, concentrates a series of other anions of
no physiological use to the gland. Lewitus e_t _al. (1961) explained
3
f


TABLE XI
ANALYSIS OF VARIANCE OF PERCENT UPTAKE OF
1-131 LABELLED T-j FOR AUGUST
Source of
Variance
D.F.
Sums of
Squares
Mean
Squares
Total
35
406.41
Breed
1
140.43
140.43**
Sex
1
116.29
116.29**
Treatment
2
54.96
27.48**
Breed X Sex
J1
1.64
1.64
Breed X Treatment
2
19.70
9.85*
Sex X Treatment
2
14.78
7.39
Breed X Sex X Treatment
2
0.41
0.205
Error
23
58.61
2.548
* Probability Less Than 0.05.
** Probability Less Than 0.01.


38
o
The higher percent uptake by Herefords in the 32 C chamber indicates
that high ambient temperatures decreased the amount of circulating
thyroid hormones.
The Brahman cattle did not follow the same ranking as the
All Animals classification. In this breed there was a reversal
between the 32 C chamber at 62.28 percent, and the 21 C chamber
at 63.03 percent. The pasture group still had the lowest percent
uptake at 60.97 percent. These data indicate that the Brahman cat
tle did not respond to heat stress by lowering the amount of
circulating thyroid hormones but were able to maintain their
secretion rate at the same level as in the 21 C chamber. This
reversal in ranking is evident in the highly significant interaction
between breed and treatment shown in Table V. Tables VI through
XV show that this interaction did not remain significant for all
months. Therefore, for three months in the experimental period
the animals did not respond the same. There may be a seasonal effect
involved as the three nonsignificant months were July, September
and October with the August collection showing a significant breed
by treatment interaction but not a highly significant difference
as in other months.
A graph of the means for each breed by months is presented
in Figure 2. This graph shows a clear distinction between the
breeds in every month but December. The same diurnal rhythm as
noted for treatment effects is evident for breeds as expected since
the data for treatment effects and breed effects are from the same
animals.
Statistical analysis of the means in Table V shows a highly
.


48
BIBLIOGRAPHY (continued)
Li, J. C. R. 1961. Introduction to Statistical Inference. Edwards
Brothers Inc. Ann Arbor, Mich.
Lucas, J. J., G. E. Brunstad and S. H. Fowler. 1958. The relationship
of altered thyroid activity to various reproductive phenomena
in gilts. J. Endocr. 17:54.
Myant, N. B. 1964. The thyroid and reproduction in mammals. The
Thyroid Gland. I. R. Pitt-Rivers and W. R. Trotter (eds.).
Butterworths Inc., Washington, D. C.
Nadler, N. J. 1962. Synthesis and release of thyroid hormones. Fed.
Proc..21:628.
Pitt-Rivers, R. and J. R. Tata. 1959. The Thyroid Hormones. Pergamon
Press, New York.
Pitt-Rivers, R. and R. R. Cavalieri. 1964. Thyroid hormone biosynthesis.
The Thyroid Gland. I. R. Pitt-Rivers and W. R. Trotter (eds.).
Butterworths Inc., Washington, D. C.
Reineke, E. P. 1946. Thyroactive iodinated proteins. Vit. and Horm.
4:207.
Reineke, E. P. and F. A. Solimn. 1953. Role of the thyroid hormone
in reproductive physiology of the female. Iowa State Coll. J.
Sci. 28:67.
Robbins, J. and J. E. Rail. 1955. Thyroxine-binding capacity of
serum in normal man. J. Clin. Invest. 34:1324.
Robertson, J. D. 1945. The effect on basal metabolism of milk from
cows fed with iodinated protein. J. Endocr. 4:300.
Ryle, M. 1961. Early reproductive failure of ewes in a hot environ
ment. I. J. Agr. Sci. 51:84.
Ryle, M. 1963. Early reproductive failure of ewes in a hot environ
ment. Ill, IV, V. J. Agr. Sci. 60:95.
Shapiro, B. and J. L. Rabinowitz. 1962. A chromatographic method
utilizing Sephadex for the separation of free iodide, protein-
bound and unbound triiodothyronine in sera. J. Nuclear Med.
3:417.
Sisson, J. C. 1965. Principles of, and pitfalls in, thyroid function
tests. J. Nuclear Med. 6:853.
Spielmann, A. A., W. E. Petersen, and J. B. Fitch. 1944. Effect of
thyroidectomy on lactation in the bovine. J. Dairy Sci. 27:441.


INTRODUCTION
In reviewing the role of the thyroid hormones in the repro
ductive physiology of the female, Reineke and Solimn (1953)
pointed out that although over half a century of investigation
indicates that the thyroid hormones are implicated in some manner,
their precise role has not been determined. There is also ample
evidence that the thyroid gland plays an important role in growth
and development. Many factors are associated with thyroid hormone
secretion rate including breed, age, sex, season of year, pregnancy,
lactation, temperature and humidity, plane of nutrition, and a variety
of diseases.
When nuclear reactor-produced 1-131 became available in 1946,
the study of the thyroid gland received new impetus. Most of the
subsequent research was in clinical diagnosis and therapy.
The introduction of an _in vitro technique in 1962 has made it
possible to study and compare circulating thyroid hormone levels
without exposing the animals to radioactive material or endogenous
thyroid hormones.
Earlier work has suggested that one of the factors influencing
adaptability is thyroid activity. Thyroid activity has been suggested
as one of the factors involved in the difference in adaptability
between European (Bos taurus) and Asiatic (Bos indicus) breeds of
beef cattle.
This project was designed to study the feasibility of an in vitro
1


parturition in the thiouracil-treated group and reduced embryonic
mortality in the thyroprotein-treated group.
Spielmann et al. (1945) noted that the main effect of thyroid
ectomy on mature cows was a'failure to manifest the physical
signs of estrus. Normal ova were produced as evidenced by the birth
of 3 calves from 3 cows thyroidectomized 161, 242, and 453 days
before breeding.
Henneman _et al. (1955) reported lactation to create increased
demand on the thyroid gland, and lactating ewes showed a signifi
cantly higher thyroid secretion rate than either nonpregnant or
pregnant ewes.
Spielmann e_t a_l. (1944) found that cows thyroidectomized prior
to gestation, during pregnancy, or during lactation would cease
lactation in about 180 days. It was also observed that incomplete
removal of the thyroid gland produced a temporary decline in
milk production, followed by a gradual return to the former
levels.
Hart (1960) reported a 9.5 percent increase in lambs born
from ewes treated with L-thyroxine pellets 10 to 11 days before
mating. Ryle (1961) found that ewes exposed to an experimentally
hot environment and ewes under normal conditions which received
thyroxine had significantly more embryos than those ewes that
received no thyroxine. In 1963, Ryle proposed that dwarfing of
fetuses exposed to high ambient temperatures, as reported by
Yeates (1958) may be prevented by thyroxine therapy.
It has been repeatedly shown that milk yield in cattle can be
increased by thyroid therapy. Graham (1934) and Folley and White


times in buffer and discard this serum.
9.After the diluted serum passes through the column, wash
with buffer until the column is again white.
10. Assay each of the 12 eluates for radioactivity as was
done for standards in step 4.
11. Determine percent uptake of protein-bound 1-131 labelled

by the following calculation procedure:
a. deduct background from all counts.
b. sum up counts in those fractions containing protein-
bound (eluates 1-4).
c. sum up counts in those fractions containing free 1-131
(eluates 5-12).
d. subtract count obtained in c. above from net counts in
serum (standard obtained in step 4) to obtain net counts
1-131 labelled in incubated serum.
e. percent uptake equals 100 X or percent uptake equals
net counts protein-bound fractions (b) divided by
net counts 1-131 labelled in standard (d) multiplied
by 100.
This technique was used to determine thyroid status in all
subsequent experiments.


20
could be used with cattle blood serum for more than one half-life
without significantly affecting the results. This would allow for
better utilization of the isotope and reduce the cost of estimating
thyroid status.
Procedure
Twenty milliliters of blood were collected from the jugular
vein of each animal and allowed to clot. The serum was decanted
and a 4 milliliter aliquot placed in each of 2 flasks. Each
sample was treated with 0.1 milliliter of 1-131 labelled Tg dil
uted to 1,000 counts per minute. One sample received 1-131 label
led Tg which had passed through 4 half-lives. The other sample
received 1-131 labelled Tg in the first half-life. For determination
of percent uptake by serum proteins, and amount of free 1-131 in
the reagent, the technique described on page 13 was employed.
Results
Data from this study (Table III) were analyzed using the
paired t test. The calculated t value was 0.676 with 7 degrees
of freedom (t = 2.365). The percent of free 1-131 in the reagent
increased with time as expected. The percent free 1-131 in the
1-131 labelled T3 increased from 1.89 percent on the date received
to 4.68 at the end of one half-life. The maximum free 1-131
after 4 half-lives was 6.84 percent. The failure to find a
significant difference in percent uptake between old and new
isotope indicates that age of isotope has no effect. This appears
to be an unusual characteristic of this technique and further work
with aged 1-131 labelled T3 is warranted to explain it.


8
(1936) were among the first to demonstrate the beneficial effects
of thyroxine on lactation in the bovine. Reineke (1946) stated that
by giving repeated small doses of thyroxine, or by adding iodinated
protein to the feed, it was possible to maintain increased milk
yield for many months without causing any apparent harm to the cow.
The main effect of the treatment, according to Myant (1964), is
upon yield of fat rather than upon yield of total solids in the milk.
Although thyroid hormones may be secreted in the milk, Robertson
(1945) stated that the amount present in the milk of cows given
iodinated proteins was insufficient to affect the metabolism of
humans. This technique is not widly practiced for it is not
economically feasible.
Temperature and Thyroid Activity
A series of experiments conducted at the Missouri Agricultural
Experiment Station have established a relationship between temper
ature and thyroid activity. The thyroid uptake of 1-131 and the
conversion ratio (ratio of blood plasma thyroxine-like 1-131 to
total 1-131) were used as parameters of thyroid activity. Blincoe
and Brody (1955) maintained Holstein, Jersey, Brown Swiss and
Brahman cows in a "comfort zone" (4-21 C). Increasing ambient
temperature to 35 C decreased the thyroid activity 30 to 65 per
cent with Holstein cows showing the greatest decrease and Brahman
the least. Decreasing ambient temperature to -8 C increased the
thyroid activity in Jersey and Brahman 60 to 100 percent but did not
increase thyroid activity in Holstein and Brown Swiss cows. Air
velocities less than 10 m.p.h. had no measurable effect on thyroid


GENERAL SUMMARY AND CONCLUSIONS
An in vitro technique employing Sephadex column chromatography
was used to estimate circulating thyroid hormone levels in beef
l
cattle. Preliminary work with this technique was conducted to as-
sertain the applicability of Sephadex chromatography to the
evaluation of thyroid status in this species. The factors studied
were: (1) the effect of different columns on percent uptake of
duplicate samples; (2) repeatability of observations on subsequent
days for estimating thyroid status; and (3) effect of age of
isotope on percent uptake of 1-131 labelled Tg by cattle serum
proteins.
It was found that there were no statistical differences
between duplicate samples placed on different columns. A highly
significant correlation was found between the two columns. It
was concluded that there were no differences between columns and
replicate determinations of a particular sample did not necessitate
the utilization of the same column. Since any residual radio
activity released by a column caused no significant differences
in the results, there was no need for a correction factor for
retention of radioactivity by the columns.
Daily blood samples obtained from an individual animal over
a 5-day period were highly correlated with a repeatability coefficient
of 0.921. Therefore, it was concluded that this technique could be
used to study an animal's thyroid secretion rate over an extended
43


11
The mechanism of Sephadex binding is not well known, but it is
known to bind compounds with aromatic rings. Thus, the Sephadex
removes the unbound T^ but apparently cannot compete with the stronger
T3 binding of blood serum, and this protein-bound T^ passes through
the column. Shapiro and Rabinowitz (1962), Cuaron and Fucugauchi
(1964) and Cowley (1965) have shown that a chromatographic column
packed with Sephadex G-25 gel can be used to separate 1-131, unbound
1-131 labelled T3 and protein-bound 1-131 labelled T^. They found
that protein-bound 1-131 labelled T3 would be eluted first, followed
by free 1-131 and the unbound 1-131 labelled T^ would remain in the
gel. This unbound T^ could then be removed by the addition of blood
serum. Jacobsson and Widstrom (1962) reported that by thorough
washing, the columns could be used over 100 times with no adverse
effects.
Shapiro and Rabinowitz (1962) compared Sephadex chromatography
with the red blood cell uptake technique of Hamolsky et a_l. (1957)
for the estimation of thyroid status. They reported that since the
Sephadex technique was rapid, uses serum instead of whole blood and
allows the user to correct for free iodide in the reagent, it
overcomes the major objections to the red blood cell uptake technique.
These objections were: (1) Lengthy multiple washings of red blood
cells; (2) No correction for free iodide in the reagent; (3) Hemoly
sis and (4) A correction for hematocrit. Cowley (1965) also compared
the two techniques and found Sephadex chromatography superior in deter
mining hyperthyroid, hypothyroid and euthyroid status in sheep.
Cuaron and Fucugauchi (1964) found diagnosis of thyroid status by
Sephadex chromatography to agree with the clinical diagnosis in 95.9


22
Effect of Temperature, Breed, and Sex
on Thyroid Secretion Rate of Beef Cattle
It has been well documented that the thyroid hormones play an
important role in an animal's ability to withstand heat stress.
It has been proposed that thyroid activity may be a factor in the
difference in ability of Bos taurus and Bos indicus cattle to with
stand heat stress. Sex differences in heat tolerance have been
noted by Myant (1964).
The purpose of this experiment was to compare both sexes of
Hereford and Brahman cattle under different environmental conditions.
Procedure
Thirty six cattle approximately one year old were used in
this study. Nine Brahman heifers, 9 Brahman bulls, 9 Hereford
heifers, and 9 Hereford bulls were allotted to 3 treatments:
pasture, hot chamber and comfort zone chamber. Twelve animals,
3 of each breed and sex, were placed on pasture exposed to normal
environmental variation. Twelve of the remaining 24 animals, 3
of each breed and sex, were placed at random in an environmental
chamber maintained at a constant temperature of 32 C and approxi
mately 95 percent relative humidity. The third group of 12 animals
was maintained in the comfort zone at 21 C and approximately 65
percent relative humidity. The animals were maintained in the
chambers for 12 months.
The cattle in the two chambers received the same diet, with
the amount of concentrate being allocated upon the basis of National
Research Council requirements for body weight for growing breeding
animals. Roughage was placed before the animals ad libitum. The
12 cattle on pasture received the same amount of concentrate but


technique to measure thyroid status in beef cattle and to measure
differences in thyroid activity between Herford and Brahman cattle
maintained in climate control chambers at 32 C and 21 C and on
pasture for 12 months.


percent of the 141 patients studied. They stated that it should be
used as a screening test for thyroid function since it is sufficiently
reliable, does not need internal administration of radioactive
material and is simple and quick to perform. Jones and Schultz (1966)
reported that gel filtration using Sephadex G-25 offers a simple,
reliable and highly reproducible means of separating iodide and the
thyroidal ibdoamino acids.


27
This would be expected for animals exposed to normal environmental
variation but its appearance in the chamber groups indicates an
overriding of the constant temperature effect by the animal's nervous
system. The cause for this rhythm is not known but the availability
of natural light through windows in the chambers may partially
explain the rhythm in the chamber animals. The difference between
the 32 C and 21 C chambers tended to disappear in the later
months (September through December) while the pasture group main
tained a lower percent uptake.
The analysis of variance for monthly collections is given in
Tables VI through XV. The treatment effect remained highly
significant throughout the experimental period even though the two
chamber groups had approximately the same percent uptake during
the last four months. This is explained by the pasture group
being significantly different from the chamber groups during this
period.
The higher percent uptake by the cattle in the 32 C chamber
indicates that heat stress affected thyroid function. The increased
number of binding sites in the blood serum available for tying up
1-131 labelled T^ indicates a reduction in the thyroid hormone
secretion rate and a reduction in the amount of circulating thyroid
hormones.
Breed effects
The mean percent uptake for the 10 month period for breeds is
presented in Table IV. The Hereford cattle had a higher percent
uptake on all treatments than the Brahman cattle. The Herefords
exhibited the same ranking by treatment as the All Animal classification.


ug and
analyzed by the paired t test using the hypothesis that u^ =
the correlation coefficient using the columns as variables (Li, 1961).
The calculated t value of 2.14 was not significant (t = 2.26).
U J
A highly significant correlation coefficient of 0.99 was found between
the columns. These data indicate that there was no significant column
effect on percent uptake of 1-131 labelled T3 by cattle blood serum,
allowing 'the worker to use either column to rerun a particular sample
without having to determine a correction factor, or reuse the same
column.
Repeatability of Observations on Subsequent
Days for Estimating Thyroid
Status in Beef Cattle
To be of significant value in any study of thyroid activity, the
technique used for estimating thyroid status must be highly repeatable.
Hocman (1966) stated that Sephadex chromatography is standardized to
the extent that it yields similar results in similar experiments
within the range of normal deviation. Shapiro and Rabinowitz (1964)
reported that on 769 patients Sephadex chromatography determinations
had a repeatability of 92 percent for estimating thyroid status when
compared with results of other laboratory diagnoses.
The objective of this study was to determine if samples collected
on different days from the same animal would give similar percent
uptake as determined by Sephadex chromatography.
Procedure
Five cattle were bled each day for five days. The blood was
allowed to clot and then 3 mililiters of serum removed. The pro
cedure given on page 13 was used to determine percent uptake of 1-131
labelled Tg by serum proteins.


47
BIBLIOGRAPHY (continued)
Deiss, W. P., E. C. Albright and F. C. Larson. 1953. Comparison of
in vitro serum protein binding of thyroxine and triiodothyronine.
Proc. Soc. Exp. Biol. Med. 84:513.
Folley, S. J. and P. White. 1936. The effect of thyroxine on milk
secretion and on the phosphotase of the blood and milk of the
lactating cow. Proc. Roy Soc. B. 120:346.
Frienkel, N., J. J. Dowling and S. H. Ingbar. 1955. The interaction
of thyroxine with plasma protein; localization of thyroxine
binding proteins in Cohn Fraction of plasma. J. Clin. Invest. 34:
1698.
Graham, W. R. 1934. The action of thyroxine on the milk and milk-fat
production of cows. Biochem. J. 28:1368.
Halmi, N. S. 1964. The accumulation and recirculation of iodide by
the thyroid. The Thyroid Gland. I. R. Pitt-Rivers and
W. R. Trotter, (eds.). Butterworths, Inc., Washington, D.C.
Hamolsky, M. W., M. Stein and A. S. Freedberg. 1957. The thyroid
hormone plasma protein complex in man. II. A new in vitro
method for study of "uptake" of labeled hormonal components of
human erythrocytes. J. Clin. Endocr. 17:33.
Hart, D. S. 1960. Fertility responses in ewes treated with thyroxine.
New Zealand J. Agr. Sci. 3:365.
Heidelberger, M. and K. 0. Pedersen. 1935. The molecular weight and
isoelectric point of thyroglobulin. J. Gen. Physiol. 19:95.
Henneman, H. A., E. P. Reineke and S. A. Griffin. 1955. Thyroid
secretion rate of sheep as affected by season, age, breed,
pregnancy, and lactation. J. Animal Sci. 14:419.
Hocman, G. 1966. The use of Sephadex in the chromatography of thyroxine
containing compounds: a critique. J. Chromatog. 21:413.
Howes, J. J. 1964. The Comparative Physiology of Bos Taurus and Bos
Indicus cattle on Two Nutritional Levels. Ph.D. dissertation,
University of Florida, Gainesville, Florida.
Jacobsson, L. and G. Widstrom. 1962. Separation of Iodine compounds
in serum by gel filtration. Scand. J. Clin. Lab. Invest. 14:285.
Jones, J. E. and J. J. Schultz. 1966. Identification of thyroidal
iodoamino acids by gel filtration and automated 1-127 determin
ation. J. Clin. Endocr. 26:975.
Lewitus, Z., M. Anbar and S. Guttman. 1961. A contribution to the
understanding of the "trapping mechanism" in the thyroid gland.
Advances in Thyroid Research. R. Pitt-Rivers (ed.). Pergamon
Press, New York.


5
(TSH) and is inhibited by excess iodide. The exact mechanism by
which the hormones pass from the thyroid tissue into the blood is
not known, but Tata (1964) suggests that passive diffusion across
membranes is the most likely process. Taurog ejt a_l. (1956) found that
both T^ and T^ were secreted into the circulatory system but neither
T2 or T^ left the thyroid gland even when the gland contained 70
percent of its 1-131 in these forms.
It has been known for many years that for proper functioning,
the thyroid gland is dependent upon the anterior pituitary secretion,
thyroid stimulating hormone (TSH). Also, the thyroid hormones them
selves are known to influence the secretion of TSH. This reciprocal
relationship provides a homeostatic mechanism which insures adequate
synthesis and secretion of thyroid hormones.
Upon release from the gland, the hormones enter the circulation
and are distributed throughout the body. The interaction between
thyroid hormones and serum proteins, which bind the hormone more
firmly than any tissue component, is the key to the present-day
understanding of hormone transport. Deiss e_t al_. (1953) reported
both T^ and T^ bound to a specific alpha globulin just ahead of
alpha-2-globulin. Frienkel e_t a_l. (1955) also reported that most
1-131 labelled T^ localized with a protein intermediate in mobility
between alpha-1 and alpha-2-globulin. They listed the globulins,
albumins, and erythrocytes as the primary, secondary and tertiary
carriers, respectively of T3 and T^.
Pitt-Rivers and Tata (1959) listed three principal types of
pathways at the organ level for the distribution of thyroid hormones
after the administration of radioactive T3 and T^. These included:


1
2
3
4
5
6
7
8
9
10
TABLE I
PERCENTAGE UPTAKE OF 1-131 LABELLED T3 BY DUPLICATE
SAMPLES OF CATTLE SERUM THROUGH
DIFFERENT COLUMNS
Co limn 1
Column 2
76.30
77.10
79.52
78.94
78.69
79.42
72.94
73.77
75.53
76.10
63.88
64.90
64.53
64.35
66.23
67.03
67.47
68.41
66.22
65.60
71.13
71.56


LIST OF TABLES (continued)
TABLE
Page
XIV. ANALYSIS OF VARIANCE OF PERCENT UPTAKE OF 1-131
LABELLED T3 FOR NOVEMBER 36
XV. ANALYSIS OF VARIANCE OF PERCENT UPTAKE OF 1-131
LABELLED T3 FOR DECEMBER... 37
V


TABLE V
ANALYSIS OF VARIANCE OF MEANS OF PERCENT UPTAKE
FOR TEN MONTH PERIOD
Source of
Variance
D.F.
Sum of
Squares
Mean
Squares
Total
35
261.71
Breed
1
34.44
34.44**
Sex
1
126.35
126.35**
Treatment
2
50.56
25.28**
Breed X Sex
1
1.77
1.77
Breed X Treatment
2
23.08
11.54**
Sex X Treatment
2
6.35
3.18**
Breed X Sex X Treatment
2
6.87
3.44**
Error
23
12.29
0.534
** Probability Less
Than 0.01.


IN VITRO MEASUREMENT OF CIRCULATING
THYROID HORMONE LEVELS IN
BEEF CATTLE
By
JERRY JENNINGS COWLEY
A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1968

ACKNOWLEDGMENTS
The author wishes to express his sincere appreciation to
Dr. Alvin Warnick, chairman of his supervisory committee, for his
counsel and assistance throughout this study.
Appreciation is also expressed to Dr. Marvin Koger, Dr. Ray
Shirley.and Dr. G. T. Edds who served as members of the supervisory
committee and to Dr. J. P. Feaster for guidance in the laboratory.
The author wishes to thank Mr. Dean Pogue and Mr. Hernando
Gutierrez for their assistance in the gathering of data and care
of experimental animals.
The author extends his deepest gratitude to his wife, Marilyn,
for her constant encouragement and untiring efforts which were
invaluable in achieving this goal.
ii

TABLE OF CONTENTS
Page
ACKNOWLEDGMENTS ii
LIST OF TABLES iv
LIST OF FIGURES vi
INTRODUCTION I
REVIEW OF LITERATURE 3
General Thyroid Physiology 3
Reproduction and Lactation 6
Temperature and Thyroid Activity. 8
Sephadex Chromatography 10
EXPERIMENTAL PROCEDURE 13
RESULTS 15
Effect of Different Columns on Percent Uptake
of Duplicate Samples 15
Repeatability of Observations on Subsequent
Days for Estimating Thyroid Status in
Beef Cattle 17
Effect of Age of Isotope on Percent Uptake of
1-131 Labelled T^ by Cattle Serum Proteins 18
^ Effect of Temperature, Breed, and Sex on Thyroid
^ Secretion Rate of Beef Cattle 22
GENERAL SUMMARY AND CONCLUSIONS 43
BIBLIOGRAPHY 46
iii

LIST OF TABLES
Page
TABLE
I.PERCENTAGE UPTAKE OF 1-131 LABELLED T BY
DUPLICATE SAMPLES OF CATTLE SERUM ?HROUGH
DIFFERENT COLUMNS 16
II.PERCENT UPTAKE OF 1-131 LABELLED T-j BY CATTLE
SERUM FOR 5 DAYS AS MEASURED BY SEPHADEX
CHROMATOGRAPHY 19
III.PERCENT UPTAKE OF OLD AND NEW 1-131 LABELLED
t3 BY CATTLE SERUM PROTEINS 21
IV.MEAN PERCENT UPTAKE OF 1-131 LABELLED Tn FOR
TEN MONTH PERIOD 24
V.ANALYSIS OF VARIANCE OF MEANS OF PERCENT UPTAKE
FOR TEN MONTH PERIOD 25
VI.ANALYSIS OF VARIANCE OF PERCENT UPTAKE OF 1-131
LABELLED T3 FOR MARCH 28
VII.ANALYSIS OF VARIANCE OF PERCENT UPTAKE OF 1-131
LABELLED T3 FOR APRIL 29
VIII.ANALYSIS OF VARIANCE OF PERCENT UPTAKE OF 1-131
LABELLED T FOR MAY ; 30
3
IX.ANALYSIS OF VARIANCE OF PERCENT UPTAKE OF 1-131
LABELLED T3 FOR JUNE 31
X.ANALYSIS OF VARIANCE OF PERCENT UPTAKE OF 1-131
LABELLED T3 FOR JULY 32
XI.ANALYSIS OF VARIANCE OF PERCENT UPTAKE OF 1-131
LABELLED T3 FOR AUGUST 33
XII.ANALYSIS OF VARIANCE OF PERCENT UPTAKE OF 1-131
LABELLED T3 FOR SEPTEMBER 34
XIII.ANALYSIS OF VARIANCE OF PERCENT UPTAKE OF 1-131
LABELLED T3 FOR OCTOBER 35
iv

LIST OF TABLES (continued)
TABLE
Page
XIV. ANALYSIS OF VARIANCE OF PERCENT UPTAKE OF 1-131
LABELLED T3 FOR NOVEMBER 36
XV. ANALYSIS OF VARIANCE OF PERCENT UPTAKE OF 1-131
LABELLED T3 FOR DECEMBER... 37
V

LIST OF FIGURES
Page
Figure
1. Monthly percent uptake of 1-131 labelled for
treatment classification ... 26
2. Monthly percent.uptake of 1-131 labelled for
breed classification 39
3. Monthly percent uptake of 1-131 labelled for
sex classification 41
vi
/

INTRODUCTION
In reviewing the role of the thyroid hormones in the repro
ductive physiology of the female, Reineke and Solimn (1953)
pointed out that although over half a century of investigation
indicates that the thyroid hormones are implicated in some manner,
their precise role has not been determined. There is also ample
evidence that the thyroid gland plays an important role in growth
and development. Many factors are associated with thyroid hormone
secretion rate including breed, age, sex, season of year, pregnancy,
lactation, temperature and humidity, plane of nutrition, and a variety
of diseases.
When nuclear reactor-produced 1-131 became available in 1946,
the study of the thyroid gland received new impetus. Most of the
subsequent research was in clinical diagnosis and therapy.
The introduction of an _in vitro technique in 1962 has made it
possible to study and compare circulating thyroid hormone levels
without exposing the animals to radioactive material or endogenous
thyroid hormones.
Earlier work has suggested that one of the factors influencing
adaptability is thyroid activity. Thyroid activity has been suggested
as one of the factors involved in the difference in adaptability
between European (Bos taurus) and Asiatic (Bos indicus) breeds of
beef cattle.
This project was designed to study the feasibility of an in vitro
1

technique to measure thyroid status in beef cattle and to measure
differences in thyroid activity between Herford and Brahman cattle
maintained in climate control chambers at 32 C and 21 C and on
pasture for 12 months.

REVIEW OF LITERATURE
General Thyroid Physiology
A fundamental knowledge of the formation and release of the
thyroid hormones is essential in any study employing thyroid physi
ology. A single layer of epithelial cells, the follicular epithelium,
encompassing a central mass of protein, the colloid, is the funda
mental parenchymal unit of the gland. The small sacs or follicles
of which the adult thyroid is composed may be considered from both
the structural and functional point of view to be the secretory
unit of the thyroid gland. Within the follicle and filling its
lumen is the homogenous, thin, clear substance, the colloid. The
synthesis of the thyroid hormones has been reported by Nadler
(1962) to take place within the colloids of the follicles. Heidel-
berger and Pedersen (1935) found that the colloid consists largely of
thyroglobulin, a large protein having a molecular weight of approxi
mately 700,000.
Iodide is the only chemical form in which the thyroid gland can
assimilate iodine for formation of the thyroid hormones. Halmi (1964)
stated that active transport is by far the most important mechanism
whereby the gland can accumulate iodine. The thyroid gland, in addi
tion to concentrating iodide, concentrates a series of other anions of
no physiological use to the gland. Lewitus e_t _al. (1961) explained
3
f

4
the "trapping mechanism" on the basis of valency and ionic size
stating that the "trapping mechanism" in the thyroid gland involves
a certain specially adapted protein in the cell membrane which will
incorporate all monovalent ions which fulfill certain requirements
of size.
Iodide that is transported into the gland from the circulation
undergoes enzymatic oxidation. The subsequent iodination of tyro
sine in the thyroglobulin to form the thyroid hormones occurs in the
colloids of the follicular cells. Pitt-Rivers and Cavalieri (1964)
gave the following sequence of events that lead to the formation of
the thyroid hormones:
1. Iodide is transported into the gland by the blood.
2. Iodide is converted by some oxidative enzyme to mono-
iodotyrosine (T^) and diiodotyrosine (T2).
3. Thyroxine (T4) is formed by the coupling of 2 molecules
of T2 and the loss of 1 alanine side chain.
4. Triiodothyronine (T^) is formed by the coupling of 1
molecule each of T and T and the loss of 1 alanine
1 2
side chain.
Reactions 2 through 4 occur within the thyroglobulin molecule
located in the follicles.
The thyroid hormones either enter the blood or are secreted
into the lumen of the follicle and stored in the colloid bound to
thyroglobulin. Because of its large molecular size, thyroglobulin
is degraded to its constituent amino acids before the thyroid hormones
are secreted into the blood stream. Pitt-Rivers and Tata (1959) state
that this step is under the influence of thyroid stimulating hormone

5
(TSH) and is inhibited by excess iodide. The exact mechanism by
which the hormones pass from the thyroid tissue into the blood is
not known, but Tata (1964) suggests that passive diffusion across
membranes is the most likely process. Taurog ejt a_l. (1956) found that
both T^ and T^ were secreted into the circulatory system but neither
T2 or T^ left the thyroid gland even when the gland contained 70
percent of its 1-131 in these forms.
It has been known for many years that for proper functioning,
the thyroid gland is dependent upon the anterior pituitary secretion,
thyroid stimulating hormone (TSH). Also, the thyroid hormones them
selves are known to influence the secretion of TSH. This reciprocal
relationship provides a homeostatic mechanism which insures adequate
synthesis and secretion of thyroid hormones.
Upon release from the gland, the hormones enter the circulation
and are distributed throughout the body. The interaction between
thyroid hormones and serum proteins, which bind the hormone more
firmly than any tissue component, is the key to the present-day
understanding of hormone transport. Deiss e_t al_. (1953) reported
both T^ and T^ bound to a specific alpha globulin just ahead of
alpha-2-globulin. Frienkel e_t a_l. (1955) also reported that most
1-131 labelled T^ localized with a protein intermediate in mobility
between alpha-1 and alpha-2-globulin. They listed the globulins,
albumins, and erythrocytes as the primary, secondary and tertiary
carriers, respectively of T3 and T^.
Pitt-Rivers and Tata (1959) listed three principal types of
pathways at the organ level for the distribution of thyroid hormones
after the administration of radioactive T3 and T^. These included:

6
(1) liver, kidney and posterior pituitary which show a high turnover
rate; (2) skeletal muscle and intestine which exhibit a slow turnover
rate; and (3) brain, spleen and gonads which accumulate only very
low levels. Albert and Keating (1952) have shown the liver to be
the most active tissue in concentrating and metabolizing thyroid
hormones. The intense enterohepatic circulation of thyroid hormones
is primarily responsible for rapid thyroid hormone catabolism and
excretion.
Robbins and Rail (1955) stated that the bilary-fecal pathway
by which most of the iodine is excreted as unchanged hormone in
the feces is the predominant pathway in rats. Albright and Larson
(1959) have shown urine to be the predominant excretory pathway
in man.
Reproduction and Lactation
Brody and Frankenbuch (1942) were among the first to show that
thyroidectomized cows fail to manifest normal physical signs of
heat, and that administration of thyroid material restores normal
estrual behavior in some thyroidectomized cows. Blaxter (1945)
reported that thyroid therapy shortened the post-partum breeding
interval.
Brunstad and Fowler (1958) found that crown-rump length of normal
embryos carried by thyroprotein-treated and control gilts were signi
ficantly larger than those of thiouracil-treated gilts. Lucas e_t al.
(1958) reported pigs born to thyroprotein-treated gilts to be heavier
than those born to control and thiouracil-treated gilts. They reported
increased embyronic mortality from the 25th day of pregnancy to

parturition in the thiouracil-treated group and reduced embryonic
mortality in the thyroprotein-treated group.
Spielmann et al. (1945) noted that the main effect of thyroid
ectomy on mature cows was a'failure to manifest the physical
signs of estrus. Normal ova were produced as evidenced by the birth
of 3 calves from 3 cows thyroidectomized 161, 242, and 453 days
before breeding.
Henneman _et al. (1955) reported lactation to create increased
demand on the thyroid gland, and lactating ewes showed a signifi
cantly higher thyroid secretion rate than either nonpregnant or
pregnant ewes.
Spielmann e_t a_l. (1944) found that cows thyroidectomized prior
to gestation, during pregnancy, or during lactation would cease
lactation in about 180 days. It was also observed that incomplete
removal of the thyroid gland produced a temporary decline in
milk production, followed by a gradual return to the former
levels.
Hart (1960) reported a 9.5 percent increase in lambs born
from ewes treated with L-thyroxine pellets 10 to 11 days before
mating. Ryle (1961) found that ewes exposed to an experimentally
hot environment and ewes under normal conditions which received
thyroxine had significantly more embryos than those ewes that
received no thyroxine. In 1963, Ryle proposed that dwarfing of
fetuses exposed to high ambient temperatures, as reported by
Yeates (1958) may be prevented by thyroxine therapy.
It has been repeatedly shown that milk yield in cattle can be
increased by thyroid therapy. Graham (1934) and Folley and White

8
(1936) were among the first to demonstrate the beneficial effects
of thyroxine on lactation in the bovine. Reineke (1946) stated that
by giving repeated small doses of thyroxine, or by adding iodinated
protein to the feed, it was possible to maintain increased milk
yield for many months without causing any apparent harm to the cow.
The main effect of the treatment, according to Myant (1964), is
upon yield of fat rather than upon yield of total solids in the milk.
Although thyroid hormones may be secreted in the milk, Robertson
(1945) stated that the amount present in the milk of cows given
iodinated proteins was insufficient to affect the metabolism of
humans. This technique is not widly practiced for it is not
economically feasible.
Temperature and Thyroid Activity
A series of experiments conducted at the Missouri Agricultural
Experiment Station have established a relationship between temper
ature and thyroid activity. The thyroid uptake of 1-131 and the
conversion ratio (ratio of blood plasma thyroxine-like 1-131 to
total 1-131) were used as parameters of thyroid activity. Blincoe
and Brody (1955) maintained Holstein, Jersey, Brown Swiss and
Brahman cows in a "comfort zone" (4-21 C). Increasing ambient
temperature to 35 C decreased the thyroid activity 30 to 65 per
cent with Holstein cows showing the greatest decrease and Brahman
the least. Decreasing ambient temperature to -8 C increased the
thyroid activity in Jersey and Brahman 60 to 100 percent but did not
increase thyroid activity in Holstein and Brown Swiss cows. Air
velocities less than 10 m.p.h. had no measurable effect on thyroid

activity but the addition of radiant energy depressed the thyroid
activity. Thus, increasing the thermal stress on cows either by
increased ambient temperature or by increased radiation reduced the
thyroid activity. As heat stress increased, the rate of clearance
of plasma iodide by the thyroid decreased and the excretion rate
increased.
Blincoe and Brody (1955) studied the influence of diurnally
variable temperatures on the thyroid activity of Jersey and Holstein
cows. A daily temperature cycle of -10 to 4 C increased thyroid
activity by 20 percent over the value of a "comfort zone" cycle.'
A temperature cycle of 21 to 38 C decreased the thyroid activity
by about 30 percent below its value in the "comfort zone" cycle.
These data roughly paralleled the heat production data for the
same animals.
Blincoe (1958) studied the influence of constant ambient
temperature on the thyroid activity of Shorthorn, Santa Gertrudis
and Brahman calves. The 3 heifers of each breed were maintained
at 10 C, 27 C, and in an open shed. The rate constant for hormone
release of the Shorthorn heifers was decreased 45 percent at 27 C.
The Brahman heifers showed no decrease and the Santa Gertrudis
were slightly decreased.
The Shorthorn calves raised at 10 C were most affected by high
temperature. At 38 C their thyroid secretory activity was reduced
60 percent below the reading at 10 C. The Santa Gertrudis were less
affected and the Brahmans practically unaffected.
Swanson e_t a_l. (1957) studied the 1-131 uptake of 8 Jersey and
Guernsey cows for 18 months. They found effects which could be

10
attributed to seasonal temperatures, but the average differences
found between seasonal measurements were relatively unimportant.
They concluded that "measuring thyroid activity of cows by this
method on an uncontrolled survey basis could not be expected to
provide reliable comparable data."
Howes (1964) measured the 1-131 uptake on 24 nonpregnant
Herefcrd and Brahman heifers. He found that the thyroid glands of
the Hereford concentrated 1-131 faster than the Brahmans and the
thyroid glands of the Herefords retained a significantly greater
percentage of the injected 1-131 at comparable time intervals than
did the Brahman heifers.
Sephadex Chromatography
Gel filtration first became an established laboratory technique
with the introduction of Sephadex in 1959. The use of a chroma
tographic method employing Sephadex gel for estimating thyroid status
was reported by Shapiro and Rabinowitz (1962).
Sephadex is a modified dextran. The dextran macromolocules
are cross linked to give a three-dimensional network of polysac
charide chains. Because of its high content of hydroxyl groups,
Sephadex is strongly hydrophilic and the Sephadex beads swell
considerably in water and electrolyte solutions giving a porus gel.
Only molecules below a certain size (below 5,000 molecular weight
with Sephadex G-25) can enter the interstices. Heavier molecules
are excluded from the pores and pass through the bed in the liquid
phase outside the particles, passing through the column more rapidly
than the smaller molecules entering the pores. Molecules are therefore
eluted from a Sephadex bed in the order of decreasing molecular size.

11
The mechanism of Sephadex binding is not well known, but it is
known to bind compounds with aromatic rings. Thus, the Sephadex
removes the unbound T^ but apparently cannot compete with the stronger
T3 binding of blood serum, and this protein-bound T^ passes through
the column. Shapiro and Rabinowitz (1962), Cuaron and Fucugauchi
(1964) and Cowley (1965) have shown that a chromatographic column
packed with Sephadex G-25 gel can be used to separate 1-131, unbound
1-131 labelled T3 and protein-bound 1-131 labelled T^. They found
that protein-bound 1-131 labelled T3 would be eluted first, followed
by free 1-131 and the unbound 1-131 labelled T^ would remain in the
gel. This unbound T^ could then be removed by the addition of blood
serum. Jacobsson and Widstrom (1962) reported that by thorough
washing, the columns could be used over 100 times with no adverse
effects.
Shapiro and Rabinowitz (1962) compared Sephadex chromatography
with the red blood cell uptake technique of Hamolsky et a_l. (1957)
for the estimation of thyroid status. They reported that since the
Sephadex technique was rapid, uses serum instead of whole blood and
allows the user to correct for free iodide in the reagent, it
overcomes the major objections to the red blood cell uptake technique.
These objections were: (1) Lengthy multiple washings of red blood
cells; (2) No correction for free iodide in the reagent; (3) Hemoly
sis and (4) A correction for hematocrit. Cowley (1965) also compared
the two techniques and found Sephadex chromatography superior in deter
mining hyperthyroid, hypothyroid and euthyroid status in sheep.
Cuaron and Fucugauchi (1964) found diagnosis of thyroid status by
Sephadex chromatography to agree with the clinical diagnosis in 95.9

percent of the 141 patients studied. They stated that it should be
used as a screening test for thyroid function since it is sufficiently
reliable, does not need internal administration of radioactive
material and is simple and quick to perform. Jones and Schultz (1966)
reported that gel filtration using Sephadex G-25 offers a simple,
reliable and highly reproducible means of separating iodide and the
thyroidal ibdoamino acids.

EXPERIMENTAL PROCEDURE
Sephadex column chromatography was used to ascertain thyroid
status of beef cattle. The assay employed the technique of Shapiro
and Rabinowitz (1962) as modified by Cowley (1965). The complete
procedure is as follows:
1. Swell 3.0 grams Sephadex G-25 medium grade in 0.2 normal
phosphate buffer for 6 hours at room temperature. Pour
the gel into a Sephadex K-9 laboratory column fitted with
*
a luer ending outlet port.
2. Add 0.1 milliliter of 1-131 labelled T^ diluted to 1,000
counts per minute in buffer solution to 3 milliliters of
blood serum.
3. Incubate sample for 15 minutes in a water bath at 37 C.
4. Remove 1 milliliter of serum and assay for activity in a
scintillation counter equipped with a sodium iodide crystal.
This serves as the standard.
5. Place 1 milliliter of sample on top of gel bed taking care
not to disturb the gel surface.
6. After the serum enters the bed, add 2 separate 1 milliliter
aliquots of buffer solution.
7. When the buffer solution completely enters the gel bed, fill
the column with buffer solution and start collection of 1
milliliter elutes until 12 eluates are obtained.
8. Wash the column with 20 milliliters of blood serum diluted 10
13

times in buffer and discard this serum.
9.After the diluted serum passes through the column, wash
with buffer until the column is again white.
10. Assay each of the 12 eluates for radioactivity as was
done for standards in step 4.
11. Determine percent uptake of protein-bound 1-131 labelled

by the following calculation procedure:
a. deduct background from all counts.
b. sum up counts in those fractions containing protein-
bound (eluates 1-4).
c. sum up counts in those fractions containing free 1-131
(eluates 5-12).
d. subtract count obtained in c. above from net counts in
serum (standard obtained in step 4) to obtain net counts
1-131 labelled in incubated serum.
e. percent uptake equals 100 X or percent uptake equals
net counts protein-bound fractions (b) divided by
net counts 1-131 labelled in standard (d) multiplied
by 100.
This technique was used to determine thyroid status in all
subsequent experiments.

RESULTS
Effect of Different Columns on
Percent Uptake of Duplicate Samples
l
Sisson (1965) stated that an undesirable characteristic of a
glass chromatographic column was the retention of radioactivity.
Release of this radioactivity from the column could inject a source
of error into the experiment. Cowley (1965) studied the repeatability
of percent uptake of 1-131 labelled of duplicate samples of sheep
serum in different glass chromatographic columns packed with Sephadex
G-25. He found no significant differences in uptake due to the
column used with a correlation between uptake by the two columns of
0.99.
The objective of this study was to determine the repeatibility
of 1-131 labelled T^ uptake by cattle serum proteins using duplicate
samples in 2 different plastic chromatographic columns.
Procedure
Ten cows were used as blood donors. Twenty-five mililiters of
blood was collected from the jugular vein of each and allowed to clot.
Five mililiters of serum was removed and treated with 0.2 mililiter
1-131 labelled T^ diluted to 1,000 counts per minute. The sample
was then placed on separate columns. The procedure described on
page 13 for application, collection and calculation was followed.
Results
The data obtained from this study, presented in Table I, were
15

1
2
3
4
5
6
7
8
9
10
TABLE I
PERCENTAGE UPTAKE OF 1-131 LABELLED T3 BY DUPLICATE
SAMPLES OF CATTLE SERUM THROUGH
DIFFERENT COLUMNS
Co limn 1
Column 2
76.30
77.10
79.52
78.94
78.69
79.42
72.94
73.77
75.53
76.10
63.88
64.90
64.53
64.35
66.23
67.03
67.47
68.41
66.22
65.60
71.13
71.56

ug and
analyzed by the paired t test using the hypothesis that u^ =
the correlation coefficient using the columns as variables (Li, 1961).
The calculated t value of 2.14 was not significant (t = 2.26).
U J
A highly significant correlation coefficient of 0.99 was found between
the columns. These data indicate that there was no significant column
effect on percent uptake of 1-131 labelled T3 by cattle blood serum,
allowing 'the worker to use either column to rerun a particular sample
without having to determine a correction factor, or reuse the same
column.
Repeatability of Observations on Subsequent
Days for Estimating Thyroid
Status in Beef Cattle
To be of significant value in any study of thyroid activity, the
technique used for estimating thyroid status must be highly repeatable.
Hocman (1966) stated that Sephadex chromatography is standardized to
the extent that it yields similar results in similar experiments
within the range of normal deviation. Shapiro and Rabinowitz (1964)
reported that on 769 patients Sephadex chromatography determinations
had a repeatability of 92 percent for estimating thyroid status when
compared with results of other laboratory diagnoses.
The objective of this study was to determine if samples collected
on different days from the same animal would give similar percent
uptake as determined by Sephadex chromatography.
Procedure
Five cattle were bled each day for five days. The blood was
allowed to clot and then 3 mililiters of serum removed. The pro
cedure given on page 13 was used to determine percent uptake of 1-131
labelled Tg by serum proteins.

18
Results
The data from this study, presented in Table II, were analyzed
by the correlation coefficient using days as variables and by an
analysis of repeatability (R) Highly significant correlation
coefficients above 0.92 were found for each comparison. The R value
was 0.921. These data indicate very good repeatability on a day to
day basis. On a long term study, differences in percent uptake could
be assumed to be due to thyroid hormone secretion rate and not to
variation in the technique employed.
Effect of Age of Isotope on Percent Uptake
of 1-131 Labelled _T3 by Cattle Serum Proteins
In routine laboratory procedure, 1-31 is usually discarded after V1
one half-life. Half-life of an isotope is defined as the length of
time required for one-half of the radioactive atoms in an isotope
to undergo decay. Chase and Rabinowitz (1964) state that the half-
life of 1-131 is 8.05 days. During the decay of 1-131 labelled T3
there is a disassociation of the 1-131 molecule from the triiodothyro
nine molecule resulting in free 1-131 in the reagent as well as a
decrease in the total amount of radioactivity.
Shapiro and Rabinowitz (1962) reported that there is no problem
with free 1-131 in the Sephadex chromatography technique because of
a correction factor employed. This correction should provide for
longer utilization of a particular sample of 1-131 labelled T3.
Cowley (1965) reported that 1-131 labelled T3 that had been through
8 half-lives did not yield significantly different percent uptake
than did the isotope in its first half-life.
The purpose of this study was to determine if 1-131 labelled T3

TABLE II
PERCENT UPTAKE OF 1-131 LABELLED T3 BY CATTLE SERUM
FOR 5 DAYS AS MEASURED BY SEPHADEX CHROMATOGRAPHY
Cow
Day 1
Day 2
Day 3
Day 4
Day 5
Mean
Variance
1
66.39
65.92
66.89
66.51
65.22
66.19
0.349
2
60.70
60.30
61.40
61.90
62.00
61.26
0.440
3
61.00
61.10
60.20
61.10
61.50
60.98
0.540
4
59.90
60.80
61.00
61.20
60.90
60.76
0.202
5
59.50
61.00
59.70
61.80
61.00
60.60
0.756
61.50
61.82
61.84
62.50
62.12
61.96
Means

20
could be used with cattle blood serum for more than one half-life
without significantly affecting the results. This would allow for
better utilization of the isotope and reduce the cost of estimating
thyroid status.
Procedure
Twenty milliliters of blood were collected from the jugular
vein of each animal and allowed to clot. The serum was decanted
and a 4 milliliter aliquot placed in each of 2 flasks. Each
sample was treated with 0.1 milliliter of 1-131 labelled Tg dil
uted to 1,000 counts per minute. One sample received 1-131 label
led Tg which had passed through 4 half-lives. The other sample
received 1-131 labelled Tg in the first half-life. For determination
of percent uptake by serum proteins, and amount of free 1-131 in
the reagent, the technique described on page 13 was employed.
Results
Data from this study (Table III) were analyzed using the
paired t test. The calculated t value was 0.676 with 7 degrees
of freedom (t = 2.365). The percent of free 1-131 in the reagent
increased with time as expected. The percent free 1-131 in the
1-131 labelled T3 increased from 1.89 percent on the date received
to 4.68 at the end of one half-life. The maximum free 1-131
after 4 half-lives was 6.84 percent. The failure to find a
significant difference in percent uptake between old and new
isotope indicates that age of isotope has no effect. This appears
to be an unusual characteristic of this technique and further work
with aged 1-131 labelled T3 is warranted to explain it.

TABLE III
PERCENT UPTAKE OF OLD AND NEW 1-131 LABELLED T
BY CATTLE SERUM PROTEINS J
Cow
4 Half-lives
First Half-life
1
64.54
66.52
2.
65.65
68.10
3
64.63
63.10
4
65.47
66.38
5
59.48
57.68
6
65.85
67.17
7
58.88
59.81
8
61.45
60.35
Mean
63.24
63.64

22
Effect of Temperature, Breed, and Sex
on Thyroid Secretion Rate of Beef Cattle
It has been well documented that the thyroid hormones play an
important role in an animal's ability to withstand heat stress.
It has been proposed that thyroid activity may be a factor in the
difference in ability of Bos taurus and Bos indicus cattle to with
stand heat stress. Sex differences in heat tolerance have been
noted by Myant (1964).
The purpose of this experiment was to compare both sexes of
Hereford and Brahman cattle under different environmental conditions.
Procedure
Thirty six cattle approximately one year old were used in
this study. Nine Brahman heifers, 9 Brahman bulls, 9 Hereford
heifers, and 9 Hereford bulls were allotted to 3 treatments:
pasture, hot chamber and comfort zone chamber. Twelve animals,
3 of each breed and sex, were placed on pasture exposed to normal
environmental variation. Twelve of the remaining 24 animals, 3
of each breed and sex, were placed at random in an environmental
chamber maintained at a constant temperature of 32 C and approxi
mately 95 percent relative humidity. The third group of 12 animals
was maintained in the comfort zone at 21 C and approximately 65
percent relative humidity. The animals were maintained in the
chambers for 12 months.
The cattle in the two chambers received the same diet, with
the amount of concentrate being allocated upon the basis of National
Research Council requirements for body weight for growing breeding
animals. Roughage was placed before the animals ad libitum. The
12 cattle on pasture received the same amount of concentrate but

had access to grass during the experiment. Treatment was confounded
with phenotype of the test animals because the 12 pasture animals
had been selected as breeding stock and were superior to the chamber
animals. However, these pasture cattle were of value in indicating
the response of good quality cattle under good nutritional conditions.
During the twelve-month experimental period, a 25 milliliter
blood sample was collected from each animal every 28 days. The
blood sample was allowed to clot and the serum withdrawn. The
blood serum was analyzed for thyroid hormones employing the Sephadex
chromatography procedure given on page 13.
Results
Due to a malfunction of the scintillation counter used to
assay for radioactivity, the first 2 months data were lost.
Therefore, data is only available for 10 consecutive months.
Treatment effects
The mean percent uptake for the 10 month period for treatment
effects is presented in Table IV. The data for all animals on
treatment shows a higher thyroid secretion rate, or lower percent
uptake, for cattle on pasture (61.67 percent uptake) than for the
cattle in either chamber. The cattle in the 32 C chamber had the
highest percent uptake, 64.67 percent, with the cattle in the 21 C
chamber being intermediate at 63.25 percent.
The analysis of variance of the 10 month means is presented
in Table V. A highly significant difference between treatments
indicates that there were different responses to the treatments.
A graph of the means for treatment effect is presented in
Figure 1. A definite diurnal rhythm was noted in all three groups.

SWi
TABLE IV
MEAN PERCENT UPTAKE OF 1-131 LABELLED T3 FOR TEN-MONTH PERIOD
All
Hereford
Brahman
All
All
Treatment
Animals
All
Heifers
Bulls
All
Heifers
Bulls
Bulls
Heifers
32 C
Chamber
64.67
66.67
63.85
69.49
62.28
61.24 '
63.84
67.23
62.55
21 C
Chamber
63.25
63.47
61.25
65.70
63.03
60.79
65.26
65.48
61.02
Pasture
61.67
62.37
61.38
63.35
60.97
59.24
62.71
63.03
60.31
to
4>

TABLE V
ANALYSIS OF VARIANCE OF MEANS OF PERCENT UPTAKE
FOR TEN MONTH PERIOD
Source of
Variance
D.F.
Sum of
Squares
Mean
Squares
Total
35
261.71
Breed
1
34.44
34.44**
Sex
1
126.35
126.35**
Treatment
2
50.56
25.28**
Breed X Sex
1
1.77
1.77
Breed X Treatment
2
23.08
11.54**
Sex X Treatment
2
6.35
3.18**
Breed X Sex X Treatment
2
6.87
3.44**
Error
23
12.29
0.534
** Probability Less
Than 0.01.

§0
N>
ON
W C

27
This would be expected for animals exposed to normal environmental
variation but its appearance in the chamber groups indicates an
overriding of the constant temperature effect by the animal's nervous
system. The cause for this rhythm is not known but the availability
of natural light through windows in the chambers may partially
explain the rhythm in the chamber animals. The difference between
the 32 C and 21 C chambers tended to disappear in the later
months (September through December) while the pasture group main
tained a lower percent uptake.
The analysis of variance for monthly collections is given in
Tables VI through XV. The treatment effect remained highly
significant throughout the experimental period even though the two
chamber groups had approximately the same percent uptake during
the last four months. This is explained by the pasture group
being significantly different from the chamber groups during this
period.
The higher percent uptake by the cattle in the 32 C chamber
indicates that heat stress affected thyroid function. The increased
number of binding sites in the blood serum available for tying up
1-131 labelled T^ indicates a reduction in the thyroid hormone
secretion rate and a reduction in the amount of circulating thyroid
hormones.
Breed effects
The mean percent uptake for the 10 month period for breeds is
presented in Table IV. The Hereford cattle had a higher percent
uptake on all treatments than the Brahman cattle. The Herefords
exhibited the same ranking by treatment as the All Animal classification.

TABLE VI
ANALYSIS OF VARIANCE OF PERCENT UPTAKE OF
1-131 LABELLED T3 FOR MARCH
Source of
Variance
D.F.
Sums of
Squares
Mean
Squares
Total
35
520.72
Breed
1
24.68
24.68**
Sex
1
253.88
253.88**
Treatment
2
116.74
58.37**
Breed X Sex
1
12.23
12.23**
Breed X Treatment
2
27.69
13.85**
Sex X Treatment
2
36.06
18.03**
Breed X Sex X Treatment
2
35.74
17.87**
Error
23
13.70
0.595
** Probability Less Than .01

TABLE VII
ANALYSIS OF VARIANCE OF PERCENT UPTAKE OF
1-131 LABELLED T3 FOR APRIL
Source of
Variance
D.F.
Sums of
Squares
Mean
Squares
Total
35
492.56
Breed
1
30.53
30.53**
Sex
1
298.42
298.42**
Treatment
2
94.67
47.34**
Breed X Sex
1
1.41
1.41
Breed X Treatment
2
10.77
5.39*
Sex X Treatment
2
30.20
15.10**
Breed X Sex X Treatment
2
4.69
2.35
Error
23
21.87
0.951
* Probability Less
Than 0.05.
** Probability Less
Than 0.01.
to
VO

TABLE VIII
ANALYSIS OF VARIANCE OF PERCENT UPTAKE OF
1-131 LABELLED T3 FOR MAY
Source of
Variance
D.F.
Sums of
Squares
Mean
Squares
Total
35
492.54
Breed
1
19.95
19.95**
Sex
1
295.84
295.84**
Treatment
2
59.15
29.56**
Breed X Sex
1
0.01
0.01
Breed X Treatment
2
55.02
27.51**
Sex X Treatment
2
11.90
5.95**
Breed X Sex X Treatment
2
28.42
14.21**
Error
23
22.25
0.967
** Probability Less Than 0.01

TABLE IX
ANALYSIS OF VARIANCE OF PERCENT UPTAKE OF
1-131 LABELLED T3 FOR JUNE
Source of
Variance
D.F.
Sums of
Squares
Mean
Squares
Total
35
527.22
Breed
1
14.83
14.83**
Sex
1
308.01
308.01**
Treatment
2
52.90
26.45**
Breed X Sex
1
0.36
0.36
Breed X Treatment
2
58.08
29.04**
Sex X Treatment
2
2.35
1.18
Breed X Sex X Treatment
2
47.30
23.65**
Error
23
43.39
1.887
** Probability Less
Than 0.01.

TABLE X
ANALYSIS OF VARIANCE OF PERCENT UPTAKE OF
1-131 LABELLED T3 FOR JULY
Source of
Variance
D.F.
Sums of
Squares
Mean
Squares
Total
35
496.68
Breed
1
87.73
87.73**
Sex
1
119.53
119.53**
Treatment
2
66.91
33.45**
Breed X Sex
1
0.33
0.33
Breed X Treatment
2
13.91
6.95
Sex X Treatment
2
80.14
40.07**
Breed X Sex X Treatment
2
42.26
21.13**
Error
23
85.87
3.733
** Probability Less Than 0.01.

TABLE XI
ANALYSIS OF VARIANCE OF PERCENT UPTAKE OF
1-131 LABELLED T-j FOR AUGUST
Source of
Variance
D.F.
Sums of
Squares
Mean
Squares
Total
35
406.41
Breed
1
140.43
140.43**
Sex
1
116.29
116.29**
Treatment
2
54.96
27.48**
Breed X Sex
J1
1.64
1.64
Breed X Treatment
2
19.70
9.85*
Sex X Treatment
2
14.78
7.39
Breed X Sex X Treatment
2
0.41
0.205
Error
23
58.61
2.548
* Probability Less Than 0.05.
** Probability Less Than 0.01.

TABLE XII
ANALYSIS OF VARIANCE OF PERCENT UPTAKE OF
1-131 LABELLED T3 FOR SEPTEMBER
Source of
Variance
D.F.
Sums of
Squares
Mean
Squares
Total
35
311.59
Breed
1
40.11
40.11**
Sex
1
169.87
169.87**
Treatment
2
26.61
13.30**
Breed X Sex
1
2.56
2.56
Breed X Treatment
2
3.89
1.95
Sex X Treatment
2
30.09
15.04**
Breed X Sex X Treatment
2
1.83
0.915
Error
23
36.63
1.593
** Probability Less Than 0.01

TABLE XIII
ANALYSIS OF VARIANCE OF PERCENT UPTAKE OF
1-131 LABELLED T3 FOR OCTOBER
Source of
Variance
D.F.
Sums of
Squares
Mean
Squares
Total
35
322.43
Breed
1
24.51
24.51**
Sex
1
100.33
100.33**
Treatment
2
59.08
29.54**
Breed X Sex
1
4.07
4.07
Breed X Treatment
2
14.31
7.15
Sex X Treatment
2
59.65
29.82**
Breed X Sex X Treatment
2
6.88
3.44
Error
23
53.60
2.33
** Probability Less Than 0.01.

TABLE XIV
ANALYSIS OF VARIANCE OF PERCENT UPTAKE OF
1-131 LABELLED T3 FOR NOVEMBER
Source of
Variance
D.F.
Sums of
Squares
Mean
Squares
Total
35
313.65
Breed
1
59.55
59.55**
Sex
1
19.81
19.81**
Treatment
2
20.61
10.30**
Breed X Sex
1
18.34
18.34**
Breed X Treatment
2
68.47
34.24**
Sex X Treatment
2
19.48
9.74*
Breed X Sex X Treatment
2
48.50
24.25**
Error
23
58.59
2.547
* Probability Less Than 0.05.
** Probability Less Than 0.01.

TABLE XV
ANALYSIS OF VARIANCE OF PERCENT UPTAKE OF
1-131 LABELLED T3 FOR DECEMBER
Source of
Variance
D.F.
Sums of
Squares
Mean
Squares
Total
35
330.82
Breed
1
0.59
0.59
Sex
1
18.20
18.20
Treatment
2
116.79
58.39**
Breed X Sex
1
1.26
1.26
Breed X Treatment
2
58.31
29.15**
Sex X Treatment
2
32.26
.16.13
Breed X Sex X Treatment
2
5.04
2.52
Error
23
116.57
5.068
** Probability Less Than 0.01.

38
o
The higher percent uptake by Herefords in the 32 C chamber indicates
that high ambient temperatures decreased the amount of circulating
thyroid hormones.
The Brahman cattle did not follow the same ranking as the
All Animals classification. In this breed there was a reversal
between the 32 C chamber at 62.28 percent, and the 21 C chamber
at 63.03 percent. The pasture group still had the lowest percent
uptake at 60.97 percent. These data indicate that the Brahman cat
tle did not respond to heat stress by lowering the amount of
circulating thyroid hormones but were able to maintain their
secretion rate at the same level as in the 21 C chamber. This
reversal in ranking is evident in the highly significant interaction
between breed and treatment shown in Table V. Tables VI through
XV show that this interaction did not remain significant for all
months. Therefore, for three months in the experimental period
the animals did not respond the same. There may be a seasonal effect
involved as the three nonsignificant months were July, September
and October with the August collection showing a significant breed
by treatment interaction but not a highly significant difference
as in other months.
A graph of the means for each breed by months is presented
in Figure 2. This graph shows a clear distinction between the
breeds in every month but December. The same diurnal rhythm as
noted for treatment effects is evident for breeds as expected since
the data for treatment effects and breed effects are from the same
animals.
Statistical analysis of the means in Table V shows a highly
.

50
5
60
5
70
5
Mar. Apr. May June July Aug. Sept. Oct. Nov. Dec.
Figure 2. -- Monthly percent uptake of 1-131 labelled T3 for breed classification.
- Brahman
- Hereford
VO

40
significant difference between breeds. There was no significant
difference between breeds in the December collection (Table XV).
This can also be seen in Figure 2.by the two lines nearly meeting
i
in December. These data tend to reinforce the assumption that
thyroid secretion rate is one of the factors accounting for the
difference in adaptability of Hereford and Brahman cattle to heat
stress., Since the Hereford cattle had a higher percent uptake on
all treatments than did the Brahman cattle, it is assumed that
the Brahman cattle had a higher amount of circulating thyroid
hormone than the Hereford cattle in this experiment. Since the
percent uptake in the 32 C chamber was 66.67 for Herefords and 62.28
for Brahmans, it may be assumed that the Brahman cattle in this
experiment were better equipped to maintain thyroid secretion rate
under heat stress than were the Hereford cattle.
Sex effects
The mean percent uptake for the 10 month period for sex
effect is presented in Table IV under All Bulls and All Heifers.
The bulls had a higher percent uptake on all treatments than did
the heifers. The All Bulls and All Heifers classifications exhibited
the same ranking by treatments as did the All Animals classification.
A highly significant difference due to sex is shown in Table V.
There was a larger mean square for sex effect than for any other source
of variation. Analysis of monthly collections, Tables VI through
XV, showed this highly significant sex effect in all but the last
month (Table XV). The graph by months, Figure 3, shows this dif
ference between sexes and a reversal in the last month that resulted
in no significant difference due to sex in December.

50
5
60
5
70
5
/
/
- All Heifers
- All Bulls
Mar. Apr. May June July Aug. Sept. Oct. Nov. Dec.
Figure 3. -- Monthly percent uptake of 1-131 labelled Tg for sex classification.
-i>

42
There was no significant interaction between breed and sex,
Table V, indicating that the sexes respond the same to the treat
ments in both breeds. Analysis by monthly collection showed two
highly significant breed by sex interactions. These occurred in
March (Table VI) and November (Table XIV). The sex by treatment
interaction in Table V was highly significant. Analysis by months
did not show this significant interaction in June (Table IX),
August (Table XI), and December (Table XV). A part of these inter
actions can be seen in the data on Table IV. There was a reversal
of ranking in response to treatment by the Brahman bulls and
heifers. The percent uptake was 63.84 in the 32 C chamber and 65.26
in the 21 C chamber for bulls and 61,24 and 60.79 respectively
for heifers. The Hereford heifers exhibited no difference between
the 21 C chamber at 61.25 percent and the pasture group at 61.38
percent. The animals with the highest uptake of all classes were
the Hereford bulls in the 32 C chamber with 69.49 percent. The
animals with the lowest uptake were the Brahman heifers on pasture
with 59.24 percent.
The fact that percent uptake for heifers was consistently
lower than the percent uptake for bulls indicates that heifers
have a higher thyroid secretion rate than bulls and are better
able to maintain their thyroid secretion levels under heat stress.

GENERAL SUMMARY AND CONCLUSIONS
An in vitro technique employing Sephadex column chromatography
was used to estimate circulating thyroid hormone levels in beef
l
cattle. Preliminary work with this technique was conducted to as-
sertain the applicability of Sephadex chromatography to the
evaluation of thyroid status in this species. The factors studied
were: (1) the effect of different columns on percent uptake of
duplicate samples; (2) repeatability of observations on subsequent
days for estimating thyroid status; and (3) effect of age of
isotope on percent uptake of 1-131 labelled Tg by cattle serum
proteins.
It was found that there were no statistical differences
between duplicate samples placed on different columns. A highly
significant correlation was found between the two columns. It
was concluded that there were no differences between columns and
replicate determinations of a particular sample did not necessitate
the utilization of the same column. Since any residual radio
activity released by a column caused no significant differences
in the results, there was no need for a correction factor for
retention of radioactivity by the columns.
Daily blood samples obtained from an individual animal over
a 5-day period were highly correlated with a repeatability coefficient
of 0.921. Therefore, it was concluded that this technique could be
used to study an animal's thyroid secretion rate over an extended
43

44
period as any significant differences noted in the percent uptake
by a particular animal would be due to a change in the amount of
circulating thyroid hormone and not to the technique employed.
Length of storage of 1-131 labelled caused no significant
difference in percent uptake by serum proteins when old (4 half-
lives) and new (first half-life) reagents were compared. The
l
percent of free 1-131 in the reagent increased and the total
activity decreased with age. It was concluded that adjustment
of all dosages to 1,000 counts per minute and correcting for free
1-131 in the reagent allowed for the complete utilization of each
shipment of reagent disregarding half-lives.
The second phase of this study was directed towards comparing
the effect of temperature, breed, and sex on circulating thyroid
.hormone levels in Hereford and Brahman beef cattle. Three animals
of each breed and sex were maintained at 32 C, 21 C and on
pasture for 12 months.
Statistical analysis revealed that the three main effects
studied; treatment, breed, and sex, were all highly significant.
A difficulty in interpretation of these significant effects arose
from the fact that the breed X treatment, sex X treatment and
breed X sex X treatment interactions were also significant.
The data from this study showed that the pasture cattle had a
lower percent uptake or higher thyroid secretion rate than the cattle
in either chamber. The cattle in the 32 C chamber had the highest
o
percent uptake with the cattle in the 21 C chamber being intermediate
between the other two groups. All three groups exhibited a diurnal
rhythm in a graph of percent uptake by months. These data indicated

that temperature variations are important in thyroid secretion rates
as even the animals maintained in the "comfort zone" at a constant
21 C had a lower thyroid output than the animals on pasture. How
ever, no conclusion could be reached on this point due to the differ
ence in quality between the pasture and chamber animals. It was
concluded from these data that the animals were stressed at 32 C
and responded with a decreased thyroid hormone secretion rate.
On all three treatments the Hereford cattle had a higher percent
uptake than the Brahman cattle. The Hereford cattle responded to
the 32 C treatment by lowering thyroid hormone secretion rate while
the Brahman cattle were able to maintain approximately the same
thyroid secretion levels in the 32 C and 21 C chambers. From
these data, it was concluded that Brahman cattle are better equipped
to withstand heat stress than Hereford cattle by maintaining cir
culating thyroid hormone levels.
The bulls had a higher percent uptake on all treatments than
did the heifers. The Hereford bulls and heifers responded to the
32 stress by lowering thyroid hormone secretion rates while the
Brahman bulls and heifers did not. Due to the lower percent uptake
by heifers on all treatments, it was concluded that the heifers had
a higher thyroid secretion rate than the bulls and were better
equipped to maintain this higher secretion rate in response to heat
stress.
The animals with the lowest percent uptake were the Brahman
heifers on pasture with a mean uptake of 59.24 percent. The animals
with the highest percent uptake were the Hereford bulls in the 32 C
chamber with a mean uptake of 69.49 percent. The range in percent
uptake for all animals on treatment was from 55.2 to 74.3 percent.

BIBLIOGRAPHY
Albert, A. and F. R, Keating. 1952. The role of the gastro-intestinal
tract, including the liver, in the metabolism of radiothyroxine.
Endocr. 51:427.
Albright, E. C. and F. C. Larson. 1959. Metabolism of L-thyroxine
by human tissue slices. J. Clin. Invest. 38:1899.
Blaxter, K. L. 1945. The effect of iodinated protein feeding on the
lactating cow. J. Endocr. 4:237.
Blincoe, C. 1958. The influence of constant ambient temperature on
the thyroid activity and iodide metabolism of Shorthorn, Santa
Gertrudis and Brahman calves during growth. Mo. Agr. Exp. Sta.
Res. Bui. 649.
Blincoe, C., and S. Brody. 1955. The influence of ambient temperature,
air velocity, radiation intensity and starvation on thyroid
activity and iodide metabolism in cattle. Mo. Agr. Exp. Sta.
Res. Bui. 576.
Blincoe, C. and S. Brody. 1955. The influence of diurnally variable
temperatures on the thyroid activity and iodide metabolism of
Jersey and Holstein cows. Mo. Agr. Exp. Sta. Res. Bui. 579.
Brody, S. and R. F. Frankenbuch. 1942. Age changes in size, energy
metabolism and cardiorespiratory activities of thyroidectomized
cattle. Mo. Agr. Exp. Sta. Res. Bui. 349.
Brunstad, G. E. and S. H. Fowler. 1959. Thyroid status and embryonic
mortality in swine. Am. J. Physiol. 196:287.
Chase, G. D. and J. L. Rabinowitz. 1964. Principles of Radioiso
tope Methodology. 2nd Edition. Burgess Publishing Co., Minnea
polis, Minn.
Cowley, J. J. 1965, A Comparison of Two jin vitro Radioisotope
Procedures for Estimating Thyroid Status in Sheep. M. S. Thesis,
Louisiana State University, Baton Rouge, La.
Cuaron, A. and M. E. Fucugauchi. 1964. The binding of 1-131-tri
iodothyronine by serum proteins as an _in vitro test of thyroid
function. Acta. Encocrinologica 46:161.
46

47
BIBLIOGRAPHY (continued)
Deiss, W. P., E. C. Albright and F. C. Larson. 1953. Comparison of
in vitro serum protein binding of thyroxine and triiodothyronine.
Proc. Soc. Exp. Biol. Med. 84:513.
Folley, S. J. and P. White. 1936. The effect of thyroxine on milk
secretion and on the phosphotase of the blood and milk of the
lactating cow. Proc. Roy Soc. B. 120:346.
Frienkel, N., J. J. Dowling and S. H. Ingbar. 1955. The interaction
of thyroxine with plasma protein; localization of thyroxine
binding proteins in Cohn Fraction of plasma. J. Clin. Invest. 34:
1698.
Graham, W. R. 1934. The action of thyroxine on the milk and milk-fat
production of cows. Biochem. J. 28:1368.
Halmi, N. S. 1964. The accumulation and recirculation of iodide by
the thyroid. The Thyroid Gland. I. R. Pitt-Rivers and
W. R. Trotter, (eds.). Butterworths, Inc., Washington, D.C.
Hamolsky, M. W., M. Stein and A. S. Freedberg. 1957. The thyroid
hormone plasma protein complex in man. II. A new in vitro
method for study of "uptake" of labeled hormonal components of
human erythrocytes. J. Clin. Endocr. 17:33.
Hart, D. S. 1960. Fertility responses in ewes treated with thyroxine.
New Zealand J. Agr. Sci. 3:365.
Heidelberger, M. and K. 0. Pedersen. 1935. The molecular weight and
isoelectric point of thyroglobulin. J. Gen. Physiol. 19:95.
Henneman, H. A., E. P. Reineke and S. A. Griffin. 1955. Thyroid
secretion rate of sheep as affected by season, age, breed,
pregnancy, and lactation. J. Animal Sci. 14:419.
Hocman, G. 1966. The use of Sephadex in the chromatography of thyroxine
containing compounds: a critique. J. Chromatog. 21:413.
Howes, J. J. 1964. The Comparative Physiology of Bos Taurus and Bos
Indicus cattle on Two Nutritional Levels. Ph.D. dissertation,
University of Florida, Gainesville, Florida.
Jacobsson, L. and G. Widstrom. 1962. Separation of Iodine compounds
in serum by gel filtration. Scand. J. Clin. Lab. Invest. 14:285.
Jones, J. E. and J. J. Schultz. 1966. Identification of thyroidal
iodoamino acids by gel filtration and automated 1-127 determin
ation. J. Clin. Endocr. 26:975.
Lewitus, Z., M. Anbar and S. Guttman. 1961. A contribution to the
understanding of the "trapping mechanism" in the thyroid gland.
Advances in Thyroid Research. R. Pitt-Rivers (ed.). Pergamon
Press, New York.

48
BIBLIOGRAPHY (continued)
Li, J. C. R. 1961. Introduction to Statistical Inference. Edwards
Brothers Inc. Ann Arbor, Mich.
Lucas, J. J., G. E. Brunstad and S. H. Fowler. 1958. The relationship
of altered thyroid activity to various reproductive phenomena
in gilts. J. Endocr. 17:54.
Myant, N. B. 1964. The thyroid and reproduction in mammals. The
Thyroid Gland. I. R. Pitt-Rivers and W. R. Trotter (eds.).
Butterworths Inc., Washington, D. C.
Nadler, N. J. 1962. Synthesis and release of thyroid hormones. Fed.
Proc..21:628.
Pitt-Rivers, R. and J. R. Tata. 1959. The Thyroid Hormones. Pergamon
Press, New York.
Pitt-Rivers, R. and R. R. Cavalieri. 1964. Thyroid hormone biosynthesis.
The Thyroid Gland. I. R. Pitt-Rivers and W. R. Trotter (eds.).
Butterworths Inc., Washington, D. C.
Reineke, E. P. 1946. Thyroactive iodinated proteins. Vit. and Horm.
4:207.
Reineke, E. P. and F. A. Solimn. 1953. Role of the thyroid hormone
in reproductive physiology of the female. Iowa State Coll. J.
Sci. 28:67.
Robbins, J. and J. E. Rail. 1955. Thyroxine-binding capacity of
serum in normal man. J. Clin. Invest. 34:1324.
Robertson, J. D. 1945. The effect on basal metabolism of milk from
cows fed with iodinated protein. J. Endocr. 4:300.
Ryle, M. 1961. Early reproductive failure of ewes in a hot environ
ment. I. J. Agr. Sci. 51:84.
Ryle, M. 1963. Early reproductive failure of ewes in a hot environ
ment. Ill, IV, V. J. Agr. Sci. 60:95.
Shapiro, B. and J. L. Rabinowitz. 1962. A chromatographic method
utilizing Sephadex for the separation of free iodide, protein-
bound and unbound triiodothyronine in sera. J. Nuclear Med.
3:417.
Sisson, J. C. 1965. Principles of, and pitfalls in, thyroid function
tests. J. Nuclear Med. 6:853.
Spielmann, A. A., W. E. Petersen, and J. B. Fitch. 1944. Effect of
thyroidectomy on lactation in the bovine. J. Dairy Sci. 27:441.

BIBLIOGRAPHY (continued)
Spielmann, A. A., W. E. Petersen, J. B. Fitch and B. S. Pomeroy.
1945. General appearance, growth and reproduction of the
thyroidectomized bovine. J. Dairy Sci. 28:329.
Swanson, E. W., F. W. Lengemann and R. A. Monroe. 1957. Factors
affecting the thyroid uptake of 1-131 in dairy cows. J.Animal
Sci. 16:318.
Tata, J. R. 1964. Distribution and metabolism of thyroid hormones.
The Thyroid Gland. I. R. Pitt-Rivers and W. R. Trotter.(eds.).
Butterworths Inc., Washington, D. C.
Taurog, A., J. 0. Wheat, and L. L. Chaikoff. 1956. Nature of the
iodine-131 compounds appearing in the thyroid vein after in
jection of iodine-131. Endocr. 58:121.
Yeates, N.T.M. 1958. Foetal dwarfism in sheep an effect of high
atmospheric temperature during gestation. J. Agr. Sci. 51:84.

BIOGRAPHICAL SKETCH
Jerry Jennings Cowley, son of Pauline Cowley and the late
Jack Cowley, was born July 14, 1940, in Marshall, Texas. He
attended Marshall public schools and was graduated from Marshall
Senior High School in 1958.
He received the degree of Bachelor of Science in Animal
Science from Texas A & M University in June, 1963. He then en
tered graduate school at Louisiana State University and received
the degree of Master of Science in Animal Science in August, 1965.
In September, 1965, he entered the University of Florida and is
now a candidate for the degree of Doctor of Philosophy.
Mr. Cowley is married to the former Marilyn Jones. He is
a member of Gamma Sigma Delta, national honorary agricultural
society.

This dissertation was prepared under the direction of the chair
man of the candidate's supervisory committee and has been approved by
all members of that committee. It was submitted to the Dean of the
College of Agriculture and to the Graduate Council, and was approved
as partial fulfillment of the requirements for the degree Doctor of
Philosophy.
December 1968
w>
Dean, Graduate School
Supervisory Committee:
Chairman



50
5
60
5
70
5
Mar. Apr. May June July Aug. Sept. Oct. Nov. Dec.
Figure 2. -- Monthly percent uptake of 1-131 labelled T3 for breed classification.
- Brahman
- Hereford
VO


TABLE VII
ANALYSIS OF VARIANCE OF PERCENT UPTAKE OF
1-131 LABELLED T3 FOR APRIL
Source of
Variance
D.F.
Sums of
Squares
Mean
Squares
Total
35
492.56
Breed
1
30.53
30.53**
Sex
1
298.42
298.42**
Treatment
2
94.67
47.34**
Breed X Sex
1
1.41
1.41
Breed X Treatment
2
10.77
5.39*
Sex X Treatment
2
30.20
15.10**
Breed X Sex X Treatment
2
4.69
2.35
Error
23
21.87
0.951
* Probability Less
Than 0.05.
** Probability Less
Than 0.01.
to
VO


BIBLIOGRAPHY (continued)
Spielmann, A. A., W. E. Petersen, J. B. Fitch and B. S. Pomeroy.
1945. General appearance, growth and reproduction of the
thyroidectomized bovine. J. Dairy Sci. 28:329.
Swanson, E. W., F. W. Lengemann and R. A. Monroe. 1957. Factors
affecting the thyroid uptake of 1-131 in dairy cows. J.Animal
Sci. 16:318.
Tata, J. R. 1964. Distribution and metabolism of thyroid hormones.
The Thyroid Gland. I. R. Pitt-Rivers and W. R. Trotter.(eds.).
Butterworths Inc., Washington, D. C.
Taurog, A., J. 0. Wheat, and L. L. Chaikoff. 1956. Nature of the
iodine-131 compounds appearing in the thyroid vein after in
jection of iodine-131. Endocr. 58:121.
Yeates, N.T.M. 1958. Foetal dwarfism in sheep an effect of high
atmospheric temperature during gestation. J. Agr. Sci. 51:84.


40
significant difference between breeds. There was no significant
difference between breeds in the December collection (Table XV).
This can also be seen in Figure 2.by the two lines nearly meeting
i
in December. These data tend to reinforce the assumption that
thyroid secretion rate is one of the factors accounting for the
difference in adaptability of Hereford and Brahman cattle to heat
stress., Since the Hereford cattle had a higher percent uptake on
all treatments than did the Brahman cattle, it is assumed that
the Brahman cattle had a higher amount of circulating thyroid
hormone than the Hereford cattle in this experiment. Since the
percent uptake in the 32 C chamber was 66.67 for Herefords and 62.28
for Brahmans, it may be assumed that the Brahman cattle in this
experiment were better equipped to maintain thyroid secretion rate
under heat stress than were the Hereford cattle.
Sex effects
The mean percent uptake for the 10 month period for sex
effect is presented in Table IV under All Bulls and All Heifers.
The bulls had a higher percent uptake on all treatments than did
the heifers. The All Bulls and All Heifers classifications exhibited
the same ranking by treatments as did the All Animals classification.
A highly significant difference due to sex is shown in Table V.
There was a larger mean square for sex effect than for any other source
of variation. Analysis of monthly collections, Tables VI through
XV, showed this highly significant sex effect in all but the last
month (Table XV). The graph by months, Figure 3, shows this dif
ference between sexes and a reversal in the last month that resulted
in no significant difference due to sex in December.


TABLE XIII
ANALYSIS OF VARIANCE OF PERCENT UPTAKE OF
1-131 LABELLED T3 FOR OCTOBER
Source of
Variance
D.F.
Sums of
Squares
Mean
Squares
Total
35
322.43
Breed
1
24.51
24.51**
Sex
1
100.33
100.33**
Treatment
2
59.08
29.54**
Breed X Sex
1
4.07
4.07
Breed X Treatment
2
14.31
7.15
Sex X Treatment
2
59.65
29.82**
Breed X Sex X Treatment
2
6.88
3.44
Error
23
53.60
2.33
** Probability Less Than 0.01.


18
Results
The data from this study, presented in Table II, were analyzed
by the correlation coefficient using days as variables and by an
analysis of repeatability (R) Highly significant correlation
coefficients above 0.92 were found for each comparison. The R value
was 0.921. These data indicate very good repeatability on a day to
day basis. On a long term study, differences in percent uptake could
be assumed to be due to thyroid hormone secretion rate and not to
variation in the technique employed.
Effect of Age of Isotope on Percent Uptake
of 1-131 Labelled _T3 by Cattle Serum Proteins
In routine laboratory procedure, 1-31 is usually discarded after V1
one half-life. Half-life of an isotope is defined as the length of
time required for one-half of the radioactive atoms in an isotope
to undergo decay. Chase and Rabinowitz (1964) state that the half-
life of 1-131 is 8.05 days. During the decay of 1-131 labelled T3
there is a disassociation of the 1-131 molecule from the triiodothyro
nine molecule resulting in free 1-131 in the reagent as well as a
decrease in the total amount of radioactivity.
Shapiro and Rabinowitz (1962) reported that there is no problem
with free 1-131 in the Sephadex chromatography technique because of
a correction factor employed. This correction should provide for
longer utilization of a particular sample of 1-131 labelled T3.
Cowley (1965) reported that 1-131 labelled T3 that had been through
8 half-lives did not yield significantly different percent uptake
than did the isotope in its first half-life.
The purpose of this study was to determine if 1-131 labelled T3


ACKNOWLEDGMENTS
The author wishes to express his sincere appreciation to
Dr. Alvin Warnick, chairman of his supervisory committee, for his
counsel and assistance throughout this study.
Appreciation is also expressed to Dr. Marvin Koger, Dr. Ray
Shirley.and Dr. G. T. Edds who served as members of the supervisory
committee and to Dr. J. P. Feaster for guidance in the laboratory.
The author wishes to thank Mr. Dean Pogue and Mr. Hernando
Gutierrez for their assistance in the gathering of data and care
of experimental animals.
The author extends his deepest gratitude to his wife, Marilyn,
for her constant encouragement and untiring efforts which were
invaluable in achieving this goal.
ii


TABLE XV
ANALYSIS OF VARIANCE OF PERCENT UPTAKE OF
1-131 LABELLED T3 FOR DECEMBER
Source of
Variance
D.F.
Sums of
Squares
Mean
Squares
Total
35
330.82
Breed
1
0.59
0.59
Sex
1
18.20
18.20
Treatment
2
116.79
58.39**
Breed X Sex
1
1.26
1.26
Breed X Treatment
2
58.31
29.15**
Sex X Treatment
2
32.26
.16.13
Breed X Sex X Treatment
2
5.04
2.52
Error
23
116.57
5.068
** Probability Less Than 0.01.


TABLE III
PERCENT UPTAKE OF OLD AND NEW 1-131 LABELLED T
BY CATTLE SERUM PROTEINS J
Cow
4 Half-lives
First Half-life
1
64.54
66.52
2.
65.65
68.10
3
64.63
63.10
4
65.47
66.38
5
59.48
57.68
6
65.85
67.17
7
58.88
59.81
8
61.45
60.35
Mean
63.24
63.64


LIST OF FIGURES
Page
Figure
1. Monthly percent uptake of 1-131 labelled for
treatment classification ... 26
2. Monthly percent.uptake of 1-131 labelled for
breed classification 39
3. Monthly percent uptake of 1-131 labelled for
sex classification 41
vi
/


BIOGRAPHICAL SKETCH
Jerry Jennings Cowley, son of Pauline Cowley and the late
Jack Cowley, was born July 14, 1940, in Marshall, Texas. He
attended Marshall public schools and was graduated from Marshall
Senior High School in 1958.
He received the degree of Bachelor of Science in Animal
Science from Texas A & M University in June, 1963. He then en
tered graduate school at Louisiana State University and received
the degree of Master of Science in Animal Science in August, 1965.
In September, 1965, he entered the University of Florida and is
now a candidate for the degree of Doctor of Philosophy.
Mr. Cowley is married to the former Marilyn Jones. He is
a member of Gamma Sigma Delta, national honorary agricultural
society.


50
5
60
5
70
5
/
/
- All Heifers
- All Bulls
Mar. Apr. May June July Aug. Sept. Oct. Nov. Dec.
Figure 3. -- Monthly percent uptake of 1-131 labelled Tg for sex classification.
-i>


EXPERIMENTAL PROCEDURE
Sephadex column chromatography was used to ascertain thyroid
status of beef cattle. The assay employed the technique of Shapiro
and Rabinowitz (1962) as modified by Cowley (1965). The complete
procedure is as follows:
1. Swell 3.0 grams Sephadex G-25 medium grade in 0.2 normal
phosphate buffer for 6 hours at room temperature. Pour
the gel into a Sephadex K-9 laboratory column fitted with
*
a luer ending outlet port.
2. Add 0.1 milliliter of 1-131 labelled T^ diluted to 1,000
counts per minute in buffer solution to 3 milliliters of
blood serum.
3. Incubate sample for 15 minutes in a water bath at 37 C.
4. Remove 1 milliliter of serum and assay for activity in a
scintillation counter equipped with a sodium iodide crystal.
This serves as the standard.
5. Place 1 milliliter of sample on top of gel bed taking care
not to disturb the gel surface.
6. After the serum enters the bed, add 2 separate 1 milliliter
aliquots of buffer solution.
7. When the buffer solution completely enters the gel bed, fill
the column with buffer solution and start collection of 1
milliliter elutes until 12 eluates are obtained.
8. Wash the column with 20 milliliters of blood serum diluted 10
13


SWi
TABLE IV
MEAN PERCENT UPTAKE OF 1-131 LABELLED T3 FOR TEN-MONTH PERIOD
All
Hereford
Brahman
All
All
Treatment
Animals
All
Heifers
Bulls
All
Heifers
Bulls
Bulls
Heifers
32 C
Chamber
64.67
66.67
63.85
69.49
62.28
61.24 '
63.84
67.23
62.55
21 C
Chamber
63.25
63.47
61.25
65.70
63.03
60.79
65.26
65.48
61.02
Pasture
61.67
62.37
61.38
63.35
60.97
59.24
62.71
63.03
60.31
to
4>


§0
N>
ON
W C


BIBLIOGRAPHY
Albert, A. and F. R, Keating. 1952. The role of the gastro-intestinal
tract, including the liver, in the metabolism of radiothyroxine.
Endocr. 51:427.
Albright, E. C. and F. C. Larson. 1959. Metabolism of L-thyroxine
by human tissue slices. J. Clin. Invest. 38:1899.
Blaxter, K. L. 1945. The effect of iodinated protein feeding on the
lactating cow. J. Endocr. 4:237.
Blincoe, C. 1958. The influence of constant ambient temperature on
the thyroid activity and iodide metabolism of Shorthorn, Santa
Gertrudis and Brahman calves during growth. Mo. Agr. Exp. Sta.
Res. Bui. 649.
Blincoe, C., and S. Brody. 1955. The influence of ambient temperature,
air velocity, radiation intensity and starvation on thyroid
activity and iodide metabolism in cattle. Mo. Agr. Exp. Sta.
Res. Bui. 576.
Blincoe, C. and S. Brody. 1955. The influence of diurnally variable
temperatures on the thyroid activity and iodide metabolism of
Jersey and Holstein cows. Mo. Agr. Exp. Sta. Res. Bui. 579.
Brody, S. and R. F. Frankenbuch. 1942. Age changes in size, energy
metabolism and cardiorespiratory activities of thyroidectomized
cattle. Mo. Agr. Exp. Sta. Res. Bui. 349.
Brunstad, G. E. and S. H. Fowler. 1959. Thyroid status and embryonic
mortality in swine. Am. J. Physiol. 196:287.
Chase, G. D. and J. L. Rabinowitz. 1964. Principles of Radioiso
tope Methodology. 2nd Edition. Burgess Publishing Co., Minnea
polis, Minn.
Cowley, J. J. 1965, A Comparison of Two jin vitro Radioisotope
Procedures for Estimating Thyroid Status in Sheep. M. S. Thesis,
Louisiana State University, Baton Rouge, La.
Cuaron, A. and M. E. Fucugauchi. 1964. The binding of 1-131-tri
iodothyronine by serum proteins as an _in vitro test of thyroid
function. Acta. Encocrinologica 46:161.
46


that temperature variations are important in thyroid secretion rates
as even the animals maintained in the "comfort zone" at a constant
21 C had a lower thyroid output than the animals on pasture. How
ever, no conclusion could be reached on this point due to the differ
ence in quality between the pasture and chamber animals. It was
concluded from these data that the animals were stressed at 32 C
and responded with a decreased thyroid hormone secretion rate.
On all three treatments the Hereford cattle had a higher percent
uptake than the Brahman cattle. The Hereford cattle responded to
the 32 C treatment by lowering thyroid hormone secretion rate while
the Brahman cattle were able to maintain approximately the same
thyroid secretion levels in the 32 C and 21 C chambers. From
these data, it was concluded that Brahman cattle are better equipped
to withstand heat stress than Hereford cattle by maintaining cir
culating thyroid hormone levels.
The bulls had a higher percent uptake on all treatments than
did the heifers. The Hereford bulls and heifers responded to the
32 stress by lowering thyroid hormone secretion rates while the
Brahman bulls and heifers did not. Due to the lower percent uptake
by heifers on all treatments, it was concluded that the heifers had
a higher thyroid secretion rate than the bulls and were better
equipped to maintain this higher secretion rate in response to heat
stress.
The animals with the lowest percent uptake were the Brahman
heifers on pasture with a mean uptake of 59.24 percent. The animals
with the highest percent uptake were the Hereford bulls in the 32 C
chamber with a mean uptake of 69.49 percent. The range in percent
uptake for all animals on treatment was from 55.2 to 74.3 percent.


44
period as any significant differences noted in the percent uptake
by a particular animal would be due to a change in the amount of
circulating thyroid hormone and not to the technique employed.
Length of storage of 1-131 labelled caused no significant
difference in percent uptake by serum proteins when old (4 half-
lives) and new (first half-life) reagents were compared. The
l
percent of free 1-131 in the reagent increased and the total
activity decreased with age. It was concluded that adjustment
of all dosages to 1,000 counts per minute and correcting for free
1-131 in the reagent allowed for the complete utilization of each
shipment of reagent disregarding half-lives.
The second phase of this study was directed towards comparing
the effect of temperature, breed, and sex on circulating thyroid
.hormone levels in Hereford and Brahman beef cattle. Three animals
of each breed and sex were maintained at 32 C, 21 C and on
pasture for 12 months.
Statistical analysis revealed that the three main effects
studied; treatment, breed, and sex, were all highly significant.
A difficulty in interpretation of these significant effects arose
from the fact that the breed X treatment, sex X treatment and
breed X sex X treatment interactions were also significant.
The data from this study showed that the pasture cattle had a
lower percent uptake or higher thyroid secretion rate than the cattle
in either chamber. The cattle in the 32 C chamber had the highest
o
percent uptake with the cattle in the 21 C chamber being intermediate
between the other two groups. All three groups exhibited a diurnal
rhythm in a graph of percent uptake by months. These data indicated


42
There was no significant interaction between breed and sex,
Table V, indicating that the sexes respond the same to the treat
ments in both breeds. Analysis by monthly collection showed two
highly significant breed by sex interactions. These occurred in
March (Table VI) and November (Table XIV). The sex by treatment
interaction in Table V was highly significant. Analysis by months
did not show this significant interaction in June (Table IX),
August (Table XI), and December (Table XV). A part of these inter
actions can be seen in the data on Table IV. There was a reversal
of ranking in response to treatment by the Brahman bulls and
heifers. The percent uptake was 63.84 in the 32 C chamber and 65.26
in the 21 C chamber for bulls and 61,24 and 60.79 respectively
for heifers. The Hereford heifers exhibited no difference between
the 21 C chamber at 61.25 percent and the pasture group at 61.38
percent. The animals with the highest uptake of all classes were
the Hereford bulls in the 32 C chamber with 69.49 percent. The
animals with the lowest uptake were the Brahman heifers on pasture
with 59.24 percent.
The fact that percent uptake for heifers was consistently
lower than the percent uptake for bulls indicates that heifers
have a higher thyroid secretion rate than bulls and are better
able to maintain their thyroid secretion levels under heat stress.


RESULTS
Effect of Different Columns on
Percent Uptake of Duplicate Samples
l
Sisson (1965) stated that an undesirable characteristic of a
glass chromatographic column was the retention of radioactivity.
Release of this radioactivity from the column could inject a source
of error into the experiment. Cowley (1965) studied the repeatability
of percent uptake of 1-131 labelled of duplicate samples of sheep
serum in different glass chromatographic columns packed with Sephadex
G-25. He found no significant differences in uptake due to the
column used with a correlation between uptake by the two columns of
0.99.
The objective of this study was to determine the repeatibility
of 1-131 labelled T^ uptake by cattle serum proteins using duplicate
samples in 2 different plastic chromatographic columns.
Procedure
Ten cows were used as blood donors. Twenty-five mililiters of
blood was collected from the jugular vein of each and allowed to clot.
Five mililiters of serum was removed and treated with 0.2 mililiter
1-131 labelled T^ diluted to 1,000 counts per minute. The sample
was then placed on separate columns. The procedure described on
page 13 for application, collection and calculation was followed.
Results
The data obtained from this study, presented in Table I, were
15


TABLE II
PERCENT UPTAKE OF 1-131 LABELLED T3 BY CATTLE SERUM
FOR 5 DAYS AS MEASURED BY SEPHADEX CHROMATOGRAPHY
Cow
Day 1
Day 2
Day 3
Day 4
Day 5
Mean
Variance
1
66.39
65.92
66.89
66.51
65.22
66.19
0.349
2
60.70
60.30
61.40
61.90
62.00
61.26
0.440
3
61.00
61.10
60.20
61.10
61.50
60.98
0.540
4
59.90
60.80
61.00
61.20
60.90
60.76
0.202
5
59.50
61.00
59.70
61.80
61.00
60.60
0.756
61.50
61.82
61.84
62.50
62.12
61.96
Means


10
attributed to seasonal temperatures, but the average differences
found between seasonal measurements were relatively unimportant.
They concluded that "measuring thyroid activity of cows by this
method on an uncontrolled survey basis could not be expected to
provide reliable comparable data."
Howes (1964) measured the 1-131 uptake on 24 nonpregnant
Herefcrd and Brahman heifers. He found that the thyroid glands of
the Hereford concentrated 1-131 faster than the Brahmans and the
thyroid glands of the Herefords retained a significantly greater
percentage of the injected 1-131 at comparable time intervals than
did the Brahman heifers.
Sephadex Chromatography
Gel filtration first became an established laboratory technique
with the introduction of Sephadex in 1959. The use of a chroma
tographic method employing Sephadex gel for estimating thyroid status
was reported by Shapiro and Rabinowitz (1962).
Sephadex is a modified dextran. The dextran macromolocules
are cross linked to give a three-dimensional network of polysac
charide chains. Because of its high content of hydroxyl groups,
Sephadex is strongly hydrophilic and the Sephadex beads swell
considerably in water and electrolyte solutions giving a porus gel.
Only molecules below a certain size (below 5,000 molecular weight
with Sephadex G-25) can enter the interstices. Heavier molecules
are excluded from the pores and pass through the bed in the liquid
phase outside the particles, passing through the column more rapidly
than the smaller molecules entering the pores. Molecules are therefore
eluted from a Sephadex bed in the order of decreasing molecular size.


TABLE IX
ANALYSIS OF VARIANCE OF PERCENT UPTAKE OF
1-131 LABELLED T3 FOR JUNE
Source of
Variance
D.F.
Sums of
Squares
Mean
Squares
Total
35
527.22
Breed
1
14.83
14.83**
Sex
1
308.01
308.01**
Treatment
2
52.90
26.45**
Breed X Sex
1
0.36
0.36
Breed X Treatment
2
58.08
29.04**
Sex X Treatment
2
2.35
1.18
Breed X Sex X Treatment
2
47.30
23.65**
Error
23
43.39
1.887
** Probability Less
Than 0.01.


IN VITRO MEASUREMENT OF CIRCULATING
THYROID HORMONE LEVELS IN
BEEF CATTLE
By
JERRY JENNINGS COWLEY
A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1968


TABLE OF CONTENTS
Page
ACKNOWLEDGMENTS ii
LIST OF TABLES iv
LIST OF FIGURES vi
INTRODUCTION I
REVIEW OF LITERATURE 3
General Thyroid Physiology 3
Reproduction and Lactation 6
Temperature and Thyroid Activity. 8
Sephadex Chromatography 10
EXPERIMENTAL PROCEDURE 13
RESULTS 15
Effect of Different Columns on Percent Uptake
of Duplicate Samples 15
Repeatability of Observations on Subsequent
Days for Estimating Thyroid Status in
Beef Cattle 17
Effect of Age of Isotope on Percent Uptake of
1-131 Labelled T^ by Cattle Serum Proteins 18
^ Effect of Temperature, Breed, and Sex on Thyroid
^ Secretion Rate of Beef Cattle 22
GENERAL SUMMARY AND CONCLUSIONS 43
BIBLIOGRAPHY 46
iii


TABLE XIV
ANALYSIS OF VARIANCE OF PERCENT UPTAKE OF
1-131 LABELLED T3 FOR NOVEMBER
Source of
Variance
D.F.
Sums of
Squares
Mean
Squares
Total
35
313.65
Breed
1
59.55
59.55**
Sex
1
19.81
19.81**
Treatment
2
20.61
10.30**
Breed X Sex
1
18.34
18.34**
Breed X Treatment
2
68.47
34.24**
Sex X Treatment
2
19.48
9.74*
Breed X Sex X Treatment
2
48.50
24.25**
Error
23
58.59
2.547
* Probability Less Than 0.05.
** Probability Less Than 0.01.


activity but the addition of radiant energy depressed the thyroid
activity. Thus, increasing the thermal stress on cows either by
increased ambient temperature or by increased radiation reduced the
thyroid activity. As heat stress increased, the rate of clearance
of plasma iodide by the thyroid decreased and the excretion rate
increased.
Blincoe and Brody (1955) studied the influence of diurnally
variable temperatures on the thyroid activity of Jersey and Holstein
cows. A daily temperature cycle of -10 to 4 C increased thyroid
activity by 20 percent over the value of a "comfort zone" cycle.'
A temperature cycle of 21 to 38 C decreased the thyroid activity
by about 30 percent below its value in the "comfort zone" cycle.
These data roughly paralleled the heat production data for the
same animals.
Blincoe (1958) studied the influence of constant ambient
temperature on the thyroid activity of Shorthorn, Santa Gertrudis
and Brahman calves. The 3 heifers of each breed were maintained
at 10 C, 27 C, and in an open shed. The rate constant for hormone
release of the Shorthorn heifers was decreased 45 percent at 27 C.
The Brahman heifers showed no decrease and the Santa Gertrudis
were slightly decreased.
The Shorthorn calves raised at 10 C were most affected by high
temperature. At 38 C their thyroid secretory activity was reduced
60 percent below the reading at 10 C. The Santa Gertrudis were less
affected and the Brahmans practically unaffected.
Swanson e_t a_l. (1957) studied the 1-131 uptake of 8 Jersey and
Guernsey cows for 18 months. They found effects which could be


had access to grass during the experiment. Treatment was confounded
with phenotype of the test animals because the 12 pasture animals
had been selected as breeding stock and were superior to the chamber
animals. However, these pasture cattle were of value in indicating
the response of good quality cattle under good nutritional conditions.
During the twelve-month experimental period, a 25 milliliter
blood sample was collected from each animal every 28 days. The
blood sample was allowed to clot and the serum withdrawn. The
blood serum was analyzed for thyroid hormones employing the Sephadex
chromatography procedure given on page 13.
Results
Due to a malfunction of the scintillation counter used to
assay for radioactivity, the first 2 months data were lost.
Therefore, data is only available for 10 consecutive months.
Treatment effects
The mean percent uptake for the 10 month period for treatment
effects is presented in Table IV. The data for all animals on
treatment shows a higher thyroid secretion rate, or lower percent
uptake, for cattle on pasture (61.67 percent uptake) than for the
cattle in either chamber. The cattle in the 32 C chamber had the
highest percent uptake, 64.67 percent, with the cattle in the 21 C
chamber being intermediate at 63.25 percent.
The analysis of variance of the 10 month means is presented
in Table V. A highly significant difference between treatments
indicates that there were different responses to the treatments.
A graph of the means for treatment effect is presented in
Figure 1. A definite diurnal rhythm was noted in all three groups.


6
(1) liver, kidney and posterior pituitary which show a high turnover
rate; (2) skeletal muscle and intestine which exhibit a slow turnover
rate; and (3) brain, spleen and gonads which accumulate only very
low levels. Albert and Keating (1952) have shown the liver to be
the most active tissue in concentrating and metabolizing thyroid
hormones. The intense enterohepatic circulation of thyroid hormones
is primarily responsible for rapid thyroid hormone catabolism and
excretion.
Robbins and Rail (1955) stated that the bilary-fecal pathway
by which most of the iodine is excreted as unchanged hormone in
the feces is the predominant pathway in rats. Albright and Larson
(1959) have shown urine to be the predominant excretory pathway
in man.
Reproduction and Lactation
Brody and Frankenbuch (1942) were among the first to show that
thyroidectomized cows fail to manifest normal physical signs of
heat, and that administration of thyroid material restores normal
estrual behavior in some thyroidectomized cows. Blaxter (1945)
reported that thyroid therapy shortened the post-partum breeding
interval.
Brunstad and Fowler (1958) found that crown-rump length of normal
embryos carried by thyroprotein-treated and control gilts were signi
ficantly larger than those of thiouracil-treated gilts. Lucas e_t al.
(1958) reported pigs born to thyroprotein-treated gilts to be heavier
than those born to control and thiouracil-treated gilts. They reported
increased embyronic mortality from the 25th day of pregnancy to


TABLE X
ANALYSIS OF VARIANCE OF PERCENT UPTAKE OF
1-131 LABELLED T3 FOR JULY
Source of
Variance
D.F.
Sums of
Squares
Mean
Squares
Total
35
496.68
Breed
1
87.73
87.73**
Sex
1
119.53
119.53**
Treatment
2
66.91
33.45**
Breed X Sex
1
0.33
0.33
Breed X Treatment
2
13.91
6.95
Sex X Treatment
2
80.14
40.07**
Breed X Sex X Treatment
2
42.26
21.13**
Error
23
85.87
3.733
** Probability Less Than 0.01.