ABSTRACT

THYROID FUNCTION IN THE LACTATING
RAT, COW, AND EWE

BY

Fritz L. Lorscheider

The author had previously demonstrated, using a direct-
output method, that thyroid hormone secretion rate (TSR) was
markedly reduced in the rat during lactation. This was
postulated to be due to a relative iodine deficiency result-
ing from the large losses of iodine occurring in milk.
Inasmuch as this TSR method was not adaptable in other than
small laboratory animals, other means were sought to further
substantiate observations on thyroid function as related to
lactation in rats and dairy cattle, to the nonbreeding cow and
heifer, and to lactation, phenothiazine treatment, and wool
growth in sheep. Thyroid function was measured by a competi-
tive protein-binding technique (Tetrasorb-lZS method) which
directly measures the serum thyroxine (T4) level.

A comparison of the serum T4 method and TSR by the direct-
output method in nonlactating and lactating rats established
that both methods detected a greater than 50% decrease in

thyroid function by 16 days lactation. In another experiment

Fritz L. Lorscheider

using heavily lactating rats suckling 12 pups each, serum T4
at 16 days lactation was reduced to one-half that of non-
pregnant, nonlactating controls. Addition of 10 times the
usual amount of iodine to the diet of another group of lac—
tating rats suckling 12 pups did not effect the serum T4 level.
In the same eXperiment lactating rats suckling 9, 6 and 5
pups had serum T4 levels that were inversely correlated to
lactational intensity. Serum T4 levels remained unchanged
in nonlactating rats when exogenous ovine prolactin was ad-
ministered. High serum prolactin, associated with nursing,
did not appear to be a direct contributing factor to the re—
duction in serum T4 observed during lactation.

Holstein cows showed no change in serum T4 during preg-
nancy when compared to dry-open cows. At peak lactation
high producing cows had serum T4 values that were only 50%
those of dry-Open and dry-pregnant cows. When milk production
had declined to 28 lbs per day, at about 5 months postpartum,
serum T4 levels returned to those of dry-Open or dry-pregnant
cows. In cows diagnosed as nonbreeders or as having cystic
ovaries, serum T4 was markedly reduced. Nonbreeding heifers
showed no reduction in serum T4. Normal heifers had higher
serum T4 levels than those of normal adult cows.

Suffolk ewes, like Holstein cows, exhibited no change
in serum T4 during pregnancy. At 30 days lactation both
Suffolk and Hampshire ewes showed a significant reduction in

serum T4 when compared with dry-pregnant or yearling ewes.

Fritz L. Lorscheider

Lactating Hampshire ewes developed a considerable amount of
wool slippage. Regrowth of wool was greatly enhanced and
serum T4 levels were significantly elevated to those of dry
controls when 0.5 g of thyroprotein was supplied daily in
the grain ration. Removal of phenothiazine from the diet
of lactating Hampshire ewes resulted in an increase in serum
T4 levels to those of dry-open controls. It appeared that
both phenothiazine and lactation may contribute to a reduc-
tion of serum T4 in the ewe and could confound the problem
of wool loss observed during advanced pregnancy and lactation.
It was concluded that thyroid function, as determined by
serum T4 assay, was significantly reduced in the heavily
lactating rat and Holstein cow. It was postulated that the
primary reason for the reduction in serum T4 during lactation
is the diversion of iodine to the mammary glands. A signifi—
cant reduction in serum T4 in cows, but not in heifers, was
associated with nonbreeding and cystic ovaries. The reduc-
tion in serum T4 in the lactating Suffolk and Hampshire ewes
may have been due in part to phenothiazine normally supplied
as an anthelmintic in the diet. Wbol loss occurring in
Hampshire ewes during late pregnancy and lactation, appeared
to be associated with a reduction in serum T4 and was be-
lieved to be due in part to lactation and in part to pheno-
thiazine treatment. After wool slippage the regrowth of
wool was greatly enhanced and the serum T4 was elevated when

thyroprotein was added to the diet.

THYROID FUNCTION IN THE LACTATING

RAT, COW, AND EWE

BY

1.\’
Fritz LP Lorscheider

A THESIS

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

DOCTOR OF PHILOSOPHY

Department of Physiology

1970

<C§JZEXQ,
/«/~70

ACKNOWLEDGMENTS

I wish to eXpress my sincere gratitude to Dr. E. P.
Reineke for his guidance and understanding throughout all
phases of my laboratory and curricular endeavors while at
Michigan State University.

I am also indebted to Drs. H. A. Henneman and W. D.
Oxender for their collaboration and assistance, without
which, the agricultural and clinical investigations reported
herein would not have been possible. Thanks are also due to
Mr. Walter Wan for his assistance with the serum thyroxine
analyses.

This work was supported by the National Institutes of
Health research grant no. AM-08513, National Institutes of
Health training grant no. GM—1121-05 and the Michigan State
University Agricultural EXperiment Station.

 

ii

TABLE OF

LIST OF TABLES. . . r .
LIST OF FIGURES . . . .

LIST OF PLATES. . . . .

INTRODUCTION. . . . . .
LITERATURE REVIEW . . .

MATERIALS AND METHODS .

CONTENTS

 

I. Rat Experiments.
II. Cow Experiments.

III.

Ewe EXperiments.

IV. Serum Thyroxine (T4) Analysis.
1) Procedure.
2) Standard Curves.

RESULTS AND DISCUSSION.

I. Rat ExPeriments.
II. Cow Experiments.

III.

SUMMARY AND CONCLUSIONS

REFERENCES CITED. . . .

APPENDICES . C C O O O O

A. Rat Feed Mixture

Ewe EXperiments.

B. M. S. U. Standard Dairy Ration

C. M. S. U. Sheep Ration.

iii

Page

vi

vii

.15
15
15
16
17
17
19
22
22
52
59
50
55
62
62
65

64

TABLE OF CONTENTS--continued Page

APPENDICES

D. Serum Thyroxine Measurements in Nonlac—
tating and'Laotating Rats . . . . . . . . . 66

E. Rat Serum T4 and PBI vs. Lactational Intens-
Bity. I O O O O O O O O O O O O O O O O O O 67

F. Serum Thyroxine Measurements, Reproduction,
and Milk Records of Holstein Cows . . . . . 69

G. KSerum Thyroxine Measurements in Yearling,
Pregnant, and Lactating Suffolk Ewes. . . . 71

H. Serum Thyroxine and FBI Measurements in

Yearling, Lactating, and Lactating Thyro-
protein-Fed Hampshire Ewes. . . . . . . . . 75

iv

TABLE

LIST OF TABLES

Page
Effect of Prolactin Injections on Serum
.Thyroxine in the Adult Female Rat. . . . . . . 27
Paired Comparison of Serum Thyroxine in
Holstein Cows During Pregnancy and Different
Stages of Lactation. . . . . . . . . . . . . . 55
Serum Thyroxine Values in Holstein Cows Diag-
nosed as Nonbreeders or as Having Cystic
ovaries O C C O O O C O O O O O O O O O O I O O 57
.Serum Thyroxine Values in Normal and Nonbreed-
ing Holstein Heifers . . . . . . . . . . . . . 58
Serum Thyroxine in Nonlactating and Lactating
Hampshire Ewes vs. Phenothiazine Treatment . . 47

LIST OF FIGURES

FIGURE Page

1. Comparison of serum thyroxine method and
thyroid secretion rate by the direct-output
method in nonlactating and lactating rats. . . 25

2. Rat serum thyroxine vs. lactational intensity
and elevated iodine intake . . . . . . . . . . 25

5. Thyroid, mammary and kidney distribution of
iodine in the nonlactating and lactating rat . 51

4. Serum thyroxine in Holstein cows during
various stages of gestation and lactation. . . 55

5. Serum thyroxine in Suffolk and Hampshire ewes

during gestation, lactation, and thyroprotein
feeding. . . . . . . . . . . . . . . . . . . . 40

vi

PLATE

1.

LIST OF PLATES

Page
Control Hampshire ewe number 421 about 2 1/2
months after lambing. . . . . . . . . . . . . . 45
Hampshire ewe number 505 about 2 1/2 months
after lambing and thyroprotein treatment. . . . 44

vii

INTRODUCTION

Studies in many Species, ranging from rat to man, have
revealed that a considerable amount of iodide is secreted
in milk. As a result of this implied thyroid-mammary
competition for iodine it had been prOposed that intense
lactation might result in a functional thyroid deficiency
(Flamboe and Reineke, 1959). The author has previously demon-
strated that rat mammary secretion of 1131 is of such magni-
tude that thyroidal Ilal uptake is reduced by as much as
eighty-six percent during lactation. These studies have
also shown that the rat thyroid hormone secretion rate (TSR),
as evaluated by a "direct-output" method, is markedly reduced,
supposedly due to the mammary diversion of iodide with the
onset of lactation (Lorscheider, 1967).

Other investigators had previously suggested that thy-
roid activity is increased during lactation (Turner and
Cupps, 1959; Monroe and Turner, 1946; Brown-Grant, 1956;
Grosvenor and Turner, 1958a), but many earlier reports have
established that thyroprotein feeding increases lactational
performance (Graham, 1954; Reineke and Turner, 1942; Reineke,
1945). The latter implies that lactation might be self-

limiting due in part to a functional deficiency of thyroid

hormone. In women either thyroid hormone or iodine therapy
has resulted in an increase in lactational performance of

up to three-hundred percent (Robinson, 1947a; Robinson,

1947b: Roche §£__l,, 1950: Miller, 1951; Romani, 1951;

Miller, 1952M. Wbol.growth, cystic ovaries, and phenothiazine
treatment have also been shown to be associated with changes
in thyroid function (Simpson, 1924; Reineke and Soliman, 1955;
Talmage gt al., 1954).

Since application of the "direct-output" method is not
as yet practical in other than small laboratory animals,
other means were sought in the present study to further sub-
stantiate observations on thyroid function as related to
-lactation in rats and dairy cattle, to the nonbreeding cow
and heifer, and to lactation, phenothiazine treatment, and
wool growth in sheep. To this end a competitive protein-
binding technique was employed which directly measures the
serum thyroxine (T4) level. .The principal analytical material
used was the commercially available Tetrasorb-125 diagnostic
kit.1 This technique was applied in the present investiga-
tions instead of the more widely used protein-bound iodine
(PBI) or butanol-extractable iodine (BEI) procedures which
are less specific, subject to iodine contamination and in-
fluenced by the level of iodine from the diet ingested

previously by the animal.

 

lAbbott RadiOpharmaceuticals, North Chicago, Illinois.

LITERATURE REVIEW

Many investigations, beginning with that of Courrier gt al.,
(1949), have revealed that a considerable amount of iodide
is secreted in milk. Subsequent reports have shown this to
occur in many Species including man (Honour §t_§l,, 1952;
Noble and Rowlands, 1955), cows (Lengeman §£_gl., 1955;
Lengeman and Swanson, 1957), goats (wright §£_§l,, 1955;
Flamboe and Reineke, 1959; Reineke, 1961), dogs (Van Middles-
worth §t_gl,, 1955; Van Middlesworth §£_al,, 1954), rabbits
(Brown-Grant, 1956; Brown-Grant, 1957; Brown-Grant and
Galton, 1958), rats (Potter and Chaikoff, 1956; Potter §£_§l,,
1959; Grosvenor, 1960), and mice (Rugh, 1951).

As reviewed by Brown*Grant (1961), in man, dogs, rabbits,
guinea pigs, rats and mice the milk:plasma ratio of 1131 is
20-40 within a few hours following administration of the
label and in the cow, goat and sheep the ratio is 2-5. In
the goat, rabbit and several other Species this milk:plasma
1131 ratio is rapidly reduced with thiocyanate treatment
(Brown-Grant, 1961; Reineke, 1961).

Iodine in the mammae of most species is present both
as free iodide and as monoiodotyrosine in peptide linkage

with milk protein (Potter and Chaikoff, 1956; Brown-Grant,

1961, Reineke, 1965). Apparently this organic binding of
iodide in the mammae does not play a significant part in

the process of iodine concentration since thiouracil does
not prevent the establishment or maintenance of a high

131 even though organification is

milk:plasma.ratio of I
inhibited. In fact, the organic binding occurs after milk
has been secreted by the alveolar cells (BrowncGrant, 1961).
.Secretion of 1131 by the functioning mammae is of such
magnitude that the maternal thyroidal accumulation of ad-
ministered 1131 is decreased by 86% in the rat (Lorscheider,
1967). Other reports have indicated similar decreases of
up to 50% (Brown-Grant, 1956; Iino and Greer, 1961). This
mammary diversion of iodine first led Flamboe and Reineke
(1959) to pr0pose that under conditions of limited iodine
supply a functional thyroid deficiency could result during
lactation. The apparent increase in thyroid activity near
the onset or during lactation as suggested by others (Turner
and Cupps, 1959; Monroe and Turner, 1946; Brown—Grant, 1956;
Grosvenor and Turner 1958a) and the lack of any specific
need for increased thyroid hormone during nursing as con-
cluded by Iino and Greer (1961) were of Special interest in
view of the well-established fact from many earlier reports
that desiccated thyroid and thyroprotein feeding increases
lactation (Graham, 1954: Reineke and Turner, 1942; Reineke,
1945). The latter investigations imply that lactation might
be self-limiting due in part to a deficiency of thyroid

hormone.

To elucidate the status of the thyroid during lactation,
thyroid hormone secretion rate (TSR) was determined in both
lactating and nonlactating rats (Lorscheider and Reineke,
1966; Lorscheider, 1967) by the thyroxine (T4) substitution
procedure (Reineke and Singh, 1955) and "direct-output"
method (Reineke and Lorscheider, 1967). Reineke and Lorscheider
(1967) showed that in nonlactating rats receiving adequate di-
etary iodine (1 ug I/g feed), TSR values determined by the two
methods were not significantly different. In iodine deficient
states, however, there was a large difference between results
obtained by the two methods. The T4 substitution method
failed to detect differences in TSR of rats receiving two dif-
ferent basal diets ranging from deficient to adequate in
their iodine content. By the "direct-output" method there
was a high positive correlation between iodine intake and TSR,
with values leveling off when iodine intake was adequate.
These results were interpreted to Show: (1) that the T4 sub-
stitution method measures the demand for thyroid hormone,
but represents true TSR only if iodine supplies are adequate,
and (2) that the "direct-output" method is more applicable
in cases where iodine supply is likely to be a limiting
factor on thyroid hormone production.

Lorscheider and Reineke (1966) and Lorscheider (1967),
using lactating rats on various levels of iodine intake,
demonstrated that the apparent TSR by the T4 substitution

method was significantly greater than that of controls.

This finding confirmed that of Grosvenor and Turner (1958a)
who employed a similar method, and their interpretation

was that TSR is elevated in the rat during lactation. But
by the recently develOped “direct-output" method (Lorscheider
and Reineke, 1966; Lorscheider, 1967) it was reported that
TSR is significantly reduced during lactation even when I
iodine is supplied at a level considered adequate for non-
lactating rats. Thus employment of the "direct-output"
method revealed the actual TSR to be significantly reduced
when there was either a nutritional or physiological limita-
tion, such as lactation, on the amount of iodine available
to the thyroid.

In view of these findings in the rat, and since adapta-
tion of the "direct-output" method is not practical in
Species other than small laboratory animals, the recently
developed Tetrasorb-125 method (Abbott Radiopharmaceuticals,
North Chicago, Illinois) was employed in the present study
to directly measure serum T4. (This method was favored over
the more widely used protein-bound iodine (PBI) determina—
tion (Barker, 1948; Barker and Humphrey, 1950) and butanol-
extractable iodine (BEI) procedure (Taurog and Chaikoff,
1948). Acland (1957) has questioned the specificity of the
PBI serum precipitation step in that no discrimination is
made between different iodothyronines. In some cases diio-
dotyrosine is also precipitated. Furthermore, several

investigators (Man and Peters, 1950; Barker et a;,, 1951;

Benotti and Benotti, 1965) have indicated that therapy with
iodine-containing compounds, which may combine with serum
protein, will yield Spuriously high results by the PBI
determination. And the PBI may remain elevated for a period
of years. Although the BEI procedure has proven somewhat
more satisfactory in separating organic from inorganic
iodine of plasma, this method, too, can present artifactual
high values due to extraction of non-thyroxine organic
iodine (Man _t_al,, 1951). Thus PBI and BEI analyses are

at best only rough estimates of thyroid function.

The development of the Tetrasorb-125 method began with
the introduction of a "saturation-analysis" technique by
Ekins (1960) for measuring total serum T4. Thyroxine was
extracted from serum in acid-butanol, and by electrophoretic
techniques a measurement was made of the globulin-to-albumin
shift of T4 caused by the addition of exogenous tracer T4.
Murphy (1964) and Murphy and Pattee (1964) simplified Ekin's
procedure which they termed "competitive protein binding".

By this method T4 was removed from serum with a single ethanol
extraction. (Serum.T4 was estimated by its effect on the
passage of a standard labeled T4-serum solution through a
gel-filtration column.

The principle of "competitive protein binding" is derived
from the fact that there is only a small amount of thyroxine-
binding globulin (TBG) in plasma. Thus the TBG binding sites

can be readily saturated by small amounts of exogeneous T4.

-1131 enables calculation of

Addition of trace amounts of T4
the protein-bound fraction of isotope. The more unlabeled

T4 which is added to the system, the greater is the decrease
in TBG-bound T4-I131 since both labeled and unlabeled T4
compete for the same binding sites. If a serum extract of
unknown T4 is substituted for purified T4 of a known amount,
the unknown T4 may be measured by the displacement of bound
T4-I131 from TBG. Murphy and Pattee (1964) provided a
Sephadex polymer gel to serve as a binding site for the dis-
placed T4-Ilal. They also used a barbital buffer and diluted
standard serum to reduce binding of T4 to prealbumin and
albumin reSpectively. Subsequently, Murphy and Jachan (1965)
further simplified their procedure by replacing the Sephadex
polymer gel with a loose anion exchange resin. Murphy §§_§l,
(1966) concluded that the intact T4 molecule is measured thus
yielding determinations unaffected by iodine, and that the
serum.T4 is clinically reliable when compared to normal human
PBI values. Furthermore, Kaplan (1966) demonstrated that
T4,-If25 could be bound to a TBG solution for several weeks
prior to a determination. Based on the foregoing studies
Abbott Laboratories in 1967 introduced the Tetrasorb-125
method which included resin impregnated Sponges and a standard
T4-1125 TBG solution. In its present form this analysis
requires an ethanol extract of serum T4, which during a fixed
incubation time and temperature, will compete with and dis-

place a quantity of T4-I125 from a prelabeled standard TBG

solution. The diSplaced labeled T4 is subsequently bound to
an anion resin sponge.

.The potential adaptability of this serum T4 assay pro-
cedure to sub-primate Species in the present investigations,
necessitated a comparison of thyroid function in lactating
rats by the "direct-output“ and Tetrasorb—125 methods. A pre-
liminary report (Lorscheider, §t_§;,, 1969) indicated that
lactating rats with reduced thyroid secretion rate (TSR)
also showed reduced serum T4. It was of interest to determine
the relationship between serum T4 and lactational intensity
in the rat and to see if a euthyroid function could be main-
tained during lactation by excess iodine supplemented in
the diet. —Miller (1951) and Miller (1952) had shown that a
high iodine intake could increase lactational performance
up to 500% in women, presumably by increasing thyroid func-
tion enough for unsupplemented feeding of young (Robinson,
1947b). In view of recent suggestions that prolactin may
inhibit thyroid activity (MacLeod gt _l., 1966; Gona, 1967;
MacLeod and Abad, 1968), the effects of prolactin on thyroid
function were also investigated.

Several studies have been conducted in attempts to
interpret PBI determinations in dairy cattle (Long §£_§l,,
1952; Lennon and Mixner,.1959; Kossila, 1967). As these
results have been somewhat inconclusive, the Tetrasorb-125

method was employed in Holstein cows to determine the status

of serum T4 during various stages of lactation. Reineke and

10

Soliman (1955) reported that hypothyroidism in mice resulted
in their ovaries being filled with large follicles but hav-
ing an absence of corpora lutea. It was of interest there-
fore to examine the serum T4 status in cows diagnosed as.
nonbreeders or as having cystic ovaries.

Previous measurements of thyroid function in sheep have
included such indices as TSR by the T4 substitution method,
thyroid histological evaluations, and thyroid uptake and out-

put of 1131 (Henneman gt. 1., 1955; Hoersch §£_al,, 1961;

Griffin §£_gl,, 1962). Henneman _£_§l, (1955) reported ele-
vated T4 substitution values in lactating sheep. ~Since the
substitution method was thought to reflect apparent TSR, or
thyroid hormone demand (Lorscheider, 1967; Lorscheider and
Reineke, 1970), the Tetrasorb-125 method was employed in
lactating Hampsire and Suffolk ewes to further assess thyroid
function. In the present study serum.T4 was also investi-
gated in lactating sheep with respect to therprotein and
phenothiazine treatments.

It was shown many years ago (Simpson, 1924) that thy-
roidectomy in sheep resulted in marked wool slippage.
Subsequently other investigators have confirmed that reduced
thyroid function in sheep decreases wool growth and causes
an alOpecia-like condition (Maqsood, 1950; Ferguson gt al.,
1956). Attempts were made to apply T4 therapy in the form

of subcutaneous pellet implants. However, these trials met

with but moderate success in reestablishing wool growth in

11

thyroidectomized sheep although in some animals wool length
increased up to 25%. The main problems encountered were
uncontrolled hormone absorption rates and the impracticality
of adapting this technique to range management conditions
(Ferguson gt a;,, 1956; Ferguson, 1958; Theriez and Rougeot,
1962).

Over the past two decades a number of agricultural
experiment stations and ranchers have noted an increased
incidence of "Spontaneous“ wool slippage (apparently undocu-
mented) in Shropshire and Southdown flocks and to a lesser
extent in Hampshire flocks. In fact, this alopecia—like
condition, of unknown etiology, has been partially reSponsible
for the discontinuation of the Shropshire breed in the Michi-
gan State University flocks. ~An investigation not reported
herein, but currently underway at the Fred and George Buckham
flock near Kalamazoo, Michigan, shows Significant wool slip-
page in nearly all ShrOpshire ewes. The interesting facet
of this condition is that wool slippage occurs near or at the
onset of lambing (Jan.-Feb.) and disappears several weeks
after weaning. As wool shearing is usually done in late fall
and late Spring, wool production is greatly impaired. Since
this alOpecia occurs in lactating Hampshire ewes to a signifi—
cant degree, their thyroid function was examined by the
Tetrasorb-125 method. ,Trials were also undertaken to supple-
ment thyroactive casein in the diet to determine its effects

on wool slippage.

12

Phenothiazines and their derivatives have found general
applicationin both veterinary and human clinics as
anthelmintics and as tranquilizers. One of the more common
phenothiazines, thiodiphenylamine, which is used in sheep
flocks, has been reported to be goitrogenic in rats (Talmage
2£.El-v 1955). This same phenothiazine was subsequently
shown to be goitrogenic in sheep (Talmage gt al., 1954).
.After further investigations it was concluded that some un-
known component or metabolite of phenothiazine was responsible
for at least partially depressing thyroid function (Nachimson
‘_E.al., 1954; Wasserman et al., 1956). Phenothiazines may
confound the problem of wool loss in lactating sheep, and in

view of this possibility, a sheep trial was set up to assess

the effects of phenothiazine on serum T4.

MATERIALS AND METHODS

I. Rat Experiments

In all experiments to be reported, female Sprague-Dawley
rats (Spartan Research Animals Inc., Haslett, Mich.) were
used. The pre-breeding weights were 200-220 9. Animal room
temperature was maintained at 76 1.20F and lights were on
14 hours daily.

The diet comprised two-thirds corn and one-third soybean
oil meal (50% protein) plus complete vitamin and mineral
salt premix supplements (Appendix A), except that KI was
added at the laboratory in the amount of 1.0 ug I per g of
feed. This diet was prepared at the Michigan State University
,Department of Animal Husbandry feed mixing plant. The diet
had previously been established as providing an adequate
iodine intake for nonlactating rats (Reineke and Lorscheider,
1967). -Rats receiving this diet without iodine supplementa—
tion deve10p enlarged thyroids within two weeks. In one
experiment the amount of KI added was increased to 10.0 ug I
per g of feed in one group of rats.

For lactating rats whose thyroid secretion rate (TSR)

was measured by the "direct-output" method, the procedure

15

14

used was that described for nonlactating rats by Reineke
and Lorscheider (1967) except that after 16 days on a

prescribed diet, carrier-free 1131

was injected 2 days post-
partum. But to approximate equivalent labeling of the
thyroid in all groups it was necessary to administer 5 uCi
of I131 to nonlactating rats and 8 uCi to lactating rats.
TSR was evaluated from 5-15 days postpartum. Pups from cer-
tain large litters were distributed between the lactating
rats to equalize the number of pUPS in all litters.
Protein-bound iodine (PBI) determinations were performed
by an adaptation of the alkaline ashing method of Barker and
Humphrey (1950). Protein was precipitated from 1.0 ml of
serum by adding 1.0 ml volumes of 10% ZnSO4 and 0.5 N NaOH.
The latter two reagents were matched by titration with phenol-
phthalein as the indicator. The precipitate was washed with
glass-distilled H20 5 times. One ml of 4 N Na2C03 was added,
and the mixture was dried overnight at 1100 C. It was
then ashed in a muffle furnace for ZQ-hours at 6050 C. The
ash was dissolved with 2 ml 2 N HCl and 2 ml 7 N H2804 and
diluted with H20 to 11.0 ml. The timed reaction with
arsenious acid and 0.58% ceric ammonium sulfate was run for
exactly 15 minutes at 570 C and then stopped with brucine
sulfate (Faulkner 25 al., 1961). Colorimetric readings were
taken with a Coleman Spectrophotometer (model 6/55) at 480
mu, set for 100% transmission (T) with a distilled-water

blank. Iodine content was read from a curve prepared under

15

identical conditions in which net percent transmittance was
plotted against iodine concentration (Lennon and Mixner,
1957). Iodine values were corrected for an average 11%
loss occurring during ashing. In all reagents, washings
and dilutions, glass distilled water was used.

.In the prolactin study nonlactating rats received sub-
cutaneous injections of either 0.9% NaCl solution or 1.0 mg
of NIH-P-S8 ovine prolactin (28.58 I.U./mg) twice daily for
14 days. Prolactin was dissolved in 0.9% NaCl solution to
a concentration of 10 mg/cc.

Blood was collected by heart puncture after 14-16 days
lactation in all experiments. Samples were placed in sili—
conized test tubes to prevent hemolysis. After 5 hours the
blood was centrifuged at 2000 r.p.m. (1000 x G) for 20
minutes. In most instances enough blood was obtained from
each rat to permit 5-4 ml of serum to be collected. Serum

thyroxine (T4) analyses were carried out as described later.

II. Cow EXperiments

All animals used in these experiments were Holstein
heifers or cows from.the Michigan State University dairy herd.
The lighting and environmental temperature for the animals
were similar throughout the herd. Cows were confined to the
barn except for about 1 hour daily. They were fed mixed
alfalfa hay and corn silage, grain, and mineral-salt supple-

ments as listed in Appendix B. The grain and mineral-salt

16

mixes were prepared at the Michigan State University Depart-
ment of Animal Husbandry feed mixing plant.

Recording of milk weights began 1 day after calving,
and milking was twice daily at 12 hour intervals. .Duration
of lactation periods averaged 505 days. Control and eXperi—
mental groups were selected as nearly as possible on a basis
of similar stages of gestation, lactation and milk yield.
Some cows and heifers suSpected or diagnosed as nonbreeders
were also sampled for T4 determinations.

Blood samples were withdrawn from the ventral coccygeal
vein into 15 ml B-D Vacutainer tubes. All sampling was done
during the period from October, 1968 to March, 1969. Serum

for T4 analysis was collected as described for rats.

III. Ewe Experiments

All sheep used in these eXperiments were Suffolk or
Hampshire ewes from the Michigan State University sheep flocks.
Animals ranged from yearlings to 5-year-olds. The ewes were
confined to barn feeding and the temperature and lighting
environment for the animals was similar throughout the flock.
The sheep were fed alfalfa hay, grain, and a mineral-salt mix
described in Appendix C. The grain and salt mixes were pre-
pared at the Michigan State University Department of Animal
Husbandry feed mixing plant. In one experiment phenothia-
zine was withdrawn from the salt mixture following lambing

for an average of 51 days. In another exPeriment therprotein

17

(Protamone, Agri-Tech, Inc., Kansas City, Mo.) was added to
the grain ration 4-6 days after lambing to provide 0.5 g
Protamone per ewe per day (Appendix H). Ewes assigned to
each control and eXperimental group were selected as nearly
as possible on a basis of similar stages of gestation, lac-
tation, and degree of wool slippage.

Blood samples were withdrawn from the jugular vein into
15 ml B—D Vacutainer tubes. All sampling was done in
NOvember, 1968 or the end of January, 1969 except for the
phenothiazine trials where blood was taken in May, 1969.
Serum was collected as described for rats and PBI determina-
tions for some Hampshire ewes were carried out as described

for rats.

IV. Serum Thyroxine (T4) Analysis

1) Procedure

The Tetrasorb-125 method (Abbott Radi0pharmaceuticals,
North Chicago, Illinois) was employed, with certain modifi-
cations, for the serum T4 analyses. The procedure was as
follows: Blood serum samples were frozen and stored for
periods up to several months. At the time of assay 1 ml of
serum was taken from the samples which had been thawed and
brought to room temperature. Two ml of 95% ethanol were
added and the solution was mixed immediately by using a Vortex
mixer for 50 seconds. To facilitate maximal T4 extraction

the resultant denatured protein precipitate was allowed to

18

stand at room temperature finrlo minutes. The mixture was
then centrifuged at 2000 r.p.m. (1000 x G) for 20 minutes.
Three-tenths ml of the alcoholic extract from the serum was
placed in polyprOpylene test tubes, and while being warmed
in a water bath (55-400 C) the extracts were evaporated to
dryness by a steady mild air stream. -After complete drying,
1.0 ml of thyroxine-binding globulin (TBG)-T4-I125 solution
was added to each tube and allowed to equilibrate with the
dried extracted thyroxine for 10 minutes at room tepmerature.
The tubes were then placed in an ice-filled Dubnoff shaker
(0.5-2.0o C) for 5 minutes. At 20 second intervals one
resin-impregnated sponge was placed successively in each tube
and saturated by lightly depressing each Sponge 5 times,
using the Special Abbott plunger.

The initial radioactivity count (I) of the total labeled
quantity of TBG-T4-I125 was obtained for each tube during the
incubation period 50 minutes after adding each Sponge. At
exactly 1 hour of incubation the reaction was stOpped in
successive tubes at 20 second intervals by adding 10 ml of
distilled water, depressing the Sponge 5 times with the
a3pirator tube and immediately drawing off the solution. The
sponge was washed 5 more times with about 10 ml portions of
distilled water to remove residual labeled TBG. The final
radioactivity count (F) was taken after 4 washes. A scintil-
lation well-type counter (Nuclear Chicago, Model DS-5), and

analyzer/sealer (Nuclear Chicago, Model 8725) was employed

19

for the radioactivity measurements. The percentage uptake of

T4-I125 on resin Sponges was obtained using the formula:

F (Cpm) - background (me)

I (Cpm) - background (Cpm) X 100

% Resin Sponge uptake =

The F and I counts were measured under constant geometric con-

ditions.

2) Standard Curves

-A standard T4 curve was run with the determinations done
on any one day. The primary standard was crystalline free
thyroxine purified by Dr. E. P. Reineke from monosodium
thyroxine pentahydrate obtained from Baxter Laboratories,
Morton Grove, Ill. The free thyroxine showed only a single
component when checked by thin layer chromatography. The
standard stock solution was prepared by dissolving exactly 10
mg of the purified .T4 in 95% ethanol with the aid of a mini-
mum of 0.1 N NaOH solution and diluting with ethanol to a
concentration of 5 ug per ml. -Concentration of the working
standard was 0.05 pg per ml. When kept refrigerated this
could be used 2-5 months without deterioration. -A series of
standards of varying concentrations was set up for each
standard curve and carried through the same procedure as the
alcoholic serum extracts, as already described.

Because a 0.5 ml aliquot of the supernatant alcoholic
serum extract was evaporated to dryness, the T4 in the dried

sample was one-tenth of the amount in 1 ml of serum, or

20

1/1,000 of the amount in 100 ml serum. Based on this ratio,
the amount of working standard solution for the standard
curve was computed by the fOllowing calculation, and eXpressed

as T4 in ug/100 ml:

 

T =v
1,000 x C (ml)
Where,

T a the T4 equivalent of the serum extract expressed
as pg per 100 ml.

C = concentration of standard solution
(0.05 ug/ml)
V = volume of standard solution

When T4, eXpressed as ug/100 ml, is plotted on the abscissa
of linear coordinate graph paper against percent I125 count
remaining in the resin Sponge, a standard curve can be plotted.
In our pilot eXperiments it was found that in nearly all of
the Species studied the serum T4 concentration did not exceed
12 ug/100 ml. In the range 0-12 ug T4 per 100 ml a perfectly
linear relationship between T4 concentration and resin sponge
count was found to exist. Thus, instead of reading values
from a plotted standard curve, the T4 standard data were
fitted by the method of least squares, and the serum T4 values
were calculated by use of the regression equation. The calcu-

lations are as follows:

21

Xu = Serum.T4 (ug/100 ml) uncorrected for recovery
Y = percent resin-Sponge uptake

a = intercept of the Y-axis

b = lepe of the standard curve

In setting up this method we found a 77.5% efficiency for
extraction of T4 from serum with 95% ethanol.

The extraction efficiency was determined by mixing 1 ml
of rat serum with 0.02 ml T4-I131 (Abbott Laboratories).
After 27 hours equilibration at -0.50 C, the first radio—
activity measurement was obtained in the well counter. Two
ml of 95% ethanol were added and the solution was treated
as described previously for serum samples. The second count
was obtained from 1 ml of the alcoholic extract, and the
percent extraction efficiency was calculated from the differ-
ence between these counts.

To obtain true T4 values, the results had to be cor-
rected for extraction efficiency, as follows:

Xc = Serum T4 (pg/100 ml) corrected for recovery. (All
equations and calculations for both the serum assay procedure
and standard curves were programmed on an Olivetti—Underwood

desk computer (Model 101).

RESULTS AND DISCUSSION

I. Rat EXperiments

A comparison of rat thyroid function as measured by the
Tetrasorb-125 and direct-output methods is illustrated in
Figure 1. .During lactation thyroid secretion rate (TSR)
falls from 2.58 to 0.99 ug T4/100 g body wt./day (p <:.001)
and serum thyroxine (T4) is reduced from 5.94 to 1.96 ug/100
ml (p <1.001). .Thus both methods show a significant reduc-
tion of more than 50% in the thyroid function of lactating
rats. It is apparent that reduced TSR by the direct-output
method will manifest itself in reduced serum T4 by day 15 or
16 postpartum, a time estimated to be peak lactation in the
rat. Individual serum T4 values for Figure 1 are presented
in Appendix D. It is interesting to note the serum T4 value
of 5.45 pg/100 ml for lactating rat no. 6. This value,
which is significantly above serum T4 levels for the other
lactating rats, may be exPlained by the fact that on the
third day postpartum 5 pups were eaten by the mother and were
not replaced with foster pups. A 5/12 reduction in lacta-
tional intensity coupled with the additional source of iodine

provided by the 5 pups would account for a higher serum T4.

22

25

 

 

 

 

 

 

 

 

    

 

 

 

 

 

.g
(n
3
DAY l6 0F LACT. T0 DAY l5 0F LACT. 3
4* + 4‘ °
‘4
\
ui 6
ed 31 10
3‘9
+I +-
2 CI
3 a: O
m a
a 5 *
K
_ 2’ + ‘0 2" i
_ F I- :4
E 0 ‘ x
c:
0 j m c o
0’ ,z 3; g h; >
- O t- 0 .(
\ I- z ¢ = 3. I:
:3 F 3 z 3 H-
0 a O a
6 j 2 z 2 g)
a o '2 0 ,m
IX l0 I0 l5
P<.OOI . P<.OOI
NOS. OF RATS/ GROUP
l n. I unto lg. nzo
Figure 1. Comparison of serum thyroxine method and

thyroid secretion rate by the direct-output
method in nonlactating and lactating rats.

24

These data by the Tetrasorb-125 method confirm the previous
interpretation by Lorscheider and Reineke (1966), Reineke
and Lorscheider (1967) and Lorscheider and Reineke (1970),
that the T4 substitution method as employed by Grosvenor and
Turner (1958a) cannot detect a decrease in thyroid hormone
production during lactation.

.Figure 2 illustrates levels of serum T4 in the rat at
several levels of lactational intensity and elevated iodine
intake. Individual serum values are listed in Appendix E.
It can be seen in groups A, B, C, and D, that as the number
of pups per litter is increased from 0 to 5, 6, and 12,
reSpectively, serum.T4 is Significantly reduced. A high in-
verse correlation exists between serum T4 and lactational
intensity (r = -0.85, p <:.001). -Even with only 5 pups per
litter the serum.T4 of a nursing rat is significantly reduced
(p <1.05) below that of nonlactating controls.

The normal level of dietary iodine necessary for euthy—
roid function in a nonlactating rat is 1.0 ug per 9 feed
(Reineke and Lorscheider, 1967). When 10 times this amount
of iodine is added to the diet of lactating rats (Group E,
Figure 2) there is no significant increase in serum T4 when
compared to that of similar rats receiving 1.0 ug of I per
9 feed (Group D). Previous studies in women have indicated
that iodine therapy could be used in the treatment of in-
adequate lactation (Robinson, 1947b; Miller, 1951; Miller,

1952). Presumably the supplemented iodine was sufficient to

25

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26

saturate the mammary iodide space thereby permitting enough
iodine to be diverted to the thyroid for increased hormone
production. However, in these circumstances some difficulty
has been encountered in quantitating the dose of iodide
(Friend, 1960). In the case of the lactating rat, either it
does not reSpond to iodine therapy by increasing T4 produc-
tion, or the rat requires an iodide supplement far in excess
of the elevated level provided in the present investigation.
‘When sufficient serum was available, PBI determinations were
run on the same rats for which data are presented in Figure 2.
As shown in Appendix E the lactating rats receiving 10 times
the normal iodine intake (Group E) had an average PBI value
of 2.65 ug I per 100 ml of serum, similar to that of nonlac-
tating rats in Group A which had a PBI of 2.70 ug I per 100
ml of serum. The fact that this similarity in PBI values
between these two groups is not reflected in a similarity in
serum T4 values further illustrates the possible error
previously discussed (Man and Peters, 1950; Barker gt_al,,
1951; Acland, 1957) in using PBI as an index of thyroid
function.

It is Shown in Table 1 that exogenous ovine prolactin
had no significant effect on serum T4 in the nonlactating
rat (p )>.90). .MacLeod _£_al, (1966) tranSplanted tumor-
bearing pituitaries secreting large amounts of prolactin and
growth hormone into rats, and reported a depression in thy-

131

roid I uptake and a decrease in host pituitary thyroid

27

TABLE 1

EFFECT OF PROLACTIN INJECTIONS ON.SERUM THYROXINE
IN THE ADULT FEMALE RAT

All rats received 1.0 ug I‘ added/g feed.

 

 

 

 

*-
NIH-P-SS Ovine Prolactin 28.58 I.U./mg (10 mg. prolactin/

1.0 ml saline)

 

Prolactin injected rats
(1 mg prolactin* injected subcuh
Saline injected rats taneously twice daily for 14 days)
H9 T4/ ”9 T4/
Rat no. 100 ml serum Rat no. 100 ml serum
1 6.17 12 6.28
2 4.95 15 5.55
5 5.80 14 5.55
4 5.85 15 6.59
5 6.02 16 5.85
6 7.05 17 5.54
7 6.17 18 5.75
8 6.17 19 6.71
9 5.52 20 6.28
10 6.81 21 6.17
X=6.05 X=6.00
S.E. = 0.19 S.E. = 0.15
N = 10 N = 10

28

stimulating hormone (TSH) content. They concluded that SUp-
pression of thyroid function was mediated by an inhibitory
effect of the tumor hormones on the pituitary of the rat.
In a subsequent study MacLeod and Abad (1968) reported free
T4 tended to be slightly lower in similar tumor bearing rats.
However, this decrease in free T4 was not statistically Sig-
nificant. Gona (1967) has suggested that prolactin has a
goitrogenic effect in the frog. Prolactin reduced thyroidal
1131 uptake and this reduction was not changed by injections
of TSH. The impairment of metamorphosis caused by prolactin
could be counteracted by T4 administration. Gona concluded
that prolactin had no direct effect on the frog pituitary
TSH content, but rather has a direct effect on the frog
thyroid. However, Grosvenor and Turner (1958b) showed by
bioassay techniques that the suckling stimulus caused a fall
in pituitary prolactin stores in the lactating rat. .More
recently, Johke (1969) has shown by radioimmunoassay an in-
creased plasma prolactin level following milking in the cow
and goat. Amenomori §£_§l, (1970) have also shown by radio—
immunoassay that serum prolactin increases are directly cor-
related to increases in suckling stimuli in rats with litter
sizes ranging from 0 to 12 pups.

In view of the earlier reports by others it could be
suggested that the progressive decrease in rat serum T4 as
lactational intensity increases (Figure 2) is due to the

concomitant increase in serum prolactin which in turn exerts

29

a progressively greater suppression on thyroid function.
However, the use of thyroid 1131 uptake as.an index of thy-
roid activity is questionable since hyper- or hypothyroid
function can be accompanied by either high or low 1131
uptake. The present study (Table 1) would indicate that
exogenous ovine prolactin, at the level administered, has

no influence on thyroid function when evaluated by serum T4
measurements.

Chen and Meites (1969) have shown that T4 has no effect
on hypothalamic prolactin inhibiting factor (PIF) content,
but does stimulate an increase in pituitary prolactin.
Thiouracil reduced pituitary prolactin levels. They con-
cluded that T4 acts directly on the anterior pituitary to
enhance prolactin production and that a lack of T4 when
thiouracil is administered will depress prolactin secretion.
This reduction in prolactin secretion in the absence of
adequate T4 would partially eXplain why lactation can be en-
hanced by the administration of thyroactive substances as
reviewed by Blaxter gt al., (1949). If prolactin were an
antithyroid agent as suggested by others (MacLeod §t_a;,,
1966; Gona, 1967; MacLeod and Abad, 1968), then it would be
inhibiting its own production via the thyroid. The evidence
to date would indicate that the primary reason for a reduc-
tion in serum T4 during lactation is the mammary diversion

of iodine coupled with a possible decrease in total serum

thyroid-binding protein (TBP). .The latter could be due to

50

the increased demand for protein synthesis during lactation.
There is also the possibility that high endogenous prolac-
tin secretion could indirectly exert an antithyroid effect
by enhancing lactation, thereby increasing the mammary drain
on iodine supplies which would ultimately further decrease
serum.T4.

Figure 5 illustrates a pr0posed schema for the thyroid-
mammary—kidney distribution of iodine. The size of the
arrows are indicative of the relative magnitude of the com-
ponents of this iodine cycle. This chart shows that sub-
normal iodine intake in the nonlactating rat will result in
less thyroid hormone production and eventually in a hypo-
thyroid condition characterized by elevated.TSH output and
thyroid gland hyperplasia. The two primary competitors for
dietary iodine in the nonlactating rat are the kidney and
the thyroid. In the lactating rat a third competitor for
dietary iodine is introduced, namely the mammary gland.

As a result a functional hypothyroidism is produced, similar
to that observed in a nonlactating rat on reduced iodine
intake, even though the lactating rat receives an iodine
allowance that would be adequate in the nonlactating state.
As indicated, some Species such as the rat, recycle iodine
via the urine of the suckling infants. This phenomenon has
been well documented by several investigators (Capek and
Jelinek, 1956; Samel gt §;,, 1965; Samel and Caputa, 1965;

Beltz and Reineke, 1968). It does not appear that the net

51

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52

Opposing factors (total thyroid I and thyroid I131 release
rate) in this maternal recycling of neonatal iodine can
appreciably influence the results obtained by direct-output
TSR in the lactating rat (Lorscheider and Reineke, 1970).
This conclusion is supported by the present study which
shows the serum T4 to be significantly reduced during lac—

tation.

II. Cow Experiments

 

Figure 4 illustrates serum T4 values in 4 groups of
Holstein cows during various stages of gestation and lacta-
tion. Individual values are listed in Appendix F. It can
be seen that serum T4 in nonlactating cows remains unchanged
during pregnancy (5.66 ug T4/100 ml) when compared to non-
lactating nonpregnant cows (6.20 ug T4/100 ml). This is
t 34., 1968)

unlike the condition found in women (Arango
where serum T4 is reported to be elevated during pregnancy.
Dairy cattle normally increase to a peak in milk yield 60
days or more after calving, then gradually decline until
dried off at about the 7th to 8th month of gestation. As
shown in Figure 4 serum T4 is markedly reduced in high pro-
ducing nonpregnant cows (5.59 ug T4/100 ml) followed by a
recovery in serum T4 to normal levels during advanced lacta—
tion and pregnancy (5.65 ughT4/100 ml). The magnitude of
serum T4 reduction in the high producing cows is similar

to the 50% reduction observed in lactating rats (Figure 1).

55

 

HOLSTEIN
COWS

 

 

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Serum thyroxine in Holstein cows during various stages

of gestation and lactation.

Figure 4.

54

These results support the trends Shown in previous studies
on Ayrshire cows (Kossila, 1967) in which protein-bound
iodine (PBI) values tended to be lower at peak lactation
with gradual increases during the first 5 months postpartum.
,Furthermore, in a comparison of five breeds of dairy cows,
PBI values tended to be lowest in Holstein cows (Long g; al.,
1952). Inasmuch as Holsteins are the highest milk producers,
the depletion of serum T4 during lactation may be the most
pronounced in this breed.

A paired comparison of serum T4 levels in Holstein cows
during pregnancy and different stages of lactation is shown
in Table 2. At 50 days lactation serum.T4 was significantly
reduced from 5.66 to 4.65 ug T4/100 ml (p <1.001). At 95
days lactation serum T4 was reduced still farther to 5.90 ug
T4/100 ml. This paired comparison was terminated in mid-
March in order to avoid the seasonal changes in light and
temperature known to influence the thyroid function of farm
animals (Henneman §£_a;,, 1955; Hoersch §t_al., 1961;

Griffin §£_al,, 1962).

One might eXpect that the high inverse relationship be-
tween lactational intensity and serum T4 as exists in the
rat, might also be present in cows. However, a close inSpec-
tion of the individual milk records in both of the present
studies (Appendix F and Table 2) does not reveal such an
inverse relationship within a given cow group. The higher

milk producers did not necessarily have the lowest serum T4

55

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56

values. {This is attributed to the fact that lactational
performance is a function of both intensity and duration.
{The overall trend between groups though, was for markedly
lower serum T4 levels during the first 5 to 4 months follow-
ing calving, a time when milk production is highest.
Tucker and Reece (1961) showed that the increase in milk
output due to therprotein feeding was much greater in
cows at peak lactation. They concluded that thyroid hormone
secretion may be a limiting factor on milk secretion at the
peak of lactation. .Sorensen (1958) reported that a high TSR
of cows was highly correlated with high milk yield. The
present investigations would tend to support the conclusions
of Tucker and Reece in that serum T4 is markedly reduced
during high lactation.

In Table 5 serum T4 values are shown for Holstein cows
diagnosed as nonbreeders or as having cystic ovaries. It
can be seen that the highest value (5.07 ug T4/100 m1) is only
half that of values for comparable nonlactating nonpregnant
control animals (Figure 4 and Appendix F). .Reineke and
Soliman (1955) reported that hypothyroidism in mice is
associated with cystic ovaries. The present study suggests
that a similar relationship may be true in cows. [Further
investigations in cows are currently underway to establish
the feasibility of employing the serum T4 assay as a diagnos-
tic tool for the detection of cystic ovaries and other causes

of reproductive failure.

57

TABLE 5

SERUM THYROXINE VALUES IN HOLSTEIN.COWS DIAGNOSED
AS NONBREEDERS OR AS HAVING CYSTIC OVARIES*

 

 

NONPREGNANT/NONLACTATING
(blood sampling 10-1—6§L

 

 

Birth
Cow No. Date Pathology, ug_T4/100 ml serum
960 7-6-65 cystic ovaries 5.07
909 11-12-62 cystic ovaries 2.94
886 6-21-66 nonbreeder 1.48
866 9-8—66 nonbreeder 1.76
709 5-7-61 nonbreeder 0.64

*-
Cystic ovaries--post mortem diagnosis.

58

TABLE 4

SERUM THYROXINE VALUES IN NORMAL AND
NONBREEDING HOLSTEIN HEIFERS

All heifers bled on

2-21-69

 

 

 

*
Etiology unknown

NORMAL HEIFERS

 

 

 

 

Heifer No. Birth Date ug T4/100 ml serum
997 11-16—67 9.58
1000 11-50-67 12.57
1005 12—12-67 .10.77
1006 12-15-67 9.98
1010 1-5-68 5.98
1011 1-4-68 9.58
1012 1-5-68 7.18
1016 1-29-68 5.19
i = 8.85
S.E. = 0.87
NONBREEDING HEIFERS*
Nos. of
Previous ung4/100
Heifer No. Birth Date Inseminations ml serum

218 5-22-67 12 11.18

255 4-9-67 8 8.58

270 7-5-67 10 9.58

286 6-21-67 11 7.18

SE = 9.08

S.E. = 0.85

59

The relationship between serum T4 and the incidence
of nonbreeding in Holstein heifers, not diagnosed as having
cystic ovaries, is somewhat different (Table 4). -Average
serum T4 values for controls and nonbreeders are 8.85 and
9.08 ug T4/100 ml reSpectively. No significant difference
was found between these two groups (p )>.90). The average
control serum T4 value is significantly higher (p <f.05) in
heifers (8.85 ug T4/100 ml) than that reported for mature
nonlactating cows (6.20 ug T4/100 ml) as shown in Figure 4
and Appendix F. This would agree with reports by others
(Long 22.§l-I 1952) that in general younger cows have a
higher PBI. However, heifers have never been through a

lactation and are therefore not directly comparable to cows.

III. Ewe Experiments

Serum T4 values in Suffolk and Hampshire ewes during
gestation, lactation and therprotein feeding are shown in
Figure 5. Individual values for the Suffolk ewes are listed
in Appendix G. Nonlactating nonpregnant Suffolk yearlings
have a serum T4 value of 15.52 ug T4/100 ml as opposed to
11.90 ug T4/100 ml for nonlactating pregnant Suffolk ewes
(p >'.10). Thus, as in the cow, pregnancy does not elevate
serum T4 of ewes. .In pregnant women, serum T4 does rise
(Arango §£__l,, 1968). At 50 days lactation, estimated to

be peak lactation in ewes, serum of T4 of Suffolks is sig-

nificantly-reduced (9.52 ug T4/100 ml) below that of pregnant

40

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41

ewes (p <1.02). In Hampshire ewes (Figure 5 and Appendix H),
serum T4 is likewise significantly reduced at 50 days lac-
tation from 8.59 ug T4/100 ml in yearling controls to 5.27
ug T4/100 ml (p ‘(.01). Thus both breeds of ewes exhibit a
reduction in serum T4 during lactation similar to that ob-
served in the rat and cow. If the serum T4 of the Suffolk
and Hampshire yearling controls is compared, 15.52 and 8.59
ug T4/100 ml reSpectively, there is a significant breed dif~
ference in thyroid hormone levels (p <7.01).

Henneman 23 El’ (1955) reported that TSR is not altered
in sheep during pregnancy. The serum.T4 values for yearling
and pregnant Suffolk ewes in the present study tend to con-
firm this finding. When the T4 substitution method was
employed in previous investigations (Henneman §£_al., 1955;
Griffin 2; al., 1962), apparent TSR was found to be elevated
in sheep during lactation. The present data, showing re-
duced serum T4 in lactating sheep, are consistent with the
previous rat studies (Lorscheider and Reineke, 1966; Reineke
and Lorscheider, 1967; Lorscheider and Reineke, 1970).

These eXperiments show that the T4 substitution method re-
flects the thyroid hormone demand, which is greater during
lactation, but cannot detect the actual decrease in thyroid
function that occurs in lactating animals. The direct-output
TSR and serum T4 methods for measuring thyroid function are
more applicable in any case where iodine availability is

likely to be a limiting factor.

42

Figure 5 shows that lactating Hampshire ewes fed thyro-
active protein had significantly higher serum T4 values
(7.01 pg T4/100 ml) than_did a comparable lactating group
not fed therprotein (5.27 pg T4/100 ml) (p (1.02).
Individual Hampshire serum T4 values for these two groups
are listed in Appendix H. It is interesting to note that
lactating ewe number 421, which was not fed thyroprotein,
has the lowest serum T4 in its group (5.54 pg T4/100 ml).
This particular animal also had the highest degree of wool
slippage at lambing in its group. Lactating ewe number 505
on the therprotein treatment also had the highest degree of
wool slippage at lambing in its group. -Plate 1 shows that
ewe number 421 has a negligible regrowth of wool 2 1/2 months
after lambing. Plate 2 shows a 2.0 cm. regrowth of wool on
ewe number 505 2 1/2 months after lambing and therprotein
treatment. Based on these data, it would appear that severe
wool slippage is associated with a marked reduction in serum
T4 during lactation. Therprotein supplemented in the diet
will significantly raise serum T4 and facilitate the regrowth
of wool. More extensive trials are currently underway in
Shr0pshire and Hampshire ewes to further assess the effec-
tiveness of thyroprotein feeding in alleviating the alopecia-
1ike condition which develOps during late pregnancy and
lactation. One theory proposed is that thyroid hormones exert
an effect on mitochondrial membranes to increase respiratory

exchange and energy transfer (Tapley and Hatfield, 1962) and

45

 

98°C

‘4‘ .1. '
vow nun IOI

        

AIIIICAN DAIIV A

,.
v0u‘ nun ovv'olow

Plate 1. Control Hampshire ewe number 421 about 2 1/2 months
after lambing. Note the negligible regrowth of wool.

44

 

Plate 2. Hampshire ewe number 505 about 2 1/2 months after
lambing and thyroprotein treatment. Note that wool
has regrown to a length of 2.0 cm.

45

that secondarily, T4 stimulates increased amino acid incor-
poration into newly synthesized protein (Sokoloff and
Kaufman, 1961) for wool growth.

Suffolk ewes have a higher serum T4 level than do
Hampshire ewes, as previously discussed. This may explain
why Suffolks rarely show wool slippage during lactation.
The fact that the incidence of wool slippage is greater in
Shropshire than Hampshire ewes could mean that the serum
T4 is lower in the Shropshire breed than in the Hampshire
breed. The study by Henneman gt_§l. (1955) would tend to
support this hypothesis. In their study the mean TSR of
nonlactating ShrOpshire and Hampshire 2—year-olds was 0.17
and 0.28 mg T4 per day, respectively, during January.
Griffin _£Mgl. (1962) also reported a significantly reduced
TSR in Shropshire rams as compared to Hampshire rams,
regardless of the season of year.

The problem of postpartum a10pecia is not exclusively
confined to Hampshire and Shropshire ewes. Morris (1969)
reported a similar condition in lactating bitches, especial-
ly in Great Danes, Beagles, and many long-haired show dogs.
The cause was hypothesized to be a low-energy maintenance
ration. The etiology of postpartum Alopecia areata in
man remains unknown and as yet no causal therapy has been
found. .Gip gt gl.(1969) reported in their study that women
with.Alopecia areata exhibited a manifest improvement during

pregnancy followed by a marked deterioration after

46

parturition. It is interesting that hair loss should be
alleviated during pregnancy (a time of elevated serum T4

in women) followed by a severe loss after parturition (at
the onset of lactation) when serum T4 is probably reduced

as it is in the rat, cow, and ewe. No records on breast—
feeding were reported in Gip's investigation. .Thyroxine
therapy has been utilized in the past as an effective treat-
ment for increasing milk yield in women suspected of having
reduced thyroid function (Robinson, 1947a; Roche §t_§l,,
1950; Romani, 1951). If the etiology of postpartum

alopecia is related to the status of the thyroid, T4 therapy
might also prove beneficial in this instance.

Table 5 shows serum T4 values in lactating Hampshire
ewes fed phenothiazine, which is routinely used as an
anthelmintic. Again, as in Figure 5, it can be seen that
serum.T4 is significantly reduced during lactation from
8.44 to 6.44 pg T4/100 ml (p <:.02). When phenothiazine was
withheld from the diet the average serum T4 level during
lactation was significantly elevated to 8.41 ug T4/100 ml
(p <L.02). While no statistical correlation was found be-
tween the number of days off phenothiazine and serum.T4,
it is interesting to note that ewe number 229 had the highest
serum T4 value in its group (10.25 ng T4/100 ml) and was off
phenothiazine treatment the longest (57 days). Ewe number
7221 had the lowest serum T4 value in this group (6.71 Mg

T4/100 ml) and was off phenothiazine the shortest period of

47

 

 

 

 

 

 

 

    

TABLE 5.
HAMPSHIRE EWES
SERUM T4 ILACTATION v: PHENOTHIAZINE TREATMENT
DAYS NO. OF DAYS OFF m1 T4IIOOml. MEAN
EWE NO. LACTATION LAMBS IIIPHENOTHIAZINE SERUM iSE

: 809 — 0 I007 844: 0.44
.3 8| 4 — — 0 8.30
3', 8| 8 —— — 0 7.44
4 833 —— — 0 8.05
§ 836 — 0 8.36

o 704 32 I 0 5.62 6.44: 0.3I
g 503 3| 2 o 7.I4
,4, 6| 9 27 2 o 6.83
605 24 2 o 5.76
535 23 l o 683

229 37 2 37 I025 8.4I $0.60
,: 7252 33 I 33 7.88
g 534 30 2 30 605
0 607 24 I 24 SIS
722I 20 I 20 an

 

 

 

* I PART PHENOTHIAZINE I SPARTS SALT - MINERAL NIX

PHENOTHIAZINE : C'ZHSNS

48

time (20 days). Based on these data it would appear that
phenothiazines influence thyroid function and may confound
the problem of wool loss in lactating sheep.

The effect of phenothiazines in blocking thyroid 1131
uptake has been observed in a number of different farm
animals (Talmage gt_§l,, 1954). In studies using the rat
and chick as experimental models (Nachimson §£_gl,, 1954;
Wasserman g£_§l,, 1956), the thyroid uptake of I131 was
likewise blocked when these animals were fed phenothiazine.
At first it was suggested that iodine, introduced as a
catalyst in the production of phenothiazine, depressed
thyroid 1131 uptake by isotOpe dilution or thyroid satura-
tion of iodide. However, when highly purified iodine-free
phenothiazine was fed, thyroid 1131 uptake was still sig—
nificantly depressed. In fact, it was shown that the
iodine adsorbed to phenothiazine is not readily dissociated
and consequently is not available in the circulation as a
free iodide ion. It was concluded in both studies that
some compound or metabolite of phenothiazine, as yet unknown,
was at least partially reSponsible for suppressing the
thyroid. The present study shows that removal of phenothia-
zine from the diet of lactating ewes will result in higher
serum T4 levels. It may be that removal of phenothiazine
from the diet of nonlactating Hampshire or ShrOpshire ewes
will raise their serum.T4 also. Trials currently underway

are designed to show whether a combination of thyroprotein

49

feeding and/or withdrawal of phenothiazine will alleviate

the problem of wool loss in lactating sheep.

SUMMARY AND CONCLUSIONS

A serum thyroxine (T4) analysis was employed to assess
thyroid function as related to lactation in rats and dairy
cattle, to the nonbreeding cow and heifer, and to lactation,
phenothiazine treatment and wool growth in sheep. The re-
sults of these studies are as follows:

1.-A comparison of serum T4 and thyroid secretion rate
(TSR) by the direct-output method in nonlactating and lac-
tating rats established that both methods detected a greater
than 50% decrease in thyroid function during lactation.

The serum T4 method was found to be valid and applicable in
cases where the direct-output method was not practical.

2. Rat serum T4 levels were inversely correlated with
lactational intensity, and the markedly reduced serum T4
observed in high-lactating rats could not be counteracted
with 10 times the normal dietary iodine.

5. Exogenous ovine prolactin, when administered to non-
lactating rats, had no effect on serum.T4. It was concluded
that high serum prolactin, associated with nursing, was not
a directly contributing factor to the reduction of serum T4

observed during lactation.

50

51

4. A schema was proposed for the thyroid-mammary-kidney
distribution of iodine. The evidence presented suggested
that the primary reason for a reduction in serum.T4 during
lactation was the diversion of iodine to the mammary glands.

5. Holstein cows showed no change in serum T4 during
pregnancy. During the period of intense lactation serum
T4 was reduced by 50%. When the intensity of lactation de-
clined, at about 5 months postpartum, serum T4 returned to
a euthyroid level.

6. In cows diagnosed as nonbreeders or as having cystic
ovaries, serum T4 was markedly reduced. This finding agrees
with previous conclusions that cystic ovaries may be associ-
ated with a hypothyroid condition.

7..Serum T4 values for heifers were higher than those
of adult cows and the difference was attributed primarily
to age. Nonbreeding heifers Showed no reduction in serum
T4. However, these animals had never lactated and therefore
were not directly comparable to cows.

8. Suffolk ewes, like the cow, exhibited no change in
serum T4 during pregnancy. During lactation both Suffolk
and Hampshire ewes showed a significant reduction in serum
T4.

9. Lactating Hampshire ewes developed a considerable
amount of wool slippage. The regrowth of wool was greatly

enhanced by therprotein treatment.

 

1'7

52

10. Removal of phenothiazine from the diet of lactat-
ing Hampshire ewes significantly raised their serum T4.
Thus both phenothiazine and lactation may contribute to a
reduction of serum T4 in the ewe and could confound the

problem of wool loss observed during advanced pregnancy and

lactation.

 

 

 

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Tapley, D. F., and Hatfield, W. B., 1962. The peripheral
action of thyroxine.
Vitam. and Horm., 99: 251-285.

Taurog, A., and Chaikoff, I. L., 1948. The nature of the
circulating thyroid hormone.
J. Biol. Chem., 176: 659-656.

Theriez, C., and Rougeot, J., 1962. Action des hormones
thyroidiennes sur la croissance en longueur du brin
de laine.

Ann. Biol. Amin. Biochim.:Eigphys., 9: 5-11.

Tucker, H. A., and Reece, R. P., 1961. Thyroid supplemen-
tation at peak lactation.
J. Dairy Sci., 44: 1751-1755.

Turner, C. W., and Cupps, P. T., 1959. .The thyrotrOphic
hormone in the pituitary of the albino rat during
growth, pregnancy, and lactation.

EndocrinoLng, 94: 650-655.

Van Middlesworth, L. A., Tuttle, A. H., and Haney, D. F.,
1954. Iodination of milk proteins by iodide.
Fed. Proc., 19: 157.

Van Middlesworth, L. A., Tuttle, A. H., and Threlkeld, A.,
1955. Iodinated protein in milk.
Science, 118: 749.

Wasserman, R. H., Trum, B. E., Monroe, R. A., Lane, J. J.,
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Am: J. Vet. Res., 11: 149-152.

61

Wright, W. E., Christian, J. E., and Andrews, F. N., 1955.
.The mammary elimination of radioiodine.
J.-Dairy15ci., 99: 151-156.

APPENDICES

APPENDIX A

RAT FEED MIXTURE

Ingredient Lbs. per 109# mix

Shelled yellow corn
ground through 1/8 screen 68.8

Soybean oil meal

(50% prot .) 28.0 L

Dicalcium phOSphate 1.8

Limestone 0.6

Dawes & Forbes Vit. B. Supplement 0.1 ;j ‘
~Dawes & Forbes Vit. B12 Supplement

(6 mg Blz/lb.) 0.2

Std. Brands 9F.yeast-

(9000 i.u. Vit. Da/gm.) 5.0 gm.

Pfizers Vit. A. Supplement

(10,000 i.u. Vit. A/gm.) 15.0 gm.

Special Mineral Salt Premix* 0.5

 

9(-
Special Mineral Salt Premix

Per cent ' Per cent
Element Element Compound Compound
Mn 0.524 MnSO4'H20 (Baker) 1.612
Cu 0.054 CuSO4 (anhyd Baker) 0.156
Fe 0.270 FeSO4-2H20 (Baker) 0.909
Zn 0.800 ZnSO4-H20 (Mallinckrodt) 2.196
NaCl (plain) (Morton's) 95.154

62

 

APPENDIX B

M. S. U. STANDARD DAIRY RATION

 

 

1ngredient 9994iof feed per batch
Shelled corn 1200
Oats 500
Soybean oil meal
(SO-55% protein) 500 1.“
Liquid molasses 150
Dicalcium phosphate 20 fl -
Trace Mineralized.Salt* .j
(Morton's) 20 i

 

*-
TMS Premix

Minimum per cent of

 

Element Compound compound in_gES
Zn Zinc oxide 1.00
Mn Manganous oxide 0.80
Fe Iron oxide 0.50
S Ferrous sulfate 0.24
Cu COpper oxide 0.10
I vCalcium iodate 0.01
Co Cobalt carbonate 0.01
Na Sodium chloride 94.00

 

Cattle had free access to mixed alfalfa hay and corn silage.

65

 

II.

III.

APPENDIX C
M. S. U. SHEEP RATION

Alfalfa brome hay fed but not weighed. Ewes consumed
approximately four pounds per day.

Grain
a.) Pre-lambing, 1 lb. per head per day of the follow-
ing ration to all lots.

 

Ingredient Per cent
Whole oats 55.00
Corn 15.75
Wheat 20.00
Wheat bran 10.00
Wet molasses- 1.00
Aurofac 10 0.25
100.00

b.) Post-lambing, 2 lbs. per head per day of the follow-
ing ration to all lots.

 

1ngredient Per cent
Whole oats 55.00
Corn 25.00
Wheat bran 15.00
Soybean oil meal 5.00
Wet molasses 2.00
100.00

Phenothiazine and trace mineral salt (TMS) SUpplementa-
tion. This mixture was weighed into self-feeding salt
boxes once each week. Average consumption of TMS is
usually 1 lb. per ewe per month.

 

1ngredient Per cent
Trace Mineral salt* 60.00
(Morton‘s)
Dicalcium phOSphate 50.00
Phenothiazine N.F.
(green) 10.00
100.00

64

L;

 

 

65

In cases where phenothiazine was removed:

 

Ingredient Per cent
Trace mineral salt* 67.00
(Morton's)
Dicalcium phOSphate 55.00
100.00

 

*
TMS Premix

Minimum per cent of

 

Element Compound compound injflgifl
Zn Zinc oxide . 1.00
Mn Manganous oxide 0.80
Fe Iron oxide 0.50
S ' Ferrous sulfate 0.24
Cu COpper oxide, 0.10
I Calcium iodate 0.01
Co Cobalt carbonate 0.01

Na Sodium chloride 94.00

 

APPENDIX D

SERUM THYROXINE MEASUREMENTS

IN NONLACTATING AND LACTATING RATS

(INDIVIDUAL VALUES)

All rats received 1.0 ug I- added per 9. feed.
Lactating rats each nursed 12 pups.
Lactators were bled at 16 days postpartum.

 

 

Rat no.

15
15

Non1actatinq rats

H9 T4/

100 ml. serum

5.94
0.15
10

5.55
5.95
5.64
5.68
4.16
4.11
5.17
4.16
4.62
4.54

 

66

Lactating rats

 

, H9 T4/
Rat no. 100 ml.4§§rum
2 2.25
5 2.47
4 2.61
6 (ate 5 pups on 5rd
day postpartum)
5.45
7 2.72
8 1.95
9 0.78
10 0.95
11 0.80
12 1.65
X = 1.96
S.E. = 0.26
N = 10

RAT SERUM T4 AND PBI VS. LACTATIONAL INTENSITY

APPENDIX E

(INDIVIDUAL VALUES)

Groups A-D received 1.0 ug_I- added/g feed.
GrouP E received 10.0 ug I added/g feed.
Lactators bled at 16 days postpartum.

 

 

no.

Rat.

OCOGNIOUUIIPCNNH

|_\

Rat.

11

15
14
15

No.

EgT41100_gd serum

pgT4/100 m1_serum

7.15
5.29
5.52
5.52
5.98
5.78
6.05
5.51
5.22
7.15

5.927

0.22

Group A

jgonlactating)

E
S.E.

GrOUp B

 

(5 pups per l1tter)

5.52
5.06
5.52
5.29
5.52

5.58
0.09

67

PBI

1ng1100 m1_serum)

1.86
2.12
1.65
2.08
5.10
5.85
4.95
2.51
2.51

. 0
6

2 7
0.5

PBI

(LI-91100 ILLS eggs).

1.85

1.66
1.56

Rat no.

16
17
18
19
20

Rat no.

21
22
25
24
25
26

Rat no.

28
29
50
51
52
55
54
55
56

(6_pnpsgper litter)

68

Group C

43 T4/100 mlgperum

5.52
4.14
4.14
2.99
2.95

5.96
0.48

Group D

(pg/100 ml serum)

PBI

 

S.E

(12 pupspper litter)

 

u91241100 ml serum

(12 pups per 11ttepi 10 x normal I- allowance)

5.44
5.91
5.44
5.21
5.21
5.68

5.48
0.11

Group E

ug_T4[100 ml serum

5.44
5.68
5.68
5.91
5.21
5.68
5.21
5.68
5.68

5.57
0.08

(pg/100 ml serwm)

X

(pg4100 ml serum)

. X

1.97
1.57
2.12
1.50

1 79
0.14

PBI

0.99
1.14
1.24
1.26
1.80
1.54

1.52
0.10

PBI

2.55
5.00
2.50

APPENDIX F

SERUM THYROXINE MEASUREMENTS, REPRODUCTION,
AND MILK RECORDS OF HOLSTEIN cows
(INDIVIDUAL VALUES)

 

 

Nonlacting-Nonpregnant

.(bloodusgppling_10-1-68L

 

 

 

 

 

 

 

Cow Birth Body Wt. No. of Mg T4/100 ml
No. Date (lbs.) Previous Calves perum
265 6-18-62 1415 2 6.52
815 5-4-64 1200 2 6.42
882 5-25-66 1260 1 5.86
"SE = 6.20
S.E. = 0.17
Nonlactating-Pregnant
(blood sampling 11-21-99)
Body No. of Subsequent
COW' Birth Wt. Previous Date Calving ug T4/100
No. Date (lbs.) Calves ‘Dried Off Date ml serum
800 12-9-64 1242 2 10-15-68 11-27-68 4.28
970 4-22-62 .1576 4 9-18-68 12-8-68 5.50
985 1-1-62 1480 4 9-1-68 12-9-68 5.40
904 10-15-64 1576 5 9-18-68 12-9-68 5.71
961 11-22-65 1568 2 .10-15-68 12-15-68 5.56
957 7-12-65 1557 2 10-15-68 12-5-68 7.59
766 10-14-65 -1554 5 10-15-68 .12-15-68 5.15
989 4-29-65 1690 1 11-1-68 12-25-68 5.59
768 12-8-65 -1796 5 10-15-68 12-26-68 5.97
801 12-25-64 1752 .1 10-1-68 12-50-68 6.81
795 10-25-64 1785 2 9-18-68 12-28-68 5.55
SE = 5.66
S.E. = 0.25

Based on drying and subsequent calving dates this

grOUp averaged 48 days dry and 255 days pregnant

at the time of blood sampling.
280 days.

69

.Gestation period

Cow
.No.

519
796
780
1027
799
716
955

Cow
No.

902
991
858
811
702
869
875
771
891
857

Birth
Date

2-15-64
11-4-64
2-26-64
6-12-65

.11-18-64

7-4-61
11-7-64

Body
Wt.
(1103;)

1560
1400
1607
1114
1498
1194
1484

Lactating-Nonpregnant

70

 

-1-
Nos. of Last Milk Rec.
Previous Calving (lbs.) ug
Calves .Date 10-1-68 ml
5 9-5-68 57.5
5 9-6-68 71.5
2 7-1-68 60.0
2 8-15-68 75.0
2 6-20-68 62.0
4 6-28-68 92.0
2 7-17-68 77.0
i =
S.E. =

T4/100
serum

5.08
5.52
5.62
5.55
5.17
5.81
5.16

5.59
0.10

Based on the last calving dates this group averaged
65 days lactation with a milk output of 68 lbs. per
day at the time of blood sampling

Birth

.Date

7-6-64
8-22-64
5-6-66

10-18-64
.11-50-60
10-28-64

5-15-64
1-6-64
6-5-65
5-4-66

Lactating-Pregnant

bl od sam

Body No. of

Wt.

1885
1550
1220
1840
1550
1815
1578
1480
1458
1122

Previous

(lbs.) Calves

l—‘l—‘(fiNNPNI—‘NN

 

 

l'n 11-26-68
Subse-
Previous quent Milk Rec.
Calving Calving (lbs.)
Date Date (11-25-68)
5-8-68 6-19-69 25.5
2-12-68 6-12-69 44.0
5-25-68 5-17-69 29.5
11-16-67 5-14-69 15.0
1-2-68 5-24-69 44.0
2-28-68 5-15-69 15.5
4-5-68 5-2-69 .18.5
2-19-68 4-20-69 17.5
1-25-68 4-26-69 15.5
2-22-68 4-7-69 55.0
X
S.E.

#9 T4/

100 ml
serum

Based on an average gestation period of 280 days and
the previous and subsequent calving dates this group
averaged 169 days pregnant and 278 days lactation

with a milk output of 28 lbs per day at the time of

blood sampling.

5.70
7.95

APPENDIX G

SERUM THYROXINE MEASUREMENTS IN YEARLING,
PREGNANT, AND LACTATING SUFFOLK EWES
(INDIVIDUAL VALUES)

 

—
-

m

Yearling Control Ewes
(blood sampling 1-29—69)

 

(Ewe NO.* ug_T4/100 ml serum
8-58 16.54
8-42 15.15
8-04 15.97
8-21 11.16
8-26 12.99

i = 13.52
S.E. = 0.84
Estimated Mid-pregnant Ewes
(b100d sampling 11-19-68)

Ewe No. ug T4/100 ml serum
4-55 10.55
7—29 410.91
4-15 8.19
4-55 12.47
7-25 15.94
4-29 12.79
5-06 11.65
6-50 10.92
7-54 15.95

i = 11.90
S.E. = 0.58

 

 

71

72

Lactating Ewes
(blood samp11ng_1-29-69)

No. of
Lambing Date Lambs

12-51-68
12-28-68
1-5-69
1-2-69
1-21-69
12-51-68

NPHPNP

ug T4/1OO
ml serum

10.85
10.85
7.71
8.75
10.49
7.56

9.52
0.67

 

*
First digit of ewe no. indicates year of birth, during

Jan. or Feb.

APPENDIX H
SERUM THYROXINE AND PBI MEASUREMENTS IN YEARLING,

LACTATING, AND LACTATING THYROPROTEIN—FED HAMPSHIRE EWES
(INDIVIDUAL VALUES)

All ewes bled on 1-29-69

 

 

Yearling Control Ewes

 

 

 

 

75

PBI
Ewe No.* ug T4/100 ml gerum ug/100 ml serum

8-25 8.05 5.82
8-09 7.01 5.06
8-56 12.57 4.94
8-44 10.14 4.01
8-18 7.56 4.67
8-55 6.66 9.69
8-14 8.40 4.17
Ef= 8.59 .§f= 5.19

S.E. = 0.80 S.E. = 0.77

Lactatingnges
Lambing No. of Mg T4/100 PBI
Ewe No. Date Lambs ml serum ug/100 ml serum

7-22 .1-22-69 1 7.56 6.47
5-06 1-15-69 2 foster 4.25 6.51
5-14 12-20-68 2 5.62 4.81
4-21a 1-11-69 1 5.54 5.61
5-22 1-9-69 1 5.62 5.10
5-26 1-25-69 5 5.27 4.87
SE = 5.27 36 = 5.55

S.E. = 0.55 S.E. = 0.50

74

Lactating Thyroprotein-Fed Ewes
(0.5 g Protamonel/ewe/day mixed with
grain bgginning 4-6 daysposppartumL

 

Lambing No. of ngvT4/100 PBI

Ewe No. Date Lambs ml serUm ug/100 ml serum
5-01 1-1-69 2 7.56 4.56
5-07 1-51-68 2 6.52 6.28
7-10 1-8-69 2 7.01 8.48
5—05b 1-9-69 2 6.66 7.59
5561 1-18-69 1 7.56 6.81
7-12 1-27-69 2 7.56 8,02
X = 7.01 X = 6.96
S.E. =0.17 S.E. = 0.58

*
First digit of ewe no. indicates year of birth, during
Jan. or Feb.

1Thyroactive casein--10X activity of U. S. P. desiccated
thyroid--contains 5% nonhormonal iodine.

aEwe 4-21, severe wool loss 1-29-69, no wool regrowth
5-25-69, see Plate 1.
b

‘Ewe 5-05, severe wool loss 1-29—69, 60% wool regrowth
5-25-69, see Plate 2.