THE HORMONAL CONTROL OF REPRODUCTIVE ACTIVITY
IN THE CYCLING AND ANESTROUS EWE

by
JACK F. WAGNER

AN ABSTRACT

Submitted to the School for Advanced Graduate Studies of
Michigan State University of Agriculture and
Applied Science in partial fulfillment of
the requirements for the degree of
DOCTOR OF PHILOSOPHY
Department of Physiology* and Pharmacology

Year

Approved

1958

Abstract

J. F. Wagner

The objectives of this investigation were to determine a more
practical method of inducing reproductive activity in the anestrous
ewe and to ascertain some of the mechanisms involved in the repro­
ductive phenomena occurring in the ewe.

The effects of various

hormones and combinations of hormones upon reproductive phenomena
in the ewe were studied.
A single injection of a microcrystalline suspension of proges­
terone in a 0*3 percent starch solution was found to exert its
action over a period of approximately 16 ^ 0.5 days in the anes­
trous ewe.

During the height of the breeding season this period

was reduced to 12 ± 0*5 days with an increase in this length of
action as both ends of the breeding season were approached.

It

was also shown that progesterone synergised with estrogen to give
a maximal estrous response.

No relationship was shown between

this action and the ovulatory mechanism.
The dosage of Pregnant Mares* Serum (PMS) given (1000 i.u.)
was adequate to induce most of the anestrous ewes to ovulate (88$) ,
and in conjunction with progesterone given 16 days previously,
induced heat in 44 to 50 percent of the ewes.

It was concluded

that the relatively low incidence of estrus in the animals treated
with progesterone-PMS used in this study, compared to daily
injections of progesterone in oil followed by PMS, was due to
the between-ewe variation in the length of inhibitory action of
the single dose of progesterone*

This would result in too much

Abstract

J. F. Wagner

or too little progesterone at the time of PMS injection.
The incidence of estrus was increased from 44 percent to 88
percent with doses of 50-200 ug. of estradiol 36 hours after the
PMS injection.

Although this treatment had no effect upon the

percentage of ewes ovulating, the length (58 hrs.) of estrus was
increased beyond the normal range (40 hrs.).

The lambing data

indicated that this increased length of estrus was detrimental
to some phase of the fertility mechanism.

In this study none

of the ewes that lambed (15) as a result of hormone treatment
had estrous periods greater than 30 hours in length.
No estrogen activity originating from an endogenous

source

was extracted from the blood of the ewe in assayable quantities.
When a microcrystalline suspension of estradiol was injected
intravenously in 500 ug. dosages, none was detected in the
blood within 5 minutes after injection.

The sodium-salt of

estradiol when injected intravenously, was extractable from the
blood but, only after the blood samples were partially hydrolyzed
in an alkaline medium prior to extraction.

It was concluded

that the microcrystals of estradiol were removed from the blood
within a very short space of time after injection, probably via
the reticuloendothelial system, and that a large proportion of
the sodium-salt of estradiol was chemically- bound to the blood
proteins shortly after entering the blood.
The progesterone starch suspension given in a single dose,
followed 16 days later by PMS, induced estrus (44-50$), ovulation

Abstract

J . F . Wagner

(88-90$), and fertility (25-50$ of ewes serviced) in the anes­
trous ewe.

The microcrystalline suspension of estradiol decreased

the lambing percentage of ewes serviced.

The sodium-salt of

estradiol at the levels used had no effect upon the incidence of
estrus in the progesterone-PMS treated ewe, but the lambing
percentage (83$, or 5 of 6 ewes) of the ewes serviced was much
higher than with the treatment using progesterone-PMS alone.

It

was inferred that the sodium-salt of estradiol had some beneficial
effect upon the reproductive mechanism other than the induction
of estrous behavior.

THE HORMONAL CONTROL OF REPRODUCTIVE
ACTIVITY IN THE CYCLING AND ANESTROUS EWE

By
JACK FRED WAGNER

A THESIS

Submitted to the School for Advanced Graduate Studies of
Michigan State University of Agriculture and
Applied Science in partial fulfillment of
the requirements for the degree of
DOCTOR OF PHILOSOPHY

Department of Physiology and Pharmacology

1959

ProQ uest Number: 10008576

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ACKNOWLEDGMENT

The author wishes to express his appreciation to the
members of the Department of Physiology and Pharmacology, Mich­
igan State University, for their contributions to the completion
of this study.

In particular I would like to express my grati­

tude to Dr. E. P. Reineke, Dr. J. E. Nellor and Dr. H. A. Henneman who gave generously of their time during the course of
experimental work and the preparation of this thesis.
The author wishes to thank Mr. Lee Bell, shepherd at
Michigan State University, for his cooperation in management
of the experimental animals throughout the experimental studies.
The services of Dr. G. H. Conner in performing the surgery
involved in the experimental work are sincerely acknowledged.

TABLE OF CONTENTS
Page
INTRODUCTION

1

REVIEW OF LITERATURE
Control of the Estrous Cycle in

the Cycling Ewe. . . .

3

The Induction of Estrus and Ovulation in the
Anestrous Ewe. . . . . .............. . . . . .

9

Hormonal Mechanisms Involved in Reproduction in the
Ewe
..............................

17

MATERIALS AND METHODS
Experimental Animals ............

. . . . . . . . . .

33

Hormone Preparations
Thyroprotein
.
Progesterone
................................
Pregnant Mare Serum (PMS)...............
Estrogens. . . . . . . . . . . . . . . . . . . . .

3^
37
38
39

Experimental Methods
Observation of Estrous Behavior................
Methods of Evaluating Slaughter Data ..........
Vaginal Smear Technique and Evaluation ........
Collection of Blood and Urine Samples for
Estrogen Assay ............................
Extraction of Estrogenic Substances from Blood and
Urine
..........................
Bioassay of Estrogen-Containing Extracts . . . .

^0
^1
^
^

^6
53

RESULTS
Determination of the Length of Progestational Activity
in the Cycling and Anestrous Ewe, Produced by a
Single Injection of Progesterone in Starch
Suspension
..................

58

The Effectiveness of PMS in Inducing Estrus and Ovula­
tion in the Anestrous Ewe......................

71

Role That Exogenous Estrogen Plays in the Induction of
Reproductive Activity in the Anestrous Ewe . . .

76

DISCUSSION

91

SUMMARY AND CONCLUSIONS

110

BIBLIOGRAPHY

H 2*

APPENDIX

127

LIST OF FIGURES
F ig ure
1.

'

The Effect of Exogenous 1 -Thyroxine and
Thyroprotein Upon Output of 1^-31 from the
Intact Ewe. . . . . . . . .
....................

2 . Flow Sheet Depicting the Extraction of the Total
Estrogenic Activity from Whole Ewes' Blood . . . .
3.

Evaporating Column Used in Tandem . . . . . . . .

4.

Uterine Weight Response to Graded Doses of
E s t r a d i o l ....................................

5-

.

Dose-Response Curve of Level of Second Injection
of PMS (16 days after first injection) Versus
Percentage of Ewes Coming in Heat at Each Level .

6 . The Urine Excretion of Estrogens Added in vivo. .

LIST OF TABLES
Table
1.

Page
Number of Anestrous Ewes Showing Estrual Activity
after Hormone Treatment. . . . . . . . . . . . . . . .

58

2 . The Return to Heat of Cycling Ewes after Injection
with 280 mg. of Progesterone in Starch Suspension. . .
3*

The Effect of the Variation of the Interval Between
the Progesterone and the PMS Injection upon the
Incidence of Estrus and Ovarian Activity in the
Anestrous Ewe.....................................

^0

63

^4-.

Synchronization, Onset of Estrus, and Breeding Data on
Cycling Ewes after Treatment with a Single Injection of
Progesterone Suspension in Starch.................
6^

5-

Distribution of Onset of Estrus after Progesterone
Treatment.........................................

66

6 . The Inhibition of Estrus by Progesterone and the
Resulting Degree of Synchronization..................
7.

Record of Estrous Behavior after Two Injections of PMS

8 . The Effects of Progesterone-PMS Treatment upon Estrual
9.

72

Behavior and Ovulation

76

Frequency of Occurrence of Heat in Ewes after Hormone
Treatment........................

77

10 . The Onset and Length of Estrus in Hormonally Treated
E w e s ................................................... 78

11 . The Effect of Exogenous Estrogen on Anestrous Ewes
Treated with Progesterone and PMS....................... 60
12.

The Effect of Hormone Treatment on the Induction of
Estrus and Fertility in the Anestrous Ewe............

®1

13 . Determination of Estrogenic Activity in the Blood of Ewes
During Induced and Natural Estrus and the Fourth Month
of P r e g n a n c y ..............................

62

1^+.

Extraction of Estrogen from the Blood of Ewes Receiving
an Intravenous Injection of Estradiol................... 65

15.

Effects of Type, Route of Administration and Level of
Estradiol upon the Occurrence of Estrus, Length of
Estrus and Conception................................

INTRODUCTION

Most breeds of ewes are sexually inactive during the spring
and summer months.

In many cases there would be an economic

advantage if the ewe could be bred during these months of anestrus. Breeding at this time would enable the farmer to obtain
one and a half or two lamb crops a year as well as spreading the
marketing of lambs throughout the year.
It is well established that the breeding cycle is under
hormonal control.

Fundamental research conducted in many labor­

atories over the past 30 years has resulted in elucidation of
many of the hormone mechanisms and hormone interrelationships
involved in the process of reproduction.

However, successful

application of these findings to the breeding of sheep has
necessitated further research on the special hormone responses
of this species.

The results obtained have been variable

and the techniques employed have been cumbersome when applied
to field conditions.
The hormone regime used .most extensively for the induction
of estrus and ovulation in the anestrous ewe involves pretreat­
ment with progesterone followed by pregnant mare serum (PMS).
Although the induction of estrus with ovulation has been
achieved with some regularity, the fertility has been low.

In

some cases the incidence of estrus has also been low and es­
trogens have been added to increase the percentage of treated
ewes coming in heat.

2

This thesis includes an analysis of some of the hormonal
factors involved in reproduction in the ewe together with a
new approach that it is hoped will contribute to the final
solution of the problem of breeding sheep during the anestrous
season.

3

REVIEW OF LITER AT IRE

Many reports have been published concerning the augmenta­
tion of fertility in the ewe.

Phillips et al. (19^5). Robin­

son (1951a), and Gordon (1958) have recently written reviews
covering the material presented in the last few years.
The emphasis in this study has been placed on three
main categories: l) the hormonal effects upon reproduction
in the cycling ewe, and 2) the hormonal effects upon reproduc­
tion in the anestrous ewe, and 3) the hormonal mechanisms involved
in reproduction in the ewe.
Control of the Estrous Cycle in the Cycling Ewe
After the initial discovery and isolation of estrogen
by Allen and Doisy (1923) > many attempts were made to utilize
estrogens to increase productivity in the ewe.

Although the

injection of estrogenic extracts would bring the ewes into
heat (Anderson, 1938; August, 19^+1; Bell et al. , 19^1; Hammond
et al., 19^ 2 ; Cpperman, 19^3) and in some cases hasten the
onset of the breeding season (Anderson, 1938 and August, 19^*1) »
the fertility was low (Bell et al., 19^1; Frank and Appleby,
19^3; Hammond et. al. , 19^-2 ; Quin et al*, 19^3; Price and Hardy,
1953; and Venzke, 1953)*

Some investigators, Anderson (1938)

and August (l9^l)> have reported that the injection of estradiol
benzoate or some other estrogenic substance in fairly large quan­
tities (0.5 to 1.0 mg.) will induce a fertile estrus in a large
percentage of the cycling or late anestrous ewes.

Except for a

4

few instances (Price and Hardy, 1953 and Venzke, 1953)» most of
the contemporary workers have abandoned the use of estrogens
alone for this purpose.
The potentialities of the gonadotropins for the increase
in ovulation rate and subsequent lambing percentage have long
been an area of interest.

Human chorionic gonadotropin (Zavad-

ovskii et al . , 1935 and Zavadovskii and Paduceva, 1935)» pitui­
tary extracts (Casida et al. , 1944; Murphree et, al., 1944) , and
PMS (Gordon, 1958; Palsson, 1956; Robinson, 1951a, 1951b, 1956;
Wallace ejs. al., 1954) have been used in attempts to increase the
lambing percentage of the cycling ewe.

The results of these

experiments varied with breed, geographical area, dosage, and
period in the cycle when treatment was given,

Zavadovskii and

Paduceva (1935) (with 150 to 500 mouse units of HCG on the first
day of heat) failed to induce superfoetation (all ewes produced
one lamb each), Injections one to four days before or two to
three days after heat also failed to increase nidation.
tion rate was not recorded in these experiments.

Ovula­

Four years

later Zavadovskii and Paduceva (1939&), with three different
gonadotropic preparations (PMS, HCG, and pituitary extracts)
and higher dosages than previously, reported an increase in the
percentage of twinning. The percentage of barren ewes also
increased with a net result of 26 percent more lambs born per
ewe bred as compared with the controls. Data from the group
treated with PMS gave the best results. In another group of
ewes (Zavadovskii et. al,. , 1939b) the lambing was increased from

5

12 to b'Z percent above that of the controls when the PMS was
given from one to three days before the onset of the normally
occurring estrus.
Casida et al. (l9^+) employed a double dose of gonadotropinpituitary FSH followed by LH

two to three days later, to induce

super-ovulation and super-fecundity.

A high embryo mortality in

the first 37 days post service was encountered with this method
of treatment, the viable fetuses recovered being no greater than
the average in the controls.

Murphree et al. (l9^) reported

that ova shed under the influence of PMS (either anestrous or
cycling ewes) are potentially incapable of being fertilized.

On

the other hand the induction of super-ovulation with pituitary
extracts of FSH during the follicular phase of the cycle resulted
in a large proportion of fertilized eggs.
Robinson (l95lb, 195^a) has used PMS exclusively as a source
of follicle stimulating hormone.

He demonstrated a dosage-ovulation

response curve when the treatment was given on the 12th day of
the cycle or early proestrus (500 i.u. equals four ovulations,

1000 i.u. equals 10 ovulations, and 2000 i.u. equals 16 ovula­
tions) . The cycles and ovaries appeared normal at all dosages
except the 2000 i.u. level where some evidence of luteal cysts
and ovulation failure were encountered.

Robinson, in contrast

to earlier reports (Casida et al. , 1 9 ^ and Murphree .et al. ,
1944) reported that ova obtained following PMS injection were
highly fertilizable. In a few ewes as many as nine to 13
embryonic attachments have been found in the uterine endometrium.

6

Although many of the ova were fertilized and a large percentage
of these attached to the endometrium, a high embryonic mortality
occurred between the seventeenth to nineteenth day of gestation.
The net result of the first study reported in this paper showed
that the lambing percentage of treated ewes was 192 percent vs.
1^7 percent for the controls.

Since some of the treated ewes

didn*t lamb, the lamb crop was considerably diminished but still
above the controls at 16? percent.

Subsequent work (Robinson,

1956) demonstrated the lack of predictability of a supposedly
successful treatment from one time to the next and one breed to
the next.

Using a smaller number of ewes (15) and a single

dosage of M S

(500 i.u.) } more lambs were born per ewe, but

fewer ewes lambed and less lambs were weaned per ewe bred as
compared with the controls.
The correct interval between the injection of PMS and the
next expected estrus was thoroughly examined by Wallace (195*0 •
He corroborated much of Robinson 5s work on ovulation rate and
fertility and showed that 650 to 1000 i.u. of PMS was suitable
for inducing multiple ovulations (two to three), but not an
excessive number (four or more) which leads to high embryonic
mortality.

He recommended that the PMS injection be given

between the twelfth to the fifteenth day of the cycle depending
upon the dose level, with the higher dosage given closer to the
next expected heat period.

Wallace ejb al. , (195**”) employing a

large number of ewes (517) > reported that the lambing percentage
of the treated ewes varied considerably from one trial to another,

7

but in all cases it was higher than in the control groups.

Two

very interesting observations, that are not supported by most
other studies (Murphree et al., 1 9 ^ ; Robinson, 1951b; and Zava­
dovskii et. al. 1939) > were: l) the number of ewes holding to the
first service were the same between treated and controls and

2) treatment did not increase the number of ewes which failed
to conceive during the breeding season.

Palsson (1956)» using

the PMS treatment previously described, reported that treating
the same ewes in two successive years maintained the increased
lambing percentage at approximately the same level for both
years.

Gordon (1958) investigated the practical application

of PMS treatment to the English breeds of sheep.

He used 2500

ewes at many levels of PMS (from 250 to 1500 i.u.) and found a
tendency for the flocks with the poorest lambing records to give
the best response to treatment, with 500 i.u* of PMS often giving
increased lambing percentages as large as those receiving higher
doses.

Although he instituted treatment without knowledge of

stage of the cycle he had a substantial increase in the lambing
percentage of the treated ewes (l67 percent vs. 139 percent).
This was not the case in most of the studies conducted, where
PMS treatment during the luteal phase of the cycle resulted in
irregular ovulation (Murphree et al., 1 9 ^ * Wallace, 195^;
Zavadovskii et al. , 1937)1 lutein cysts (Wallace, 195^; Zavadov­
skii et a^L., 1937; Zavadovskii et al. , 1939) » irregular estrous
behavior (Wallace, 195^; Zavadovskii et al., 1939)* a^d low
lambing percentages (Murphpee et al. , 1 9 ^ ; Wallace, 195^;

8

Zavadovskii and Paduceva, 1939a).
The first workers to investigate the ability of progesterone
treatment to synchronize the estrous cycle in the ewe were Dutt
and Casida, (19^8), O ’Mary et, al. , (1950)» Hunter, (195^)» and
Robinson, (1956).

Dutt and Casida (19^8) reported that daily

subcutaneous injections of 10 mg. of progesterone in oil effec­
tively suppressed ovulation in the ewe irrespective of the stage
of the cycle at which the treatment was started.

This treatment

was carried out for 1*+ consecutive days and estrus occurred with­
in three to 3*5 days after the last injection.

Ova were recovered

after insemination and fertility was found to be unaffected by the
treatment.

At the 5

* dosage it was found that although estrus

is suppressed, ovulation occurred in a few cases and cystic
follicles developed in some which did not ovulate.

O'Mary et al.,

reported that 70 percent of the ewes came into heat three days
after progesterone withdrawal resulting in a lambing percentage
that was not significantly different from the control group.
Using the treatment described by Dutt and Casida, Hunter, (195^)t
found that in one group of Border Leicester ewes, 25 percent
failed to come into heat on the first estrus after progesterone
withdrawal, while none missed the second.

In another group of

ewes ^+7 percent came into heat in less than three days after
progesterone withdrawal, 37*5 percent came into heat after more
than three days and 15*5 percent failed to come into heat.
Robinson, (1956) used both progesterone and BIS in the
cycling ewe in order to produce both synchronization of estrus

9

and multiple ovulation.

Daily injections of 10 mg. progesterone

in oil were given for 16 days, with one group receiving an addi­
tional 500 i.u. PMS the day after the final progesterone injec­
tion.

The PMS injection after progesterone withdrawal signifi­

cantly advanced and improved the precision of the onset of
estrus* and presumably of ovulation.

However, contrary to the

previous reports that PMS increased lambing percentage (Robinson,
1951b, 195^a)* the progesterone-FMS treatment had no significant
effect either upon the percentage of ewes lambed or upon the total
lamb crop.

Data were presented to indicate that the lambing

percentage was highest in those ewes inseminated on the third day
after the progesterone withdrawal (regardless of PMS treatment)
and that it fell off sharply in those ewes inseminated within
the next 2^ hours.

The New Zealand Department of Agriculture

(1957) conducted a similar experiment on a field trial basis and
obtained essentially the same results as did Robinson.

They

indicated that a possible explanation for the lack of increase
in lambing percentage in the treated groups was that the PMS
dosage was too low.
The Induction of Estrus and Ovulation in the Anestrous Ewes
Phillips et al. (19^5) and Robinson (1951a) have published
two reviews on the hormonal induction of estrus, ovulation and
fertility in the anestrous ewe.

Robinson (1950) reported an

extensive series of experiments involving the study of the
various hormonal treatments and their effect upon the induction
of pregnancy in the anestrous ewe.

10

The initial attempts at hormonal induction of reproductive
activity in the anestrous ewe involved the use of estrogenic
substances.

Cole and Hiller, 1933 and Zavadovskii et al. (1935)

were the first to apply the use of hormones to induce fertility
in the anestrous ewe.

Although they met with little success,

some observations were made in this early work which have
remained as guideposts in the studies which followed.

They

observed that estrogen alone did not induce ovulation in the
ewe with any degree of certainty and especially in the anestrous
ewe where the presence of a mature follicle is rare.

Estrogens

produced heat when given in large enough quantities, but appar­
ently had little effect upon ovulation and induction of cyclic
activity in the anestrous ewe.
Bell et al. (l9^l) using oestradiol benzoate (ODB) , Price
and Hardy (1953) and Venzke (1953) using estradiol cyclopentylpropionate (ECP) and Frank and Appleby (19^3) using stilbestrol,
consistently produced heat in anestrous ewes, but neither concep­
tion nor hastening of the breeding season was observed.

Hammond

et al, (19^ 2) injected ten ewes with stilbestrol and reported six
ewes ovulating and six ewes exhibiting estrus, but only three ewes
showed estrus and ovulation together.

In view of this limited

success using estrogen to induce estrus and ovulation, these
workers postulated that perhaps stilbestrol was inducing ovula­
tion by stimulating the release of LH from the anterior pitui­
tary.

However, they recognized the need for a mature follicle

to be present at the time of treatment, and pointed out that their

experiments were carried out towards the end of the anestrous
period and the possibilities were high for the occurrence of
’’silent heat” along with mature follicles.

Quin and VanJerWath

(19^ 3) also used stilbestrol (1-5 ^6 •) and did not induce estrus
in a significant proportion of anestrous ewes.

They also reuor-

ted that stilbestrol (l mg.) would induce estrus 100 percent
of the time when injected into ewes 16 months after spaying.
They postulate that the anestrous ovary decreases the physiolog­
ical

response of the uterus, vagina, and estrual behavior

patterns to estrogen.
Tn direct contradiction to the preceding reports, Anderson
(1938) observed that a single injection of oestradiol benzoate
(ODB) in anestrous ewes would induce, not merely a single heat
period, but caused the resumption of the normal estrous cycle
in approximately 75 percent of the treated animals.

August

(19^1), using large numbers of anestrous ewes, stated that a
single injection of ODB caused 75-2 percent of them to show
heat and 60 percent of those mated produced lambs.
Cole and Miller (1933) and Frank and Appleby (19^3) reported
that the addition of FMS to estrogen showed little evidence of
inducing estrus along with ovulation in a significant oortion of
the treated ewes.

However, Cole and Miller laid some of the

initial groundwork for the use of FMS as a gonadotropic agent
for the stimulation of follicular growth and ovulation in the
anestrous ewe.

They demonstrated that the injection of FMS on

several successive days did not produce estrus, nor did a single

12

large dose of PMS produce estrus, although In either case the
majority of the ewes ovulated approximately four days after the
last injection.

When three injections of PMS were given in the

late anestrous period (between July 12 and August 11) , the begin­
ning of normal cycling activity was hastened by approximately
one month as compared to the controls.
Many investigators

(McKenzie and Terrill, 1937; Varadin,

1957; Zavadovskii et al.. , 1935; Zavadovskii et al., 1937 to 1939)
have attempted forced mating or artificial insemination two to
four days after the administration of PMS.

Although ovulation

occurred in a large percentage of the treated animals, few if any
became pregnant.

Zavadovskii (1939^), used single injections

of PMS, HCG, or pituitary extracts, and obtained conflicting
results between the various extracts and with different trials
using the same extracts.
effect than with PMS.

He stated that HCG had a quicker FSH

In one case, Zavadovskii et. al,. (1939a)

obtained a 55 percent lamb crop when the ewes were artificially
inseminated two days after HCG treatment.

Zavadovskii (1939b)

also demonstrated that nutritional factors (both pasture quality
and severity of weather which affects ability to forage) played
a more important role in the induction of reproductive activity
in the anestrous ewe than they did under normal breeding condi­
tions ,
Cole and Miller (1933) introduced the double injection of
PMS spaced at approximately a 1 6 day interval.

Ten out of 15

anestrous ewes came into heat in one to seven days after the

13

second Injection, were bred, and had lambs the following fall.
Varadin (1957) reported that a single injection of FMS induced
no estrous periods and upon insemination 24 to 48 hours after
the PMS injection, none of the ewes conceived.

However, using

two doses of PMS spaced at a 14 day interval, 65 percent of the
ewes came into heat and 41,7 percent conceived.

Cole and Miller

(1945) conducted a survey in California to determine the effective­
ness of PMS in producing heat and pregnancies in anestrous ewes.
The single dose of PMS resulted in seven percent of the ewes
coming into heat and no ewes became pregnant.

The double dose

of PMS induced 34 percent of the treated ewes to come in heat
and one-third of these or nine percent became pregnant.
Since the double dose of PMS spaced at a 16 day interval
was not as effective as the preliminary studies indicated, a
number of investigators (Cole et. al*, 1945; Davis and Dun, 1957;
Dutt, 1952; Lambourne, 1955; Robinson, 195^a) instituted a differ­
ent hormone sequence that involved pretreatment with progesterone
prior to the FMS injection.

Dutt (1952) compared the PMS alone,

progesterone alone (five injections of 30 mg. at three day inter­
vals) and progeste'rone-PMS with regard to their ability to induce
estrus and ovulation at approximately the same time in the anes­
trous ewe.

PMS alone induced ovulation in all animals, but none

came in heat.

Five injections of progesterone without FMS resul­

ted in four out of nine ewes coming into heat and ovulating within
four to five days after the progesterone withdrawal.

When five

injections of progesterone were given prior to the PMS all nine

1^

ewes came into heat and ovulated.

Forty-nine percent of the ova

recovered from the progesterone treated ewes were fertilized.

As

the number of the progesterone injections decreased, the treat­
ment became less effective with regard to heat, ovulation, and
fertility.

In a later study Dutt (1953a) , using five proges­

terone injections at three day intervals followed by PMS, induced
heat in 100 percent of the treated ewes within ^+8 hours after the
PMS injection and 50 percent (ll) of these ewes lambed. Robinson
(I95^a, 195^b) has used essentially the same procedure with two
exceptions: l) the progesterone given twice daily (25 mg./day)
for three days, and 2) followed two days later by 1000 i.u. of
PMS.

The results obtained by Robinson in this study were similar

to those of Dutt.

Robinson

(l95^b) reported that the time rela­

tionships were physiologically normal, estrus commencing Zh to
36 hours after PMS and preceding ovulation by 10 to 20 hours.
Hysterectomized and intact ewes responded alike, suggesting that
the conditioning effect of progesterone is a central phenomena.
A field trial was conducted by Lambourne (1955) using five injec­
tions of 25 mg. progesterone followed by 1000 i.u. of PMS in the
late anestrous season.

The results obtained in this trial were

very satisfactory in inducing heat and ovulation, but the objec­
tive of this experiment was primarily to hasten the onset of the
breeding season, and not to obtain pregnancies at the first
induced heat period.

The treatment had no effect upon the lambing

percentage, but the control animals lambed later in the spring
than did the treated group.

A progesterone pellet inserted

15

under the skin also was used to determine if it would exert
an effective progestational activity over a period of 12 to 1^+
days.

This treatment was ineffective in altering the onset of

estrus.

Re-weighing the pellets after removal showed that only

about 15 m g . had been absorbed from the 100 m g . pellets. Davis
and Dun (1957) gave daily doses of 10 mg. progesterone for 16
days and on the seventeenth day 500 i.u. of FM5.

A majority of

the ewes came into heat (3^ out of 36), 22 hours after the FMS
injection, but none of the ewes serviced at this estrus conceived.
They postulated that early occurrence of estrus prior to ovula­
tion accounted for the infertility.

Averill (1958) in an attempt

to produce fertilized sheep eggs for ova transplant, used both
PMS in the cycling ewe, and progesterone-FMS in the anestrous
ewe.

The results obtained concerning ovulation and estrus were

similar to those previously cited.

He observed that the fewer

the ovulations (two to four) the greater the percent recovery
and that 78 percent of the anestrous ewes treated had fertilized
ova.

The proportion of ewes in which fertilization occurred

appeared to decline when the number of ovulations per ewe was
above ten.

They were very successful in transplanting 30 ova

to 1^ recipient ewes which subsequently bore Zh lambs.

It was

made clear that some of the recipient ewes were in the anestrous
season and had been conditioned by progesterone-FMS,

These ewes

did as well as the conditioned recipients receiving fertilized
ova during the breeding season.
Although much of the preliminary work indicated that the

16

progesterone-PMS treatment would induce estrus and ovulation in
a large percentage of the treated anestrous ewes, later attempts
at duplicating these results were unsuccessful (Robinson, 1955b).
Using the progesterone-PMS treatment, Robinson (1955b) found that
the percentage of ewes coming in heat ranged from 37 to 72 per­
cent.

Attempting to increase the incidence of induced heat in

the anestrous ewe Robinson injected various amounts of oestradiol
benzoate (ODB) simultaneously with the FMS injection.

When the

full treatment (progesterone-FMS-estrogen) was given, 100 percent
of the ewes came into heat.

However, the percent of the ewes

which lambed was very low in all groups regardless of treatment.
It was also shown that pretreatment with progesterone for three
days was marginal for the induction of the estrous response
whereas, progesterone pretreatment for six to twelve days will
produce a satisfactory response.

The length of progesterone

treatment was also reflected in the minimal effective dose of
ODB necessary to induce estrus in spayed ewes
1956).

(Robinson et al.,

An important point illustrated in this study was the

high sensitivity of the ewe to very small amounts of estrogen.
In much of the work done in the past (Anderson, 1938; August,
19^1; Price and Hardy, 1953; Quin and VahDerWath, 19^3; Venzke,

1953) estrogen has been used in such quantity that the physiolog­
ical

mechanisms of the reproductive tract would be affected out

of proportion to their normal function during the breeding period.
Robinson (1955b) found that as little as 15 to 25 ug. of ODB
coupled with progesterone pretreatment would induce estrus in a

17
large percent of the anestrous ewes, whereas the investigators
previously cited used estrogen of various types in milligram
quantities.
Hormonal Mechanisms Involved in the Reproductive Processes in
the Ewe
The ewe, unlike most other domestic animals, has an anes­
trous period lasting from six to nine months depending upon the
breed and geographical location.

The study of the hormonal actions

and interactions during the interphase period between the anes­
trous and breeding season has been of great interest to many
investigators.

The transition from the anestrous period to the

breeding period is not an abrupt occurrence, but rather one
taking four to eight weeks.

According to Robinson (l95^a) it

appears probable that photoperiodic control of the onset of the
breeding season is effected either by the release of gonado­
tropin by the anterior pituitary or by a change in the proportions
of the two components (LH and FSH) of the gonadotropin complex.
It has been reported by Gordon (1958), McKenzie and Terrill
(1937)> and Robinson (l95^a) that the first ovulation of- the breed­
ing season is not usually accompanied by heat. It is also apparent
that the first ovulation of the breeding season would be in the
absence of a corpus luteum. Many investigations have shown (Boling
and Blandau, 1939; Hammond £t. al., 19^ 2 ; Hertz et al., 1937; Melampy, et al., 1957; Moore and Robinson, 1957a; Robinson, 195^b;
Robinson et al,, 1956; Smith and Smith, 19^6) that progesterone con­
ditioning is necessary ^.n many species for the full manifestation

18

of the estrogen response.

Hertz et al. (1937) demonstrated the

necessity of progesterone for the induction of the copulatory
reflex in the estrogen treated spayed guinea pig.

Boling and

Blandau (1939) gave progesterone after, rather than before, the
estrogen in spayed rats.

They reported that this procedure was

more effective in producing a normal heat period than the injec­
tion of relatively large quantities of estrogen alone.

They hypo­

thesized that in the normal rat, sexual receptivity is caused by
the synergistic action of estrogen and progesterone produced in
the still unruptured follicles.
Smith and Smith (19^6) studying the cause and purpose of
menstruation, hypothesized that progesterone exerts a sparing
action upon estrogen by preventing the destruction of estrone to
oxidative inactivation products and these, rather than estrogens
per se are responsible for the release of the gonadotropins.
Campbell (1957) showed that the impaired liver lost some of its
ability to conjugate and inactivate estrogens and supplementary
to this it was shown that increased estrogen levels stimulated
repair processes in the liver. Szego and Roberts (1956) also
demonstrated that the estrogen-serum protein binding capacity
was correlated with the functional state of the liver indicating
that the liver also plays a role in the activation or carrying
of estrogens as well as inactivation.

Consequently, the proges­

terone sparing effect could be acting in one or both areas of
estrogen metabolism.
Robinson (195^ c ) determined the quantitative requirements

19

of progesterone and estrogen for estrous behavior and vaginal
cornifioation in the spayed ewe.

He found that at no level of

estrogen administration was it possible to induce estrus regu­
larly in a high proportion of the treated ewes.

When proges­

terone preceded estrogen, regular cycles of estrous behavior
were readily induced at all levels of estrogen treatment down to
40 ug. resulting in a 50 percent response.

Progesterone pre­

treatment also advanced the time of onset of estrus after estrogen
injection by 12 to 24 hours.

Robinson (1954b) and Moore and

Robinson (1957a) demonstrated that high levels of estrogens given
to spayed ewes induced a refractory condition.

After two or

three injections at seven and fourteen day intervals, few ewes
continued to show normal estrous behavior, with some exhibiting
male-like mounting behavior.

Robinson (1955a) plotted the estro­

gen dosage vs. the percent treated at each dose that came into
heat.

The effective dose to bring 50 percent into heat (ED^q )

was 22 ug. for those pretreated with progesterone and 64 ug. for
those which received ODB alone.

However, Robinson (1955a) could

demonstrate no dose-response curve when ODB was given alone.
Progesterone pretreatment resulted in a. steeper dose-response
line for estrus.

It was also demonstrated by Robinson et al.

(1956) that the duration of progesterone treatment (12 to 13 days)
is of more importance in sensitizing the body to estrogen than
the amount of progesterone used (3 to 24 mg. daily).

The sensi­

tivity appears to be linearly related to the log-duration of
progesterone influence.

20

The estrogen dosage to give an ED50 for vaginal cornification
was much less, 14 ug. for the progesterone pretreated and 24 ug.
for the ODB alone.

A positive smear was free of leucocytes, but

contained an appreciable number of cornified cells either alone
or with nucleated epithelial cells.
state that there is considerable

Robinson and Moore (195&)

variation between sheep con­

cerning the time after the occurrence of estrual behavior that
the positive cornification smears appear.
Melampy et al. (1957) reported that progesterone at the
proper dosage can induce estrous behavior in the estrogenconditioned ovariectomized cow.

Maximal synergistic action of

progesterone was observed when this hormone was injected 12
hours before, simultaneously with, and 12 hours following a
conditioning level of estrogen.

It was suggested that after

conditioning with estrogen, normal sexual receptivity in the cow
may be caused by the action of progesterone produced during the
period of the preovulatory development of the Graafian follicle.
Ewes and cattle differ in their responses to the progesteroneestrogen treatment.

Melampy et al. (1957) showed that the proges­

terone could be given at any time within 12 hours of the estrogen
treatment and still get a maximal estrous response.

However,

Moore and Robinson (1957b) reported that when ODB was injected
before or during the period of progesterone treatment no heat
was observed and the vaginal response was partially suppressed.
Maximal behavioral responses were observed when ODB was injected
24 to 48 hours after the final injection of progesterone.

21

The role of progesterone in the ovulatory mechanism has
been given a great deal of study. With the initial discovery
and isolation of progesterone from the corpora lutea in 1929
by Corner and Allen, they demonstrated that the major function
of progesterone in the estrous cycle was antagonistic to estro­
gen.

Large doses of progesterone inhibited estrual behavior,

vaginal cornification, and ovulation.

Makepeace et al. (1937)

reported that progesterone also inhibited ovulation in the
rabbit if given within one hour after mating.

Post-partum

estrus was also inhibited in the rabbit by injection of proges­
terone.

However, ovulation was induced with an LH preparation

in the progesterone treated rabbit and this led to the hypothesis
that the action of progesterone in the ovulatory mechanism was
at some site other than at the Graafian follicle.

Since that

t ime, much work has been done concerning the neural-humoral
mechanisms of gonadotropin release (Harris, 1950; Everett and
Sawyer, 19^0-^9; and Hansel, 1951)*
As the study of the hormonal interactions in the ovulatory
mechanism became more precise, it was found that small amounts of
progesterone were necessary in some species just prior to the rup­
ture of the follicle in order that ovulation take place. Everett
(l9^0-^3) demonstrated that appropriately timed injections of
sub-inhibitory quantities of progesterone in persistent estrous
rats restored the ovulatory cycle.'

The initial effect of the

treatment was the appearance of diestrus which was followed by
follicular development and ovulation if the progesterone treat­

22

ment was continued up to the ovulatory period, otherwise the rats
would lapse into persistent estrus at the end of the induced
dlestrous period.

In a related study Everett (19^3) determined

the optimum time for the second injection of progesterone after
the first injection had induced a diestrous condition in the
persistent-estrous rat.

The effectiveness of a second proges­

terone injection in producing ovulation and corpus .luteum form­
ation was slight in early diestrus,

rose to a maximum on the

first day of estrus, and declined gradually to a negligible value
when estrus had persisted longer than 8 days.

It was also shown

by Everett (19^8) that when estrogen levels were elevated above
normal, a normally inhibitory level of progesterone facilitated
ovulation, which indicated that this was a relative rather than
an absolute phenomena.

This evidence suggested that in the norm­

ally cycling rat, progesterone began to be secreted in the pre­
ovulatory phase of the estrous cycle and its source was the
Graafian follicle (Edgar 1953b)*
Sawyer

al# (1950) were able to induce spontaneous ovula­

tion in four out of ten intact rabbits by using an estrogenprogesterone treatment.

Although this is not a large proportion

of the treated animals, the other two groups which received only
estrogen or progesterone had no ovulations occurring.

Simpson

(1952) found that ovulation time in estrual ewes was shortened
by progesterone given early in estrus, but this reduction in
time was not significantly different from the controls.

Hammond

et al. (19^2) reported that in the absence of a corpus luteum,

23

ovulation will occur in the cycling ewe, but without any evidence
of heat.

In cattle Hansel and Trimberger (1952) found that the

subcutaneous injection of small doses (5 to 10 mg.) of proges­
terone at the beginning of estrus hastens the ovulatory process.
Both the length of estrus and the time from the end of estrus to
ovulation were significantly reduced.

If this progesterone treat­

ment was started early in the follicular phase, abnormal luteal
development, ovulation failure, and cystic follicle resulted
(Trimberger and Hansel, 1955)•

Ulberg et al. (1951) found with

gilts that when varying levels of progesterone were given to
synchronize estrus, ovulation occurred regularly after cessation
of progesterone treatment at the 100 mg. level.

However, when

12.5* 25 and 50 mg. dosages were used, a cystic follicular
condition resulted with very few ovulations occurring. Although,
these lower dosages were given over a long period of time (six
to 15 days) they were capable of inhibiting estrus and ovulation,
but at their withdrawal ovulation was not facilitated.

Rothchild

and Fraps (19^9) observed that small amounts of progesterone given
12 to 24- hours before the next expected ovulation will shorten
the time to that ovulation.

Pfeiffer (1950) using anovulatory

monkeys, was able to stimulate ovulation in three out of five
adult females by injecting 0.5 nig. progesterone daily for three
to six days beginning during the early follicular phase (tenth
to fourteenth day of menstrual cycle). It was suggested by most
of the investigators working on this problem that progesterone
synergizes with estrogens effecting a release of LH from the ante-

2k

rior

pituitary.

The action of these two hormones was thought

to be mediated through the hypothalamus (Harris, 1955» Everett
et al. , 19^9). Dutt (1953b) and Hansel (1953) give very good
reviews on the role of estrogens and progesterone upon ovula­
tion.
The source of progesterone during the pre-ovulatory period
has been given some study.

A chemical means for the extraction

and assay of progesterone in the blood has been developed by
Edgar (1953a) .

Although the limit of sensitivity of this proc­

edure was 0.1 ug./ml. of sample, a great deal of information was
gained concerning changes in progesterone levels and secretion
rates in the normally cycling, anestrous, and pregnant ewe.
Edgar (1953b) reported that the follicular fluid in the cow
and the sow contained no discernable progesterone during the
estrous, metestrous, and diestrous periods, but 300-800 ug. per
100 ml. of progesterone was found in the fluid of follicles just
prior to ovulation.

When samples of jugular vein blood in the

ewe were assayed for progesterone none could be detected (Edgar,
1953b, 195**; Edgar and Ronaldson, 1958)* Fifteen to 40 ml. samples
of blood taken from the ovarian vein of an active ovary contained
varying amounts of progesterone.

Edgar (195*0 and Edgar and Ronald­

son (1958) reported that detectable amounts of progesterone in the
blood appeared on the third day of the estrous cycle and the mean
concentration increased to about 180 ug. percent on the seventh
day.

This level was maintained till the sixteenth day, and fell

to approximately 15 ug. percent on the seventeenth day.

In the

25

course of one day (seventeenth day of the cycle) the progesterone
concentration in the blood leaving the ovary was reduced better
than ten fold.

It was also shown by Edgar (1958) that the right

ovary which contained two 8 mm. follicles was secreting 100 ug.
of progesterone into each 100 ml. of blood.

Although this ewe

was 105 days pregnant, the right ovary contained no corpora
lutea.
During the course of the experiments to be reported it be­
came necessary to extract and bioassay estrogens from the blood
of the ewe to gain further insight on their mode of action.
Whether or not estrogens are bound to proteins, to what extent
they are bound, and the nature of the binding has recently been
a subject of great interest and study.

Observations made in this

particular study indicate that a large percentage of the estrogens
are bound to plasma (or red cell) proteins and to such an extent
that extraction of whole blood with ethyl alcohol or alcohol and
ether will not remove them from the proteins*
In general, the research and its interpretation as found in
the literature concerning the binding of estrogens to protein can
be divided into three basic theories.

Bischoff and Katherman

(19^8) and Bischoff and Pilhorn (19^8) conducted A q vitro studies
to determine the distribution of steroid hormones in various
chemical and biological systems.

They concluded that the distri­

bution of estradiol in the blood is governed solely by the forc­
es

which affect its solubility and that there is no indication

of protein binding (other than simple adsorption) or inactivation

26

thereby.

Although Iontc strength and pH affect this solubility,

estradiol solubility is not appreciably affected by pH change
within the range of 5»3 to 8.

Boettiger (19^6), Bischoff and

Katherman (1952), and Bischoff

al,, (1951) have examined

the dispersing properties of various blood proteins upon estra­
diol, estrone, and estriol.

In the .in vitro experiments bovine

serum albumin (BSA) and human serum albumin (HSA) at equilibrium
with the added estrogens contained approximately 0.03 to 0,06 ug./
100 mg. of a six to eight percent albumin solution.

Although this

is approximately 100 times the amount that would be soluble in
saline solution, it is still below the levels found in blood of
other domestic animals during reproductive activity (see pageT03) •
When they added estradiol in vitro to rabbit serum containing all
the proteins except fibrogen the amount of estrogen dissolved in
the solution at equilibrium was 1^.5 ug./ml, with 73 ug. remaining
undispersed for each ml. of serum.

Even though their starting

concentration was quite high (100 ug./ml.), an appreciable
amount of the estrogen became dispersed in the medium.

In in

vivo experiments they found 0*92 ug./ml. distributed in the serum
after five minutes.

Their injection material carried estradiol

in a supoosedly soluble form (17*1 mg. estradiol dissolved in 0.1
ml. ethanol and transferred to 30 ml. of rabbit serum) , but this
much estradiol probably did not all remain soluble in that small
volume of serum.

Also, using such large doses (l.l to 1.2 mg./

kilogram) they would be expected to find some estrogenic activity
in any tissue in the body.

The kidney and uterus had the highest

27

concentration of estrogen.

Bischoff et al. (1952) and Bischoff

and Stauffer (195^) determined the dispersing power of lecithin,
Cohn's III-O lipoprotein fraction, and a lecithin-protein mixture
upon estradiol and found that these solutions were relatively
weak dispersants compared to albumin and blood serum.

Bischoff

Si. al. (195^) summarized most of this work by stating that estrogen
dispersed by albumin solution and serum proteins is not protein
bound, even though it may be adsorbed or oriented on the protein.
Xn evaluating Bischoff and co-workers observations on the
physiochemical state of circulating estrogens, two factors must
be taken into consideration: l) In determining the physiochemical
state, all work was carried out in vitro and the only enzyme
systems available for metabolism of estrogen were those in serum
or whole blood.

2) Reports of other investigators which have

attempted to describe the physiochemical state of estrogens when
added to blood In vivo. have given much evidence to show that
estrogens are bound to proteins by some sort of chemical bonding
rather than weak van-der-Waal1s forces.

It may be admitted that

the blood and other tissues are capable of carrying "effective"
levels of estrogens in the soluble form and not bound to proteins,
but other reports will show that this is not necessarily the case.
Rakoff et, al* (19^+3) made the observation that very little
of the estrogenic activity in late pregnancy serum was extractable with an ether-alcohol mixture and that almost all of the
hormone was found in the protein residue.

The principal propo­

nents of the theory that estrogen is carried in a large part in

?.

the protein-bound form are Szego and Roberts (19^6, 1956) .

8

Roberts

and Szego (19^6) reported that practically all the protein-bound
estrogen was found in Cohn's III-O lipoprotein fraction in the
ester if led form.

Szego and Roberts (19^6, 19^*7) showed that

approximately two-thirds of the plasma estrogens were bound to
protein.

These protein-bound estrogens were present in alcohol

and acetone precipitates of plasma proteins.

However, the bound

estrogens were quantitatively dialyzable across a collodian mem­
brane.

They postulated that they were available for physiological

action by dissociation from the protein at the cell membrane,
It has been demonstrated that liver tissue homogenates will
bind estrogens to blood proteins when incubated jin vitro from 0 2 hours (Szego, 1953)*
of

This

particular study employed the use

labeled estrone and approximately 88 percent of the radio­

activity was found bound to the serum proteins.

Homogenates from

regenerating liver were found to be even more active in binding
the estrogen to the protein.

This binding to serum protein is

thought to occur after the estrogen conjugates with a glucuronic
acid (Szego and Wolcott, 1955) and it may be bound to the liver
proteins after conjugating with some other anionic radicle such
as sulfate.

Paper electrophoretic studies of protein-bound

estrogen formed in vitro have been conducted (Szego and Roberts,

1955) which show that

labeled estrogens migrate with the

albumin boundary while the control samples ( C ^ labeled estro­
gen incubated in serum alone or with tissues which were inactive
in facilitating protein binding) had no

radioactivity in the

29

protein areas of the strip.

Although these data discouraged

the argument that co-orecipitation of the estrogens and the
protein might occur during the fractionation procedures (Bischoff
et al,, 195^0 » it does throw some doubt as to whether an aporeciable-

amount of estrogen is bound to the III-O fraction.
The functional state of the liver was correlated with its

ability to bind estrogens to plasma proteins (Szego, 1955 and
Szego and Roberts, 1956).

It was found that regenerating and

normal liver tissue had the greatest capacity to bind proteins
to estrogens whereas hepatomatous tissue and cirrhotic livers
showed a decreased binding capacity as well as aberrations in the
metabolic scheme.
serum albumins.

Again the estrogens were found attached to the
Cortisol was shown to inhibit the binding of

estrogens to serum albumin in the presence of liver homogenates
explaining to some extent the competitive phenomena exhibited
by estrogens and certain corticosteroids tn vivo.
An attempt has been made to isolate the fractions in the
liver homogenates that are responsible for the protein binding
of estrogens (Riegel and Mueller, 195^* Rumney, 1956).

One of

the primary prerequisites for maximal binding is an oxidizable
substrate (electron donor) which was found to be triphosphopyridine nucleotide (TFN). This, reduced under aerobic conditions,
gives the steroid molecule a free negative charge.

However, this

bound estrogen was found to have little biological activity.

It

is also believed that the estrogens are converted to an estrone­
like metabolite before being bound to the protein.

30

Two widely divergent interpretations have been presented
concerning the significance of protein binding of estrogen and
its transport and availability throughout the body.

There is

another group of investigators which take the "middle road"
and present data which indicate that although all estrogens
are bound to protein, the percentage bound is much less than
reported by Szego and Roberts.

Antoniades

a]*. (l956) using

Cohn fractionation and subsequent bioassay found estrogenic
activity in the albumin fraction, but most of the activity was
found in fraction VI or the supernatant which indicated free
estrogens.

Sandberg et a]*. (195?) state

that the binding of

estrogens to plasma proteins is probably used as a means of
hormone transport in the body.

Their data indicate however,

that the binding is weak and of a reversible nature with most
of the estrogen circulating in some other form.

They also

found the estrogen bound to the albumins (Cohn fractions TV-1,
XV-h', and V) in both the free and unconjugated forms and they
believe that the liver plays a small role in the in vivo evolu­
tion of a blood protein-steroid complex, although in vitro
strongly bound prote in-steroid complexes may form.

In one in­

stance they found that the red blood cell carried a large por­
tion of the C ^ labeled estrone.
The study of the hormonal control of reproductive activity
in the ewe has given impetus to many attempts to induce estrus,
ovulation and fertility in the anestrous ewe.

When only PMS is

used In the anestrous ewe, ovulation occurs, but estrous behavior

31

is not induced and forced mating or artificial insemination do
not result in conception.

The double dose of PMS spaced at 16

day intervals was partially successful in inducing estrus and
ovulation, but only a small percentage of the ewes lambed.

Pre-

treatment daily for five to sixteen days with progesterone in
oil subsequent to the PMS injection produced variable results.
In some experiments the lambing percentage was good, but the
results were unpredictable from one flock to the next and from
one year to the next.

In many trials using the progesterone-PMS

treatment, the incidence of estrus was low and exogenous estrogen
was added to increase the number of ewes coming in heat.

The

fundamental research which was concerned with the hormonal mech­
anisms and interrelationships of reproduction in the ewe has
provided a basis for the hormone sequences used to control repro­
ductive activity.
The injection of progesterone in oil daily has been used
extensively in order to maintain a level of circulating hormone
which will inhibit subthreshold cycling or cyclic activity in
the ewe.

However, this procedure is not practical when applied

to large numbers of ewes under field conditions.

A major objec­

tive of the present investigation was to determine a method
which would produce prolonged progestational inhibition of a
specified length with a single injection of progesterone.
The role of PMS in the induction of reproductive activity
in the ewe has been investigated by many workers.

Its effec­

tiveness in combination with a single injection of progesterone

o

of prolonged duration has not been reported.

In the present

investigation the ability of PMS to induce estrus and ovulation
in conjunction with the single injection of progesterone was
studied.
After collection of preliminary data it was found that the
incidence of estrus was low with the progesterone-PMS treatment.
The use of exogenous estrogen in the hormone sequence to increase
the incidence of estrus introduced another variable vihich had to
be considered.

The physical state, route of administration and

level o^ estrogen necessary to produce the most effective resu3_ts
in the induction of reproductive activity in the anestrous ewe
were investigated. Some of the factors involved in the metabolism
of estrogen by the ewe were also studied.

33

MATERIALS AMD METHODS

Experimental animals:

The sheep used in the various exper­

iments reported in this thesis can be divided into three groups:
(l) the first group consisted of 85 broken-mouthed Western ewes
most of which were probably over six years old, (2) the second
group included 35 three-year old Western ewes which were donated
by the Upjohn Company.

These ewes were of very good quality,

uniform type, and their breeding capabilities proven by a pre­
vious lamb crop, (3) the third group was not a particular flock
of sheep, but rather those ewes owned by the University which
were available for various field trial experiments.

These sheep

represented both grade Westerns and purebred mutton breeds.
The husbandry of all ewes was under the direction of the
Animal Husbandry Department at Michigan State University.

The

ewes were fed hay, grain, silage, and mineral throughout the
winter months, and pasture only during the summer months, except
when a grain concentrate was used as a part of the experiment in
a flushing procedure, or as a means of introducing a hormone
orally.

The ewes were housed under various conditions depending

upon the season, type of experiment, and space available.

The

ewes were sheared once each year, in the early spring, at which
time they were paint-branded in large 4 inch numbers on both sides
for easy identification in the pasture.

All ewes were drenched

and dipped each spring and fall prior to any experimentation.
Vasectomized rams were used to check the occurrence and
duration of estrus and all breeding was by natural service.

During

the anestrous season (spring and summer months) the rams were fed
200 ug. of thyroprotein daily, starting at least two weeks before

3k
their services were needed and continued throughout the experiment.
Reineke (19^6) and Bogart and Mayer (19^6) reported that in bulls
and rams, respectively, the feeding of thyroprotein improved the
breeding performance*

In this experiment, libido, sperm motility

and concentration were enhanced during thyroprotein feeding as
compared to the pre-treatment period.
Hormone Preparations:
Thyroprotein:

An iodinated casein containing approximately

a one percent equivalent of 1-thyroxine by chemical analysis was
used in some of the experiments.

It was given orally at the rate

of 200 mg. daily, beginning approximately two weeks before any
other hormone therapy.

It had been previously reported (Henneman

et al. (1955) that the thyroid secretion rate in ewes during the
summer months was significantly lower (0.0^ mg./day of 1-thyroxine)
than during the breeding season (0.17 to 0.2^ mg./day).
Since it had been decided to give thyroprotein orally instead
of 1-thyroxine by injection, it was necessary to determine the
thyroprotein equivalent of 1-thyroxine.

This was accomplished by

using an in vivo measurement of thyroxine secretion (Henneman,
Reineke, and Griffin, 1955) • Approximately 50 uc. of I ^ l were
injected intramuscularly and seven days were allowed for maximum
thyroid uptake of the isotope.

Counts were made over the thyroid

every other day for a period of 18 days.

A total of 12 ewes were

in this preliminary trial, six receiving five graded doses of 1thyroxine and the remainder receiving five graded doses-of thyro­
protein over a period of ten days during the counting period.
The uptake of I
injection.

Ill

was determined on the seventh day after

The average percentage uptake at this time was 25.75

35
percent

7*1.

The maximum uptake could have been higher since

the peak uptake had been passed by the seventh day after injection
of il31.
The secretion rates in terms of 1-thyroxine were calculated
for the control ewes in the usual manner, except that the correc­
tions for physical decay were only made back to the previous count
and not back to zero time.

This procedure tends to minimize the

physical errors of counting which are unduly magnified towards the
end of the experiment because counts are relatively small compared
to those counts at the beginning.
When all six sheep in each group were used to calculate the
secretion rate, it required 0.17 mg. of 1-thyroxine or 200 mg. of
131
thyroprotein to completely inhibit the output of I ^ , with the
effective ratio of 1-thyroxine to thyroprotein of 1:1176.

However,

when three sheep which failed to produce a significant response to
the exogenous thyroxine were eliminated from the calculations
(Henneman, Reineke, and Griffin, 1955)* the effective ratio was
lowered to 1:900. (Figure l) .
Without entering into a discussion of the precision of the
experimental technique, it may be stated that 1-thyroxine is approx­
imately 900 times as active as oral thyroprotein. This figure agrees
very closely with the chemically determined 1-thyroxine in thyro­
protein and its estimated absorption from the gastro-intestinal
tract.

Thyroprotein contains about a one percent of 1-thyroxine

equivalent (Reineke, 195*+) an^ ^

previously was reported that 12.5

percent of this was absorbed from the GI tract (Turner and Reineke,
19^6).

Therefore, the response obtained when 180 mg. of thyroprotein

were given orally, would be approximately equal to that of 0.20 mg.

36

FIGURE 1.

THE EFFECT OF EXOGENOUS 1-THYROXINE AND THYROPROTEIN
UPON OUTPUT OF I1^! FROM THE INTACT EWE

100

COUNT

90

80

6

8

10

12

,08

.12

.16

8

10

12

120

160

DAYS
L-THYROXINE (mg,)

.0^

.20

100

7o

PREVIOUS

2

90

80

2

6
DAYS

THYROPROTEIN (mg.)

^0

80

200

37
0:^ 1-thyroxine when injected intramuscularly.

This level of 1-thyroxine

is within the physiological range of the daily secretion rate in the
cycling ewe.
From the data we can also compute the percentage of the thyrox­
ine

contained in the thyroprotein that was absorbed.
180 x 0.01 = 1.8 mg. (Thyroxine fed)^"
0.20 m g .(Thyroxine injected) x ^ q o = 11.1 percent absorbed^
1.80 mg (Thyroxine fed)
Progesterone:

An aqueous starch suspension of microcrystal­

line progesterone was prepared using the method of Nellor and Cole
(1956).

This solution was made up in two batches of 1000 mL. each.

Seventy grams (70 mg./ml.) of crystalline progesterone was homogen­
ized in a ground glass, 50 ml. test tube and pestle.

Ten grams of

progesterone was added to 25 ml. of a 0.3. percent starch solution
and run through the motor driven homogenizer for five minutes.

This

was repeated until the 70 grams of progesterone was in a uniform
microcrystalline suspension.

The seven homogenates were then placed

In a liter volumetric flask and brought up to volume with the
remaining 0.3 percent starch solution.

The mixture was then poured

into 100 ml. injection bottles for convenient withdrawal by syringe.
During any transfer

of this suspension, care was taken to keep it

thoroughly mixed at all times, because it settled out very rapidly.
This precaution was also necessary when withdrawing the suspension
from the bottle for injection.
The injection site for this progesterone starch suspension was
in the inguinal region in an area free of wool and dirt where four
ml., totaling 280 mg. of progesterone were injected subcutaneously.
Care was taken to avoid nicking or scratching the muscle layer.
In view of previous work (Robinson, 1955; Nellor and Cole, 1957;

38
Gordon, 1958) it was hoped that 280 mg. of progesterone in starch
suspension would impose a reasonable duration (10 to 20 days) of
orogestational inhibition.

The progesterone-starch suspension was

given at the same dosage in all trials and the effect of variation
in this dosage level was not studied.

One of the objectives of this

work was to determine the length of progestational inhibition of
the subthreshold cycling in the anestrous ewe using 280 mg. of the
progesterone-starch suspension in a single injection.
Three other progesterone or progestational compounds were
tested.

Progesterone in oil (10 mg./ml.)* was used in daily sub­

cutaneous injections as a standard for the inhibition of cycling
or subthreshold cycling in the ewe.

An aqueous suspension of pro­

gesterone in isotonic saline, containing methyl cholate as an emul­
sifier, and polyoxyalkylene sorbitan monolaurate as an absorbent
was used in comparison with the progesterone-starch suspension in
regard to its effectiveness over a period of time when given in a
single dose.

Prodox (17-acetoxyprogesteron)*, an orally active

progestational compound, was also tested for its duration of action
when injected subcutaneously.

The Prodox which is usually dispensed

in crystalline form, was put into an aqueous suspension at the Upjohn
Laboratories.

The concentration of the aqueous suspension and the

Prodox was 50 mg./ml. and five ml. were used as the dose level.
Pregnant Hare Serum (PMS):*

This protein fraction which is

extracted from the serum of mares between the second and fifth
month of pregnancy is known to have predominantly FSH activity
although it apparently has a much smaller proportion of LH activ­
ity

as well (Cole and Hart* 1930).

This hormone was received in

♦Donated by the Upjohn Company through the courtesy of Dr. Gordon
Stocking.

39
one ml. vials bearing the lyopholized protein containing 1000
i.u. of FMS.
Estrogens;

A microcrystalline suspension of estradiol con­

taining two mg. of estradiol per ml. with ethylene sorbitan monooleate as a stabilizer was obtained from the Haver-Glover Labor­
atories.

This preparation was used as a stock solution from which

microcrystalline suspensions were made containing 1.0, 10.0, 20,0,
50.0, and 100.0 ug./ml of estradiol.

These suspensions were used,

both for treatment purposes and as standard solutions in the deter­
mination of estrogenic activity in blood and urine.
Estradiol, in the same concentrations as listed above, was
also prepared as the sodium-salt. Upon microscopic examination of
the weakest suspensions of estradiol (l ug./ml.), the rhomboid
crystals characteristic of the highly purified hormone were found
to be fairly large (1-2 microns in length) .

Considering the poss­

ibility that the crystal size might lower the efficiency of
utilization, the sodium-salt was prepared to determine whether
estradiol in a water soluble form would be more suitable for induc­
tion of estrus. This sodium-salt of estradiol was prepared by adding
the desired amount of hormone to 5 ml* of N NaOH. This was diluted
with distilled water up to 100 ml. containing the sodium-salt of
estradiol in a 0.05 N NaOH solution. This solution was easily in­
jected intravenously or intramuscularly without any tissue damage.
Another estrogen, estradiol cyclopentylpropionate (EQP) was
also used to induce estrous behavior in the anestrous ewes.

This

compound was reported (Upjohn Company) to have a longer lasting
affect compared to other estrogenic substances dissolved in oil.
It was dispensed in 10 ml. vials containing 1 mg./ml.

Uo
EXPERIMENTAL METHODS
Observation of estras behavior:
to determine the occurrence of estras*

Two different methods were used
When only the determination

of the onset of estrus was necessary a marking harness was used on
the rams.

Attached to the harness at the brisket is an oil base

crayon that marks the ewe on the rump when mounted by the ram#

In

cold weather some difficulty is encountered when using this method
because the crayon becomes hard and does not always leave a mark on
the ewe#

In determining onset of estrus by this means it was nec­

essary to turn the rams in with the ewes three to four days prior
to application of the marking harness#

This was done to prevent

over-anxious rams from marking ewes that were not in heat.
When it was necessary to determine both the onset and duration
of estrus the ewes were checked twice daily with rams kept separate
from the ewes during the rest of the day.

Each ram checked from

eight to sixteen ewes at each check period.

This took approximately

one half to one hour depending upon the number of ewes in heat#
Both vasectomized and fertile rams were used.

When fertile rams

were used for the insemination of the ewes as well as checking the
onset and duration of estrus, both methods of checking were used.
One ram wearing a marking harness was turned out to pasture with
16 to 32 ewes and two rams were kept at the barn and used to handbreed the ewes twice daily when driven in from pasture.

In this

way the capacity of the rams, and the length of time the ewes were
exposed to the rams during estrus was not a limiting factor.

Con­

sequently a ewe in heat only a few hours would be serviced by t he
ram in pasture, whereas she might have been missed by hand breeding.
The duration of estrus was determined with a precision of plus

41
or minus 12 hours.

Since each ewe was checked twice daily, the

interval between check periods was 12 hours.

The number of check

periods was then multiplied by 12 hours and this was the calculated
duration of estrus. • The error involved in this calculation is
apparent, however, when this system is strictly adhered to, the
results between experiments can be favorably compared.
Methods of evaluating slaughter data:
a) Ovulation data and response to PMS:

In order to

determine the effects of PMS on the ovary and the occurrence of
ovulation, the ewes were slaughtered five to seven days after the
injection of PMS.

The presence of fresh corpora lutea at this time

was evidence that ovulation had taken place as a result of the hor­
mone treatment.

Fresh corpora lutea were so determined by the

following criteria: l) relatively high protuberance above the sur­
face of the ovary, 2) more pointed at the apex than the older cor­
pora lutea, 3) presence of a stigma or small opening at the apex,
and 4) presence of some hemorrhagic material (incomplete reabsorp­
tion of the corpus hemorrhagic urn) depending upon the age (l hr. to
3 days) of the corpus lute urn.
Since all of the ewes were slaughtered during the period of
anestrus most of the ovarian activity was due to the follicle stim­
ulating properties of the injected PMS.

Therefore an arbitrary sys­

tem of rating ovarian activity, one through four was devised using
the following criteria:
Rating 1

No response - follicular growth and absence of
fresh CL indicating no ovulation.

Rating 2

Limited response - some follicular growth and absence
of fresh CL.

Rating 3

Positive response - definite follicular growth with
either one large follicle and/or a fresh corpus
luteum.

42
Rating 4

Maximum response - excessive follicular growth and
multiple fresh corpora lutea indicating super­
ovulation#

b) Fertility data in slaughter animals:

Approximately 35

days post service, a proportion of the ewes treated with various
hormone combinations, were slaughtered to determine the presence
of a viable fetus.

Since few miscarriages occur after 35 days

(Brarabell, 1948) , a check at this time gave a good indication of
the lambing percentage that would have been obtained if the gesta­
tion had been allowed to go to term*

The gross anatomical structure

of the reproductive system was also checked at this time and any
obvious abnormalities were noted.

In some of the older ewes such

conditions as occluded cervical os, occluded fallopian tube, tumorous
growths, and pyometria were found.
c) Supplemental data obtained from slaughtered ewes:

The

uterus was excised and grossly examined for cotyledonary grcwth and
endometrial proliferation and vascularization.

The presence of

pyometria or intrauterine hemorrhage was also noted.

Other endo­

crine organs including the thyroid, adrenals, and the pituitary
were also removed and weighed.

Ova recovery was also attempted in

those animals slaughtered for ovulation data.

This was accomplished

by tying off the distal portion of the fallopian tubes and the
uterine horn at their bifurcation.

Both sections were then flushed

with physiological saline by inserting the needle in the distal end
of the fallopian tube and rinsing the saline through the tract into
a small watch glass.
Vaginal smear technique and evaluation:

The vaginal smears were

obtained from ewes which had been treated with various hormonal com­
binations during their anestrual period.

The smears were composed

43
of the exfoliative cells from both the entire vaginal wall and por­
tions of the anterior cervix.

A clean sterile metal spatula was

used to procure the cells with no attempt to locate the area In
the vagina from which the smear was taken.
The smear was placed on a glass slide and fixed in a solution
containing 50 percent ethyl ether and 50 percent absolute ethyl
alcohol.

It was then stained using the exfoliative tissue staining

method of Papanicolaou, 1942.

This method allows the observer to

differentiate between various degrees of cornification of the
epithelial cells.

As Herrich (1951) and Casida (1936) have shown,

the epithelial cells of the vaginal wall of the ewe do not lose
their nuclei upon cornification.

The cells in general become more

angular and crenated as cornification progresses, but the difference
between the cornified epithelial cell and the metestrous epithelial
cell is less apparent in the ewe than in the rodent.
The vaginal smears were placed in two general classifications,
the luteal or progestational phase and the follicular or estrogenic
phase.

The progestational smear was characterized by an abundance

of leucocytes, and many varicolored epithelial cells.

The epithe­

lial cells in this type of smear ranged In color from light green-to
brown-to pink-to bright red with no particular color in excess of
the others* The follicular smear has many of the same components as
the luteal smear except that the proportion of red epithelial cells
is very high and in some cases all the cells are red.

As mentioned

before, these red, cornified cells show some crenation and may dis­
integrate and lose their nuclei.

In most of the smears during the

follicular phase leucocytes can readily be seen.

Usually they are

less abundant than in the progestational smears, but, in the oase

44
of a slight endometrial or vaginal infection they will appear in
great numbers and have to be disregarded as an indicator of the
stage of the estrous cycle.
Collection of blood and urine samples for estrogen assay:
Blood was collected from sheep, both during induced and naturally
occurring estrus and immediately after hormone treatment.

One

hundred ml* was drained from the external Jugular into a vacuum
bottle containing 1 ml. of heparin.

The sample was then refrig­

erated and it was extracted for estrogenic substances within 24
hours after collection.
In two ewes that had been given progesterone in oil for ten
days and 1000 i.u. of PMS the day after the last progesterone
injection, a blood sample was taken from the ovarian vein two
days after the PMS injection.

The ewes were given a spinal block

and local anesthetic in the region of the proposed operation. They
were also given 10 mg., intramuscularly, of the tranquilizer thorazine, which was very effective in keeping the ewe quiet during the
whole procedure which lasted about 3*5 hours.

An incision approx­

imately five inches long was made on the left side, parallel to the
last rib in the paralumbar fossa.

The left ovary was exposed at the

surface of the incision by gently working the whole reproductive
tract away from its normal position without damaging any of the
uterine or ovarian blood supply or connective tissue attachments.
After exposure of the ovarian vein, but prior to its cannulation the ewe was given 500 mg. of heparin intravenously in 50 ml.
volume.

The first dose of heparin lasted for approximately an

hour after which time a similar dose was given which lasted for
about two hours more.

The cannula and the collecting bottle were

45
also heparinized.
Three separate veins could be seen leaving the ovary at its
attachment to the broad ligament. Two of these were tied off and
the third was cannulated.

The cannula used was Irrfcramedic polyethy­

lene tubing, FE 60 with an inside diameter of 0.030” and an outside
diameter of 0.048” . One end of an 18 inch length of this tubing
was

inserted into the vein with very little difficulty after a

small slithad been made in the vein approximately 1.5
the

ovary.

was

placed around the vein and tube and tightened.

cm. from

After the tube had been inserted (l cm.) a ligature

The blood flowed immediately after the cannula was in place
with no apparent leakage or other means of venous blood escape
from that ovary.

Blood was collected from this ovary continuously

for a period of 135 minutes at a fairly constant rate of 20 drops
per minute.

These drops were small and the total volume at the

end of this period was approximately 100 ml.

It was necessary

to keep the site of the cannulation exposed at all times in order
that the tubing would not cause a kink in the fragile vein and
stop the flow of blood.
Urine samples were also collected from two ewes after the
intravenous administration of estrogenic compounds.

The ewes

were given seven rag. of the tranquilizer thorazine and restrained
in a ci*ate which allowed them to lie down and have other limited
movements.

An anchored catheter (with an inflatable bulb at

the tip) was inserted into the bladder and all of the urine present
was eliminated and the bladder thoroughly washed with distilled
water.

An extension was then placed on the catheter, taped to

the tail of the ewe and led outside of the crate to a collection

46
bottle.

After the ewe was thoroughly quieted and the collection

system in operation the estrogen was given and urine samples were
collected continuously over a period of 12 to 24 hours.

The

collection bottles were changed at various intervals and the bladder
washed with distilled water between each collection period and the
washings were added to the previously collected sample.
The extraction of estrogenic substances from blood and urine;
The extraction procedure used was designed to remove and activate
all of the estrogenic substances from the blood sample. No attempt
was made to separate the various metabolites of estradiol or to
determine the fraction of the total estrogens present in the free,
conjugated (glycuronides or sulfates), or the protein bound form.
The experimental results had to be divided into two groups because
the initial extraction of estrogens (Step A, Fig. 2) from the blood
had to be changed after about half the data were taken due to inad­
equacy of the initial extraction procedure when applied to blood
containing estrogen that, had been added in vivo.
It has been reported in the literature (Smith and Smith, 1934;
Bischoff s£ & 1 * » 1951 i Diczfalusy, 1953 and Diczfalusy and Lindkvist, 1956) that repeated extraction of the whole blood and placental
tissues with 95$ ethyl alcohol will quantitatively remove the estro­
genic substances including the conjugates.
It occurred to the author that extraction with boiling alco­
hol would further insure the removal of the protein bound estro­
gens from the sample by virtue of a mild hydrolysis. The recovery
experiments were very satisfactory when estradiol was added to rams'
blood in vitro.

However, this eatrogen was not recoverable by this

procedure when added to the blood jja vivo indicating that a large

47
percentage of the estradiol was either protein bound or removed
from the blood to such an extent that it was not available for
extraction* It was, therefore, decided to employ a mild alkaline
hydrolysis using equal volumes of 2N NaCH and blood for a period of
12 hours. This step coupled with the standard procedure employed
for the remainder of the extraction gave similar results when extract­
ing estradiol added to rams' blood ijj vitro. It also enabled the
extraction of estradiol when added in the soluble form to rams'
blood _in vivo.
The remaining steps in the extraction procedure together with
the rationale for their use are described below:'
l) The extraction of the alkaline hydrolysate (B) with a 50-50
alcohol-ether mixture removes both the free and the conjugated
estrogens, the former being more soluble in ether and the latter
more soluble in alcohol. 2) The alcohol-ether mixture is evaporated
from the extract (C) under vacuum and nitrogen to reduce the poss­
ibilities of oxidation (Diczfalusy, 1953) of the estrogen by reducing
the temperature at which the mixture will boil and eliminating the
presence of oxygen in the closed system. The initial volume at the
beginning of the evaporation was approximately 450 ml. and a one
liter flask equipped with a glas-col heater was used* The one liter
flask was necessary to prevent the froth which forms from boiling
into the condenser and the glas-col heater was necessary for uniform
heating, reduction of bumping, and safety from ignition of the ether
fumes which might be present.
was shown in Figure 3.

A diagram of the evaporating setup

After the alcohol-ether mixture was removed

approximately 100 ml. remains. This fraction contains the estrogens,
other steroids, lipids, and chromogens in a water medium. 3) This

48
Figure 2

FLCW SHEET DEPICTING THE EXTRACTION OF THE TOTAL
ESTROGENIC ACTIVITY FROM WHOLE EWES* BLOOD
A. 100 ml. of whole ewes blood
plus
100 ml. of 2N NaOH
overnight at room temperature
B.

Extract 4 times with a 50:50
alcohol-ether mixture
______________ I_______________
Protein residue_,
Evaporate off the alc.~
discard.
ether in a closed condenser
system under a vacuum and
under nitrogen
Discard ale.-ether

D. Dilute water residue to
200 ml. with dist. water
and add 30 ml. of conc. HC1
Reflux for 1 hr. under N 2

E. Extract 1 time with 150 ml. toluene
Extract 3 times with 100 ml. Ethyl ether
F. Evaporate off the ether as in step C.
G. Extract the toluene
3 times with 60 ml. of
' 1.25 N NaOH

Discard ether

r

Discard toluene

H. Adjust pH of NaOH to 3 i 2
with conc. HC 1 and extract
4 times with 100 ml. ethyl ether

Discard NaOH residue

I. Wash ether 4 times with
40 ml. of dist. water.

Discard water wash

Evaporate ether to dryness on
slide warmer under hood at 37°0
1

K. Take up residue in 20 ml, ether
add cottonseed oil and evaporate
ether as in J.

2+9

suspension is transferred to a 500 ml. flask along with the dis­
tilled water rinses to bring it up to a total volume of 200 ml.
This suspension

is then placed in a reflux system and brought to

a boil, at which time 30 ml. of concentrated HCl
the mixture

is added and

is refluxed for one hour (D). This step was necessary

to hydrolyze the estrogen conjugates.

This procedure for the

hydrolysis of estrogen conjugates has been shown (Sandberg et al.,
1957; Brown, 1955; and Diczfalusy, 1953) to be very effective in
the hydrolysis with a minimum destruction of the estrogens. After
completion of the hydrolysis the hydrolysate is immediately cooled
in tap water and is allowed to stand a minimum amount of time (no
more than three hours) before the next step is initiated.

This

was thought advisable because of the possibility of oxidation of
estrogen in the acid mixture in the presence of oxygen (air). The
sample

is refluxed under a constant stream of nitrogen.

After

the hydrolysis all of the estrogenic substances are in the organic
solvent form. 4) Consequently, the extraction of this hydrolysate
with toluene and ether (E) removes all estrogens as well as other
steroids, lipids, and chromogens. 5) Tie ether is removed from the
toluene (F) via the evaporating column leaving the above mentioned
substances in the toluene. 6) The next step involves the extraction
of the phenolic steroids (mainly estrogens) from the toluene with
1.25 N NaOH (G) .

The chemistry involved in this reaction is the

formation of the sodium-salt of the phenolic steroid rendering it

+
Toluene soluble

Water soluble

H0H

figure 3.

^PORATI eq
Iw TANDEM

H‘Tf{06EM

C0i® w USED

ac
LU
t—

water soluble.

This phase of the procedure separated all of the

other steroid hormones, including adrenocorticoids, androgens, and
progesterone from the estrogenic compounds which are of the phenolic
steroid variety. The rationale involving the use of toluene is two­
fold; a) the phenolic steroids are less soluble in toluene than in
ether and are more easily extracted from the former with NaOH, and
b) most of the lipids contained in the original sample are present
in the toluene extract in which they are less subject to saponifica­
tion by the NaOH as compared to their ability to be saponified in
some other organic solvent (Diczfalusy and Lindkvist, 1956).

Since

a large amount of fatty* material was undesirable in the sample from
the standpoint of toxicity and physical consistency of the final
extract, the use of toluene in this respect was of critical impor­
tance.

7) The NaOH solution containing the sodium-salt of the phen­

olic steroids is acidified with concentrated HC1 to a pH somewhere
below five.

Lowering the pH to this level was essentially a safety

precaution to insure the complete de-salting of the phenolic steroids
It has been reported (Diczfalusy and Lindkvist, 1956) that at pH 10
the phenolic steroids partition readily into the ether phase.

How­

ever, any phenolic steroid in an acid medium would have a partition
coefficient highly in favor of an organic solvent rather than an
aqueous solvent.

8) After the acidification of the NaOH solution

the phenolic steroids were extracted with ethyl ether (H).

9) This

ether was then washed with distilled water to remove all traces of
the acid which would be present (I) in small amounts since most
solutions were slightly soluble in one another.

10) After the

water wash of the ether extract, which still contains many chromogens
the ether was placed in a ^00 ml. beaker on a slide warmei* (at 37°C)

52
in a hood and evaporated to dryness (J). The ether, which amounts to
approximately 380 ml. , is rapidly evaporated in 1-2 hours leaving 0.5
to 1 ml, of water which was dissolved in the ether. It takes about

1-2 hours more for this water to completely evaporate because it has
a thin film of lipoidal nature, covering it and therefore, reducing
therate of evaporation. Care should be taken

to insure the control

of the temperature at or below 37°C to reduce the possibility of
oxidation of the phenolic compounds. It also should be pointed out
that this evaporation was not carried out in an atmosphere of nitrogen.
However, this should not appreciably affect the percent recovery of
active estrogens as demonstrated by Engel (1950). He reported that
refluxing urine in 15 volumes percent HG1 under a stream of oxygen
did not affect the recovery of estrogens added to the urine. All the
other evaporations were carried out under a stream of nitrogen be­
cause: a) they were carried out at a higher temperature, and b) this
precautionary measure was easily arranged in the preceding phases
of the extraction procedure. 11) The evaporation of ether to dry­
ness, as called for in this last step (K) , was best accomplished
in an open system in order to eliminate the danger of explosion.
A dry residue was obtained in the bottom of a 400 ml. beaker
and

this was subsequently dissolved in 10 ml. of redistilled ethyl

ether and transferred to a 50 ml* erlenmeyer flask with a 1 ml.
teat protruding from its base. The beaker was rinsed with 10 ml.
more of redistilled ethyl ether which was added to the flask. The
desired volume of cottonseed oil (1.5 to 3*0 ml.) was then added
to the ether and mixed. The flasks were then placed on top of 50 ml.
beakers containing water and set on the slide warmer overnight.
After the ether in the sample was completely evaporated the flasks

53
containing the estrogen extracts in oil were stoppered and reserved
for bioassay.
The extraction of estrogenic substances from urine was similar
to that used for whole blood except that the initial step was
omitted.

The urine sample was diluted to 200 ml. and started in

the procedure at the acid hydrolysis step.

If the urine samples

were larger than 200 ml., an adequate amount of acid was added to
bring the hydrolysate up to 15 volumes percent of HCl and a larger
flask was used for refluxing.

In most cases this procedure was

adequate to give an extract acceptable to the assay animal, however,
the extract from a few samples
ed

was very toxic.

Brown (1955) report­

a procedure in which this toxicity could be removed by a steam

distillation.

The urine extractions were not re-run however, be­

cause enough of the samples were free of toxicity to give an
adequate picture of the excretion of estrogen.
The bioassav of the estrogen containing extracts:

The bioassay

of estrogenic activity proposed by Rubin et al. (1951) was used for
all blood and urine extracts.

This procedure entailed the use of

21-day old female mice which were injected with 0.1 ml. of the
extract in oil for three consecutive days and were killed on the
fourth day.

The uteri were removed anterior to the cervix and

weighed without removing the fluid in the lumen.

A wet weight

determination of the uterus was thought advisable, since one of
the primary functions of estrogen is an increase in water content
in the lumen of the uterus and its tissues as well.
Usually the uterine weight is corrected for variations in
body weight in the following manner:

5^

It was found however, that the uterine weight was not significantly
correlated with the body weight in the single age group of mice
used.. The samples for this correlation came from control animals
weighing 12 +, 0.5 grams that were received in various shipments.
Therefore, the standard curve was computed using the uterine weights
alone and not the ratio of uterine weight to body weight.
The standard curve was established for the response of the
uterus to known amounts of estradiol which had been added in
vivo to rams* blood and extracted.

In this manner the unknowns can

be read directly off the standard curve without correcting for
extraction efficiency.

Some error was introduced by this method

because of the assumption it makes, that all extractions will be
of the same efficiency.

However, the variability of the extraction

efficiency was not determined due to the labor involved in numerous
extractions.

It can be seen from the standard curve in Figure k

that the relationship of uterine response to graded doses is a
straight line on linear graph paper within a certain range of
doses.

Most of the unknowns fell within this range.

However,

when they were above or below this range it was recorded in the
results.

Although the precision of this assay is not as great as

could be desired, the between-variation of the unknowns is large
enough to give indications of true differences between experimental
studies (P < 0 .01).

Consequently the precision of the assay in

these preliminary studies was not critical.
In most assays of this kind, the statistics described by Bliss
in the "slope ratio" assay are used.

However, this procedure

requires different levels of the unknown which is then compared
to the standard and its relative potency is determined.

In this

55

FIGURE

UTERINE WEIGHT RESPONSE TO GRADED DOSES OF ESTRADIOL.

Oo

X
Oo

or

Uj

/U6

Ul

estr ad io l

CrOJ

Ul

or
Ui

<r\j

3S N O d S 3 y

3 N IU 3 ± n

56
procedure the parallel response of the unknown to the standard
was checked and thereby the validity of the assay was determined.
However, very little assayable material was available and it was
necessary to use only one level of the unknown to compare to the
standard at two levels*

As Bliss has stated, "the potency computed

from such an assay may be of limited validity.

It represents a

single level of response and there is no evidence that the potency
would be the same, within limits of the sampling error, at larger
or smaller doses*

In extending this result to other dosage levels,

It is assumed that, if determined, the slope of the dosage-response
curve for the unknown would have been the same as that of the stan­
dard.

In other words it is assumed that under these assay conditions

the unknown differs from the standard only in its concentration of
the same active constituent."
In assaying a single unknown the most efficient distribution
is to divide the animals equally between the standard and the unknown.
If there are "g" unknowns the total number of responses at all
dosage levels of the standard should be N ^g~ for the greatest pre­
cision over the entire assay, (N = number of responses at same level
for each unknown).

Therefore if there are four unknowns and five mice

used to assay each unknown, then the standard curve should contain
a total of ten responses over all the dosage levels.
assays this requirement was met.

In most of the

The formula for computing the

relative potency for one level of unknown is:
M = T s - X u 4^°s
M * Relative potency*

=5 Mean dosage given for standard.
X u = Dosage given for unknown (only one) .

57
Y u » Mean response for unknown.
Tg a Mean response for standard*
b s a Slope of the standard curve.

58

EXPERIMENTAL RESULTS

Determination of the length of follicular inhibition in the
cycling and anestrous ewe* produced by a single injection of 280
mg. of microcrystalline
starch solution:

progesterone in ^ ml. of a 0.3 percent

In the spring and summer of 1956 an experiment

was conducted with 63 ewes of various breeds to determine what
effect protamone, progesterone* and PMS would have upon the
induction of estrus in the anestrous ewe.

Since no informa­

tion was available concerning the length of activity of the
starch suspension of progesterone in ewes* the dosage used in
cattle by Nellor

aX.» (1956) was cut in half and the 13 day

inhibition period found in cattle by these workers was used as
a basis for this experimental procedure.
TABLE 1
NUMBER ANESTROUS EWES SHOWING ESTRUAL
ACTIVITY AFTER HORMONE TREATMENT
Treatment
Standing
Not
_________________________ Heat______ Standing
Controls
Protamone only
Protamone-Progesterone
Protamone-Prog.-PMS^
Progesterone-PMS
Total

0
0
2
0
3
5

1
2
1
2
1
7

No
Response
6
12
11
12
10
51

IProtamone fed, 200 mg. daily, starting 2 weeks before
the progesterone injection.
^Progesterone injeoted subcutaneously, 280 mg. in a
starch suspension.
3pMS injeoted subcutaneously, 1000 i.u., 13 days after
the progesterone injection.

The data in Table 1 shows no significant differences be­
tween experimental groups.

None of the treatments were effect­

ive in inducing estrus in the ewes, although some very important
problems were brought to light,

l) The estimated 13 day period

of progestational activity aoparently was considerably off the
true mean value; and 2} the effect of prptamone upon the inci­
dence of estrus could not be ascertained unless the sheep which
received the progesterone-PMS treatment gave a fairly predict­
able

response.

It has been pointed out in the literature

review that from 50-100 percent of anestrous ewes of various
breeds in various geographical locations, when given the proges­
terone in oil daily, followed by PMS could be expected to show
estrus one to three days after the PMS injection.

It was

therefore assumed that the single injection of the microcrys­
talline suspension cf progesterone was either exerting its
effect over a longer or shorter period than the 13 days origin­
ally estimated.

The next experiment was designed to estimate

this length of activity with more precision.
Fifteen ewes (l Hampshire and 1^4- Westerns) started their
cycling activity early in the season (July 31 to August 13).
These ewes were injected with the progesterone starch suspen­
sion (280 mg.) on August thirteenth and checked for the
occurrence of the next heat period with a vasectomized ram
wearing a marking harness.
The average interval between the injection of the proges­
terone suspension in starch solution and the occurrence of the

6o

TABLE 2
THE RETURN TO HEAT OF CYCLING EWES AFTER INJECTION
WITH 280 M3. OF PROGESTERONE IN STARCH SUSPENSION
Day of
Cycle
Injected

11
9
7

10
8
11
1
5
4
5
k

Date of 1st
Heat Period
After Inj.

Interval Betw.
In j . and 1st
Heat After Inj.*
(days)

Interval Betw. 1st
Heat Before Irij. and
1st Heat After Inj *
(days)
27

8-29
8-30

16
17

26

8-31

18

8-31
9-1
9-1
9-1
9-1
9-2
9-2
9-3

18
19
19
19
19

25
28
27
30

Kean i, Std. error

20
2^

20
20
21

25
25

18.7 ±, 0.^3

25.5 ± 0.78

*The progesterone was injected in all sheep on August 13.
first estrus after injection in these 11 cycling ewes was 18.7
days (Table 2) . Assuming a follicular phase in these ewes of two
to three days (time necessary for the Graafian follicles to
mature and ovulate after the progestational activity has been
reduced to a level compatible with follicular growth), the average
length of activity of the single injection of progesterone was
approximately 16 days (18.7 - 2*5 = 16.2} .

These data afford

some explanation for the failure in the first experiment where
a 13 day period was estimated as the length of inhibition of the
single progesterone injectionThe treatment of these ewes also produced a fair degree of
synchronized estrus, with eight out of eleven ewes coming into
heat within a three day period.

However, four of the fifteen

6l

ewes treated did not show any signs of heat within 25 days after
the progesterone injection.

They subsequently were bred later

that fall season, but the treatment caused them to skip one or
more estrous periods even though they had spontaneously exhibited
their first estrus of the season prior to the progesterone
treatment.
Twenty-three ewes were given this same progesterone treat­
ment in the latter part of the breeding season (Jan. 9-25» 1957)*
Only four of the 23 ewes returned to heat after the progesterone
treatment.

Apparently this late in the breeding season, high

levels of progesterone activity over a 16 day period will cause
many of the ewes to terminate their cycling processes.

It should

be noted that a few similar ewes, not being treated with proges­
terone, were already in anestrus, starting the first of January.
Consequently some of the ewes under treatment probably would
have stopped cycling without any treatment.

However, 80 percent

of the control ewes were still cycling at the time that 83 per­
cent of the treated ewes were showing an anestrous condition.
These 19 anestrous ewes along with six other anestrous ewes were
used in a further study to determine the length of activity of
the single progesterone injection.
In this experiment a different approach was used to deter­
mine the length of progestational activity from a single
injection of the starch suspension.

The optimum interval be­

tween the progesterone injection and the PMS injection was
determined as measured by the incidence of estrus in each group

62
and ovarian activity data obtained by slaughter.

The results

(Table 3) indicate that the 16 day interval between the proges­
terone and the PMS injection was optimal.

Four of the five ewes

in this group came into heat with an index of ovarian response
to PMS of 3-75*

Ewe number 67 was omitted from this calculation.

This animal showed an endometritus, tumorous growths in the
uterus and complete absence of any follicles on the ovary. These
observations indicated that this ewe was incapable of a normal
response to PMS and therefore should be eliminated from the cal­
culations.

Most of the ewes (10 out of 12) which had received

PMS had ovulated regardless of the interval between injections.
In the latter part of the breeding season (Nov. 14 to Dec.
If 1956) another trial was run with fertile rams with the
primary objective of determining the effects of this single
injection of progesterone upon conception and fertility in
the cycling ewe, at the first service after treatment.
Without adequate fertility at this time of the normal breeding
season, there would be a small probability of the anestrous
ewe being fertile after progesterone-PMS treatment.

Twenty

ewes were injected with 280 mg. of progesterone in starch
solution on Nov. 14.

A single fertile ram equipped with a

marking harness was turned in with the ewes on Nov. 18 and the
ewes were checked daily to determine the initial onset of

TABLE 3
THE EFFECT OF THE VARIATION OF THE INTERVAL BETWEEN THE
PROGESTERONE AND THE PMS INJECTION UPON THE INCIDENCE
OF ESTRUS AND OVARIAN ACTIVITY IN THE ANESTROUS EWE
Ewe
No.

Treatment

35
55

Prog. only
11
it
rt
ti
11
it
No prog..

26
39
38

Interval betw.
Injections
(days)
—
_

Prog.--PMS

11

11

it

it
it
ti

11
11

49
50
52
7^

Prog.--PMS
ti
11
it
it

11
11

11
it

Prog.-PMS
it
11
11
ti
n
11

11

11

-

-

1
3

-

1. 67

20
20
20
20
20

18
18
18
18
18

16
16

16
16
16

Averag e
31
51
25
80
65

Prog.-PMS
ti
11
11
ti
1
1
ii
1
1
it
Averag e

_
—

Average
72
59
67
69
44

1

—

_

Average
21

—

PMS
Resoonse*

_

Average
28
66
83
73
81

Estrus-hrs.
after PMS

14
14
14
14
14

—
52
-

168

2
3
3
-

110

2. 67

-

4
3
-

-

3. 50

_

48
36
- omitted
96
72

4
3
1
4
4

63

3.75

48
52

3
2
4

50

3. 00

♦Since every ovary was assigned a rank-order depending
upon its activity, it can be seen that only a few
animals in each group were selected for slaughter. Cr
terion for this ranking was given in Materials and
Methods.

64

estrus.

These data (Table 4) also showed the degree of synch­

ronization obtained with the single injection of progesterone
as well as the length of progestational activity and the effects
of treatment on fertility.

TABLE 4
SYNCHRONIZATION, ONSET OF ESTRUS, AND BREEDING DATA ON
CYCLING EWES AFTER TREATMENT WITH A SINGLE INJECTION
OF PROGESTERONE SUSPENSION IN STARCH*
Ewe
No.

Date Serviced
After Inject.

1
2

11-29

3
4
5

12-1
12-1
11-30

6

11-29
12-7

7

8
9

10
11
12

12-1

Interval betw.
Inject, and 1 st
Service after
(daysT
17
15
17
17

—

12-1
-

12-1

16
15
23

Expected Date
Lambing Lambed
Date
5-4
5-2
5-4
5-4
5-3
5-2
5-11

-

5-4

17

-

-

5-4

17

—

-

-

-

-

-

13
14
15

11-29

16

12-1

12-2

17
18
19

12-1
12-1

20

-

_

Mean jt Std* error

18
15
17

5-5
5-2
5-4
-

-

5-4
5 —4
—

17
17
—

17*0

5-12
5-1
—

died
5-11
died
—

5-5
5-3
-

5-4
5-14
-

5-4
-

5-16
—

0.52

♦Ewes injected Nov. 14 with 280 mg. of progesterone
suspension in starch solution.
Three ewes lambed which did not mark the previous D«
It was observed thati at this time of year the marking hai
was not adequate to insure a proper check on the estrual

65

of the ewes.

During this time of year the temperature varies

greatly from day to day and also during the morning and evening
periods.

At temperatures appreciabely below freezing it was

found that even the winter marking crayons did not leave a recog­
nizable mark with just one or two mounts.

In the following

experiments both marking harness and hand checking were used to
determine onset and length of estrus.
The percentage of the ewes lambing of those bred was appreci­
ably lower than could normally be expected (50 percent or nine
out of eighteen ewes lambed) . However, those ewes which did lamb,
apparently conceived during the first estrous period after treat­
ment since they all lambed within a 12 day period . Also subse­
quent slaughter of ewes that did not lamb showed that a large
percentage of them had occluded cervix, tumorous growths In the
uterus and ovaries, and endometritus. Taking these factors
into consideration the conception rate in the reproductively
normal ewes was not significantly affected by the progesterone
treatment.
The average interval between injection of progesterone
and the onset of estrus was 17 days which was approximately two
days shorter than previously shown under similar conditions
(Table 2).

The next experiment also shows a progestational

inhibition lasting approximately two days less than previously
determined.
Except for one ewe which came into heat 23 days after the
injection of progesterone, 13 ewes came into heat within a three

66

day period according to the marking harness data.

Using the

lambing data (150 days gestation) coupled with the marking
harness data, (Table 5)» 16 ewes came into heat within a three
day period.

It can be seen from the data in Table 5 that the

degree of synchronization was very good, giving a mean interval
between injection and onset of first estrus after injection of

17 days with a standard error of only +, 0.^3.
TABLE 5
DISTRIBUTION OF ONSET OF ESTRUS AFTER PROGESTERONE TREATMENT
Interval betw. Inj. and
Onset of Estrus after
Injection

N o . of Animals
(Marking Harness
Data)

23 days
18
17
16
15

11
"
"
"

Average interval

No. of Animals
(Marking Harness
+■ Lambing Data)

1
1
8
1
3
17.0 ± 0.53

1
2
10
1
3
,17.1 ± 0.^3

All of the experimental animals used in the previous exper­
iments have been broken-mouthed Western ewes probably over six
years of age.
The next study on the effects of progesterone upon the
estrual behavior in the cycling ewe was carried out on 35 head
of Western ewes received from the Upjohn Company.
ular

In this partic­

experiment a single injection of the progesterone starch

suspension was compared with two Upjohn products and run simul­
taneously with a daily injection (10 mg.) of progesterone in
oil which served as a control.
In this experiment the data were obtained by the use of

67

two vasectomized rams which were turned in with the ewes twice
daily during the observation period.

In Table 6, three critical

observations were made on each ewe:
a) The stage of the cycle at which they received the
progesterone injections.
b) The interval lapsed between the progesterone injection
and the onset of the first estrus after injection.
c) The interval between the last estrus before the injec­
tion of progesterone and the first estrus after the
injection of progesterone. (The figures in oolumn C
are equal to the sum of columns A «- B.)
The mean interval between the progesterone injection and the
onset of the first estrus after injection, minus two to three
days for follicular maturation and ovulation, measures the
average length of activity of the progesterone compound given in
a single injection.

In this experiment, however, an estimation

of the time necessary for follicular maturation and ovulation
can be made (assuming ovulation occurs soon after the onset of
estrus, Robinson, 195^e)•

The ewes which received daily subcu­

taneous injection progesterone in oil for 16 days came into
heat approximately two days after cessation of treatment, indi­
cating a two to two and one-half day period was necessary for
follicular maturation.

Consequently only two days need be

subtracted from the interval between the injection of proges­
terone and the onset of estrus to determine the length of
progesterone inhibition of follicular growth.

The variation

68

TABLE 6
THE INHIBITION OF ESTRUS BY PROGESTERONE AND
THE RESULTING DEGREE OF SYNCHRONIZATION

Ewe
No.

Stage of
Date of Normal Cycle TreatLast
Cycle
ment Started
Estrus
Length* (Days Since
_________________ Last Estrus)
(days)
a. Control - Progesterone in

Interval
Interval
Between
Betw. Last
Injection and
Estrus be1st Estrus
fore Inject.
Post Inject.
and Last After
(days)
(days)
oil daily for 16 days

64

10-21

17-5

14

19

33

84

10-22

17-0

13

17

30

91

10-24

19-5

11

-

-

102

10-24

18.5

11

18

29

42

10-25

19.0

10

-

-

79

10-29

18.0

6

18

24

94

10-31

17.5

4

18

22

32

11-3

17.0

1

18

19

18.0 jt; 0.26 days

Mean ± Std. Error
b.

Prodox

20

82

10-20

17.5

15

5

48

10-22

18.5

13

-

75

10-23

17.0

12

12

24

66 10-24

18 .5

11

-

-

12

10-25

17.5

10

-

-

76

10-27

18.0

8

-

-

53

10-30

16.0

5

-

-

33

11-1

17.5

3

-

-

Mean +■ Std. Error

8.5 days

-

69

A

Ewe
No.

B

Stage of
Normal Cycle Treat­
Cycle
ment Started
Length* (Days Since
Last Estrus)
(days)
Sterile Aqueous Suspension

Date of
Last
Estrus

c.

C

Interval
Between
Injection and
1st Estrus
Post. Inject.
(days)
of Progesterone

Int erval
Betw. Last
Estrus be­
fore Inject,
and Last After
(days)

22

10-20

17.0

15

13

28

24

10-22

18.0

13

24

37

87

10-23

17.0

12

10

22

92

10-24

16.0

11

-

26

10-25

19.5

9

Pregnant

20

10-29

17.0

6

14

20

35

10-31

16.0

4

20

24

47

11-2

17.0

2

15

17

-

16 ± 2.08 days

Mean +, Std. Error
d.

-

Progesterone suspension in 0.3 percent starch solution

10

10-19

18.0

16

89

10-21

16.5

68

10-23

25

(Omit)

3

19

14

16

30

17.0

12

15

27

10-24

18.5

11

13

24

63

10-24

18.0

11

13

24

96

10-25

18.0

10

12

22

21

10-30

16.5

5

13

18

108

10-31

17.5

4

15

19

Mean ± Std. Error

13.9 * 0.55 days

♦Normal cycling data found in appendix Table 1.

70

about this mean interval (Column B) is a good index of the degree
of synchronization of estrus induced by the progesterone treat­
ment,

This variation within groups-also indicates the degree of

predictability of length of effective exogenous progesterone
action that can be expected.
The majority of those ewes receiving Prodox apparently were
thrown into anestrus
the treatment.

(in the middle of the breeding season) by

Six out of eight ewes showed no sexual activity

subsequent to the Prodox injection and two showed five abnormal
cycles throughout the rest of the breeding season.

Three out

of seven ewes which received Prodox (250 mg.) at the middle of
the breeding season failed to produce lambs.
The sterile aqueous suspension of progesterone (Upjohn)
induced a certain measure of inhibition of estrual activity
(Column G, Table 6) and, although the mean interval between the
injection and the onset of the post-injection estrus was 16 days,
the variation about this mean (Std. error » 2.1 days) was too
great, indicating lack of synchronization.
In the third group the microcrystalline suspension of pro­
gesterone in a 0.3 percent starch solution produced an average
length of inhibition of estrual activity of 14 days (actually
m, « 2 - 12 days) with a standard error of 0*55 days.

The

synchronization of estrus in this group was approximately four
times as effective than in the preceding group.

Ewe number 10

was omitted from these calculations because the treatment was
given during the follicular phase, which is approximately equal

71

to the two day period prior to the onset of the next estrus.
When given during the follicular phase the progesterone starch
suspension apparently is not capable of suppressing estrus in
the cycling ewe.

This treatment gave a fair degree of synchron­

ization with seven out of eight 'ewes showing estrus within a five
day period.

However, the length of progestational inhibition of

estrus during the height of the breeding season was approximately
four days shorter than its inhibitory effect upon the subthresh­
old

cycling during the anestrous season.

In preceding experi­

ments (Tables 2 and 3) the length of inhibition of estrus was
approximately 16 days.
The control group which received 10 mg./day of progesterone
for 16 days, came in heat, on the average, two days after the
sixteenth injection.

In all eight ewes, the 10 mg. daily was

sufficient to inhibit estrus regardless of the stage of the
cycle at which the treatment was started.

Two of'the eight ewes

(those with the longest normal cycles, 19.0 and 19*5 days - Table
1 in the Appendix) were completely inhibited and thrown into
anestrus by the 16 day treatment.
The next study involved the effectiveness of FMS in induc­
ing

estrus and ovulation in the anestrous ewe, both alone and

in conjunction with other hormone treatments:
In an attempt to determine the effectiveness of the double
dose of FMS spaced at a 16 day interval upon the induction of
estrus, a preliminary experiment was conducted in the latter
part of July and the first half of August.

Eighteen ewes

72

received 1000 i.u. of PMS (Upjohn's Gonadogen) . For the second
injection the ewes were divided into three groups of six each and
were given graded doses of FMS (125, 250, and 500 i.u. per ewe>.
Ten days after the first injection a vasectomized ram with a
marking harness was turned in with the ewes and they were observed
twice daily for signs of estrual activity.
Twelve of the 18 ewes came into heat (Table 7) within a
three day period. Three ewes came into heat in that group
which received 125 i.u., four in the 250 group, and five out of
six in the group receiving 500 i.u. of FMS on the second injec­
tion.

Although there is an apparent high correlation (r = 0.98)

between dosage level and estrus response, more data are necessar;
TABLE 7
RECORD CF ESTROUS BEHAVIOR AFTER TWO INJECTIONS OF PMS

Ewe
No.

43
50
56
4
17
25
29
7
10
33
27
2

Date in
Heat

8-13
8-13

8-13
8-14
8-14
8-14
8-14

8-15
8-15

8-15
8-15
8-15

Days
After
1st
Inject.
17
17
17
18
18
18
18
19
19
19
19
19

Total
Mean i Std.
Error

Days
Group 1 Group 2 Group 3
After
2nd
125 i.u . 250 i.u. 500 i.u.
Inject.
1
1
1
2
2
2
2
3
3
3
3
3

X
X
X
X
X
X
X
X
X
X
X
X
3

18.17 + 0.24

2.17 +: 0.24

4

5

73

before much reliability can be attached to this
curve (Figure 5)*

dose-response

Also, it has been shown by Hammond, (19^ 2),

that with the hormone treatment used in this experiment, there
was no significant difference in response to graded doses of
PMS at the second injection.

The exact dosage of PMS is not too

critical as long as it is sufficiently high to induce at least
one ovulation and estrual behavior as well.

Consequently the 500

i.u. dosage was doubled (1000 i.u.) and used at this level through
all other experiments.
The average interval between the second FMS injection and
estrus was 2.17 days (18.17 - 16 = 2.17) or approximately 52
hours. This figure is in agreement with those reported by Lambourne (1955) and Robinson (l954e) • When the average interval
was broken down into the various groups (Group 1 - 2.67 days;
Group 2 = 2.25 days; Group 3 = 1.80 days) a trend was noticed,
with the higher dosages requiring a shorter time to induce
estrus than the lower dosages (r = - 0.43

P<0.05)*

Although

the data are limited, the indications are that the ewes do show
a graded behavioral response to the graded dose of PMS.
When a single injection of progesterone was given at various
intervals before 1000 i.u. of FMS (Table 3) both the incidence
of estrus and the ovulation (slaughter) data were used to deter­
mine the optimum interval between the progesterone and the FMS
injection.

However, a few observations can be made in this

study concerning the effectiveness of PMS in inducing ovulation.
Only the fourth group (l6 day interval) had a high percentage of

74

FIGURE 5

DOSE-RESPONSE CURVE OF LEVEL OF 2ND INJECTION OF PMS
(16 DATS AFTER FIRST INJECTION) VERSUS PERCENT EWES
COMING IN HEAT AT EACH LEVEL.

K
$
O

o
K
Cj
Ul
"a

U;
U
Ui

Uj

CM

uj
oc
CL
UJ

or
Uj

Ui

£
Uj
UJ
--4

O
O

o
o

o
CXb

M V 3 H Al/

o

O

o

o

vX>

^

75

the ewes coming into heat, but of the twelve ewes slaughtered in
this study ten (83 percent) had ovulated, but only seven (58
percent) had shown estrus prior to slaughter.

This particular

observation determined the pattern followed in the subsequent
experiments when this hormone sequence was used.
Between June 20 and July 13 an experiment was conducted
with five groups of ewes that were treated as shown in
Table 8.

Inasmuch as thyroprotein apparently had no signifi­

cant effect on the results, groups 1 and 2 and groups 3 and
4, respectively, should be combined in analyzing the results.
Out of the eight animals in each group four were slaughtered
five days after the PMS injection and the other four were slaugh­
tered at 35 days after the PMS injection.

Since the 35-day slaughter

was primarily to determine the conception rate, ewes which did not
come into heat were selected for slaughter to determine ovulation.
The ewes in the first two groups (Table 8) had a fairly low
incidence of estrus (44 percent or seven out of 16 ewes) .

How­

ever, of the eight ewes slaughtered for observation of response
to PMS, seven or 87.5 percent had ovulated.

The one that failed

to ovulate was in that group which received the thyroprotein
along with the progesterone-PMS and even those ewes in that
group which did ovulate had less follicular activity than in the
other groups receiving PMS (See Appendix Table 2).
If it is assumed that the same response (or greater) to
PMS would be found in those ewes which came into heat, but were
not slaughtered at 5 days post PMS injection, then it becomes

76

TABLE 8
THE EFFECTS OF PROGESTERONE-FMS TREATMENT
UPON ESTRUAL BEHAVIOR AND OVULATION
Treatment

No. Ewes No.
Total No.
Slaught.
Slaught. Ovarian
No* of Ewes
in Heat at *3 Davs (in Heat)Resr»onse
Ewes
0

2.75

b

0

3-75

6

b

2

3.50

8

8

h

8

5

1.

Thy.-Prog.-FMS1

8

3

2.

Prog.-PMS

8

h

3.

Thy.-Prog.-FMS-Est.2

8

4.

Prog.-PMS-Est.

5.

Thy.-Prog•-Est.

3.75
2

1.75

1-Two hundred mg. of thyroprotein was fed, starting two weeks
before the progesterone injection. Two hundred and eighty
mg. of progesterone in starch suspension was injected
subcutaneously followed 16 days later by 1000 i.u. of FMS.
^Two hundred mg* of crystalline estradiol was injected
subcutaneously 36 hours after the FMS injection or 17.5
days after the progesterone injection.
apparent that FMS following progesterone will only induce estrus
in approximately
occur in 88 percent.

percent of the ewes whereas ovulation will
This ovulation figure may even be a little

low since the experimental ewes in this study were broken-mouthed
Westerns with many reproductive abnormalities showing in the
slaughter data (Appendix Table 3)•
The role of exogenous estrogen in the induction of estrus,
ovulation and fertility in the anestrous ewe:
In order to increase the incidence of estrus, exogenous
estrogen was added to the treatment sequence (estradiol).
Exogenously added estrogen increased (Table 8) the percentage

77

coming in heat from seven out of 16 (44 percent) in groups one
and two, which received no estrogen, to 1^ out of 16 (87*5 percent)
in groups three and four, which received 200 ug. of a suspension
of crystalline estradiol'.

All ewes which showed estrus came

into heat within a four day period.

TABLE 9
FREQUENCY OF OCCURRENCE OF HEAT IN EWES
AFTER HORMONE TREATMENT

Treatment

1. Thy•-Prog.-PMS

Days after FMS
Inject, that Ewes Hrs. in
Heat
Came in Heat
1
4
2
3
0

2

1

°1 *30.8
oJ

No. Ewes Total
Ovulated Ewes

3 (it-)*

8

4 (4)

8
8
8
8

i 6*.

1

1

2

3. Thy.-Prog.-FMS-E. 0

5

1

4. Prog.-PMS-Est.

0

2

6

3 (4)
*48.0 x 5*.
1
4 (4)
oJ1

5. Thy.-Prog.-Est.

0

3

1

1

0 (4)

1

13

11

1

14 (20)

2. Prog•-PMS

Total

40

♦Total number of ewes checked for ovulation - see Table 8
for a ranked ovarian response to PMS.
♦♦There is a significant difference between these means
(P < 0.05).
It can be seen from the data in Table 9 that the addition
of high physiological levels of thyroid-active protein (200 mg.
protamone) had little if any effect upon the reproductive phe­
nomena of these hormonally treated ewes.

Even though the groups

receiving thyroprotein (groups one and three) showed lower ovula­
tion and estrous responses, the number of ewes and the differences

78

involved are too small to be of any great significance.
A very interesting observation was made concerning the
relationship between progesterone and estrogen in an experiment
conducted at the Upjohn-Richland farms upon lactating Western
ewes*

Although the lactating ewes presented another unknown

factor which further complicated the problem, some interesting
results were obtained.

Robinson (1954 b and c) and a number of

other investigators reported that the presence of snail amounts
of progesterone were necessary for the optimal effect of estrogen
upon the estrual behavioral pattern.

The data in Table 10 support

this observation even though the induoed behavior was more intense
and of longer duration than the physiological norm (See Appendix
Table l ) .
TABLE 10
THE ONSET AND LENGTH OF ESTRUS IN HORMONALLY TREATED EWES

Treatment

Total
No. of
Ewes

No. of
Ewes
Serviced

Ave. Hrs.
in Heat
0

Occurrence of
Estrus (hrs.
after ECP)

1.

Control

15

0

2.

PMS

20

2

3-

Prog.-PMS

21

0

4.

PMS-ECP*

20

13

53 «5 jt 5.2

46.2 i 4.5

5*

Prog•-PMS-ECP*

21

20

75.0 ± 6.7

32.1 ± 3.9

22.4 £ 14
0

0
0

*There are significant differences between the groups four
and five (P < 0.05) where each ewe received one mg. of
estradiol cyclopentylpropionate (ECP).

79

Although no ovulation data were obtained in this experiment,
two observations can be made:

1) when progesterone preceded

estrogen, a significantly greater estrual behavior response was
obtained (compare groups five and four), which indicated that
some progestational activity from the treatment was present just
prior to and/or at the time of estrogen injection, 2) the single
injection of the microcrystalline suspension of progesterone when
given to lactating ewes has a longer period during which it is
present in effective concentration inhibiting any estrual response
in all ewes treated with PMS (group three) compared to nonlactating ewes injected during the anestrous period (group two,
Table 9).
Aside from the ovulation and estrous behavioral data presented,
little has been said concerning the number of ewes which lambed
as a result of the hormone treatment.

In a previously cited

experiment (estrus and ovulation data given in Table 9) the
length of estrus was shown to be closely correlated with concep­
tion.

The three out of 11 ewes (Table 11) which were found with

viable fetuses 35 days post insemination, all had estrous periods
lasting 24 hours or less whereas the other eight ewes all had
estrous periods of 36 hours or more.
Sixteen purebred Hampshire ewes from the University flock
were given two hormone treatments on a field trial basis.

Although

the duration of estrus was not measured, some interesting lambing
results were obtained.

A ram with a marking harness was turned

in with these ewes and they were checked for only three days after

80

TABLE 11
THE EFFECT OF EXOGENOUS ESTROGEN* ON ANESTROUS
EWES** TREATED WITH PROGESTERONE AND PMS
Treatment

Hrs. in Heat No. in Heat

Prog.-FMS

30.8 +, 5.8

Prog.-PMS-Est.

48.0

± 4.9

No. Pregnant No. Ovulated

7 (16)

2 (4)

7 (8)

14 (16)________1 (7)________ 7 (8)

*Microcrystalline suspension given subcutaneously.
**Broken-mouthed Westerns

the PMS injection.

A comparison of the lambing dates with the

breeding dates (Table 12) shows that two ewes lambed (ewes number
2 and 100) as a direct result of the hormone treatment, but the
other three ewes (ewes number 31 , 206, and 531) which lambed did
so, 17 to 25 days after it would normally be expected if they
were bred within the three day check period.

In fact ewe number

31 was not even marked during the breeding period within the three
day interval after the PMS injection.

Therefore, it can be assumed

that the hormone treatment had more than its initial effect upon
reproductive behavior and function, and caused further cyclic
activity in these three ewes.

It should be mentioned that the

initial breeding of these ewes took place around July 5 which is
towards the end of the anestrous season.

At thxs time it is

possible to hasten the onset of regular cyclic activity with
proper hormone treatment (Gordon, 1958) •
Although the estrous season was apparently hastened in these
three ewes, this was not the primary objective of this work.

81

TABLE 12
THE EFFECT OF HORMONE TREATMENT ON THE INDUCTION
OF ESTRUS AND FERTILITY IN THE ANESTROUS EWE

Number

Onset of Estrus
Days after FMS

Date
Lambed

Number
of Lambs

Progesterone-FMS-Estrogen*
205
265
2
102
531
172
267

2.0
3.0
1.0
3.0
1.0
1.0

206

3.0

12-11

1

2.0 days

3 Ewes

b Lar

—

8 Ewes

_

11-25
_

12-19

—

2
—

1
_

—

—

Pr og es terone-PMS

268

2.0

2^6
31
219
120
100
258
273

—
-

1.0
0.5
-

3-0

8 Ewes

1.6 days

—

12-19
-

11-25

—

2
-

-

2
-

_

—

2 EWes

^ Lai

*200 ug. of crystalline estradiol given subc utane ously
Since no observations were made to determine further occurrence
of estrus in these ewes, it cannot be definitely stated that
cyclic activity was influenced.

Therefore, any studies to deter­

mine the effects of hormone treatment to induce estrus, ovulation,
and fertility in the anestrous ewe, should be started early
enough in the anestrous season to eliminate the possibility of
further resumption of estrous periods after the initial effect.

82

It appeared that the supplemental estrogen might be reducing
conception and implantation.
ed
ewe.

Consequently a study was institut­

to determine some phases of metabolism of estrogen in the
The preliminary work was designed in order to determine

whether assayable quantities of estrogenic substances could be
extracted from 100 ml. samples of blood taken from ewes in the
first 12 hours of natural estrus.

The extraction procedure was

the same as outlined in the Methods section except that no initial
protein alkaline hydrolysis was used (the extraction efficiency
was approximately 75 percent, both with and without the initial
protein hydrolysis, using both crystalline estradiol and the
sodium-salt of estradiol added to rams? blood in vitro at a rate
of 1.5 ug./lOO ml.).

Under these particular circumstances little

estrogenic activity was found in the blood.

These data are

briefly presented in Table 13*
TABLE 13
DETERMINATION OF ESTROGENIC ACTIVITY IN THE
BLOOD OF EWES DURING INDUCED AND NATURAL
ESTRUS AND 4TH MONTH OF PREGNANCY

Condition of Ewe

Total No.
of Ewes

Level of
Activity
(conc.)

No. of Ewes
Showing
Activity

Induced estrus

11

0

0

Natural estrus

14

0.54 ug./lOO

1

Induced estrus (ovarian
vein blood)

2

0

0

4th month of Pregnancy

3

0

0

83
The extraction technique as employed here would quantitatively
remove estradiol from rams' blood when it was added in minute
quantities (0.5 - 1.5 ug./lOO ml.) in vitro. However, neither
naturally occurring nor injected estrogens could be detected in
ewes' blood from the jugular or ovarian vein.

Consequently the

next study was designed to measure the biological half-life in
the blood of an intravenously injected dose (200 to 500 ug.) of
a crystalline suspension of estradiol.

However, even with as much

as 500 ug. of estradiol given intravenously, no assayable amounts
of estrogen were found in the blood, including those samples of
blood collected as early as five minutes after the injection.
It can be seen from the standard curve that the sensitivity of
the estradiol assay requires only as little as 0.0^ ug. of estra­
diol to elicit a significant response in the assay animal.

Since

only five mice were used for any of these unknowns, only 0.20 ug.
of estradiol or its equivalent was necessary in any blood sample
(50 to 100 ml.) to give a measurable

response.

A few rough

calculations point out the significance of this failure to find
estrogenic activity in the blood when it was added in vivo,.
Assume:

Given:

A.

Injected estradiol was distributed evenly
throughout every tissue in the body within
5 minutes.

B.

Specific gravity of body (and blood) equals
one.

A.

Ewe weighs 60 kilo

B.

Dose equals 300 ug./ewe.

Calculations:

Ewe volume B 60,000 cubic centimeters

84

Each ml. would then contain 0.005 ug.
100 ml. of blood would then contain 0.50 ug.
of estradiol.
Although the assumptions made in these calculations are far
from what actually would happen, it was noted that if most of the
injected estrogen was passively handled within the first five
minutes after injection, there would be more than enough in the
blood to assure an assayable concentration.

Since this was not

the case, there are three possible explanations of this obser­
vation: l) The estradiol was very rapidly metabolized to inactive
nonphenolic or less active phenolic steroids (not conjugates),
2) it was actively removed intact in some manner from the blood,
3) the estrogen was bound to some protein in the blood so that
the alcohol extraction would not remove it.
At this time it seemed probable that the micro crystalline
estradiol might be handled differently by the ewe as compared to
the water soluble sodium-salt of estradiol (oil solutions were
not used because they are not readily injectable in the venous
system). An experiment was designed to determine the ability
of various initial extraction procedures to extract estradiol
from blood when added in vivo in its two physical forms.
The initial extraction procedure C as shown in Table 14
was designed to partially hydrolyze the blood proteins and in
so doing, remove from the protein any estrogens that might be
bound to them.

Therefore, the data in Table 14 indicate that

the microcrystalline estradiol has been removed from the blood

85

TABLE 14
EXTRACTION OF ESTROGEN FROM THE BLOOD OF EWES
RECEIVING AN INTRAVENOUS INJECTION OF ESTRADIOL
300 ug.
Crystalline Suspension
conc. (ug$)
No obs.

Initial Extraction*
Procedure
A. 4 X with 1:1 ethyl
alcohol

0

20

B. 4 X with 1:1 ethyl
alcohol-ether mixture

0
0

C. Overnight hydrolysis
with 2N NaOH and
extract as in B.

300 ug.
Sodium-salt Estradiol
No obs.
conc. (ug$)

0.17

1

5

0

1

8

0.462**

4

*Blood samples taken nine minutes after injection.
**Signif icant at the *001 level. The concentration column
represents one determination, but in four ewes significant
quantities were found.
stream to the extent that assayable quantities could not be detect­
ed.

On the other hand, when the water-soluble sodium-salt of

estradiol was injected intravenously significant quantities were
found in 100 ml. samples of blood within nine minutes after
injection provided the sample was initially treated with a mild
alkaline hydrolysis.

Although some estrogen was extracted without

the hydrolysis the indications are that most of the estrogen was
bound in some manner and in a very short time after injection.
The sodium-salt of estradiol existed in the blood for a much longer
period of time which allowed for blood samples to be taken at
reasonable lengths of time after injection (five to nine minutes)
and the estrogen concentration was maintained at levels which were

86

easily assayable.

However, the rate of disappearance of the

sodium-salt from the blood was also quite rapid.

The greatest

recovery obtained, based on the percentage of the injected dose,
was (sodium-salt of estradiol given intravenously and extracted
with method C - Table 1^) 8.62 percent left in the total blood
volume (based on 8 percent of body weight) at the end of nine
minutes.
The difference in the excretion rate in urine of the intra­
venously injected (l.O mg.) microcrystalline suspension and the
water-soluble sodium-salt of estradiol was measured in two ewes.
The pattern of estrogen excretion was similar in both ewes,
except that the ewe receiving the microcrystalline suspension had
twice the rate of excretion of estrogen as the ewe receiving the
sodium-salt of estradiol (Figure 6).

Compared to the micro­

crystalline suspension, the sodium-salt of estradiol was appar­
ently more quickly converted in vivo to relatively impotent
excretion products that would not be detected by the assay.
Although the amounts of estrogen excreted in the urine declined
very rapidly within the first hour after injection, the sodium-salt
of estradiol fell to a much lower level, indicating a shorter
duration of effective concentration in the body compared to the
microcrystalline suspension.
The preceding interpretations of estrogen metabolism are
supported in part by the following study in which estrus and sub­
sequent fertility was induced in the anestrous ewe.

All the ewes

received the same pretreatment hormone regime consisting of the

87

FIGURE 6.

THE URINE EXCRETION OF ESTROGENS ADDED IN VTVO.
THE EXTRACTION PRODUCT W AS ASSAYED BY THE UTERINE
RESPONSE IN IMMATURE MICE AND WAS COMPUTED AS
ESTRADIOL EQUIVALENT.

CO
— J

o
Q
Q:
K

3

Uj

C£

Q:
o

O

O

c q

— >

o

*

Uj
O
^

L

Cq
CM

i

s

3J.DNIW / 031330X3 -&W

88

progesterone suspension in starch solution given in a single
injection followed 16 days later by 1000 i.u. of PMS.

Thirty-

six hours after the PMS injection the ewes received the estrogen
treatment which varied as to the physical qualities of the estro­
gen, level of estrogen, and its route of administration.

Fertile

rams were used to check for the occurrence and duration of estrus
and the ewes were given a chance to lamb if bred.

The data are

presented in Table 15.
It is apparent that the crystalline suspension of estradiol
and the subcutaneous route of administration was the most effec­
tive means of inducing estrous behavior in the ewe as compared
to the other three treatments (Table 15)*

The ewes receiving

the sodium-salt of estradiol apparently were not affected by the
estrogen treatment as far as the induction of estrus was concerned.
The data in Tables 11 and 12 show that 50 percent of the ewes
which received the progesterone-PMS treatment came into heat
without receiving exogenous estrogen.

As shown in Table 15 the

groups receiving the sodium-salt of estradiol by either route
of administration failed to exceed this control level.

However,

the sodium-salt apparently hastened the onset of estrus in those
ewes which did come into heat.

The average interval between the

injection of FMS and the onset of estrus was ^2.7 hours in the
ewes receiving the sodium-salt of estradiol and 59*2 hours in
ewes receiving the microcrystalline suspension of estradiol.

The

difference between these means was significant (P < 0.02).
Those ewes which received the sodium-salt of estradiol and

89

TABLE 15
EFFECTS OF TYPE, ROUTE OF ADMINISTRATION AND
LEVEL OF ESTRADIOL UPON THE OCCURRENCE OF
ESTRUS. LENGTH OF ESTRUS AND CONCEPTION*

Level
in ug.

Intravenous
Crystalline
Estrus Lambed
hours

Intravenous
Sodium-Salt
Estrus Lambed
hours

Sub. Q
Crystalline
Estrus Lambed

Sub. Q
Sodium-Salt
Estrus Lambed
hours

hours

200

6

0

6

1

6

0

0

0

200

6

0

0

0

24

0

12

0

150

6

0

0

0

0

0

24

1

150

0

0

0

0

12

0

0

0

100

0

0

0

0

6

0

0

0

100

0

0

6

2

24

0

0

0

50

30

1

0

0

6

1

30

1

50

6

0

0

0

60

0

24

1

in heat

25.0

62.5

$ lambed

12.5

% lambed that
were bred

20.0

50.0

87.5

25.0
100

12.5

37.5

14.3

75.0

♦For control data (progesterone-PMS only) refer to Tables
11 and 12.

90

did come into heat had a very high lambing percentage (five out
of six) as compared to those receiving the crystalline suspension
(two out of twelve).

Although the data are somewhat limited, they

indicate that the sodium-salt of estradiol was having some effect
upon the fertility of the ewe at the time of fertilization of the
ovum and its descent down the reproductive tract.

91

DISCUSSION

The need has been shown for progestational participation
at various levels throughout a period prior to and during the
follicular phase if ovulation and estrus was to occur in a normal
fashion (Hammond et al. , 19^2; Dutt and Casida, 19^ 8 ; C'Mary et
a]_. , 1950; Robinson, 1950; Everett, 19^-0).

Although it has not

been definitely established that progesterone is necessary for
ovulation in the ewe, it does have a significant effect upon
estrual behavior and subsequent fertility if given just prior to
and during the follicular phase of the cycle.

Two basic reasons

have been cited for the need of a period of progestational activ­
ity in the anestrous ewe prior to gonadal stimulation with exog­
enous hormone for the purpose of inducing estrus, ovulation, and
fertility; l) In the normally cycling ewe, estrus and ovulation
occur in the presence of a regressing corpus luteum and ferti­
lization, migration of ova, and the beginning of implantation
take place in a reproductive tract that has been under the pre­
dominant influence of progesterone for approximately 13 days.
The normal hormonal sequence present in the cycling ewe should
be established in the anestrous ewe if successful mating is to
occur. 2) Subthreshold cycling has been observed by Robinson
(1950) and Dutt (1952) in the anestrous ewe and it is postulated
that a suppression of this cycling is necessary in order for the
normal estrous response to be induced.
Since the primary objective of the progesterone phase of
this study was to measure the length of activity of a single

92

injection of progesterone, and thus avoid daily injections over
a long period, a knowledge of the effects of progesterone just
prior to, and during the follicular phase is of great importance.
In the work conducted on the ewes at the Upjohn-Richland Farms*
progesterone, when injected in a single dose to lactating ewes^
exhibited a higher level of activity at the end of 16 days than
was the case when it was injected in non-lactating anestrous
ewes.

This was shown when the progesterone-PMS treatment (Group

three, Table 10) induced no heat periods in the 20 treated ewes,
whereas all other cases in which this treatment was given to nonlactating anestrous ewes, the incidence of estrus was approxi­
mately 44 to 50 percent*

However, when this same treatment

(Progesterone-PMS) in the lactating ewe was supplemented with
exogenous estrogen (progesterone-PMS-estrogen), the progesterone
increased both the incidence of estrus as well as the duration
of estrus when compared to the PMS-estrogen treatment (Table

10) *
These results indicate that the absolute concentration of
progesterone in the effector organs was not as important as the
relative level of progesterone with regard to the concentration
of estrogen, insofar as the induction and duration of estrual
behavior were concerned*

If the absolute level of progesterone

were the deciding factor it would be expected that the FMSestrogen treatment would cause a greater percentage of the ewes
to come in heat for a longer period when compared to the proges­
terone-PMS-estrogen treatment.

Since the reverse was observed

93

it is proposed that the exogenous estrogen was able to not only
override the primary effects of progesterone, but caused a utili­
zation of its secondary effects to increase the estrous behav­
ioral response.
The secondary effect of progesterone, mentioned above, refers
to the augmentation of the estrual response by progesterone when
the behavioral effectors are under the dominant influence of
estrogens.

This synergism of progesterone with the primary

estrogen behavioral response, was quantitated by Moore and Robin­
son (1957b) using ovariectomized ewes.

However, the vaginal

response to estrogen was inhibited regardless of the relative
level of progesterone to estrogen.

Therefore, since the syner­

gistic action of progesterone with estrogen does not manifest
itself in all phases of the reproductive phenomena, care should
I
be taken in extrapolating this synergism to other phenomena such
as ovulation, endometrial proliferation, and motility patterns
in the reproductive tract.

Everett and co-workers (19^0, 1950)*

using the persistent estrous rat have definitely established the
role of progesterone in induction of ovulation, however, this
particular effect has not been shown with any degree of signi­
ficance in the ewe.
It has been reported (Cole and Miller, 1933 and McKenzie
and Terrill, 1937) that anestrous ewes will ovulate when treated
with a single injection of PMS even though an endogenous or
exogenous source of progesterone was not present. It appeared
in this study however, that progesterone was playing some part

9^

in the initial reproductive mechanisms other than enhancing the
induction of estrus*

McKenzie and Terrill (1937) and Zavadov-

skii (1939^) have shown that artificial insemination without
heat, at approximately three days after FMS injection without
progesterone pretreatment gave very poor conception rates,
indicating a function of progesterone in the reproductive proc­
esses other than in the behavioral response*
It has been established (Robinson, 1956; Dutt and Casida,
19^8; Dutt, 1953a) that 10 mg* of progesterone per day will
suppress both ovulation and estrus in the normally cycling ewe.
Assuming that this level of progesterone was necessary to inhib­
it subthreshold cycling in the anestrous ewe and prepare the
reproductive tract for implantation, then a single dose of
progesterone makes available to the ewe at least 10 mg./day over
the desired or measured length of progestational inhibition*
Robinson demonstrated that optimal length of progestational
inhibition was 12 to 13 days if the response to a given level of
estrogen after progesterone treatment was to be maximal.

There­

fore, a single injection of 280 mg. progesterone suspension in
starch solution should make available to the system of the ewe
an equivalent of 10 mg. of progesterone per day when given in
oil over a period of at least 12 days.

The data indicate that

the period of progestational inhibition lasts for approximately
16 days (Table 2 and 3) in the anestrous ewe and only 12 days
(Table 6) in the normally cycling ewe.

It also appeared that

toward the end of the breeding season the length of progesta-

95
ttonal inhibition from this treatment was somewhere between 12
and 16 days (l^ to 15 days; Table 5)*

Consequently, if it was

assumed that the rate of exit of progesterone from the injection
site was exponential with time and constant from one season to
the next it appears that the level of progesterone necessary to
maintain progestational inhibition (inhibit follicular grcwth
and ovulation) decreases as the ewe progesses towards anestrus.
Although no observations have been made concerning the rate
of withdrawal of progesterone from the subcutaneous depot, it
was assumed that it was exponential with time.

The crystalline

suspension of progesterone is essentially a micropellet implant.
The rate of withdrawal of progesterone from this implant should
be proportional to the surface area of the progesterone crystals
and the surface area of the crystals should grow smaller exponentially with time.

In the single injection, therefore, much higher

levels of progesterone would be available to the ewe during the
first part of this treatment than would be required for progesta­
tional inhibition.

Therefore, at the end of the progestational

inhibition period of a single injection of progesterone in
starch suspension, the level of progesterone would be higher as
compared to the end of progesterone treatment when given daily
in oil.

Any error in the estimate of the length of the proges­

tational inhibition of the single injection of progesterone
(Table 1) due to suoh factors as lactation (Table 10, group
three) or between ewe variation would result in a failure of
the ewe to respond optimally to the PMS treatment.

However, the

96

daily injection of progesterone in oil allows for a more abrupt
cessation of progestational activity and thereby increases the
predictability of response to treatment.

The primary reason for

using the single injection of a progesterone starch suspension
was to increase the practicality of the treatment .
Although most of the interest has been centered on various
methods used to hasten or duplicate breeding activity in the
anestrous ewe, some observations were made concerning the role
that progesterone might play in inducing or hastening the onset
of the anestrous season.

It was observed that 19 out of 23

ewes failed to come into heat after receiving a single injection
of progesterone suspension in the latter part of the breeding
season (Jan. 9) *

In ewes receiving 'Prodoa? (Table 6) during the

middle of the breeding season (Nov. *0 only two out of eight
ewes came into heat after the treatment.

In the same experi­

ment* two ewes failed to come into heat after receiving 16 daily
injections of 10 mg. of progesterone.

These two ewes had lorger

cycle lengths (19.0 and 19*5 days - compared to a 17 day aver­
age) than any of the other ewes in the experiment.

It appeared

that ewes with relatively long cycles possibly are more suscep­
tible to progestational inhibition of cyclic activity.

This

also indicates that "Prodox11 produced a period of progestational
inhibition of at least 16 days and probably longer because of
the number of ewes in this group which were thrown into anestrus.
When FMS was given after progesterone treatment the inci­
dence of estrus was reported to vary from 50 (Dutt* 1953; Lam-

97

bourne, 1955; Gordon, 1958) to 90 percent (Dutt, 1952; 1953;
Robinson, 195^d)•

Usually the lower response was due to daily

infection of progesterone for short periods of time (less than
five days) * In most cases when the daily injection of proges­
terone in oil was given to anestrous ewes for a period of seven
to sixteen days followed by PMS, the incidence of estrus was
approximately 90-100 percent.

The progesterone starch suspen­

sion exerted its effect over a period of 12 to 16 days and
therefore, it was expected that the percentage of ewes coming
in heat with the progesterone-PMS treatment would be around 90
to 100 percent.
age

However, this was not the case and the percent­

of anestrous ewes coming in heat with this treatment was

only ^5 to 50 percent.

The 16 day interval was the optimum

interval at which the progesterone-PMS treatment should be
given, but between ewe variation in the rate of availability,
need, and assimilation of the progesterone would cause many
ewes to have too little or too much progesterone in their sys­
tem, and estrus would not occur.

Geographical location and the

breed of the ewe may also be factors involved in the low percent­
age

of ewes coming in heat with this treatment when used at

Michigan State.

Since Rambouillet and Dorse "blood" will

increase the incidence of spontaneous cycles during the spring
and summer months, the use of grade ewes of mixed breed is sub­
ject to false interpretation of the data unless they are compared
with control ewes.

It is also difficult to compare this type

of data from various geographical locations.

98

Dosage levels of PMS used by other investigators range from 125
to 2000 i*u. with the most common dosages being 500 i«u for
increasing lambing percentages in normally cycling ewes and
1000 i.u. coupled with the progesterone treatment to produce
reproductive activity in the anestrous ewe.

Super-ovulation

apparently does not affect the ability of the ewe to give
birth to at least one lamb (Robinson, 1951b) .

The use of PMS

to increase the lambing percentage of ewes bred during the
estrous season requires precise information concerning the
optimal dose level (Palsson, 1958; Wallace, 195^0 because if too
many ova are shed embryonic mortality will cause the lambing
percentage to drop below normal (Wallace, 195^)*

Since this

work was concerned with the percentage of the ewes lambing,
rather than with the lambing percentage, embryonic mortality
as a result of super-ovulation was not considered when selec­
ting the PMS dosage.

If the more immediate problems connected

with the hormonal induction of reproductive activity in the anes­
trous ewe are solved, the refinement of the PMS dosage to in­
crease lambing percentage would then merit further consideration.
The lambing percentage of the ewes lambing as a result of
progesterone-PMS-estrogen treatment (Table 15) was below normal
(seven ewes had eight lambs for 11^ percent) as compared to the
overall record of this same group of ewes the previous spring
(135 percent).

However, comparing the seven ewes that lambed

as a result of hormone treatment with their previous year's
record the lambing percentage is exactly the same.

In another

99

group of Hampshire ewes lambing in the fall of 1958 (Table 12)
five ewes had eight lambs (l60 percent) which was the same
lambing percentage of these five ewes in the spring of 1958.
The average percent lamb crop for this flock over the past
three years was 152 percent. From these data it appears that
the dose level of PMS did not affect the lambing percentage.
The progesterone-PMS treatment as described in this study
was successful in inducing ovulation (88 percent), but the
incidence of estrus (^5-50 percent) left much room for improve­
ment.

In order to increase the number of ewes coming into

heat, exogenous estrogen was given to help supplement the endog­
enous estrogen resulting from PMS stimulation.

Cole and Miller

(1933) and Robinson (1955b) used estrogen to supplement endog­
enous estrogen from PMS stimulation and the estrogen was given
simultaneously with the PMS.
given

36

In this study the estrogen was

hours after the PMS injection.

It was reasoned that

this time interval between the two injections would allow the
PMS to stimulate follicular growth before the estrogen was given.
Also, the onset of estrus usually occurs within 12 to 18 hours
after the administration of estrogen.

If estrogen were to be

given simultaneously with the PMS, there would not be enough
time for follicular maturation and ovulation before the onset
of estrus and ovulation might be inhibited entirely (Robinson,
1955b) . It was important to keep the exogenous dosage level
of estrogen to a minimum and its duration of action short
(length of induced estrous period should not be more than 36

100

hours).

Therefore, if the follicles are given time to develop

they will also add endogenous estrogen at such a time as to give
maximal supplementation of the exogenously added estrogen.
Although, no data were collected in this study to determine the
ovulation time after PMS injection, it has been reported by
Robinson (1956) that ewes ovulated approximately ^8 hours after
PMS injection and they came into heat shortly before this at
^+0 hours after PMS injection (Robinson, 1956; Gordon, 1958).
The ewes in this study that were not given estrogen also came
into heat at approximately ^+8 hours after PMS injection (Appen­
dix Tables

5» and 6).

Therefore, the exogenous estrogen was

given 36 hours after the PMS injection and entered the system
of the ewe at what was hoped to be the peak of the endogenous
production of estrogen (four hours before the ewe would normally
come in heat from progesterone-PMS alone) and thus result in a
single peak of estrogen available to the initiating mechanisms
of estrual behavior in the ewe.

This statement assumes that the

peak of estrogen production in the follicle occurs just prior to,
or during the onset of a progesterone-PMS induced estrus.
The level of estrogen used to increase the incidence of
estrus in the progesterone-PMS treated anestrous ewes varied
between 0.05 and 1.0 mg.

The first experiment in which estrogen

(ECP) was given, was at the Upjohn-Richland Farms in which the
1.0 mg. dosage was used.

This level of estrogen was in excess

of the amount needed to produce a physiological estrus of normal
duration.

This is evidenced by the excessive length of estrus

101

(50 to 72 hours) and the fact that none of the ewes lambed
as the result of breeding during this estrous period.

Since

ECP is an estrogenic substance of prolonged activity, the nat­
ural estrogen, estradiol, was used in order to give a more
precise and limited period of action.

Robinson (1956) and

Moore and Robinson (1937a) had shown that 20 to 40 ug. of QDB
(oestradiol benzoate), given at the end of five to 16 days of
progesterone pretreatment would elicit estrous behavior in 90
percent of ovariectomized ewes.

They were using daily injec­

tions of progesterone in oil and could depend upon less variation
in the level of progestational activity at the time of the es­
trogen treatment as commred to the single dose of progesterone
used in this study.

An estrogen of limited duration of activity

was necessary because estrogens affect uterine motility, and
abnormal motility could have a detrimental effect upon descent
of ova down the tract, ascent of sperm up the tract, fertili­
zation of the ova, and/or subsequent implantation of the tyrophoblast.
In the second experiment in this study, 200 ug. of estra­
diol (microcrystalline suspension) were used and the incidence
of estrus was increased from

percent in the progesterone-PMS

treatments to 88 percent in the progesterone-FMS-estrogen treat­
ments.

However, of eleven ewes which were checked for pregnancy

at 35 days post service only three were with lamb.

Out of these

eleven ewes, seven received the progesterone-PMS-estrogen treat­
ment and only one of these was with lamb.

Of those ewes receiving

102

the progesterone-PMS treatment two out of* four contained viable
fetuses.

As mentioned previously, these three pregnant ewes

all had heat periods of 2^ hours or less.

It appeared there­

fore, that the 200 ug. level of estradiol maintained the
duration of estrus past the optimal time for fertilization
to occur.
In considering the importance of exogenous estrogen as a
means of increasing the incidence of estrus in the progesteronePMS treated anestrous ewe, a study was conducted to determine
various aspects of estrogen metabolism in the ewe.

The first

objective was to determine the level of circulating estrogens
during both natural and induced estrus from both jugular and
ovarian vein blood*

Jugular vein blood from three ewes during

the fourth month of pregnancy was also extracted to determine
its level of estrogen.

Out of a total of 30 ewes (Table 13) ,

either pregnant or in estrus, only one ewe in a naturally
occurring estrus showed an assayable quantity of estrogenic
activity.

In cattle it has been reported that the circulating

estrogens range from 0.35 (Szego and Roberts, 19^6) to 0.55 ug.
of alpha-estradiol/100 ml. of whole blood (Albritton, 1951) and
estrogens have been extracted from cow feces (Stob, 1956; Levin,

1945) and urine (Gorski, 1957) in relatively large amounts with
variation associated with the stage of pregnancy or the estrous
cycle.

Rakoff and Cantarow (1950) found relatively low, but

assayable quantities of estrogen in the ovarian vein blood of
the PMS treated dog and even higher levels in the spermatic vein

103

blood of the stallion*

Many investigators have found significant

quantities of the three primary estrogens, estriol, estrone, and
estradiol in human placenta (Diczfalusy, 1953* Diczfalusy and
Lindkvist, 1956; Mulbock, 1939)* semen (Diczfalusy, 195^)* and
circulating blood (Mulbock, 1939; Rakoff et al., 19^3; Roberts
and Szego, 19^6; Szego and Roberts, 19^6 and 19^7).

With all

of this information on the cow, bitch, stallion, and human under
various physiological conditions, no data could be found concern­
ing the level of circulating estrogens in the ewe.

Since no

estrogenic activity had been found in the blood of the ewe under
relatively normal physiological estrous and pregnancy conditions,
estrogens were added to blood in vivo* extracted and assayed to
determine their concentration at a given interval of time after
injection.

Although in the preliminary studies, the extraction

method was found to be reliable, no estrogen (microcrystalline
suspension) added in vivo could be extracted from the blood when
as much as 200 to 500 ug. were injected intravenously.
It was then decided to place the crystalline estradiol in
a 0.05 N NaOH solution and thus form the sodium-salt of estra­
diol which is water soluble.

The two physical forms of estra­

diol were then injected intravenously into two ewes and blood
studies conducted to compare various aspects of their metabolism
in the ewes.

Since in previous work no estrogen added in vivo

could be extracted from blood, three different initial extraction
procedures were also tested to determine their efficiency in
extracting jin vivo added estrogens from ewes'blood.

The blood

10^

samples were withdrawn from the ewe approximately nine minutes
after the estrogen was injected and the results are given in
Table 14-.

The fact that no estrogen could be detected in the

blood of the ewe which received the microcrystalline suspension
regardless of the initial extraction procedure was not surprising
since previous work using these various methods had also met
with failure (approximately half of the number of ewes in Table
10 represent ”initial extraction procedure” C in Table 14).
When the sodium-salt of estradiol was used, estrogens were
extractable in relatively large quantities from ewes? blood after
overnight hydrolysis in 2 N NaOH.

The obvious inference was,

that not only was the estrogen in a large part protein bound,
but also that the microcrystalline estradiol was not present in
the blood in assayable quantities within nine minutes after
intravenous injection*

Although no studies were conducted to

determine the fate of the microcrystalline estradiol, the size
of the crystals (one to two microns) was such that phagocytosis
of these crystalline particles could readily occur.

In this

manner the added estrogen could have been removed from the blood
in a relatively short space of time (within five minutes) and
released more slowly into the systemic circulation over a period
of time.

Heller e£ al* (1957) have shown that estradiol and

estriol have a stimulatory effect upon the reticuloendothelial
system (RES) of the rat, however, this does not support the
theory given, to explain the disappearance of estradiol crystals
from ewes'blood.

However, it does show a relationship between

105

estrogenic hormone and the RES which could be interpreted as the
RES having a certain affinity for crystalline estrogens.
As reported in this thesis* the only method which would
extract estrogens added iji vivo involved an initial alkaline
hydrolysis.

In all cases where 300 ug. or more of the sodium-

salt of estradiol were injected intravenously, this procedure
extracted estrogenic activity from the blood.

When ethyl alco­

hol of alcohol-ether mixtures was used to extract directly from
blood, estrogen was extracted when doses as high as 500 ug. were
injected intravenously.

These observations indicate that the

estrogen was bound in some manner to the protein which rendered
it un-extractable with simple organic solvents (ether and/or
alcohol).

However, partial degradation of the blood proteins

(alkaline hydrolysis) apparently frees the estrogen so that
these same solvents will extract them.

These data imply that

the "binding" is of a chemical nature rather than an adsorptive
or dispersing function of the proteins.

As mentioned previously,

the red cell may play an important role in the binding of estro­
gens.

However, no attempt has been made to determine the exact

nature of the binding nor to what specific protein it was bound.
The clearance of added estrogen from the blood by the kidney
indicated that the two physical forms of estradiol were initially
handled by the ewe in a different manner.

The amount of estro­

genic activity appearing in the urine within an hour after intra­
venous injection of the sodium-salt of estradiol was only about
half as much as was detected after administration of the micro­

lo 6

crystalline suspension of estradiol*

Either the sodium-salt of

estradiol was inactivated by the liver (not conjugated) or a
relatively large portion of it was bound to the extracellular
and/or intracellular body proteins.

The clearance of micro-

crystalline estradiol declined very rapidly in the first hour
as did that of the sodium-salt of estradiol„ but it remained at
a much higher level compared to the sodiurn-salt„ throughout
the remainder of the collection period.

This observation

supports the theory that the reticuloendothelial system (RES)
or some other depot area acts as a reservoirP trapping the
crystals before they are metabolized to an appreciable extent.
These depot areas would then dole out small quantities of estra­
diol over a relatively long period of time which would be metab­
olized and excreted in the urine.

In this way the mechanism

responsible for estrual behavior to which estrogens act as a
stimulus would be under the primary influence of estrogens for
a longer period of time as compared to treatment when the sodiumsalt of estradiol was used.

This concept was further illustrated

by the fact that the microcrystalline suspension of estradiol
was much more effective in inducing estrus in the progesteronePMS-primed ewe (Table 15)* than was the sodium-salt of estradiol
regardless of route of administration.
It was also apparent that the intravenous route of adminis­
tration

of estradiol was much less effective in inducing estrus

than subcutaneous injection.

This emphasizes the importance of

a depot area for estrogen storage to facilitate the maintenance

107

of a sustained® relatively low, level of estrogenic substances
in the blood which can act as a stimulus to the estrous response
centers in the ewe.

The necessary level in the blood and how

long it must be maintained are not known® but once the estrogenic
components reach the blood® their course of action is determined
and they are quickly metabolized and excreted.

No apparent

storage of estrogen takes place once it leaves its point of
origin either at its endogenous source (ovary or possibly the
adrenal cortex) or the injection site.

The use of crystalline

estradiol could be considered as a micro-pellet implant which is
removed from the blood and deposited at various sites (RES)
throughout the body.
Although 50-200 ug. of microcrystalline suspension of
estradiol was quite effective in producing estrous behavior in
the progesterone-PMS treated ewe, the lambing results were poor
(two ewes lambed of 12 serviced, Table 15) . On the other hand,
the sodium-salt of estradiol was ineffective in producing estrus
in the treated ewes, but, of those ewes serviced 83 percent (five
of six) lambed.

Although no control group (progesterone-PMS

only) was present in this particular study, previous and later
studies during the same season have shown that progesterone-PMS
treatment will cause approximately it-5-50 percent of the ewes to
come into heat.

In the treated groups receiving the sodium-salt

of estradiol only 37*5 percent of the ewes came into heat, indi­
cating that this physical form of estradiol at the 50-200 ug.
level had no effect in the induction of estrous behavior,

It

103

was shown in lour ewes that very large doses (500 to 1000 ug*)
of the sodium-salt of estradiol itfould, by itself, induce estrus
in the anestrous ewe.
It was not known if the sodium-salt of estradiol had any
effect upon the ability of the anestrous ewe to conceive, implant,
maintain pregnancy and finally produce a lamb.

In the control

group which was treated six weeks after the study involving the
use of the sodium-salt of estradiol four out of eight progester­
one -PMS

treated

ewes came into heat and only one lambed (Table

12) as a direct result of the treatment.

Although the number of

ewes involved are small, the differences between the control
group and the group receiving the sodium-salt of estradiol appear
to be significant® indicating that the sodium-salt of estradiol
was exerting a beneficial effect upon some function in the
reproductive mechanism other than its influence on behavior.
Larger doses of the sodium-salt of estradiol will effect the
estrous behavioral mechanisms in the ewe, but whether or not
the beneficial effect as postulated will be maintained at these
higher dosages is a matter for future study.
Another group of eight ewes treated at the same time as.the
control group received the microcrystalline suspension of estra­
diol after the progesterone-PMS treatment.

Although seven ewes

came into heat, only one lambed as a direct result of the treat­
ment (Table 12).

However, two ewes in the estrogen treated

group lambed 16 to 22 days after the mean lambing date of those
ewes bred earlier.

This experiment was started toward the end

109

of the anestrous season and apparently a normal cycling pattern
was hastened by the hormone treatment.

One of these three ewes

that lambed later in the fall had not shown any signs of heat
within three days after the IMS treatment.

no

SUMMARY AND CONCLUSIONS

In summarizing^ the objectives are again stated as follows?
A.

To determine the effectiveness of various hormonal
regimes upon the induction of estrus, ovulation,
and fertility in the anestrous ewe.

B.

To determine the length of primary action of a
subcutaneous dose of a microcrystalline suspension
of progesterone in 0*3 percent starch solution in
the ewe.

C*

To measure the effectiveness of pregnant mare serum
(PMS) given in conjunction with progesterone at a
given interval (as measured in B) t upon the induc­
tion of estrus9 ovulation, and fertility.

D.

To determine the effectivenss of exogenous estrogen
in increasing the incidence of estrus in conjunction
with the progesterone-PMS treatment and to show
what effects, if any, it would have on other repro­
ductive mechanisms.

E.

To determine some of the aspects of estrogen metab­
olism in the ewe.

The single injection of progesterone starch suspension was
found to exert its inhibition over a period of approximately
16 ± 0.5 days in the anestrous ewe.

During the height of the

breeding season this period was reduced to 12

0.5 days with an

increase in this length of action as both ends of the breeding

Ill
season were approached.

It was also shown that progesterone had

a secondary effect which involved 4a synergism with estrogen to
give a maximal estrous response.

No relationship was shown be­

tween this secondary action and the ovulatory mechanism.
Progesterone given in a single injection followed 16 days
later by 1000 i.u. of PMS was adequate to induce the majority
of the anestrous ewes to ovulate (88 percent), but it induced
heat in only 44-50 percent of the ewes.

The level of PMS used

had no effect upon the lambing percentage of those ewes' lambed,
although in some cases it produced super-ovulation.

It was

concluded that the relatively low incidence of estrus in the
progesterone-PMS treatment used in this study compared to daily
injections of progesterone in oil followed by PMS, was due to
the between-ewe variation in primary length of action of the
single dose of progesterone.

This would allow for too much or

too little progesterone at the time of PMS injection.
The incidence of estrus was increased from 44 percent to

88 percent with doses of 50-200 ug. of estradiol 36 hours after
the PMS injection.

Although this treatment had no effect upon

the percentage of ewes ovulating, the length (58 hrs.) of estrus
was increased beyond the normal (40 hrs.) .

The lambing data

indicated that this increased length of estrus was detrimental
to some phase of the fertility mechanism.

In this study all the

ewes that have lambed (15) as a result of hormone treatment, none
have estrous periods greater than 30 hours in length.
No estrogen activity originating from an endogenous source

112

(naturally occurring follicle, PMS induced follicle, or areas of
estrogen production in the pregnant ewe) was extracted from the
blood of the ewe in assayable quantities.

When a microcrystal­

line suspension of estradiol was injected intravenously in 500
ug. dosages, none was detected in the blood within five minutes
after injection.

However, when the water soluble sodium-salt

of estradiol was injected intravenously, estrogenic activity
was found in the blood only after partial alkaline hydrolysis of
the blood proteins before the initial extraction procedure was
started.

It was concluded that the micro-crystals of estradiol

were removed from the blood within a very short space of time
after injection, probably via the reticuloendothelial system
(RES) and that a large portion of the sodium-salt of estradiol
was chemically bound to the blood proteins shortly after entering
the blood.

The crystalline estradiol was then released slowly

from the RES back into the blood.
The progesterone starch suspension given in a single dose
followed 16 days later by PMS induced estrus (44-50 percent),
ovulation (88-90 percent), and fertility (25=50 percent of ewes
serviced) in the anestrous ewe.

Exogenous estrogen was then

added to the treatment in order to increase the incidence of
estrus with the hope of maintaining or increasing the lambing
percentage of the ewes serviced.

The microcrystalline suspension

of estradiol was very effective in increasing the incidence of
estrus (88 percent), but decreased the lambing percentage of the
ewes serviced.

The sodium—salt of estradiol at the levels used

113

had no effect upon the induction of estrus in the progesteronePMS treated ewe„ but the lambing percentage (83 percent or five
of six ewes) of the ewes serviced was much higher than the treat­
ment using progesterone-PMS alone.

It was concluded that the

sodium-salt of estradiol had some beneficial effect upon the
reproductive mechanism other than the induction of estrous
behavior.

114

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Zavadovskii# M. M. (1939&) Proizvoj-jna ja reguljacija polovogo
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"mertvyi" sezon kak metod stimuljacii Movuljacii"# "tecki"
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VH.
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at will of the sexual cycle in ewes. I. The use of gonado­
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lambing.) Trud. Dinam. Razvit., 11s 24-31 (Abstracted in
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Zavadovskii# M. M. , Margulis# Z. S.# Wunder# P. A. and Lingart,
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(Control at will of the sexual cycle in ewes. VTII. An
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Zavadovskli# M. M. and Paduceva# A. L. (1935) Neljzja li,
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125

prisedsth v ohotu. (Control at will of the sexual cycle in
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Zavadovskii, M. M . , Paduceva, A. L. and Wunder, P. A. (l939a)
Proizvoljnaja reguljacija polpvogo cikla ovec. II. Opyt
nasiljstvennogo osemenija ovec, ovulirovavsih, no ne
prisedsih v ohotu posle in’ekcii gonadotropnyh agentov.
(Control at will of the sexual cycle in ewes. H . An experi­
ment on the forced insemination of ewes which ovulated with­
out coming on heat after the injection of gonadotropic
agents.) Trud. Dinam. Razivit. 11s 32-41. (Abstracted in
Anim. Br. Abstr. 12: 87 , 1944).
Zavadovskii, M. M. , Paduceva, A. L. and Wunder, P. A. (1939b)
Proizvoljnaja reguljacija polovogo cikla ovec. V. Funkcija
l,cikliceskihtl i "eksperimentaljnyh" zeltyh tel u ovec.
(Control at will of the sexual cycle in ewes. V. The func­
tion of cyclic and experimental corpora lutea in sheep).
Trud. Dinam. Razvit. 11: 53“58. (Abstracted in Anim. Br.
Abstr. 12s 87, 19^)Zavadovskii, M. M. , Paduceva, A. L. , Wunder, P. A. and Muruev,
K. M. (1939) Proizvoljnaja reguljacija polovogo cikla
ovec. IV. Priciny nizkogo okata pri nasiljstvinnom pokrytii
ovec, stimulirovannyh prolanom i syvorotkoi. (Control at
will of the sexual cycle in ewes. IV. The causes of low
percentage of lambings following forced mating of ewes
treated with prolan or FMS) Trud. Dinam. Razvit., 11:
50-52. (Abstracted in Anim. Br. Abstr. 12: 87=*88, 1944).
Zavadovskii, M. H . , Paduceva, A. L. , Wunder, P. A., Muruev, K.
M. , Grigorjeva, M. P., Margvelasvili, S. G., and Sokolova,
L. V. (1939) Proizvoljnaja reguljacija polovogo cikla ovec.
III. Frodvizenie jaica cerez jaicevody ovey, nahodjasceisja
v ohote i vne ohoty. (Control at will of the sexual cycle in
ewes. H I . The passage of the ovum through the oviducts of
ewes which either are or are not on heat) Trud. Dinam.
Razvit. 11: 42-49, (Abstracted in Anim. Br. Abstr. 12: 88 ,
1944).
Zavadovskii, M. M . , Wunder, P. A., Paduceva, A. L. and Margve­
lasvili, S. G. (1935) Mozno li proizvoljno upravljatj
ovuljaciei, teckoi, i projavleniem ohoty u ovec. (Is it
possible to control ovulation, oestrus and the manifestation
of heat in sheep?) . Trud Dinam. Razvit* , 9* 21—74. (Abstract­
ed
in Anim. Br. Abstr., 4s 51-52, 1936).

126

Zavadovskii, M. M . , Paduceva, A. L. , and Wunder, F. A. (1937)
The junction of cyclic and experimental corpora lutea in
sheep. Bull. Biol. Med. Expt. USSR., ki 203-20^. (Abstract­
ed in Anim. Br. Abstr. 7' 131-32 , 1939).

APPENDIX

127

APPENDIX TABLE 1
THE NORMAL CYCLIC ACTIVITY OF 35 WESTERN EWES
DURING A PERIOD LASTING FROM
SEPTEMBER l«j TO NOVEMBER 1
Ewe
No.

63
10
12
20
21
22
24

25
26
32
33
35
42
47
48

49
53
64

66
68
75
76
79
82
84
87
89
91
92

Date 1st
Heat

9-18
9-15
9-20

9-26
9-28
9-18
9-17
9-19
9-19
9-29

9-28
9-29
9-18
9-29
10-4

10-1
9-28
9-16
9-18

9-20
9-21
9-22
9-24

10-3
9-20
9-18
9-18
10-4

9-21

94

9-27

96
98

9-20

102

9-30
9-18

103 10-21

108

9-26

Mean
Std. error

Date 2nd Heat
and Length
of Estrus

PM)
10-6 AM)
AM)
10-1 PM)
PM)
10-8 AM)
AM)
10-12 AM)
AM)
10-14 PM)
AM)
10-3 PM)
AM)
10-4 AM)
AM)
10-6 AM)
AM)
10-6 AM)
AM)
10-17 AM)
AM)
10-15 AM)
AM)
10-15 AM)
AM)
10-6 AM)
AM)
10-16 AM)
AM)
10-22 PM)
AM)
10-18 AM)
AM)
10-14 PM)
10-4 AM)
AM)
AM)
10-5 PM)
AM)
10-6 AM)
AM)
10-6 AM)
PM)
10-9 AM)
AM)
10-11 AM)
AM)
10-20 PM)
AM)
10-6 PM)
AM)
10-5 AM)
10-4 PM)
AM)
PM)
10-24 AM)
PM)
10-8 PM)
AM)
10-13 PM)
AM)
10-? PM)
AM)
10-17 AM)
AM)
10-6 AM)
PM) 12
10-14(AM)
AM)

36
24
36
36
24
24
36
48
36
36
24
36
48
48
36
48
36
36
36
36
48
36
36
48
48
48
24
36
48
36
36
36
36

Interval

Date 3rd Heat
and Length
of Estrus

17.5

10-24 (AM)

16.0

10-19 (PM)
10-25 (PM)

17.5

16.0
16.0
15.5
17.0
17.0

17.0
18.0

17.0
16.0
18.0
17.0
18.5
17.0
16.5
18.0
17.5

24
36
36
24
36
36
36
36
24
36
24
36
36
48

10-29 (AM)

10-30 (PM)
10-20 (PM)
10-22 (AM)
10-24 (PM)
10-25 (PM)
11-3 (AM)
11-1 (PM)
10-31 (AM)
10-25 (AM)
11-2 (AM)
11-4

(PM)

Interval
18.0
18.0
12.5
17.0
16.5
17.0
18.0
18.5
19.5
17.0
17.5

16.0
19.0
17.0

(a m )
(PM)
(AM)
(AM)
(AM)

36
36
36
24
24
36
36
36

10-23 (PM)
10-22 (AM)
10-21 (AM)

36
36
24

17.0
17.0

(PM)
(AM)
(PM)
(AM)
(PM)

24
36
24
36
36

16.0

18.0

10-24
10-31
10-25
11-3
10-24

17.5
18.0
17.0
18.5

24

18.0

10-31 (PM)

24

17-5

37

17.0

32.5

17.5

1 .37

16.0
15.0
16.5
17.0
17.5
16.5
17.0
16.5
19.5

17*0
16.5
17.5
17.0

0.49

10-30 (PM)
10-21 (PM)
10-24
10-23
10-23
10-27
10-29

1.14

17.5

16.0
17.5
18.5

17.0
17.0
18.0
18.0

16.5

0.48

128

APPENDIX TABLE 2
SLAUGHTER DATA (5 DAYS AFTER FMS INJECTION)*

Ewe Date
No. Estrus

Length of
Ovaries
Estrus
Right Left
(hours)
gm.
gm.

Thyroid
gm.

Adrenal
gm.

Ovarian
Response

Control

9
18

22
75

0
0
0
0
0

0
0
0
0
0

0.2

0.2

0.5

0.3
0.5
0,7
0,45

18.1
4.5
6.3
5,0
8.5

1.2
0 .6

20.8
8,8

0.5

15*1
7.4

0.5
0,6
0.42

6.0

1
1
2
1

5.6

1.25

4.9
5-3
7-1
3.2
5-1

2
1
1
1

6.4
6.7
3.5

Thvroorotein

20

0
0
0

0
0
0
0
0

1.0

0
0
0
0

0,8
1,0
1.2
0.92

0,4
0.5
o
0.3
0
o.6o
Thvroorotein-■orogest.-PMS
1
0
0
0.5
31
34
74

36
55

68

0
0
0
0

Thvroo.-Prog.-PMS-Estrogen
48
2.0
16
7-9
60
51
7-8
1.5
58
0
0
0,7
0,8
0
79
0
2
5b
1.25
Prog.-FMS

2
30
50

0
o
o

61

7-8

1

0
0
0
60
6o

0.3
0.60

13-0

0,7

10,1

0,8
1.0
1.2
0.88

7.5
13.4

1.3
1.5

10.0

1.2
1.0

6.2

1.25

1.5
1.6
1.0
0.7 _.
1.20

1.5

0.9
4.0

0.8
1.6

5*2

4.0

1 .2
2.82

0.8
1.80

1.6
1.2
1.1
1.35

6.2
9.3

9-1
5.2
7.7
9.0
11.3
7.5
. 6,1
8.5

1.25

4,2
5.5
51
4.1
4.7

3
2.75

9-0
7.1
4,2
4.5
6 .2

4
4
4
3.50

6.1

3
3

2

2

4.2
7.4
3.5
5-3

4
4
3
4
3.75

3-9
5.2
5.7

4
4
4

Proe.-FMS-Estrogen

7

7-9

2b

7-9
7-9
7-9
b

26
78

b8
2b
b8
2b
36

♦Slaughtered July 13

7.3
6.4
8.4
22.4

11.1

....7.-8 ,_
5.6

3.75

129

APPENDIX TABLE 3
SLAUGHTER DATA ( 3 ^ 2 DAYS AFTER FMS INJECTION)*

Ewe
No.'

Subject

Notes

17

Ovaries
Uterus
Pathology

Non functional, no sign of follicular growth.
No gross signs of stimulation.
Body of uterus closed-cystic growth in uterus.

56

Ovaries
Uterus
Pathology

One corpus luteum of pregnancy on both ovaries
One viable fetus in each horn.
None.

Thyrop.-Prog.-PMS^Estrogert
28

Ovaries
Uterus
Pathology

Right ovary contained a regressing C,L.
Some Cotyledonary development.
None.

37

Ovaries
Uterus
Pathology

Infantile or anestrous ovaries
No stimulation.
None.

70

Ovaries
Uterus
Pathology

Right ovary contained a C.L. of pregnancy.
One fetus on right side.
None.

77

Ovaries
Uterus
Pathology

Abnormal cystic growths on ovaries.
Inflamed with endometritus as apparent cause.
Tumorous growths on the ovaries.

Prog.-PMS
6

Ovaries
Uterus
Pathology

13

Ovaries
Uterus
Pathology

^2

Ovaries
Uterus
Pathology

Left ovary contained a mature follicle and fresh
corpus luteum.
Stimulated with cotyledonary areas well stimulated
Black exudate present in uterus.
2 large follicles„ left - 1 funct
Right ovary
C .L •
No stimulation
None.
Four corpora lutea on the ovaries
1 viable fetus in left horn and one dead and
one hemorrhagic fetus in the right horn.
None.

130

APPENDIX TABLE 3 (cont.)
Ewe
No.

Subject

Notes

Prog .-^PMS^Estrogen

8

Ovaries
Uterus
Pathology

Follicular activity on both ovaries.
Some stimulation.
None.

10

Ovaries
Uterus
Pathology

Large fibroid cyst and 1 large reg. C.L.
Cotyledonary area well stimulated.
Ten cm. cyst on right ovary.

53

Ovaries
Uterus
Pathology

3 regressing corpora lutea.
No stimulation.
Black exudate present in lumen of uterus.

5^

Ovaries
Uterus
Pathology

Infantile or anestrous ovaries.
No stimulation.
Black exudate present in lumen of uterus.

Thyrop. .-Prog..-Estrogen
li+

Ovaries
Uterus
Pathology

h-1

Ovaries

hS

Ovaries not recognizable due to tumorous growths.
Uterus also not recognizable.
Metastatic tumor filled reproductive area.

Uterus
Pathology

High degree of follicular activity on both
ovaries.
Highly stimulated.
None.

Ovaries
Uterus
Pathology

Some follicular activity.
Highly stimulated.
None.

♦Slaughter dates were on August 9 and 16.

131

APPENDIX TABLE 4
THE OCCURRENCE OF ESTRUS IN HORMONALLY TREATED ANESTROUS EWES
Ewe
No.

Date in
Heat

Interval (Hrs.)
betw . PMS & Heat
Thvron.-Pro2 .-FMS**
17
56
57.

7-8(PM)
36
7-8 (PM)
367-9 (PM)
60
44
3
Thyrop, -Prog.-FMS-Estrogen
16
7-9(AM)
48
28
7-9 (AM)
48
48
37
7-9(«0
51
7"8(FM)
36
70
7-9(PM)
60
77
7-8(FM)
36
6
46
P-goe.-PMS
6
7-9(PM)
60
48
13
7-9(AM)
60
42
7-9(PM)
24
61
7-8 (AM)
48
4

Hrs. in
Heat

Date of
Slaughter*

36
12
36
28

8-16
8-9

8-16

48
72

7-13

60
60

8-9
7-13

24
84
58

8-16

48
48
12

8-16
8-9

60

7-13. __

8-16

8-9

8-9

42

Pros.-FMS-Estroaen
7
8
10
24
26
53

54
78

7“9(PM)
7 “10 (AM)
7-9(PM)
7-9 (PM)
7“9(PM)
7-10 (AM)
7 “9 (AM)
7-9(AM)
8

60
72

60
60
60
72
48
48

60

Thvroo. -P-roe •-Estrogen
96
14
7 “11 (AM)
48
41
7“9(AM)
48
45
7-9 (AM)
72
46
7-10 (AM)
60
81
7-9(PM)
65
5

48
48
36
24
48
36

60
24
40
24
36

60
24
12
31

7=13

8-16
8-9
7-13
7-13
8-9
8-16
7.-13....

8-9

8-16
8-16
7-13
7-13

♦July slaughter dates for ovulation data and August dates
for pregnancy data.
♦♦There were a total of 8 animals in each group.

132

APPENDIX TABLE 5
THE EFFECTS OF EXOGENOUS ESTROGEN UPON THE
PROGES TERCNE-PMS TREATED ANESTROUS EWE
Level of
Ewe Estrogen
ug-*-—
V
-

■

.. .

Length of
Estrus
0
Hrs.
•

Interval (Hrs.)
betw.PMS and
wiio“
1* ofEstrus
Onset

Crystalline Estradiol
Intravfinnns
200
47
6
200
53
9
108
150
6
42
150
0
22
100
0
48
100
0
94
50
30
62
6
50
11.4

Interval (Hrs.)
betw. Estrogen
and
aria Onset
unseu of
oj. Heat
neau

-

Sod ium~-Salt of Estradiol
89
10
82
68
21
92
33
84

200
200
150
150
100
100

6
0
0
0
0
6

50

0
0

^0

-

_

48
72
58.4

16
40
26.4

48

16

=.

-

-

-

_

-

-

-

16
_

48
=

—

0

48

16

Subcutaneous

6
24
0
26
9
6
75
18
35
6
87
57
68
18.0
Sodium-Salt of Estradiol
0
200
20
12
200
98
18
150
76
0
24
150
0
100
79
0
100
12
24
32
50
24
50
103
19.5
25
64
49

—

22
27
27

Intravenous

6
Crvstalline Estradiol

54
59
59

200
200
150
150
100
100
50
50

-

72
54
54
72
48
84
35
59.8
Subcutaneous

♦Lambing data in Table 13*

=

48
48
-

40
22
22
40

16
52
3
27.8
-

16
16

=

32
32
40.0

2
2
9.0

133

APPENDIX TABLE 6
THE EFFECT OF THE INTERVAL BETWEEN PROGESTERONE AND PMS
UPON THE REPRODUCTIVE RESPONSE IN THE ANESTROUS EWE
Interval (Hrs.)
Ewe betw. PMS and
N o . Onset of Estrus

PMS
Response

Progesterone - Control
35
55

1

Ovarian
Weight
jght Left

0.8

0.8

Thyroid Adrenal
Weight Weight

6.7
=

26

—

5-0
=,
=

1.2
1.0
0
1.0
1.67
Proses terone-=PMS (20 dav Interval)
28
66
2
1.6
0.7
83
52
3
1.4
73
3
81
168
2.6?
1.2
112

0.8
1.2
0.9

4.3
6.2
5-7

4.1
7.8
5-6

2.2
1-5
1.4

—

4.9

4o4
6.6

21
49
50
52
Zt

39
38

1

=

3.1
5.4

=,

—

1.7

5-5

4.5

1.4

3-2

7-7

6.0

0.9

0,9

6.2

3-4

2.0

7.0

4.7

1.2
1.2
0.9

7.4
4.8
7.2
6.4

4.6
5.7
4.1
5-2
SO,

<=>

3.5
1.2
Progesterone- -PMS (16 day Interval)
1.2
4
0.6
3
0.7
1
Omitted
4
2.3
it
JUZ_
1.3
3-75
14 day Interval)

1.8
2.1
1-4

JL3-.
6.2

4.9