THE EFFECTS OF GONADOTROPINS
IN INDUCENG MULTIPLE OVULATIONS
IN THE BEEF COW

Thesis for the Degree of M. S.
MICHIGAN STATE UNIVERSITY
DAVID EDWIN WILDT
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ABSTRACT

THE EFFECTS OF GONADOTROPINS IN INDUCING
MULTIPLE OVULATIONS IN THE BEEF COW

BY
David Edwin Wildt

Ovulation induction in the beef cow was studied to
determine the effects of various FSH-P and HCG levels in
achieving a high incidence of double and triple ovulations.
Experimental design allowed for examination of the effects
of repetitive gonadotropin administration on behavioral and
ovarian activity.

The study was conducted in three phases. Ovarian acti—
vity was monitored both before and after ovulation by rectal
palpation.

Preliminary studies in Phase I included three gonadof
tropin regimes administered in consecutive cycles. Animal
numbers and treatments were as follows: I) 7, 25 mg FSH-P
on day 16, 2,000 IU HCG on day of estrus; II) 6, 25 mg
FSH-P on day 16, 1,000 IU HCG on day of estrus; III) 6,
12.5 mg FSH-P on day 16, 1,000 IU HCG on day of estrus.
Treatment III, utilizing low levels of FSH-P and HCG,
resulted in the highest incidence of double ovulations with-
out overstimulating the ovary. No refractory effect due to

the repetitive gonadotropin treatment was observed, but the

David Edwin Wildt

incidence of standing estrus was reduced.

Phase II involved the utilization of two progestins in
an attempt to induce overt estrus and to study the effects
of the repetitive gonadotropin treatment of Phase I on the
synchronized estrus. Our regime of 17ac-hydroxyprogesterone-
17 acetate administration was ineffective in synchronizing
estrus within 8 days after withdrawal. The progestin, 170c-
hydroxy-6-c-methylprogesterone acetate, proved to be an
adequate synchronizing agent but was only partially effective
in increasing the incidence of standing heat.

During the post synchronized estrual cycle developed
from the l7ec-hydroxy-6oc-methylprogesterone acetate, gonado-
tropins were administered to three animals each in the
following two treatments: I) 25 mg FSH-P on day 16, 1,000
IU HCG on day of estrus; II) 12.5 mg FSH-P on day 16, 1,000
IU HCG on day of estrus. The animals were mated, slaughtered
7 to 10 days later, and the reproductive tracts were recovered.
Treatment I resulted in excessiVe ovarian stimulation in one
of the treated animals. The low levels of gonadotropin uti-
lized in Treatment II resulted in multiple ovulations with
no overstimulation of the ovary. Mean corpus luteum size
was reduced in the animals frequently treated with gonado-
tropins when values were compared to those cited in liter-
ature.

Low level gonadotrOpin treatment was studied on a
larger scale in Phase III. Sixteen postpartum cows receiv-

ing 12.5 mg FSH-P on day 16 and 1,000 IU HCG on day of estrus

David Edwin Wildt

achieved a highly significant increase (p<:0.01) in ovula-
tion rate in comparison to 30 controls.’ Over 56 percent of
the gonadotropin treated cows achieved double ovulatiOns
while 69 percent ovulated two or three ova. Fertility was
not affected at a later estrus in gonadotropin treated

animals.

THE EFFECTS OF GONADOTROPINS IN
INDUCING MULTIPLE OVULATIONS

IN THE BEEF COW

BY

David Edwin Wildt

A THESIS
Submitted to
Michigan State University
in partial fulfullment of the requirements

for the degree of
MASTER OF SCIENCE
Department of Animal Husbandry

1973

ACKNOWLEDGMENTS

The author wishes to acknowledge the many people
involved in the research presented in this study. Deepest
appreciation is expressed to Dr. W. Richard Dukelow for
providing assistance and sound advice throughout the
course of this project.

Special thanks is due to Dr. Ronald Nelson, Chairman,
Department of Animal Husbandry, for the facilities and
financial aid that contributed to the study. Special
mention is due to Dr. Harlan Ritchie for providing the
research animals required in the project.

Special thanks is due to H. Dee Woody who assisted in
much of the data collection and Richard M. Harrison for
his willing advice and counsel.

Gratitude is expressed to all members of the Endocrine
Research Unit for their thoughtfulness and cooperation
during the term of this study.

Sincerest appreciation is due to my wife, Brenda,
not only for assistance in preparing this manuscript but

for her perpetual encouragement and understanding.

ii

TABLE OF CONTENTS

LIST OF TABLES . . . . . . . . . . . . . . . .
LIST OF FIGURES O O O O O O O O O O O O O O O 0
INTRODUCTION 0 O O O O O O O O O O O O O O O 0
LITERATURE REVIEW . . . . . . . . . . . . . . .
Incidence of Natural Twinning . . . . . .
Ovulation Induction and Superovulation . .
Early Gonadotropin Research . . . . . . .
The Ovulatory Induced Response . . . . .
Induced Ovulation of the Prepuberal Calf
Ovulation Induction in Postpuberal Females
Refractoriness to Gonadotropins . . . . .
Estrus Synchronization Prior to
GonadotrOpin Treatment . . . . . . . . .
GENERAL MATERIALS AND METHODS . . . . . . . . .
PHME I O O O O O O O O O O O O O O I O O O O 0

Materials and Methods . . . . . . . . . .
Results and Discussion . . . . . . . . . .

PHME II 0 O O O O O O O O O O O O O O O O O 0

Materials and Methods . . . . . . . . . .
Results and Discussion . . . . . . . . . .

PHASE III . . . . . . . . . . . . . . . . . . .

Materials and Methods . . . . . . . . . .
Results and Discussion . . . . . . . . . .

SUMMARY AND CONCLUSIONS . . . . . . . . . . . .

LITERATURE CITED . . . . . . . . . . . . . . .

iii

Page

vii

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TABLE OF CONTENTS

(Continued)
Page
APPENDIX . . . . . . . . . . . . . . . . . . . . .
A. LAPAROSCOPY IN THE COW . . . . . . . . . . 66
B. PUBLICATIONS BY THE AUTHOR . . . . . . . . 68
C. ABSTRACTS. . . . . . . . . . . . . . . . . 69
VITA 73

iv

LIST OF TABLES

Table Page

1. Summary of Gonadotropin Treatments in
Prepuberal Bovine Females . . . . . . . . . . 10

2. Summary of Gonadotropin Treatments in
Postpuberal Bovine Females . . . . . . . . . . 12

3. Summary of Synchronization and
Gonadotropin Treatments in Bovine Trials . . . 23

4. Ovulation Numbers Achieved from Three
Gonadotropin Treatments . . . . . . . . . . . 29

5. Ovulation Rates of Cows Administered
with Three Gonadotropin Treatments . . . . . . 31

6. Ovulatory Follicle Numbers on Day of
Estrus and Ovulation Rate in Relation
to Three Gonadotropin Treatments . . . . . . . 33

7. Percentage of Follicles Greater Than 12
mm on Day of Estrus that Subsequently
Ovulated . . . . . . . . . . . . . . . . . . . 34

8. Two Synchronization Treatments and the
Effectiveness of Each . . . . . . . . . . . . 42

9. Ovulation Achieved in Presynchronized
Gonadotropin Treated Heifers . . . . . . . . . 44

10. Ovarian Size and Weights of Gonadotropin
Treated Heifers O O O O O I O C O O O O O O O 45

11. Number and Size of Corpora Lutea on Left
and Right Ovaries of Gonadotropin Treated
Heifers O O O O O O O O O O O O O O O O O O O 47

Table

12.

13.

14.

LIST OF TABLES
(Continued)

Page

Number of Follicles of Various Sizes
on the Left and Right Ovaries . . . . . . . 49

Ovulation Data on Control and
Gonadotropin Treated Postpartum Cows . . . . 54

Fertility in Control and Earlier

Gonadotropin Treated Cows as Measured

by Failure to Return to Estrus after

Artificial Insemination . . . . . . . . . . 56

vi

LIST OF FIGURES

Figure Page

1. Prodox (17°C—hydroxyprogesterone-l7
acetate) . . . . . . . . . . . . . . . . . . . 38

2. Provera (17sC-hydroxy-6oC-methyl-
progesterone acetate) . . . . . . . . . . . . 38

3. Ovum recovered from right oviduct
(Maginified 200x) . . . . . . . . . . . . . . 51

vii

INTRODUCTION

The efficiency of commercial cattle production can be
vastly augmented by increasing the incidence of multiple
births in the beef cow. Numerous studies have demonstrated
that the administration of exogenous gonadotropic hormones
at various levels and stages in the estrual cycle of the
cow will result in enhanced follicular development and,
consequently, increased ovulation numbers. Although some
success has followed these endeavors, the application of
most ovulation induction regimes is not practical on a
commercial basis at the present time.

Many problems are inherent in attempting to regulate
reproductive functions and fetal numbers. Previous re-
search has demonstrated the importance of limiting calf
numbers to two or three per cow, emphasizing the necessity
of developing techniques for limiting the ovulation rate
to one which will result in a high incidence of twin or
triplet births. In addition, there is a need to examine,
characterize, and determine effective dosage levels and
stages of administration of available follicle stimulating
and luteinizing agents. Finally, since follicle stimulat-
ing source administration must be timed precisely during

the follicular phase of the cycle, extensive study and

development of accurate estrus synchronization techniques,
coupled with proper gonadotropin treatment, will aid in
accomplishing the goal of total regulated reproduction.
The objectives of these investigations were:

1) To develop a treatment for inducing a high incidence
of double or triple ovulations;

2) To study the effects of various FSH dosages in
combination with low levels of HCG;

3) To observe the refractoriness of cattle to repeated

doses of FSH and HCG.

LITERATURE REVIEW

Incidence of Natural Twinning

The frequency of natural twin births is low in all
types of cattle. The summarized records of nearly a
million parturitions in dairy cattle demonstrated that
only 1.88 percent of all births resulted in twins. The
corresponding rate for beef breeds was even less, being
only 0.44 percent (Lush, 1945).

Summaries of cattle twinning rates based on the
number of calvings revealed marked variations between
breeds (Meadows and Lush, 1957), herds within breeds
(Meadows and Lush, 1957), and families within herds
(Lush, 1925).

Although twinning ability is inherited to some extent,
selection is ineffective in increasing the incidence of
natural twinning due to the low heritability of the trait
(Lush, 1925; Lush, 1945; Pfau et al., 1948; Erb et al.,
1960). Mechling and Carter (1964) studied the incidence
of twinning in an Angus beef herd in Virginia which had
been selected entirely on the basis of twinning since the
19303. A systematic selection program utilized twins,
daughters of twins, dams of twins, and cows with histories

of twinning. Sires consisted only of twin Angus bulls or

Jai’

 

 

 

bulls with records of twin offspring. After almost 30 years
of intensive selection, the incidence of natural twinning

in the selected herd was only 1.71 percent in comparison to
two non-selected Virginia herds which had twinning frequen-

cies of 1.64 and 0.81 percent respectively.

Ovulation Induction and Superovulation

Ovulation induction of prepuberal and postpuberal
cattle, coupled with future advancements in ovum transfer,
has potential for practical application. The benefits
have been reviewed by Foote and Onuma (1970) and include:
(a) reduction of the generation interval; (b) early
progeny testing of females; (c) utilization of outstanding
females as ovum donors; (d) transfer of embryos over great
distances when the embryos have specific genetic distinctions;
(e) isolation of prenatal maternal effects; (f) examination
of the requirements for peak prolificacy and normal matur-
ation; (g) a greater understanding of the effects of stress
on conception and gestation; and, (h) the regulation of
progeny numbers per female. In addition, the time interval
between parturition and occurrence of estrus and ovulation
can be shortened with the administration of gonadotropins
(Oxenreider, 1968). The following review considers the
significant advancements and knowledge achieved in the field
of ovulation induction as it concerns the bovine specie. Due
to the similarity in technique, gonadotropin research will
be reviewed in both the prepuberal and postpuberal animal.

The discussion will also be concerned with estrus

5

synchronization coupled with exogenous gonadotropin
administration since the advantage of successful synchro-
nization increases the potential of total regulated

reproduction in the beef cow.

Early Gonadotropin Research

 

Gonadotropins were first investigated by Zondek and
Aschheim (1927) and Smith and Engle (1927). These groups,
utilizing laboratory animals, demonstrated the relation-
ship of the anterior pituitary to the development and
regulation of the genital system. Subsequent experimen-
tation established that extracts of the anterior pituitary
could be utilized to induce follicular stimulation and
ovulation in immature female rats and rabbits (Casida,
1934). It was Casida et al., (1943), who between 1934 and
1941, first induced ovulation in cows and calves by sub-
cutaneous injections of unfractionated ovine and bovine
pituitary extracts or by subcutaneous injections of follicle
stimulating extracts succeeded by an intravenous inoculation
of luteinizing extract. Although this study met with
limited success, it initiated experimentation with a variety
of exogenous hormonal regimes to induce ovulation in the

bovine.

The Ovulatory Induced Response
The ovulatory induced response is basically concerned
with the stimulation of the follicular tissue of the ovary

by.a source of follicle-stimulating hormone (FSH) that will

result in a higher total level of FSH in the system than
would normally be contributed by endogenous sources. In
the past, the most commonly utilized source of exogenous
FSH gonadotropin has been pregnant mare's serum (PMS) dis-
cussed by Cole and Hart (1930) and by Zondek (1930). More
recently FSH—P, a partially purified pituitary extract, has
received wide acceptance as an FSH source.

To further aid the ovulatory process, a form of
luteinizing hormone is administered either intravenously or
intramuscularly. The conventional preparation employed has
been human chorionic gonadotropin (HCG) first described by

Aschheim and Zondek (1927).

Induced Ovulation of the Prepuberal Calf

 

Superovulation of the prepuberal calf holds several
distinct advantages when coupled with the successful trans-
fer of ova including reduction of the generation interval
by permitting early progeny tests for females and the pro-
duction of the greatest possible number of progeny from
outstanding genetic superiors (Onuma et al., 1969; Foote
and Onuma, 1970). Such experimentation also allows the
researcher to utilize an animal with none of the cyclic
divergence or luteal characteristics of his mature counter—
parts (Foote and Onuma, 1970).

High ovulation rates, 40 to 80 percent, in prepuberal
heifers have been obtained from exogenous gonadotropin
treatments (Hafez, 1969); however, individual responses

have been quite variable (Avery et al., 1962; Howe et al.,

1962; Jainudeen et al., 1966; Onuma et al., 1970) and
fertilization depressed (Black, et al., 1953; Marden,
1953; Howe et al., 1962; Jainudeen et al., 1966; Onuma
et al., 1969; Lineweaver and Hafez, 1970). Nutritional
and management factors do not affect the widespread
variability observed in numbers of follicles obtained
(Onuma and Foote, 1969; Onuma et al., 1970). There is

a reduced fertilization rate due to a large extent to the
difficulties experienced when attempting to deposit semen
beyond the cervical entrance of treated calves (Onuma

et al., 1969).

Several attempts have been made to enhance the ovarian
response of superovulated calves by administering exogenous
steroids. Onuma et al., (1969) demonstrated that the
administration of l7B-estradiol benzoate had no effect on
follicle numbers or ovulation rate. Seidel et al., (1971)
reported that 17B-estradiol injected after PMS adminis-
tration reduced ovarian response to PMS but increased the
numbers of fertilized ova recovered in calves. Additional
evidence has indicated suppressed follicular growth and
decreased ovulation rate in calves receiving this specific
estrogen (Howe et al., 1962; Onuma et al., 1970). Howe and
Black (1963) found that the addition of PMS and HCG with
or without progesterone or a combination of progesterone and
estrogen had no effect on sperm transport in the treated calf.

Calves rectally inseminated achieved significantly

higher fertilization rates than younger calves which were

inseminated by other methods (Avery and Graham, 1962).
Cleaved ova and recovery rate were lowered significantly
in the presence of corpora lutea. Higher fertility was
achieved when liquid semen was utilized in comparison to
frozen semen and three inseminations were completed in
contrast to two inseminations (Onuma et al., 1970).

Hafez (1969) has speculated that an essential hormonal
balance or component is missing in the reproductive system
of these prepuberal animals treated with PMS and HCG.

The recovery rate of fertilized ova from calves induced
to ovulate is low (Jainudeen et al., 1966; Lineweaver and
Hafez, 1970; Seidel et al., 1971). This, in part, appears
to be due to the inability of the oviductal fimbria to pick
up all of the ova ovulated, possibly because of the large
ovaries produced in relation to the small, normal sized
fimbriae (Onuma et al., 1969; Seidel et al., 1971).
Apparently, however, the infantile condition of the ovi-
duct does not interfere with the passage of ova through the
reproductive tract (Marden, 1953).

Seidel and Foote (1969) and Seidel et al., (1971) have
studied the age-ovarian response relationship after gonado-
tropin treatment. Calves superovulated at birth, 1, and 2
months of age had an average of 0, 9.4, and 28.2 ovulations
and 0, 77.7, and 100 percent ovarian response, respectively.

Neville and Williams (1973) have recently reported
attempts to induce estrus, ovulation, and conception in

heifers averaging 327 days of age. Heifers received progestin

 

I‘l'l’.‘ III III

treatment with dihydroxyprogesterone acetophenide (DHPA) or
medroxyprogesterone acetate (MAP). On the second day of
progestin feeding, each animal received estradiol valerate
and near the end of progestin feeding either PMS or FSH-P.
Induction of estrus ranged from 50 to 100 percent and con-
ception from 0 to 50 percent. PMS was found to result in
higher ovulation rates and more mature follicles than FSH-P
in these prepuberal animals.

Various hormonal regimes to induce multiple ovulations
in the calf have been attempted (Casida et al., 1943; Black
et al., 1953; Marden, 1953; Jainudeen et al., 1966; Onuma
et al., 1969; Seidel and Foote, 1969; Lineweaver and Hafez,
1970; Onuma et al., 1970; Seidel et al., 1971; Neville and
Williams, 1973). A summation of the most successful of

these is shown in Table l.

Ovulation Induction in Postpuberal Females

 

To achieve multiple ovulations, most research has been
directed toward detection of the appropriate levels and
sources of gonadotropins and the prOper stage of the cycle
for treatment. Numerous hormonal preparations and injection
schedules have been utilized (Casida et al., 1943; Hammond
and Bhattachanya, 1944; Dowling, 1949; Umbaugh, 1949; Avery
et al., 1962; Avery and Graham, 1962; Gordon et al., 1962;
Arbeiter, 1963; Hafez et al., 1963; Hafez et al., 1963;
Schilling and Holm, 1963; Denny, 1964; Hafez et al., 1964;
Peli and Castelli, 1964; Scanlon et al., 1968; Lamond and

Hill, 1969; Schwartz and Shelby, 1969; Roussel and Beatty,

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11

1970; Turman et al., 1971; Foote and Radmall, 1972; Johnson
et al., 1972; Scanlon, 1972). The more successful treatments
are shown in Table 2.

In the past, PMS has been the most common follicle
stimulating preparation utilized. Because of its large
molecular size and slow metabolism (Folley and Malpress,
1944), PMS exerts its effect over a longer period of time
than purified FSH extracts (Rowson, 1951; Brock and Rowson,
1952). Hammond (1955) postulated that a single PMS injec-
tion is potent for four to five days compared to anterior
pituitary FSH which is quickly metabolized and consequently
requires priming dosages every 12 hours.

Disadvantages are evident with PMS use. Gordon et al.,
(1962) observed a high incidence of "silent heats" (absence
of overt signs of estrus) when PMS was administered at low
doses or late in the cycle. No effect on cycle length or
mating behavior was noted. The tendency for PMS to result
in refractoriness in treated cows will be discussed in
detail later.

Although PMS and HCG have been the most extensively
used gonadotropins, other preparations have been used such
as unfractionated sheep pituitary extracts (Casida et al.,
1943; Umbaugh, 1949; Black et al., 1953), beef pituitary
extracts (Casida et al., 1943), horse anterior pituitary
extracts (Dowling, 1949), and partially purified luteinizing
hormone (Avery et al., 1962; Jainudeen et al., 1966; Seidel

and Foote, 1969; Seidel et al., 1971). Recently FSH-P, a

12

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pituitary extract with much of the LH activity removed, has
been used extensively as a follicle stimulating source
(Bellows et al., 1969; Onuma et al., 1969; Laster, 1972;
Rich and Johnson, 1972). Some preliminary research has been
completed using gonadotropin releasing hormone (GnRH) (Mauer
and Rippel, 1972) as a stimulant of follicular growth.
Results utilizing this particular preparation are incon-
clusive and require additional study.

The presence of a functional corpus luteum has a
modifying effect on the ovarian response to gonadotropins
and subsequent fertilization in the bovine (Hammond and
Bhattachanya, 1944; Dowling, 1949; Avery et al., 1962;
Hafez et al., 1963; Lamond, 1964). When the corpus luteum
is present, ovulation is significantly decreased or com-
pletely obstructed (Dowling, 1949; Hafez et al., 1963;
Laster, 1972). Furthermore, when ovulation does occur the
ova do not become fertilized (Rowson, 1951), probably as a
result of the inhibition of capacitation (Dukelow, 1971).
Enucleation of the corpus luteum has been found to initiate
estrus in cows prior to treatment for superovulation (Avery
et al., 1962). In addition, this expression results in a
higher fertility rate (Lamond, 1964). The reduction in the
interval between enucleation treatment and subsequent
estrus was superior by almost two days over daily subcu-
taneous injections of progesterone (Avery et al., 1962).

Contrary to some of the benefits postulated above from

the expression of the corpus luteum, Hafez et al., (1965)

 

14

found no advantage to enucleating this structure and spec-
ulated that this process may have a detrimental effect on
conception.

Ovarian response is also influenced by previous or
subsequent treatment with estrogen during the superovula—
tory process. The administration of 17B-estradiol intra-
muscularly prior to PMS injection tended to reduce the
number of developed follicles while treatment after PMS
injection significantly increased the number of developed
follicles and fertilized ova recovered. The estrogen had
no effect on ovulation rate (Hafez et al., 1963).

Gonadotropin treatment is most effective when adminis-
tered during the follicular phase, but no benefit is ob-
tained by initiating single gonadotropin treatments prior
to day 16 of the cycle (Casida et al., 1943; Dowling, 1949;
Hafez et al., 1963). Hafez (1969) indicated that treat-
ment during the luteal phase led to a high incidence of
cystic follicles.

Follicular growth rate from a diameter of l to 2 mm is
continual and independent of cyclic hormonal stimulation
during the luteal state of the estrual period (Choudary et
al., 1968; Marion et al., 1968). However, several studies
have been completed in which gonadotropin injections have
been given both in the luteal and follicular phase of the
cycle. Schilling and Holm (1963) hypothesized that ripe
follicles begin development during or just after the last

heat period with only one continuing to maturation while the

 

15

others become atretic. iThey attempted to prevent this normal
occurrence of atresia by injecting 1,000 to 1,500 IU PMS on
day 5, e.g., during the luteal period. Enucleation of the
corpora lutea and an additional injection of PMS between
days 16 and 18 were followed by HCG administration on the
day of estrus. This procedure resulted in 73 percent of

the cows producing the desired number of two or three ovu-
lations. Laster et al., (1971) confirmed this study when
using a comparable treatment following post-estrus synchro-
nization. Of the 57 animals treated, 40 percent had 2
corpora lutea and 33.3 percent single corpora lutea. Con-
ception rate was only 36 percent. Schwartz and Shelby
(1969), using similar regimes, obtained excessive stimula-
tion (average 6.56 ovulations) and consequently low fertili-
zation. When 2.5 mg of ovine FSH was administered on day

5 and 3 mg ovine FSH on day 16, a 1.39 ovulation rate and

44 percent conception rate were obtained.

Concerning the optimum number of injections of LH for
highest induced ovulatory efficiency, Avery et al., (1962)
found that a single injection of luteinizing hormone was
superior to two consecutive daily doses of the same prepar-
ation.

It seems that a definite linear dose-response relation-
ship exists for PMS (Gordon et al., 1962; Denny, 1964),
although Hammond and Bhattachanya (1944) found no obvious
correlation when doses from 800 to 1,500 IU PMS were uti-

lized after enucleation of the corpora lutea. Bellows

 

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16

et al., (1969) indicated a dose-response for porcine FSH and
also noted that dosages in excess of 12.5 mg FSH-P resulted
in overstimulation of the ovary. Lineweaver and Hafez (1970)
found a relationship between the amount of HCG administered
and the ovarian response. As HCG levels increased from
2,000 to 10,000 IU, the number of large follicles developing
decreased but the ovulation rate of these follicles increased
significantly. Ovarian size increased as both the dose of
HCG and time interval between PMS and HCG injection increased.
For the practical application of superovulation to
cattle, a sequence of gonadotropin injections or mechanical
measures is required that will result in a consistent per-
centage of two or three ovulations (Schilling and Holm,
1963; Laster et al., 1971). The results for achieving this
goal have been variable. Evidence indicates that not only
the level of treatment but other factors are involved in
attaining only double ovulations. Laster et al., (1971)
indicated that HCG injected on day 3 after PMS treatment
(compared to the day of estrus) induced more double ovu-
lations. Gordon et al., (1962) demonstrated that the sur-
vival rate of twin ova was significantly higher when one
ovum was ovulated by each ovary in comparison to a double
ovulation from a single ovary. Cows which ovulated two ova
experienced a high rate of embryonic mortality during the
first six weeks of gestation, and cows which carried three
fetuses at six weeks usually aborted all of them prior to

parturition.

17

No correlation between body weight and ovarian response
to gonadotropin treatment exists (Gordon et al., 1962;
Bellows et al., 1969). Gordon et al., (1962) found that the
season of the year had no effect on inducing multiple
ovulations. However, others have reported that variable
seasonal factors such as temperature and humidity are
definite determining factors in inducing ovulation (Stott
and Williams, 1962; Hafez, 1969). The superovulatory
response is also affected by the time interval from the
injection of the gonadotropin to the subsequent estrus
(Hammond, 1949; Scanlon et al., 1968). Hammond (1949)
achieved the best results when estrus occurred three, four,
or five days after the gonadotropin injection. Scanlon et
al., (1968) reported a substantially higher ovulation rate
as the interval between PMS and estrus increased from two to
five days. Although limited evidence is available, it has
also been reported that the induced ovulatory response is
also affected by breed, genetic constitution, previous
levels of hormonal injections, the postpartum interval, and

the plane of nutrition (Hafez, 1969).

Refractoriness to Gonadotropins

 

Refractoriness to repeated injections of various gonado-
tropins in the bovine has occurred (Willet et al., 1952a;
Willet et al., 1953; Hafez et al., 1964; Hafez et al., 1965;
Jainudeen et al., 1966; Laster et al., 1971).

This condition is not common to all mammalian species.

Fertility and litter size were typical when rabbits, after

18

weaning their previous litter, were again treated with
purified luteinizing hormone and inseminated (Hafez et al.,
1964).

Willet et al., (1952a) experienced a reduced number
of corpora lutea with successive treatments of sheep and
swine pituitary FSH or equine gonadotropin plus HCG.

Hafez et al., (1964) reported reduced superovulatory
response to 2,000 IU PMS injected during the second estrual
cycle following first cycle treatment with 1,500 IU.
Reports using similar treatments found complete refractor-
iness when the injections were repeated at 18 to 40 day
intervals (Jainudeen et al., 1966), while no refractori-
ness was found 5 to 7 months after original treatment of
PMS (Jainudeen et al., 1966). Gordon et-al., (1962)
reported that PMS treatment did not affect the super-
ovulatory response if only one treatment was utilized per
year. Willet et al., (1953), observing refractory cows,
concluded that long periods of rest without gonadotropin
treatment failed to reduce or eliminate the condition.

Concerning refractoriness due to LH preparations,
Slyter et al., (1965) reported that follicle numbers were
not significantly affected by daily 500 IU injections of
HCG for 3 to 17 days. However, corpora lutea weight did
decrease significantly during the longer treatments.

Refractoriness appears to be caused by the development
of antibodies to the gonadotropins (Willet et al., 1953;

Jainudeen et al., 1966). It is reported that the levels of

19

antigonadotropic activity obtained from PMS increases with
successive treatments after the second injection and
achieves maximal values 16 days after the fourth injection
(Jainudeen et al., 1966). Other postulated factors attrib-
uted to the cause of refractoriness include the exhaustion
of primary follicles in the ovary and varying degrees of
damage to the ovaries as a result of treatment or surgery.

Excessive stimulation of the ovary can also have
other effects. Hafez et al., (1963) noted that levels of
5,000 IU PMS caused a substantial number of hemorrhagic
follicles. Hafez (1969) indicated that overstimulation of
the ovary caused a reduced rate of ovum recovery by:

(a) the endogenous estrogen effect on ovum transport rate;
(b) the trapping by the follicles of some ova; and,

(c) undersize fimbriae to surround the ovarian surface.
Estrus Synchronization Prior

to Gonadotropin Treatment

A combination of estrous cycle control and gonadotropin
treatment is an important prerequisite for making the induc-
tion of multiple births more suitable for practical appli-
cation.

One of the earliest studies in this field, completed by
Nellor and Cole (1956), involved administration of various
levels of crystalline progesterone followed by an injection
of equine gonadotropin. This particular regime resulted in
a 17 percent conception rate. In a later study, it was

noted that cows which underwent progesterone synchronization

20

produced an average of 7.9 ovulations more than were pro-
duced by individuals superovulated but not synchronized
(Avery et al., 1962). Osland and Ellington (1971) were
successful by injecting two separate progesterone treat-
ments of 750 mg each and 1,500 IU PMS on day 29. Estrus
was demonstrated within 9 days in 86 to 91 percent of the
test animals. The conception rate was 65 percent and
three sets of twins and one set of triplets resulted from
the treatment.

Several trials have been completed in which various
levels and injection sequences of norethandrolone and
FSH-P were utilized to induce multiple ovulations (Vincent
and Mills, 1972). Of the FSH-P treated cows, 49 percent
were estimated to have multiple ovulations (with an average
of 1.9 corpora lutea per cow). Of the cows calving from
first breeding, 24 percent achieved multiple births for a
126 percent calving rate. Vincent and Mills (1969), using
norethandrolone to synchronize cycles and similar regimes
of FSH-P, reported 55 percent of the cows attaining multiple
ovulations with only 7 percent having more than 3 corpora
lutea.

Lamond and Hill (1969) presented data that suggested
that melengestrol acetate (MGA) and PMS could be used
together to produce concurrent synchronization of the
estrual cycle followed by subsequent multiple ovulation and
satisfactory fertility. MGA, fed at the rate of 0.5 mg per

animal daily for 18 days with PMS administered on day 14,

21

resulted in a corpora lutea average of 6.7 and an average
fertilization rate of 95 percent for the recovered ova.

Bellows et al., (1969) fed medroxyprogesterone acetate
(MAP) for 11 days with estradiol valerate administered on
day 2 followed by various levels of FSH-P injected twice
daily on days 8 through 12. This treatment resulted in the
most predictable ovarian response. A total of 6.25 mg
FSH-P resulted in the most desirable number of ovulations
(2.1) and a fertilization rate of 93.8 percent.

Graves and Dziuk (1968) have studied the induction of
ovulation in cattle with HCG after MAP treatment. An inter-
val of 60 hours between withdrawal of MAP to the HCG injec-
tion followed 24 hours later by insemination gave optimum
results: a 79 percent ovulation and 55 percent conception
rate were achieved.

In comparing the effectiveness of MAP versus MGA,
Reynolds et al., (1969) superovulated cows with FSH-P on
days 8 through 12. In addition, 5 mg estradiol valerate
was injected on day 2. The percentage of cows showing heat
within 5 days after withdrawal and the conception rate at
first and second cumulative services were: MAP 81.8, 55.6,
and 100; MGA 78.6, 45.4, and 65.4. Subsequent to first
service, the MAP treated cows carrying twins was 33 percent
to 20 percent for the MGA cows.

Laster et al., (1971) successfully synchronized estrus
with chloroacetoxy progesterone (CAP) and then used PMS and

HCG in the subsequent cycle. Administration of 1,500 IU PMS

22

on day 5 and 2,000 IU PMS on day 17, after CAP-induced
estrus, and with 2,500 or 4,000 IU HCG on day 3 following
the second PMS injection gave the most repeatable ovu-
lation rates. This treatment in 57 cows and heifers
resulted in almost 53 percent of the animals achieving 2
or 3 ovulations. However, conception rate was only 36 per-
cent to post treatment insemination.

A summary of successful synchronization and gonado-

tropin treatment is listed in Table 3.

3
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GENERAL MATERIALS AND METHODS

This study was conducted in three consecutive stages
from February to November 1972 at the Endocrine Research
Unit facilities at Michigan State University. Although
some methods and procedures were unique in each stage of
the study, a number of factors were common to all phases
and will be discussed in this section.

The experimental animals included a total of 6 heifers
and 39-cows. The heifers and a single cow were maintained
on a standard roughage diet consisting of approximately 35
pounds mixed hay daily from February through May. From
June through August, this group was maintained on grass
pasture. All cows in the Phase III trial were sustained
on grass pasture and supplemented with sorghum silage daily.

The follicle stimulating source, FSH-P, was purchased
from Armour-Baldwin Laboratories, Omaha, Nebraska. The
luteinizing agent, HCG (A.P.L.), was donated by Ayerst
Laboratories, New York, New York. Progestins were fur-
nished by Upjohn Company, Kalamazoo, Michigan. The gonado-
tropins were.lyophilized, standardized preparations. The
biological activity of the HCG was 782 IU per milligram.

The FSH-P and HCG were reconstituted with 10 m1 of

physiological, isotonic 0.15 M sodium chloride prior to

24

25

injection. All gonadotropic injections were intramuscular.

Estrual behavior in all trials was determined by visual
observations made twice daily, at approximately 8 a.m. and
5 p.m. During more critical periods (when the animals were
scheduled to return to estrus or after progesterone with-
drawal) visual observations were made at least three times
daily.

Ovulation rate was detected in all animals by rectal
palpation. Palpation schedules during the estrual cycle
were unique to each stage of the study and will be dis-
cussed later. Ovarian activity was monitored closely both
before and after ovulation. Rectal palpation, as described
by Zemjanis (1962), detected both follicular and corpora
lutea development and activity. Development of the pal-
pation technique was undertaken in February 1972 and was
facilitated by intense utilization of 15 heifers and 1 cow
that had failed to conceive during a previous 90 day
breeding season. Of these animals, seven were reproductively
normal while nine contained abnormal characteristics such
as small or cystic follicles, adhesions, or freemartinism.
Extensive palpation of these animals provided a broad per-
spective of ovarian morphology and activity in both the
normal and abnormal state.

Disposable shoulder length palpation gloves (Haver-
Lockhart Laboratories, Shawnee, Kansas) and Roccal-D
(Winthrop Laboratories, New York, New York), a disinfectant

detergent compound, were used at each palpation.

26

The Student's t test was used for statistical analysis
of ovulation data. The Chi Square procedure was employed

for analysis of fertility results.

PHASE I

OVULATION INDUCTION WITH VARIOUS GONADOTROPIN LEVELS
IN A REPETITIVE EXPERIMENTAL DESIGN

Materials and Methods

 

This trial was conducted from February 1972 through
May 1972 and utilized six nulliparous heifers of Angus,
Hereford-Angus, and Holstein-Angus descent and one pluri-
parous Angus cow. All animals had failed to conceive in
a previous breeding season but were cycling normally and
were anatomically and reproductively sound. None of the
animals had received gonadotropin or other hormone treat-
ment previously. All animals were observed cycling at
least twice prior to the start of hormone administration.
Ovarian activity was monitored by rectal palpation on day
16 of the estrual cycle, day of estrus, and three consecu-
tive days post-estrus.

The gonadotrOpin injections were timed from a non-
synchronized estrus. FSH-P and HCG were administered in
a repetitive experimental design with both gonadotropins
administered to the same experimental group for three
consecutive estrual cycles.

The first treatment in Phase I was: (a) 25 mg FSH-P
on day 16 of the estrous cycle; and, (b) 2,000 IU of HCG
on day of scheduled or exhibited estrus.

Treatment 2, administered in the next cycle to the
same experimental group, consisted of: (a) 25 mg FSH-P on

day 16; and, (b) 1,000 IU HCG on the day of estrus.

27

28

Treatment 3, administered in the third cycle, was:
(a) 12.5 mg FSH-P on day 16; and, (b) 1,000 IU HCG on day

of estrus.

Results and Discussion

 

Phase I was designed to determine the effects of
various gonadotropin levels on ovulation numbers. In
addition, the effect of repetitive gonadotropin treatment
on ovarian activity was studied.

Ovulation rates for each animal in each trial are
presented in Table 4. All animals responded to gonado-
tropins administered in Trial 1. Heifer number 67 was
excessively stimulated, producing five ovulations. This
animal demonstrated an 11 and a 7 day cycle subsequent to
the gonadotropin treated cycle. In addition to decreased
cycle lengths, intense standing estrus and lengthened estrus
periods for as long as six days were observed. Ovarian
activity was quite variable, ranging from small cystic
protrusions on the ovarian perimeter to an absence of any
palpable structures on the ovarian surface. The raised
tailhead, lowered loin characteristic was also observed.
This condition persisted for approximately six weeks after
the initial gonadotropin treatment after which time the
animal's ovarian function became normal. The absence of
overt signs of estrus became evident at this time. Due to
difficulty experienced with this animal, heifer 67 was
excluded from the subsequent two trials in Phase I.

Gonadotropin treatment in Trial 2, administered to the

29

 

 

 

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30

same experimental group, resulted in four of six animals
ovulating with one of six producing double ovulations. In
comparing Trials 1 and 2, it was apparent that either:

(1) antigonadotropic activity reduced ovulation; or,

(2) the reduced level of HCG was preventing ovulation of
follicles that could be ovulated with higher levels of HCG.

This may be further examined in the results of Trial
3 in the subsequent cycle. Only one of six animals failed
to ovulate while three of six produced the desired double
ovulations, two of these heifers being those that had failed
to ovulate in Trial 2.

Although average ovulation rate was reduced after treat-
ment in Trial 1, it was apparent that low levels of gonado-
tropin, and not antigonadotropic activity, was responsible
for reduced ovulation.

Because another objective of this study was to develop
a treatment regime that would result in a high incidence of
double or triple ovulations, the numbers of ovulations were
tabulated and are presented in Table 5. Ovulation rate in
each trial is expressed as the number of treated cows with
each number of corpora lutea. Based on data from the total
of 19 treated heifers, 9 (47%) had 2 or 3 corpora lutea.

In comparing individual treatments, Trial 1 resulted
in the highest incidence of double or triple ovulations. It,
was in this group that severe behavioral and reproductive
abnormalities were observed in one of the treated heifers.

Trial 2, which also utilized a high dosage level of FSH

31

 

 

 

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32

but reduced LH, gave variable results. The incidence of
double or triple ovulations was only one in six while two
of six animals were found with no corpora lutea on their
ovaries following treatment.

Treatment in Trial 3 resulted in 50 percent of the
heifers having double ovulations with no excessive ovarian
stimulation.

Table 6 depicts the number of follicles greater than
12 mm in diameter on the day of estrus and subsequent
number of ovulations in each of the treatment groups. With
the high level of 25 mg FSH and 2,000 IU HCG, a large pro-
portion of the follicles developed on day of estrus
ovulated. This resulted in an abnormally high incidence of
ovulations in one of the treated heifers. It seemed
apparent in the subsequent two treatments that even though
considerable numbers of follicles had developed from the
25 and 12.5 mg FSH treatment, the proportion of these
follicles which ovulated was limited by the reduced dosage
level of HCG utilized. This is further illustrated in
Table 7. The 2,000 IU dose of HCG resulted in 86 percent
ovulation of the follicles developed on the day of estrus.
This is higher than the ovulatory rate (73%) observed in
Trials 2 and 3 utilizing 1,000 IU HCG.

An apparent variable number of follicles on the day
of estrus was observed in Trial 2 which consisted of 25 mg
FSH in comparison with the even distribution of ovulatory

follicles detected in Trial 3 which utilized only 12.5 mg

 

33

 

 

 

 

 

 

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34

 

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35

FSH. It could be postulated that this irregularity was
caused by some minor antigenic activity alteration in
follicle numbers due to the high levels of FSH used in
Trial 1. Even so, the elevated dosage of FSH in Trial 2
had no effect on the distribution of ovulatory follicles
observed in Trial 3.

Although ovulation numbers were not affected by any
refractory response, standing estrus became less evident.
The observance of standing heat was less apparent in the
cycle following Trial 1 and completely absent after Trial
2 forcing estrus detection by the presence of vaginal dis-
charge and rectal palpation. It may be speculated that
repetitive gonadotropin administration can upset the deli-
cate hormonal balance in the cycling heifer and reduce the
incidence of behavioral estrus. Exogenous gonadotropic
sources, specifically LH sources such as HCG, may have an
inhibitory effect on estrogen release from the mature
follicles thus reducing the intensity of standing estrus.

The results of Phase I indicate that multiple ovula-
tions can be achieved with restricted levels of gonado-
tropins.

The data agree with earlier results (Bellows et al.,
1969) which indicate that dosage levels in excess of 12.5
mg FSH-P result in overstimulation of the ovary. Refrac-
toriness observed when PMS was repeatedly used (Willet et
al., 1952a; Hafez et al., 1964; Jainudeen et al., 1966)

was not observed with repetitive FSH-P administration.

36

Finally, these data agree with Slyter et al., (1965) who
reported no refractory effects due to frequent injections

of HCG.

PHASE II

ESTRUS SYNCHRONIZATION, OVULATION INDUCTION,
AND SLAUGHTER DATA COLLECTION

Materials and Methods

 

These trials were conducted from June through August
1972. The experimental animals consisted of six heifers
and one cow utilized in Phase I. The animals were main-
tained on grass pasture throughout the synchronization pro-
cedures and supplemented with mixed hay when necessary.
During ovulation induction and breeding, heifers were con-
fined to the feedlot and maintained on mixed hay.

Two progestins were used as synchronizing agents.

The structure of each is shown in Figures 1 and 2.

Prodox (17uc-hydroxyprogesterone-l7 acetate) was
administered to individual heifers at the rate of 50 mg per
head daily for seven consecutive days. A stock solution of
the progestin was prepared by dissolving 2.8 gm Prodox in
140 m1 of corn oil. Each dose consisted of a 2.5 ml injec-
tion in the rump region.

All animals were observed twice daily during progestin
administration and 3 times daily for 14 days following
hormone withdrawal.

Four weeks after the final Prodox injection, Provera
(l7-c-hydroxy—6oC-methylprogesterone acetate) was adminis-
tered at the rate of 5 mg per head daily for 14 days. A
standard solution of Provera was prepared by dissolving 525

mg of the progestin in 262.5 ml corn oil. The injection

37

38

Synchronizing Agents

 

 

 

Prodox
Figure l. 17c‘bhydroxyprogesterone-l7 acetate

 

Provera
Figure 2. l7a<-hydroxyf6u=Dmethylprogesterone acetate

39

dosage was 2.5 m1 and was given in the same area as des-
cribed for Prodox. Estrus observations were similar to
those made during Prodox administration.

The post synchronized estrual cycle developed from the
Provera was utilized in the final gonadotropin treatment
which occurred 158 days after the initial gonadotropin
treatment in Phase I.

The final treatment consisted of either 25 or 12.5 mg
FSH-P (i.m.) on day 16 of the cycle. On day 18 of the
cycle and each day thereafter, heifers were checked for.
standing estrus by being exposed to a proven Charolais bull
and observed for one hour. On the day of scheduled or
exhibited estrus, all heifers received 1,000 IU HCG (i.m.)
and were allowed to mate naturally. On the day of HCG
administration, each heifer was palpated. Because the
animals were to be killed for tract recovery, palpation~
time was kept minimal. However, each ovary was palpated
to determine the presence of mature follicles.

A11 heifers were killed at a local packing company
7 to 10 days after scheduled ovulation. The reproductive
tracts were recovered, tagged, placed in damp towels, and
transported to the Endocrine Research Unit within one hour
of killing. The ovaries were removed and described ‘
relative to the shape, number and size of follicles, and
number and size of existing corpora lutea. The length and
width of each ovary was measured.

Oviducts were removed and flushed with 5 m1 of 0.15 M

40

NaCl (in a 5 ml syringe, 24 gauge needle) into an 85 mm
watchglass. The needle was inserted through the infundi-
bular portion of the oviduct. Immediate examination of the
flushings was made under a dissecting microscope.

Uterine horns were flushed with 25 ml of 0.15 M NaCl
into individual 95 x 15 mm Petri dishes. Recovery pro-
cedures utilized a 50 m1 syringe and 14 gauge needle
directed from the tubouterine junction toward the oviduct.
Flushings were immediately examined with a dissecting micro-
scope for the presence of ova.

Recovered ova were removed from the medium by micro-
pipette and transferred to a 75 x 25 mm glass slide. The
slide was mounted on a Wild Heerbrugg inverted microscope
with camera attachment. The presence of an ovum was docu-

mented with 8.5 x 10.5 cm black and white film.

Results and Discussion.

 

Phase II of the study was designed to study the effect
of various gonadotropin levels on ovulation rate following
earlier progesterone administration. In addition, develop-
ment of a successful ovum recovery technique for large
animals was desired.

Estrus synchronization procedures were carried out in
an effort to achieve three objectives: (1) to determine if
progestin administration followed by withdrawal could induce
a more observable estrus; (2) to identify any effects of
previously administered exogenous gonadotropins on the

effectiveness of the synchronized estrus; (3) to determine

41

if progestin sources facilitate gonadotropin treatment.

The results of the two progestin treatments are
summarized in Table 8. The effectiveness of Prodox was
poor, with only one of seven animals demonstrating any
evident signs of standing estrus within eight days after
termination of treatment. Copious vaginal discharge
similar to that observed during estrus and normal cyclic
ovarian activity was observed in one of the heifers during
Prodox administration. This particular progestin is
experimental and positive results have not been reported in
literature.

It is evident that the ineffectiveness of Prodox was
due to either inadequate daily levels of progestin or the
period of administration. Studies have shown that reducing
the levels of dihydroxyprogesterone acetophenide (DHPA) from
400 mg daily to 75 to 100 mg has rendered the progestin
inadequate in synchronizing estrus (Wiltbank et al., 1967;
Tripathi and Howel, 1969). Results with melengestrol
acetate (MGA) show reduced effectiveness when the compound
is administered for only 10 days in comparison with 14 day
administration (DeBois and Bierschwal, 1970; Britt and
Ulberg, 1970).

Provera resulted in four of seven heifers demonstrating
evidence of estrus within seven days while all animals demon-
strated indications of estrus within ten days of progestin
withdrawal. Standing estrus was observed in only three of

seven heifers. Estrus was confirmed in all animals by the

42

 

meme OH in»?
msHumm :H D\D

 

mmsw D HHEDHB when eH HOH
msHumm sH D\¢ H.E.Hv DHHMU DE m D AMH0>0HHV
mumumom msoumummDoum
-Hsfimzlu. Susaonesmuuoz
name m cHnuH3 mast D How D Axoooumv mumumom DH
msuumm EH D\H H.E.Hv DHHmo DE om IDEOHmummDOHmDXOHUDDIUDDH
mm0s0>HuomHHm usmEDmmHB mHmnEsz sHumoDOHm

HmquH

 

moflm ho mmmzm>HBUmmmm WEB
32¢ mBZmZBdmma ZOHBdNHZOMNUZNm 038

m mqm<fi

43

presence of a clear vaginal discharge on the day of estrus
and the observance of metestrus bleeding within three days
after estrus. Normal ovarian activity was noted at this
synchronized estrus.

It is suggested from these data, and in conjunction
with the treatments and results obtained in Phase I, that
repetitive gonadotropin administration of FSH-P and HCG,
while having little effect on ovulation, may reduce the
incidence of standing estrus. In addition, estrus syn-
chronization with progestins is only partially effective in
counteracting this effect and inducing a typical, prominent
estrus.

Gonadotropin treatment levels and ovulation rates in
the post synchronized estrual cycle are shown in Table 9.
One ovary of one of the heifers was lost in the killing
procedure and was excluded from the trial.

Observance of recent corpora lutea on the recovered
ovaries demonstrated that animals receiving 25 mg FSH—P
ovulated 2, 2, and 5 ova respectively while heifers
receiving 12.5 mg ovulated 0, 3, and 2 ova. Increased
levels of FSH, although producing a high incidence of double
ovulations, again resulted in overstimulation of the ovaries
of one of the experimental animals.

Ovarian size and weights are recorded in Table 10. No
observable ovarian activity was noted upon examination of
the ovaries of heifer number 91. These ovaries were reduced

in size and weight in comparison to the majority of those

 

44

H eoD DH coo.H Dumas De m.~H own

 

m Dom DH ooo.H mummm ms m.mH mm

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45

 

 

 

 

 

 

 

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DD.mH m.m x m.e om.D o.~ m.m CNN
-.~H m.m x m.m oo.oH m.~ m.m mm
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Dum>o DEDHm Hmm>o uHmH HmsHDH

 

r)

mmthmm DMHflMMB
ZHHOMBOQdZOU m0 mfimeNS QZ¢ HNHm Z¢Hm¢>0

0H mflmda

46

recovered from other treated animals.

There does not seem to be a relationship between
amounts of FSH-P administered and subsequent ovarian size
and weight. Right ovaries were heavier as reported by
Asdell (1955), but the measured dimensions of both the
right and left ovary suggest no differences in total size.

Ovarian weights for both ovaries are reduced when com-
pared to results from earlier studies. Asdell (1955)
reported individual ovaries weigh 15 to 20 gm while
McDonald (1969) reported a range of 10 to 20 gm. Although
control animals were not available, it is possible that the
frequency of gonadotropin administration in our study
reduced ovarian mass.

The number and dimension of corpora lutea on left and
right ovaries of gonadotropin treated heifers are reported
in Table 11. There was a definite increase in the percen-
tage of corpora lutea on the right ovary in comparison to
the left. This confirmed earlier reports citing 60 to 65
percent of ovulations from the right ovary (Asdell, 1955;
Salisbury and Vandemark, 1961; McDonald, 1969).

There was no significant difference in mean corpus
luteum size between ovaries. Corpora lutea ranged from 0.4
to 1.5 cm with a mean size of 0.9 cm based on 14 ovulations.
Earlier studies have reported a mean mature corpus luteum
size of 2.0 to 2.5 cm on day 10 of the estrual cycle (Asdell,
1955; McDonald, 1969). The reduced corpus luteum size may

have been due to immaturity of the corpus, the younger age

47

 

 

 

 

 

 

 

mm. ouHm Ho new: HmuoH

am.o mm.o muHm Ho cums

m.vo D.mm Ho Hmuou mo unmoumm
mm. m u o omm
oo.H H oo.H H mm
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HH mqmda

48

of the cattle, and/or the gonadotropin treatment.

Follicle numbers of various sizes on each ovary are
shown in Table 12. The number of follicles was variable
with no evidence of a correlation between administered FSH
levels and total number of follicles. This was equally true
for follicles one cm or more in diameter. Laster (1972)
demonstrated a significant increase in numbers of follicles
greater than one cm with increased FSH-P. This dose-
response was not significant with follicles 0.5 cm or
greater.

Reduced follicle numbers were observed in animal 91
which failed to ovulate following treatment. Even so,
heifer 032 with feW'follicles had several fresh corpora
lutea on the ovarian perimeter.

The follicle destined to ovulate in the cycling cow is
1.0 to 1.2 cm in diameter (Rajakoski, 1960; Marion et al.,
1968). A total of 15 ovulatory follicles within this range
were observed in 4 of the 6 heifers on days 7 to 10 of the
cycle. Follicular development during the subsequent cycle
would not be so advanced as to produce follicles of such
dimensions. It can thus be postulated that these follicles
were unovulated remnants of the gonadotropin treated cycle.
This supports earlier discussion which speculated that
reduced ovulation rate and increased incidence of double
and triple ovulations were due to the decreased amounts of
administered LH.

Ova recovery rate was less than 10 percent. One of 14

49

 

 

 

 

 

 

 

 

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DD mH a u u . Hmuoa

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Eu DH mNHm mHoHHHoH Hmchm

 

 

 

mMHm4>O BmUHm 62¢ Emma mmB ZO
mmNHm mDOHm¢> m0 mMHUanom m0 mmmZDz

NH Hands

50

ova was recovered from the right oviduct of animal 220
and is shown in Figure 3. The ovum resembled fertilized
ova at a similar age and stage of development recovered
from the horse (Oguri and Tsutsumi, 1972).

Recoveries of ova from gonadotropin treated cows range
from 25 to 75 percent (Dowling, 1949; Avery and Graham,
1962; Hafez et al., 1963; Rowson and Moor, 1966). Optimum
time for ovum recovery is two to six days after estrus or
LH administration (Foote and Onuma, 1970). Because mature
corpora lutea were to be observed in this study, slaughter
and ovum recovery occurred 7 to 10 days after LH adminis-
tration. Consequently, most of the ovulated ova had entered
the uterus and were difficult to recover.

This phase of the study supported earlier results demon-
strating that low levels of gonadotropins can produce
restricted ovulation. The effectiveness of such treatment
was not influenced by earlier repeated gonadotropin adminis-
tration. Results indicated an inverse relationship between
frequency of gonadotropin administration and corpora lutea
size.

Estrus synchronization was not totally effective in
increasing the incidence of overt estrus that is lost

following frequent gonadotropin use.

51

 

Figure 3. Ovum recovered from right oviduct
(Magnified 200x)

PHASE III

OVULATION INDUCTION WITH LOW DOSE GONADOTROPINS
FROM A NON-SYNCHRONIZED ESTRUS

Materials and Methods

 

Phase III, conducted from September through October
1972, used 38 pluriparous cows experiencing their first or
second estrus after parturition. None of the animals had
previously received gonadotropin or other hormone treat-
ment. All cows were checked for estrus two or three times
daily. Estrus was confirmed in all animals by standing
heat, the mounting of other animals, and/or the presence
of clear vaginal discharge.

Two groups of control cows were utilized. Control:
Group I consisted of a random group of 15 cows demonstrating
their first postpartum estrus. Ovarian data were collected
by rectal palpation on day of observed heat and on the
seventh day after estrus.

Control Group II consisted of 15 cows selected
randomly that had experienced at least one estrus prior to
being admitted to the experiment. Three cows used in
Control Group I were also entered in Control Group II.
These animals were palpated three to five days prior to
scheduled estrus, on the day of the second observed estrus,
and on the seventh day following estrus.

Sixteen of the postpartum cows were assigned to a

treated group and received 12.5 mg FSH-P on day 16 and an

52

53

ovulatory dose of 1,000 IU HCG on the day of estrus. Five
cows in this group were previously included in Control Group
I. All treated cows were rectally palpated on day 16 of the
cycle, on the day of estrus, and on days 3 and 7 following
estrus.

Thirty of the cows used in Phase III were utilized in
a subsequent study by another investigator. Estrus was r
synchronized in one to three cycles following the use of the
cows in the treated or control groups of Phase III. Animals

were artificially.inseminated and then exposed under pasture

 

conditions to 5 bulls for approximately 90 days. '

Results and Discussion

 

Ovulation results of control and gonadotropin treated
postpartum cows are presented in Table 13. Nine of 15
(60%) of the cows in Control Group I ovulated at the first
postpartum estrus. Mean ovulation rate for all cows was
0.6 while the average for those cows ovulating was 1.0.

In Control Group II, 10 of 15 (66.6%) ovulated with 9
of 15 producing one ovulation. One cow produced a double
ovulation. The mean ovulation rate was 0.73 and the mean
of those cows ovulating 1.10. Ovulation rate between the
first and second estrus control groups was not significant.

Of the 16 gonadotropin treated cows, 9 (56.3%) had
double ovulations. Eleven of 16 (68.7%) had 2 or 3 corpora
lutea on their ovaries on day 7. One of the 16 treated
animals failed to ovulate and no excessive ovarian stimu-

lation was observed. Mean ovulation rate for all cows was

54

 

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55

1.75 and the mean of cows ovulating was 1.87.

Fertility was not measured at the gonadotropin treated
estrus. Fertility was measured in 30 of the cows one to
three cycles following palpation of control or gonadotropin
treated animals. First service conception rates for
treated and control groups are presented in Table 14. Of
the 16 control cows in which fertility was measured, 5
(31.2%) failed to conceive at first service. Fertility was
measured in 14 of the gonadotropin treated cows. Three
of 14 (21.6%) failed to conceive at first breeding.
Statistical analysis demonstrated no significant difference
between fertility in the treated group and fertility achieved
in the combined control groups.

The results of Table 14 also indicate that the time
interval from gonadotropin administration to breeding was;
not related to the ability of the cow to conceive at first
service.

Phase III demonstrated the effectiveness of low level
gonadotropin in inducing a high incidence of double and
triple ovulations on a large scale in postpartum cows.
Earlier gonadotropin treatments were found to have no effect
on conception rate, even as early as the cycle following

gonadotropin administration.

56

 

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+ mm me I mD
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mBOU Dmadmma ZHAQmBOdeow mmHH24m Q2¢ HQmEZOU 2H NBHHHBmmh

DH mamfla

SUMMARY AND CONCLUSIONS

A practical, reliable gonadotropin regime was developed

to induce a high incidence of double and triple ovulations

in the beef cow.. In addition, the study was designed to

determine the effects of repetitive gonadotropin adminis-

tration on ovulation. Estrus synchronization was utilized

in portions of the study to determine the effects of

progestins in combination with gonadotropins. The following

conclusions resulted from the data obtained:

1.

low level administered gonadotropins, specifically
12.5 mg FSH-P on day 16 and 1,000 IU HCG on day of
estrus, resulted in 68.7 percent of the treated
cows achieving double or triple ovulations;
fertility of cows, mated as early as the first
cycle following low dose gonadotropin treatment,
was not affected;

FSH-P levels greater than 12.5 mg resulted in
overstimulation of the ovary;

although the number of ovulations was not affected
by repeated gonadotropin administration, the
incidence of standing heat was reduced;

previous repetitive gonadotropin treatment was not

a factor in determining the effectiveness of

57

58

exogenous progestins;

synchronizing agents were only partially
effective in inducing overt signs of estrus
lost following frequent gonadotropin admin-
istration;

mean corpus luteum size in animals frequently
receiving gonadotropins was reduced when
compared to values cited in literature;

the right ovary was consistently heavier

and produced more ovulations than the left

ovary.

LITE RATURE CI TED

LITERATURE’CITED

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Aschheim, S., and B. Zondek. 1927. In Wilfrid R. Butt.
1967. The Chemistry of the Gonadotropins. Charles
C. Thomas Co., Springfield.

 

 

Asdell, S. A. 1955. Cattle Fertility and Sterility.
Little, Brown and Co., Boston and Toronto.

 

Avery, T. L., M. L. Fahning, and E. F. Graham. 1962.
Investigation associated with the transplantation
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Fertil. 3:212.

Avery, T. L., and E. F. Graham. 1962. Investigation
associated with the transplantation of bovine
ova. III. Recovery and fertilization. J. Reprod.
. Fertil. 3:218.

Avery, T. L., C. L. Cole, and E. F. Graham. 1962.
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J. Reprod. Fertil. 3:206.

 

Bellows, R. A., D. C. Anderson, and R. E. Short. 1969.
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Black, W. G., L. C. Ulberg, R. E. Christian, and L. E.
Casida. 1953. Ovulation and fertilization in the
hormone stimulated calf. J. Dairy Sci. 36:274.

 

Britt, J. H., and L. C. Ulberg. 1970. Melengestrol
Acetate for controlling days open in dairy cows.
J. Anim. Sci. 30:315 (abstr.).

 

Brock, H., and L. E. Rowson. 1952. The production of
viable bovine ova. J. Ag . Sci. 42:179.

Casida, L. E. 1934. Production of ovulation by gonado-
tropin extracts. Endocrinol. 18:714.

 

59

60

Casida, L. E., R. K. Meyer, W. H. McShan, and W. Wisnicky.
1943. Effect of pituitary gonadotropins in the
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cattle. Amer. J. Vet. Res. 4:76.

Choudary, J. B., H. T. Gier, and G. B. Marion. 1968.
Cyclic changes in bovine vesicular follicles.

J. Anim. Sci. 27:468.

Cole, H. H., and G. H. Hart. 1930. The potency of blood
serum of mares in progressive stages of pregnancy in
effecting the sexual maturity of the immature rat.
Amer. J. Physiol. 93:57.

 

DeBois, C. H. W., and C. J. Bierschwal. 1970. Estrus
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ment of melengestrol acetate. Amer. J. Vet. Res.
31:1545.

Denny, J. E. F. M. 1964. Ovulation control as a first
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Dowling, D. F. 1949. Problems of the transplantation of
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Dukelow, W. R. 1971. Bioassay techniques related to
sperm capacitation. >Acta Endocrinol. 66:503.

 

Erb, R. E., W. R. Anderson, P. M. Hinze, and E. M.
Gildow. 1960. Inheritance of twinning in a herd
of Holstein-Friesian cattle. J. Dairy Sci. 43:393.

 

Folley, S. J., and F. H. Malpress. 1944. Proc. Roy. Soc.
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Foote, R. H., and H. Onuma. 1970. Superovulation, ovum
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Foote, W. D., and M. B. Radmall. 1972. Beef and dairy
cow response to gonadotrophin. ‘J. Anim. Sci.
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Gordon, 1., G. Williams, and J. Edwards. 1962. The use
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Graves, C. N., and P. J. Dziuk. 1968. Control of ovula-
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61

Hafez, E. S. E. 1969. Superovulation and preservation of
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Hafez, E. S. E., M. R. Jainudeen, and D. R. Lindsay. 1965.
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Hafez, E. S. E., E. Rajkoski, P. B. Anderson, 0. L. Frost,
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Hafez, E. S. E., T. Sugie, and I. Gordon. 1963. Super-
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Hafez, E. S. E., T. Sugie, and W. L. Hunt. 1963. Super-
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Hammond, J. 1949. Induced twin ovulations and multiple
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Hammond, J., and P. Bhattachanya. 1944. Control of
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62

Lamond, D. R. 1964. Hormonal synchronization of ovulation
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63

Neville, W. W., and D. J. Williams. 1973. Estrus,
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Oguri, N., and Y. Tsutsumi. 1972. Non-surgical
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affecting superovulation, fertilization, and
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Onuma, H., J. Hahn, R. R. Maurer, and R. H. Foote. 1969.
Repeated superovulation in calves. J. Anim. Sci.
28:634.

Osland, R. B., and E. F. Ellington. 1971. Regulation of
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Oxenreider, S. L. 1968. Effects of suckling and ovarian
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Peli, I., and F. Castelli. 1964. Multiple births obtained
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Reynolds, W. L., R. A. Bellows, T. M. DeRouen, and D. C.
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Rich, T. D., and C. L. Johnson. 1972. Increasing the
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64

Roussel, J. D., and J. F. Beatty. 1970. Pregnant mare's
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65

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P '-

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APPENDIX A

LAPAROSCOPY OF THE COW

 

APPENDIX A

LAPAROSCOPY OF THE COW

The laparoscopy technique was attempted in the cow F1
to assist in confirmation and documentation of ovarian
activity detected by rectal palpation. Laparoscopy has

been successfully used in many species including human and

 

nonhuman primates, rabbits, rodents, and, more recently, L,
the pig. Five attempts were made to laparoscope individual
cows at the Endocrine Research Unit facilities in February
1972.

The laparoscope apparatus included a 135 degree pedi-
atric laparoscope, 5 mm in diameter, a trocar-cannula, a
fiber optic cable, and a Model 4000 projector light source
(Richard Wolf, GMBH, 7134 Knittlingen, West Germany).

Cows were placed in a squeeze chute and lightly
tranquilized (i.m.) with Sparine (Promazine Hydrochloride,
Wyeth Laboratories, Inc.) at 1.2 mg/kg of body weight. The
site of the laparoscope insertion was between the last rib
and the external angle of the ilium. The hair in the area
of incision was clipped and the area was washed with
Zephiran (benzalkonium chloride). Xylocaine (2%) (Lidocaine
Hydrochloride, Jensen-Salsbery Laboratories), a local

anesthetic, was injected into the skin and underlying tissues

66

67

in the immediate incision area. A 2 cm incision was

made, the trocar-cannula inserted, followed by insertion
of the laparoscope through the cannula. Observations were
facilitated by admitting 5 percent COz/air into the
abdominal cavity.

Unsuccessful attempts indicated that the inadequate
length of the laparoscope was preventing observation of
internal structures. This was confirmed in another attempt
in which rectal palpation and laparoscopy were employed
simultaneously. The ovary was grasped and moved to the
lateral abdominal wall in an effort to position it in
proximity to the inserted laparoscope. Observation of the
ovary was unsuccessful and the end of the laparoscope was
not detected protruding through the inner abdominal wall.

Although limited success was achieved in this study,
the potential of laparoscopy may be beneficial in the
study of ovarian activity in the smaller or younger bovine

animal.

1"“-

 

APPENDIX B

PUBLICATIONS BY THE AUTHOR

___~£7

 

APPENDIX B

PUBLICATIONS BY THE AUTHOR

Controlled Ovulation in Beef Using FSH and HCG. D. E.
Wildt and W. Richard Dukelow. J. Anim. Sci. 35:1125.

Control of Ovulation in Squirrel Monkeys. R. M. Harrison,
D. E. Wildt, and W. R. Dukelow. Fed. Amer. Soc. Exp.

Biol. Proceedings, Vol. 32; March 1973.

68

 

APPENDIX C

ABSTRACTS

 

W‘Afl
V
v u. .2.

CONTROLLED OVULATION IN BEEF
USING FSH AND HCG1

D. E. Wildt and W. Richard Dukelow2
The objective of this study was to determine the
effects of porcine-FSH and low dosages of HCG in inducing

multiple ovulation in mature Angus, Angus-Hereford, and
All animals were observed for two

The number

 

Angus-Holstein heifers.
estrous cycles before treatments were begun.

of animals and the treatments employed were as follows:
(I) 7, 25 mg FSH-P, 2,000 IU HCG; (II) 6, 25 mg FSH-P,
1,000 IU HCG; (III) 6, 12.5 mg FSH-P, 1,000 IU HCG. The

heifers were rectally palpated on day 16 of the cycle and

injected with a single dose of FSH-P (i.m.) on that day.
They were

The animals were checked for estrus twice daily.
rectally palpated and injected (i.m.) with HCG on the day

of scheduled estrus (or earlier if signs of estrus were
The heifers were then

evident prior to the let day).
the

The number of ovulations,

palpated daily for 3 days.
and the number of

number of animals with twin ovulations,

 

1Presented at the Midwestern Section-American Society
of Animal Science Meetings, Chicago, 111., Nov. 24-25, 1972.

2Endocrine Research Unit (Ctr. Lab. Animal Res. ,

Depts. An. Hus. & Physiol.).

69

70

animals excessively stimulated (>3 ovulations) for each

group were as follows: (I) 2.6, 3/7, 1/7; (II) 1.3, 1/6,

l/6; (III) 1.3, 3/6, 0/6. The above data demonstrate that

controlled multiple ovulations may be produced with

restricted levels of FSH-P and HCG and that the desirable

twin ovulation objective may be attained without excessive

ovarian stimulation. (This work supported by NIH Research

Career Development Award No. l-K4-HD35, 306-01 and the

Michigan Agricultural Experiment Station.)

 

CONTROL OF OVULATION IN SQUIRREL MONKEYSl

R. M. Harrison, D. E. Wildt,
and W. Richard Dukelow2

Ovulation induced in squirrel monkeys with proges-

terone-FSH-HCG can be inhibited by megestrol acetate (MA)

injections. We studied MA administration by silastic

implants, disposition i_n vivo, and rate of release. The

effectiveness of MA implants depends on induction pre-

treatment and time in situ. Monkeys receiving progester-

one pretreatment ovulated at a lower rate (2/8; 25%) than

those not receiving progesterone (12/20; 60%). Monkeys

with implants 11 days prior to FSH ovulated at a rate of

62% (10/16) compared to 33% (4/12) for those with implants

29 days. Using MA—H3 implants, concentration of the pro-

gfimtin was highest in ovaries and oviducts, less in adrenals,
IuerusIand vagina, and not found in the pituitary. J3 vivo
release rate was determined with 40 implants in 20 monkeys.

Toémtermine in_vitro release rate, each implant was incu-

bated in 10 m1 of TC-199 at 37°C for 5 weeks. One ml

 

1Presented at the Fed. of Amer. Soc. for Exp. Biol.
hmefimg, Atlantic City, N.J., April 16-20, 1973.
2

Endocrine Research Unit (Ctr. Lab. Animal Res.,
Depts. An. Hus. SI Physiol.) .

71

 

72

aliquots were removed and replaced daily and implants were

transferred each week to fresh medium. The i_n vivo release“

rate was 56 1' 12}ng/day, whereas the in vitro rate was 13 1’ 5
lug/day. In vitro release was influenced by build-up in the

medium within 48 hours, and the rate was not linear over

the five week period.

 

lflame:
Born:
Birthplace:

Formal Education:

Degrees Received:

VITA

David Edwin Wildt
March 12, 1950
Jacksonville, Illinois

Chandlerville High School
Chandlerville, Illinois

Illinois State University
Normal, Illinois

Michigan State University
East Lansing, Michigan

Bachelor of Science,

Illinois State University, 1972

73

" his. .
_ ‘7' .

 

 

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