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thesis entitled

MANIPULATING FOLLICLE AND CORPUS LUTEUM
FUNCTION TO IMPROVE EFFICIENCY OF SYNCHRONIZATION
OF OVULATION IN LACTATING DAIRY CATTLE

presented by

Michael W. Peters

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Masters . Science
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MANIPULATING FOLLICLE AND CORPUS LUTEUM FUNCTION TO
IIVIPROVE EFFICIENCY OF SYNCHRONIZATION OF OVULATION IN
LACTATING DAIRY CATTLE.

By
Michael W. Peters

A THESIS

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

MASTER OF SCIENCE
Department of Animal Science

2000

Abstract
MANIPULATING FOLLICLE AND CORPUS LUTEUM FUNCTION TO IMPROVE
EFFICIENCY OF SYNCHRONIZATION OF OVULATION IN LACTATING DAIRY
CATTLE
By

Michael W. Peters

The synchronization of ovulation (Ovsynch) program is an effective tool for dairy
managers to control calving interval. However, the program may be more widely
adopted if conception rate following the program was higher or if Ovsynch required less
labor. Objectives of this thesis were to: 1) determine if initiating Ovsynch at mid-cycle
increased conception rate, 2) determine if conception rate and synchronization rate after
treatment with Ovsynch are depressed when the second gonadotropin releasing hormone
(GnRH) and the prostaglandin F20l are administered simultaneously, 3) determine the
optimal time between the prostaglandin F20l injection and the second GnRH injection of
the Ovsynch protocol for maximal conception rates. To address objective one, a novel
program was developed (OVPLUS). OVPLUS succeeded in synchronizing cows in a
mid-luteal phase before Ovsynch. However OVPLUS did not increase conception rates.
For objective two, a program was tested that administered the prostaglandin F2Cl injection
at the same time gonadotropin releasing hormone was administered. When these two
injections are administered simultaneously synchronization rate was similar but
conception rate was reduced when compared to administering the two injections 48 h
apart. For objective three, four time periods between the prostaglandin F2Cl and the final
GnRH injection of Ovsynch were tested. Conception rate was greatest when

prostaglandin F20, was injected 36 h before last GnRH.

ACKNOWLEDGEMENTS

I would like to thank the following people for their help and guidance during my
graduate training at Michigan State University.

First, I would like to thank my major professor, Dr. Richard Pursley. His
guidance, mentoring, and patience have helped me develop scientific thinking while
obtaining greater knowledge of dairy management. I also thank the members of my
guidance committee, Drs. Roy Fogwell, Thomas Herdt, and George Smith. Their
guidance and input has been invaluable in development of my thesis.

I would also like to thank Ms. Leanne Bakke, Ms. Carolyn Cassar, Mr. Mark
Dow, and Mr. Isam Qahwash for their help in data collection. Large on farm projects
like these would be impossible without such great pe0ple. I thank Mr. Roy Radcliff,
Mr. Larry Chapin, and Dr. Chris McMahon for training and assisting me with lab work.

I express deepest gratitude to the five farms used in my projects, especially
Nobis Dairy Farm. Their constant support of dairy research at Michigan State
University is impressive and is to be commended.

Finally, I thank my family Tonia and Kylie for their continued love,

encouragement, and understanding. Without their support, completing a masters degree

would never have been possible.

iii

TABLE OF CONTENTS

LIST OF TABLES ................................................................................ v
LIST OF FIGURES .............................................................................. vi
INTRODUCTION ................................................................................. 1
CHAPTER 1
REVIEW OF LITERATURE ................................................................... 2
Rationale for controlling ovulation ................................................... 3
Folliculogenesis in the bovine .......................................................... 5
Bovine follicle growth ........................................................... 5
Endocrinology of follicle waves in the bovine .............................. 9
Pharmacological control of the bovine estrous cycle ........................... 10
History ........................................................................... 10
Prostaglandiana (PGFZQ) programs ....................................... 11
Using GnRH to synchronize ovulation .................................... 15
Conclusion ................................................................................ 18
CHAPTER 2

STARTING OVSYNCH IN THE MID-LUTEAL
PHASE OF THE ESTROUS CYCLE FAILS TO IMPROVE

FERTILITY OF LACTATING DAIRY COWS .......................................... 19
Introduction .......................................................................... .~....20
Materials and Methods ................................................................. 21
Results ..................................................................................... 22
Discussion ................................................................................. 26

CHAPTER 3

EFFECT OF DIFFERENT TIMES BETWEEN PGan
AND SECOND GnRH OF OVSYNCH ON CONCEPTION
RATES, OVULATORY F OLLICLE SIZE,

AND SUBSEQUENT PROGESTERONE LEVELS ..................................... 30
Introduction ............................................................................. 31
Materials and Methods ............................................................... 32
Results .................................................................................... 35
Discussion ............................................................................... 41

CHAPTER 4 .

GENERAL DISCUSSION ..................................................................... 45

BIBLIOGRAPHY ................................................................................. 48

iv

Table 1.

Table 2.

Table 3.

LIST OF TABLES

Conception rates in lactating dairy cows

following Ovsynch initiated at a random stage of the

estrous cycle (control) or during the mid-luteal phase of the

estrous cycle (OV+) .................................................................. 23

Effect of milk production on conception
rates in lactating dairy cows ......................................................... 24

Comparison of synchronization rate,
CL regression, follicle size and pregnancy rate
between OV (control) and MOV groups ........................................... 36

LIST OF FIGURES

Figure 1. Description of the timing for
each of the injections used in the 0V
group (control) and the OVPLUS group ......................................... 21

Figure 2. Proportion of lactating dairy cows
with P4 concentrations > 1 ng/ml during the Ovsynch
protocol at time of the lst GnRH of the standard
Ovsynch protocol ((1 0), 3 d after lst
GnRH (d 3), and at the time of PGFZQ of the standard
Ovsynch protocol (d 7). ............................................................ 25

Figure 3. Description of the timing for each of the
injections used in the Ovsynch
and the modified Ovsynch protocol .............................................. 32

Figure 4. Description of the timing for each of the injections used in the
36 h (control), 24 h, 12 h, and 0 h treatments .................................... 34

Figure 5. Synchronization of ovulation rates in lactating dairy
cows treated with GnRH at specific times after PGF;Cl .................................. 37

Figure 6. Size of the ovulatory follicle in lactating
dairy cows at time of final GnRH at specific times
after PGan .......................................................................... 38

Figure 7. Conception rates in lactating dairy cows following
treatment with GnRH at specific times after PGan ....................................... 39

Figure 8. Mean serum progesterone concentrations in
lactating dairy cows in the estrous cycle afier modified
versions of Ovsynch. The modified versions involved
injections of GnRH at specific times (0, 12, 24, 36 h)
after exogenous PGan. ............................................................ 40

vi

INTRODUCTION

Pregnancy is the greatest stimulator of lactation (68). However achieving
pregnancy in lactating dairy cows is becoming increasingly difficult. In the 1950’s
conception rates for lactating dairy cattle were approximately 66 % (65). In the 1970’s
conception rates declined to 50 % (65). In the early 1980’s conception rates were
reported to be 46 % (21). Presently, conception rates are at an all time low of
approximately 40 % (49). During this time period, estrus detection rates have also
declined dramatically (59). Traditionally artificial insemination of cows requires
detection of estrus. To facilitate use of artificial insemination methods have been
developed to synchronize estrus in cows (35), facilitate detection of estrus behavior (I 6),
and synchronize ovulation to circumvent estrus detection and allow for timed
insemination. Synchronizing ovulation is useful because it removes the need to detect
estrus, thus increasing number of cows inseminated. Currently synchronization of
ovulation results in conception rates that are similar to conception rates after estrus
detection. However, increasing conception rates after treatment with Ovsynch, or
reducing labor needs to perform the hormone injections, may increase the number of

dairy producers who adopt Ovsynch. This thesis test adaptations to Ovsynch that may

accomplish the two ideas listed previously.

Chapter 1

Review of Literature

Rationale for controlling ovulation

It is possible for a dairy cow to produce milk for up to four years
following one parturition. However, at some point milk production drops below a
profitable level. There is no profit when the maintenance costs for a low producing cow
exceeds the income from milk sold. To maintain a profitable dairy herd, milk production
must stop and be restarted. The best method for restarting lactation is a cow completing
a healthy gestation followed by parturition (68). However achieving a pregnancy in
lactating dairy cattle is becoming increasingly difficult. In the 1950’s, conception rates
for lactating dairy cattle were approximately 66 % (65). In the 1970’s, conception rates
declined to 50 % (65). In the early 1980’s, conception rates were reported to be 46 %
(21). Presently, conception rates are at an all time low of approximately 40 % (49).
Conception rates are affected negatively by shorter days to first service and high milk
production (25). It is also difficult to detect estrus in lactating dairy cows (5 9). A cow
only displays estrus for an average of 7.1 h (9). During the 7.1 h period cows have an
average of 8.5 occurrences of standing to be mounted (9). Consequently the average
estrus detection rate in dairy cattle is below 50% (59). Failure to detect estrus or false
diagnosis of estrus results in an estimated loss of over $300 million to the US dairy
industry each year (5 9). Since the inception of artificial insemination (AI), dairy
producers rely on estrus detection to know when to AI cows. These low estrus detection
rates and conception rates lead to an estimated pregnancy rate (PR) of 20%. (A definition
of PR is estrus detection rate x conception rate). Maintaining an efficient dairy operation
is difficult with a 20% PR, because cows that do not become pregnant are forced to have

long inefficient lactations. Farm management may decide to cull cows instead of

continuing to try to inseminate them. A replacement must then be raised or purchased to
maintain lactating cow numbers.

To gain a better understanding of poor reproductive performance in lactating
cows, previous research has focused on the basic mechanisms of dairy cattle
reproduction. Applied research then focused on ways to employ basic findings to assist
producers in achieving pregnancy in dairy cattle. A key application of basic reproduction
research is the ability to synchronize ovulation in dairy cattle. Controlled ovulation
allows for timed insemination without the need to observe cows for estrus activity. By
synchronizing ovulation in cows scheduled for A1, 100% of the cows can be inseminated
on the first day of the breeding period without waiting for the cow to display estrus. This
is equivalent to a 100% estrus detection rate for first AI, and results in a 100% service
rate. When applied to the formula above; a 40% PR can be attained. The benefits from
synchronization of ovulation are demonstrated in a 1997 paper from Pursley et a1. They
demonstrated the power of breeding all cows on a predetermined day. In this study the
control cows were inseminated only after visual detection of estrus. The treated cows
had ovulation synchronized with a program called Ovsynch. Treated cows received their
first insemination by 50 to 57 days in milk (DIM), with an average of 54 d. The control
cows averaged 83 DIM at first insemination and ranged from 50 to 160 DIM. The
second and third inseminations also occurred earlier and with less variation compared to
the control group. The earlier times of AI in the Ovsynch group resulted in a shorter
median interval to conception for Ovsynch (99 C!) vs. the control group (118 d; 47).

This literature review will address the basic and applied research that formed the

foundation for development of the synchronization of ovulation method. The first section

will summarize the literature on the basic biology of follicular growth, also known as
folliclulogenesis. The second section discusses applied dairy cattle reproduction research
and current pharmacological techniques used to control estrous cycle and ovulation in

cattle.

Bovine Follicnlogenesis

In 1672 Regnier de Graaf first discovered and described antral ovarian follicles.
However, de Graff thought the whole follicle was the egg, instead of an oocyte within the
follicle (60). Since the time of de Graff, the knowledge of follicular function and growth
has evolved immensely. It is now well understood that bovine follicles grow in wave-

like pattern that is under strict hormonal control.

Bovine Follicle Growth

In 1960, Rajakoski did histological studies of bovine ovaries (50). Cows and
heifers were slaughtered on a known day of an estrous cycle. Ovaries of the slaughtered
cattle were collected and examined. All antral follicles were counted, measured, and
recorded. Based on these results he proposed that two waves of follicular activity occur
during each estrus cycle. Rajakoski reported that a “crop” of follicles appeared at the
beginning of the cycle and by day 5 one follicle continued to grow while the other
follicles within the cohort became atretic. The second wave appeared on approximately
day 12 of the cycle and resulted in recruitment of the ovulatory follicle. In 1972, Dufour
et a1. (10) used a laproscope to mark the largest and the second largest follicle on the

ovary with India ink. Follicles on the ovaries of heifers were marked on days 9 through

21 of the estrous cycle. Ovaries were then removed 3 days afier subsequent estrus and
examined. A new corpus luteum surrounded by India ink was an indicator that one of the
marked follicles ovulated. They reported that after day 18 postestrus, the largest follicle
on the ovary ovulated. However, before day 18, the largest follicle on the ovary was
never the ovulatory follicle. While unknown to them at the time, these data helped foster
the theory of follicular waves by showing that the ovulatory follicle does not become the
largest follicle on the ovary until late in the cycle. The idea of follicular waves was tested
again in 1981, when Matton et a1. (38) published histological data augmenting
Rajakoski’s theory of two follicular waves. The author used a laproscope to mark the
largest follicles in 20 animals on either day 3, 8, 13, or 18 of the estrous cycle. The other
20 of the animals had the largest follicle destroyed and their next largest follicle marked
with India ink on the same days of the cycle as above. Heifers in the day 3, 8, and 13
groups were slaughtered 5 days after surgery. Heifers in the day 18 group were
slaughtered one day afier estrus. Ovaries were examined post-mortem for follicular
changes or ovulation. Matton’s results supported the theory of rapid follicle turnover.
Also, they postulated that continued growth of medium sized follicles occurs only after
the largest follicle has been destroyed. In 1983, more data was published that supported
the theory of follicular waves (28). Ireland and Roche used blood steroid levels,
follicular measurements, and follicular fluid steroid levels to argue that two or three
waves of growth occurred during each estrous cycle in heifers. Thirty heifers were
ovariectomized on day 5, 7, 9, l 1, 13, or 15 of the estrous cycle. Follicles larger than 6
mm were categorized as estrogen active or estrogen inactive based on estradiol

concentrations within the follicular fluid. They reported that at least two waves of

follicle. growth occurred during days 3 to 13 of each estrous cycle. On days 9, 11, and 13
of the cycle only one estrogen inactive follicle was reported for each heifer, all other
follicles were estrogen active. On days 5, 7 and 15 of the cycle one follicle was estrogen
active while all others were estrogen inactive. Their paper provided endocrinological
evidence for follicle waves by demonstrating that a group or cohort of estrogen active
follicles emerge at the beginning of each follicle wave. From this cohort of estrogen
active follicles a single estrogen active follicle emerges. These data augment the theory
that on approximately day 5 of a follicle wave only one follicle is estrogen active. This
phenomena is referred to as dominance. Then on approximately day 9 of the wave the
dominant follicle becomes estrogen-inactive and with atresia (loss of dominance) another
wave of estrogen active follicles begin to grow. The hormonal control of follicle
dominance will be discussed further in the section entitled Endocrinology of Follicle
Waves in the Bovine (p 14).

Prior to the mid-1980’s, progress in the study of follicular dynamics was limited
because animals had to have invasive surgery or be slaughtered to observe ovarian
changes. These techniques can be expensive and make it diffith to acquire large
amounts of sequential data. A 1984 publication reported the use of transrectal
ultrasonography to monitor growth of ovarian structures in the bovine (43). This
noninvasive technological discovery allowed researchers to observe and monitor growth
and atresia of antral follicles, ovulation, and corpus luteum growth and regression.
Ultrasonography of the bovine ovary could be done as frequently as needed to address
objectives. In the mid 1980’s, the capabilities of ultrasound were expanded. The

equipment manufacturers developed ultrasound emitting probes that could display

pictures of 2 mm follicles on the ovaries. Also, users of ultrasound technology became
more adept at collecting and analyzing data derived from ultrasound sessions. Articles
describing daily tracking of follicle growth flourished. Many of these papers reported
that a majority of heifers had 3 follicular waves per cycle (56, 63). Others reported that
the majority of heifers had 2 follicular waves per cycle (19). Above and beyond
identifying the number of waves per cycle, daily ultrasound observation of ovaries
allowed researchers to characterize the follicle turnover that occurs during follicular
waves (19). Independent of discrepancies in number of waves per estrous cycle,
published data agreed on several key points regarding follicle waves. Ultrasound allowed
researchers to determine that a new wave starts as a cohort of small follicles near the time
of ovulation. Approximately three days into a wave, one follicle will become dominant
and the others will become subordinate. Subordinate follicles become atretic. A second
wave emerges around day 10 of the estrous cycle. If an animal exhibits three waves, the
third and final wave emerges around day 16 of the estrous cycle. The ovulatory follicle
develops from the final follicular wave (1 7,20). Ultrasound has also allowed researchers
to demonstrate that follicle waves occur during pregnancy (44) and that growth hormone
and diet influence follicle growth (36).

Applications of ultrasound technology for study of folliculogenesis are still
expanding. Ultrasound technology has been used to identify atretic follicles by the pixel
intensity of the image displayed on the screen (62). In addition to allowing researchers
the ability to visually characterize follicle waves, ultrasonography has also given

researchers a valuable tool to help correlate follicle growth and turnover with changes in

serum hormone concentrations.

Endocrinology of Bovine Follicle Waves

The pituitary gonadotropins, follicle stimulating hormone (FSH) and luteinizing
hormone (LH) play a key role in regulation of follicle growth and atresia. Adams et al.
demonstrated in cattle that an increase in plasma FSH is necessary to have a new follicle
wave begin. Adams et al. inhibited FSH secretion in a group of heifers treating them
with a proteinaceous fraction of bovine follicular fluid. This inhibition of F SH delayed
the onset of a new wave of follicle growth. They also reported that an increase in serum
FSH is detected at the onset of a new follicle wave (1). The increase in FSH causes a
cohort of follicles to grow on both ovaries until one of the follicles becomes dominant.
Shortly after one of the follicles establishes dominance the other subordinates become
atretic (32,33). This point in the wave is referred to as deviation or selection of the
dominant follicle. Coincident with deviation, the dominant follicle expresses LH
receptors, responds to LH and continues to grow. In concurrence with dominance being
established, F SH secretion from the pituitary decreases (1,39). Estradiol and the
endogenous action of inhibin mediate the decline in FSH (29). The dominant follicle
requires basal levels of LH to continue to grow. Also, once a dominant follicle acquires
LH receptors on the granulosa cells it gains the ability to ovulate in response to an LH
surge. If the dominant follicle grows in a proestrus wave it will ovulate. If the dominant

follicle grows in a wave that occurs during metestrus or diestrus, it will become atretic

and regress.

As knowledge of follicle growth continues to grow, a lot is still unknown about it.
Mechanisms that dictate which follicles enter the cohort of a wave, and which follicle

will become dominant are still unclear.

Pharmacological Control of the Bovine Estrous Cycle

History

The first published reports of successfirl AI in cattle were in 1902 by a Russian
scientist Ivanoff (76). Ivanoff adapted techniques done earlier in the horse and human.
Semen was collected by placing a sponge in the vagina of a cow. The ejaculate was then
squeezed out of the sponge. The semen was then placed into the vagina of another cow.
Thirty years later another Russian scientist, Milovanov, demonstrated the power of this
technology by artificially inseminating (AI) 19,800 cows using 100 cows per bull.
Suddenly one bull could have hundreds of offspring believed to have high genetic merit.
The power of Al was expanded when cryopreservation of sperm became possible in
1949. The discovery of glycerol as a cryoprotectant was accidental. But frozen semen
could be transported all over the world to expand the genetic impact of superior bulls
( 76).

While AI has many advantages, there are challenges. The most obvious challenge
is the need for the farm manager to detect the animals in estrus. As stated earlier, estrus
detection in dairy cattle is difficult. It is generally accepted that the estrus detection rate
on dairy farms is below 50% (5 9). To address the challenge to detect cows in estrus,

there are several non-pharmacological methods to assist dairy managers. These include

10

(but are not limited to): measurement of electrical impedance in vaginal mucus (74),
detection of estrus by a trained dog (23,31, 75), pedometers that measure cow activity
(30), devices that detect mounting activity in dairy cattle (9,22), milk progesterone
analysis (67), and allowing cows access to dirt lots (3). While these non-phannacological
methods benefit the ability of dairy producers to detect cows in estrus, this literature
review will not review them in depth. Rather, the following sections will focus on the
pharmacological methods that are used to control the timing of estrus, timing of

ovulation, and ultimately the timing of AI.

Prostaglandin F20, (PGan) programs

In the 1970’s Prostaglandin F 20, (PGFZQ) was discovered. Early in PGFZa
research, it became known that exogenous PGFZQ causes a 5 day old or older bovine
corpus luteum (CL) to regress. On days 1-4 of the bovine estrous cycle, PGan will not
cause CL regression (34,55). CL regression means serum progesterone concentrations
decline. Declining progesterone and elevating estradiol produced from the dominant
follicle will cause the cow to display estrus. It was reported that estrus occurs between 2
to 4 days after an injection of PGan (34). With this information, researchers developed
programs to control estrus. In 1973 Inskeep reported on the potential uses of PGFZQ (26).
With emphasis on several methods that would cause timed expression of estrus in cattle,
Inskeep further forecasted development of methods that would allow for timed AI.

Timing estrus in dairy cattle is advantageous for several reasons. A manager can

focus estrus detection during a shorter period and specifically on the animals that were

11

treated. Also, if more animals are in estrus at one time it is easier to ascertain which

animals are in heat, because they will display the signs of estrus with each other (16).
In 1974, Lauderdale et al. tested the fertility of cattle after exogenous PGan.

Cattle with 3 CL were assigned to one of three treatments. Treatment I (controls) were

observed for estrus activity twice daily. Cattle detected in estrus were inseminated 12 h
after detected estrus. Cattle for treatment 11 received an injection of PGan and were
observed for estrus twice daily. They were inseminated 12 h after of estrus. Cattle for
Treatment 111 cattle were time inseminated 72 and 90 h after injection of PGFZQ. The
percent animals pregnant at 35-60 days after AI did not differ among treatments and
averaged 54%. Therefore, Lauderdale et al. proposed that PGFZG could be used for timed
AI if two inseminations were administered. Lauderdale et al. also reported that cattle
would display estrus 1-7 (1 after PGan injection. One to 7 d was longer than the period
that he had previously reported (35). The 1-7 (1 window for cattle to exhibit estrus after a
PGF2(ll injection was confirmed in another study in 1978 (2).

In 1979, PGan became available for commercial use. This spurred a number of
researchers to test the applicability of programs that utilized PGFzCl on dairy farms.
Dailey et al. showed that a single timed AI afier a single injection of PGFZa will not yield
conception rates comparable to those achieved with estrus detection (7). However, two
injections of PGan administered 10 to 14 d apart resulted in a greater percentage of
animals in heat 1-7 d after the second injection than when one injection of PGFZQ was
used. The greater percentage of animals synchronized happens because at the time of the
second injection all animals will have a CL that is responsive to PGan and will regress.

However, afier a timed insemination this treatment regime does not yield conception

12

rates that are as great as conception rates after detected estrus (64). This is because
estrus occured over a period of 6 d among treated animals. If estrus is displayed over a
period of 6 d, ovulation does not occur over a short enough period of time to allow timed
AI. Successfiil timed AI requires a synchronous ovulation. In spite of timed AI not
being efficient with PGan injections, PGan is still a powerful tool for estrus
synchronization. Seguin et al. reported on the applicability of using PGan in a milking
dairy herd (58). Half the cows of three herds were assigned to the control group
(traditional estrus detection with no PGan injection). The experimental group of cows
received an injection of PGan if a CL was present. After the injection of PGan, cows
were observed for estrus. Cows in the experimental group received first A1 at fewer days
in milk when compared to the control (70 d vs 81 (1 respectively). Cows in the
experimental group also conceived an average of 18 days sooner than the controls (93 d
vs 111 d). It is also important to realize that with the use of PGan to synchronize estrus,
managers must avoid common pitfalls that will counteract the benefits of estrus
synchronization. These include: thawing too many units of semen at once (13), falsely
identifying animals in heat due to anticipatory zeal following PGF2Cl injection (58), and
relying on untrained help to detect estrus.

To decrease the period in which estrus is expressed after a PGF;0L injection,
researchers combined PGan with other exogenous hormone treatments. If estrus could
be synchronized more precisely than over a 6 d period, timed AI may be possible.
Estradiol was tested in conjunction with PGF2(ll in 194 heifers. The hypothesis was that
an injection of estradiol would cause a timed LH surge. A timed LH surge would lead to

a timed ovulation, and thus a timed insemination would be possible. All heifers with a

13

CL present in an ovary were injected with PGFZG. Half the heifers were treated
intramuscularly with 400 pg of estradiol benzoate 40 to 48 h after PGFZQ. Timed
insemination occurred at 72 or 80 h after the injection of PGan. Control animals were
inseminated 12 h after detected estrus. Exogenous estradiol did not improve the number
of heifers that were observed in estrus (86.7% for PGFZQ only vs 84.3% for PGFZa and
estrogen) or reduce the variability in time to estrus. In Dailey’s study, conception rates
were lower for animals that were inseminated at 72 h post-PGan than controls (7).
Exogenous progesterone has also been hypothesized to assist in synchronization
of estrus. Progesterone can be administered by way of a progesterone releasing
intravaginal device (PRID). PRIDs allow for a sustained release of progesterone over
the period of a week or more. This allowed for the maintenance of serum progesterone
levels at a constant concentration. Fogwell et al. inserted PRIDs into heifers for 10 d.
Seven d following PRID insertion, heifers were treated with PGFZG. On (1 10 the PRID
was removed. Treated heifers had a more homogeneous display of estrus, and a more
synchronous LH surge. All treated animals inseminated at estrus did not have reduced
fertility when compared to animals bred without progesterone manipulation (12). It was
suggested from these findings that PRID’s with PGFZQ may allow for a timed
insemination of dairy heifers, but this program was not tested in this study. Other
researchers tested the applicability of combining progesterone with PGFZQ injections.
Smith et al. inserted PRIDs for 7 d and administered PGF2(ll one day before PRID
removal. Smith et al. also demonstrated reduced variability in occurrence of estrus after
exogenous PGFZQ. Heifers were either time inseminated at 84 h after PGFZQ or

inseminated after detected estrus. Conception rate in heifers that received timed Al was

14

66 % and did not differ from the control group that was inseminated 12 h after detected
estrus. These studies demonstrated that progesterone in conjunction with PGFz0L allow for
successful timed insemination of heifers. However, PRIDs have not received FDA
approval for use in the United States. Therefore, this option is not legally or

commercially available to cattle managers at this time.

Using GnRH to Synchronize ovulation

Gonandotropin releasing hormone (GnRID is FDA approved for use in lactating
dairy cattle. GnRH has been proven to be effective in the treatment of ovarian follicular
cysts (8). When GnRH is injected into cattle, a surge of LH begins within an hour (6).
An LH surge causes a dominant follicle with LH receptors on the granulosa cells to
ovulate (4 7). In 1995 a method (Ovsynch) was developed that uses GnRH to synchronize
ovulation in cattle. The method was based partially on the theory that lactating dairy
cows display two follicular waves per estrous cycle. Pursley et al. used ultrasound to
monitor daily growth and atresia of follicles in lactating dairy cows and heifers. An
injection of GnRH was administered at a random time of an estrous cycle. If a cow had
an LH responsive follicle, it would ovulate, a new CL would form, and a new follicle
wave would begin. Ninety percent of lactating dairy cows injected randomly with GnRH
will ovulate a follicle (47). If a cow did not have an LH responsive follicle, the injection
would do nothing. Seven (1 after the GnRH injection, an injection of PGFZQ was given to
regress any CL present in the ovaries. Two (1 after the PGFZG , a second injection of
GnRH was given. Cows that responded to the first injection of GnRH should now have a

follicle that is 8 to 9 (1 old. If the animal did not respond to the first GnRH injection, she

15

is between d 10 to 12 of a follicle wave. Regardless of the earlier response an animal
should have a dominant follicle that would ovulate to the second GnRH injection.
Ovulation occurred 28 hours after the second GnRH injection, with a range of only 4
hours. This time to ovulation is similar to what is seen afier estrus (73). In a study
published by Pursley et a1, 20 of 20 cows had a synchronous ovulation after treatment
with Ovsynch. However in heifers, only 18 of 24 had a synchronous ovulation after
treatment with Ovsynch (4 7). Because ovulation was synchronized, a timed insemination
was possible after cows were treated with Ovsynch. In 1997, data was published that
demonstrated applicability of Ovsynch to reproductive management on commercial dairy
farms (46). At calving, cows were assigned to either a control (reproductive management
protocol for each farm) or treatment (Ovsynch) group. Cows in the Ovsynch group could
only be inseminated after treatment with Ovsynch. Ovsynch reduced days to first AI (54
treatment vs 83 controls) compared to controls. Days to second and third AI were also
reduced. Conception rates for the first AI were the same for control and treatment, in
spite of the fact that Ovsynch cows were bred at an average of 19 days sooner. Ovsynch
led to more cows pregnant at 60 d post-partum (37% vs 5%) and more cows pregnant at
100 d post-partum (53% vs 35%) compared to controls. Another study published in 1997
(49) substantiated the previous reported results for lactating cows, but found unacceptable
pregnancy rates in Ovsynch treated heifers (35.1% for Ovsynch vs 74.4% for controls).

A Florida lab has further substantiated Ovsynch to be beneficial in controlling PR in
lactating dairy cattle (5). The same lab has also reported that Ovsynch yields lower

conception rates when used on heifers (46% Ovsynch vs 61% for AI 12 h after estrus)

l6

(57). So while Ovsynch was a viable alternative for reproductive management of
lactating cows, it should not be used for management of heifer reproduction.

The economic advantage of Ovsynch over traditional estrus detection remains
controversial. One study demonstrated that Ovsynch provides economic advantages over
traditional estrus detection (4). However, a 1998 paper on the use of synchronized estrus

using PGFZQ injections and the Ovsynch protocol argued otherwise (41). They reported

that the mean cost of a pregnancy using the PGan program with a 40% estrus detection
rate was $29.61. This was substantially lower than the mean cost of pregnancy from
synchronization of ovulation, which was reported to be $46.10. The majority of the cost
for the Ovsynch protocol was the GnRH injections. The average cost for a lOOug
injection of GnRH was $7.27. To assist in making Ovsynch more economical, Fricke et
al. tested the efficacy of using SOug (half dose) of GnRH for each injection (18). They
found that a half dose of one form of GnRH yielded the same synchronization rate and
conception rate as a full GnRH dose. Now Ovsynch can be utilized at a decreased cost.
Further work with modifying the Ovsynch protocol has not demonstrated that changing
the sequence of or time between the injections yields conception rates higher than those
of Ovsynch (42, 66). Also there is no reported advantage in using hCG to ovulate a
follicle instead of GnRH (57). Publications show that timing AI after treatment with
Ovsynch may allow for alterations in the gender ratio and decreased pregnancy loss (48).

Further research needs to be done on greater numbers of animals to substantiate these

results.

Summary

Low estrus detection rates, coupled with low conception rates have led to
difficulties managing reproduction of lactating dairy cows. To combat low fertility
ultrasound and endocrinology research was done to gain a greater understanding of
follicle growth. Years of work and many researchers have contributed to the knowledge
of follicle waves in the bovine. Basic discoveries allowed researchers to define when a
follicle would become responsive to an LH surge. With basic knowledge of follicle
growth, CL formation and regression, and ovulation, there is an informed rationale for
applied research to synchronize estrus and ovulation. Synchronization of estrus is a
powerfirl management tool to control reproduction in cows and heifers. Synchronization
of ovulation is a management tool that dairy mangers can use to control reproduction in
lactating cows. Ovulation synchronization allows managers to control cow reproduction,

and is a key to proactive versus reactive management.

18

Chapter 2
Starting Ovsynch in the mid-luteal phase of the estrous cycle

failed to improve fertility of lactating dairy cows

19

Introduction

Synchronization of ovulation (Ovsynch) is a management tool to control time to first
artificial insemination (AI) in dairy cattle (46). This in turn allows farm management to
have more control of calving interval. Ovsynch accomplishes control of calving interval
by allowing for timed insemination (4 7). Ovsynch creates the opportunity for managers
to inseminate at a preset time with no need to observe cows for estrus. Compared to
Prostaglandin F 20, (PGFZQ) programs, Ovsynch is more advantageous because the need to
observe cattle for estrus can be eliminated and service rate is 100 %. Even with diligent

visual observation for estrus after synchronization of estrus with PGFz0L some cows will

still not be detected in estrus (58). Thus, service rate after PGan is almost always less
than 100%. Within a herd, conception rate following treatment with Ovsynch is similar
to inseminations after observed estrus (4, 46, 49). Improving synchronization and
conception rates after Ovsynch would elevate the percent of cows that become pregnant
to an insemination. In lactating dairy cows conception rates were increased when
progesterone (P4) levels were increased 10-12 (1 prior to insemination (14, 15, 52). From
these data it appears that if lactating cows were synchronized into the luteal stage of the
estrous cycle (d 5 to 17) at the start of Ovsynch conception rates may be increased. Also,
synchronization rates after Ovsynch are highest when Ovsynch is initiated d 1 through 12
of the estrous cycle (71). Increasing synchronization rate after Ovsynch could increase
the number of cows conceiving after an AI. This study tested whether lactating dairy

cows in luteal phase ((1 5 to 179) of an estrous cycle at the time of the first injection of the

20

Ovsynch program will have greater fertility than cows that begin Ovsynch at a random
stage of an estrous cycle.

Materials and Methods
From February to December 1997 lactating dairy cows (n=530) from two commercial
dairy farms in Michigan were utilized. All cows were housed in freestall barns. All cows
between 49 to 55 d in milk were assigned to either the control or the treatment group. All
cows received A1 at 69 to 75 d in milk. Only cows receiving their first postpartum AI

were used in the study. The control cows received the traditional Ovsynch program

 

 

 

 

[ OV-Protocol I GnRH PGF2 GnRH AI

7 d 48 h 16 h

I 3 d I 7 d 7 d 48 h 16 h
PGFZG GnRH GnRH PGFM GnRH AI

 

{OVPLUS-Protocol J

 

Figure 1. Description of the timing for each of the injections used in the 0V
group (control) and the OVPLUS group.

 

 

 

(Figure l). The treated cows received PGan 10 d and GnRH 7 d before Ovsynch (Figure
1). This program was labeled Ovsynch plus (OVPLUS). Laboratory staff administered
all injections. The GnRH injections were Cystorellin® (Merial, Ltd., Iselin, NJ)
administered at 100 jig/dose. The PGan was Lutalyse® (The Pharmacia-Upjohn Co.,
Kalamazoo, MI) administered at 25mg/dose. Farm personnel performed all Al. Control

and treated cows received injections in sequences that allowed for Al on the same

21

calendar date and the same week postpartum. The herd veterinarian diagnosed pregnancy
at 36 d post AI by rectal examination of uterus for a conceptus. Conception rate data
were analyzed using Chi Square analysis. Within treatments, a randomly selected subset
of cows (n=84) had blood sampled fiom a coccygeal vessel. Blood was sampled at three
times: at the time of the first GnRH of Ovsynch, 3 d after the first GnRH of Ovsynch, and
at the time of the final PGan. Progesterone (P4) in serum was quantified by
radioimmunoassay (P4 Coat-a-count kit, Diagnostic Products Cooperation, Los Angeles,
CA). Intra- and interassay coefficients of variation were 5.6 % and 9.1 %, respectively.
Milk production data were available from one of the farms used in this study. The
average milk production nearest time of AI (milk weight measurement taken every two
weeks) within each parity was determined to compare conception in rates in cows above
the mean versus conception rates in cows producing below the parity mean. The average
milk production was 75 pounds/day for first pairity, 109 pounds/day for second parity,
and 110 pounds/day for third and greater parities. Cows that had an infirmity that
negatively impacted milk production were removed from the analysis. Conception rates
within milk production groups were examined by Chi Square analysis for effects of
treatment and parity.

Results

There was no effect of farm on conception rates therefore data were pooled. Conception
rates for OVPLUS (39.7 %) did not differ from Ovsynch (39.1 %). Conception rates by
parity did not differ between treatments (Table 1). However, conception rates for first
parity cows (48.2 %) were greater than cows in parity three or greater (33.9 %, p =

0.008). Effects of milk production on conception rates are reported in Table 2.

22

 

 

 

Table 1. Conception rates in lactating dairy cows
following Ovsynch initiated at a random stage of the

estrous cycle (control) or during the mid-luteal phase of the
estrous cycle (OV+).

 

Paritya OV OV+ Totalc
First (%) 43.5 (n=69) 52.8 (n=73) 48.2 (n=142)
Second (%) 40.0 (n=50) 37.9 (n=58) 38.9 (n=108)
Third + (%) 33.3 (n=90) 34.5 (n=87) 33.9 (n=177)
Totalb (%) 38.3 (n=209) 41.5(n=218) 39.9 (n=427)

aNo difference in parity*treatment (P = 0.332)
1)No difference in treatments (P = 0.87)

cFirst parity different than third+ (P = 0.008), all others
similar (P > .14)

 

 

 

 

 

 

 

 

 

 

23

 

 

Table 2. Effect of milk production on conception
rates in lactating dairy cows.a

 

Parityb High Low Total

 

Firstc(%) 59.7 (n=67) 37.3 (n=75) 48.2(n=142)

 

Secondd(%) 35.6 (n=55) 43.4 (n=53) 38.9(n=108)

 

Third +e(%) 42.4 (n=92) 24.7 (n=85) 33.9(n=177)

 

 

 

 

 

 

Totalf 45.8 (n=214) 33.8 (n=213) 39.9 (n=427)

 

 

a Data grouped by peak daily milk production above
the parity mean (High) or below parity mean (Low).
Parity milk production means:
First parity < 75 pounds/day = Low
2 75 pounds/day = High
Second parity < 109 pounds/day = Low
_>_ 109 pounds/day = High
Third+ parity < 110 pounds/day = Low
2 110 pounds/day = High

bTreatment data pooled since no difference in treatments
cHigh and low producers different within parity (P < 0.01)
dHigh and low producers trend towards difference

within parity (P < 0.10)

eHigh and low producers different within parity (P <0.005)
fHigh and low producers different (P < 0.025)

 

24

 

 

 

 

_L
O

on
O.

 

 

- 0V
- OV+

 

 

 

.h
0.

Percent of cows with > 1 ng/ml
N a)
O. O
93
c.

 

 

 

0 d 0 d 3 d 7
Day of standard Ovsynch program

Figure 2. Proportion of lactating dairy cows

with P4 concentrations > 1 ng/ml during the Ovsynch
protocol at time of the 1sthRH of the standard
Ovsynch protocol (d 0), 3 d after lst

GnRH (d 3), and at the time of PGF20L of the standard
Ovsynch protocol (d 7).

“Day 0 treatments different (P = 0.022)
bDay 3 treatments different (P = 0.013)
cDay 7 treatments similar (P = 0.352)

 

25

 

Independent of parity, conceptions rates of cows that had higher milk production around
the time of A1 (45.8%) had a greater conception rate compared to cows that produced
below the parity mean (33.8%, p < 0.025). High producing first parity cows (59.7 %) had
higher conception rates than low producing cows (37.3 %, p < 0.01). Low producing
second parity cows tended (p < 0.10) to have greater conception rates than high
producing cows (43.4 % vs 35.6 %). Conception rates for high producing cows (42.4 %)
in third plus parity was greater (p < 0.005) than conception rates in low producing cows
(24.7 %) of the same parity. Percent of cows with a fimctional CL are summarized in
Figure 2. At first injection of GnRH during Ovsynch, percentage of cows with greater
than 1 ng/ml serum P4 was greater for OVPLUS than Ovsynch (93.0 % vs 56.1 %, p =
0.02). Three days after the first injection of GnRH during Ovsynch, percentage of cows
with greater than 1 ng/ml serum P4 was greater for OVPLUS (90.7 %) than Ovsynch
(51.2 %, p = 0.013). On the d of PGFZQ the percentage of cows with greater than lng/ml
serum P4 did not differ for OVPLUS (82.9 %) and Ovsynch (95.3 % , p = 0.352). The
OVPLUS group had a higher percentage of cows greater than 2 ng/ml serum P4 compared

to Ovsynch (95.3 % vs 63.4 %, p = 0.049) on d of PGFZQ.

Discussion
This study was designed to test if increasing the number of cows in the mid-luteal
phase of the estrous cycle at commencement of Ovsynch program would improve fertility
in lactating dairy cows. To have cows in mid-luteal phase at the beginning of treatment

with Ovsynch, cows were injected with PGFZO, 10 d and GnRH 7 d before start of

Ovsynch. Gonadotropin releasing hormone was injected three days after the first PGFz,ll

26

to ensure that all cows would ovulate and start formation of a CL at least 6 (1 prior to the
initiation of Ovsynch. Cows responding to the PGFZO, (CL regression) should have either
had an estrus induced endogenous LH surge or a follicle large enough to ovulate at the
time of the GnRH (45). Interestingly compared to Ovsynch, OVPLUS increased the
percentage of cows above 1 11ng serum P4 at the time of first GnRH during Ovsynch
and three days later. OVPLUS also increased the number of cows with more than 2
ng/ml serum P4 at the time of the final injection of PGan during Ovsynch. The percent
of cows with greater than 1 ng/ml serum P4 in the Ovsynch group at first GnRH was
similar to a previous study (49). Therefore, differences in Ovsynch and OVPLUS are due
to increase by OVPLUS and not spurious decrease in cows treated with Ovsynch. The
increase in number of cows in the mid-luteal phase of the estrous cycle did not affect
conception rate following treatment with OVPLUS. Conception rates in this study were
39 % for OVPLUS and Ovsynch.

Previous studies established that lactating cows with serum P4 greater than
controls 11 to 13 (1 prior to AI have increased conception rates (I 4, I 5, 52). Reasons for
the differences in our data and previous findings could be in other studies serum P4 was
increase with exogenous sources (14). Folman et al used progesterone releasing
intravaginal devices (PRID) to elevate the serum P4 concentrations prior to insemination.
This treatment increased conception rates 27 %. Other studies reported higher serum P4
levels were the result of a CL from an endogenous LH surge (15, 52). While OVPLUS
did not elevate serum P4 concentrations in the treated animals, OVPLUS did synchronize

cows to a mid-luteal phase of the estrous cycle.

27

Starting Ovsynch at specific stages of an estrous cycle increases the percentage of
synchronized ovulations (71). Therefore, OVPLUS may have elevated synchronization
rate as well as increased percentage of cows in the mid-luteal phase of the estrous cycle.
Previous reports indicate that beginning Ovsynch 5 to 9 d postestrus resulted in 96 % of
the cows ovulating a follicle to the first GnRH of Ovsynch (71). Sixty six percent of the
cows treated with OVPLUS should be d 5 to 9 postestrus. The other one third would be
10 to 15 d postestrus. Vasconcelos et al. also reported that cows 1 to 12 d postestrus at
the time of the first GnRH of Ovsynch had a higher percentage of synchronized
ovulations than cows 13 to 20 d postestrus. (71). Approximately 90 % of the cows in the
OVPLUS treatment would be between 5 to 12 d postestrus. Vasconcelos et al. also
reported that cows 5 to 9 d postestrus at the commencement of Ovsynch had the higher
average serum P4 concentrations at the time of PGan than cows that were in any other
stage of the estrous cycle (71). This is consistent with the findings of the current study.

These data demonstrate that conception rates decline as parity increases. This
trend is consistent with literature reports where synchronization of ovulation was not used
(25). However the effect of parity on conception rate in this study is at variance with
previous data on synchronization of ovulation (48). In the previous report in which
synchronization of ovulation was used, second parity cows had a higher conception rate
than first or 33 parity. The reason for the disparity in my data and the other published
reports is unclear.

The positive association of milk production with conception rate in the current
study is contrary to previous reports. When cows are inseminated following a detected

estrus, it has been reported previously that milk production negatively affects conception

28

rates (1 1, 15, 25, 65). In my data, after treatment with Ovsynch cows that produce more
milk in their first parity or third and greater parity are more likely to conceive compared
to cows with lower milk yield. However this positive effect of yield on fertility is not
seen in second parity animals. A difference between experiments is that cows in the
present study were all inseminated without regard for estrus following synchronization
with Ovsynch. If high yield cows experienced higher synchronization rate, this would be
consistent with greater conception rate. However, this experiment was not designed to
test whether a difference in synchronization rates exists between low and high producing
cows. No data exists regarding the effect of milk yield on follicle growth and subsequent
synchronization rates following Ovsynch. If low producing cows have a lower
synchronization rate, they would have a lower conception rate following treatment with
Ovsynch. An additional possible explanation for the effect of milk yield on conception
rate observed in this study is that the high milk producing cows may be healthier at the
time of AI. These cows may have experienced less body condition loss after calving and
experienced fewer metabolic problems and greater health should support greater fertility.
In summary, the OVPLUS protocol increased the percentage of cows in luteal
phase of the estrus cycle before starting Ovsynch. However timing cows into a luteal

phase did not increase conception rates after treatment with Ovsynch.

29

Chapter 3
Effect of various schedules to inject Prostaglandin F20, and
second GnRH during Ovsynch on conception rate, ovulatory

follicle size, and subsequent progesterone

30

Introduction

Detecting estrus in lactating dairy cows is a difficult and labor intensive responsibility for
dairy producers (5 9). Average success to detect estrus is only 50 % and severely limits
the use of Artificial Insemination (AI) and reproductive performance of dairy cows. To
assist with this problem, a program to synchronize ovulation (Ovsynch) was developed
(4 7). Lactating cows were inseminated prior to an ovulation that was controlled by
exogenous hormones injected during a 10 d period before Al. The specific schedule of
injections for Ovsynch is inject gonadotropin releasing hormone (GnRH) —wait 7 d-inj ect
Prostaglandiana (PGan)-wait 2 d-inject GnRH-wait 24 h-AI. Ovsynch is effective to
manage pregnancy rate (5) and to decrease days to first and subsequent AI (4 6).
However, the Ovsynch program requires that dairy farm managers must perform
injections on three separate occasions. A program for synchronization of ovulation with
two injections is appealing because there would be fewer times to handle animals and less
farm labor. Because it is possible to increase efficiency, this advantage may increase the
adoption of synchronized breeding. Furthermore, the optimal time between the
Prostaglandian0L (PGFZQ) injection and the second gonadatropin releasing hormone
(GnRH) for maximal conception rates is unknown. Two experiments were conducted to
test whether synchronized ovulation could be achieved when cows receive GnRH and
PGFZG simultaneously and to determine the optimal time between PGan and the final
GnRH of Ovsynch. Experiment 2 of this thesis was designed to test whether the second
GnRH injection of the Ovsynch program can be moved and given concurrently with

PGqu and allow to similar conception rate compared to Ovsynch. Experiment 3 of this

31

thesis was designed to test for the optimal time between the injection of PGan and the
second GnRH during Ovsynch to maximize fertility.

Materials and Methods

Experiment 2
To evaluate the effects on conception rate of administering PGan and the second GnRH

of the Ovsynch protocol (Figure 3) simultaneously, 218 non-pregnant lactating dairy

 

 

Ovsynch (OV)-control
GnRH For,“ GnRH AI

.1 7. l 1
1 ~ r—

GnRH PGF2m & GnRH Al
Modified Ovsynch (MOV)

 

 

 

 

 

 

 

Figure 3. Description of the timing for each of the injections used in the Ovsynch
and the modified Ovsynch protocol.

 

 

cows >60 d in milk were assigned randomly to the control or treatment group. The study
occurred February through May 1998. All cows were located at Nobis Dairy (St. Johns,
MI) and the rolling herd average is greater than 23,000 pounds of milk. All cows were
housed in freestall barns with free access to feed and water. Controls were treated with
the standard Ovsynch program (OV) and the treatment group received a modified version

of Ovsynch (MOV--Figure 3-1). All GnRH injections were 100 jig/dose of Cystorellin®

(Merial, Ltd., Iselin, NJ) and all PGan injections were 25mg/dose of Lutalyse®

32

 

(Pharmacia-Upjohn Co., Kalamazoo, MI). The farm owner performed all Al. The herd
veterinarian diagnosed pregnancy 36 d after AI by rectal examination of the uterine horns
and ovaries. Synchronization rates were analyzed in a randomly selected subset of cows
(n=85). To determine synchronization, serum progesterone (P4) levels and ovarian
ultrasonography data were collected. An ovulation was categorized as synchronized if
the CL regressed (indicated by serum P4 concentration decreasing to below 1 ng/ml) and
if the dominant ovulatory follicle disappeared (indicated by ultrasound). To determine
CL regression, blood was sampled from a coccygeal vessel from the subset cows
concurrent with and two days after injection of PGFZQ. Progesterone concentration was
quantified in blood serum by RIA Coat-A-Count kits from Diagnostic Products
Corporation (Los Angeles, CA). Intra- and interassay coefficients of variation were 5.6
%and 9.1 %, respectively. Ultrasonography was performed at the time of the injection of
GnRH and 48 h post-injection to determine ovulation of the dominant follicle(s). All
ovarian structures were measured and mapped using a Pie Medical 200 ultrasound
machine with a 7.5 MHz transducer. Disappearance of a follicle and appearance of a new
CL in the location of the antecedent follicle by ultrasound were considered an ovulation.
In previous experiments, ovulation occurred 24 to 32 hours after the second injection of
GnRH (4 7). Differences in pregnancy and synchronization rates were tested for
significance using Chi square analysis. Size of the ovulatory follicle at the time of the

second injection of GnRH and P4 concentrations were analyzed using Student’s T-test.

33

Experiment 3
This experiment was conducted to determine the optimal period of time between the
injection of PGFZCl and the second injection of GnRH to maximize conception rates after

Ovsynch. During May and June of 1999, 457 cows from five Michigan dairy farms were

 

 

 

 

GnRH PGFZG GnRH
24 h t 7 d '36:
GnRH PGF2m GnRH
GnRH “POP,“ GnRH
12 h I 7 d I 12 h
0 h t 7 d 16 h
GnRH PGFZa & GnRH AI
Figure 4. Description of the timing for each of the injections used in the
36 h (control), 24 h, 12 h, and 0 h treatments.

 

 

 

 

 

 

used for this study. All non-pregnant cows that were beyond the voluntary waiting
period were assigned randomly to one of four treatment groups (Figure 4). Each farm
was allowed to use its own VWP. The 36 h treatment served as our control because it
provided conception rates similar to AI 12 h after detected estrus (4 9). The other three
treatments had different periods of time (24 h, 12 h, and 0 h) between the PGFZQ and the
second GnRH of Ovsynch (Figure 4). Interval from first injection of GnRH to PGFzCl and
interval from second GnRH to AI were identical for all treatments. All injections were
scheduled so Al for all groups would occur on the same calendar date. Laboratory

personnel administered all injections. Pregnancy diagnoses were performed using

ultrasound at 28 d after AI. Differences in pregnancy rate among groups were examined

34

by Chi Square analysis within the Proc GENMOD function of SAS (27). Cows from two
farms in the study were selected for determination of synchronization rate (n=102). This
subset of cows was prescribed to the same schedule to sample blood and to examine
ovaries as described in experiment 2. In this experiment an Aloka SSD-900 ultrasound
machine with a 7.5 MHz transducer was used. Also, a group of cows (n=63) that
ovulated in response to the second GnRH injection had blood sampled on 4, 6, 8, 11, 13,
15, and 18 d postestrus of the cycle subsequent to induced ovulation. All serum samples
were analyzed for P4 by the RIA method described in experiment 1. Differences in size
of ovulatory follicle were examined by AN OVA using the general linear model
procedure of SAS. Synchronization rate was examined for differences among groups
using Chi Square within the Proc GENMOD function of SAS (2 7). Serum P4 levels

during the subsequent cycle were analyzed using the Proc MIXED firnction of SAS (2 7).

Results

Experiment 2

Results of experiment 2 are shown in Table 3. Synchronization rate and percent of cows
<1 ng/ml P4 two days following PGFZa were not different (p > 0.1) between 0V and
MOV. Diameter of the ovulatory follicle at the time of second GnRH was greater in OV

than MOV (p < 0.05). Conception rate in OV was greater than for MOV (p < 0.025).

Experiment 3
As in experiment 2, synchronization rate was not different among all groups (P > 0.28;
Figure 5). Size of the ovulatory follicle had a significant linear effect. As time increased

between PGFZG and the second GnRH follicle size increased (p < 0.05; Figure 6).

35

 

 

 

 

 

 

 

Table 3--Comparison of synchronization rate, CL regression, follicle size and
pregnancy rate between OV (control) and MOVl groups

OV MOV P value
Synchronization rate (%) 89 (nflS) 85 (n=40) > 0.10
Regress CL to PGF2(ll 97.5 (n=45) 100 (n=40) > 0.10
Injection (%)
Average Ovulatory 14.5 (n=45) 13.3 (n=40) < 0.05
Follicle Size (mm)
Conception Rate (%) 31.3 (n=109) 14.7 (n=109) < 0.025
‘Mov =GnRIl—7d—PGF2a and GnRH—16 h--Al

 

 

 

However, the ovulatory follicle size at O-hour was significantly smaller than the follicle
size in the 36-hour group (p < 0.05). All other pair comparisons of follicle size were not
different within the linear trend (p > 0.17). Pregnancy data had a significant linear trend
(p < 0.0001; Figure 7). As time between PGFZQ and GnRH injection increased
conception rate increased, with 36 h having the highest conception rate. Pregnancy rates
were different when the 0 h group was compared to 24 and 36 h (p < 0.01); significant
difference was also detected between the 12 h and 36 h treatment group (p < 0.01).
Serum P4 levels following A1 are reported in Figure 8. Cows in the 0 and 36 h treatment
groups had similar mean serum P4 throughout the estrous cycle subsequent to AI. On d
11, 0 h group was different than 12 and 24 h groups, and 36 h group was different than 24
h group (p < 0.05). On d 13, 0 and 36 h groups had significantly greater serum

progesterone concentrations than 24 h group (p < 0.02).

36

 

 

 

_s
O
O

85.74 87-80

95 80

Q)

.N .

2:3 60

.C

0

g _

U, 40

.\°

N
O .

 

 

 

0 12 24 36
Times for exogenous GnRH after PGFth b

Figure 5. Synchronization of ovulation rates in lactating dairy
cows treated with GnRH at specific times after PGFZa

aSynchronization=CL regression and disappearance of dominant
follicle

t’No differences in treatments (P>0.24) and no significant linear
trend (P>0.16)

 

37

 

 

 

14.58

14-

12-

Follicle Size (mm)

 

 

10 -
0 12 24 36
Times for exogenous GnRH after PGFth

Figure 6. Size of the ovulatory follicle in lactating
dairy cows at time of final GnRH at specific times
after PGFZab.

aTreatments different from each other (P < 0.05)
bData has significant linear effect (P < 0.05)

 

38

 

 

 

30 l 27.94

% Pregnant

 

 

0 ' 0 12 24 36
Treatment

Figure 7. Conception rates in lactating dairy cows following
treatment with GnRH at specific times after PGFZab.

aBars with different superscript letters are significantly
different (P < 0.01)
bData significantly linear (P < 0.0001)

 

39

 

 

 

P4 nglni

 

 

 

 

n=63
li’oifirl

group ‘
i ...12hourl
l group
+24h0ll'

group
l *36hour
i 9MP .

 

 

 

 

 

 

 

 

 

 

 

Dayonyde

Figure 8. Mean serum progesterone concentrations in
lactating dairy cows in the estrous cycle after modified
versions of Ovsynch. The modified versions involved
injections of GnRH at specific times (0, 12, 24, 36 h)

after exogenous PGFZa.

80 h mean concentration different than 12 and 24 h mean
concentration, and 36 h mean concentration

different than 24 h mean concentration (P < 0.05)

b0 and 36 h mean concentration different than 24 h
mean concentration (P < 0.02)

 

40

 

Discussion
This study was designed to determine the effect of different intervals from CL regression
to the LH surge on reproductive performance in dairy cows. To our knowledge, this is
the only study that directly compared the effect of different intervals from PGan to LH
surge on conception rates in lactating dairy cattle. This study tested a GnRH-induced LH
surge at 0, 12, 24, or 36 h following PGan induced CL regression. This was facilitated
by the Ovsynch program (4 7). Ovsynch will synchronize follicle growth and luteal
function using GnRH and PGFZQ. From previous data after Ovsynch, the first injection of
GnRH ovulates a follicle and initiates a new follicle wave 90 % of the time (47). The
injection of PGFZQ regressed the CL 97 % of the time (49). The second injection of
GnRH caused ovulation 24 to 32 hours after injection. In previous studies,
synchronization rate was 83 to 100 percent of lactating cows (18,47). In the current
experiments, synchronization rates for different treatment groups ranged from 75 to 89
percent. In previous studies with Ovsynch, the second injection of GnRH occurred at 36
to 48 h afier PGan. In previous studies, conception rates after Ovsynch were not
different from conception rates achieved with AI 12 h after detected estrus (46,48). Thus
the basic Ovsynch program with modifications provides an ideal opportunity to
manipulate occurrence of an LH surge at various times after luteal regression. Our
question was whether different timing of an LH surge will affect conception rates. To
avoid bias between treatments, follicle growth was synchronized to provide a follicle of
similar age in all groups at the time of PGan. Another procedure to avoid bias was that
cows in all groups were inseminated on the same calendar date, at the same time of day,

and the inseminator did not know treatments assigned to individual cows. Selection of 36

41

h as our control group was based on similar conception rates for cows inseminated 12 h
after standing estrus to conception rate in cows inseminated afier Ovsynch when PGFZa
was injected 30 to 36 h before the second injection of GnRH (49). It is also reported that
48 h between PGan and second GnRH injection provided similar conception rates to
cows bred from a standing estrus (46). Based upon these data we were confident that 36
and 48 h would have similar results. In previous Ovsynch studies, the time of 36 to 48 h
between PGan and the second GnRH was selected because this was the maximum
amount of time that could pass afier PGF2Cl before cows begin to display signs of estrus
and to exhibit endogenous spontaneous preovulatory secretion of LH (35, 69). Thus, any
modifications of Ovsynch that still allow AI without estrus must shorten, not lengthen,
the interval between PGFzCl and second GnRH injection of Ovsynch. That is why all
intervals tested in Experiment 3 were greater than or equal to 36 h.

Previous studies evaluated the fertility of lactating dairy cows following
synchronization of ovulation. Conception rates varied from 27 to 39 percent (5,46, 48).
However, none of these studies directly compared different time periods between PGFZQ
and second GnRH injection. Data from our study proves that shortening the interval
between PGF2Cl and the second injection of GnRH to less than 36 h will reduce
conception rate.

This study demonstrates that the negative impact of increasing time between
PGFzCl and the second GnRH injection on conception rates was not due to decreased P4
production from the CL resulting from a smaller ovulatory follicle. This is contrary to

published reports from sheep (40) in which decreased P4 production from CL formed

from a smaller ovulated follicle of the ewe. Our ultrasound scans demonstrate that the 0

42

h group had a smaller follicle. However, the P4 concentrations after AI did not indicate
any form of luteal insufficiency for the smallest follicle group (0 h). On d 11, 0 h group
actually had higher serum P4 than the 12 h or the 24 h group. At all times during the
subsequent cycle the 0 h cows had serum P4 concentrations similar to the 36 h group.
From these data, there is no evidence that follicle size (13.23 mm to 14.58 mm) at the
time of the LH surge affects subsequent serum P4 levels.

For cows in 0 h group, PGFZG began CL regression at the same time an LH surge
was being induced. In contrast, when cows were injected with PGFZQ and a GnRH
agonist at the same time CL regression was delayed by 3 to 6 d (37). In this study, all
cows injected with PGan and GnRH concurrently had serum P4 levels at sub-luteal
levels within 48 h. Concurrent injection of GnRH with PGFz0L did not block luteal
regression induced by exogenous PGan.

In the current study, decreased time between decline of P4 until the LH surge
decreased conception rates. It is not clear why conception rates were different between
treatments. Conception rates are not associated with P4 levels in estrous cycle subsequent
to AI. Possible explanations for the difference in conception rates are impaired oviduct
contractility (53,54), impaired oocyte transport (72), impaired sperm transport (24), and a
less than ideal endometrial environment (61).

In addition, decreased fertility in the treatment groups with shorter time between
PGan and the final GnRH may be a result of ovulating a smaller less mature follicle.
This is contrary to previous reports in which ovulating a smaller follicle resulted in higher
conception rates (70) and a more competent oocyte (51). Perhaps, a critical time exists in

the dominant follicle growth pattern when oocytes reach maximal fertility potential.

43

In conclusion, ovulation can be synchronized when the GnRH and the PGFzCl
injections are administered together. However, injecting GnRH 24 h or less afier PGan
will reduce conception rate. Luteal insufficiency does not cause the decreased
conception rates in the treatment groups with less than 36 h between the PGFz0L and the

final GnRH.

44

CHAPTER 4

General Discussion

45

General Discussion

The results presented in this thesis add to our understanding of synchronization of
ovulation in lactating dairy cattle. The modifications of the Ovsynch program tested in
this thesis did not increase conception rates compared to standard Ovsynch.
Manipulating lactating dairy cows into a mid-luteal phase of the estrous cycle at the
beginning of Ovsynch provided conception rates that were similar to cows that were
started on Ovsynch at a random time of the cycle. Another option to increase conception
rates after treatment with Ovsynch is to use exogenous progesterone before beginning

Ovsynch. Exogenous progesterone sources during the Ovsynch program has not been

reported.

When the last two injections of Ovsynch were given concurrently conception rate
was reduced and there was no effect on synchronization rate. The final experiment in this
thesis tested the time between the PGan and the final GnRH of Ovsynch. Results
demonstrate that as time between the PGFZQ and the final GnRH increase, the conception
rate increases with 36 h between PGFz0L and the final GnRH of Ovsynch yielding the
highest conception rates in my study. The lowered conception rate with less than 36 h
between the PGFZQ and the second GnRH was not due to luteal insufficiency. Cows with
less time between the PGan and the second GnRH did ovulate a smaller follicle and this
is possible cause for the reduced fertility. The final two experiments of this thesis
provide insight into the physiological mechanism needed to establish pregnancy. It is
now established that to achieve acceptable conception rates progesterone concentrations
must be declining before an ovulatory follicle releases the oocyte. This thesis did not

address why serum progesterone concentrations must be declining prior to ovulation, but

46

the discussion in chapter 3 presented some plausible reasons. Testing the reasons
mentioned in chapter 3 of this thesis may provide insight into the problems with dairy
cattle fertility.

Synchronization of ovulation is an effective method to control reproduction of
lactating dairy cattle, but current methods are not perfect. This thesis pursued some
opportunities to increase success and applicability of synchronized ovulation. However,
the modifications of Ovsynch tested in this thesis were not effective and should not be

employed by dairy producers.

47

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58

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