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Response to prostaglandin Fâ‚‚[subscript alpha] (PGFâ‚‚[subscript alpha]) and gonadotropin-releasing hormone (GnRH) in Bos taurus, Bos indicus, and Bos taurus x Bos indicus cattle

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Title:
Response to prostaglandin Fâ‚‚[subscript alpha] (PGFâ‚‚[subscript alpha]) and gonadotropin-releasing hormone (GnRH) in Bos taurus, Bos indicus, and Bos taurus x Bos indicus cattle
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Portillo, German E
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English
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xiii, 286 leaves : ill. ; 29 cm.

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Subjects / Keywords:
Breeding ( jstor )
Cattle ( jstor )
Estrus ( jstor )
Estrus cycle ( jstor )
Heifers ( jstor )
Luteolysis ( jstor )
Ovulation ( jstor )
Pregnancy rate ( jstor )
Prostaglandins ( jstor )
Zebu ( jstor )
Animal Sciences thesis, Ph.D ( lcsh )
Dissertations, Academic -- Animal Sciences -- UF ( lcsh )
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bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

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Thesis:
Thesis (Ph.D.)--University of Florida, 2003.
Bibliography:
Includes bibliographical references.
General Note:
Printout.
General Note:
Vita.
Statement of Responsibility:
by German E. Portillo.

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University of Florida
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University of Florida
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Copyright [name of dissertation author]. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
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RESPONSE TO PROSTAGLANDIN F2a (PGF2a) AND GONADOTROPIN-
RELEASING HORMONE (GnRH) IN Bos taurus, AND Bos taurus x Bos indicus
CATTLE













By

GERMAN E. PORTILLO


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

UNIVERSITY OF FLORIDA


2003

























This dissertation is dedicated to my parents Mary and Heberto, for their
unconditional support; to my loving wife Marisol and my children Valeria,
Eduardo, and Vivian, for inspiring me and giving me reason to achieve; and
above all to God, for giving me the desire and ability to accomplish my goals.














ACKNOWLEDGMENTS

I would like to express my sincere gratitude to the chairman of my

supervisory committee, Dr. Joel V. Yelich, for his guidance, knowledge and

patience throughout my graduate program. Appreciation is also extended to the

members of my supervisory committee (Drs. William W. Thatcher, Maarten

Drost, and Ramon C. Littell) for their valuable insight and important contributions.

Special thanks go to my lab partners and friends (Eric Hiers, Christin

Barthle, Janis Fullenwider, Mary-Karen Dahms, Jennifer Araujo, Joseph Kramer,

and Tim Dickerson). They devoted their time and skill in the field and in the lab,

and truly made this an enjoyable experience. I would also like to thank the crew

at the Santa Fe Beef Research Unit and the Beef Teaching Unit for their

cooperation and facilitation during the experiments. Particular thanks go to Bar-L

Ranch, Mariana, FL, and Deseret Cattle and Citrus, Deer Park, FL, for cattle

used in this research. I would also like to thank InterVet, Inc.; Pharmacia-Animal

Health; and Schering-Plough for donation of drugs used in the different

experiments. Thanks to Dr. Neal Shrick at University of Tennessee for doing the

LH and estrogen assays. I am also grateful to the entire Department of Animal

Sciences for their excellent graduate program.

I thank Dervin Dean, a friend who has been like a brother for many years.

I also would like to express my sincere appreciation to all my friends in








Gainesville, for their companionship and shared laughs. They will be esteemed

for a lifetime.

I especially thank my parents, Mary and Heberto Portillo for their love,

wisdom and support. Without them none of this would have been possible. I

would also like to thank my sisters and their respective families for lifting me up

when I thought I could go no farther.














TABLE OF CONTENTS
apaqe

ACKNOWLEDGMENTS ............. ................................................ iii

LIST O F TABLES........................ .................................................................. viii

LIS T O F F IG U R E S .................... ..................................................................... x

A B S T R A C T ..................... ....................... .............................................. xii

CHAPTER

1 IN T R O D U C T IO N ................................................................... ................... 1

2 REV IEW O F LITERATURE .................................................... .................. 6

Introd uctio n ............................... .... .................................................. 6
Hypothalamic-Pituitary-Ovarian Axis and Primary Hormones of
R e p ro d u ctio n ......................................................................... .................. 7
Gonadotropin Releasing Hormone (GnRH) ..........................................7...
Follicle Stimulating Hormone (FSH) and Luteinizing Hormone (LH) ........8
Estrogens and Progesterone .......................................... .................. 11
P ro stag la nd ins ................................................................ .......... ......... 17
Estrous Cycle in Cattle ................... .................................................. 18
Estrus ................ .......... .. ........... ........ ...... ...... ............ 18
M e testrus ............................................... .......................................... 19
Diestrus ................... ................. .................20
P ro e strus............................................................................................ 2 0
Regulation of Ovarian Follicle Growth, Atretic Demise and the Ovulatory
Response .......... .............................. .... 21
Ovulation, Corpus Luteum Development, and Luteolysis..........................27
Exogenous Control of Ovarian Follicles and Corpus Luteum Dynamics ......38
E stro g e n s ..................................................................... .................... 3 9
Progesterone and Progestagens ......................................................40
Combination of Estrogens and Progestagens........................................43
Gonadotropin Releasing Hormone (GnRH) .........................................45
Prostaglandin F2a (PG F2a)................................................................... 52
Estrus Synchronization Systems and Time Artificial Insemination ...............55
S um m ary ....................... ......................... 72








3 RESPONSE TO A PROSTAGLANDIN F2a INJECTION ON EITHER DAY
SIX OR SEVEN OF THE ESTROUS CYCLE IN ANGUS AND
BRAHMAN x ANGUS HEIFERS ........................................ ..................... 73

Introduction................................................................................ 73
Materials and Methods ............ ................................................. 74
Results .. .................. ......... .......... ....................... 80
D iscussio n .................................................. ............................................ 87
Im plicatio ns ................................................ .......................................... 10 2

4 ENDOCRINE AND REPRODUCTIVE RESPONSES OF ANGUS,
BRANGUS, AND BRAHMAN x ANGUS HEIFERS TO A GNRH
INJECTION ON DAY 6 OF THE ESTROUS CYCLE FOLLOWED BY
PROSTAGLANDIN F2a 7 DAYS LATER..................... ........................ 103

Introduction............. ..................................... 103
Materials and Methods ................... ................................................. 104
Results ................ .... .......... ......... ............ ........... ... 111
D iscussio n ................................................. ........................................... 12 0
Im plicatio ns ................................................ .......................................... 136

5 EFFECT OF PLASMA PROGESTERONE CONCENTRATIONS ON A
PROSTAGLANDIN F2a INDUCED LUTEOLYSIS IN ANGUS AND
BRAHMAN x ANGUS HEIFERS ...................................... ....................... 137

Intro d u ctio n ......................................................................... .................. 13 7
Materials and M ethods ................... ................................................. 138
Results .......................................... .............. ... 142
D iscu ss io n ......................................................................... ........... ....... 14 8
Im plicatio ns ................................................ .......................................... 16 3

6 EFFICACY OF A SINGLE VERSUS SPLIT INJECTIONS OF PGF2a IN A
GNRH + PGF2a SYNCHRONIZATION PROTOCOL IN COMBINATION
WITH MELENGESTROL ACETATE (MGA) IN CROSSBRED Bos
INDICUS C A TT LE ............. .............................................. ...................... 164

Introduction................................ .............................. 164
M materials and M ethods ........................................................ ................. 165
Results ................. ...... .. ........ ...... ......... ...... .......... 171
Discussion .................................................... 182
Im p licatio ns ................................................ .......................................... 2 04

7 CONCLUSIONS AND IMPLICATIONS .................................................... 205








APPENDIX


A ABSTRACT FOR EXPERIMENT 1...... ........ .. ................... 219

B ABSTRACT FOR EXPERIMENT 2.......................................................... 221

C ABSTRACT FOR EXPERIMENT 3.................................... .................... 224

D ABSTRACT FOR EXPERIMENT 4.................... .............. .................... 226

E CORPUS LUTEUM REGRESSION IN CYCLING ANGUS AND
BRAHMAN x ANGUS HEIFERS (EXPERIMENT 1)................................. 228

F ODDS RATIOS AND CONFIDENCE INTERVALS (EXPERIMENT 1).......229

G PRE-SYNCHRONIZATION ESTRUS RESPONSE IN CYCLING ANGUS,
BRAHMAN, BRAHMAN X ANGUS AND BRANGUS HEIFERS
(EXPERIM ENT 2)........ ............................. ............ ................... 230

H PLASMA LH CONCENTRATIONS FROM GNRH INJECTION FOR
HEIFERS IN THE ANGUS, BRANGUS, AND BRAHMAN X ANGUS
BREED GROUPS (EXPERIMENT 2)...................................... 231

I REGRESSION OF TOTAL CORPUS LUTEUM VOLUME WITH
PROGESTREONE CONCENTRATIONS AT PGF2a INJECTION IN
ANGUS, BRANGUS AND BRAHMAN X ANGUS HEIFERS TREATED
WITH GnRH FOLLOWED BY PGF2a 7 DAYS LATER
(EX P ER IM E NT 2)............................................................. ..................... 237

J ODDS RATIOS AND CONFIDENCE INTERVALS (EXPERIMENT 3).......238

K ESTROUS, CONCEPTION, AND PREGNANCY RATES OF CYCLING
AND NONCYCLING Bos taurus X Bos indicus COWS AND SURVIVAL
ANALYSIS DESCRIBING THE PROPORTION OF CYCLING AND
NONCYCLING Bos taurus x Bos indicus COWS THAT DID NOT EXHIBIT
ESTRUS AFTER BEING SYNCHRONIZED WITH GNRH AND EITHER
SINGLE OR SPLIT DOSES OF PROSTAGLANDINF2a (PGF2.) IN
COMBINATION WITH MELENGESTROL ACETATE (MGA)
(EXPERIM ENT 4) ......................................... ...... .................... 240

LIST OF REFERENCES..... ........................................ 245

BIOGRAPHICAL SKETCH .................. ......................... 286














LIST OF TABLES


Table page

3-1 Corpus luteum (CL) regression and ovarian characteristics in Angus and
Brahman x Angus heifers treated with prostaglandin F2. (PGF2.) on
either d 6 or 7 of the estrous cycle ................................... ..................... 81

3-2 Estrous response and behavioral estrus characteristics in Angus and
Brahman x Angus heifers treated with prostaglandin F2a (PGF2a) on
either d 6 or 7 of the estrous cycle ................................... ..................... 85

4-1 Follicle size, estradiol and progesterone concentrations prior to a GnRH
treatment (100 jg) on d 6 of the estrous cycle and ovulation rate after
GnRH in Angus, Brangus, and Brahman x Angus heifers...................... 112

4-2 Mean LH concentrations, LH peak-height, and interval from GnRH to LH
peak in Angus, Brangus and Brahman x Angus heifers in response to
GnRH treatment (100 [tg) on d 6 of the estrous cycle ............................ 117

4-3 Estrous response, follicle size at prostaglandin F2a (PGF2a) and
behavioral estrous characteristics after PGF2a for Angus, Brangus, and
Brahman x Angus heifers in response to a GnRH + PGF2a
synchronization protocol.................................................. ................... 118

4-4 Total corpus luteum (CL) volume, volume of the original and accessory
CL and plasma progesterone concentrations at prostaglandin F2a
(PGF2a) injection for Angus, Brangus, and Brahman x Angus heifers in
response to a GnRH + PGF2. synchronization protocol......................... 119

5-1 Breed and progesterone group effects on follicle size at GnRH,
ovulation rate after GnRH, and progesterone concentration at GnRH in
Angus and Brahman x Angus heifers in the High and Low progesterone
groups ................ ......... ........................................ 145

5-2 Breed and progesterone group effects on heifers with two corpora lutea
(CL) at PGF2a, CL regression rate after PGF2a, follicle diameter at PGF2a,
progesterone concentration at PGF2a, and estrous response after PGF2a
in Angus and Brahman x Angus heifers in the High and Low
progesterone groups. ...... ..... ................... .. .......... ................... 147








6-1 The effect of treatment and cycling status on estrous, conception and
pregnancy rates of Bos taurus x Bos indicus cows synchronized with a
modified GnRH + prostaglandin F2. (PGF2a) protocol in combination
with melengestrol acetate (MGA) .............................. 172

6-2 Estrus, conception, and pregnancy rates of Bos taurus x Bos indicus
cows synchronized with GnRH and either a single or split doses of
prostaglandin F2a (PGF2,) in combination with melengestrol acetate
(MGA) treatment for cows with progesterone 1 ng/ml at PGF2a............ 176

6-3 Estrus, conception, and pregnancy rates of Bos taurus x Bos indicus
cows synchronized with GnRH and either single or split doses of
prostaglandin F2a (PGF2a) in combination with melengestrol acetate
(MGA) treatment for cows with progesterone < 1 ng/ml at PGF2 ............177

6-4 Estrus, conception and pregnancy rates of Bos taurus x Bos indicus
cows synchronized with GnRH and either single or split doses of
prostaglandinF2, (PGF2a) in combination with melengestrol acetate for
cows classified by cycling status and progesterone (P) status
(H = >1 and L = <1 ng/mL) on d -10, 0, and 7 of the experiment..............178

6-5 Conception and timed-Al pregnancy rates by Al sire in Bos taurus x Bos
indicus cows synchronized with GnRH and either a single or split doses
of prostaglandin F2a (PGF2.) in combination with melengestrol acetate
(M G A ) treatm ent .................. .. ....................................... .......... 181














LIST OF FIGURES


Figure page

3-1 Experimental protocol Experim ent 1................................ ...................... 76

3-2 Progesterone concentration profiles in Angus and Brahman x Angus
heifers that either regressed or did not regress their CL after a single
PGF2. injection on either d 6 or 7 of the estrous cycle............................ 82

3-3 The effect of corpus luteum (CL) volume on progesterone concentrations
on either d 6 or 7 of the estrous cycle in Angus and Brahman x Angus
heifers. ........... .......................... .... .................. 84

3-4 Distribution of mounts received during 3 h periods -.hroughCou the duration
of estrus after an injection of PGF2, on either d 6 or 7 of the estrous cycle
in Angus and Brahman xAngus heifers............................. .................... 86

3-5 Effect of number of heifers in estrus within a 3 h period on number of
m ounts received during a period ...................................... ..................... 88

3-6 Survival analysis representing the percentage of Angus and Brahman x
Angus heifers that did not exhibit estrus during the 168 h after PGF22 on
either d 6 or 7 of the estrous cycle .................................... .................... 89

4-1 Experimental protocol Experiment 2....................... ........................ 106

4-2 Intensive blood sampling protocol to determine luteinizing hormone,
progesterone, and estradiol concentrations in cycling Angus, Brahman,
Brangus and Brahman x Angus heifers receiving GnRH on d 6 of the
estrous cycle. ......................................... .... ............. ... ................... 107

4-3 Plasma progesterone concentrations in cycling Angus, Brangus and
Brahman x Angus heifers receiving 100 pig GnRH on d 6 of the estrous
cycle. ............... .. .. .. ............................ ..... .. ............. 113

4-4 Plasma LH concentrations in cycling Angus, Brangus and Brahman x
Angus heifers receiving 100 pg GnRH on d 6 of the estrous cycle ..........115

4-5 Progesterone concentrations after a PGF2a treatment administered on d 6
of the estrous cycle in cycling Angus, Brangus and Brahman x Angus,








which received GnRH on d 6 of the estrous cycle followed by PGF2,
treatm ent 7 d latter. ....... .. ........ ........... .......... ................... 121

5-1 Experimental protocol Experiment 3........................ ......................... 139

5-2 Progesterone concentrations for the two days before GnRH (d 6 of the
estrous cycle) for heifers in the high progesterone group n.:lu'irng Angus
and Brahman x Angus and heifers in the low progesterone group
including Angus and Brahman x Angus heifers..................................... 143

5-3 Progesterone concentrations between GnRH and PGF2. treatment for
heifers in the high progesterone group including Angus and Brahman x
Angus and heifers in the low progesterone group including Angus and
Brahm an x Angus heifers................................................. .................. 146

5-4 Regression of progesterone concentrations at PGF2. on corpus luteum
regression in Angus and Brahman x Angus heifers in the Low
progesterone group ..................... .......... ................... 149

6-1 Experimental protocol Experiment 4............................ 166

6-2 Survival analysis describing the proportion of Bos taurus x Bos indicus
cows with progesterone concentrations > 1 ng/mL at PGF20 compared to
cows with progesterone concentrations < 1 ng/ mL that did not exhibit
estrus after being synchronized with GnRH and either single or split
doses of PGF2a system in combination with melengestrol acetate........... 174

6-3 Timed-Al and total CL regression rates in Bos taurus x Bos indicus cows
synchronized with GnRH and either a full or two consecutive split .,
injections of PGF2a in combination with MGA treatment ......................... 180













Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

RESPONSE TO PROSTAGLANDIN F2a (PGF2a) AND GONADOTROPIN-
RELEASING HORMONE (GnRH) IN Bos taurus, AND Bos taurus x Bos indicus
CATTLE

By

German E. Portillo

December 2003

Chair: Joel V. Yelich
Major Department: Animal Sciences

A series of experiments were conducted to evaluate the response of Bos

taurus, Bos indicus, and Bos taurus x Bos indicus cattle to PGF2a and GnRH

treatments. Experiment 1 was replicated twice and cycling Angus (AN) and

Brahman x Angus (BA) heifers were used to evaluate the effectiveness of a

single injection (25 mg i.m.) of PGF2, administered during the early estrous cycle

(d 6 or 7) to initiate corpus luteum (CL) regression as measured by progesterone

concentrations. Breed had no effect on PGF2, induced luteolysis on d 6 or 7 of

the estrous cycle. In the second experiment, Angus (AN), Brahman (B), Brangus

(BR), and Brahman x Angus (BA) heifers were used to evaluate secretary

patterns of LH and associated ovarian events in response to administration of

GnRH (100 lig i.m.) on d 6 of the estrous cycle followed by a single injection of

PGF2a 7 d later. There was a breed-dependent response to a GnRH challenge








administered on d 6 of the estrous cycle. As the percentage of Bos indicus

breeding increased, the amount of luteinizing hormone released to GnRH

decreased. In Experiment 3, AN, B, and BA heifers were used to evaluate the

effectiveness of a single injection of PGF2. administered 7 d after a GnRH

treatment on d 6 of the estrous cycle, to initiate luteolysis of an accessory CL,

which had been exposed to either low (heifers treated with 15 mg PGF2J i.m. at

12-h intervals on d 4 and 5 of the estrous cycle, followed by a single injection of

PGF2a on d 6) or high (no PGF2, treatment on d 4, 5 and 6) progesterone

concentrations before PGF2a. Progesterone exposure before PGF2. had no

effect on luteolysis. In Experiment 4, cycling and noncycling crossbred Bos

indicus lactating cows were used to evaluate the effectiveness of a single versus

split (12.5 mg i.m.) dose of PGF2,a in a 7 d GnRH/MGA/ PGF2. protocol.

Modifying the delivery of PGF2a from a single to two consecutive split injections 7

d after GnRH had no effect on subsequent estrous response and pregnancy rate.













CHAPTER 1
INTRODUCTION

Cattle of Bos indicus breeding are used extensively for beef production in

subtropical regions of the United States, and tropical and subtropical regions all

over the world. Environmental adaptability is a crucial factor in beef cattle

production in tropical and subtropical environments. Cattle of Bos indicus

breeding are characterized by their adaptation to elevated temperatures and

humidity, tolerance to internal and external parasites and the ability to use

forages of high fiber content (Frisch and Vercoe, 1984; Randel, 1994). However,

problems associated with estrus detection and response to different estrus

synchronization drugs often compromise the effectiveness of Al and estrus

synchronization protocols in cattle of Bos indicus breeding. Additionally, most

estrus synchronization research studies have been conducted using beef and

dairy cattle of Bos taurus breeding and not cattle of Bos indicus breeding.

Artificial insemination (Al) allows genes from superior bulls to be distributed

among many females without incurring the expenses of buying the animals.

Therefore, the genetic potential of a calf crop can be improved by using Al.

However, this practice takes a lot of time and effort. For Al to be effective and

efficient for beef producers, estrus synchronization and timed-Al (TAI) are

important tools.

Gonadotrophin releasing hormone (GnRH) has been combined with

prostaglandin F2a (PGF2a) to develop what is known as the GnRH+ PGF2,








estrous synchronization protocol. The GnRH is used to synchronize follicle

development so that a majority of cattle will have dominant follicle present on the

ovaries when PGF2u is administered 7 d after the GnRH treatment. In most

cases, the GnRH + PGF2, protocols are effective for synchronizing estrus in

cattle of Bos taurus breeding, but appear to be less effective in cattle of Bos

indicus breeding. Moreover, direct comparisons of the endocrine and ovarian

responses of cattle to the GnRH plus PGF2. protocol have not been made

between cattle of Bos taurus and Bos indicus breeding. Lemaster et al. (2001)

reported a very low estrous response in Bos indicus cows treated with GnRH +

PGF2., and hypothesized that the decrease was due to incomplete regression of

the accessory CL formed as a result of the GnRH treatment. Few reports in the

literature support this hypothesis. However, Hiers (2001) reported that crossbred

Bos indicus x Bos taurus cows treated with GnRH on d 18 of the estrous cycle

with PGF2. administered 7 d later tended to have a decreased luteolysis and 5-d

estrous response, compared to cows injected with GnRH on d 2, 5 and 12 of the

estrous cycle with PGF2. administered 7 d later.

There is enough literature referring the use of PGF2. and its analogues for

estrus control in both Bos taurus cattle (Watts and Fuquay, 1985; Maurer et al.,

1989; Morbeck et al., 1991; Smith et al., 1998), and cattle of Bos indicus breeds

(Landaeta et al., 1999; Rekwot et al., 1999; Williams et al., 1999; Mattoni and

Ouedraogo, 2000). The use of these synchronizing agents in cattle of Bos

indicus breeding appears to be primiarn, related to problems with estrus detection

(Orihuela et al., 1983; Pinheiro et al., 1998; Rekwot et al., 1999). However, the








effectiveness of PGF2aand its analogues on the induction of luteolysis, estrus

s,,nchr.:.r, z iion, and fertility is also highly variable (Hardin et al., 1980a, Pinheiro

et al., 1998; Rekwot et al., 1999; Mattoni and Ouedraogo, 2000).

Agonists to GnRH have been given at various stages of the estrous cycle to

induce an LH surge which induces ovulation of the dominant follicle resulting in

emergence and synchronization of a new follicular wave. The GnRH agonist

initiates an acute secretion of LH and FSH such that circulating concentrations

are elevated for a 3- to 5-h period, which stimulates ovulation (Thatcher et al.,

1993; Gong et al., 1996; Vizcarra et al., 1997; D'Occhio and Aspden, 1999). The

effectiveness of GnRH agonist to induce ovulation appears to be affected by the

stage of follicular development at the time of treatment (Prescott et al., 1992;

Silcox et al., 1993; Pursley et al., 1995; Moreira et al., 2000) and only induces

ovulation in follicles greater than 9 mm that are in the gro,.in phase of a follicle

wave (Zaied et al., 1980; Macmillan and Thatcher, 1991; Vasconcelos et al.,

1999; Moreira et al., 2000).

Information about the concentrations of LH in the peripheral circulation of

cycling cows and/or heifers after treatment with GnRH during the estrous cycle

are numerous in cattle of Bos taurus breeding (Mori and Takahashi, 1978;

Chenault et al., 1990; Williams and Stanko, 1996; Fajersson et al., 1999).

However, direct comparisons of the endocrine and ovarian responses of cattle

administered GnRH between cattle of Bos taurus and Bos indicus breeding are

few (Griffin and Randel 1978; Irvin et al., 1978; Randel, 1994).








To implement the most effective estrus synchronization system in tropical

and subtropical environments, it is necessary to develop a complete

understanding of how GnRH affects the endocrine and reproductive patterns of

cattle of Bos indicus breeding; and also how cattle of Bos indicus breeding

respond to a luteolytic injection of PGF2a. Therefore, the literature review

discusses the similarities and differences during the estrous cycle including

follicle development and luteolysis in cattle of Bos taurus, Bos indicus, and Bos

taurus x Bos indicus breeding. Furthermore, it discusses factors that influence

the effectiveness of synchronization agents GnRH and PGF2. and effectiveness

of the GnRH and PGF2, estrus synchronization system in cattle of Bos taurus,

Bos indicus, and Bos taurus x Bos indicus breeding.

The overall experimental objective was to evaluate the reproductive

response of Bos taurus, Bos indicus, and Bos taurus x Bos indicus cattle to

exogenous administration of GnRH and PGF2. in order to develop a better

understanding of these compounds for developing effective but practical estrus

synchronization protocols. There are three main objectives of this dissertation.

The first objective was to determine the effectiveness of a single injection of

PGF2a administered during the early estrous cycle (d 6 or 7) to initiate corpus

luteum regression in cycling Angus and Angus x Brahman (5/8 Angus x 3/8

Brahman and 3/8 Angus x 5/8 Brahman) heifers.

The second objective was to evaluate acute ovarian responses and LH

secretary profiles during and after administration of an exogenous GnRH agonist

on d 6 of the estrous cycle in Angus, Brahman, Brangus, and Brahman x Angus








heifers. We also evaluated the effectiveness of PGF2a to induce luteolysis when

injected 7 d after GnRH, along with the subsequent estrous response.

A third objective was to evaluate the effectiveness of a single injection of

PGF2. administered 7 d after a GnRH treatment, to initiate luteolysis of an

accessory CL exposed to either high or low progesterone concentrations in

Angus, and Brahman x Angus heifers. The low progesterone group was

achieved by njeciing PGF2a at 12-h intervals on d 4 and 5 of the estrous cycle,

followed with an injection of PGF2a on d 6. The high progesterone group did not

receive PGF2. on d 4, 5, and 6. All heifers received GnRH on d 6 and a luteolytic

injection of PGF2a 7 d later. The effects of progesterone treatment group and

breed on ovulation rate after GnRH, PGF2a induced luteolysis, and estrous

response were analyzed.

Finally, a field trial was conducted to evaluate the effectiveness of either a

single or two consecutive split injections of PGF2a administered 7 d after GnRH

treatment with melengestrol acetate administered between the GnRH and PGF2.

to synchronize estrus in postpartum lactating cows of Bos indicus breeding.

Furthermore, the effectiveness of the GnRH + PGF2. system to synchronize

estrus in either cycling or noncycling cows was determined by blood

progesterone concentrations taken before administering GnRH.













CHAPTER 2
REVIEW OF LITERATURE

Introduction

The use of cattle with varying percentages of Bos indicus breeding for

beef production in subtropical regions of the United States, and tropical and

subtropical regions of the world is widespread. However, the effectiveness of

estrus synchronization systems is often compromised because of problems

associated with estrus detection and response to different estrus synchronization

drugs in cattle of Bos indicus breeding. Moreover, development of predictable

estrus synchronization systems oriented toward cattle of Bos indicus breeding

has seldom been the primary focus of researchers. Most estrus synchronization

research has been conducted using beef and dairy cattle of Bos taurus breeding.

This review considers the hypothalamic-pituitary-ovarian axis and the

primary hormones of reproduction in the cow. The estrous cycle in the female

bovine, including regulation of ovarian follicle growth, atretic demise of follicles,

ovulation and luteolysis are briefly discussed. Exogenous control of ovarian

follicle dynamics and estrus synchronization protocols are also discussed.

Because most available research is from Bos taurus cattle, these are presented

together with appropriate reference to Bos indicus and Bos indicus x Bos taurus

cattle when data are known, or when important differences have been described.








Hypothalamic-Pituitary-Ovarian Axis and Primary Hormones of
Reproduction

In the presence of a hypothalamic releasing factor, the anterior pituitary and

ovarian hormones exert mutual control over the circulating concentrations of one

another. The complex interactions among pituitary, ovarian, and uterine

hormones involve further control by positive and negative feedback mechanisms

to sustain the estrous cycle of the cow. The primary hormones secreted by these

reproductive structures are gonadotropin releasing hormone (GnRH), the

gonadotropins follicle stimulating hormone (FSH) and luteinizing hormone (LH),

estradiol 17p, progesterone, and prostaglandin F2a (PGF2 ).

Gonadotropin Releasing Hormone (GnRH)

The neuropeptide GnRH is released from the hypothalamus to the anterior

pituitary, inducing de novo synthesis and release of LH and FSH, which control

ovarian function (D'Occhio et al., 2000).

Smith and Jennes (2001) summarized the anatomical organization of the

GnRH neuronal system. Two loose networks of neurons of the hypothalamus

control the secretion of GnRH. One network of neurons is located in the

ventromedial and arcuate nuclei of the hypothalamus. These neurons include

the tonic GnRH center, responsible for the basal secretion (small, frequent

pulses) of GnRH throughout the estrous cycle. The other groups of neurons are

located in the anterior hypothalamic area, which includes the suprachiasmatic

and preoptic nuclei. These nuclei comprise the surge center, responsible for the

preovulatory release of GnRH that stimulates the surge of LH, which initiates the

process of ovulation. Axonal projections of GnRH neurons are extended toward








many sites in the brain; however, major projections responsible for the control of

the anterior pituitary function end in the median eminence. At this point, GnRH is

released into the fenestrated capillaries of the hypophyseal portal system, which

carries GnRH to the anterior pituitary to regulate gonadotrophin secretion.

Several neurotransmitters, neuropeptide receptor mRNA, and proteins are

expressed by GnRH neurons, which regulate GnRH neuronal activity (for review,

see Parvizi, 2000; Smith and Jennes, 2001). Several neurotransmitters and

neuropeptides including catecholamines, gamma-aminobutyric acid (GABA),

glutamine, neuropeptide Y, neurotensin, vasoactive intestinal polypeptide (Smith

and Jennes, 2001), and nitric oxide (Parvizi, 2000) mediate the stimulatory

effects of estradiol on GnRH secretion. Estradiol may act directly on certain

GnRH neurons through specific nuclear receptors (Roy et al., 1999). However,

most studies have not been able to detect estrogen receptors in GnRH neurons,

or to detect estrogen accumulation in nuclei of GnRH neurons (Smith and

Jennes, 2001). For estradiol to stimulate secretion of GnRH from the GnrRH

neurons, estradiol apparently must also stimulate the neurotransmission of other

afferent neuronal systems controlling GnRH neurons (Parvizi, 2000; Smith and

Jennes, 2001). Parvizi (2000) and Smith and Jennes (2001) also suggest that

progesterone may also restrict GnRH secretion by regulating the different

neuronal systems controlling GnRH neurons.

Follicle Stimulating Hormone (FSH) and Luteinizing Hormone (LH)

Gonadotropin releasing hormone released from the hypothalamus

regulates gonadotrophin secretion from the anterior pituitary. Follicle stimulating

hormone is involved in the recruitment and development of follicles. In contrast,








LH leads to the maturation of follicles, induces ovulation, formation of the corpus

luteum (CL), and maintains the synthesis and secretion of progesterone by the

CL (Peters and Lamming, 1983). The secretary nature of FSH and factors

regulating its control are not as well understood as they are for LH

(Padmanabhan and McNeilly, 2001). Compared to LH (half-life: 30 min), the

longer half-life (4 h) and molecular heterogeneity of FSH makes it difficult to

assess its secretary patterns in the blood stream (Ulloa-Aguirre et al., 1995,

Padmanabhan and Sharma, 2001). Since LH and FSH share a common a-

subunit (Padmanabhan and McNeilly, 2001) and the gonadotropin a-subunit is

secreted in an episodic pattern (Hall et al., 1990), it is unclear whether the

pulsatile secretion of FSH is just an indication of gonadotropin a-subunit

secretion or cross-reactivity with LH. Secretion of FSH appears to be regulated

by two mechanisms. One mechanism controls the basal secretion, which

appears to be the major portion of FSH released, and the other mechanism

controls its pulsatile release (Padmanabhan and McNeilly, 2001; Padmanabhan

and Sharma, 2001). There is also evidence supporting the presence of a

separate FSH-releasing factor (FSH-RF) in the hypothalamus (Mizunuma et al.,

1983; Lumpkin et al., 1987; McCann et al, 2001). However, a variant form of

GnRH (Montaner et al., 2001) may be the putative FSH-releasing factor

(Padmanabhan and McNeilly (2001); Padmanabhan and Sharma 2001). Along

with this theory, other studies suggest the existence for separate hypothalamic

areas conIr.oIirng LH and FSH secretion (Chappel and Barraclough, 1976;








Lumpkin and McCann, 1984; Lumpkin et al., 1989), supporting the existence of a

separate FSH-releasing factor.

Factors other than GnRH have been implicated for stimulain._ the secretion

of FSH and LH. In the pituitary, local -e.gul.cors such as follistatins, activins,

inhibins and other neuroendocrine factors modulate GnRH and the subsequent

differential secretion of FSH and LH (Padmanabhan and McNeilly, 2001;

Padmanabhan and Sharma, 2001). Additionally, estradiol and inhibin secreted

by the ovaries act via negative feedback to regulate FSH (Walczewska et al.,

1999; Padmanabhan and McNeilly, 2001), with estradiol (Walczewska et al.,

1999) being the central effector. Furthermore, the adipocyte hormone leptin

induces the secretion of FSH and LH (Walczewska et al., 1999; McCann et al.,

2001).

The stage of the estrous cycle differentially influences the secretary pattern

of LH. A GnRH pulse from the hypothalamus precedes each LH pulse (Schams

et al., 1974). In addition, changes in concentrations of circulating progesterone

and estradiol affect the episodic secretion of LH (Wolfe et al., 1992; Stumpf et al.,

1993). In dairy cattle (Rahe et al., 1980), LH pulses are classified as low

amplitude (0.3 1.8 ng) and high frequency (20 30 pulses/24 h) on d 3 or early

luteal phase of the estrous cycle. In contrast, LH pulses are classified as high

amplitude (1.2 7.0 ng) and low frequency (6 8 pulses/24 h) on d 10 to 11 or

mid-luteal phase of the estrous cycle. A pulse of estradiol follows each LH pulse

during the early and mid-luteal stages of the estrous cycle (Walters et al., 1984).

Similar patterns of LH secretion have also been reported in sheep (Baird and








McNeilly, 1981, Wallace et al., 1988). Moreover, LH concentrations between d 2

and 4 of the estrous cycle did not differ among Brahman (Bos indicus), Senepol

(tropical Bos taurus) and Angus (temperate Bos taurus) cows (Alvarez et al.,

2000). On d 18 to 19 or late luteal phase of the estrous cycle, LH secretion is

variable (Rahe et al., 1980) with an increase in the frequency of LH pulses (Cupp

et al., 1995). Furthermore, the late luteal phase of the estrous cycle is

characterized by a decline in luteal progesterone followed by an increase in the

concentrations of estradiol, which initiates the preovulatory surge of LH (Kesner

et al., 1981). Moreover, estradiol acts on the hypothalamus to increase secretion

of GnRH and (or) at the pituitary to increase its sensitivity to GnRH (Parvizi,

2000; Smith and Jennes, 2001) to enhance the preovulatory surge of LH.

Breed differences between Bos taurus and cattle of Bos indicus breeding in

the amplitude and timing of the LH surge have been reported (Randel, 1976).

The interval from the onset of estrus to LH surge is approximately 0.4 + 3.4 h in

Brahman, 6.8 2.1 h in Brahman x Hereford, and 5.3 1.3 h in Hereford cows.

In addition, the interval between the LH surge and ovulation is 18.5 + 3.1 h in

Brahman, 22.2 2.6 h in Brahman x Hereford, and 23.3 2.1 h in Hereford

cows. Thus, Bos indicus cows have decreased intervals from estrus to the LH

surge and from the LH surge to ovulation than Bos taurus and Bos indicus x Bos

taurus cows.

Estrogens and Progesterone

The preovulatory release of GnRH occurs in the presence of increased

estrogen and decreased progesterone concentrations in the circulation. As








follicles grow and develop, they secrete increased amounts of estrogen, which

acts positively on the surge center, resulting in increased quantities of GnRH

being released.

Ovarian steroids also act as local regulators of follicular and luteal activity

and are classified according to either their chemical structure or their principal

biological functions. Steroids are grouped into three major classes including

progesterone, androgens, and estrogens. Estrogens (estrone and estradiol-17p)

are the most physiologically important of the follicular steroids; and progesterone

is the most important of the luteal steroids (Scham and Berisha, 2002).

Estrogens are directly involved in several ovarian processes such as

folliculogenesis, steroidogenesis, ovulation, and CL formation and function

(Scham and Berisha, 2002). Theca cells produce androgens, which are taken up

by the granulosa cells (GC) and converted by the enzyme aromatase P450 to

estradiol-17p (Fortune and Hansel, 1979; Roberts and Skinner, 1990; Scham

and Berisha, 2002). Adequate secretion of progesterone by luteal cells is critical

for establishing the physiological duration of the estrous cycle and for maintaining

a successful pregnancy (Scham and Berisha, 2002). Numerous factors are

produced within and outside the CL that control the development and secretary

function of the CL and progesterone and estradiol may act within the bovine CL

as autocrine and/or paracrine regulators (Scham and Berisha, 2002).

Ovarian estrogen receptors (ERs) must be present in specific cell types in

the ovary for estrogens to act and for estrogen-induced gene activation to occur

(Berisha et al., 2002). Estrogen receptor-a (ERa) and estrogen receptor-P (ERp)








expression have been located in the bovine follicle (Rosenfeld et al., 1999;

Schams and Berisha, 2002; Van Den Broeck et al., 2002). Additionally, Berisha

et al. (2000a) found expression of progesterone receptor (PR) in the bovine

follicle, which was generally decreased in theca internal cells and granulosa cells

compared to expression of ERs in these cells. Both types of ERs were found in

both types of cells with greater concentrations in theca internal than in granulosa

cells (Berisha et al., 2000a). The significance of these findings is that the

expression of these steroid receptors may affect folliculogenesis, expression of

other hormone receptors, steroid production in the ovary, gap junctions between

granulosa cells, and apoptosis in granulosa cells (Schams and Berisha, 2002).

Estrogen has been reported to increase expression of FSH and LH

receptors in rat granulosa cells (Richards et al., 1976) and oxytocin receptors in

bovine granulosa cells (Uenoyama and Okuda, 1997). The induction of LH

receptor mRNA in granulosa cells depends on the synergistic effects of estradiol

and FSH in the bovine (Berisha et al., 2000a). Production of androgen and

progesterone in bovine ovaries can be regulated by estradiol-1713 and

catecholestrogens (Schams and Berisha, 2002). Some research suggests that a

local feedback regulation may be present in ovarian follicles (Fortune and

Hansel, 1979; Leung and Armstrong, 1980; Fortune, 1986; Roberts and Skinner,

1990). Androgens produced by theca cells undergo aromatization into estrogens

within granulosa cells, which may feedback to stimulate theca cell production of

androgens. In dominant bovine follicles, the major steroid produced by

granulosa cells is pregnenolone, which is exported to the theca cells for








conversion to androgens. However, high concentrations of progesterone reduce

peripheral concentrations of estradiol due to inhibition of granulosa cell

aromatase activity. Cells cannot convert androgen to estrogens, as

demonstrated in anestrous ewes (Hunter and Southee, 1987) and cycling

nonlactating cows (Taylor et al., 1994). Similarly, heifers of Bos indicus breeding

have a -iqni.,n-ant reduction in circulating concentrations of estradiol after

administration of high doses of progesterone (Cavalieri et al., 1998a; 1998c).

In bovine CL, progesterone and estradiol act as autocrine and/or paracrine

regulators of its formation and function (Berisha et al., 2002; Schams and

Berisha, 2002). Specific binding sites for estradiol are present in the bovine CL

(Kimball and Hansel, 1974). Additionally, progesterone receptor mRNA was up

regulated in vitro by forskolin in bovine granulosa cells (Lioutas et al., 1997).

Schams and Berisha (2002) reported the profile for PR, ERa, and ERp mRNA

expression by RT-PCR in bovine luteal tissue. The PR expression was greatest

during the early stage of the estrous cycle and decreased significantly in the late

luteal phase and during CL regression. The ERa expression was greatest during

the early luteal phase and decreased significantly in the late luteal phase. On the

contrary, ERp showed no clear regulatory changes during all stages of the

estrous cycle examined (Schams and Berisha, 2002). In addition, progesterone

binding to membranes of large luteal cells was greater than progesterone binding

to small luteal cells, and concentrations were similar in membranes prepared

from CL at all stages of the luteal phase. Schams and Berisha (2002) suggested

that membrane bound steroid receptors might be involved in the








autocrine/paracrine regulation of follicular and luteal function by progesterone.

Similarly, specific membrane binding sites for progesterone in granulosa and

thecal membranes from bovine follicles of different sizes and in luteal cell

membranes have been described (Rae et al., 1998a; 1998b). The secretion of

and possible role of estradiol-17p in the bovine CL are unclear (Schams and

Berisha, 2002). Nevertheless, specific binding sites of estradiol-17p are present

in the bovine CL (Kimball and Hansel, 1974). Okuda et al. (2001) reported that

mRNA for the aromatase P450 is present in the bovine CL with a clear up-

regulation during the mid and late luteal phase of the estrous cycle and positive

correlation with the up-regulation of LH receptors (Kobayashi et al., 2001;

Schams and Berisha, 2002). Basal release of estradiol within the bovine CL from

an in vitro microdialysis system did not change during the estrous cycle (Okuda

et al., 2001). Okuda et al. (2001) reported that in luteal cell culture, estradiol

stimulated only PGF2a secretion, while it did not affect progesterone and oxytocin

secretion. In contrast, PGF20 did not stimulate estradiol secretion from cultured

bovine luteal cells. The functionality of the early bovine CL is affected by

progesterone in an autocrine and paracrine manner (Skarzynski and Okuda,

1999; Skarzynski et al., 2001). Secretion of progesterone, oxytocin, and PGF2a

and PGE2 was reduced after treatment with a specific progesterone antagonist

(onapristone) in the early luteal cells (Schams and Berisha, 2002). In addition,

the antagonist inhibited oxytocin secretion in midcycle luteal cells, even "-r,,ugn it

stimulated PGF2a secretion. These results show that progesterone stimulates

secretion of progesterone, oxytocin, and PGs in the early CL. However, in the








mid-cycle CL progesterone inhibits PGF2a secretion. Another study (Pate, 1988)

confirmed the inhibiting effect of progesterone on PGF2a secretion by bovine

luteal cells in the mid-cycle CL, but not in the late CL.

Progesterone has a luteotropic action by siimulatirig the synthesis of LH

receptors in bovine luteal cells (Jones et al., 1992). In addition, progesterone

stimulates its own basal secretion by activating 3p3-hydroxysteroid

dehydrogenase (Pate, 1996). It is possible that progesterone prevents luteal

regression by inhibiting apoptosis (Rae et al., 1998b; Friedman et al., 2000),

which may occur through a PR-dependent mechanism. Furthermore, Fiedman et

al. (2000) reported that cells producing progesterone are protected from

apoptosis while the same authors reported that a prerequisite for the initiation of

apoptosis in CL endothelial cells is a decline in progesterone, preceding

structural luteolysis. During early to mid-diestrus in cows, exposure to or

inhibition of progesterone by a progesterone antagonist controlled the initiation of

release of PGF2o from uterine endometrium (Garrett et al., 1988; Schams and

Berisha, 2002). Exposure of progesterone during these stages causes a

reduction of the interestrous interval, while inhibition of progesterone causes an

increase of the estrous interval (Garrett et al., 1988; Schams and Berisha, 2002).

Therefore, progesterone regulates the life span of the CL. Desensitization of

endometrial tissue to progesterone correlates with the down regulation of

estrogen, oxytocin and progestin binding in luteal tissue (Meyer et al., 1988).

The authors showed that receptors were minimal on d 12 and increased again

toward d 21 of the estrous cycle. These results indicate sensitivity of the








endometrium for the inhibitory action of progesterone on PGF2a secretion and

down regulation of receptors for steroids and oxytocin for about 10-11 d. After

this time, desensitization of receptors for progesterone and up-regulation of

receptors for steroids and oxytocin are essential for initiation of luteolysis in

cattle.

Prostaglandins

Prostaglandins play a broad role in mammalian physiology and metabolism,

and they are local regulators usually synthesized near cells on which they have

an effect (Staples et al., 1998). In cattle, uterine tissue is the most important

source of the F series prostaglandins (e.g., PGF2a) during the first weeks after

calving (Guilbault et al., 1984; Madej et al., 1984; De Fries et al., 1998).

Concentrations of 13, 14-dihydro-15-keto- PGF2. (PGFM)--PGF2a metabolite--in

plasma increase dramatically, reaching 1800 pg/mL by 3 to 4 d post partum,

which is associated with the regression of CL at the end of the pregnancy and

during postpartum uterine involution (De Fries et al., 1998; Staples et al.; 1998).

During the early postpartum period, PGFM slowly returns to baseline

concentrations. After the first estrus, the uterus releases PGF2a recurrently to

induce regression of CL to initiate a new estrous cycle if the cow does not

conceive (Madej et al., 1984; Staples et al., 1998). If the cow becomes pregnant,

the uterine release of PGF2a is inhibited and the CL is preserved to maintain

pregnancy (Helmer et al., 1989). Thus, concentrations of plasma progesterone

are related inversely to concentrations of PGF2a when CL regression occurs in

late diestrus. However, progesterone priming of the uterus is necessary to

stimulate uterine lipids for synthesis of PGF2a (Staples et al., 1998).








The luteolytic mechanism of PGF2. is described in detail in the section

"Ovulation, Corpus Luteum Development, and Luteolysis."

Estrous Cycle in Cattle

The bovine estrous cycle ranges from 18 to 24 d and comprises a

sequence of predictable reproductive events beginning with estrus (period of

sexual receptivity) and ending at the subsequent estrus. Differences in estrous

cycle length between Zebu (Bos indicus) and European breeds (Bos taurus)

have been reported. In Bos taurus breeds, the length of the estrous cycle is

typically 21 d (Hansel et al., 1973), with little variation. In contrast, the length of

the estrous cycles in Bos indicus breeds varies considerably, reported to average

28 d in Brahman heifers (Plasse et al., 1970) and 23 d in Boran cows (Llewelyn

et al., 1987). In a study by Moreira-Viana et al. (2000), mean estrous cycle

length was 21.7 d in Gir cows. Alvarez et al. (2000) reported similar estrous

cycle lengths among Senepol (Tropical adapted Bos taurus; 20.4 d), Angus (19.5

d), and Brahman (19.7 d) cows. However, it should be noted that in the later

study, the three breeds were housed as a single group throughout the duration of

the experiment.

Estrus

Estrus is characterized by sexual receptivity and mating and it is the most

identifiable stage of the estrous cycle. Estradiol is the hormone responsible for

inducing estrous behavior in cattle (Short et al., 1973; Randel, 1990). In addition,

estradiol is the main stimulus for induction of the preovulatory surge of LH in the

bovine (Henricks et al., 1971; Christensen et al., 1974; Randel, 1990).

Behavioral estrus is shorter in duration and less evident in Bos indicus breeds








(Plasse et al., 1970; Galina et al., 1982; Pinheiro et al., 1998) compared with Bos

taurus breeds (Stevenson et al., 1996). In Bos taurus breeds, the duration of

estrus ranges from 3 to 26 h with an average of 14 h (Schams et al., 1977), while

the duration of estrus in Bos indicus breeds ranges from 2 to 22 h with an

average of 7 h (Plasse et al., 1970; Rae et al., 1999).

Metestrus

Metestrus is the period between ovulation and the formation of a

functional CL, lasting 3 to 5 d. Ovulation occurs 24 to 36 h from beginning of

estrus in Bos taurus beef cows (Looper et al., 1998; Rorie et al., 1999). Similarly,

ovulation in Bos indicus cows occurs approximately 25 h after the onset of estrus

(Lamothe-Zavaleta et al., 1991; Pinheiro et al., 1998). Additionally, 26% of

ovulations in Bos indicus cows occurred without visible signs of estrus (Plasse et

al., 1970). The newly ovulated follicle undergoes cellular and structural

remodeling resulting in the formation of the CL. During CL formation, granulosa

and theca cells luteinize in response to LH and the walls of the follicle collapse,

mixing the theca and granulosa. The basement membrane becomes the

connective tissue network of the CL and the breakage of small blood vessels

leads to a blood clot termed the corpus hemorrhagicum. Two types of luteal cells

have been described; large and small luteal cells. Large and small luteal cells

are formed from granulosa and theca cells undergoing a process of luteinization.

The newly formed CL is rapidly invaded by blood vessels, which supply the

necessary substrate (cholesterol) for the production of progesterone.








Diestrus

Diestrus is characterized by the presence of a functional CL and increased

concentrations of progesterone. Diestrus is the longest stage of the estrous

cycle, lasting 10 to 14 d. Diestrus ends with the release of prostaglandin F2c

(PGF2a) from the uterus, which results in luteolysis and a reduction in

progesterone production. Whether pregnancy results or not, the CL develops

into a fully functional organ producing large amounts of progesterone. If an

oocyte is fertilized and reaches the uterus, the CL will be maintained throughout

the pregnancy. In contrast, if the oocyte is not fertilized, the CL remains

functional until d 17 or 18 and it degenerates after luteolysis, thereby permitting a

new estrous cycle to be initiated.

Proestrus

Proestrus is characterized by follicular growth and estradiol production

(Chenault et al., 1975; Kesner et al., 1982) and it occurs 2 to 3 d before the onset

of estrus in the cow. Proestrus is characterized by a major endocrine transition

from a period of progesterone dominance to a period of estrogen dominance

(Chenault et al., 1975; Kesner et al., 1982). Proestrus begins when blood

progesterone concentrations decline due to luteolysis (Auletta and Flint, 1998).

Concomitant with the decrease in progesterone is an increase in circulating

concentrations of estradiol, as a dominant follicle prepares for ovulation (Ireland

et al., 1984). Gonadotropins LH and FSH are the primary hormones responsible

for this transition (Kesner et al., 1982).








Regulation of Ovarian Follicle Growth, Atretic Demise and the Ovulatory
Response

Follicle development is initiated during the fetal life. The process of follicle

development can be summarized based on several reviews (Fortune, 1994;

Ginther et al., 1996b; Ireland et al., 2000). By mid-gestation, the bovine ovary

contains its entire lifetime supply of oogonia. Subsequent to termination of

mitotic proliferation during gestation, the oogonia enter meiosis. At birth, the

oogonia are arrested in the first meiotic division and are called oocytes.

Primordial follicles contain oocytes surrounded by a single granulosa cell layer

consisting of 14 to 29 flattened granulosa cells (Erickson, 1966; Van Wezel and

Rodgers, 1996; Van den Hurk et al., 1997). As the follicle grows and develops, it

acquires a cuboidal layer of 20 to 50 granulosa cells and it becomes a primary

follicle. The'pool of primordial and primary follicles is considered part of the

ovarian reserve of oocytes. The initial stages of folliculogenesis occur

independently of gonadotrophic hormones (Roche, 1996). During the

reproductive lifespan of the animal, individual follicles are recruited as actively

growing follicles (Marion and Gier, 1971). Formation of the zona pellucida occurs

when primary follicles develop into secondary follicles and granulosa cells

multiply and form several layers. In addition, theca cells are formed and the

follicle becomes gonadotropin dependent as secondary follicles develop

(Scaramuzzi et al., 1993; Mihm et al., 2002). The tertiary follicle is formed with a

separation of the granulosa cell layers forming a cavity or antrum. Subsequent

enlargement and accumulation of follicular fluid in the antrum takes place, and

the follicle continues to grow and it is designated as a mature Graafian follicle.








Ovarian follicular development in cattle is a dynamic progression of events

that has been described to occur in a wave-like fashion (Pierson and Ginther,

1984; Savio et al., 1988; Sunderland et al., 1994) and is characterized by waves

of follicular growth and regression during the estrous cycle (Taylor and

Rajamahendran, 1991). In cattle, growth of ovarian antral follicles from

approximately 300 pm in diameter to 3 to 5 mm is calculated to take more than

30 d (Lussier et al., 1987). Cattle usually have 2 (Ginther et al., 1989c; Taylor

and Rajamahendran, 1991) or 3 waves (Savio et al., 1988; Sirois and Fortune,

1988) of follicular development during an estrous cycle; however, estrous cycles

consisting of either 1 or 4 waves have also been reported (Savio et al., 1988;

Sirois and Fortune, 1988). The main characteristics of follicular growth and

atresia can change among animals due to factors like energy balance and body

condition score (Rhodes et al., 1995; Burke et al., 1998), reproductive stage

(Roche and Boland, 1991), and breed (Figueiredo et al., 1997). The proportion

of beef heifers having 3 follicular waves was increased with low dietary intake

compared to 2 follicular waves during normal nutrition (Murphy et al., 1991). In

contrast, parity and negative energy balance during lactation in dairy cows has a

negative effect on the number of waves per estrous cycle (Lucy et al., 1992).

Additionally, follicular waves also occur in prepubertal heifers (Evans et al.,

1994), early postpartum cows (Savio et al., 1990), and throughout pregnancy

(Ginther et al., 1989a; Ginther et al., 1996a). Apparently, there is no difference in

the number of follicular waves during an estrous cycle between cattle of Bos

indicus breeding (Figueiredo et al., 1997; Gomez-Alves et al., 2002; Henao et al.,








2000) and Bos taurus breeds (Alvarez et al., 2000). However, Gir (Bos indicus)

cows had a greater incidence of estrous cycles with three (60.0%) and four

(26.7%) waves (Moreira-Viana et al., 2000).

The dynamics of a follicular wave during the estrous cycle consist of

recruitment, selection, dominance, and either atresia or ovulation. Recruitment is

a process where a cohort of small follicles (2 to 4 mm) reaches the tertiary stage.

Increased concentrations of FSH promote the growth of the cohort of follicles.

The stimulated growth of this cohort of follicles occurs 2 to 4 d after a surge in

FSH (Adams et al., 1992a) r.uiltng in follicles 4 to 5 mm in diameter as

determined by transrectal ultrasound (Adams et al., 1992a). Moreover, mRNA

for FSH receptor was localized in granulosa cells of pre-antral primary follicles

(Xu et al., 1995) supporting the integral role of FSH in follicular recruitment.

Follicles that are recruited, but not selected for continued growth regress in a

process known as follicular atresia. Atresia continues until one follicle remains,

and under a favorable hormonal environment obtains the ability to ovulate. This

process is known as selection. The follicle remaining is termed dominant, while

all other follicles are termed subordinate. The dominant follicle and the largest

subordinate follicle (second largest follicle) initially grow in parallel (Ginther et al.,

1996b; Kulick et al., 1999). The point when growth of the largest subordinate

follicle slows, resulting in a difference in the growth rate of the two largest follicles

is known as deviation (Ginther et al., 1996b). The selected follicle exerts its

dominance through inhibition of the recruitment of follicles of the next follicular

wave (Lucy et al., 1992).








Deviation marks the completion of follicular selection and recent research

has focused on understanding the endocrine control of deviation. It has been

proposed that the control of deviation involves a two-way functional coupling

between FSH and follicles (Ginther et al., 2000a). Growing follicles of the cohort

attain the capacity to suppress FSH secretion when they are 5 mm in diameter

(Gibbons et al., 1999), and the dominant follicle is 8.5 mm in diameter at the time

of deviation (Ginther et al., 1996b). Estradiol produced by the future dominant

follicle is involved in the suppression of FSH (Ginther et al., 2000b). The

declining concentration of FSH fails to support the continued growth of the

subordinate follicles. However, even with the decreased concentrations of FSH,

it is required and remains at sufficient concentrations for the dominant follicle to

continue to grow (Turzillo and Fortune, 1993). After the decline in FSH, primary

gonadotropic support shifts to LH. Although suppression of LH did not affect the

time of deviation, the diameter of the largest follicle was decreased when LH

decreased after deviation (Ginther et al., 2001). Consequently, an increase in

responsiveness to LH is crucial for continued development of the dominant

follicle. Furthermore, granulosa cells of the dormnni follicle express more LH

receptor mRNA than granulosa cells in the largest subordinate follicle before the

time of deviation (Beg et al., 2001). The same authors concluded that acquisition

of LH receptors allows for continued dominant follicle growth after the decline in

FSH. After deviation, the dominant follicle continues to mature in preparation for

ovulation. The most important aspect of maturation of the dominant follicle is its

ability to secrete increasing amounts of estradiol, which ultimately induce a surge








of LH for ovulation (Fortune, 1994). Estradiol enhances the release of GnRH and

increases sensitivity of the pituitary to GnRH (Kesner et al., 1981), but only if

concentrations of progesterone are relatively low or have declined ,rgnific:ranil,'

(Kesner et al., 1982) since progesterone inhibits gonadotropin surges (Kesner et

al., 1982). Therefore, if the CL does not regress during the first and some

second wave dominant follicles, an LH surge and ovulation will not occur.

Instead, the dominant follicle undergoes atresia, giving rise to a new wave of

follicular irochn In concordance, Savio et al. (1993b) proposed that increased

concentrations of luteal progesterone decreased LH pulse frequency and initiated

turnover of nonovulatory dominant follicles. If the CL is regressed LH pulse

frequency increases causing the dominant follicle or future dominant follicle to

grow and secrete more estradiol (Auletta and Flint, 1998). Increasing secretion

of estradiol will induce a surge of LH and sub-equErii, ovulation (Kesner et al.,

1982; Ireland et al., 1984).

The length of the estrous cycle in Bos taurus cattle with two foiljcular Va,, e

averages 20.4 d (Ginther et al., 1989c) comparable to the interovulatory period of

Bos taurus x Bos indicus (Perea et al., 1998; Gomez-Alves et al., 2002) and Gir

(Bos indicus) cows (Moreira-Viana et al., 2000). The first wave dominant follicle

can be identified on d 3 to 4 of the estrous cycle in Bos taurus (Ginther et al.,

1989b; Savio et al., 1993b) and Bos taurus x Bos indicus cattle (Perea et al.,

1998; Gomez-Alves et al., 2002). However, the first wave dominant follicle was

identified earlier (d 1 of the estrous cycle) in Gir cows (Moreira-Viana et al.,

2000). According to Ginther et al. (1989b) and Savio et al. (1993b), dominance








in Bos taurus cattle is established by d 5 and the dominant follicle reaches its

maximal size (> 10 mm) on d 6 or 7. Similar results were observed in Bos taurus

x Bos indicus dual-purpose cows (Perea et al., 1998). Size of the dominant

follicle in both Bos taurus and Bos taurus x Bos indicus cows remains constant

until d 9 to 12 of the estrous cycle at which time it begins to regresses in size

(Ginther et al., 1989b; Perea et al., 1998; Savio et al. 1993b). In contrast,

establishment of dominance was between 2 to 3 d in Gir cows (Moreira-Viana et

al., 2000), and maximal size of the dominant follicle (> 10 mm) was reached on d

4. However, as described for Bos taurus cattle, size of the dominant follicle

remained constant until d 9 to 12 of the estrous cycle. The less predictable

second wave in Bos taurus (Ginther et al., 1989b; Savio et al., 1993b), Bos

taurus x Bos indicus (Perea et al., 1998; Gomez-Alves et al., 2002), and Bos

indicus (Moreira-Viana et al., 2000) cattle is usually detected between d 9 and 14

of the estrous cycle.

The average length of an estrous cycle with three follicular waves appears

to be shorter (< 21 d) in Bos indicus (Moreira-Viana et al., 2000) than Bos taurus

and Bos taurus x Bos indicus cattle (22 to 25 d; Ginther et al., 1989c; Gomez-

Alves et al., 2002). In Bos taurus cattle (Ginther et al., 1989c), the first wave

dominant follicle was detected on d 4 of the estrous cycle, reached its maximal

size on d 6 and maintained its size until d 10. In contrast, in Bos indicus cows

(Moreira-Viana et al., 2000) the first wave dominant follicle was detected on d 1

of the estrous cycle, reached its maximum size on d 6 (similar to Bos taurus

cattle), and maintained its size for only two days (until d 8). Regression of the








first wave dominant follicle occurred on approximately d 12 to 13 of the estrous

cycle in both Bos taurus and Bos indicus cattle (Ginther et al., 1989c; Moreira-

Viana et al., 2000). The second wave dormnr,.ri ollicle emerged on d 9 and

reached maximal size on d 16 in Bos taurus cattle (Ginther et al., 1989c).

However, in Bos indicus cows the second wave dominant follicle emerged on d 7

and reached its maximal size on d 13 (Moreira-Viana et al., 2000). The third

wave dominant follicle is usually detected on d 16 with continued development

until ovulation in Bos taurus cattle (Ginther et al., 1989c), but it was detected

earlier (d 13) in Bos indicus cows (Gomez-Alves et al., 2002). In any case, the

average diameter of preovulatory follicles range from 12 to 15 mm for Bos taurus

and Bos indicus breeds (Dufour et al., 1971; Figueiredo et al., 1997; Alvarez et

al., 2000).

Ovulation, Corpus Luteum Development, and Luteolysis

Ovulation is defined as the degradation of the follicular basement

membrane and the fragmentation of the extracellular matrix (ECM) at the apex of

the follicle wall, resulting in the release of the oocyte (Richards et al., 1998;

Richards et al., 2000). The vascular, structural and metabolic events leading to

the rupture of the follicle wall share many similarities with tumor formation and an

acute inflammatory reaction (Smith et al., 1994). Remodeling of the follicle wall

involves degradation of extracellular matrix components by proteases and is

associated with prominent changes in vascular architecture and leukocyte

infiltration. Immune cells have been implicated in several physiological

processes occurring in the ovary, and their effects are largely mediated by locally

produced cytokines (Machelon and Emilie, 1997).








Dissolution of tissue and subsequent tissue remodeling are important

processes associated with rupture of follicles at ovulation and eventual formation

of the CL (Dow et al., 2002; Gottsch et al., 2002). The production of proteinases

initiated by the LH surge mediate the degradation of the extracellular matrix and

cellular remodeling required for ovulation and CL formation (Dow et al., 2002).

Proteins from the plasminogen activator family (tissue-type and urokinase) of

serine proteinases are implicated in ovulation and CL formation due to their

ability to convert plasminogen to plasmin (Dow et al., 2002). Plasmin-directed

ovarian extracellular matrix remodeling during follicle rupture and CL formation is

regulated through inhibition of plasminogen activation by the plasminogen

activator inhibitors (PAI)-1 and PAI-2. The PAl-1 and PAI-2 mRNAs are up

regulated in preovulatory bovine follicles after the gonadotropin surge in a cell-

specific manner (Dow et al., 2002). Matrix metalloproteinase (MMP) -2 is also

associated with follicular rupture and CL formation (Gottsch et al., 2002). Matrix

metalloproteinase 2 belongs to a family of endopeptidases that cleaves

extracellular proteins, which act on the basement membrane that support

epithelial cells and endothelium.

Angiogenesis, the establishment of new capillary blood vessels from pre-

existing ones, plays a major role in CL formation and development. In a rat

model of hormonally induced ovulation, treatment with truncated soluble

macrophage colony-stimulating factor 1- Like tyrosine kinase 1 receptors (Flt-1),

which inhibit vascular endothelial growth factor (VEGF) bioactivity, resulted in

virtually complete suppression of CL angiogenesis. This effect was associated








with inhibition of CL development and progesterone release (Ferrara et al.,

1998). In the bovine CL, there are increased concentrations of VEGF transcript

and proteins, and increased VEGF receptor-2 (VEGFR-2) expression during the

early luteal phase of the estrous cycle, which coincides with luteal vascularization

(Berisha et al., 2000b). These results suggest an important role of VEGF in

angiogenesis of the newly formed CL. Other angiogenic factors belonging to the

fibroblast growth factor (FGF) family (Redmer and Raynolds, 1996) and ovarian

cell proliferation factors like transforming growth factor P, platelet derived Jrr.,:vh

factor, hepatocyte growth factor, and insulin like growth factor biding protein-3

(Smith et al., 1999) are essential for angiogenesis of the developing CL.

Thr,:,ugr,ouT ihe- first third of the ovarian cycle, developing endothelial cells

invade the developing CL and continue to grow (Augustin et al., 1995). The

mature CL is characterized by a dense system of blood vessels with

progressively decreasing blood vessel density.

The initial development of the CL takes approximately 3 d in cattle (d 2 to 5

of the estrous cycle), with angiogenesis playing an important role in development

and maintenance of the CL. Size of the CL increases more than 20 fold during

luteal development (Gottsch et al., 2002) and has one of the highest rates of

blood flow per unit of tissue of any organ in the body (Reynolds and Redmer,

1999). Baumgartner et al. (1998) concluded that luteal vascularization in the

bovine increases during early diestrus and remains constant until luteolysis.

Early studies (Ursely and Leymarie, 1979; Chegini et al., 1984; Rodgers et

al., 1986; Weber et al., 1987) identified two functionally distinct cell populations,








large and small luteal cells, in the bovine CL. The CL is comprised of

approximately 40% large luteal and 28% small luteal cells (O'Shea et al., 1989).

Large luteal cells are derived from granulosa cells of the preovulatory follicle and

small luteal cells are derived from thecal cells (Meidan et al., 1990). The

distinctive characteristics of the two luteal cell types have been well defined in

ruminants. Not only are the diameter and morphological characteristics different

between these two cells, but differences in receptors for LH (generally greater in

small cells), estradiol (much greater in large cells), and PGF2a (much greater in

large cells) have also been reported (Wiltbank, 1994; Diaz et al., 2002).

Treatment with LH increased progesterone production by small, but not large

luteal cells, whereas, treatment with PGF2a inhibited progesterone production by

large, but not small luteal cells (Wiltbank et al., 1993).

The ovaries in cattle of Bos indicus breeding are smaller than cattle of Bos

taurus breeding (Moreno et al., 1986; Rentfrow et al., 1987; Soto et al., 1999).

Similarly, luteal tissue area tends to be decreased in cattle of Bos indicus

breeding compared with Bos taurus cattle (Castilho et al., 2000; Moreira-Viana et

al., 2000). The maximum CL diameter for Bos taurus heifers has been reported

to range from 25 to 30 mm (Adams et al., 1993). In contrast, maximum CL

diameter for Brahman and Nelore (Bos indicus) heifers was 17 and 18 mm,

respectively (Rhodes et al., 1995; Figueiredo et al., 1997). Likewise, maximum

CL diameter for Gyrolando (Bos indicus x Bos taurus) dairy heifers was 19 to 20

mm (Castilho et al., 2000), similar to maximum CL diameter of 18 mm and 19

mm observed in Bos taurus x Bos indicus dual purpose cows and heifers,








respectively (Perea et al., 1998). The mean diameter of CL was even smaller

(9.3 mm) for Bos indicus cows ranging from 7 to 11.7 mm (Ruiz-Cortes and

Olivera-Angel, 1999). Similarly, CL weight on d 8 and 13 of the estrous cycle for

Brahman heifers (2.5 and 2.7 g) was decreased compared to Brahman x

Hereford (4.6 and 3.8) or Hereford heifers (4.0 and 3.6 g), respectively (Irvin et

al., 1978). Likewise, Segerson et al. (1984) reported that CL weight on d 17 of

the estrous cycle was less for Brahman (2.4 g) than Angus cows (4.1 g). In

agreement, progesterone concentrations in CL from Bos indicus heifers and

cows were decreased compared to Bos taurus heifers and cows (Irvin et al.,

1978; Segerson et al., 1984). Related to these findings, progesterone

concentrations are less in cattle of Bos indicus breeding compared to Bos taurus

cattle (Randel, 1977; Segerson et al., 1984). According to Irvin et al. (1978),

progesterone concentrations of CL from Brahman (216.9 pg/CL) and Brahman x

Hereford (217.7 pg/CL) was decreased compared to Hereford heifers (334.6

pg/CL). Similarly, Segerson et al. (1984) observed that CL from Brahman had

decreased progesterone concentrations (190.8 pg/CL) compared to Angus cows

(266.3 pg/CL). Furthermore, Brahman and Brahman x Hereford heifers had

decreased circulating progesterone concentrations from 2 toll11 d after estrus

compared to Hereford heifers (Randel et al., 1977). Similar results have been

observed when comparing Brahman and Angus cows from 7 to 17 d after estrus

(Segerson et al., 1984). These observations indicate that both Bos indicus and

Bos indicus x Bos taurus cattle have decreased progesterone concentrations

during diestrus compared with Bos taurus cattle.








During late diestrus, both functional and structural luteolysis occurs in order

for the non-pregnant female to return to estrus (Auletta and Flint, 1998). The

endometrium follows a default program to release luteolytic pulses of PGF20 and

if a concepts is present it sends the appropriate antiluteolytic signals to block

PGF2a production (Binelli et al., 2001). Regulation of luteolysis is particularly

complex and numerous reviews are available for consideration (Silvia et al.,

1991; McCracken et al., 1999; Binelli et al., 2001). Luteolysis is a local

mechanism involving a countercurrent transfer of PGF2a from the uterine vein to

the ovarian artery (Hixon and Hansel, 1974). Uterine derived PGF2Q is the

hormone responsible for luteal regression in ruminants (McCracken, 1971;

McCracken et al., 1981). It appears that PGF2a exerts its luteolytic effect not only

on the steroidogenic cells of the CL, but also on other cell types such as

endothelial cells (Auletta and Flint, 1998). During late diestrus, progesterone

concentrations began to decrease and estradiol increases (Meyer et al., 1988;

Mirando et al., 1993). The increase in estradiol enhances the oxytocin pulse

generator and oxytocin receptors in the endometrium (Meyer et al., 1988;

Mirando et al., 1993). The loss of progesterone inhibition on oxytocin receptor

formation and subsequent oxytocin receptor formation induced by estradiol is

thought to be an initiating factor in luteolysis (Wathes and Lamming 1995).

Oxytocin from both the neurohypophysis and the CL is responsible for the

pulsatile secretion of PGF2a from the endometrium (Hooper et al., 1986; Zarco et

al., 1988). Hypothalamic oxytocin acting on uterine oxytocin receptors induces

the production of concentrations of PGF2a, which act on PGF2a receptors in luteal








cells to stimulate the release of oxytocin (Hooper et al., 1986; Zarco et al., 1988).

The aforementioned cascade of events induces the secretion of high

concentrations of PGF2a by the uterus, which stimulates and increases release of

oxytocin to induce luteolysis.

Some of the earliest histological alterations of the CL that occur during

luteolysis take place in the vascular components of the CL (Azmi and O'Shea,

1984). These changes include hypertrophy and hyperplasia of endothelial cells

in the arteriolar wall, accumulation of elastic fibers in the blood vessels,

degeneration of the intima, protrusion of some endothelial cells in the lumen of

capillaries, and formation of adherent junctions across the lumen. These result in

a decrease in the vascular diameter and a reduction in the blood flow within the

CL (Azmi and O'Shea, 1984; Knickerbocker et al., 1988). Decreased luteal blood

flow occurs during a spontaneous and PGF2a-induced luteolysis (Azmi et al.,

1982; Knickerbocker et al., 1988; Acosta et al., 2002). During the midluteal

phase CL, an acute increase in blood flow is induced by PGF2a, which is followed

by a decrease in blood flow (Acosta et al., 2002). Similar changes in blood flow

do not occur in the early developing CL (Acosta el al., 2002). Acting directly on

endothelial cells, PGF2a stimulates the secretion of vasoactive substances such

as endothelin-1 and angiotensin II (Othani et al., 1998; Hayashi etal., 2001;

2002), which play important roles in the luteolytic cascade (Othani et al., 1998;

Hayashi et al., 1999).

Endothelin 1 binds specific endothelin type A receptors (Mamluk et al.,

1999) located on large and small luteal cells to inhibit progesterone synthesis








and stimulate luteal PGF2a production (Milvae, 2000). Prostaglandin F20 acts

directly on large luteal cells inducing an acute release of oxytocin, which increase

endothelin 1 secretion by endothelial cells (Ohtani et al., 1998). Angiotensin II

inhibits progesterone production in bovine luteal cells (Stirling et al., 1990;

Hayashi and Miyamoto, 1999). Angiotensin II release and the expression of

angiotensin-converting enzyme mRNA in the CL after injection of PGF20

analogue have been reported in the cow (Hayashi et al., 2001). Reduction of

progesterone production by the CL results in a processes known as functional

luteolysis.

Functional luteolysis is followed by a process known as structural luteolysis,

which involves the physical breakdown of the CL. Induction of functional

luteolysis activates cytokine secretion from endothelial cells, which induces the

recruitment of macrophages (Penny, 2000; Webb et al., 2002). Macrophages

have a phagocytic role on luteal cells during structural regression of the CL

(Webb et al., 2002). Furthermore, endothelin-1 stimulates the production of local

factors including cytokines like tumor necrosis factor-a (TNF-a) and interleukin-p

(IL-p) not only by endothelial cells, but also by monocytes and T lymphocytes

(Penny, 2000; Webb et al., 2002). Monocyte chemoattractant protein 1 (MCP-1),

a member of the chemokine family of cytokines involved in leukocyte physiology

and trafficking, is a potent chemoattractant for both monocytes and T

lymphocytes (Tsai et al., 1997; Penny et al., 1998). Another immunological

mechanism that has been hypothesized to potentially regulate luteal regression

involves the proteins that make up the major histocompatability complex (MHC),








especially MHC-11 (Penny et al., 1999; Webb et al., 2002). Major

-istoc.cmpaat 3rIt, complex (MHC) are cell surface markers required for antigen

recognition by T-lymphocytes (Webb et al., 2002). Additionally, tumor necrosis

factor-a (TNF-a) secreted by macrophages induces programmed cell death

(apoptosis) of small and large luteal cells, and endothelial cells (Nagaosa et al.,

2002).

Other cellular mechanisms are involved in structural luteolysis. For

instance, when PGF2o receptors are activated there is an influx of free calcium

(Ca") into luteal cells, which is mediated by angiotensin-ll (Pepperell et al.,

1993). The increased intracellular concentration of free calcium (Ca++) is

cytotoxic and it induces apoptosis and degeneration of luteal cells destroying the

structural integrity of the CL.

The number of PGF2a receptors in bovine luteal cells gradually increases

from the early to the late luteal stages of the estrous cycle, parallel with the

expression of PGF2a receptor mRNA (Sakamoto et al., 1995; Skarzynski et al.,

2001). However, concentration and affinity of PGF2a receptors were similar

during the early (d 2 4) and mid (d 6 10) stages of the estrous cycle (Wiltbank

et al., 1995; Mamluk et al., 1998; Skarzynski et al., 2001). The fact that

concentration and affinity of PGF2o receptors were similar during the early and

mid stages of the estrous cycle suggested that PGF2a induced luteolysis is

regulated by different mechanisms involved in the regulation of the

responsiveness and activation of PGF2a receptors in the bovine CL during the

entire luteal phase (Skarzynski et al., 2001).








Several processes have been implicated in the regulation of the

responsiveness of PGF2a receptors (Sharzynski and Okuda, 1999; Sharzynski et

al., 2000; Sharzynski et al., 2001). Since a single class of high-affinity PGF2a

receptors is present in the bovine CL by 2 d after ovulation (Sakamoto et al.,

1995; Wiltbank et al., 1995; Sharzynski et al., 2001), neither the lack of

responsiveness of the early CL to PGF2a nor the decreased sensitivity of the mid-

luteal CL to PGF2a (Sharzynski et al., 1997) can be attributed to a deficiency of

PGF2a receptors (Sharzynski et al., 2001). Sharzynski and Okuda (1999)

proposed that the responsiveness of PGF2a receptors may be due to homologous

desensitization of PGF2a receptors in the CL due to long-lasting stimulation by

PGF2a produced locally in the bovine ovary. Furthermore, some researchers

concluded that oxytocin and progesterone (Sharzynski and Okuda, 1999;

Sharzynski et al., 2001) and also noradrenaline (Sharzynski et al., 2000;

Sharzynski et al., 2001; Kotwica et al., 2002) through their luteotropic actions on

the early and mid luteal phase CL may indirectly (via PGF2a) or directly

(heterologus desensitization) affect the functionality of PGF20 receptors and/or

formation of second messengers. Moreover, treatment of bovine luteal cells with

progesterone decreased PGF20 production in a dose-dependent manner (Pate,

1988); therefore, it appears that high intraluteal progesterone inhibits luteal

PGF2a production (Diaz et al., 2002). Similarly, the effect of PGF2a on the early

luteal phase CL was greater in luteal cells receiving pretreatment with a

progesterone antagonist (onapristone), an oxytocin antagonist (atosiban) and a

cyclooxygenase inhibitor (indomethacin) compared to control cells (Sharzynski








and Okuda, 1999). Thus, luteal oxytocin, progesterone and prostaglandins are

components of an autocrine/paracrine positive feedback in early to mid cycle CL,

which may be responsible for the resistance of the early luteal phase CL to the

exogenous PGF2a.

Progesterone may also have an active role in the inhibition of luteal

regression by preventing apoptosis (Rae et al., 1998; Friedman et al., 2000;

Schams and Berisha 2002). Kobayashi and Miyamoto (2000) reported that

PGF2a is capable of enhancing lipoprotein utilization by luteal cells for

progesterone synthesis in early luteal phase CL, thus supporting the concept that

luteal PGF2a acts as a luteotropic agent in the early luteal phase CL.

The neurotransmitter noradrenaline has been reported to stimulate

progesterone, oxytocin, PGF2a and PGE2 secretion (Sharzynski et al., 2000;

Sharzynski et al., 2001; Kotwica et al., 2002). ConsequenII,. noradrenaline may

be indirectly involved in resistance of the CL against premature luteolysis by

stimulating these luteotropic factors in cattle. In any case, functional regression

of the CL occurs prior to any morphological change in luteal cells and it is likely a

reversible step if sufficient luteotropic support is provided (Pate and Townson,

1994). There is growing evidence suggesting that a PGF20-induced luteolysis

involves altered gene expression in the CL (Tsai et al., 2001). As indicated by

Tsai and Wiltbank (1998), lack of PGF2a-induced luteolysis in the early luteal

phase CL may be due to changes in gene expression, particularly prostaglandin

synthase-2 (COX-2) that probably prevents intraluteal PGF2a production and

possibly other luteolytic processes.








Some researchers have suggested that the lack of endothelin-1 synthesis

and might make the early luteal phase CL refractory to the luteolytic action of

PGF2a (Mamluk et al., 1998; Levy et al., 2000). Therefore, blood vessel

endothelium of the early luteal phase CL would be non-responsive to the action

of PGF2a.

There is a positive correlation between the amount of vascularization and

mean diameter of the CL (Baumgartner et al., 1998). Wiltbank et al. (1995)

suggested that incomplete vascularization in early luteal phase CL may be

responsible in part for the lack of luteolytic capacity of the early luteal phase

bovine CL. In general, the ovaries and CL in cattle of Bos indicus breeding are

smaller than cattle of Bos taurus breeding (Moreno et al., 1986; Rentfrow et al.,

1987; Soto et al., 1999). Therefore, these findings suggest that the reduced

response of the early luteal phase CL to PGF2a in cattle of Bos indicus breeding

compared to Bos taurus cattle may be associated with luteal size and lack of

vascularization. Brito et al. (2002) reported that the efficacy of PGF2a for initiating

luteolysis and ovulation in buffalo cows (Bubalus bubalis) at different stages of

the estrous cycle was dependent upon CL size at PGF2a treatment. Only buffalo

cows with CL > 189 mm2 responded to PGF2a treatment. Whether differences in

luteal vascularization occur between cattle of Bos indicus and Bos taurus exist is

unclear, but the hypothesis is an interesting one and requires further

investigation.

Exogenous Control of Ovarian Follicles and Corpus Luteum Dynamics

To accurately control the estrous cycle it is essential to control the life

span of the CL and the follicle development in the bovine. Most estrus








synchronization systems initiate CL regression using the luteolytic agent

prostaglandin F2. or one of its potent analogs. However, the resulting estrus is

highly variable and can occur over a 7 d period. However, administration of

exogenous estrogen or GnRH can be used to control new follicle wave

emergence and increase the synchrony of the subsequent estrus (Diskin et al.,

2002).

Estrogens

Administration of exogenous estrogens can induce follicular turnover

(Rajamahendran and Manikkam, 1994; Bo et al., 2000; Burke et al., 2000) and

(or) atresia of persistent follicles (Yelich et al., 1997; Fike et al., 1999; Bo et al.,

2000), which results in emergence of a new follicular wave. Bo et al. (1993)

reported that cycling cows injected with 5 mg of estradiol valerate on d 3 of the

estrous cycle had emergence of a second follicular wave between d 9 and 14 of

the estrous cycle. Similarly, Bo et al. (1994) reported cows injected with 5 mg of

estradiol 173 on d 3 of the estrous cycle had emergence of a second follicular

wave on d 6 to 7 of the estrous cycle. Delayed emergence of the second

follicular wave in cows treated with estradiol valerate was a result of the

prolonged suppressive effects of estradiol valerate (Bo et al., 1995). Circulating

concentrations of estradiol in treated cows are greater than normally present

during the follicular phase of the estrous cycle and act to suppress LH and FSH

release, which initiates follicle atresia and emergence of a new follicle (Butler et

al., 1983; Price and Webb, 1988; Wolfe et al., 1992).








The effects of estrogen on synchronizing follicular wave emergence

depends on the stage of follicular wave development when it is administered

(Lane et al., 2001). Estradiol administered to heifers during the luteal phase of

the estrous cycle suppresses growth of the dominant follicle (Bo et al., 1993,

1994; Burke et al., 2000) and thereby synchronizing follicular development.

Whereas, estrogen administered during the follicular phase of the estrous cycle

would result in estrus and ovulation of the do::mranir illicle (Ulberg and Lindley,

1960; Peters et al., 1977; Lammoglia et al., 1998; Lemaster et al., 1999).

Moreover, estrogen given in the follicular phase of the estrous cycle decreases

the interval to estrus (Nancarrow and Radford, 1975; Ryan et al., 1995) and the

variation in its onset (Ulberg and Lindley, 1960; Nancarrow and Radford, 1975).

Estrogens can also have luteolytic effects when administered during the

estrous cycle (Wiltbank et al., 1971; Thatcher et al., 1986; Diskin et al., 2002).

Thatcher et al. (1986) administered estradiol-17 p during the second half of the

estrous cycle that resulted in spikes of 15-keto-13, 14-dihydro-prostaglandin F2a

(PGFM) in the circulation. They concluded that estradiol-17P induced luteolysis

by provoking a release of PGF2a from the uterus. Although, others have reported

that estrogens actions as a luteolytic agent are less clear (Lemon, 1975; Burke et

al., 1999).

Progesterone and Progestagens

The rationale for utilizing exogenous progestogens in estrus

synchronization protocols is to mimic the action of the CL by suppressing estrus

and ovulation (Hansel et al., 1961; Wiltbank et al., 1967; McDowell et al., 1998).

Sirois and Fortune (1990) used a controlled intravaginal progesterone-releasing








device (CIDR) to artificially lengthen estrous cycles and to characterize follicular

development in dairy heifers. The CIDRs were inserted on d 14 to 28 after an

observed estrus. Control heifers (group 1) received a blank CIDR containing no

progesterone, while heifers in groups 2 and 3 received either one or two CIDRs,

respectively. Ovulatory follicles in group 2 had a longer persistence, reached a

larger maximal size, and were associated with a complete absence of follicular

recruitment compared to heifers in groups 1 and 3. The authors concluded that

the increased progesterone concentrations in heifers receiving two CIDRs (group

3) had a negative effect on LH secretion, which resulted in regression of

dominant non-ovulatory follicles. They concluded that increased concentrations

of progesterone regulate dominant follicle turnover through negative feedback by

decreasing LH pulse frequency. This observation was supported by other

studies (Savio et al., 1993a; Stock and Fortune, 1993). Furthermore, exogenous

progesterone administered during early metestrus suppressed the diameter of

the first-wave dominant follicle (Adams et al., 1992b; Burke et al., 1994; biskin et

al., 2002) by suppressing LH pulse frequency (Burke et al., 1994; Diskin et al.,

2002).

In contrast, when progestogens are administered in the absence of a

functional CL, the wave-like pattern of follicular development does not occur

(Sirois and Fortune, 1990). As a result, there is prolonged development of the

dominant follicle and increased concentrations of estradiol than usually observed

with growing dominant follicles (Sirois and Fortune, 1990; Cupp et al., 1995;

Kojima et al., 1992). The abnormally large follicle is termed a "persistent follicle"








and it remains on the ovary throughout the duration of the progestogen treatment

(Savio et al., 1993a; Stock and Fortune, 1993). Development of the persistent

follicle is typically observed when exogenous progestogens are administered for

period > 10 d. The persistent dominant follicle is supported by the high

frequency, low amplitude LH secretion during the progestogen treatment in the

absence of a functional CL (Roberson et al., 1989; Kojima et al., 1992). In

addition, fertility of subsequent estrus after progestogen withdrawal is

compromised due to ovulation of the persistent follicle. Oocytes ovulated from

persistent follicles are fertilized but subsequent development is compromised

resulting in increased embryonic mortality (Mihm et al., 1994; Ahmad et al., 1995;

Revah and Butler, 1996). Additionally, due to the increased concentrations of

estradiol produced by the persistent follicle, the altered hormonal environment

effects oviductal protein synthesis and secretion that may contribute to the

decreased fertility (Binelli et al., 1999).

The most common progestogen used for estrus synchronization is

melengestrol acetate (MGA). In most cases, MGA-treated cattle develop

persistent dominant follicles that do not ovulate during the duration of the MGA

treatment (Anderson and Day, 1994; Custer el al., 1994; Yelich et al., 1997)

because MGA blocks the preovulatory surge of LH (Kojima et al., 1995; Imwalle

et al., 2002). While 0.5 mg of MGA per day is enough to block the estradiol-

induced surge of LH, between 1.5 to 5.0 mg of MGA are necessary to suppress

LH pulses (Kojima et al., 1995; Hageleit et al., 2000). However, even at the








greatest MGA concentrations, there is still not enough suppression of LH pulses

to initiate dominant follicle turnover.

Exogenous progesterone can induce regression of persistent follicles

allowing for recruitment of a new wave of follicular growth (Savio et al., 1993a;

Anderson and Day, 1994; Fike et al., 1997). The mechanism by which

regression of dominant follicles is induced most likely is due to reduction of

pulsatile secretion of LH induced by increasing peripheral concentration of

progesterone or progestogens (Stock and Fortune, 1993; Anderson and Day,

1994; Taylor et al., 1994). Acute administration of progesterone during a period

of progestogen treatment has also been reported to initiate atresia of dominant

follicles and synchronize emergence of a new follicular wave in cattle of Bos

indicus breeding (Cavalieri et al., 1997; Cavalieri et al., 1998a; 1998c).

Combination of Estrogens and Progestagens

Administration of estrogens during progesterone-based treatments at

different stages of follicle development also alters follicle development (Bo et al.,

1995; Tribulo et al., 1995; Diskin et al., 2002). Bo et al., (1995) administered 5

mg of estradiol 17p at the initiation of a norgestomet implant at varying stages of

dominant follicle development and observed emergence of a new follicle wave

4.3 d after treatment. Emergence of a new follicular wave was similar for heifers

treated on d 3 (growing phase), 6 (early static phase), or 9 (regressing phase) of

the estrous cycle. The authors concluded that treatment with estradiol 170 in

combination with a norgestomet implant is effective in synchronizing follicle

emergence regardless of the stage of dominant follicle development.








Administration of an exogenous estrogen (Yelich et al., 1997; Diskin et al.,

2002) or estrogen plus progesterone (Fike et al., 1999) during administration of

an exogenous progestogen also induces regression of persistent follicles and

permits ovulation of a newly recruited follicle after progestogen withdrawal.

However, the estrogen treatments were only effective in initiating atresia in 71

and 77% of the persistent follicles in the Yelich et al. (1997) and Diskin et al.

(2002) studies, respectively.

It appears to be more difficult to control follicle wave dynamics with an

exogenous estrogen in animals that are at early stages of the estrous cycle (d 1

and 6) (Diskin et al., 2002). Besides, it is against the physiological

concentrations of estradiol that the use of estrogens for follicle wave regulation is

considered (Diskin et al., 2002). Furthermore, endogenous estradiol has a

marginal negative effect on LH secretion, requiring the simultaneous use of

progesterone to synchronize new follicle emergence (Bo et al., 1995).

Alternatively, estrogen may induce follicular atresia by altering LH secretion and

(or) FSH suppression (Price and Webb, 1988). Estrogen induced suppression of

LH may be apparent only during a progesterone dominated phase, whereas

estradiol and progesterone may have a synergistic suppressive effect on both LH

and FSH (Price and Webb, 1988; Bo et al., 2000). Estradiol in combination with

progestogen, suppress FSH secretion (Barnes et al., 1981; Bolt et al., 1990);

however, suppression was more prolonged in progesterone-implanted heifers

(Bolt et al., 1990). An additional reason for combining progesterone with

estradiol to cattle at random stages of the estrous cycle is to prevent the








exogenous estradiol from inducing ovulation when administered during the

follicular phase of the estrous cycle (Bolt et al., 1990).

Treating cattle of Bos indicus breeding (Cavalieri et al., 1997) with a

combination of progesterone and estradiol to regulates ovarian follicular

development similar to the same treatments in Bos taurus cattle (Bo et al., 1994;

Rajamahendran and Manikkam, 1994; Bo et al., 2000). Cavalieri et al. (1997)

treated crossbred Brahman cows with a sub-cutaneous implant containing

estradiol 1713 on d 8 of a 10 d CIDR treatment. Estradiol regulated the

emergence of the ovulatory follicle and prevented the ovulation of dominant

follicles present at the time of treatment. The mean interval between initiation of

estradiol treatment and day of emergence of the ovulatory follicle was 3.3 d.

Gonadotropin Releasing Hormone (GnRH)

Administration of GnRH to cattle at various stages of the estrous cycle has

been shown to alter follicular development as evidenced by changes in the

distribution of follicles among different size classes and induction of an L"H surge,

which induces either ovulation or luteinization of the dominant follicle (Thatcher et

al., 1989; Macmillan and Thatcher, 1991). The result is emergence and

synchronization of a new follicular wave (Twagiramungu et al., 1995). The GnRH

initiates an acute secretion of LH and FSH such that circulating concentrations

are elevated for 3 to 5 h, which stimulates ovulation or luteinization of large

follicles (Thatcher et al., 1993; Vizcarra et al., 1997; D'Occhio and Aspden,

1999). Consequently, removal of the dominant follicle permits an endogenous

surge of FSH, which initiates recruitment of a new follicular wave. The

effectiveness of a GnRH agonist to induce ovulation appears to be affected by








the stage of follicular development at GnRH treatment (Prescott et al., 1992;

Silcox et al., 1993; Pursley et al., 1995; Moreira et al., 2000a). Furthermore,

GnRH appears to be effective in inducing ovulation in follicles greater than 9 mm

and during the growing phase of follicle development (Macmillan and Thatcher,

1991; Vasconcelos et al., 1999; Moreira et al., 2000). Martinez et al. (1999)

reported that administration of a GnRH agonist 3, 6, or 9 d after estrus did not

induce atresia of the dominant follicle or alter the interval to new wave

emergence in cattle that did not ovulate in response to the GnRH treatment. In

another experiment, Moreira et al. (2000) reported that the ovulatory response to

GnRH (0, 100, 25, 60, and 100%) was affected by day of the estrous cycle (d 2,

5, 10, 15, and 18, respectively) when treatment was administered. The authors

concluded that heifers treated on d 2 did not have an established dominant

follicle, while the low response in the d 10 group was attributed to the small size

of the newly emerged second wave dominant follicle. Similarly, Kohram et al.

(1998) reported that GnRH could induce ovulation during the early (d 4 to 7) and

late (d 15 to 18) phases of the estrous cycle. Therefore, it appears that GnRH

synchronizes new wave emergence only when administered in the presence of a

dominant follicle, which ovulates to GnRH. However, before a follicle obtains

dominance, it appears not to affect the subsequent progress of that wave,

presumably because of lack of LH receptors on the granulosa cells of the

growing follicles (Diskin et al., 2002).

In contrast, there are reports that suggest that there is considerable

variation in the ability of a GnRH to induce ovulation and initiate the emergence








of a new follicle wave. In presynchronized beef heifers treated with a GnRH

agonist on d 3, 6 or 9 of the subsequent estrous cycle, ovulation occurred within

36 h after GnRH in 89, 56 and 22% of the heifers, respectively (Martinez et al.,

1999). Diameter of the largest follicle on d 3 was greater in follicles that ovulated

(9.6 mm) than those not ovulating (7.5 mm). The authors concluded that their

results did not support the hypothesis that the administration of GnRH at known

stages of the follicular wave in cycling heifers would consistently induce ovulation

or atresia and emergence of a new follicular wave at a predictable interval. New

wave emergence was induced consistently (1.3 d after GnRH) only in heifers that

ovulated in response to GnRH. However, 44% of the heifers treated with GnRH

failed to ovulate. In contrast, Rajamahendran et al. (1998) reported that the first

wave dominant follicle ovulated in all the nonlactating Holstein cows treated with

8 [ig of buserelin on d 5 of the estrous cycle. Vasconcelos et al. (2000)

comparing Holstein and Holstein-Gyr crossbred cows receiving an Ovsynch

protocol during winter and summer, reported that ovulation rate after the first

GnRH injection was greater in the winter than summer, with ovulation rates 61.4

and 37.8% for Holstein, and 58.3 and 45.2% for crossbred cows, respectively.

The variability in GnRH ability to induce ovulation at a known stage of the estrous

cycle between studies is unclear. These differences could be attributed to follicle

wave differences that could be associated with physiological state and (or) age of

the animals being used. Whether there is a breed effect (i.e., Bos indicus vs.

Bos taurus) is even less clear.








Chronic administration of GnRH agonists has also been used to suppress

estrous cycles in beef cattle (Gong et al., 1996; D'Occhio et al., 1996; 2000).

Alterations of the CL or follicle induced by GnRH seem to be indirect through

changes in LH and FSH secretion (Thatcher et al., 1993). Furthermore, a single

injection of GnRH late in diestrus will cause acute increases in plasma

progesterone and a delay in CL regression (Thatcher et al., 1993).

Administration of GnRH also appears to influence subsequent CL function.

Ambrose et al. (1998) showed that a GnRH agonist implant (Deslorelin) in dairy

cattle altered CL function and follicular dynamics when administered on d 0 (d 1=

ovulation). The increase in plasma progesterone between d 0 and 15 was

greater for the GnRH agonist group than either a non-treated or a standard

GnRH injection. Furthermore, development of the first wave dominant follicle

was delayed. These data support the hypothesis that GnRH agonist induces

development of a more functional corpus luteum. Apparently, early LH secretion

was stimulated in the GnRH agonist group to increase CL development (Mattos

et al., 2001; Thatcher et al., 2002). However, subsequent secretion of FSH and

LH was probably reduced due to the desensitization of the pituitary in a way that

delayed development of the dominant follicle but did not compromise CL function

(Mattos et al., 2001; Thatcher et al., 2002). Induction of ovulation, stimulation of

development of a more functional CL, and the delay in the functional maturation

of dominant follicles until after development of the antiluteolytic mechanisms, are

potential means to improve conception rates and embryo survival (Mattos et al.,

2001; Thatcher et al., 2002).








Information about LH concentrations in the peripheral circulation in cattle

after a GnRH treatment during the estrous cycle is numerous. The data available

refer to description of LH response following administration of GnRH or its

analogs in a nymphomaniac cow (Mori et al., 1974), in normal cows at

insemination (Mori and Takahashi, 1978), in cows with ovarian follicular cysts

(Mori et al., 1979; Tanaka et al., 1979), in beef heifers following treatment with

GnRH within hours after the onset of estrus (Coleman et al., 1988), in dairy

heifers following injection of various dosages (Chenault et al., 1990), in beef

cows treated during the postpartum period alone (Williams and Stanko, 1996;

Fajersson et al., 1999), or in combination with PGF2a (Cruz et al., 1997), in early

postpartum crossbred cows (Deen et al., 1995) and dairy cows (Vural et al.,

1999), in cattle following chronic administration for controlled, reversible

suppression of estrous cycles (Gong et al., 1996; D'Occhio et al., 1996; Vizcarra

et al., 1997; Rajamahendran et al., 1998; D'Occhio et al., 2000), in

ovariectomized Brahman and Hereford cows challenged with GnRH (Griffin and

Randel 1978), and in Brahman and Angus cows on d 17 and 34 after calving

(Stahringer et al., 1989). In summary, most of these data have been generated

in cattle of Bos taurus breeding and very little in cattle of Bos indicus breeding.

There is little information where direct comparisons between cattle of Bos taurus

and Bos indicus breeding have been made relative to endocrine and ovarian

responses of cycling cattle to a single injection of GnRH.

In reference to LH response after exogenous GnRH administration in cattle,

Coleman et al. (1988) reported that Fertirelin acetate (FA; synthetic GnRH








agonist) administered to crossbred Bos taurus beef heifers 12 h after the onset of

estrus increased plasma concentrations of LH without affecting subsequent luteal

function. Mean LH concentration was 18.6 ng/ml at 120 min after FA; however,

the variation in LH concentrations was significant and ranged form 5.3 to 115.9

ng/ml. Chenault et al. (1990) performed a study to describe the changes in

serum LH and FSH concentrations in Holstein heifers following intramuscular

injection of various dosages of FA and other GnRH agonist at their labeled

dosages. The authors reported that mean LH concentrations increased within 15

min after treatment for the different dosages of FA, and LH concentrations

remained above baseline for approximately 180 to 360 min, dependent on the

dosage of GnRH. The interval to peak LH and the mean LH peak after GnRH for

all products and dosages ranged from 36 to 158 min and 2.5 to 25.5 ng/ml,

respectively. In a study by Cruz et al. (1997), the effect of GnRH on LH secretion

and ovulation in anestrous and cyclic postpartum beef cows were evaluated.

Mean peak LH values were similar (P> 0.20) for the cows that ovulated (107.7 +

11.0 ng/ml) compared with cows that failed to ovulate (103.6 + 14.5 ng/ml) after

GnRH administration. Vural et al. (1999) working with Holstein cows injected

with GnRH on d 14 post partum reported that the LH concentrations (0.47 to 24.6

ng/ml) peaked within two hours after GnRH, and returned to preinjection

concentrations within six hours. The variation between animals in LH

responsiveness to exogenous GnRH under standardized physiological conditions

is evident, and has been reported by others (Williams and Stanko, 1996;

Fajersson et al., 1999).








Data comparing the induction of LH secretion by GnRH administration

between Bos indicus and Bos taurus females are variable and may be effected

by the dose and (or) reproductive status of the animal. Griffin and Randel (1978)

comparing LH responses to 500 pg GnRH in ovariectomized Brahman and

Hereford cows, and reported that all cows responded with increased serum LH

concentrations within 15 min of administration of GnRH. However, mean LH

response and average peak LH concentrations were decreased in the Brahman

(34 4 ng/ml and 94 7 ng/ml, respectively) versus Hereford cows (67 20

ng/ml and 185 68 ng/ml, respectively). In contrast, Stahringer et al. (1989)

reported that Brahman cows that were 17 to 34 d post partum and challenged

with a low dose (4.5 ng/lb BW) of GnRH had increased basal LH, increased

mean LH concentrations, increased GnRH-induced LH pulse amplitude, and an

increased GnRH-induced pulse height versus Angus cows. These data suggest

that pituitary function in Bos indicus cattle is different from pituitary function in

Bos taurus animals.

In progesterone-based treatments for the control of ovarian activity, GnRH

has been used to induce turnover of large follicles that would otherwise have the

potential to develop into persistent follicles. Schmitt et al. (1996a) using

nonlactating Holstein cows treated with a norgestomet implant on d 7 of the

estrous cycle and PGF2a at implant withdrawal observed that injection of GnRH

on d 9 of the estrous cycle induced ovulation of dominant follicle and a newly

recruited dominant follicle was induced to grow and ovulate following removal of

the norgestomet implant. Similarly, Ryan et al. (1998) observed that








administration of GnRH was effective in initiating follicle turnover in cows with

dominant follicles at CIDR insertion, resulting in emergence of a new follicular

wave. However, GnRH did not initiate follicle turnover when administered before

dominant follicle selection. These data indicate that GnRH is effective in induced

turnover of dominant follicles in progesterone based treatments, preventing the

potential development of persistent follicles that fail to ovulate due to the

suppressive action of progesterone on LH secretion.

Prostaglandin F2a (PGF2a)

The luteolytic actions of PGF2a and its analogues (Cloprostenol, Alfaprostol,

and Luprostiol) after d 4 of the estrous cycle and rirougnoul tr-e remainder of

luteal phase of the estrous cycle have been well documented (Rowson et al.,

1972; Kiracofe et al., 1985; Godfrey et al., 1989). Several researchers have

reported that after d 5 of the estrous cycle, the CL was responsive to a PGF2a

treatment, but the degree of luteolysis was dependent on the stage of the estrous

cycle when PGF2a was administered (Tanabe and Hahn, 1984; Watts and

Fuquay, 1985). Consequently, the ineffectiveness of a single dose of PGF2. to

initiate luteolysis before d 5 of the estrous cycle limits the overall effectiveness of

using PGF2a for the synchronization of estrus in cattle random stages of the

estrous cycle (Beal et al., 1980). Administration of PGF2a to cycling cattle of Bos

taurus breeding yields consistent results relative to induction of luteolysis,

synchronization of estrus, and fertility of the synchronized estrus. However,

similar results in cattle of Bos indicus breeding treated with PGF2a have not been

observed. Initially, the inconsistent results in cattle of Bos indicus brseejng








appeared to be associated with compromised expression of estrus (Orihuela et

al., 1983; Pinheiro et al., 1998; Rekwot et al., 1999). However, the decreased

estrous response after PGF2a appears to be associated with incomplete

luteolysis, which resulted in blood progesterone concentrations that are great

enough to prevent the expression of estrus (Hardin et al., 1980a, 1980b; Pinheiro

et al., 1998; Rekwot et al., 1999; Mattoni and Ouedraogo, 2000).

In Bos taurus heifers treated with PGF2. between d 5 and 8 of the estrous

cycle, 85 to 100% of the heifers regressed their CL (King et al., 1982; Tanabe

and Hann 1984; Kiracofe et al., 1985). On the contrary, in Bos taurus x Bos

indicus cattle, Hardin et al. (1980a) observed a 64% estrous response after an

injection of cloprostenol between d 6 and 9 of the estrous cycle. Williams et al.

(1999) reported estrous rates of 54 and 63% in Brahman x Hereford heifers

treated with PGF2. on d 6 and 10 of the estrous cycle, respectively. Likewise,

Cornwell et al. (1985) reported that only 67% of Brahman heifers expressed

estrus after being treated on d 7 of the estrous cycle with PGF2a, while 100% of

the heifers expressed estrus when PGF2a was injected on d 14 of the estrous

cycle. In a second study using Brahman heifers, Cornwell et al. (1985) reported

that only 50% of heifers treated with PGF2a on d 7 of the estrous cycle expressed

estrus, while 67% injected on d 10 of the estrous cycle expressed estrus. In the

later study, blood progesterone concentrations decreased within 12 h after PGF2a

injection in all heifers; however, for heifers that failed to express estrus, blood

progesterone concentrations began to increase within 48 h after PGF2,. In a

similar study, Santos et al. (1988) treated Brahman heifers with PGF2a on d 7 and








10 of the estrous cycle and observed only 50 and 67% estrus expression,

respectively. Additionally, for all heifers treated on d 7 and 10 blood

progesterone concentrations decreased within 12 h after treatment, but in heifers

not expressing estrus the decrease in blood progesterone concentrations were

temporary and progesterone concentrations recovered to pre-treatment

concentrations. However, it should be noted that the "rebounding effect" of

progesterone observed in heifers failing to undergo CL regression has also been

observed in cattle of Bos taurus breeding (Chenault et al., 1976; Copelin et al.,

1988). Hansen et al. (1987) concluded that in Simmental x Brahman x Hereford

heifers treated on d 8 to 10 of the estrous cycle required a greater dose of

alfaprostol to elicit luteolysis and estrus than heifers treated on d 11 to 13 of the

estrous cycle.

The aforementioned data demonstrate that luteolysis appears to be

incomplete and quite variable when PGF2a is injected between d 6 to 7 of the

estrous cycle in cattle of Bos indicus breeding. Moreover, when cattle of Bos

indicus breeding are administered PGF2a later in the estrous cycle (d 10),

luteolysis and estrous response are improved but still highly variable. In

conclusion, cattle of Bos indicus breeding appear to be less responsive to PGF2a

at all stages of the estrous cycle than cattle of Bos taurus breeding. However,

there are insufficient data to indicate what the difference in magnitude for CL

regression and estrous response are between Bos taurus and Bos indicus cattle

and there are no studies where direct comparisons have been made. If luteolysis

is significantly compromised in cattle of Bos indicus breeding, this could have








significant implications on the type of estrus synchronization systems used

relative to how and when PGF2a is used to induce luteolysis.

In general, when CL regression is induced by exogenous PGF2. treatment

at random stages of the estrous cycle, estrus will be displayed within 2 to 7 d.

The large variation for the interval to estrus is likely due to differences in follicle

development at luteolysis (Fogwell et al., 1986; Sirois and Fortune, 1988; 1990).

Sirois and Fortune (1988) observed that size of the dominant follicle at luteolysis

was negatively correlated with the interval to the LH surge, suggesting that the

interval to estrus may be determined by the size of the preovulatory follicle at

luteolysis. The interval to estrus in cows and heifers administered PGF2a early in

the estrous cycle (d 5 to 9) is shorter than in cattle injected late in the estrous

cycle (King et al., 1982; Tanabe and Hann, 1984; Watts and Fuquay, 1985).

Therefore, cattle early in their estrous cycle are likely to have a dominant follicle

from the first follicular wave that is capable of ovulation immediately after

luteolysis. Unlike the first follicular wave, the second and (or) third follicular

waves do not emerge in a synchronous fashion resulting in longer and more

variable intervals from PGF2a treatment to estrus.

Estrus Synchronization Systems and Time Artificial Insemination

The main purpose for synchronizing estrus in cattle is to decrease the

number of days detecting estrus from approximately 21 d without synchronization

to approximately 5 to 7 d with synchronization so that all cattle have an

opportunity to be artificially inseminated (Al). Decreasing the numbers of days

spent detecting estrus makes Al more practical. An estrus synchronization








system should elicit a fertile, closely synchronized estrus in a high percentage of

treated females (Odde, 1990). Additionally, an ideal estrus synchronization

system should require minimal cattle handling with minimal economic inputs for

the producer. However, to attain accurate control of the estrous cycle and the

subsequent synchronized estrus, life span of the CL and follicle wave status must

be regulated by the synchronization system. Synchronization systems regulating

the lifespan of the CL and follicle wave development increase the synchrony of

the synchronized estrus and ovulation. If estrus and ovulation can be tightly

synchronized, estrus detection can be eliminated and cattle can be inseminated

at a designated time known as timed-Al.

The elimination of estrus detection is probably the key to increasing the

effectiveness of estrus synchronization systems in cattle of Bos indicus breeding.

The effectiveness of estrus synchronization systems in cattle of Bos indicus

breeding is often compromised because of problems associated with estrus

detection and response to different estrus synchronization drugs. Moreover,

development of predictable estrus synchronization systems oriented toward

cattle of Bos indicus breeding has seldom been the primary focus of researchers.

Most estrus synchronization research has been conducted using beef and dairy

cattle of Bos taurus breeding. Also, certain differences in the reproductive

performance of heifers in response to different estrus synchronization systems

have been reported when compared with lactating postpartum cows (Williams et

al., 2002).








The knowledge that progestogens prevented the occurrence of estrus and

ovulation was the basis for the early attempts to synchronize estrus in cattle

(Christian and Casida, 1948). Several forms of progestogens have been used in

estrus synchronization systems including norgestomet, melengestrol acetate

(MGA), injections of progesterone, or administration of progesterone via the

vaginal inserts like the progesterone releasing intravaginal device (PRID) and

controlled intravaginal progesterone-release device (CIDR).

Melengestrol acetate is an orally active progestogen, which when

administered at a rate of 0.5 mg/head/d suppresses estrus in beef (DeBois and

Bierschwal 1970) and dairy cattle (Roussel and Beatty, 1969). Beef cattle

producers like to use MGA because it is inexpensive, easy to administer, and can

induce estrous cycles in some postpartum cows (Beal and Good, 1986;

McDowell et al., 1998) and peripubertal heifers (Fike et al., 1999). However,

there are several disadvantages to using MGA. In grazing cattle, it is dirt,:uit it.

assure adequate consumption of MGA during the treatment period. Second,

fertility of the estrus after long-term (> 10 d) MGA administration is significantly

compromised due to the development of a persistent follicle. The etiology and

reason for the decreased fertility were discussed earlier in this review. Athough,.

the reduction in fertility is temporary and it returns to normal at the subsequent

estrus. Researchers used this later observation to their advantage to develop a

synchronization system that used long-term MGA administration.

Brown et al. (1988) administered MGA for 14 d followed by the

administration of PGF2. 17 d after the last day of MGA treatment or the








MGA/PGF2. estrus synchronization system. Administering PGF2a 17 d after

MGA, allowed the researchers to inject PGF2. when a majority of heifers were in

the later stages of the estrous cycle where PGF2a is more effective in inducing

CL regression (Tanabe and Hahn, 1984; Watts and Fuquay, 1985). The

MGA/PGF2. estrus synchronization system induced a tightly synchronized estrus

that resulted in excellent synchronized pregnancy rates in beef heifers (Brown et

al, 1988). Later research also reported that the MGA/PGF2a estrus

synchronization system also was effective in suckled postpartum beef cows

(Patterson et al., 1989; Yelich et al., 1997).

One of the problems with the MGA/PGF2a estrus synchronization system is

that it takes so long to implement. Therefore, another approach was developed

to deal with the reduced fertility after MGA withdrawal. This included

administration of an exogenous estrogen (Kastelic et al., 1996; Yelich et al.,

1997) or progesterone (Anderson and Day, 1998) during MGA feeding to initiate

regression of the persistent follicle, which resulted in improved fertility at the

synchronized estrus after MGA withdrawal.

To circumvent the problems with reduced fertility with long-term MGA

treatments, the approach was taken to reduce the duration of progestogen

treatment to approximately 7 d. Several studies (Patterson et al., 1986; Beal et

al., 1988) evaluated the effectiveness of reduced periods of progesterone

exposure (7 or 9 d) by combining MGA with a luteolytic dose of PGF2a at the end

MGA administration. Feeding MGA for short periods resulted in improved fertility

when feeding was started early in the estrous cycle, but conception rates were








s.gni.ic aniil, reduced when the MGA treatment was initiated late in the estrous

cycle. This is probably due to the development of persistent dominant follicles

(Custer et al. 1994; Kojima et al. 1995), which are associated with reduced

fertility (Patterson et al., 1989; Custer et al., 1994; Ahmad et al., 1995), likely due

to ovulation of aged oocytes (Mihm et al., 1994). Other researches have used

the PRID and CIDR to deliver progesterone via the vagina for the duration of 7 to

12 d. Luteolysis is achieved by administering PGF2a at or just prior to device

removal. Lucy et al. (2001) reported that treatment with a CIDR for 7 d with

PGF2. on d 6 resulted in a increased synchronized estrous response compared

to PGF2, alone and untreated controls in postpartum beef cows, beef heifers,

and dairy heifers. Differences in fertility between MGA and CIDR treatments are

probably due to differences in circulating levels of progesterone between the two

treatments. Plasma progesterone concentrations have been reported to increase

by 2 h after CIDR-B insertion (Burke et al., 1999). However, progestin levels

may increase more slowly with an oral preparation such as MGA (Kojima et al.,

1995). Administration of exogenous progesterone to animals lacking a functional

CL, results in the development of large follicles that persist on the ovaries (Sirois

and Fortune, 1990; Savio et al., 1993a; Yelich et al., 1997). The development of

the persistent follicle is associated with an increased LH pulse frequency due to

the lack of negative feedback effects of luteal progesterone (Savio et al., 1993a;

Kojima et al., 1995). This problem may be overcome by strategically

synchronizing follicular wave emergence at the start of the short-term

progesterone treatment. Estrogens and GnRH have been compared in both








CIDR based programs and short-term (7-d) MGA treatments (Martinez et al.,

2001; 2002) for synchronization of wave emergence and ovulation and therefore,

to regress and prevent the ovulation of a persistent dominant follicle.

As previously discussed in this chapter, administration of exogenous

estrogens initiates turnover of dominant follicles that might develop into

persistent follicles during both long and short-term progestogen treatments.

Recently, researchers have focused on administering estrogens at the initiation

of short-term progestogen treatments to synchronize follicle development, which

leads to a more synchronous estrus and ovulation after progestogen removal.

Tribulo et al. (1995) reported that administration of estradiol-171 either at CIDR

insertion or within 2 d after CIDR insertion resulted in a more synchronous estrus

and ovulation than two injections of PGF2a or CIDR alone. Estrogens

administered after CIDR treatment are very effective in inducing estrus and

ovulation. Administration of estradiol benzoate 24 to 30 h following a 7-d CIDR

treatment in cows (Fike et al., 1997) and heifers (Johnson et al., 1997; Lemaster

et al., 1999) increased the estrous response and the synchrony of estrus

compared to a CIDR treatment alone in cattle of Bos taurus breeding. Similarly,

Lammoglia et al. (1998) reported that over 90% of cows and heifers exhibited

estrus within 48 h after a 7-d CIDR with PGF2a at CIDR removal and estradiol

benzoate administered 24 to 30 h after CIDR removal in heifers of Bos taurus

breeding. Lemaster et al. (1999) also reported that administration of estradiol

benzoate after CIDR removal was effective in inducing a tightly synchronized

estrus and ovulation in Bos indicus x Bos taurus heifers. In addition, exogenous








estrogens have been used after progestogen treatment as a treatment regime for

postpartum anestrous cows (McDougall et al., 1992; Fike et al., 1997) in order to

eliminate cows that ovulate without an observed estrus (M.-Dcugall et al., 1992).

Consequently, the addition of estrogen at the termination of a progestogen

treatment increases the number of animals detected in estrus and submitted for

Al.

Similar to results obtained by administering estrogens at the initiation of

short-term progestogen treatments to synchronize follicle development (Tribulo et

al., 1995; Martinez et al., 2001), administration of GnRH at the initiation of a

CIDR treatment improved pregnancy rates in heifers (Martinez et al., 2001;

2002).

In general, progestogens treatments in postpartum anestrous Bos indicus x

Bos taurus dual-purpose cows (Soto et al., 1998; de Ondiz et al., 2002; Soto et

al., 2002) have shown satisfactory results in the induction of estrus. Percentage

of anestrous cows that exhibited estrus was 75 to 81% and 60.7% (Hernandez et

al., 1995; de Ondiz et al., 2002; Soto et al., 2002) when using norgestomet

implants (Sincro-Mate B and Crestar, respectively); and 83.3% for CIDR (Soto et

al., 1998). Conception rates at the synchronized estrus after progestogen

withdrawal are quite variable (28 to 68%; Hernandez et al, 1995; Soto et al.,

1998; 2002). However, treatment of postpartum dual purpose crossbred Bos

indicus x Bos taurus cows with intravaginal sponges (Medroxiprogesterone

acetate) 40 d post partum (Palomares et al., 2002) have resulted in decreased

induction of estrus when combined with either GnRH (65.2%) or estradiol








(47.6%) at sponge insertion, with ovulation rates of 57.1 and 25.0%, respectively.

Very low conception rates at first estrus after these treatments were observed

(26.7% and 10.0% for sponges + GnRH and sponges + estradiol, respectively).

Thatcher et al. (1989) were the first to propose a protocol to synchronize

follicular development using GnRH followed by 7 d later with an injection of

PGF20 to initiate luteal regression. A surge of LH in response to GnRH treatment

causes ovulation when a dominant follicle is present (Thatcher et al., 1989;

Macmillan and Thatcher, 1991) while the PGF2a or its analogues induced

regression of the newly formed CL and other CL present on the ovaries.

Following administration of the GnRH/PGF2. system, cattle can be inseminated

either after a detected estrus or timed-Al in combination with GnRH in dairy

(Pursley et al., 1995; Schmitt et al., 1996b) and beef cattle (Geary et al., 1998a;

Stevenson et al., 2000) resulting in very acceptable pregnancy rates. The

GnRH/PGF2a systems appear to be more effective in cows of Bos taurus

breeding (Pursley et al., 1995; Geary et al., 1998a) compared to cows of Bos

indicus breeding (Lemaster et al., 2001). Although, no direct breed comparisons

have been made.

Macmillan and Thatcher (1991) synchronized estrus in cattle with GnRH

followed 7 d later with an injection of PGF2a compared to a single injection of

PGF2a. Estrous response and synchrony of estrus were more favorable with the

combination of GnRH and PGF2a compared to PGF2a alone, with estrus being

expressed 2 to 3 d after PGF2a injection. In addition, there were no differences in

fertility between the two groups. The combination of GnRH and PGF2,, with








estrus detection for 5 to 7 d, has been designated as the SelectSynch system

(Geary and Whittier, 1999). Cattle are typically inseminated 8 to 12 h after the

observed estrus. Furthermore, the percentage of synchronized cattle is

increased and the variation in the interval from PGF2. to the onset of estrus is

reduced compared to a single injection of PGF2a (Thatcher et al., 1989;

Twagiramungu et al., 1992). The mean interval from PGF2a injection to estrus is

51 h in Bos taurus cattle (Twagiramungu et al., 1992; Downing et al., 1998).

Conception rates for SelectSynch are similar and (or) increased compared to

cattle inseminated after a single injection of PGF2a (Twagiramungu et al., 1992;

Stevenson et al., 2000).

The SelectSynch system was modified to eliminate the need for estrus

detection. It consisted of a GnRH injection followed 7 d later by an injection of

PGF2a. A second dose of GnRH was injected 48 h after the injection of PGF2a

and all cows are timed-Al 8 to 18 h later, with maximum pregnancy rate achieved

when cows are inseminated 16 h after the second GnRH (Pursley et al., 1998).

The second GnRH injection was administered to synchronize ovulation of the

newly recruited dominant follicle prior to timed-Al. The system was called the

OvSynch system and was first tested in lactating dairy cows and dairy heifers

(Pursley et al., 1995; Burke et al., 1996; Schmitt et al., 1996b). Dairy cows

synchronized with the OvSynch and SelectSynch systems have similar

pregnancy rates (Burke et al., 1996).

Postpartum Bos taurus beef cows synchronized with either SyncroMate-B

(SMB) or OvSynch have also been compared (Geary et al., 1998a). In the Geary








et al. (1998a) study timed-AI took place 24 h after the second GnRH injection or

48 h after removal of the SMB implant. In addition, calves were removed from

cows either at SMB implant removal or at PGF2, injection until timed-AI was

completed. The OvSynch protocol resulted in greater timed-Al pregnancy rates

(54%) than SMB treated cows (42%). For cows determined to be cyclic at the

start of the treatments, timed-Al pregnancy rates were greater for OvSynch

(59%) than SMB (38%). Furthermore, timed-Al pregnancy rates for cows

determined to be anestrous at the start of the treatments tended to favor the

OvSynch protocol. The initial GnRH injection of OvSynch system probably

induced estrous cycles in more cows than the SMB treatment. One

disadvantage of the OvSynch system is that cattle are handled four times. This

has lead to the development of the Cosynch protocol, where cattle are

inseminated at the time of the second GnRH injection (Geary et al., 1998b). Both

the OvSynch and CoSynch systems resulted in similar timed-Al pregnancy rates

(48%) in Bos taurus beef cows (Geary and Whittier, 1997).

Stevenson et al. (2000) treated Bos taurus beef cows with SelectSynch,

CoSynch, or a modified SelectSynch system called the HybridSynch system. In

the HybridSynch system, cows were inseminated after a detected estrus for 54 h

after PGF2~ and all cows not exhibiting estrus by 54 h were administered GnRH

and timed-AI. In agreement with a previous study by Thompson et al. (1998),

pregnancy rates of anestrous cows were similar among treatments; however,

pregnancy rates in cycling cows were greater for the SelectSynch (56%)

compared with CoSynch (40%) or HybridSynch (39%).








Geary and Whittier (1999) carried out two experiments to evaluate the

efficacy of using variations of the HybridSynch protocol in Bos taurus cows. In

the first experiment, all cows received the GnRH and PGF2. injections and

inseminated 12 h after an observed estrus for 72 h after PGF2a. At 72 h post-

PGF2a, cows that had not been inseminated were divided into two groups and

were timed-AI concomitant with GnRH either 72 or 84 h after PGF2a injection.

The percentage of cows that exhibited estrus within 72 h following PGF2a

injection was 48% and their conception rate was 56%. In contrast, conception

rates for cows timed-Al at 72 and 84 h were 21 and 24%, respectively. In the

second experiment, cows received the GnRH and PGF2. injections and were

artificially inseminated 12 h after an observed estrus for 48 h after PGF2a

injection. At 48 h post-PGF2z, cows that had not been inseminated were timed-

Al concomitant with GnRH either 48 or 64 h after PGF2a injection. Only 18% of

the cows were observed in estrus within 48 h after PGF2a; however, their

conception rate was high (68%). Pregnancy rate of cows that were timed-Al

either 48 or 64 h were 43 and 39%, respectively. Overall Al pregnancy rates for

the two groups were 48 and 44%, respectively. These data suggest that optimal

pregnancy rates were obtained when GnRH and timed-Al were conducted 48 h

following PGF2, injection. However, because of the increased conception rates

of cows inseminated after an observed estrus compared to the timed-AI,

extending estrus detection from 48 to 72 h after PGF2a might be justified in the

HybridSynch system.








Limited research with any of the GnRH and PGF2a systems for estrous

synchronization in cattle of Bos indicus breeding has been conducted.

Compared to results obtained in suckled post partum cows of Bos taurus

breeding synchronized with the three GnRH + PGF2a systems (Twagiramungu et

al., 1992; Geary and Whittier 1999; Stevenson et al., 2000), synchronized

pregnancy rates appear to be decreased in suckled postpartum cows of Bos

indicus x Bos taurus breeding (Lemaster et al., 2001). In the study conducted by

Lemaster et al. (2001), pregnancy rates were greater for CoSynch (31%) and

HybridSynch (36%) treated cows than for SelectSynch (21%) cows. For cows

determined to be cycling at the initial GnRH injection, estrous response the

SelectSynch and HybridSynch cows during the 72 h after PGF2. were decreased

by greater than 30% compared to cycling, lactating Bos taurus cows

synchronized with the same protocols (Geary and Whittier, 1999; Stevenson et

al., 2000). Lemaster et al. (2001) speculated that the low estrous response might

have been due to either incomplete regression of accessory CL formed resulting

from the first GnRH injection or a compromised estrous expression by the Bos

indicus influenced cattle.

One of the disadvantages of the GnRH + PGF2a systems is that depending

of the stage of the estrous cycle the cattle are in, not all cattle will respond to the

initial GnRH (Moreira et al., 2000). Recent studies have also shown stage of

cycle effects the Ovsynch protocol in dairy cattle (Vasconcelos et al., 1999;

Moreira et al., 2000). Vasconcelos et al. (1999) reported ovulation rates to the

first GnRH injection of 23, 96, 54, and 77% for lactating dairy cows between d 1








to 4, 5 to 9, 10 to 16, and 17 to 21 of the estrous cycle, respectively. The low

ovulation rate during the early (d 1 to 4) and mid (d 10 to 16) estrous cycle was a

result of no dominant follicle available for ovulation (Vasconcelos et al., 1999;

Moreira et al., 2000). Overall, 87% of cows responded to the second GnRH

injection, which varied according to the response to the first GnRH. For cows

ovulating to the first GnRH, 92% of these ovulated to the second GnRH, whereas

for cows did not ovulate to the first GnRH injection, 79% of these ovulated to the

second GnRH. Consequently, the decreased rate of ovulation to the first GnRH

injection appears to generate a decreased synchronization rate following the

second injection of GnRH.

If the initial GnRH injection fails to ovulate a follicle in cattle at the late

stages of the estrous cycle, these cattle will exhibit a spontaneous estrus before

the PGF2a injection (Thatcher et al., 1996). Approximately 5 to 15% of the cows

may be detected in estrus either on or before the day of PGF2a injection, thus

reducing the number of animals inseminated during the synchronized period

(Schmitt et al., 1996b; Downing et al., 1998; Moreira et al., 2000). According to

Geary and Whittier (1999), cows that exhibit estrus early are usually between d

14 and 17 of their estrous cycle at the initial GnRH treatment and do not respond

to GnRH. Several studies have confirmed that the response to the GnRH +

PGF2a protocol differs according to the day of the estrous cycle at the initial

GnRH treatment (Downing et al., 1998; Vasconselos et al., 1999; Moreira et al.,

2000). Downing et al. (1998) reported that 88% of the cows treated with GnRH

at different stages of the estrous cycle responded to PGF2a as noted by complete








regression of the original and (or) induced CL. However, only 14% of the cows

between d 15 and 17 at GnRH injection responded to PGF2a with the majority of

these cows exhibiting estrus prior to PGF2,. Therefore, cows treated with GnRH

between d 15 and 17 of the estrous cycle were most likely undergoing normal

luteal regression prior to PGF2,. In addition, as the day of cycle at initiation of

GnRH treatment progressed, interval to estrus also increased. These data

suggest that the GnRH + PGF2a protocols are effective in synchronizing estrus,

but maximum response is achieved when estrus detection begins 1 to 2 d prior to

PGF2,. The current recommendation is that detection of estrus begin as early as

4 d after GnRH injection and continue through 5 d after PGF2. in cows treated

with GnRH + PGF2, (Kojima et al., 2000). However, this is impractical since it

increases the number of days required to detect estrus. Therefore, alternative

methods such as administering a progestogen between the GnRH and PGF2a

injections would eliminate the need for the extra estrus detection.

Thundathil et al. (1999) conducted an experiment to evaluate the efficacy of

short-term progestogen treatments for estrus synchronization in beef cows.

Cattle were administered MGA for 7 d with 1 or 5 mg of estradiol-17p and 100

mg of progesterone or 100 pg of GnRH at the initiation of MGA with 500 pg of

cloprostenol on 7 d later. Cattle were Al approximately 12 h after detected

estrus. There were no significant differences among treatments for estrous

response or conception rates suggesting short-term progestogen treatments are

effective estrus synchronization systems in lactating cows.








Other researchers have combined a long-term MGA treatment with the

SelectSynch system for estrus synchronization (Patterson et al., 2002).

Postpartum cows received either a 14 d MGA treatment with SelectSynch

(MGASelect) initiated 12 d after MGA withdrawal compared to the

MGA/PGF2,.system with PGF2a 19 d after MGA. Cows were observed for estrus

for 7 d after PGF2a and inseminated 12 h after estrus. Estrous response was

greater in MGASelect (87%) than MGA- PGF2a (76%) cows. Mean interval to

estrus after PGF2. was longer for MGA/ PGF2a (80.4 h) than MGASelect (74.5 h).

Synchronized pregnancy rate did not differ between MGASelect (66.0%) and

MGA/ PGF2a (58.0%). Furthermore, the MGASelect improved the estrous

response and subsequent pregnancy rates in cows determined to be anestrous.

In the effort to find the appropriate estrus synchronization and (or) ovulation

system for Bos taurus beef cattle various studies have been conducted to

compare different protocols. Richardson et al. (2002) evaluated fertility of beef

heifers after synchronization of estrus using three different protocols:

(GnRH+PGF) GnRH on d -7 + PGF2a on d -1; (P4+PGF) CIDR on d -7, PGF2a on

d -1, and CIDR removal on d 0; and (P4+GnRH+PGF) CIDR + GnRH on d -7 +

PGF2a on d -1. Estrus rates were greater in heifers treated with CIDR (P4+PGF

and P4+GnRH+PGF) compared to GnRH+PGF. Pregnancy rates were greater

for both GnRH treatments (GnRH+PGF and P4+GnRH+PGF) compared to CIDR

insertion without GnRH. Johnson et al. (2002) compared intervals of 48 or 60 h

between PGF2a and TAI, and administration of GnRH or saline at TAI in a GnRH-

CIDR-PGF synchronization protocol in cycling and non-cycling beef cows. It was








concluded that TAI at 48 or 60 h after PGF in a GnRH-CIDR-PGF protocol was

equally effective and administration of GnRH at TAI improved fertility in all cycling

cows, but not in non-cycling cows.

The same attempt to find the appropriate estrus synchronization and (or)

ovulation system for cattle of Bos indicus breeding, comparing different protocols

has been made by some researchers. Williams et al. (2002) compared the

relative efficacies of three protocols (Syncro-Mate-B, SMB; norgestomet-PGF2a,

NP; and Ovsynch) for synchronizing ovulation for timed-Al of predominantly

Brahman-influenced cows and heifers. Animals in the NP treatment were

implanted with SMB without the injection of estradiol valerate/norgestomet. In

cows, administration of estradiol (SMB) or GnRH (Ovsynch) to synchronize

follicle development resulted in increased TAI pregnancy rates compared with

the NP group. Conversely, in heifers TAI conception rate was greater in NP

compared with SMB and Ovsynch. Synchronization of ovulation using SMB or

Ovsynch in Bos indicus influenced beef cows in this study resulted in TAI

conception rates comparable to each other and to those reported for treatments

in Bos taurus cows (Geary et al., 1998b). Fernandes et al. (2001) conducted a

series of experiments to evaluate a 7 d GnRH + PGF2a protocol with or without

GnRH or estradiol benzoate after PGF2a for timed-Al in cycling or anestrous,

lactating or nonlactating Nelore (Bos indicus) cows. Percentage of animals

detected (44.5 to 70.3%) in heat and pregnancy rates (20 to 42%) varied

according to the number of animals with corpus luteum at the beginning of

treatment. Administration of a second dose of either GnRH or estradiol benzoate








was effective in synchronizing ovulation in cycling Nelore cows and improved

pregnancy rates after TAI. It was concluded that the estradiol benzoate

treatment after PGF2a injection is a promising alternative to the use of GnRH in

GnRH-PGF-TAI protocols for Nelore cattle due to the low cost of estradiol

benzoate compared to GnRH agonist.

Hiers et al. (2003) evaluated the effectiveness of three different PGF2a

treatments in a GnRH + PGF2a protocol combined with MGA to synchronize Bos

indicus x Bos taurus cows for a TAI. All cows received GnRH at the start of the

experiment (d 0) and were administered MGA on d 1 to 7. On d 7, cows received

either a single injection of 25 mg PGF2a, a single injection of 500 pg cloprostenol,

or half (12.5 mg) the recommended dose of PGF2a on d 7 and 8. On d 10, all

cows were timed-Al concomitant with GnRH. Administering MGA along with an

injection of GnRH has been shown to reduce the ability of GnRH to induce

ovulation in cows with dominant follicles (Pancarci et al., 1999); therefore, in this

study cows were not administered MGA on the day of GnRH injection.

Additionally, as some cows do not have a functional CL at PGF2a injection in the

GnRH + PGF2a systems (Twagiramungu et al., 1995; Schmitt et al., 1996a;

Downing et al., 1998; Moreira et al., 2000), MGA was fed until the day of PGF2.

to prevent early expression of estrus and probably tighten the synchrony of

estrus, follicle development and ovulation after MGA withdrawal. Timed-Al and

30-d pregnancy rates were similar between the single (36 and 77%), split (39 and

74%), and cloprostenol (41 and 75%) treatments. These data suggested that








two injections of PGF2a were not necessary and that it does not seem to matter

whether a single injection of PGF2a or cloprostenol was used.

Summary

In order for estrus synchronization programs to have a high rate of success,

maximizing the synchrony of estrus is essential. Simultaneously, the

synchronization programs must be inexpensive and easy to administer in order

for producers to adopt them. In order to develop such a system, regulation of

follicular development as well as the lifespan of the CL must be undertaken.

Current studies have focused on synchronizing ovulation in conjunction with

timed-Al and eliminating estrus detection. Several estrus synchronization and/or

ovulation protocols include the use of GnRH in combination with PGF2a.

Generally, protocols combining GnRH and PGF2. are effective for synchronizing

estrus in cattle of Bos taurus breeding but less effective in cattle of Bos indicus

breeding. Moreover, few studies have made direct breed comparisons of the

endocrine and ovarian responses of cattle synchronized with the GnRH plus

PGF2. systems. The limited data available and the decreased pregnancy rates

already achieved in Bos indicus x Bos taurus cattle justify additional research.

Therefore, the following experiments were conducted to characterize endocrine,

ovarian and reproductive responses to the administration of GnRH and (or)

PGF2, in Bos indicus x Bos taurus cattle, as well as to make direct comparisons

of these responses between Bos taurus cattle and animals of Bos indicus

breeding.













CHAPTER 3
RESPONSE TO A PROSTAGLANDIN F2a INJECTION ON EITHER DAY SIX OR
SEVEN OF THE ESTROUS CYCLE IN ANGUS AND BRAHMAN x ANGUS
HEIFERS

Introduction

Most estrus synchronization protocols used today include the use of

prostaglandin F2. (PGF2.) or its analogues. Prostaglandin F2a is used to control

the length of the estrous cycle by shortening the life span of the corpus luteum

(CL) through the induction of luteolysis and subsequent expression of estrus

(Beal et al., 1980; Hardin et al., 1980a; Lauderdale et al., 1981). However, it

appears that an injection of PGF2a early in the estrous cycle (d 6 to 8) is not as

effective in regressing the CL in cattle of Bos indicus breeding (Santos et al.,

1988; Pinheiro et al., 1998; Rekwot et al., 1999) compared to cattle of Bos taurus

breeding (King et al., 1982; Tanabe and Hann, 1984; Kiracofe et al., 1985). This

is important since many estrus synchronization systems include administration of

PGF2. to cattle with a CL that is approximately 5 to 6 d old including the GnRH

plus PGF2. system, which consists of administralior of GnRH followed 7 d later

by an injection of PGF2. (Thatcher et al., 1989).

The effectiveness of the GnRH plus PGF2. estrus synchronization

systems are consistent in cattle of Bos taurus breeding (Pursley et al., 1995;

Geary et al., 1998a) but not in cattle of Bos indicus breeding (Lemaster et al.,

2001). Initially, the inconsistent results in cattle of Bos indicus breeding treated








with PGF2. appeared to be associated with compromised expression of estrus

(Orihuela et al., 1983; Pinheiro et al., 1998; Rekwot et al., 1999). However, the

decreased expression of estrus may also be associated with incomplete

luteolysis after PGF2a resulting in the blood progesterone concentrations that are

great enough to prevent the expression of estrus (Pinheiro et al., 1998; Rekwot

et al., 1999; Mattoni and Ouedraogo, 2000). In order for cattle producers in

tropical and subtropical environments to implement the most effective estrus

synchronization systems, a complete understanding of how PGF2. functions in

cattle of Bos indicus breeding is necessary.

The objective of this experiment was to determine the effectiveness of a

single injection of PGF2a administered during the early estrous cycle (d 6 or 7) to

initiate luteolysis in Angus and Brahman x Angus heifers.

Materials and Methods

The experiment was replicated twice during the fall (September to

November) of 2000 at the Santa Fe Beef Research Unit, Department of Animal

Sciences, University of Florida. In replication 1, cycling Angus (Bos taurus; n =

13; mean BW = 379 kg), and Brahman x Angus (Bos indicus x Bos taurus 3/8

Brahman x 5/8 Angus and 5/8 Brahman x 3/8 Angus; n=16; mean BW = 451 kg)

heifers that were approximately 18 to 20 months old were initially synchronized

with a modified two-injection PGF2a protocol. Angus heifers were moved from an

extensively managed cattle operation (Deseret Cattle and Citrus, Deer Park, FL)

to the Santa Fe Beef Research Unit approximately two weeks prior to the start of

the experiment and were co-mingled with Brahman x Angus heifers, which were








maintained at the Santa Fe Beef Research Unit. Heifers were pastured in a

single group and received 25 mg PGF2a i.m. (LutalyseSterile Solution;

Pharmacia Animal Health, Kalamazoo, MI) on d -14 and the second and third

injections of 12.5 mg PGF2. i.m. on d -3 and -2 of the experiment (Figure 3-1).

Heifers were fitted with electronic estrus detection devices (HeatWatch, DDx,

Boulder, CO) on d -3 to determine the onset of estrus during the 5 d after PGF2.

(d -3). The onset of estrus was defined as 3 or more mounts within a 4 h period

(Landaeta et al., 1999; Lorton et al., 1999). After the expression of estrus (d 0),

heifers of each breed type received 25 mg of PGF2,. i.m. on either d 6 or 7 of the

estrous cycle. At PGF2. (d 0) both ovaries were examined via ultrasonography

using a real time, B-mode ultrasound (Aloka 500V, Corometrics Medical

Systems, Wallingford, CT) equipped with a 7.5 MHz transducer. At ultrasound

examination, follicles > 5 mm, height and width of all luteal structures, diameter of

any luteal cavities and their respective locations on the ovaries were measured

with the internal calipers of the ultrasound machine and recorded. Volume of the

CL was calculated using the formula for the volume of a sphere (nd3/6). When a

luteal cavity was present, its volume was subtracted from the volume of the outer

sphere resulting in net luteal volume (CL volume) represented by luteal tissue.

Daily blood samples were collected via jugular venipuncture from PGF2. (d 0)

until heifers either exhibit estrus or for 7 d after PGF2. for heifers that did not

exhibit estrus. After collection of blood samples, they were immediately placed

on ice and centrifuged (3000 rpm) within 4 h. Plasma was separated and stored

at -20 C











Daily blood sample collection to
determine if PGF2a regressed
the CL as determined by
progesterone concentrations.
Estrus
25 mg 12.5 mg 25 mg
PGF, PGF2, PGF2.



-14 -3 -2 0 6/7 8 9 10 11 12 13 14

Estrus detection using HeatWatch


Figure 3-1. Experimental protocol evaluating the effectiveness of a single
injection of prostaglandin F2a (PGF20) administered on d 6 or 7 of the
estrous cycle to initiate corpus luteum (CL) regression in Angus and
Brahman x Angus heifers. Heifers were presynchronized with a single
PGF2a injection followed 11 d later by two half injections of PGF2.
administered 24 h apart.








for later analysis. Progesterone concentrations were determined using RIA

(Seals et al., 1998) in multiple assays with intra- and interassay CV of 2.3 and

13.9 %, respectively. Sensitivity of the assay was 0.02 ng/tube.

The presence of a CL at PGF2a (d 0) as detected by ultrasound was

defined as functional if progesterone concentrations were > 1.0 ng/mL. Corpus

luteum regression was defined as progesterone concentrations < 1.0 ng/mL in

two consecutive blood samples following PGF2a. During the treatment phase of

the experiment, estrus was also monitored using HeatWatch. Estrous response

was defined as the percentage of heifers exhibiting estrus within 7 d after PGF2a

divided by the total treated. The onset of estrus was defined as 3 or more

mounts within a 4 h period while the end of estrus was defined as the last mount

recorded prior to a period of extended inactivity of at least 8 h (Landaeta et al.,

1999; Lorton et al., 1999). Duration of estrus was defined as the total time from

the initiation of estrus to the end of estrus. Distribution of mounting activity

during estrus was defined as the number of mounts received during consecutive

3 h periods starting at the initiation of estrus to the end of estrus. Interval from

PGF2a to the onset of estrus was calculated as the time from PGF2a

administration to the first mount as detected by HeatWatch. Heifers were

ultrasounded to determine the presence of a CL 10 d after expression of estrus

to confirm the site of ovulation.

After the completion of replication 1, heifers were allowed to go through a

normal estrous cycle before the start of the second replication. The same

experimental protocol was used for the second replication. At the initiation of








each replication, body condition scores (Richards et al., 1986) and BW were

recorded on each heifer.

For statistical analysis, the effects of breed, replication, and replication x

breed on PGF2. induced CL regression, estrous response and estrous response

in heifers that regressed their CL were analyzed with logistic regression modeling

computing likelihood ratio test for type 3 contrasts for each term in the model

using GENMOD procedure of SAS (SAS Inst. INC., Cary, NC). When there were

no replication and replication x breed effects (P > 0.10) data were pooled and

presented as such. Largest follicle size at PGF2a was included as a covariate in

the estrous response model. Progesterone concentrations and CL volume at

PGF2a were included as covariates in the CL regression model. Corpus luteum

regression was also analyzed with logistic regression modeling using the Logistic

procedure of SAS. To determine the degree of association between the

independent variables and the outcome variable, odds ratio and Wald 95%

confidence intervals for adjusted odds ratios were calculated. Progesterone

concentrations following PGF2. were analyzed as repeated measures analysis

using the GLM procedure of SAS. The model included fixed effects of breed,

replication, CL regression, time, and all appropriate interactions with random

effect of heifer nested within breed as the error term. Effects of breed and CL

regression were separated using contrasts for Angus vs. Brahman x Angus, and

CL regression vs. no-CL regression. Effects of breed, replication and breed by

replication interaction on the interval from PGF2a to the onset of estrus, duration

of estrus, total number of mounts received during estrus, size of the largest








follicle, and CL volume at PGF2. were analyzed by ANOVA using GLM

procedures of SAS. Follicle size at PGF2, was included as a covariate in the

model for the analysis of interval to estrus. When there were no replication and

replication x breed effects (P < 0.10) data were pooled and presented as such.

Additionally, the effect of breed on interval from PGF2a to the onset of estrus was

evaluated using the LIFETEST procedure (survival analysis) of SAS. The

survival analysis regressed the proportion of heifers not observed in estrus

during the 7 d after PGF2,. Data for heifers that were never observed in estrus

were included in the survival analysis as censored observations. Differences

between survival curves were tested with the Wilcoxon test (Klein and

Moeschberger, 1997).

The distribution of mounting activity during estrus was analyzed by

repeated measures analysis using the MIXED procedure of SAS where breed,

replication, period, and all appropriate interactions were included in the model,

with heifer nested within breed as the error term, which was subjected to three

covariance structures: autoregressive order 1, compound symmetry, and

unstructured covariance. The covariance that resulted in the smallest Akaike's

Information Criterion was used. Period was defined as sequential 3 h periods

from the initiation to the end of estrus. Total number of heifers in estrus during

each 3 h period was also included in the model as a covariate. Slice mean

comparisons were used to examine differences between breeds within 3 h

periods. Data were subsequently analyzed with regression analysis and

differences between breed response curves were analyzed using homogeneity of








regression. The effect of the number of heifers in estrus during each 3 h period

on the number of mounts received during each period and the effect of CL

volume on progesterone concentrations at PGF2, were analyzed by regression

analysis using the GLM procedure of SAS.

Results

Corpus luteum regression was greater (P = 0.04) for Angus than Brahman

x Angus heifers when the statistical analysis was conducted using GENMOD

(Table 3-1). Using the GENMOD procedure, there was not significant breed x

replication effect on CL regression; therefore, the model was analyzed without

the interaction and breed effect tended to be significant (P = 0.06). However,

when data were analyzed with Logistic Regression there was no breed effect (P

= 0.10).

Mean progesterone concentrations for heifers that did not regress their

CL, including one Angus and six Brahman x Angus heifers after PGF2a, were

different (P < 0.0001) compared to Angus (n = 25) and Brahman x Angus (n =

25) heifers that regressed their CL (Figure 3-2). Mean progesterone

concentrations one day after PGF2a were greater (P < 0.05) for heifers that did

not regress their CL (1.1 0.2 ng/mL) than heifers that regressed their CL (0.5

0.1 ng/mL). For heifers that regressed their CL, progesterone concentrations

after PGF2a were < 1.0 ng/mL by 24 h after PGF2. and remained < 1.0 ng/mL

through 96 h. In contrast, progesterone concentrations of heifers that did not

regress their CL declined to concentrations > 1.0 ng/mL by 24 h after PGF2. and

remained > 1.0 ng/mL until 96 h (Figure 3-2). Neither CL volume nor







Table 3-1. Corpus luteum (CL) regression and ovarian characteristics (LS means
SE) in Angus and Brahman x Angus heifers treated with
prostaglandin F2a (PGF2,) on either d 6 or 7 of the estrous cycle

Breed

Variable Angus Brahman x Angus
(n = 26) (n = 31)

CL regression, % a. b 96.2 80.6

CL volume, mm3 3231.2 + 226.7 2914.3 + 207.9

Progesterone, ng/mL 3.1 + 0.3 3.2 + 0.3

Largest follicle diameter, mm 10.5 + 0.40 12.0 +0.4d

a CL regression defined as two consecutive daily blood samples after a 25 mg
injection of PGF2, with progesterone < 1 ng/mL.
bBreed with GENMOD (P = 0.04) and with logistic regression (P = 0.10).
cdValues lacking a common superscript differ (P < 0.01).












AN (CL regress) n = 25

3.0 1' BR x AN (CL regress) n = 25


... AN (no CL regress) n = 1
- BR x AN (no CL regress) n = 6


a.-
-. #


u.u -
0 1 2 3 4
Days from PGF2,



Figure 3-2. Progesterone concentration profiles in Angus (AN) and Brahman x
Angus (BR x AN) heifers that either regressed or did not regress their
CL after a single prostaglandin F2a (PGF2a) injection on either d 6 or 7
of the estrous cycle. Breed (P > 0.05), Breed x Time (P > 0.05), CL
regression x Time (P < 0.0001), CL regression vs No-CL regression (P
< 0.0001).


-1
E 2.5


4T 2.0
C
S2.0
o

a 1.5
U)
4)

1.0
a.







progesterone concentrations at PGF2, affected (P > 0.10) total CL regression.

Corpus luteum volume and progesterone concentrations at PGF2. were similar

(P >0.10) between breeds (Table 3-1). Diameter of CL at PGF2, was similar (P >

0.05) between Angus (18.5 0.6 mm) and Brahman x Angus (18.3 0.5 mm)

heifers. Additionally, CL volume and CL diameter at PGF2a were positively

correlated (Pearson correlation = 0.85; P < 0.0001). Across breeds,

progesterone concentrations at PGF2. were influenced (P < 0.05) by CL volume

(Figure 3-3). For every 1000 mm3 increase in CL volume, there was a 1.28

ng/mL increase in progesterone concentrations.

Estrous response after PGF2a, mean interval from PGF2.to the onset of

estrus, and duration of estrus were not influenced (P > 0.10) by either breed or

replication (Table 3-2). Among heifers that regressed their CL, estrous response

was not affected (P > 0.10) by replication, but more (P < 0.05) Brahman x Angus

heifers expressed estrus compared with Angus heifers (Table 3-2). Across

breeds, 72% (36/50) of heifers that regressed their CL expressed estrus,

whereas 28% (14/50) failed to express estrus. None of the heifers that failed to

regress their CL expressed estrus. Total number of mounts received during

estrus was not affected (P > 0.10) by replication, but was greater (P < 0.05) in

Brahman x Angus than Angus heifers (Table 3-2). The distribution of mounting

activity during estrus was affected by breed (P < 0.05), period (P < 0.0001), and

breed x period (P < 0.05; Figure 3-4). Furthermore, the breed response curves

were different (P < 0.05) between the Angus and Brahman x Angus heifers

throughout the duration of estrus. The number of heifers in estrus within a period













8-- -- .--- --- -

o4


y = 0.00128x + 0.7637
2

a. 2
R = 0.12

0

1000 2000 3000 4000 5000

CL volume, mm3

Figure 3-3. The effect of corpus luteum (CL) volume on progesterone
concentrations (P < 0.05) on either d 6 or 7 of the estrous cycle in
Angus and Brahman x Angus heifers.








Table 3-2. Estrous response and behavioral estrus characteristics (LS means
SE) in Angus and Brahman x Angus heifers treated with prostaglandin
F2. (PGF2a) on either d 6 or 7 of the estrous cycle

Breed

Variable Angus Brahman x Angus


Estrous response, % a 15/26 = 57.7 21/31 = 67.7

Estrous response in
heifers that regressed 15/25= 60.0 21/25= 84.0
their CL, % b 15/25 = 60.c 21/25 84d

Interval from PGF2a
injection to onset of 51.5 3.8 (n = 26) 52.4 3.1 (n = 31)
estrus, h

Duration of estrus, h 10.6 + 1.4 (n = 26) 12.9 1.1 (n = 31)

Total number of mounts 28.9 + 9.2c(n = 26) 51.1 + 7.1d(n = 31)
received during estrus
aThe number of heifers observed in estrus for 7 d after PGF2Q divided by the total
treated.
bThe number of heifers observed in estrus for 7 d after PGF2a divided by the
number of heifers that regressed their CL.
cdValues within a row lacking a common superscript differ (P < 0.05).











14

12 ----.....





a0





3 6 9 12 15 18 24
Hours from onset of estrus




Figure 3-4. Distribution of mounts received during 3 h periods throughout the
duration of estrus after an injection of PGF2a on either d 6 or 7 of the
estrous cycle in Angus (white bars) and Brahman xAngus (black bars)
heifers. Breed (P < 0.05). Period (P < 0.0001). Breed x Period (P <
0.05). a'bMeans within periods without a common letter differ (P <
0.05).







tended (P = 0.08) to affect the distribution of mounting activity during estrus, but it

did not change the significance values for breed, period, and breed x period

when included in the model as a covariate. As the number of heifers in estrus

during a 3 h period increased, the number of mounts received increased in a

linear manner (P < 0.0001; Figure 3-5). The Angus and Brahman x Angus

heifers had similar (P > 0.05) mounting activity during the first 6 h of estrus and

during all periods for durations exceeding 15 h. However, Brahman x Angus

heifers had more (P < 0.05) mounting activity between 9 and 15 h after the

initiation of estrus than Angus heifers.

Diameter of the largest follicle on the ovaries at PGF2a was smaller (P <

0.01) for Angus than Brahman x Angus heifers (Table 3-1). Although, largest

follicle size at PGF2a did not influence (P > 0.10) either the expression of estrus

or interval from PGF2, to the onset of estrus. There was no effect (P > 0.10) of

breed on the survival curves for heifers not observed in estrus (Figure 3-6).

Discussion

A PGF2~ induced CL regression on either d 6 or 7 of the estrous cycle was

15.6% greater for Angus than Brahman x Angus heifers in the present

experiment. Whether the decrease in luteolysis of the Brahman x Angus heifers

was significant depends on the method of statistical analysis used. Not

withstanding, results from the present experiment warrant the need for additional

research with larger experimental numbers to provide unequivocal evidence that

the early developing CL (< d 7) in cattle of Bos indicus breeding are less

responsive to administration of PGF2a.




Full Text
69
Other researchers have combined a long-term MGA treatment with the
SelectSynch system for estrus synchronization (Patterson et al., 2002).
Postpartum cows received either a 14 d MGA treatment with SelectSynch
(MGASelect) initiated 12 d after MGA withdrawal compared to the
MGA/PGF2a.system with PGF2a 19 d after MGA. Cows were observed for estrus
for 7 d after PGF2ct and inseminated 12 h after estrus. Estrous response was
greater in MGASelect (87%) than MGA- PGF2a (76%) cows. Mean interval to
estrus after PGF2ct was longer for MGA/ PGF2a (80.4 h) than MGASelect (74.5 h).
Synchronized pregnancy rate did not differ between MGASelect (66.0%) and
MGA/ PGF2ci (58.0%). Furthermore, the MGASelect improved the estrous
response and subsequent pregnancy rates in cows determined to be anestrous.
In the effort to find the appropriate estrus synchronization and (or) ovulation
system for Bos taurus beef cattle various studies have been conducted to
compare different protocols. Richardson et al. (2002) evaluated fertility of beef
heifers after synchronization of estrus using three different protocols:
(GnRFI+PGF) GnRH on d -7 + PGF2a on d -1; (P4+PGF) CIDR on d -7, PGF2a on
d -1, and CIDR removal on d 0; and (P4+GnRH+PGF) CIDR + GnRH on d -7 +
PGF2a on d -1. Estrus rates were greater in heifers treated with CIDR (P4+PGF
and P4+GnRH+PGF) compared to GnRH+PGF. Pregnancy rates were greater
for both GnRH treatments (GnRH+PGF and P4+GnRH+PGF) compared to CIDR
insertion without GnRH. Johnson et al. (2002) compared Intervals of 48 or 60 h
between PGF2a and TAI, and administration of GnRH or saline at TAI In a GnRH-
CIDR-PGF synchronization protocol in cycling and non-cycling beef cows. It was


Table 6-4. Estrus, conception and pregnancy rates of Bos taurus x Bos indicus cows synchronized with GnRH and either
single or split doses of prostaglandinF2a (PGF2a) in combination with melengestrol acetate for cows classified by cycling
status and progesterone (P) status (H = >1 and L = <1 ng/mL) on d -10, 0, and 7 of the experiment
Category (d -10, 0, 7)
Three day estrous
response, %a (n)
Conception rate,
%b (n)
Timed-AI pregnancy
rate, %c (n)
Synchronized
pregnancy rate, %d (n)
Cycling + HP at PGF2a
26.9e (130)
57.1 (35)
53.5e (95)
54.6e (130)
HHH
31.1 (61)
47.4(19)
61.9 (42)
57.4 (61)
HLH
13.3(30)
75.0 (4)
42.3 (26)
46.7 (30)
LHH
30.8 (39)
66.7(12)
51.9(27)
56.4 (39)
Cycling + LP at PGF2a
62.2' (37)
47.8 (23)
14.3'(14)
35.11'9 (37)
HHL
70.6 (17)
50.0(12)
20.0 (5)
41.2(17)
HLL
25.0 (4)
100.0 (1)
33.3 (3)
50.0 (4)
LHL
62.5 (16)
40.0(10)
0(6)
25.0(16)
Noncycling + LP at PGF2a(LLL)
39.3e (61)
37.5 (24)
16.2' (37)
24.69(61)
Noncycling + HP at PGF2(LLH)
24.4s (41)
70.0 (10)
35.5e' (31)
43.9e'(41)
aPercentage of cows displaying estrus 3 d after PGF2a of the total treated.
Percentage of cows pregnant that exhibited estrus and were Al.
Percentage of cows pregnant of the total that were timed-AI.
Percentage of cows pregnant of the total treated.
e ,'9Values with different superscript in a column are different (P < 0.05).


CHAPTER 1
INTRODUCTION
Cattle of Bos indicus breeding are used extensively for beef production In
subtropical regions of the United States, and tropical and subtropical regions all
over the world. Environmental adaptability is a crucial factor In beef cattle
production in tropical and subtropical environments. Cattle of Bos indicus
breeding are characterized by their adaptation to elevated temperatures and
humidity, tolerance to Internal and external parasites and the ability to use
forages of high fiber content (Frisch and Vercoe, 1984; Randel, 1994). However,
problems associated with estrus detection and response to different estrus
synchronization drugs often compromise the effectiveness of A! and estrus
synchronization protocols in cattle of Bos indicus breeding. Additionally, most
estrus synchronization research studies have been conducted using beef and
dairy cattle of Bos taurus breeding and not cattle of Bos indicus breeding.
Artificial insemination (Al) allows genes from superior bulls to be distributed
among many females without incurring the expenses of buying the animals.
Therefore, the genetic potential of a calf crop can be improved by using Al.
However, this practice takes a lot of time and effort. For Al to be effective and
efficient for beef producers, estrus synchronization and timed-AI (TAI) are
important tools.
Gonadotrophin releasing hormone (GnRH) has been combined with
prostaglandin F2a (PGF2o) to develop what is known as the GnRH+ PGF2ct
1


58
MGA/PGF2a estrus synchronization system. Administering PGF2a 17 d after
MGA, allowed the researchers to inject PGF2o! when a majority of heifers were in
the later stages of the estrous cycle where PGF2a is more effective in inducing
CL regression (Tanabe and Hahn, 1984; Watts and Fuquay, 1985). The
MGA/PGF2a estrus synchronization system induced a tightly synchronized estrus
that resulted in excellent synchronized pregnancy rates in beef heifers (Brown et
al, 1988). Later research also reported that the MGA/PGF2a estrus
synchronization system also was effective in suckled postpartum beef cows
(Patterson etal., 1989; Yelich etal., 1997).
One of the problems with the MGA/PGF2cl estrus synchronization system is
that it takes so long to implement. Therefore, another approach was developed
to deal with the reduced fertility after MGA withdrawal. This included
administration of an exogenous estrogen (Kastelic et al., 1996; Yelich et al.,
1997) or progesterone (Anderson and Day, 1998) during MGA feeding to initiate
regression of the persistent follicle, which resulted in improved fertility at the
synchronized estrus after MGA withdrawal.
To circumvent the problems with reduced fertility with long-term MGA
treatments, the approach was taken to reduce the duration of progestogen
treatment to approximately 7 d. Several studies (Patterson et al., 1986; Beal et
al., 1988) evaluated the effectiveness of reduced periods of progesterone
exposure (7 or 9 d) by combining MGA with a luteolytic dose of PGF2o at the end
MGA administration. Feeding MGA for short periods resulted in improved fertility
when feeding was started early in the estrous cycle, but conception rates were


176
Table 6-2. Estrus, conception, and pregnancy rates of Bos taurus x Bos indicus
cows synchronized with GnRH and either a single or split doses of
prostaglandin F2ci (PGF2a) in combination with melengestrol acetate
T reatment3
Variable
Single PGF2a
Split PGF2a
Three day estrus rate, %b
27/85 = 31.8
24/102 = 23.5
Conception rate, %c
20/27 = 74.19
10/24 = 41.7h
Timed-AI pregnancy rate, %d
26/58 = 44.8
39/78 = 50.0
Synchronized pregnancy rate, %e
46/85 = 54.1
49/102 = 48.0
30 day pregnancy rates, %f
71/85 = 83.5
88/103 = 85.4
mg PGF2a on d 7 or 12.5 mg PGF2c(on d 7 and 8. Cows that exhibited estrus
were Al approximately 8-12 h later and all cows not exhibiting estrus by d 10 of
the experiment were timed-AI with 100 pg GnRH. Cows received 0.5 mg
MGA'day'1 on d 1 to 6 of experiment.
bPercentage of cows displaying estrus 3 d after PGF2a of the total treated.
Percentage of cows pregnant that exhibited estrus and were Al.
Percentage of cows pregnant of the total that were timed-AI.
Percentage of cows pregnant to estrus Al and timed-AI of the total treated.
Percentage of cows pregnant during the first 30 d of the breeding season of the
total treated.
9,hValues with different superscript In a row differ (P < 0.05).


248
Berisha, B., D. Schams, M. Kosmann, W. Amselgruber, and R. Einspanier.
2000b. Expression and tissue concentration of vascular endothelial growth
factor, its receptors, and localization in the bovine corpus luteum during
estrous cycle and pregnancy. Biol. Reprod. 63:1106-1114.
Binelli, M., J, Hampton, W.C. Buhi, and W.W: Thatcher. 1999. Persistent
dominant follicle alters pattern of oviductal secretory proteins from cows in
estrus. Biol. Reprod. 61: 127-134.
Binelli, M., W.W. Thatcher, R. Mattos, and P.S. Baruselli. 2001. Antiluteolytic
strategies to improve fertility in cattle. Theriogenology 56:1451-1463.
Blache, D., C.J. Fabre-Nys, and G. Venier. 1991. Ventromedial hypothalamus as
a target for oestradiol action on proceptivity, receptivity and luteinizing
hormone surge of the ewe. Brain Res. 546: 241-249.
Bo, G.A., G.P. Adams, R.A. Pierson, and R.J. Mapleloft. 1995. Exogenous
control of follicular wave emergence in cattle. Theriogenology 43: 31-40.
Bo, G.A., G.P. Adams, R.A. Pierson, H.E. Trbulo, M. Caccia, and R.J. Mapleloft.
1994. Follicular wave dynamics after estradiol-17p treatment of heifers with
or without a progestogen implant. Theriogenology 41: 1555-1569.
Bo, G.A., D.R. Bergfelt, G.M. Brogliatti, R.A. Pierson, G.P. Adams, and R.J.
Mapletoft. 2000. Local versus systemic effects of exogenous estradiol-17p
on ovarian follicular dynamics in heifers with progesterone implants. Anim.
Reprod. Sci. 59: 141-157.
Bo, G.A., L.F. Nasser, G.P. Adams, R.A. Pierson, and R.J. Mapleloft. 1993.
Effect of estradiol valerate on ovarian follicles, emergence of follicular
waves and circulating gonadotropins in heifers. Theriogenology 40: 225-
239.
Bolados, J.M., J.R. Molina, and M. Forsberg. 1997. Effect of blood sampling and
administration of ACTH on cortisol and progesterone levels in
ovariectomized zebu cows (Bos indicus). Acta Vet. Scand. 38: 1-7.
Bolt, D.J., V. Scott, and G.H. Kiracofe. 1990. Plasma LH and FSH after estradiol,
norgestomet, GnRH treatment in ovariectomized beef heifers. Anim.
Reprod. Sci. 23:263-271.
Braden, T.D., M.E. King, K.G. Odde, and G.D. Niswender. 1989. Development of
preovulatory follicles expected to form short-lived corpora ltea in beef
cows. J. Reprod. Frtil. 85: 97-104.


183
used in the present experiment. Data from the present study and that reported
by Hiers et al. (2003) do not support the hypothesis that CL regression is the
primary reason for the decreased synchronized pregnancy rates in mature cows
of Bos indicus breeding synchronized with GnRH + PGF2a estrus synchronization
systems (Lemaster et al., 2001).
Cycling status at GnRH did affect the response of the synchronization
systems but there were no treatment by cycling status effects on any variable
analyzed. Cycling status had no influence on estrous and conception rates, but
cycling cows had improved timed-AI, synchronized and thirty-day pregnancy
rates. Cycling cows were approximately two times more likely to become
pregnant to either the timed-AI, synchronized breeding or during the first 30 d of
the breeding season than noncycling cows. Similarly, other studies (Stevenson
et al., 2000; Lemaster et al., 2001; Moreira et al., 2001) have reported that
cycling cows had increased pregnancy rates to GnRH + PGF2a protocols than
'i
noncycling cows. Alternatively, MGA administration and split injection of PGF2a
failed to stimulate pregnancy rates to a level of cycling cows.
The overall mean BCS was 4.2 at the start of the experiment and it did not
affect any of the variables analyzed in the present study. These findings are in
disagreement with other reports in cows synchronized with either Select Synch,
CoSynch, or Select Synch + Norgestomet protocols (Stevenson et al., 2000) or
cows treated with a similar protocol used in the present experiment (Hiers et al.,
2003). According to Kunkle (1996), a minimum BCS of 5.0 (scale 1 to 9) at
breeding appears to be required for cows to become pregnant early in the


Gainesville, for their companionship and shared laughs. They will be esteemed
for a lifetime:
I especially thank my parents, Mary and Heberto Portillo for their love,
wisdom and support. Without them none of this would have been possible. I
would also like to thank my sisters and their respective families for lifting me up
when I thought I could go no farther.
IV


which received GnRH on d 6 of the estrous cycle followed by PGF2a
treatment 7 d latter 121
5-1 Experimental protocol Experiment 3 139
5-2 Progesterone concentrations for the two days before GnRH (d 6 of the
estrous cycle) for heifers in the high progesterone group Including Angus
and Brahman Angus and heifers in the low progesterone group
Including Angus and Brahman Angus heifers 143
5-3 Progesterone concentrations between GnRH and PGF2a treatment for
heifers In the high progesterone group including Angus and Brahman x
Angus and heifers In the low progesterone group Including Angus and
Brahman x Angus heifers 146
5-4 Regression of progesterone concentrations at PGF2a on corpus luteum
regression In Angus and Brahman x Angus heifers in the Low
progesterone group 149
6-1 Experimental protocol Experiment 4 166
6-2 Survival analysis describing the proportion of Bos taurus x Bos indicus
cows with progesterone concentrations > 1 ng/mL at PGF2a compared to
cows vyith progesterone concentrations < 1 ng/ mL that did not exhibit
estrus after being synchronized with GnRH and either single or split
doses of PGF2a system In combination with melengestrol acetate 174
6-3 Tlmed-AI and total CL regression rates in Bos taurus x Bos indicus cows
synchronized with GnRH and either a full or two consecutive split ;
injections of PGF2o in combination with MGA treatment 180


236
Interval from GnRH, min
Figure H-6. Plasma LH concentrations (LS Means) in all Brahman x Angus
heifers receiving 100 pg GnRH on d 6 of the estrous cycle.


32
During late diestrus, both functional and structural luteolysis occurs In order
for the non-pregnant female to return to estrus (Auletta and Flint, 1998). The
endometrium follows a default program to release luteolytic pulses of PGF2a and
if a conceptus is present it sends the appropriate antiluteolytic signals to block
PGF2a production (Binelli et al., 2001). Regulation of luteolysis is particularly
complex and numerous reviews are available for consideration (Silvia et al.,
1991; McCracken et al., 1999; Binelli et al., 2001). Luteolysis is a local
mechanism Involving a countercurrent transfer of PGF2a from the uterine vein to
the ovarian artery (Hixon and Hansel, 1974). Uterine derived PGF2a is the
hormone responsible for luteal regression in ruminants (McCracken, 1971;
McCracken et al., 1981). It appears that PGF2a exerts Its luteolytic effect not only
on the steroidogenic cells of the CL, but also on other cell types such as
endothelial cells (Auletta and Flint, 1998). During late diestrus, progesterone
concentrations began to decrease and estradiol Increases (Meyer et al., 1988;
Mirando et al., 1993). The Increase In estradiol enhances the oxytocin pulse
generator and oxytocin receptors in the endometrium (Meyer et al., 1988;
Mirando et al., 1993). The loss of progesterone Inhibition on oxytocin receptor
formation and subsequent oxytocin receptor formation induced by estradiol is
thought to be an initiating factor in luteolysis (Wathes and Lamming 1995).
Oxytocin from both the neurohypophysis and the CL is responsible for the
pulsatile secretion of PGF2a from the endometrium (Hooper et al., 1986; Zarco et
al., 1988). Hypothalamic oxytocin acting on uterine oxytocin receptors induces
the production of concentrations of PGF2a, which act on PGF2a receptors in luteal


CHAPTER 4
ENDOCRINE AND REPRODUCTIVE RESPONSES OF ANGUS, BRANGUS,
AND BRAHMAN x ANGUS HEIFERS TO A GNRH INJECTION ON DAY 6 OF
THE ESTROUS CYCLE FOLLOWED BY PROSTAGLANDIN F2a 7 DAYS
LATER.
Introduction
Several estrus synchronization and/or ovulation protocols include the use of
GnRH plus prostaglandin F2ot (PGF2a). Early experiments (Thatcher etal., 1989;
Macmillan and Thatcher, 1991; Twagiramungu et al., 1995) reported that
administration of GnRH altered follicular development by inducing ovulation of
large follicles resulting in emergence and synchronization of a new follicular
wave. Administration of exogenous GnRH initiates acute secretion of LH and
FSH for three to five hours, which initiates ovulation (Thatcher et al., 1993;
Vizcarra et al., 1997; DOcchio and Aspden, 1999). Prostaglandin F2ci controls
and regulates the life span of the corpus luteum (CL) through the induction of
luteolysis (Rowson et al., 1972; Kiracofe et al., 1985; Godfrey et al., 1989)
resulting in estrus and ovulation. Administration of GnRH followed 7 d later by
PGF2a is effective for synchronizing estrus in cattle of Bos taurus breeding
(Pursley et al., 1995; Burke et al., 1996; Geary and Whittier, 1999) but appears to
be less effective in cattle of Bos indicus breeding (Lemaster et al., 2001).
Although, data relative to responses to GnRH and/or GnRH agonist are
numerous (Coleman et al., 1988; Chenault et al., 1990; Cruz et al., 1997), most
data have been generated in cattle of Bos taurus breeding. Few direct
103


79
follicle, and CL volume at PGF2a were analyzed by ANOVA using GLM
procedures of SAS. Follicle size at PGF2a was included as a covariate In the
model for the analysis of interval to estrus. When there were no replication and
replication breed effects (P < 0.10) data were pooled and presented as such.
Additionally, the effect of breed on interval from PGF2a to the onset of estrus was
evaluated using the LIFETEST procedure (survival analysis) of SAS. The
survival analysis regressed the proportion of heifers not observed in estrus
during the 7 d after PGF2a. Data for heifers that were never observed in estrus
were included in the survival analysis as censored observations. Differences
between survival curves were tested with the Wilcoxon test (Klein and
Moeschberger, 1997).
The distribution of mounting activity during estrus was analyzed by
repeated measures analysis using the MIXED procedure of SAS where breed,
replication, period, and all appropriate interactions were included In the model,
*
with heifer nested within breed as the error term, which was subjected to three
covariance structures: autoregressive order 1, compound symmetry, and
unstructured covariance. The covariance that resulted In the smallest Akaikes
Information Criterion was used. Period was defined as sequential 3 h periods
from the initiation to the end of estrus. Total number of heifers In estrus during
each 3 h period was also included in the model as a covariate. Slice mean
comparisons were used to examine differences between breeds within 3 h
periods. Data were subsequently analyzed with regression analysis and
differences between breed response curves were analyzed using homogeneity of


147
Table 5-2. Breed and progesterone group effects on heifers with two corpora
ltea (CL) at PGF2a, CL regression rate after PGF2a, follicle diameter at
PGF2a, progesterone concentration at PGF2a, and estrous response
after PGF2a In Angus and Brahman Angus heifers in the High and
Low progesterone groups.
Breed
Progesterone group
Brahman x
Angus
Angus
High
Low
Variable
(n = 7)
(n = 19)
(n = 13)
(n = 13)
Heifers with two CL
at PGF2a, %
42.9
42.1
84.6a
0b
CL regression rate,
%
85.7
89.5
100.0
76.9
Follicle size at
PGF2ci, mm
15.5 2.0
11.8 1.0
12.7 1.4
14.7 1.4
Progesterone at
PGF2a, ng/mL
5.4 0.9
6.4 0.5
8.9 0.7e
2.9 0.7f
Estrous response,
%
28.63
84.2b
69.2
69.2
a,bValues within a breed or progesterone group and within a row lacking a
common superscript differ (P < 0.001).
cd Values within a group and within a row lacking a common superscript differ (P
< 0.05).
e,f Values within a group and within a row lacking a common superscript differ (P
<0.0001).
Breed Progesterone group (P > 0.10).


259
Helmer, S. D., T. S. Gross, G. R. Newton, P. J. Hansen, and W. W. Thatcher.
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39
synchronization systems initiate CL regression using the luteolytic agent
prostaglandin F2a or one of its potent analogs. However, the resulting estrus is
highly variable and can occur over a 7 d period. However, administration of
exogenous estrogen or GnRH can be used to control new follicle wave
emergence and Increase the synchrony of the subsequent estrus (Diskin et al.,
2002).
Estrogens
Administration of exogenous estrogens can induce follicular turnover
(Rajamahendran and Manlkkam, 1994; Bo et al., 2000; Burke et al., 2000) and
(or) atresia of persistent follicles (Yellch et al., 1997; Flke et al., 1999; Bo et al.,
2000), which results In emergence of a new follicular wave. Boetal. (1993)
reported that cycling cows injected with 5 mg of estradiol valerate on d 3 of the
estrous cycle had emergence of a second follicular wave between d 9 and 14 of
the estrous cycle. Similarly, Bo et al. (1994) reported cows Injected with 5 mg of
estradiol 17(3 on d 3 of the estrous cycle had emergence of a second follicular
wave on d 6 to 7 of the estrous cycle. Delayed emergence of the second
follicular wave In cows treated with estradiol valerate was a result of the
prolonged suppressive effects of estradiol valerate (Bo et al., 1995). Circulating
concentrations of estradiol In treated cows are greater than normally present
during the follicular phase of the estrous cycle and act to suppress LH and FSH
release, which initiates follicle atresia and emergence of a new follicle (Butler et
ai 1983; Price and Webb, 1988; Wolfe et al 1992).


157
the early to mid cycle CL may indirectly (via PGF2a) or directly (heterologous
desensitization) affect the functionality of PGF2a receptors and/or formation of
second messengers. Furthermore, treatment of bovine luteal cells with
progesterone decreased PGF2q production in a dose-dependent manner (Pate,
1988) suggesting that high intraluteal progesterone inhibits luteal PGF2a
production (Diaz et al., 2002). Similarly, Sharzynski and Okuda (1999) observed
that the effect of PGF2a on the early luteal phase CL was greater In luteal cells
receiving pretreatment with a progesterone antagonist (onapristone), oxytocin
antagonist (atoslban), and cyclooxygenase Inhibitor (indomethacin) compared to
untreated control cells. Thus, luteal oxytocin, progesterone and prostaglandins
are components of an autocrine/paracrine positive feedback mechanism in early
to mid luteal phase CL and could be responsible for the resistance of the early
developing CL to an exogenous PGF2o treatment. Additionally, progesterone may
play an active role In the Inhibition of luteal regression by preventing apoptosis
(Rae et al., 1998; Friedman et al., 2000; Schams and Berisha 2002). Although
not conclusive from results of the current study, the previous remarks along with
the observation that progesterone concentrations at PGF2a had a negative linear
effect on CL regression In LP heifers support the hypothesis that progesterone
may play an active role In the inhibition of luteal regression in the early
developing CL. On the contrary, a report by Pate (1988) confirmed that
progesterone did not Inhibit PGF2a secretion by bovine luteal cells from the late-
diestrous CL compared with the mid-cycle CL, which may have been the case for
the HP heifers. Additionally, progesterone priming of the uterus is required for


269
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Kobayashi, A. Miyamoto, and D. Schams. 2001. Estradiol-17a is produced
in bovine corpus luteum. Biol. Reprod. 65: 1634-1639.
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following prostaglandin F2 alpha injection in zebu cattle under continuous
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OShea, J.D., R.J. Rodgers, and M.J. DOcchio. 1989. Cellular composition of the
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Padmanabhan, V., and T.P Sharma. 2001. Neuroendocrine vs. paracrine control
of follicle-stimulating hormone. Arch. Med. Res. 32: 533-543.
Palomares, R., A. de Ondiz, J. Sandoval, R. Romn, R. Gonzlez, and E. Soto.
2002. Estrus induction and fertility in noncyclinc cebu crossbred cows
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progestagens. Rev. Cientfica, FCV-LUZ 12: 371-378.
Pancarci, S.M., E.R Jordan, C.A. Risco, M.J. Schouten, F.L. Lopes, F. Moreira,
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85: 122-131.


198
The thirty-day pregnancy rates of the present study were similar to other reports
in Bos taurus cows synchronized with different GnRH + PGF2a protocols
(Twagiramungu et al., 1992; Geary et al., 1999; Stevenson et al., 2000). The
GnRH + PGF2a + MGA protocol used in the present study resulted in a high
percentage of both cycling and noncycling cows pregnant early in the breeding
season. Therefore, there is an indirect benefit on enhanced reproductive
performance later in the breeding season when the GnRH + PGF2a + MGA
protocol was used in both cycling and noncycling lactating crossbred Bos indicus
cows. Increasing the number of beef cows pregnant early in the breeding
season results in more calves born early in the subsequent calving season,
which warrants heavier and more uniform calves at weaning resulting in
significant economic returns for the producer. Consequently, proper
management (supplementation to maintain most of the cows postpartum with a
body condition score of 5 or 6, temporary calf removal, etc.) is critical in order to
increase the number of cycling cows in the herd when trying to implement a
synchronization and timed-AI protocol in beef cattle.
Both the timed-AI and the total CL regression rates were similar between
the split and single PGF2o treatments, providing evidence that the split-PGF2a
treatment on d 7 and 8 after the first GnRH administration was not advantageous
in enhancing luteolysis in mature Bos indicus x Bos taurus cows compared to the
single injection of PGF2a 7 d after the GnRH. The similar CL regression rates
obtained with the single and split PGF2a treatments in the present experiment are
in disagreement with a report by Cornwell et al. (1985), which observed


116
Mean LH concentrations (Table 4-2) were greater (P < 0.05) in Angus
compared with Brangus and Brahman x Angus heifers. Mean LH peak-height in
Brangus heifers was similar (P > 0.10) to that for Angus and Brahman x Angus
heifers. However, LH peak-height was greater (P < 0.05) in Angus compared
with Brahman x Angus heifers (Table 4-2). Interval from GnRH to LH peak was
similar (P > 0.10) between Angus, Brangus and Brahman x Angus heifers (Table
4-2).
Estrous response after PGF2a, follicle size at PGF2a, interval from PGF2a to
the onset of estrus, duration of estrus, and total number of mounts received
during estrus (Table 4-3) were not influenced (P > 0.10) by breed. According to
contrast procedures, follicle size at PGF2a, interval from PGF2a to the onset of
estrus, duration of estrus, and total number of mounts received during estrus
were similar (P > 0.05) for Angus compared with Brangus and Brahman x Angus
heifers and for Brangus compared with Brahman x Angus heifers.
All heifers had a functional CL and progesterone > 1 ng/mL at PGF2a. All
heifers, except the single Angus heifer that did not ovulated after GnRH, had an
accessory CL at PGF2a. Mean volume of the original CL and accessory CL, as
well as total CL volume and mean progesterone concentrations at PGF2a were
similar (P > 0.10) between Angus, Brangus and Brahman x Angus heifers (Table
4-4). According to contrast procedures, mean volume of the original CL and
accessory CL, as well as total CL volume and mean progesterone concentrations
at PGF2c, were similar (P > 0.10) for Angus compared with Brangus and Brahman
x Angus heifers and for Brangus compared with Brahman x Angus heifers.


201
was actually high In the present study. Alternatively, animals with lower
progesterone concentrations (< 4 ng/ml) at PGF2a could have also included cows
that were already regressing their CL and would not have had decreased CL
regression rates. Instead, cows with progesterone concentration > 7 ng/mL were
most likely cows with either a mature functional CL or cows with two CL at PGF2a
administration and had an increased response to PGF2a treatment. This is
supported by results obtained in previous experiments (See chapter 5), where
results suggest that high progesterone concentrations at PGF2a can increase CL
regression.
Of the cows determined to be noncycling at the first GnRH treatment in the
present experiment, GnRFI Induced ovulations in 40% of the cows, similar to
other reports jn Select Synch-treated cows (Stevenson et al., 2000), but
considerably greater than those In beef cows after a Selct Synch + norgestomet
protocol (Stevenson et al., 2000). In contrast, GnRH induced ovulations for
noncycling cows In the present study was decreased compared to the 90%
induced ovulation in noncycling beef cows treated with the Select Synch system
(Troxel et al., 1993). These observations show the great variability existing in the
ovulatory response of anestrous cows to GnRH among different experimental
studies and estrus synchronization protocols. Early studies demonstrated that
administration of GnRH (Twagiramungu et al., 1995; Geary et al., 1998b;
Thompson et al., 1999) or treatment with MGA (Beal and Good, 1986; Kojima et
al., 2000) induces ovulation in both cycling and noncycling females. The inability
of the first dominant follicle to ovulate In anestrous beef cows Is thought to be


CHAPTER 2
REVIEW OF LITERATURE
Introduction
The use of cattle with varying percentages of 80s indicus breeding for
beef production in subtropical regions of the United States, and tropical and
subtropical regions of the world Is widespread. However, the effectiveness of
estrus synchronization systems Is often compromised because of problems
associated with estrus detection and response to different estrus synchronization
drugs In cattle of 60s indicus breeding. Moreover, development of predictable
estrus synchronization systems oriented toward cattle of 60s indicus breeding
has seldom been the primary focus of researchers. Most estrus synchronization
research has been conducted using beef and dairy cattle of 60s taurus breeding.
This review considers the hypothalamic-pituitary-ovarian axis and the
primary hormones of reproduction in the cow. The estrous cycle In the female
bovine, including regulation of ovarian follicle growth, atretic demise of follicles,
ovulation and luteolysls are briefly discussed. Exogenous control of ovarian
follicle dynamics and estrus synchronization protocols are also discussed.
Because most available research Is from 60s taurus cattle, these are presented
together with appropriate reference to 60s indicus and 80s indicus 80s taurus
cattle when data are known, or when important differences have been described.
6


171
procedure (survival analysis) of SAS. Furthermore, cows with progesterone
concentrations > 1.0 ng/ml_ at PGF2a were compared to cows with progesterone
concentrations < 1.0 ng/mL at PGF2a using the LIFETEST procedure (survival
analysis) of SAS. The survival analysis procedure regressed the proportion of
cows not observed in estrus during the 3 d following PGF2a. Data for cows that
were never observed in estrus were included in the survival analysis as censored

observations. Differences between the survival curves were tested with the
Wilcoxon test (Klein and Moeschberger, 1997). The effect of progesterone
concentrations at PGF2a on the effectiveness of PGF2a treatment to induce total
CL regression was analyzed by regression analysis using the GLM procedures of
SAS. Three-day estrous response, conception rate, timed-AI and synchronized
pregnancy rates were also analyzed in cows categorized by progesterone
concentrations on d -10, 0, and 7 using GENMOD procedures of SAS.
Results
There was no evidence of treatment x cycling status effects (P > 0.10) for
any variables analyzed so data were pooled. Furthermore, when BCS was
Included as a covariate there were no (P > 0.10) BCS effects on any of the
variables analyzed.
Three-day estrous, conception, timed-AI pregnancy, synchronized
pregnancy, and thirty-day pregnancy rates were similar (P >0.10) for the single
and split PGF2a treatments (Table 6-1). When PGF2a treatments were combined,
mean three-day estrous, conception, timed-AI pregnancy, synchronized
pregnancy, and 30-d pregnancy rates were 34, 53, 37, 43, and 85%,


Cl regression,
APPENDIX E
CORPUS LUTEUM REGRESSION IN CYCLING ANGUS AND BRAHMAN x
ANGUS HEIFERS (EXPERIMENT 1)
Figure E-1. Corpus luteum regression after an injection of PGF2a on either day 6
or 7 of the estrous cycle in Angus and Brahman x Angus heifers.
Statistical analysis was conducted using GENMOD procedure (P <
0.05). However, there was no difference (P > 0.05) when data were
analyzed with Logistic Regression.
228


127
2000) of the estrous cycle. Diameter of the largest follicle at GnRH on d 6 of the
estrous cycle for Angus, Brangus and Brahman x Angus heifers is similar to
results reported by Moreira et al. (2000) In dairy heifers. However, diameter of
the largest follicle at GnRH on d 6 Is greater compared to that reported for non-
lactating Angus and Brahman cows (Alvarez et al., 2000). The effectiveness of
GnRH to induce ovulation Is affected by the stage of follicular development at
GnRH treatment (Prescott et al., 1992; Silcox et al., 1993; Moreira et al., 2000a).
Furthermore, GnRH appears to be effective in inducing ovulation in follicles
greater than 9 mm during the growing phase of follicular growth (Macmillan and
Thatcher, 1991; Vasconcelos et al., 1999; Moreira et al., 2000a) when follicles
have a sufficient number of LH receptors (Lucy et al., 1992). This is in
agreement with the diameter of all the follicles that ovulated to GnRH in the
present experiment (range 10 to 14 mm). Similar results were reported in Bos
taurus x Bos indicus dual purpose cows (Perea et al., 1998). The Angus heifer
that did not ovulate when treated with GnRH on d 6 of the estrous cycle in the
present study appears to be a result of GnRH induced atresia (Macmillan and
Thatcher, 1991; Twagiramungu et al., 1995) as noted by the decrease In
diameter of the largest follicle from d 6 to 8 of the experiment.
In the pre-synchronization phase of the present experiment, only one
Brahman heifer expressed estrus, which was significantly less than the Angus,
Brangus, and Brahman x Angus heifers. Similarly, Galina et al. (1987) observed
estrus in only 30% of Bos indicus cattle treated with PGF2a after detection of a CL
by rectal palpation. Pinheiro et al. (1998) synchronized Nelore (Bos indicus)


271
Perea, F., R. Gonzlez, R. Cruz, E. Soto, E. Rincn, C. Gonzlez, and P.
Villamediana. 1998. Ultrasonographic evaluation of the follicular dynamics
in crossbred dairy cows and heifers. Rev. Cientfica, FCV-LUZ 8: 14-24.
Perry, R.C., L.R. Corah, G.H. Kiracofe, J.S. Stevenson, and W.E. Beal. 1991.
Endocrine changes and ultrasonography of ovaries In suckled beef cows
during resumption of postpartum estrous cycles. J. Anim. Sci. 69: 2548-
2555.
Peters, A. R., and G. E. Lamming. 1983. Hormone patterns and reproduction in
cattle. In Practice 5: 153-157.
Peters, J.B., J.A. Welch, J.W. Lauderdale, and E.K. Inskeep. 1977.
Synchronization of estrus in beef cattle with F2a and estradiol benzoate. J.
Anim. Sci. 45:230-235.
Peterson, L.A., S.F. Mares, E.A. Henderson, and M.E. Davenport. 1979. Effect of
calf separation time on pregnancy rate of cows synchronized with
Synchromate B (SMB). J. Anim. Sci. 49 (Suppl. 1): 326.
Picard, D. 1998. Molecular endocrinology. Steroids tickle cells inside and out.
Nature 392: 437-438.
Pierson, R.A., and O.J. Ginther. 1984. Ultrasonography of the bovine ovary.
Theriogenology 21:495-505.
Pinheiro, O.L., C.M. Barros, R.A. Figueiredo, E.R. do Valle, R. Oliveira-
Encarnagao, and C.R. Padovani. 1998. Estrous behavior and the estrus-to-
ovulation interval in Nelore cattle (Bos indicus) with natural estrus or estrus
induced with prostaglandin F2aor norgestomet and estradiol valerate.
Theriogenology 49: 667-681.
Pinto Neto, A., J.M. Silva Filho, J.F. Fonseca, M.F. Mota, H. Belisario, W.S.
Pardini, and M.T.T. Alvim. 2000. Performance of donor cows from Nelore
breed in an embryo transfer program. Arquivos da Faculdade de Veterinria
UFRGS 28 (Suppl. 1): 311 (Abstr.).
Plasse, D., A.C. Warnick, and M. Koger. 1970. Reproductive behavior of Bos
indicus females in a subtropical environment. IV. Length of estrous cycle,
duration of estrus, time of ovulation, fertilization, and embryo survival in
grade Brahman heifers. J. Anim. Sci. 30: 63-72.
Pool, S.H., R.A. Godke, and R.H. Ingraham. 1979. Effect of dexamethasone and
confinement stress on estrous cycle length in beef heifers. Theriogenology
11: 106.


APPENDIX B
ABSTRACT FOR EXPERIMENT 2
Cycling Angus (AN, n = 6), Brangus (BR, 5/8 Angus x 3/8 Brahman, n = 6),
and Brahman x Angus (BA, 3/8 Angus x 5/8 Brahman, n = 6) heifers were used
to evaluate secretory patterns of LH and associated ovarian events in response
to an administration of GnRH on d 6 of the estrous cycle followed by a single
Injection of PGF2a7 d later, Heifers were pre-synchronlzed with a modified two-
Injectlon PGF2a (Lutalyse Sterile Solution) protocol (25 mg I.m. on d -14 and
12.5 mg i.m. on d -3 and -2 of the experiment). Heifers received 100 pg I.m. of
GnRH (Fertagyl) on d 6 of the estrous cycle (Estrus d 0). Blood samples were
collected at 60, 30, and 1 min before GnRH and 15, 30, 60, 90, 120, 150,180,
240, 300, 360, 420, and 480 min after GnRH for determination of LH and
progesterone concentrations. Just prior to GnRH (d 6) and 48 h later, ovaries
were examined by ultrasonography to determine follicular and corpus luteum
(CL) characteristics. Seven days after GnRH, heifers were ultrasounded to
confirm the site of ovulation by the presence of an accessory CL. On d 13,
heifers received 25 mg of PGF20 i.m. and blood samples were collected at 0, 6,
12, 24, 36, and 48 h after PGF2a and dally thereafter until either the expression of
estrus or until d 20 of the experiment from heifers not exhibiting estrus to
determine progesterone concentrations. Estradiol concentrations were
determined in blood samples collected at -60, -30, and -1 min before GnRH.
221


40
The effects of estrogen on synchronizing follicular wave emergence
depends on the stage of follicular wave development when It Is administered
(Lane et al., 2001). Estradiol administered to heifers during the luteal phase of
the estrous cycle suppresses growth of the dominant follicle (Bo et al., 1993,
1994; Burke et al., 2000) and thereby synchronizing follicular development.
Whereas, estrogen administered during the follicular phase of the estrous cycle
would result in estrus and ovulation of the dominant follicle (Ulberg and Lindley,
1960; Peters etal., 1977; Lammoglia etal., 1998; Lemaster et al., 1999).
Moreover, estrogen given in the follicular phase of the estrous cycle decreases
the Interval to estrus (Nancarrow and Radford, 1975; Ryan et al., 1995) and the
variation In Its onset (Ulberg and Lindley, 1960; Nancarrow and Radford, 1975).
Estrogens can also have luteolytic effects when administered during the
estrous cycle (Wlltbank et al., 1971; Thatcher et al., 1986; Dlskin et al., 2002).
Thatcher et al. (1986) administered estradiol-17 p during the second half of the
estrous cycle that resulted In spikes of 15-keto-13,14-dlhydro-prostaglandln F2a
(PGFM) in the circulation. They concluded that estradiol-17p Induced luteolysls
by provoking a release of PGF2a from the uterus. Although, others have reported
that estrogens actions as a luteolytic agent are less clear (Lemon, 1975; Burke et
al., 1999).
Progesterone and Progestagens
The rationale for utilizing exogenous progestagens In estrus
synchronization protocols is to mimic the action of the CL by suppressing estrus
and ovulation (Flansel et al., 1961; Wlltbank etal., 1967; McDowell et al., 1998).
Sirols and Fortune (1990) used a controlled Intravaglnal progesterone-releasing


192
at PGF2a had timed-AI pregnancy rates that were 37% greater than both cycling
and noncycling cows with low progesterone concentrations at PGF2a. Decreased
timed-AI pregnancy rates observed In cows with low progesterone concentrations
at PGF2a is probably associated with the stage of development of dominant
follicles at timed-AI in conjuction with GnRH injection. Stage of development of
the dominant follicle affects its ability to ovulate in response to GnRH (Silcox et
al., 1993); therefore, some of the cows may not have had a follicle large enough
for ovulation. In suckled crossbred cows synchronized witha GnRH +
cloprostenol + shortterm MGA protocol (Thundathil et al., 1999) similar to the
present study, only 35% of the cows were in estrus within 96 h after cloprostenol
administration. These results suggest that in cows with low progesterone
concentrations at PGF2a, timed-AI at 72 h after PGF2a in the present experiment
may have been initiated too late relative to the developmental competence of the
dominant follicle, resulting in the decreased pregnancy rates observed for this
particular group of cows. Furthermore, this group could also be cows that did not
show estrus and therefore, ovulated an aged oocyte In response to GnRH.
Follicles with longer period of dominance and aged oocytes have been reported
to have decreased timed-AI pregnancy rates (Mlhm et al., 1994; Macmillan et al.,
2003).
In contrast, cows classified as cycling and having high progesterone
concentrations at PGF2a had improved timed-AI pregnancy rates compared with
cycling and noncycling cows with low progesterone at PGF2a. This improvement
may be associated with the stage of development of dominant follicles at the time


APPENDIX K
ESTROUS, CONCEPTION, AND PREGNANCY RATES OF CYCLING AND
NONCYCLING Bos taurus X Bos indicus COWS AND SURVIVAL ANALYSIS
DESCRIBING THE PROPORTION OF CYCLING AND NONCYCLING Bos
taurus x Bos indicus COWS THAT DID NOT EXHIBIT ESTRUS AFTER BEING
SYNCHRONIZED WITH GNRH AND EITHER SINGLE OR SPLIT DOSES OF
PROSTAGLANDINF2cl (PGF2ct) IN COMBINATION WITH MELENGESTROL
ACETATE (MGA) (EXPERIMENT 4)
Table K-1. Estrous, conception, and pregnancy rates of cycling Bos taurus x Bos
indicus cows synchronized with GnRH and either single or split doses
of prostaglandinF2cl (PGF2a) in combination with melengestrol acetate
T reatment3
Variable
Single PGF2ct
Split PGF2a
Three day estrous rate (%)b
32/82 = 39.0
30/100 = 30.0
Conception rate (%)c
21/32 = 65.6h
12/30 = 40.01
Timed-AI pregnancy rate (%)d
22/50 = 44.0
34/70 = 48.6
Synchronized pregnancy rate (%)e
44/82 = 53.7
46/100 = 46.0
30 day pregnancy rates (%)f
73/83 = 88.0
84/101 =83.2
3 Cows received 100 pg GnRH (Fertagyl) on d 0 of the experiment and either 25
mg PGF2a on d 7 or 12.5 mg PGF2on d 7 and 8. Cows that exhibited estrus
were Al approximately 8-12 h later and all cows not exhibiting estrus by d 10 of
the experiment were timed-AI with 100 pg GnRH. Cows received 0.5 mg
MGA*day"1 on d 1 to 6 of experiment.
Percentage of cows displaying estrus 3 d after PGF2a of the total treated.
Percentage of cows pregnant that exhibited estrus and were Al.
Percentage of cows pregnant of the total that were timed-AI.
Percentage of cows pregnant to estrus Al and timed-AI of the total treated.
Percentage of cows pregnant during the first 30 d of the breeding season of the
total treated.
240


This dissertation is dedicated to my parents Mary and Heberto, for their
unconditional support; to my loving wife Marisol and my children Valeria,
Eduardo, and Vivian, for inspiring me and giving me reason to achieve; and
above ail to God, for giving me the desire and ability to accomplish my goals.


49
Information about LH concentrations in the peripheral circulation in cattle
after a GnRH treatment during the estrous cycle is numerous. The data available
refer to description of LH response following administration of GnRH or its
analogs in a nymphomaniac cow (Mori et al., 1974), in normal cows at
insemination (Mori and Takahashi, 1978), in cows with ovarian follicular cysts
(Mori et al., 1979; Tanaka et al., 1979), in beef heifers following treatment with
GnRH within hours after the onset of estrus (Coleman et al., 1988), in dairy
heifers following injection of various dosages (Chenault et al., 1990), in beef
cows treated during the postpartum period alone (Williams and Stanko, 1996;
Fajersson etal., 1999), or in combination with PGF2a(Cruz et al., 1997), In early
postpartum crossbred cows (Deen et al., 1995) and dairy cows (Vural et al.,
1999), in cattle following chronic administration for controlled, reversible
suppression of estrous cycles (Gong et al., 1996; DOcchio et al., 1996; Vizcarra
et al., 1997; Rajamahendran et al., 1998; DOcchio et al., 2000), in
ovariectomized Brahman and Hereford cows challenged with GnRH (Griffin and
Randel 1978), and in Brahman and Angus cows on d 17 and 34 after calving
(Stahringer et al., 1989). In summary, most of these data have been generated
in cattle of Bos taurus breeding and very little in cattle of Bos indicus breeding.
There is little information where direct comparisons between cattle of Bos taurus
and Bos indicus breeding have been made relative to endocrine and ovarian
responses of cycling cattle to a single injection of GnRH.
In reference to LH response after exogenous GnRH administration in cattle,
Coleman et al. (1988) reported that Fertirelin acetate (FA; synthetic GnRH


141
determination of progesterone concentrations daily starting on d 4 of the
experiment either until heifers exhibit estrus or until d 20 of the experiment if
heifers failed to exhibit estrus (Figure 5-1). After collection of blood samples,
they were immediately placed on ice and centrifuged (3000 rpm) within 4 h.
Plasma was separated and stored at -20 C for later analysis. Progesterone
concentrations were determined in multiple assays using RIA (Seals et al., 1998).
The intra- and inter-assay CV were 2.1% and 13.6 %, respectively. Sensitivity of
the assay was 0.02 ng/tube. The presence of a CL at PGF2ci as detected by
ultrasound was defined as functional If progesterone concentrations were > 1.0
ng/mL. Corpus luteum (CL) regression was defined as progesterone
concentrations were > 1.0 ng/mL PGF2ol followed by two consecutive blood
samples with, progesterone concentrations <1.0 ng/mL. The onset of estrus was
defined as 3 or more mounts within a four-hour period (Landaeta et al., 1999;
Lorton et al., 1999; Landaeta-Flernandez et al., 2002).
For statistical analysis, the effects of progesterone group (HP vs. LP), breed
and the interaction were analyzed for ovulation rate after GnRH, percentage of
heifers with an accessory CL (d 13 PGF2a injection), CL regression (d 13 PGF2o
injection) and estrous response with logistic regression modeling computing
likelihood ratio test for type 3 contrasts for each term in the model using the
GENMOD procedure of SAS (SAS Inst. INC., Cary, NC). Estrous response was
defined as the percentage of heifers exhibiting estrus within 7 d following the d
13 PGF2a induced luteolysis. Progesterone concentrations for the 2 d prior to
GnRH administration (experimental d 4) and until GnRH (experimental d 6), and


109
min before GnRH were not included in the statistical analyses but were plotted in
each figure. Peak-height was defined according to Chenault et al. (1990) as the
maximum concentration (ng/ml) of LH detected following GnRH treatment.
Heifers were fitted with electronic estrus detection devices (HeatWatch,
DDx, Boulder, CO) on d -3 of the pre-synchronization period to monitor estrus
throughout the remainder of the experiment. The onset of estrus was defined as
3 or more mounts within a 4-h period while the end of estrus was defined as the
last mount recorded prior to a period of extended inactivity of at least 8 h
(Landaeta et al., 1999; Lorton et al., 1999). Duration of estrus was calculated by
subtracting the time of the initial mount from the time of the last mount. Interval
to estrus after PGF2a (d 13) was calculated as the time from PGF2a to first mount
as detected by HeatWatch. Total number of mounts received during estrus was
also determined.
The effect of breed on pre-synchronization estrous response, ovulation rate
after GnRH, and estrous response after PGF2ol, were analyzed with logistic
regression modeling computing likelihood ratio test for type 3 contrasts for each
term in the model using the GENMOD procedure of SAS (SAS Inst. INC., Cary,
NC). Pre-synchronization estrous response was defined as the percentage of
heifers exhibiting estrus within 5 d after the split PGF2o, treatment. Treatment
estrous response was defined as the percentage of heifers exhibiting estrus
within 7 d after PGF2a, which followed the GnRH treatment by 7 d. Effects of
breed on LH peak-height, time to LH peak, interval from PGF2a to the onset of
estrus, duration of estrus, total number of mounts received during estrus, size of


132
variability in response to PGF2a and extended intervals to the onset of estrus
after PGF2a treatments. A possible reason for the conflicting results of the
present study is that all heifers were managed together; thus, breed effects were
confounded. Therefore, the excellent estrous response in the present study can
probably be attributed to the fact that the heifers were trained to the stanchions
for three weeks prior to the beginning of the experiment to reduce the stress
associated with the daily working of animals. Stress can affect the interval from
PGF2a to the onset of estrus (Hein and Allrich, 1992). Frequent training before
the initiation of the present study may have prevented the stress related
response. Additionally, follicle size at PGF2a in the current study was similar
among breeds. This could indicate that heifers from the present experiment had
a synchronized follicular wave at PGF2a that resulted in all of the heifers with a
similar size ovulatory follicle at PGF2q and coming into estrus at one time.
Administration of GnRH to cattle given at various stages of the estrous cycle has
been reported to alter follicular development as evidenced by changes in the
distribution of follicles among different size classes and induction of an LH surge,
which induces either ovulation or luteinization of the dominant follicle (Thatcher et
al., 1989; Macmillan and Thatcher, 1991). The end result is emergence and
synchronization of a new follicular wave (Twagiramungu et al., 1995).
Synchronizing follicle development helps to improve estrous response and
provides a more synchronous estrus (Fike et al., 1997; Johnson et al., 1997).
In the present experiment, duration of estrus or total number of mounts
received after PGF2a, as determined using radiotelemetric estrus detection


261
Kesler, D.J, T.S. Dyson, R.N. Summers, T.L. Steckler, and T.G. Nash. 1997.
Effect of prostaglandin F20 treatment before norgestomet and estradiol
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Kesner, J.S., E.M. Convey, and C.R. Anderson. 1981. Evidence that estradiol
induces the preovulatory LH surge In cattle by increasing pituitary sensitivity
to LHRH and then increasing LHRH release. Endocrinology 108: 1386-
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Kesner, J.S., V. Padmanabhan, and E.M. Convey. 1982. Estradiol induces and
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follicle-stimulating hormone in heifers. Biol. Reprod. 26: 571-578.
Kieborz-Loos, K.R., H.A. Garverlck, D.H. Keisler, S.A. Hamilton, B.E. Salfen,
R.S. Youngquist, and M.F. Smith. 2003. Oxytocin-Induced secretion of
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endometrium and corpus luteum. Biol. Reprod. 11: 566-577.
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of persistent ovarian follicles in cattle. J. Anim. Scl. 74: 1424-1440.
King, M.E., G.H. Kiracofe, J.S. Stevenson, and R.R. Schalles. 1982. Effect of
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Theriogenology 18: 191-200.
Kiracofe, G.H., L.E. Keay, and K.G. Odde. 1985. Synchronization of estrus in
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Kirby, C.J., M.F. Smith, D.H. Keisler, and M.C. Lucy. 1997. Follicular function in
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Klein, J.P., and M.L. Moeschberger. 1997. Survival Analysis: techniques for
censored data and truncated data. Springer-Verlag, New York.
Knickerbocker, J.J., M.C. Wiltbank, and G.D. Niswender. 1988. Mechanisms of
luteolysis in domestic livestock. Domest. Anim. Endocrinol. 5: 91-107.
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151
endogenous estrogen to provoke the standing response (Britt et al., 1986). After
a threshold concentration (> 7 pg/mL) of estradiol is reached (Chenault et al.,
1975; Lyimo et al., 2000; Lopez et al., 2002) in a relative absence of
progesterone (Blache et al., 1991), induction of estrous behavior is initiated.
However, once the concentration of plasma estradiol is sufficient to induce
estrus, additional amounts have no further stimulatory influence on expression of
estrous behavior (Coe and Allrich, 1989). Luteolysis was initiated in heifers in
the LP group on d 4 of the estrous cycle when follicles have been reported to be
in the growing phase (about 4.0 mm) of the first follicular wave (Adams et al.,
1992b; Ginther et al., 1997) and serum estradiol concentrations are about 1.3
pg/mL (Austin et al., 2001). Additionally, progesterone concentrations were
above 1 ng/mL on d 4 for this group of heifers.
Progesterone concentrations for the 7 days after GnRH administration (d 6
to 13 of the estrous cycle) were similar between Angus and Brahman x Angus
heifers, regardless of progesterone group in the current study. These results
contradict a study by Randel (1977) where Brahman x Hereford heifers had
decreased serum progesterone concentrations during the estrous cycle
compared with Hereford heifers (Randel, 1977). Similarly, Brahman cows had
lower serum progesterone concentrations from d 7 to 17 of the estrous cycle
compared with Angus cows (Segerson et al., 1984). The differences between
results from this study and those reported by others (Randel, 1977; Segerson et
al., 1984) are unclear.


266
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secretion of LH, ovarian activity and milk yield of postpartum dairy cows.
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Sower, and W.H. Yu. 2001. Control of gonadotropin secretion by follicle-
stimulating hormone-releasing factor, luteinizing hormone-releasing
hormone, and leptin. Arch. Med. Res. 32: 476-485.
McCracken, J.A. 1971. Prostaglandin F2a and corpus luteum regression. Ann.
New York Acad. Scl. 10: 456-461.
McCracken, J.A., E.E. Cruster, and J.C. Lamsa. 1999. Luteolysis: A
neuroendocrine-mediated event. Physiol. Rev. 79: 263-324.
McCracken, J.A., W. Schramm, B. Barclkowski, and L. Wilson. 1981. The
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197
differences in timed-AI pregnancy rates. As mentioned before, the overall
conception rate observed in the current study was satisfactory and was not
affected by treatment, cycling status, and (or) progesterone concentrations at
PGF2a- Therefore, if cows expressed estrus the likelihood that they would
conceive to that estrus is very likely. These observations indicate that to
continue to improve the effectiveness of the synchronization protocol used in the
present study, it would be beneficial to have as many cows expressing estrus as
possible during the post-synchronization period. This should result in greater
estrous rates during the period of estrus detection, thereby reducing the number
of cows that are timed-AI and decreasing the variability observed among cycling
and noncycling cows with either low or high progesterone concentrations at
PGF2.
The overall thirty-day pregnancy rate (80.4%) in the present experiment
was at least 6.0% greater than others reported in different experiments using an
intravaginal progesterone-releasing device (Beal, 1983), norgestomet (Peterson
et al., 1979), or either the CoSynch or Hybrid Synch protocols (Lemaster et al.,
2001). Thirty-day pregnancy rates were greater for cycling (85%) cows
compared with noncycling (74%) cows in the present study. Using Bos indicus x
Bos taurus cows synchronized with the CoSynch protocol, Lemaster et al. (2001)
reported a 30-d pregnancy rate of 74% when 65% of the cows were cycling at
the initiation of the CoSynch treatment. Hiers et al. (2003) reported 30-d
pregnancy rates > 75% for Bos taurus x Bos indicus cows synchronized with a
GnRH + PGF2c + MGA protocol, similar to the one used in the present study.


128
females with two injections of PGF2a 11 d apart and observed no estrus after the
first injection and only 46% of the cows exhibited estrus after the second
injection. In general, administration of PGF2? in cycling cattle of Bos indicus
breeding yields inconsistent results relative to induction of luteolysis and
synchronization of estrus. The inconsistent results obtained after PGF2a
administration in cattle of Bos indicus breeding appears to be primarily
associated with compromised expression of estrus (Orihueia et al., 1983;
Pinheiro et al., 1998; Rekwot et al., 1999). Furthermore, cattle of Bos indicus
breeding can ovulate without exhibiting any visible signs of estrus (Plasse et al.,
1970), which is probably due to social effects (Dobson and Smith, 2000) having a
negative effect on estrous expression. The decreased estrous response in
Brahman heifers after the pre-synchronization treatment in the present
experiment is unclear. Part of the reason could be attributed to social
interactions between breeds (Landaeta-Plernandez et al., 2002) where Brahman
and Angus cattle were managed together in a single group, similar to the present
study. Genotype, environmental and social factors have been implicated as
factors that can influence behavioral estrus (Hurnick et al., 1975; Galina and
Arthur, 1990; Rodtian et al., 1996; Landaeta-Fiernandez et al., 2002). Flowever,
it is unclear from the present study if these factors acted directly or in concert
with each other to have a negative affect on estrous expression in the Brahman
heifers. Whether managing the four different genotypes represented in the
present experiment as separate breed groups would have increased estrous
expression is unclear, but needs to be investigated further.


LH, ng/mL
115
25
a
Figure 4-4. Plasma LH concentrations (LS Means) in cycling Angus (diamonds),
Brangus (squares) and Brahman Angus (triangles) heifers receiving
100 ng GnRH on d 6 of the estrous cycle. Breed (P < 0.05). Time (P <
0.0001). Breed x Time (P < 0.0001). Angus vs. Brangus and Brahman
x Angus (P < 0.01); Brangus vs. Brahman x Angus (P < 0.01),. a,b,c
Different letters within each time category differ (P < 0.05).


118
Table 4-3. Estrous response, follicle size at prostaglandin F2a (PGF2a; LS Means
SE) and behavioral estrous characteristics (LS Means SE) after
PGF2a for Angus (AN), Brangus (BR), and Brahman x Angus (BA)
heifers in response to a GnRH + PGF2o synchronization protocol3.
Variable
n
Estrous
response15,
%
Mean
diameter
of largest
follicle,
mm
Mean
estrous
duration, h
(range)
Mean
interval to
the onset of
estrus after
PGF2a, h
(range)
Mean
number
of total
mounts
(range)
Breed
NS
NS
NS
NS
NS
Angus
6
5/6=83
8.2 0.9
16 3
(8-28)
64 4
(60-73)
31 6
(17-48)
Brangus
6
5/6=83
9.5+ 0.9
15 + 3
(8-23)
60 4
(52-68)
20 6
(17-22)
Brahman
x Angus
6
6/6=100
9.5 0.9
14 + 3
(9-18)
68 4
(47-77)
31 6
(10-65)
Contrasts
AN vs. BR
and BA
-
NS
NS
NS
NS
BR vs. BA
-
NS
NS
NS
" NS
aHelfers received 100 pg GnRH on d 6 of the estrous cycle and 25 mg PGF2o 7 d
latter.
bPercentage of heifers displaying estrus 7 d after PGF2o of the total treated.


44
Administration of an exogenous estrogen (Yelich et al., 1997; Diskin et al.,
2002) or estrogen plus progesterone (Fike et al., 1999) during administration of
an exogenous progestogen also induces regression of persistent follicles and
permits ovulation of a newly recruited follicle after progestogen withdrawal.
However, the estrogen treatments were only effective in initiating atresia in 71
and 77% of the persistent follicles in the Yelich et al. (1997) and Diskin et al.
(2002) studies, respectively.
It appears to be more difficult to control follicle wave dynamics with an
exogenous estrogen in animals that are at early stages of the estrous cycle (d 1
and 6) (Diskin et al., 2002). Besides, it is against the physiological
concentrations of estradiol that the use of estrogens for follicle wave regulation is
considered (Diskin et al., 2002). Furthermore, endogenous estradiol has a
marginal negative effect on LH secretion, requiring the simultaneous use of
progesterone to synchronize new follicle emergence (Bo et al., 1995).
Alternatively, estrogen may induce follicular atresia by altering LH secretion and
(or) FSH suppression (Price and Webb, 1988). Estrogen induced suppression of
LH may be apparent only during a progesterone dominated phase, whereas
estradiol and progesterone may have a synergistic suppressive effect on both LH
and FSH (Price and Webb, 1988; Bo et al., 2000). Estradiol in combination with
progestogen, suppress FSH secretion (Barnes et al 1981; Bolt et al., 1990);
however, suppression was more prolonged in progesterone-implanted heifers
(Bolt et al., 1990). An additional reason for combining progesterone with
estradiol to cattle at random stages of the estrous cycle is to prevent the


117
Table 4-2. Mean LH concentrations, LH peak-height, and interval from GnRH to
LH peak In Angus (AN), Brangus (BR) and Brahman Angus (BA)
heifers In response to GnRH treatment (100 pg) on d 6 of the estrous
Variable
n
Mean LH
concentrations,
ng/mL
Mean LH peak-
height, ng/mL
Mean interval
to LH peak,
min
Breed
P < 0.05
P < 0.05
NS
Angus
6
7.0 0.8a
21.9 3.4a
90.0 + 14.7
Brangus
6
4.6 0.8b
13.9 3.4a,b
77.5 14.7
Brahman x
Angus
6
2.9 0.8b
8.0 3.4b
55.0 14.7
Contrasts
AN vs.
BR and BA
P < 0.05
P < 0.05
NS
BR vs. BA
P < 0.05
P < 0.05
NS
Values within a column lacking a common superscript differ (P < 0.05)


182
conception rates that were at least two times greater than the lowest timed-AI
conception rate. Additionally, two sires had a 13% and 29% decrease in the
timed-AI conception compared with their conception rates, whereas the other two
sires had a decrease of only 1.7 and 7.3% in the timed-AI pregnancy rates
compared to the conception rate.
Discussion
Results from the present study suggest that modifying the delivery of PGF2a
from a single to two consecutive split doses initiated 7 d after GnRH did not
improve the estrus, conception, timed-AI pregnancy, and synchronized
pregnancy rates in lactating crossbred Bos indicus cows synchronized with a
Hybrid Synch estrus synchronization system combined with MGA. Therefore, it
does not appear that the additional PGF20 injection had any significant effect on
enhancing CL regression, which is supported by the observation that the timed-
AI and total CL regression rates of the present experiment were similar between
PGF2a treatments. Approximately 85% of cows with a functional CL at PGF2a
treatment had a PGF2a induced CL regression, similar to reports in 60s taurus
cattle (85 to 100%) treated with PGF2ot between days 5 and 8 of the estrous cycle
(King et al 1982; Tanabe and Hann, 1984; Kirakofe et al 1985). Fernandes et
al. (2001) reported similar estrous, conception and timed-AI pregnancy rates In
Nelore cows treated with either single or split doses of PGF2,, in an OvSynch
protocol. Similarly, Hiers et al. (2003) reported similar timed-AI pregnancy rates
between a single and two consecutive split doses of PGF2a in nonlactating 60s
indicus x 60s taurus cows using an estrus synchronization protocol like the one


181
Table 6-5. Conception and tlmed-AI pregnancy rates by Al sire in Bos taurus x
Bos indicus cows synchronized with GnRH and either a single or split
doses of prostaglandin F20 (PGF2a) in combination with melengestrol
acetate (MGA) treatment3,
Al Sire
Conception rate0, %
Timed-AI pregnancy rate0, %
A
4/13 = 30.8
6/34 = 17.6d
B
16/33 = 48.5
21/51 = 41,2e
C
12/24 = 50.0
29/60 = 48.3e
D
20/32 = 62.5
17/50 = 34.0e
a Cows received 100 pg GnRFI (Fertagyl) on d 0 of the experiment and either 25
mg PGF2tl on d 7 or 12.5 mg PGF20on d 7 and 8. Cows that exhibited estrus
were Al approximately 8-12 h later and all cows not exhibiting estrus by d 10 of
the experiment were timed-AI with 100 pg GnRFI. Cows received 0.5 mg
MGA'day'1 on d 1 to 6 of experiment.
Percentage of cows pregnant of the total detected in estrus. Al Sire (P = 0.06).
Percentage of cows pregnant of the total that were timed-AI.
d,eValues with different superscript in the same column differ (P < 0.05).


160
CIDR + PGF2a group (Lucy et al., 2001). Although luteolysis was not evaluated
In the aforementioned studies, the results suggest that the CIDR may enhance
the PGF2a Induced luteolysis. Another option to improve fertility is the pre
synchronization of the estrous cycle before starting the GnRH + PGF2c, system
so the majority of cattle are in a common stage of the estrous cycle when the
GnRH + PGF2a system is initiated (Moreira et al., 2001; Pancarci et al., 2002).
However, this is impractical for beef producers who prefer to limit the number of
cattle handlings involved In a synchronization system.
Size of the largest follicle at PGF2a was similar between the HP and LP
heifers similar to a report by Shaham-Albalancy et al. (2000) In beef cows on d
15 of the estrous cycle with a low ascending progesterone curve Induced by
three PGF2 Injections on d 3 to 4 of the estrous cycle. Similarly, Moreira et al.
(2000a) did not observe any significant differences In diameter of the dominant
follicle In dairy heifers synchronized with the Ovsynch protocol when the first
GnRH treatment was Initiated on d 5 of the estrous cycle compared to d 18. The
GnRH treatment on d 6 of the estrous cycle In the present study resulted in
synchronization of the emergence of a new follicular wave regardless of breed
and progesterone concentration.
Estrous response after PGF2a was affected by breed but not by
progesterone group in the present study. Additionally, among heifers that
regressed their CL, 74% expressed estrus after PGF2a regardless of
progesterone group. Brahman x Angus heifers were 13.9 times more likely to
exhibit estrus after PGF2a than Angus heifers In the current experiment. Estrous


20
Diestrus
Diestrus is characterized by the presence of a functional CL and increased
concentrations of progesterone. Diestrus is the longest stage of the estrous
cycle, lasting 10 to 14 d. Diestrus ends with the release of prostaglandin F2a
(PGF2a) from the uterus, which results in luteolysis and a reduction in
progesterone production. Whether pregnancy results or not, the CL develops
into a fully functional organ producing large amounts of progesterone. If an
oocyte Is fertilized and reaches the uterus, the CL will be maintained throughout
the pregnancy. In contrast, if the oocyte Is not fertilized, the CL remains
functional until d 17 or 18 and it degenerates after luteolysis, thereby permitting a
new estrous cycle to be initiated.
Proestrus
Proestrus is characterized by follicular growth and estradiol production
(Chenault et al., 1975; Kesner et al., 1982) and it occurs 2 to 3 d before the onset
of estrus in the cow. Proestrus is characterized by a major endocrine transition
from a period of progesterone dominance to a period of estrogen dominance
(Chenault et al., 1975; Kesner etal., 1982). Proestrus begins when blood
progesterone concentrations decline due to luteolysis (Auletta and Flint, 1998).
Concomitant with the decrease in progesterone is an increase in circulating
concentrations of estradiol, as a dominant follicle prepares for ovulation (Ireland
et al., 1984). Gonadotropins LH and FSH are the primary hormones responsible
for this transition (Kesner et al., 1982).


113
GnRH
> 'V3 < Interval from GnRH, min
Figure 4-3. Plasma progesterone concentrations (LS Means) in cycling Angus
(diamonds), Brangus (triangles) and Brahman x Angus (squares)
heifers receiving 100 pg GnRH on d 6 of the estrous cycle. Breed (P >
0.10). Time (P < 0.0001). Breed x Time (P < 0.0001). Angus vs.
Brangus and Brahman x Angus (P > 0.10); Brangus vs. Brahman x
Angus (P > 0.10). a,b Different letters within each time category differ
(P< 0.001).


no
the largest follicle at GnRH and PGF2o CL volume at GnRH, original CL volume
at PGF2a, accessory CL volume at PGF2a, total CL volume at PGF2a, and
progesterone concentrations at PGF2o were analyzed by ANOVA using GLM
procedures of SAS. Additionally, breed effects were separated using contrasts
comparing Angus vs, Brangus and Brahman x Angus, and Brangus vs. Brahman
x Angus. The effect of total CL volume on progesterone concentrations at PGF2a
was analyzed by regression analysis using the GLM procedure of SAS.
Progesterone concentrations after GnRH and PGF2a, estradiol
concentrations before GnRH, and LH concentrations after GnRH were analyzed
In a by repeated measures analysis using the MIXED procedure of SAS. The
model Included fixed effects of breed, time, and breed x time, and random effect
of heifer nested within breed was the error term, and was subjected to three
covariance structures: autoregressive order 1, compound symmetry, and
unstructured covariance. The covariance that resulted in the smallest Akaikes
Information Criterion was used. Follicle size before GnRH was included as a
covariate in the model for the analysis of estradiol concentrations before GnRH.
Also, follicle size and estradiol concentrations before GnRH were tested
separately as covariates In the model for the analysis of LH concentrations after
GnRH. The LH response curves were analyzed by homogeneity of regression
procedures (Wilcox et al 1990). The LH curves were also analyzed using
contrasts comparing Angus vs. Brangus and Brahman x Angus, and Brangus vs.
Brahman x Angus to examine breed differences.


180
Figure 6-3. Timed-AI and total CL regression rates in Bos taurus x Bos indicus
cows synchronized with GnRH and either a full (white) or two
consecutive split injections of PGF2a (grey) in combination with
melengestrol acetate (MGA) treatment. PGF2q treatment (P > 0.10).


250
Castilho, C., A.L.G. Gambini, P. Fernandez, L.A. Trinca, A.B. Teixeira, and C.M
Barros. 2000. Synchronization of ovulation in crossbred dairy heifers using
gonadotropin-releasing hormone agonist, prostaglandin F2a and human
chorionic gonadotropin or estradiol benzoate. Braz. J. Med. Biol. Res. 33:
91-101.
Cavalieri, J., C. Coleman, J.E. Kinder, and L.A. Fitzpatrick. 1998a. Comparison
of three methods of acute administration of progesterone on ovarian
follicular development and the timing and synchrony of ovulation in Bos
indicus heifers. Theriogenology 49: 1331-1343.
Cavalieri, J., J.E. Kinder, G. Death, and L.A. Fitzpatrick. 1997. Effect of 48 h
treatment with estradiol or progesterone on follicular wave emergence and
synchrony of ovulation in Bos indicus cows when administered at the end of
a period of progesterone treatment. Anim. Reprod. Sci. 46: 187-201.
Cavalieri, J., J.E. Kinder, and L.A. Fitzpatrick. 1998c. Effect of acute treatment
with progesterone on the timing and synchrony of ovulation in Bos indicus
heifers treated with a norgestomet implant for 17 days. J. Reprod. Frtil.
112: 249-258.
Chappel, S.C., and C.A. Barraclough. 1976. Hypothalamic regulation of pituitary
FSH secretion. Endocrinology 98: 927-935.
Chegini, N., N. Ramani, and C.V. Rao. 1984. Morphological and biochemical
characterization of small and large bovine luteal cells during pregnancy.
Molec. Cell. Endocrinol. 37: 89-102.
Chenault, J.R., D.D. Kratzer, R.A. Rzepkowski, and M.C. Goodwin. 1990. LH and
FSH response of Holstein heifers to fertirelln acetate, gonadorelin nd
buserelin. Theriogenology 34: 81-98.
Chenault, J.R., W.W. Thatcher, P.S. Kalra, R.M. Abrams, and C.J. Wilcox. 1975.
Transitory changes in plasma progestlns, estradiol, and luteinizing hormone
approaching ovulation in the bovine. J. Dairy Sci. 58: 709-717.
Chenault, J.R., W.W. Thatcher, P.S. Kalra, R.M. Abrams, and C.J. Wilcox. 1976.
Plasma progestlns, estradiol, and luteinizing hormone following
prostaglandin F2 alpha Injection. J. Dairy Sci. 59: 1342-1346.
Christensen, D.A., J.N. Wiltbank, and M.L. Hopwood. 1974. Levels of hormones
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17p and estrous behavior in heifers. J. Anim. Sci. 67: 1546-1551.


126
higher doses (350 to 500 III) of the same product frequently used for
superovulation of Bos taurus cattle. Superovulation response was similar
between the reduced doses and the higher doses, suggesting that Bos indicus
cattle require less FSH than Bos taurus to reach optimal superovulatory
response. Although, studies using decreasing concentrations of FSH and
comparing concurrently superovulatory response of Bos indicus and Bos taurus
cattle have not been currently reported. Further investigations must be
conducted to determine if ovaries of cattle of Bos indicus breeding have an
increased sensitivity to LH release compared to Bos taurus cattle.
Estradiol concentrations and follicle size were similar prior to the GnRH
challenge between the Angus, Brangus, and Brahman Angus heifers in the
present study. Although It Is interesting that mean plasma estradiol
concentrations were numerically greater in Brangus and Brahman x Angus
heifers compared with Angus heifers, which is similar to a previous report
(Alvarez et al., 2000) in Angus and Brahman cows on d 5 of the estrous Cycle.
Furthermore, mean LH concentrations induced by GnRH were not affected by
either estradiol concentrations or follicle size prior to the GnRH challenge.
The GnRH administration induced ovulation and formation of a new CL in
nearly all of the Angus, Brangus, and Brahman x Angus heifers in the present
study, which are considerably greater than the results previously reported
(Fernandes et al., 2001) In Bos indicus cattle treated with GnRH at random
stages of the estrous cycle, but similar to studies In Bos taurus cattle
administered GnRH on d 5 to 9 (Vasconcelos et al., 1999) or d 5 (Moreira et al.,


61
estrogens have been used after progestogen treatment as a treatment regime for
postpartum anestrous cows (McDougall et al., 1992; Fike et al., 1997) in order to
eliminate cows that ovulate without an observed estrus (McDougall et al., 1992).
Consequently, the addition of estrogen at the termination of a progestogen
treatment increases the number of animals detected in estrus and submitted for
Al.
Similar to results obtained by administering estrogens at the Initiation of
short-term progestogen treatments to synchronize follicle development (Trbulo et
al., 1995; Martinez et al., 2001), administration of GnRH at the initiation of a
CIDR treatment improved pregnancy rates in heifers (Martinez et al., 2001;
2002).
In general, progestagens treatments in postpartum anestrous Bos indicus x
Bos taurus dual-purpose cows (Soto et al., 1998; de Ondiz et al., 2002; Soto et
al., 2002) have shown satisfactory results in the induction of estrus. Percentage
of anestrous cows that exhibited estrus was 75 to 81 % and 60.7% (Hernandez et
al., 1995; de Ondiz et al., 2002; Soto et al., 2002) when using norgestomet
implants (Slncro-Mate B and Crestar, respectively); and 83.3% for CIDR (Soto et
al., 1998). Conception rates at the synchronized estrus after progestogen
withdrawal are quite variable (28 to 68%; Hernndez et al, 1995; Soto et al.,
1998; 2002). However, treatment of postpartum dual purpose crossbred Bos
indicus x Bos taurus cows with intravaginal sponges (Medroxiprogesterone
acetate) 40 d post partum (Palomares et al., 2002) have resulted in decreased
induction of estrus when combined with either GnRH (65.2%) or estradiol


Progesterone, ng/mL
143
-2 -1 0
Days from GnRH
Figure 5-2. Progesterone concentrations for the two days before GnRH (d 6 of
the estrous cycle) for heifers in the high progesterone (HP) group
including Angus (diamonds) and Brahman Angus (squares) and
heifers in the low progesterone (LP) group including Angus (triangles)
and Brahman Angus (X) heifers. Breed (P > 0.10), Day (P <0.0001),
Breed x Group (P > 0.10), Group Day (P < 0.0001), Breed x Day (P
> 0.10), Breed x Group x Day (P > 0.10). HP vs. LP (P < 0.0001).


275
Rosenfeld, C.S., X. Yuan, M. Manikkam, M.D. Calder, H.A. Garverick, and D.B.
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Roussel, L.E., and J.F. Beatty. 1969. Effect of melengestrol acetate on
synchronization of estrus, subsequent fertility and milk constituents of
lactating dairy cows. J. Dairy Sci. 52: 2020-2023.
Rowson, L.E., R. Tervlt, and A. Brand. 1972. The use of prostaglandins for
synchronization of oestrus in cattle. J. Reprod. Frtil. 29: 145.
Roy, D., N.L. Angellni, and D.D. Belsham. 1999. Estrogen directly represses
gonadotropin-releasing hormone (GnRH) gene expression in estrogen
receptor-alpha (ERalpha)- and ERbeta-expressing GT1-7 GnRH neurons.
Endocrinology 140: 5045-5053.
Ruiz-Corts, Z.T., and M. Olivera-Angel. 1999. Ovarian follicular dynamics In
suckled zebu (Bos indicus) cows monitored by real time ultrasonography.
Anim. Reprod. Sci. 54: 211-220.
Ryan, D.P., S. Snijders, A. Aarts, and K.J. Farrell. 1995. Effect of estradiol
subsequent to Induced luteolysis on development of the ovulatory follicle
and interval to estrus and ovulation. Theriogenology 43: 310 (Abstr.).
Ryan, M., M. Mihm, and J.F. Roche. 1998. Effect of GnRH given before or after
dominance on gonadotropin response and the fate of that follicle wave in
postpartum dairy cows. J. Reprod. Frtil. Abstract Series 21: 61 (Abstr.).
Saacke, R.G. 2002. Factors affecting fertilization in estrus-synchronized .cattle. J.
Anim. Sci. 80 (Suppl. 2): 85-86 (Abstr.).
Sakamoto, K., K. Mlwa, T. Ezashi, E. Okuda-Ashitaka, K. Okuda, T. Houtanl, T.
Suglmoto, S. Ito, and O. Hayaishi. 1995. Expression of mRNA encoding the
prostaglandin F2a receptor in bovine corpora ltea throughout the oestrous
cycle and pregnancy. J. Reprod. Frtil. 103: 99-105.
Sanchez, T., M.E. Wehrman, F.N. Kojima, A.S. Cupp, E.G. Bergfeld, K.E. Peters,
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endogenous secretion of 17p-estradiol in heifers. Biol. Reprod. 52: 464-469.
Santos, E.A., A.C. Warnlck, J.R. Chenault, D.R. Wakeman, and M.J. Fields.
1988. A novel approach for Prostaglandin F2ctfor estrous synchronization In
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LIST OF TABLES
Table page
3-1 Corpus luteum (CL) regression and ovarian characteristics In Angus and
Brahman Angus heifers treated with prostaglandin F2a (PGF2cc) on
either d 6 or 7 of the estrous cycle 81
3-2 Estrous response and behavioral estrus characteristics in Angus and
Brahman Angus heifers treated with prostaglandin F2ot (PGF2(X) on
either d 6 or 7 of the estrous cycle 85
4-1 Follicle size, estradiol and progesterone concentrations prior to a GnRH
treatment (100 pg) on d 6 of the estrous cycle and ovulation rate after
GnRH In Angus, Brangus, and Brahman Angus heifers 112
4-2 Mean LH concentrations, LH peak-height, and interval from GnRH to LH
peak In Angus, Brangus and Brahman x Angus heifers in response to
GnRH treatment (100 pg) on d 6 of the estrous cycle 117
4-3 Estrous response, follicle size at prostaglandin F2a (PGF2o) and
behavioral estrous characteristics after PGF2ol for Angus, Brangus, and
Brahman Angus heifers in response to a GnRH + PGF2a <
synchronization protocol 118
4-4 Total corpus luteum (CL) volume, volume of the original and accessory
CL and plasma progesterone concentrations at prostaglandin F2a
(PGF2a) injection for Angus, Brangus, and Brahman x Angus heifers in
response to a GnRH + PGF2a synchronization protocol 119
5-1 Breed and progesterone group effects on follicle size at GnRH,
ovulation rate after GnRH, and progesterone concentration at GnRH in
Angus and Brahman x Angus heifers in the High and Low progesterone
groups 145
5-2 Breed and progesterone group effects on heifers with two corpora ltea
(CL) at PGF2a, CL regression rate after PGF2a, follicle diameter at PGF2a,
progesterone concentration at PGF2a, and estrous response after PGF2a
In Angus and Brahman x Angus heifers in the High and Low
progesterone groups
147


107
Estrus
(d 0) d 6
Presynchronization
100 pg
GnRH
-60 -30-1 15 30 60 90 120 150 180 240 300 360 420 480
{ {{}}} IHHH H
0 0 0
{* Blood sample collection for LH and progesterone determination.
() Blood sample collection for estradiol determination
Figure 4-2. Intensive blood sampling protocol (minutes before and after GnRH
administration) to determine luteinizing hormone (LH; stars),
progesterone (stars), and estradiol (diamonds) concentrations in
cycling Angus, Brahman, Brangus and Brahman Angus heifers
receiving GnRH on d 6 of the estrous cycle.


2
estrous synchronization protocol. The GnRH is used to synchronize follicle
development so that a majority of cattle will have dominant follicle present on the
ovaries when PGF2a is administered 7 d after the GnRH treatment. In most
cases, the GnRH + PGF2ct protocols are effective for synchronizing estrus in
cattle of Bos taurus breeding, but appear to be less effective in cattle of Bos
indicus breeding. Moreover, direct comparisons of the endocrine and ovarian
responses of cattle to the GnRH plus PGF2a protocol have not been made
between cattle of Bos taurus and Bos indicus breeding. Lemaster et al. (2001)
reported a very low estrous response in Bos indicus cows treated with GnRH +
PGF2o. and hypothesized that the decrease was due to incomplete regression of
the accessory CL formed as a result of the GnRH treatment. Few reports In the
literature support this hypothesis. However, Hiers (2001) reported that crossbred
Bos indicus x Bos taurus cows treated with GnRH on d 18 of the estrous cycle
with PGF2a administered 7 d later tended to have a decreased luteolysis and 5-d
estrous response, compared to cows injected with GnRH on d 2, 5 and 12 of the
estrous cycle with PGF2c, administered 7 d later.
There is enough literature referring the use of PGF2a and its analogues for
estrus control in both Bos taurus cattle (Watts and Fuquay, 1985; Maurer et al.,
1989; Morbeck et al., 1991; Smith et al., 1998), and cattle of Bos indicus breeds
(Landaeta et al., 1999; Rekwot et al., 1999; Williams et al., 1999; Mattoni and
Ouedraogo, 2000). The use of these synchronizing agents in cattle of Bos
indicus breeding appears to be primarily related to problems with estrus detection
(Orihuela et al., 1983; Pinheiro et al., 1998; Rekwot et al., 1999). However, the


Proportion of cows not
observed in estrus
244
Interval from PGF2a injection to onset of estrus, h
Figure K-2. Survival analysis describing the proportion of Cycling (triangles) and
noncycling (squares) Bos taurus x Bos indicus cows that did not
exhibit estrus after being synchronized with GnRH and either a single
or split doses of prostaglandin F2a (PGF2a) in combination with
melengestrol acetate (MGA) treatment. Cycling status (P > 0.10).


200
Approximately 15% of the cows in the present experiment did not respond
to the luteolytic action of PGF2c, resulting in a decreased estrous response and
fewer cows being inseminated. Moreover, because the negative effect of
progesterone was not removed, dominant follicles present on the ovaries would
not ovulate in response to the GnRH (Sartori et al., 2001) administered at timed-
Al and thereby reducing timed-AI pregnancy rates.
Progesterone concentrations at PGF2a significantly influenced the total CL
regression in a quadratic manner. Reasons why total CL regression rate
decreased as progesterone concentrations at PGF2ct increased from 1.0 ng/mL to
proximately 6.0 ng/mL and then total CL regression rate increased as
progesterone concentrations increased over 7.0 ng/mL are unclear. These
observations disagree with other reports (Santos et al., 1988; Pinheiro et al.,
1998; Rekwot et al., 1999), since some of the animals with lower progesterone
concentrations (< 4 ng/ml) at PGF2o were probably cows that were early in the
estrous cycle, resulting in a CL that is less responsive to PGF2a as reported.
Alternatively, some of these cows could also have been noncycling cows that
had a CL from the GnRH induced ovulation developed under a low progesterone
environment. Results obtained in previous experiments (See chapter 5) and
those reported by Howard and Britt (1990) and Moreira et al. (2000a), indicate
that progesterone exposure prior to PGF2a administration influences a PGF2o
induced luteolysis 7 d after a GnRH treatment. Therefore, it is expected that this
group of cows would have a reduced total CL regression. However, induced CL
regression in noncycling cows that had a CL from the GnRH induced ovulation


158
oxytocin receptors to become functionally linked to PGF2a secretion (Lamming
and Mann, 1995; Kieborz-Loos et al., 2003) and is necessary to stimulate uterine
lipids for the synthesis of PGF2a (Staples et al., 1998) in cows, allowing the
normal cascade of hormonal and receptor changes required to induce luteolysis
(Meyer et al., 1988; Mirando et al., 1993). Since HP heifers had greater
progesterone concentrations between GnRH and PGF2a treatments than LP
heifers, uterine tissue from HP heifers may have had a greater release of PGF2c,
in response to the stimulation of the oxytocin released by the CL following PGF2a
induced luteolysis compared with LP heifers. The fact that progesterone inhibits
PGF2a secretion in the early- to mid-cycle CL, but not in the late CL (Pate, 1988)
along with the evidence that progesterone priming of the uterus is necessary to
stimulate uterine synthesis of PGF2cl (Lamming and Mann, 1995; Staples et al.,
1998; Kieborz-Loos et al., 2003) and the normal changes required to induce
luteolysis (Meyer et al., 1988; Mirando et al., 1993) may partially explain the
significant increase in CL regression of the HP compared to LP heifers in the
present study. The inhibitory effects of progesterone on PGF2a secretion in the
early- to mid-cycle CL, but not in the late CL may also explain the observation
that progesterone concentrations at PGF2a had a negative linear effect on CL
regression in the LP heifers, but not in the HP heifers.
Naturally low circulating progesterone concentrations prior to a PGF2a
induced estrus similar to that in the LP heifers in the present experiment have
been reported to result in low fertility in Bos indicus heifers (Singh et al., 1998)
and in beef cows (Munro, 1989) and dairy cows (Moreira et al., 2000b). One


148
breed x progesterone group effect (P > 0.10) on CL regression. Corpus luteum
regression was similar (P > 0.10) between Angus and Brahman x Angus heifers
(Table 5-2). However, more (P < 0.05) HP heifers regressed their CL after
PGF2a compared to LP heifers (Table 5-2). Progesterone concentrations at
PGF2a were greater (P < 0.0001) for HP than LP heifers (Table 5-2). There were
neither breed nor breed x progesterone group effects (P >0.10) on progesterone
concentrations at PGF2a administration. Within HP heifers, progesterone
concentrations at PGF2a did not influence (P > 0.10) CL regression (data not
shown). However, within LP heifers, there was a negative linear effect (P < 0.05)
of progesterone concentrations at PGF2a on CL regression (Figure 5-4). For
every 1.0 ng/mL increase in progesterone concentrations, there was a 14%
decrease in QL regression for LP heifers.
There were no breed, progesterone group, or breed x progesterone group
effects (P > 0.10) on the size of the largest follicle at PGF2a (Table 5-2). Estrous
response following PGF2a was greater (P < 0.001) in Brahman x Angus (Odds
ratio, OR = 13.87; 95% confidence interval, 95% Cl = 1.72-111.65; P < 0.05)
than Angus heifers (Table 5-2). However, estrous response after PGF2a was not
influenced (P < 0.10) by progesterone group or breed x progesterone group.
Discussion
Results from the present experiment indicate that the experimental
approach used to induce low progesterone concentrations in Angus and
Brahman x Angus heifers early in the estrous cycle was effective. Similar
methods have successfully been used to induce CL regression by delivering


52
administration of GnRH was effective in initiating follicle turnover in cows with
dominant follicles at CIDR insertion, resulting in emergence of a new follicular
wave. However, GnRH did not initiate follicle turnover when administered before
dominant follicle selection. These data indicate that GnRH is effective in induced
turnover of dominant follicles in progesterone based treatments, preventing the
potential development of persistent follicles that fail to ovulate due to the
suppressive action of progesterone on LH secretion.
Prostaglandin F2o (PGF2a)
The luteolytic actions of PGF2ol and its analogues (Cloprostenol, Alfaprostol,
and Luprostiol) after d 4 of the estrous cycle and throughout the remainder of
luteal phase of the estrous cycle have been well documented (Rowson et al.,
1972; Kiracofe et al., 1985; Godfrey et al., 1989). Several researchers have
reported that after d 5 of the estrous cycle, the CL was responsive to a PGF2
treatment, but the degree of luteolysis was dependent on the stage of the estrous
cycle when PGF2a was administered (Tanabe and Hahn, 1984; Watts and
Fuquay, 1985). Consequently, the ineffectiveness of a single dose of PGF2a to
initiate luteolysis before d 5 of the estrous cycle limits the overall effectiveness of
using PGF2o(forthe synchronization of estrus in cattle random stages of the
estrous cycle (Beal et al., 1980). Administration of PGF2o to cycling cattle of Bos
taurus breeding yields consistent results relative to induction of luteolysis,
synchronization of estrus, and fertility of the synchronized estrus. However,
similar results in cattle of Bos indicus breeding treated with PGF2a have not been
observed. Initially, the inconsistent results in cattle of Bos indicus breeding


272
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Rajamahendran, R., and M. Mannlkkam. 1994. Effects of exogenous steroid
hormones on the dominant follicle maintained by a norgestomet implant In
heifers. Can. J. Anlm. Sci. 74: 457-464.
Ramlrez-Godinez, J.A., G.H. Klracofe, R.M. McKee, R.R. Shalles, and R.J.
Kittok. 1981. Reducing the incidence of short cycles In beef cows with
norgestomet. Theriogenology 15: 613-623.
Randel, R.D. 1976. LH and ovulation in Brahman and Brahman x Hereford and
Hereford heifers. J. Anim. Sci. 43: 300 (Abstr.).


21
Regulation of Ovarian Follicle Growth, Atretic Demise and the Ovulatory
Response
Follicle development is initiated during the fetal life. The process of follicle
development can be summarized based on several reviews (Fortune, 1994;
Ginther et al., 1996b; Ireland et al., 2000). By mid-gestation, the bovine ovary
contains its entire lifetime supply of oogonia. Subsequent to termination of
mitotic proliferation during gestation, the oogonia enter meiosis. At birth, the
oogonia are arrested in the first meiotic division and are called oocytes.
Primordial follicles contain oocytes surrounded by a single granulosa cell layer
consisting of 14 to 29 flattened granulosa cells (Erickson, 1966; Van Wezel and
Rodgers, 1996; Van den Hurk et al., 1997). As the follicle grows and develops, it
acquires a cuboidai layer of 20 to 50 granulosa cells and it becomes a primary
follicle. The'pool of primordial and primary follicles is considered part of the
ovarian reserve of oocytes. The initial stages of folliculogenesis occur
independently of gonadotrophic hormones (Roche, 1996). Duringthe
reproductive lifespan of the animal, individual follicles are recruited as actively
growing follicles (Marion and Gier, 1971). Formation of the zona pellucida occurs
when primary follicles develop into secondary follicles and granulosa cells
multiply and form several layers. In addition, theca cells are formed and the
follicle becomes gonadotropin dependent as secondary follicles develop
(Scaramuzzi et al., 1993; Mihm et al., 2002). The tertiary follicle is formed with a
separation of the granulosa cell layers forming a cavity or antrum. Subsequent
enlargement and accumulation of follicular fluid in the antrum takes place, and
the follicle continues to grow and it is designated as a mature Graafian follicle.


71
was effective in synchronizing ovulation in cycling Nelore cows and improved
pregnancy rates after TAI. It was concluded that the estradiol benzoate
treatment after PGF2a injection is a promising alternative to the use of GnRH In
GnRH-PGF-TAI protocols for Nelore cattle due to the low cost of estradiol
benzoate compared to GnRH agonist.
Hlers et al. (2003) evaluated the effectiveness of three different PGF2a
treatments In a GnRH + PGF2a protocol combined with MGA to synchronize Bos
indicus x Bos taurus cows for a TAI. All cows received GnRH at the start of the
experiment (d 0) and were administered MGA on d 1 to 7. On d 7, cows received
either a single Injection of 25 mg PGF2a, a single Injection of 500 pg cloprostenol,
or half (12.5 mg) the recommended dose of PGF2q on d 7 and 8. On d 10, all
cows were tlmed-AI concomitant with GnRH. Administering MGA along with an
Injection of GnRH has been shown to reduce the ability of GnRH to induce
ovulation In cows with dominant follicles (Pancard et al., 1999); therefore, in this
study cows were not administered MGA on the day of GnRH Injection.
Additionally, as some cows do not have a functional CL at PGF2c, Injection In the
GnRH + PGF2a systems (Twagiramungu etal., 1995; Schmitt et al., 1996a;
Downing et al., 1998; Morelra et al., 2000), MGA was fed until the day of PGF2a
to prevent early expression of estrus and probably tighten the synchrony of
estrus, follicle development and ovulation after MGA withdrawal. Timed-AI and
30-d pregnancy rates were similar between the single (36 and 77%), split (39 and
74%), and cloprostenol (41 and 75%) treatments. These data suggested that


7
Hypothalamic-Pituitary-Ovarian Axis and Primary Hormones of
Reproduction
In the presence of a hypothalamic releasing factor, the anterior pituitary and
ovarian hormones exert mutual control over the circulating concentrations of one
another. The complex interactions among pituitary, ovarian, and uterine
hormones Involve further control by positive and negative feedback mechanisms
to sustain the estrous cycle of the cow. The primary hormones secreted by these
reproductive structures are gonadotropin releasing hormone (GnRH), the
gonadotropins follicle stimulating hormone (FSH) and luteinizing hormone (LH),
estradiol 17(3, progesterone, and prostaglandin F2a (PGF2cl).
Gonadotropin Releasing Hormone (GnRH)
The neuropeptide GnRFI Is released from the hypothalamus to the anterior
pituitary, inducing de novo synthesis and release of LH and FSH, which control
ovarian function (DOcchio et al., 2000).
Smith and Jennes (2001) summarized the anatomical organization of the
GnRH neuronal system. Two loose networks of neurons of the hypothalamus
control the secretion of GnRH. One network of neurons Is located in the
ventromedial and arcuate nuclei of the hypothalamus. These neurons Include
the tonic GnRH center, responsible for the basal secretion (small, frequent
pulses) of GnRH throughout the estrous cycle. The other groups of neurons are
located in the anterior hypothalamic area, which Includes the suprachiasmatic
and preoptic nuclei. These nuclei comprise the surge center, responsible for the
preovulatory release of GnRH that stimulates the surge of LH, which Initiates the
process of ovulation. Axonal projections of GnRH neurons are extended toward


APPENDIX.A
ABSTRACT FOR EXPERIMENT 1
Cycling Angus (AN, n = 13; Mean BW = 379 kg) and Brahman x Angus
(BA) heifers (5/8 Angus x 3/8 Brahman and 3/8 Angus x 5/8 Brahman; n = 16;
mean BW = 451 kg) were used to evaluate the effectiveness of a single injection
of PGF2a administered during the early estrous cycle to Initiate corpus luteum
(CL) regression as measured by progesterone concentrations. Heifers were pre
synchronized with a modified two-Injectlon PGF2a (Lutalyse Sterile Solution)
protocol (25 mg I.m. on d -14 and 12.5 mg I.m. on d -3 and -2 of the experiment).
Estrus was designated as d 0 of experiment. On d 6 or 7 of the subsequent
estrous cycle, heifers were injected with 25 mg PGF2a i.m. Estrus was monitored
using HeatWatch for the pretreatment synchronization and treatment phase of
the experiment. At PGF2a both ovaries were examined via ultrasonography to
determine follicle and CL characteristics. Blood samples were collected from
PGF2a until heifers either exhibit estrus or for 7 d after PGF2a for heifers that did
not exhibit estrus. The experiment was replicated twice using the same pre-
synchronization and treatment protocols and heifers. There were no replication
or replication x breed effects (P > 0.10) so data were pooled. CL regression was
greater for AN (25/26 = 96.2%) than BA (25/31 = 80.6%) heifers. Whether the
decrease in luteolysls of the BA heifers was significant depended on the method
of statistical analysis of SAS (SAS Inst. INC., Cary, NC) used (GENMOD, P <
219


264
Llewelyn, C.A., C.D. Munro, A.G. Luckins, T. Jordt, M. Murray, and E. Lorenzini.
1987, Behavioral and ovarian changes during the estrous cycle in the Boran
(Bos indicus). Br. Vet. J. 143: 75-82.
Looper, M.L., R.P. Wettemann, T. Pardo, and G.L. Morgan. 1998. Estrus
behavior and time of ovulation of beef cows in summer and winter. J. Anim.
Sci. 76 (Suppl. 1): 215 (Abstr.).
Lopez, H., T.D. Bunch, and M.P. Shipka. 2002. Estrogen concentrations in milk
at estrus and ovulation in dairy cows. Anim. Reprod. Sci. 72: 37-46.
Lorton, L.L., T.D. Lester, C.F. Roseukrans, and R.W. Rorie. 1999. Effect of
estrous parameters and time of insemination on pregnancy rate in beef
cattle. J. Anim. Sci. 77(Suppl. 1): 15 (Abstr.).
Lucy, M.C., H.J. Billings, W.R. Butler, L.R. Ehnis, M.J. Fields, D.J. Kesler, J.E.
Kinder, R.C. Mattos, R.E. Short, W.W. Thatcher, R.P. Wettemann, J.V.
Yelich, H.D. Hafs. 2001. Efficacy of an intravaginal progesterone insert and
an injection of PGF2a for synchronizing estrus and shortening the interval to
pregnancy in postpartum beef cows, peripubertal beef heifers, and dairy
heifers. J. Anim. Sci. 79: 982-995.
Lucy, M.C., J.D. Savio, L. Badinga, R.L. De La Sota, and W.W. Thatcher. 1992.
Factors that affect ovarian follicular dynamics in cattle. J. Anim. Sci. 70:
3615-3626.
Lumpkin, M.D., and S.M. McCann. 1984. Effect of destruction of the dorsal
anterior hypothalamus on follicle-stimulating hormone secretion in the rat.
Endocrinology 115: 2473-2480.
Lumpkin, M.D., J.K. McDonald, W.K. Samson, and S.M. McCann. 1989.
Destruction of the dorsal anterior hypothalamic region suppresses pulsatile
release of follicle stimulating hormone but not luteinizing hormone.
Neuroendocrinology 50: 229-235.
Lumpkin, M.D., J.H. Moltz, W.H. Yu, W.K. Samson, and S.M. McCann. 1987.
Purification of FSFI-releasing factor: its dissimilarity from LFHRH of
mammalian, avian and piscian origin. Brain Res. Bulletin 18: 175-178.
Lussier, J.G., P. Matton, and J.J. Dufour. 1987. Growth rates of follicles in the
ovary of the cow. J. Reprod. Frtil. 81: 301-307.
Lyimo, Z.C., M. Nielen, W. Ouweltjes, T.A.M. Kruip, and F.J.C.M. van
Eerdenburg. 2000. Relationship among estradiol, cortisol and intensity of
estrous behavior in dairy cattle. Theriogenology 53: 1783-1795.


38
Some researchers have suggested that the lack of endothelin-1 synthesis
and might make the early luteal phase CL refractory to the luteolytic action of
PGF2a (Mamluk et al., 1998; Levy et al., 2000). Therefore, blood vessel
endothelium of the early luteal phase CL would be non-responslve to the action
of PGF2a.
There is a positive correlation between the amount of vascularization and
mean diameter of the CL (Baumgartner et al., 1998). Wlltbank et al. (1995)
suggested that incomplete vascularization in early luteal phase CL may be
responsible In part for the lack of luteolytic capacity of the early luteal phase
bovine CL. In general, the ovaries and CL in cattle of Bos indicus breeding are
smaller than cattle of Bos taurus breeding (Moreno et al., 1986; Rentfrow et al.,
1987; Soto et al., 1999). Therefore, these findings suggest that the reduced
response of the early luteal phase CL to PGF2a In cattle of Bos indicus breeding
compared to Bos taurus cattle may be associated with luteal size and lack of
vascularization. Brito et al. (2002) reported that the efficacy of PGF2a for Initiating
luteolysls and ovulation in buffalo cows (Bubalus bubalis) at different stages of
the estrous cycle was dependent upon CL size at PGF2q treatment. Only buffalo
cows with CL > 189 mm2 responded to PGF2a treatment. Whether differences In
luteal vascularization occur between cattle of Bos indicus and Bos taurus exist Is
unclear, but the hypothesis is an interesting one and requires further
investigation.
Exogenous Control of Ovarian Follicles and Corpus Luteum Dynamics
To accurately control the estrous cycle It is essential to control the life
span of the CL and the follicle development in the bovine. Most estrus


175
Three-day estrous, timed-AI pregnancy, synchronized and thirty-day
pregnancy rates were similar (P > 0.10; Table 6-2) between single and split
PGF2a treatments. Furthermore, conception rate was greater (P < 0.05) for the
single PGF2o (OR = 0.30; 95% Cl = 0.62 2.47; P < 0.05) than split PGF2o:
treatment (Table 6-2). There were no treatment effects (P > 0.10) on three-day
estrous, conception, timed-AI pregnancy, synchronized and thirty-day pregnancy
rates for cows with progesterone concentrations < 1 ng/mL (Table 6-3).
Of cows determined to be noncycling on d 0 of experiment, GnRH induced
ovulation or luteinization of follicles In 40.2% (41/102) as determined by
progesterone concentrations > 1 ng/mL on d 7 of the experiment. Progesterone
profiles on d -10, 0 and 7 of the experiment affected (P < 0.05) three-day estrous,
tlmed-AI and synchronized pregnancy rates, but did not affect (P > 0.10)
conception rates (Table 6-4). Cows classified as cycling and having low
progesterone at PGF2a had an increased (P < 0.05) estrous rate compared with
cows classified as either cycling and with high progesterone at PGF2a or
noncycling with high and low progesterone at PGF2a; and estrous rate was
similar (P >0.10) for the latter three treatments.
Timed-AI pregnancy rates were greater (P < 0.05) for cycling cows with
high progesterone at PGF2a compared with cycling and noncycling cows with low
progesterone at PGF2ci, which had similar (P > 0.10) timed-AI pregnancy rates.
Regardless of cycling status, cows with high progesterone at PGF2a had similar
(P > 0.10) timed-AI pregnancy rates. Among cows with high progesterone at
PGF2a, synchronized pregnancy rates were similar (P > 0.10) between cycling


30
large and small luteal cells, In the bovine CL. The CL Is comprised of
approximately 40% large luteal and 28% small luteal cells (OShea et al., 1989).
Large luteal cells are derived from granulosa cells of the preovulatory follicle and
small luteal cells are derived from thecal cells (Meldan et al., 1990). The
distinctive characteristics of the two luteal cell types have been well defined in
ruminants. Not only are the diameter and morphological characteristics different
between these two cells, but differences in receptors for LH (generally greater in
small cells), estradiol (much greater in large cells), and PGF2a (much greater in
large cells) have also been reported (Wiltbank, 1994; Diaz et al., 2002).
Treatment with LH Increased progesterone production by small, but not large
luteal cells, whereas, treatment with PGF2a Inhibited progesterone production by
large, but not small luteal cells (Wiltbank et al., 1993).
The ovaries In cattle of Bos indicus breeding are smaller than cattle of Bos
taurus breeding (Moreno et al., 1986; Rentfrow et al., 1987; Soto et al., 1999).
Similarly, luteal tissue area tends to be decreased in cattle of Bos indicus
breeding compared with Bos taurus cattle (Castilho et al., 2000; Morelra-Vlana et
al., 2000). The maximum CL diameter for Bos taurus heifers has been reported
to range from 25 to 30 mm (Adams et al., 1993). In contrast, maximum CL
diameter for Brahman and Nelore (Bos indicus) heifers was 17 and 18 mm,
respectively (Rhodes et al., 1995; Flgueiredo et al., 1997). Likewise, maximum
CL diameter for Gyrolando (Bos indicus x Bos taurus) dairy heifers was 19 to 20
mm (Castilho et al., 2000), similar to maximum CL diameter of 18 mm and 19
mm observed in Bos taurus x Bos indicus dual purpose cows and heifers,


233
Interval from GnRH, min
Figure H-3. Plasma LH concentrations (LS Means) in Brahman x Angus heifers
receiving 100 gg GnRH on d 6 of the estrous cycle.


260
Hurnick, J.F., G.J. King, and H.A. Robertson. 1975. Estrous and related behavior
in postpartum Holstein cows. Appl. Anlm. Ethol. 2: 55-68.
Imwalle, D.B., D.L. Fernandez, and K.K. Schillo. 2002. Melengestrol acetate
blocks the preovulatory surge of luteinizing hormone, the expression of
behavioral estrus, and ovulation In beef heifers. J. Anim. Sci. 80: 1280-
1284.
Inskeep, E.K., T.D. Braden, P.E. Lewis, M. Garcia-Winder, and G.D. Niswender.
1988. Receptors for luteinizing hormone and follicle-stimulating hormone in
largest follicles of postpartum beef cows. Biol. Reprod. 38: 587-591.
Ireland, J.J., R.L. Fogwell, W.D. Oxender, K. Ames, and J.L. Cowley. 1984.
Production of estradiol by each ovary during the estrous cycle of cows. J.
Anim. Sci. 59: 764-771.
Ireland, J.J., M. Mlhm, E. Austin, M.G. Diskin, and J.F. Roche. 2000. Historical
perspective of turnover of dominant follicles during the bovine estrous cycle:
key concepts, studies, advancements, and terms. J. Dairy Sci. 83: 1648-
1658.
Irvin, H.J., R.D. Randel, W.E. Haensley, and A.M. Sorensen. 1978. Reproductive
studies of Brahman cattle III. Comparison of weight, progesterone content,
histological characteristics, and 3-hydroxysteroid dehydrogenase activity in
corpora ltea of Brahman, Hereford and Brahman x Hereford heifers.
Theriogenology 10: 417-427.
Johnson, S.K., M.L. Day, J.M. Lynch, J.E. Kinder, R. Rasby, R.E. Short, R.P.
Wettemann, and H.D. Hafs. 1997. Onset of estrus and luteal function in
peripubertal heifers given an intravaginal progesterone releasing insert with
or without a subsequent injection of estradiol benzoate. J. Anim. Sci. 75
(Suppl. 1): 231 (Abstr.).
Johnson, S.K., K.R. Harmoney, and J.S. Stevenson. 2002. Synchronization of
ovulation in suckled beef cows with GnRH-CIDR-PGF and timed
insemination at 48 or 60 h after PGF2a- J. Anim. Sci. 80 (Suppl. 2): 86
(Abstr.).
Jones, L.S., J.S. Ottobre, and J.L. Pate. 1992. Progesterone regulation of
luteinizing hormone receptors on cultured bovine luteal cells. Mol. Cell.
Endocrinol. 85: 33-39.
Kastelic, J.P., D.H. McCartney, W.O. Olson, A.D. Barth, A. Garcia, and R.J.
Mapletoft. 1996. Estrus synchronization in cattle using estradiol,
melengestrol acetate and PGF. Theriogenology 46: 1295-304.


41
device (C1DR) to artificially lengthen estrous cycles and to characterize follicular
development in dairy heifers. The CIDRs were inserted on d 14 to 28 after an
observed estrus. Control heifers (group 1) received a blank CIDR containing no
progesterone, while heifers in groups 2 and 3 received either one or two CIDRs,
respectively. Ovulatory follicles in group 2 had a longer persistence, reached a
larger maximal size, and were associated with a complete absence of follicular
recruitment compared to heifers in groups 1 and 3. The authors concluded that
the increased progesterone concentrations in heifers receiving two CIDRs (group
3) had a negative effect on LH secretion, which resulted in regression of
dominant non-ovulatory follicles. They concluded that increased concentrations
of progesterone regulate dominant follicle turnover through negative feedback by
decreasing LH pulse frequency. This observation was supported by other
studies (Savio et al., 1993a; Stock and Fortune, 1993). Furthermore, exogenous
progesterone administered during early metestrus suppressed the diameter of
the first-wave dominant follicle (Adams et al., 1992b; Burke et al., 1994; Diskin et
al., 2002) by suppressing LH pulse frequency (Burke et al., 1994; Diskin et al.,
2002).
In contrast, when progestagens are administered in the absence of a
functional CL, the wave-like pattern of follicular development does not occur
(Sirois and Fortune, 1990). As a result, there is prolonged development of the
dominant follicle and increased concentrations of estradiol than usually observed
with growing dominant follicles (Sirois and Fortune, 1990; Cupp et al., 1995;
Kojima et al., 1992). The abnormally large follicle is termed a persistent follicle


199
increased incidences of incomplete luteolysis after a single injection of PGF2o in
heifers of Bos indicus breeding. However, the same authors observed that by
giving two injections of PGF2a 24 h apart, more heifers expressed estrus,
indicative of increased luteolysis. Other experiments have reported that
luteolysis was decreased in cattle of Bos indicus breeding resulting in a
decreased estrous response following a single injection of PGF2a (Cornwell et al.,
1985; Patterson et al., 1989; Pinheiro et al., 1998). Lemaster et al. (2001)
hypothesized that decreased pregnancy rates of Bos indicus x Bos taurus cows
compared to Bos taurus cows may have been due to an incomplete PGF2a-
induced luteolysis of CL developed after the first GnRH. However, results from
the present study do not support this hypothesis.
Total CL regression rate in cows with progesterone concentration > 1.0
ng/mL at PGF2ol in the present study (87%) were similar to reports in Bos taurus
heifers (85 to 100%) treated with PGF2ci between d 5 and 8 of the estrous cycle
(King et al., 1982; Tanabe and Hann, 1984; Kiracofe et al., 1985). In contrast,
Williams et al. (1999) reported CL regression rates of 54% and 63% in Brahman
x Hereford heifers treated with PGF2a on d 6 or 10, respectively. In addition, a
recent report by Bridges et al. (2003) indicated that yearling Bos taurus x Bos
indicus heifers between d 12 to 15 of the estrous cycle had a CL regression rate
to a single injection of PGF2o of 82% compared to 97% in two-year-old Bos
indicus x Bos taurus heifers and 96% in yearling Bos taurus heifers. Therefore, it
is unclear if animal age has an effect on the luteolytic actions of PGF2a but it does
warrant further investigation.


209
receiving different GnRH analogues (Chenault et al., 1990). The relative
differences observed in mean LH concentrations in the Angus (greatest) versus
Brangus (intermediate) and Brahman Angus (smallest) heifers and mean LH
peak-height in Angus versus Brahman x Angus heifers observed in the present
experiment are in agreement with other studies; as the percentage of Bos indicus
breeding increased, the LH response to GnRH (Griffin and Randel, 1978) or an
exogenous estrogen treatment (Rhodes et al., 1978) also increased.
Additionally, mean Intervals from GnRH to LH peak for all breed groups in the
current experiment were greater compared with Holstein heifers following an
injection of 100 pg of Gonadorelin (Chenault et al., 1990). It is unclear whether
the decreased LH response to a GnRH challenge in cattle of Bos indicus
breeding compared to Bos taurus females Is due to smaller LH stores, less LH
being synthesized by the pituitary, decreased sensitivity of the hypothalamus and
pituitary, and (or) a combination of these. Although there are no apparent reports
In the literature that have determined pituitary content of LH in Bos indicus
females, the observation that young Bos indicus bulls have decreased pituitary
content of LH (Aspden et al., 1997) compared with Holstein bulls (Macmillan and
Hafs, 1968) may Indicate that cattle of Bos indicus breeding may have less
available LH to be released from the pituitary than cattle of Bos taurus breeding,
which could be part of the reason for the differential release of LH in response to
GnRH between breeds In the present study. Furthermore, the differential LH
release between breeds in the current experiment could also be explained by
differences in sensitivities of the hypothalamus and pituitary between Angus,


16
mid-cycle CL progesterone inhibits PGF2a secretion. Another study (Pate, 1988)
confirmed the inhibiting effect of progesterone on PGF20 secretion by bovine
luteal cells in the mid-cycle CL, but not in the late CL.
Progesterone has a luteotropic action by stimulating the synthesis of LH
receptors in bovine luteal cells (Jones et al., 1992). In addition, progesterone
stimulates its own basal secretion by activating 3p-hydroxysteroid
dehydrogenase (Pate, 1996). It is possible that progesterone prevents luteal
regression by inhibiting apoptosis (Rae et al., 1998b; Friedman et al., 2000),
which may occur through a PR-dependent mechanism. Furthermore, Fiedman et
al. (2000) reported that cells producing progesterone are protected from
apoptosis while the same authors reported that a prerequisite for the initiation of
apoptosis in CL endothelial cells is a decline in progesterone, preceding
structural luteolysis. During early to mid-diestrus in cows, exposure to or
inhibition of progesterone by a progesterone antagonist controlled the initiation of
release of PGF2a from uterine endometrium (Garrett et al., 1988; Schams and
Berisha, 2002). Exposure of progesterone during these stages causes a
reduction of the interestrous interval, while inhibition of progesterone causes an
increase of the estrous interval (Garrett et al., 1988; Schams and Berisha, 2002).
Therefore, progesterone regulates the life span of the CL. Desensitization of
endometrial tissue to progesterone correlates with the down regulation of
estrogen, oxytocin and progestin binding in luteal tissue (Meyer et al., 1988).
The authors showed that receptors were minimal on d 12 and increased again
toward d 21 of the estrous cycle. These results indicate sensitivity of the


101
onset of estrus. Additionally, the number of heifers in estrus at one time in the
present study affected the number of mounts that heifers received. As more
heifers were in estrus at a particular time, the number of mounts received during
estrus also increased. It is clearthat social factors (Hurnick et al., 1975;
Macmillan, 1992; Landaeta-Hernandez et al., 2002) influence the characteristics
associated with a behavioral estrus. Furthermore, synchronization of estrus
increases the number of animals in estrus at a particular time. Consequently, the
coincidence of several females in estrus at a particular time due to
synchronization of estrus, leads to the formation of sexually active groups
(Macmillan, 1992; Landaeta-Hernandez et al., 2002), which promote increased
mounting activity in Bos taurus and Bos indicus cattle (Hurnick et al., 1975;
Macmillan, 1992; Galina et al., 1994).
In summary, CL regression was numerically greater for Angus than
Brahman x Angus heifers. Progesterone concentrations in heifers that regressed
their CL were < 1.0 ng/mL by 24 h after PGF2a and remained below these
concentrations. In contrast, progesterone concentrations never decreased <1.0
ng/mL in heifers that did not regress their CL. Additionally, as CL volume at
PGF2ol increased, progesterone concentrations increased. Estrous response,
interval from PGF2a to the onset of estrus, and duration of estrus were similar
between Angus and Brahman x Angus heifers. However, Brahman x Angus
heifers had more mounts during estrus than Angus heifers. Additionally, as the
number of heifers in estrus at one time increased, the number of mounts
received also increased.


144
Progesterone concentrations at GnRH were greater (P < 0.0001) for HP than LP
heifers (Table 5-1). There were no breed and breed progesterone group
effects on progesterone concentrations at GnRH treatment. There were no
breed, breed x progesterone group, breed x day, and breed x progesterone
group x day effects (P > 0.10) on progesterone concentrations from GnRH
administration (experimental d 6) until PGF2ct treatment (experimental d 13).
However, progesterone group, day, and progesterone group x day affected (P <
0.0001) progesterone concentrations from GnRH until PGF2ct administration.
Heifers in the HP group had a linear increase in progesterone concentrations
from GnRH treatment until PGF2a 7 d later (Figure 5-3). In contrast, LP heifers
had progesterone concentrations that remained below 1 ng/mL until
approximately 4 d after GnRH. After d 4, progesterone concentrations for the LP
group increased above 1.0 ng/mL and the ascending progesterone curve
remained parallel but of decreased concentrations than the HP group until PGF2o
i
treatment.
Diameter of the largest follicle at GnRH was similar (P > 0.10) between
breeds and progesterone groups (Table 5-1) and there were no breed *
progesterone group effects (P > 0.10). There were no effects (P > 0.10) of
breed, progesterone group, and breed progesterone group (Table 5-1) on
ovulation rate after GnRH.
All heifers had a functional CL at PGF2ct treatment (experimental d 13; data not
shown). However, 85% of HP heifers had an additional CL at PGF2cl and LP
heifers only had a new induced CL at PGF2Ci (Table 5-2). There was no


213
formation of second messengers and (or) other cellular mechanisms inducing
luteolysis. These experiments may provide some insight on what role
progesterone may play in the inhibition of luteal regression in the early
developing CL.
From the standpoint of implementing estrus synchronization systems, if
luteolysis of the early CL fails to occur more than 90% of the time, it could
decrease the overall effectiveness of GnRH with PGF20 estrus synchronization
protocols. This could be particularly true when a GnRH + PGF2a system is used
in herds with an increased proportion of anestrous cattle. Anestrous cattle would
not have had progesterone exposure prior to a GnRH induced ovulation, which
could result in a young CL that is not completely responsive to PGF2ci
administered 7 d after the GnRH induced ovulation. If luteolysis does not occur
in a high percentage of cattle, subsequent pregnancy rates could be significantly
compromised. The anestrous cow hypothesis would be further evaluated in
Experiment 4.
The objectives of Experiment 4 were to evaluate the efficacy of either a
single or two consecutive split injections of PGF2a administered 7 d after GnRH
treatment to synchronize estrus in cycling and noncycling postpartum lactating
cows of Bos indicus breeding. There were no effects of PGF2n treatments on the
PGF2a induced luteolysis, estrous response, conception, and pregnancy rates.
Therefore, splitting the dose of PGF2 to two consecutive injections did not
provide any additional benefit of enhancing CL regression and increasing
subsequent pregnancy rates compared to the single injection similar to a recent


104
comparisons between cattle of Bos taurus and Bos indicus breeding
(Vasconcelos et al., 2000) have been made comparing the endocrine and
ovarian responses to an exogenous administration of GnRH at doses typically
used in estrus synchronization protocols. Therefore, It is necessary to develop a
complete understanding of how GnRH affects the endocrine and reproductive
patterns of cattle of Bos indicus breeding.
The objectives of this experiment were to evaluate secretory patterns of LH
and associated ovarian events in response to an administration of GnRH on d 6
of the estrous cycle followed by an Injection of PGF2a 7 d later In Angus,
Brahman, Brangus, and Brahman x Angus heifers.
Materials and Methods
The experiment was conducted during the fall (September through October)
of 2001 at the North Beef Teaching Unit, Department of Animal Sciences,
University of Florida. To reduce the effect of management stress from the
intensive bleeding and transrectal ultrasonography, heifers were trained and
acclimated to stanchions for three weeks prior to the beginning of the
experiment. Heifers were fed grass hay ad libitum and provided approximately
0.45 to 1.36 kg of a high energy/protein supplement daily to maintain body weight
(BW) and body condition score (Richards et al., 1986), which were recorded
weekly throughout the experiment. Heifers were managed as a single group and
had access to water and shade during all phases of the experiment. Cycling
Angus (n = 7), Brangus (n = 10; 5/8 Angus 3/8 Brahman), Brahman x Angus (n
= 9; Va Brahman x % Angus) and Brahman (n = 11) heifers were pre


97
studies (Vaca et al., 1985; Voh Jr. et al., 1987) have reported considerable
variation in estrous responses and intervals to the onset of estrus following
PGF2c treatments in Bos indicus cattle. There are several factors that can affect
the interval from PGF2a treatment to the onset of estrus including stress (Hein
and Allrich, 1992), social interactions (Galina et al., 1996), PGF2a system used
and stage of the estrous cycle at PGF2a treatment (Stevenson et al., 1998), type
and effectiveness of the luteolytic agent (Rowson et al., 1972), and stage of
follicle development at PGF2a (Fogwell et al., 1986; Sirois and Fortune, 1988).
Diameter of the largest follicle at PGF2a was significantly larger for
Brahman x Angus than Angus in the present experiment. Although, follicle size
at PGF2a did not influence either estrous response or the interval from PGF2a
treatment to the onset of estrus. Size of the largest follicle in the Angus and
Brahman x Angus heifers in the present study was similar to those reported on d
6 or 7 of the estrous cycle in Bos taurus (Ginther et al., 1989b; Savio et al.,
1993b) and Bos taurus x Bos indicus (Perea et al., 1998) cattle, respectively.
However, it is unclear if breed affected estradiol concentrations since they were
not determined in the present study. Segerson et al. (1984) reported decreased
concentrations of circulating estradiol between d 7 and 17 of the estrous cycle in
Brahman compared to Angus cows. Additionally, Rhodes and Randel, (1978)
observed that Bos indicus cows required increased concentrations of
exogenously administered estradiol in order to induce estrus compared to Bos
taurus cows. Consequently, if follicles produce decreased concentrations of
estradiol in cattle of Bos indicus breeding, it may take longer for estradiol to attain


CHAPTER 3
RESPONSE TO A PROSTAGLANDIN F2a INJECTION ON EITHER DAY SIX OR
SEVEN OF THE ESTROUS CYCLE IN ANGUS AND BRAHMAN x ANGUS
HEIFERS
Introduction
Most estrus synchronization protocols used today include the use of
prostaglandin F2a (PGF2a) or Its analogues. Prostaglandin F2a Is used to control
the length of the estrous cycle by shortening the life span of the corpus luteum
(CL) through the Induction of luteolysis and subsequent expression of estrus
(Beal et al., 1980; Hardin et al., 1980a; Lauderdale et al., 1981). However, it
appears that an Injection of PGF2ct early In the estrous cycle (d 6 to 8) Is not as
effective In regressing the CL in cattle of Bos indicus breeding (Santos et al.,
1988; Plnheiro et al., 1998; Rekwot et al., 1999) compared to cattle of Bos taurus
breeding (King et al., 1982; Tanabe and Hann, 1984; Kiracofe et al., 1985). This
Is Important since many estrus synchronization systems include administration of
PGF2a to cattle with a CL that Is approximately 5 to 6 d old including the GnRH
plus PGF2a system, which consists of administration of GnRH followed 7 d later
by an injection of PGF2a (Thatcher et al., 1989).
The effectiveness of the GnRH plus PGF2a estrus synchronization
systems are consistent in cattle of Bos taurus breeding (Pursley et al., 1995;
Geary et al., 1998a) but not In cattle of Bos indicus breeding (Lemaster et al.,
2001). Initially, the Inconsistent results in cattle of Bos indicus breeding treated
73


17
endometrium for the inhibitory action of progesterone on PGF2a secretion and
down regulation of receptors for steroids and oxytocin for about 10-11 d. After
this time, desensitization of receptors for progesterone and up-regulation of
receptors for steroids and oxytocin are essential for initiation of luteolysis in
cattle.
Prostaglandins
Prostaglandins play a broad role in mammalian physiology and metabolism,
and they are local regulators usually synthesized near cells on which they have
an effect (Staples et al., 1998). In cattle, uterine tissue is the most important
source of the F series prostaglandins (e.g., PGF20) during the first weeks after
calving (Guilbault et al., 1984; Madej et al., 1984; De Fries et al., 1998).
Concentrations of 13, 14-dihydro-15-keto- PGF2a (PGFM)~PGF2a metabolite-in
plasma increase dramatically, reaching 1800 pg/mL by 3 to 4 d post partum,
which is associated with the regression of CL at the end of the pregnancy and
during postpartum uterine involution (De Fries et al., 1998; Staples et al.'; 1998).
During the early postpartum period, PGFM slowly returns to baseline
concentrations. After the first estrus, the uterus releases PGF2a recurrently to
induce regression of CL to initiate a new estrous cycle if the cow does not
conceive (Madej et al., 1984; Staples etal., 1998). If the cow becomes pregnant,
the uterine release of PGF2a is inhibited and the CL is preserved to maintain
pregnancy (Helmeret al., 1989). Thus, concentrations of plasma progesterone
are related inversely to concentrations of PGF2a when CL regression occurs in
late diestrus. However, progesterone priming of the uterus is necessary to
stimulate uterine lipids for synthesis of PGF2a (Staples et al., 1998).


130
progesterone concentrations after GnRH. The increased progesterone
concentrations were coincident with the movement of the heifers into the
stanchions and the initiation of the intensive bleeding and could have been
associated with a stress related response of moving the heifers into the
stanchions. Increased concentrations of adrenocorticotropic hormone in blood
following stress can increase circulating concentrations of both cortisol and
progesterone (Hein and Allrich, 1992; Bolados et al., 1997). Furthermore, it does
not appear that the acute increase in progesterone concentrations of the two
heifers had a negative effect on the LH released in response to the GnRH
challenge since all heifers in the Brahman x Angus group had similar LH
response curves.
All heifers that ovulated to GnRH in the present study had an accessory CL
at PGF2a and had progesterone concentrations > 1 ng/mL indicative of a
functional CL. Progesterone concentrations at PGF2o were not influenced by
breed but were considerably greater than what is normally observed during the
late luteal phase (Kesler et al., 1997) of the estrous cycle. Progesterone
concentrations after a GnRH treatment are increased, which is attributed to the
formation of an accessory CL due to the induced ovulation by GnRH (Schmitt et
al., 1996a, 1996b). Heifers in the present study had an increase in plasma
progesterone concentrations after GnRH similar to another study where GnRH
was administered during the early luteal phase of the estrous cycle (Foster et al.,
1980). Volume of the original CL of the estrous cycle, accessory CL, and total
CL volume at PGF2a were similar between breeds. Data regarding CL volume in


166
-10
A
Figure 6
GnRH
Treatments:
PGF2a
Split PGF2a
TAI &
GnRH

0.5 mg MGA/d
i
0123456 7 8 9 10
A
Estrus detection & Al
A Blood sample collection
-1. Experimental protocol. Cows received GnRH (100 pg) on d 0 of the
experiment and melengestrol acetate (MGA) on d 1 to 6. Cows
received either a single injection of prostaglandin F2a (PGF2tI; 25 mg)
on d 7 or two consecutive injections of PGF2a (split-PGF2a; 12.5 mg) on
d 7 and 8 of the experiment. Cows that did not exhibit estrus by d 10
of the experiment were timed-AI (TAI) with GnRH (100 pg) on d 10 of
the experiment. Blood samples were collected on d -10. 0, 7, and 10
to determine progesterone concentrations.


99
decreased for Brahman Angus heifers in the present study compared with non-
lactating Brahman cows synchronized with PGF2a (Landaeta-Hernandez et al.,
2002). Behavioral estrus has typically been described as shorter in duration and
less evident in Bos indicus breeds (Plasse et al., 1970; Galina et al., 1982;
Pinheiro et al., 1998) compared with Bos taurus breeds (Stevenson et al., 1996).
However, such differences were not detected in the present study. It should be
noted that heifers in the present study were managed in a single pasture.
Therefore, it is quite possible that some of the behavioral characteristics
associated with estrus, including duration of estrus, are confounded because the
breed effects are being masked. Social environment, physical environment, and
breed have all been implicated as factors affecting the duration of estrus (Hurnick
et al., 1975; Galina and Arthur, 1990; Rodtian etal., 1996). Nevertheless, it is
unclear from the present experiment whether breed or the social and physical
environments acted directly or interacted with each other to influence the
duration of estrus, since the experimental design was not developed to evaluate
these variables.
Although the duration of estrus was similar between breeds, the total
numbers of mounts received during that duration was greater for Brahman *
Angus than Angus heifers. Total number of mounts received during estrus by the
Brahman x Angus heifers in the present study is consistent with observations by
Stevenson et al. (1996) but significantly greater than reports by Lemaster et al.
(1999) and Rae et al., (1999) in synchronized Bos taurus x Bos indicus heifers.
The total number of mounts received for Angus heifers in the present study was


129
Progesterone concentrations prior to the GnRH treatment were not affected
by breed in the present experiment. These results are in contrast with other
studies (Randel, 1977; Rhodes et al., 1982; Segerson et al., 1984) where
Brahman and Brahman x Hereford heifers had decreased serum concentrations
of progesterone compared with Hereford heifers (Randel, 1977). Similarly,
Brahman cows had lower serum concentrations of progesterone on d 7 of the
estrous cycle than Angus cows (Segerson et al., 1984). Furthermore, luteal cells
from Bos indicus heifers incubated with LH in vitro produced less progesterone
and were less responsive to LH than cells from Bos taurus heifers (Rhodes et al.,
1982). The conflicting reports between the present study and those reported by
others (Randel, 1977; Rhodes et al., 1982; Segerson et al., 1984) are puzzling,
but may be partially explained by the fact that heifers in this study were trained to
the stanchions prior to the beginning of the experiment to reduced stress
compared to no acclimation of animals to the working facilities in the reported
studies. It Is important to note that two Brahman x Angus heifers had
considerably greater progesterone concentrations prior to GnRH treatment
compared to the other heifers within their respective breed group and the other
breeds. Their progesterone concentrations remained increased for
approximately 2 h after GnRH and decreased to concentrations that were similar
to pretreatment concentrations across breeds. Furthermore, CL volume of the
two heifers at GnRH administration was similar to the all other heifers regardless
of breed. The increased progesterone concentrations of the two heifers are
probably responsible for the significant breed x time effect observed for


125
from occurring in cattle of Bos indicus breeding administered GnRH as part of an
estrus synchronization system. Acute stress has been reported to negatively
affect hypothalamic function (Dobson et al., 2001). Pulsatile patterns of GnRH
release and frequency and amplitude of LH pulses from the pituitary are
decreased by exposure to acute stressors such as transport (Dobson and Smith,
2000; Dobson et al., 2001). Similarly, chronic administration of ACTH resulted In
effects similar to those of acute stressors (Dobson and Smith, 2000; Dobson et
al., 2000). Dobson et al. (2000) observed that the pulsatile secretory pattern of
LH was disrupted, the LH surge was delayed, and ovulation rate was decreased
in response to ACTH administration. Therefore, further investigations must be
conducted to determine if acute stress will have a negative effect on a GnRH
induced LH release and possibly prevent ovulation in non-accllmated Bos indicus
cattle.
As aforementioned, although the Bos indicus heifers had decreased LH
concentrations in response to a GnRH challenge, LH concentrations were still
adequate enough to induce ovulation under the conditions of the present
experiment. However, one could question if ovaries of cattle of Bos indicus
breeding were more sensitive, and therefore, more responsive to the decreased
LH concentrations compared to Bos taurus cattle. There Is no evidence that this
may be the case. However, in cattle of Bos indicus breeding several
experiments (Barros and Nogueira, 1997; Gradela and Esper, 1998; Pinto et al.,
2000) compared the superovulatory response to reduced doses (200 to 300 IU)
of a commercial drug (Pluset, Serano, Italy) with relatively high LH:FSH ratio to


administered on d 6 of the estrous cycle. As the percentage of Bos indicus
breeding increased, the amount of luteinizing hormone released to GnRH
decreased. In Experiment 3, AN, B, and BA heifers were used to evaluate the
effectiveness of a single injection of PGF2a administered 7 d after a GnRH
treatment on d 6 of the estrous cycle, to initiate luteolysis of an accessory CL,
which had been exposed to either low (heifers treated with 15 mg PGF2i.m. at
12-h intervals on d 4 and 5 of the estrous cycle, followed by a single Injection of
PGF2a on d 6) or high (no PGF2a treatment on d 4, 5 and 6) progesterone
concentrations before PGF2a. Progesterone exposure before PGF2a had no
effect on luteolysis. In Experiment 4, cycling and noncycling crossbred Bos
indicus lactatlng cows were used to evaluate the effectiveness of a single versus
split (12.5 mg i.m.) dose of PGF2 in a 7 d GnRH/MGA/ PGF2a protocol.
Modifying the delivery of PGF2o, from a single to two consecutive split injections 7
d after GnRH had no effect on subsequent estrous response and pregnancy rate.
xiii


239
Table J-2. Odds ratio and confidence intervals using high progesterone as the
reference for ovulation rate after GnRH and estrous response after
PGF20! in Angus and Brahman x Angus heifers in the high or low
progesterone groups
Progesterone group
Variable
High
Low
P
value
Odds
ratio
Confidence
interval
Ovulation rate after
11/13 =
11/13 =
>0.10
1.00
0.06-16.14
GnRH, %
84.6
84.6
Estrous response, %
9/13 = 69.2
9/13 =
>0.10
1.00
0.54-1.84
69.2
Breed x Progesterone group (P > 0.10).


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3 RESPONSE TO A PROSTAGLANDIN F2ci INJECTION ON EITHER DAY
SIX OR SEVEN OF THE ESTROUS CYCLE IN ANGUS AND
BRAHMAN x ANGUS HEIFERS 73
Introduction 73
Materials and Methods 74
Results 80
Discussion 87
Implications 102
4 ENDOCRINE AND REPRODUCTIVE RESPONSES OF ANGUS,
BRANGUS, AND BRAHMAN x ANGUS HEIFERS TO A GNRH
INJECTION ON DAY 6 OF THE ESTROUS CYCLE FOLLOWED BY
PROSTAGLANDIN F2cc 7 DAYS LATER 103
Introduction 103
Materials and Methods 104
Results 111
Discussion 120
Implications 136
5 EFFECT OF PLASMA PROGESTERONE CONCENTRATIONS ON A
PROSTAGLANDIN F2c, INDUCED LUTEOLYSIS IN ANGUS AND
BRAHMAN x ANGUS HEIFERS 137
Introduction 137
Materials and Methods 138
Results 142
Discussion .'. 148
Implications 163
6 EFFICACY OF A SINGLE VERSUS SPLIT INJECTIONS OF PGF2 IN A
GNRH + PGF2ct SYNCHRONIZATION PROTOCOL IN COMBINATION
WITH MELENGESTROL ACETATE (MGA) IN CROSSBRED Bos
INDICUS CATTLE 164
Introduction 164
Materials and Methods 165
Results 171
Discussion 182
Implications 204
7 CONCLUSIONS AND IMPLICATIONS 205
vi


57
The knowledge that progestagens prevented the occurrence of estrus and
ovulation was the basis for the early attempts to synchronize estrus In cattle
(Christian and Casida, 1948). Several forms of progestagens have been used In
estrus synchronization systems including norgestomet, melengestrol acetate
(MGA), injections of progesterone, or administration of progesterone via the
vaginal inserts like the progesterone releasing intravaginal device (PRID) and
controlled Intravaginal progesterone-release device (CIDR).
Melengestrol acetate is an orally active progestogen, which when
administered at a rate of 0.5 mg/head/d suppresses estrus in beef (DeBois and
Bierschwal 1970) and dairy cattle (Roussel and Beatty, 1969). Beef cattle
producers like to use MGA because it is inexpensive, easy to administer, and can
induce estrous cycles in some postpartum cows (Beal and Good, 1986;
McDowell et al., 1998) and perlpubertal heifers (Fike et al., 1999). However,
there are several disadvantages to using MGA. In grazing cattle, it is difficult to
assure adequate consumption of MGA during the treatment period. Second,
fertility of the estrus after long-term (> 10 d) MGA administration is significantly
compromised due to the development of a persistent follicle. The etiology and
reason for the decreased fertility were discussed earlier in this review. Although,
the reduction in fertility is temporary and it returns to normal at the subsequent
estrus. Researchers used this later observation to their advantage to develop a
synchronization system that used long-term MGA administration.
Brown et al. (1988) administered MGA for 14 d followed by the
administration of PGF2a 17 d after the last day of MGA treatment or the


123
under the LH curve, Brahman Hereford were intermediate, and Hereford cows
had the greatest amount of LH released.
Regarding the ability of the pituitary to respond to treatments to initiate an
LH response, results from the present study and others in intact females injected
with GnRH (Griffin and Randel, 1978) and ovariectomized animals treated with
exogenous estrogen (Rhodes et al., 1978) clearly indicate that cattle of Bos
indicus breeding have a decreased LH response compared with cattle of Bos
taurus breeding. Whether the decreased LH response to a GnRH challenge in
cattle of Bos indicus breeding compared to Bos taurus females is due to lower
LH stores, less LH being synthesized by the pituitary, decreased sensitivity of the
hypothalamus and pituitary, and(or) a combination of these is unclear. There are
no apparent reports in the literature that have determined pituitary content of LH
in Bos indicus females. However, 14-month-old Brahman (Bos indicus) bulls
have decreased pituitary content of LH (553 ng/mg tissue; Aspden et al., 1997)
compared with 12-month-old Holstein bulls (1960 ng/mg tissue; Macmillan and
Hafs, 1968). Although, values between studies are probably not comparable
since LH pituitary content in the Aspen et al. (1997) study was determined by
RIA, while in the Macmillan and Hafs (1968) was determined by a Bioassay.
Therefore, cattle of Bos indicus breeding may have less available LH to be
released from the pituitary than cattle of Bos taurus breeding, which could be part
of the reason for the differential release of LH in response to GnRH between
breeds in the present study. Additionally, the differential LH release between
breeds in response to GnRH in the current experiment could also be explained


102
Implications
Although not conclusive from results from the present study, It appears
that breed effects the ability of PGF2ct to regress the early (d 6 or 7) developing
CL since more Angus than Brahman x Angus heifers had CL regression. This
observation warrants the need for further investigations in this area.
Furthermore, the compromised luteolytic response could have significant
implications for producers when cattle of Bos indicus breeding are involved,
because the overall effectiveness of estrus synchronization systems that Include
PGF2o, may be compromised. Additionally, the decreased estrous response
observed after PGF2ol treatment in the co-mingled Angus and Brahman x Angus
heifers stresses the need for development estrus synchronization systems that
Incorporate timed-AI.


114
Brahman x Angus heifers had progesterone concentrations that were
considerably greater than all other heifers in their breed group prior to GnRH and
for two hours after GnRH. The progesterone concentrations for the two heifers
decreased to concentrations that were similar to the other heifers In their group
by 150 min after GnRH (Data not shown) resulting In similar progesterone
concentrations for all breeds by 150 min after GnRH (Figure 4-3).
Mean plasma LH concentrations for the 480 min after GnRH for the Angus,
Brangus, and Brahman x Angus heifers are presented in Figure 4-4. There were
breed (P < 0.05), time (P < 0.0001) and breed x time (P < 0.0001) effects on LH
concentrations after the GnRH Injection. Homogeneity of regression procedure
Indicated that LH response curves were affected (P < 0.05) by breed. According
to contrast procedures, LH concentrations for the 480 min after GnRH were
different (P < 0.05) for Angus compared with Brangus and Brahman x Angus
heifers and for Brangus compared with Brahman x Angus heifers. Plasma LH
concentrations increased within 15 min after GnRH for all heifers and remained
elevated above baseline for approximately 360 min. For the Brangus and
Brahman x Angus heifers an initial LH peak was apparent 15 min after GnRH,
followed by a small decrease by 30 min and a subsequent increase until
maximum concentrations. In contrast, Angus heifers had a linear increase in LH
concentrations after GnRH until they reached maximum concentrations. Neither
estradiol concentrations before GnRH nor follicle size at GnRH affected (P >
0.10) LH response curves In the Angus, Brangus, and Brahman x Angus heifers.


APPENDIX H
PLASMA LH CONCENTRATIONS FROM GNRH INJECTION FOR HEIFERS IN
THE ANGUS, BRANGUS, AND BRAHMAN X ANGUS BREED GROUPS
(EXPERIMENT 2)
Figure H-1. Plasma LH concentrations (LS Means) In cycling Angus heifers
receiving 100 pg GnRH on d 6 of the estrous cycle.
231


281
Thompson, K.E., G.C. Lamb, D.M, Grieger, L.R. Corah, and J.S. Stevenson.
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215 (Abstr.).
Thompson, K.E., J.S. Stevenson, G.C. Lamb, D.M. Grieger, and C.A. Loest.
1999. Follicular, hormonal and pregnancy responses of early postpartum
suckled beef cows to GnRH, norgestomet, and prostaglandin F2a- J- Anlm.
Sci. 77: 1823-1832.
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Troxel, T.R., L.C. Cruz, R.S. Ott, and D.J. Kesler. 1993. Norgestomet and
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Turzlllo, A.M., and J.E. Fortune. 1993. Effects of suppressing plasma FSH on
ovarian follicular dominance in cattle. J. Reprod. Frtil. 98: 113-119.
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3151.


77
for later analysis. Progesterone concentrations were determined using RIA
(Seals et al., 1998) In multiple assays with Intra- and interassay CV of 2.3 and
13.9 %, respectively. Sensitivity of the assay was 0.02 ng/tube.
The presence of a CL at PGF2o (d 0) as detected by ultrasound was
defined as functional if progesterone concentrations were a 1.0 ng/mL. Corpus
luteum regression was defined as progesterone concentrations < 1.0 ng/mL in
two consecutive blood samples following PGF2a. During the treatment phase of
the experiment, estrus was also monitored using HeatWatch. Estrous response
was defined as the percentage of heifers exhibiting estrus within 7 d after PGF2a
divided by the total treated. The onset of estrus was defined as 3 or more
mounts within a 4 h period while the end of estrus was defined as the last mount
recorded pripr to a period of extended Inactivity of at least 8 h (Landaeta et al.,
1999; Lorton et al., 1999). Duration of estrus was defined as the total time from
the Initiation of estrus to the end of estrus. Distribution of mounting activity
during estrus was defined as the number of mounts received during consecutive
3 h periods starting at the Initiation of estrus to the end of estrus. Interval from
PGF2a to the onset of estrus was calculated as the time from PGF2a
administration to the first mount as detected by FleatWatch. Flelfers were
ultrasounded to determine the presence of a CL 10 d after expression of estrus
to confirm the site of ovulation.
After the completion of replication 1, heifers were allowed to go through a
normal estrous cycle before the start of the second replication. The same
experimental protocol was used for the second replication. At the Initiation of


265
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Mamluk, R., D.B. Chen, Y. Greber, J.S. Davis, and R. Meidan. 1998.
Characterization of messenger ribonucleic acid expression for
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Mamluk, R., N. Levy, B. Rueda, J.S. Davis, and R. Meidan. 1999.
Characterization and regulation of type A endothelin receptor gene
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effectiveness of PGF2aand its analogues on the induction of luteolysis, estrus
synchronization, and fertility is also highly variable (Hardin et al., 1980a, Pinheiro
et al., 1998; Rekwot et al., 1999; Mattoni and Ouedraogo, 2000).
Agonists to GnRH have been given at various stages of the estrous cycle to
induce an LH surge which induces ovulation of the dominant follicle resulting in
emergence and synchronization of a new follicular wave. The GnRH agonist
initiates an acute secretion of LH and FSH such that circulating concentrations
are elevated for a 3- to 5-h period, which stimulates ovulation (Thatcher et al.,
1993; Gong et al., 1996; Vizcarra et al., 1997; D'Occhio and Aspden, 1999). The
effectiveness of GnRH agonist to induce ovulation appears to be affected by the
stage of follicular development at the time of treatment (Prescott et al., 1992;
Silcox et al., 1993; Pursley et al., 1995; Moreira et al., 2000) and only induces
ovulation in follicles greater than 9 mm that are in the growth phase of a follicle
wave (Zaied et al., 1980; Macmillan and Thatcher, 1991; Vasconcelos et al.,
1999; Moreira et al., 2000).
Information about the concentrations of LH in the peripheral circulation of
cycling cows and/or heifers after treatment with GnRH during the estrous cycle
are numerous in cattle of Bos taurus breeding (Mori and Takahashi, 1978;
Chenault et al., 1990; Williams and Stanko, 1996; Fajersson et al., 1999).
However, direct comparisons of the endocrine and ovarian responses of cattle
administered GnRH between cattle of Bos taurus and Bos indicus breeding are
few (Griffin and Randel 1978; Irvin et al., 1978; Randel, 1994).


47
of a new follicle wave. In presynchronized beef heifers treated with a GnRH
agonist on d 3, 6 or 9 of the subsequent estrous cycle, ovulation occurred within
36 h after GnRH in 89, 56 and 22% of the heifers, respectively (Martinez et al.,
1999). Diameter of the largest follicle on d 3 was greater In follicles that ovulated
(9.6 mm) than those not ovulating (7.5 mm). The authors concluded that their
results did not support the hypothesis that the administration of GnRH at known
stages of the follicular wave in cycling heifers would consistently induce ovulation
or atresia and emergence of a new follicular wave at a predictable interval. New
wave emergence was induced consistently (1.3 d after GnRH) only in heifers that
ovulated in response to GnRH. However, 44% of the heifers treated with GnRH
failed to ovulate. In contrast, Rajamahendran et al. (1998) reported that the first
wave dominant follicle ovulated in all the nonlactating Holstein cows treated with
8 gg of buserelin on d 5 of the estrous cycle. Vasconcelos et al. (2000)
comparing Holstein and Holsteln-Gyr crossbred cows receiving an Ovsynch
protocol during winter and summer, reported that ovulation rate after the first
GnRH injection was greater in the winter than summer, with ovulation rates 61.4
and 37.8% for Holstein, and 58.3 and 45.2% for crossbred cows, respectively.
The variability in GnRH ability to induce ovulation at a known stage of the estrous
cycle between studies is unclear. These differences could be attributed to follicle
wave differences that could be associated with physiological state and (or) age of
the animals being used. Whether there is a breed effect (i.e., Bos indicus vs.
Bos taurus) is even less clear.


LIST OF FIGURES
Figure page
3-1 Experimental protocol Experiment 1 76
3-2 Progesterone concentration profiles in Angus and Brahman Angus
heifers that either regressed or did not regress their CL after a single
PGF2a injection on either d 6 or 7 of the estrous cycle 82
3-3 The effect of corpus luteum (CL) volume on progesterone concentrations
on either d 6 or 7 of the estrous cycle in Angus and Brahman x Angus
heifers 84
3-4 Distribution of mounts received during 3 h periods throughout the duration
of estrus after an injection of PGF2cl on either d 6 or 7 of the estrous cycle
in Angus and Brahman Angus heifers 86
3-5 Effect of number of heifers in estrus within a 3 h period on number of
mounts received during a period 88
3-6 Survival analysis representing the percentage of Angus and Brahman
Angus heifers that did not exhibit estrus during the 168 h after PGF2ot on
either d 6 or 7 of the estrous cycle 89
4-1 Experimental protocol Experiment 2 106
4-2 Intensive blood sampling protocol to determine luteinizing hormone,
progesterone, and estradiol concentrations in cycling Angus, Brahman,
Brangus and Brahman Angus heifers receiving GnRH on d 6 of the
estrous cycle 107
4-3 Plasma progesterone concentrations in cycling Angus, Brangus and
Brahman Angus heifers receiving 100 pg GnRH on d 6 of the estrous
cycle 113
4-4 Plasma LH concentrations in cycling Angus, Brangus and Brahman
Angus heifers receiving 100 pg GnRH on d 6 of the estrous cycle 115
4-5 Progesterone concentrations after a PGF2a treatment administered on d 6
of the estrous cycle In cycling Angus, Brangus and Brahman Angus,
x


145
Table 5-1. Breed and progesterone group effects on follicle size at GnRH,
ovulation rate after GnRH, and progesterone concentration at GnRH in
Angus and Brahman x Angus heifers in the High and Low
Breed
Progesterone group
Variable
Angus
(n = 7)
Brahman x
Angus
(n = 19)
High
(n = 13)
Low
(n = 13)
Follicle size at
GnRH, mm
13.7 + 1.3
12.2 0.7
12.0 + 1.0
13.9 0.9
Ovulation rate after
GnRH,%
85.7
84.2
84.6
84.6
Progesterone at
GnRH, ng/mL
1.9 0.2
2.0 0.1
3.6 0.1a
0.3 0.1b
superscript differ (P < 0.0001),
Breed Progesterone group (P > 0.10).


Ill
Results
Only one (1/11 = 9.1%) Brahman heifer exhibited estrus after the pre
synchronization protocol; therefore, Brahman heifers were removed from the
experiment. Pre-synchronization estrous response was similar (P > 0.10)
between Angus (6/7 = 85.7%), Brahman Angus (6/9 = 66.7%), and Brangus
(7/10 = 70.0%) heifers.
There was no effect (P >0.10) of breed on size of the largest follicle at
GnRH, mean plasma estradiol concentrations before GnRH, or ovulation rate
after GnRH (Table 4-1). Mean CL volume at GnRH was similar (P > 0.10)
between Angus (1973.6 323.8), Brangus (2125.2 323.8) and Brahman x
Angus (1605.8 323.8) heifers. According to contrast procedures, the size of
the largest follicle, mean CL volume at GnRH, and mean plasma estradiol
concentrations before GnRH were similar (P > 0.10) for Angus compared with
Brangus and Brahman x Angus and for Brangus compared with Brahman x
Angus heifers. Furthermore, CL volume and CL diameter at GnRH were'
positively correlated (Pearson correlation = 0.86; P < 0.0001) and all heifers had
progesterone concentrations > 1 ng/mL at GnRH (Data not shown).
Progesterone concentrations at GnRH and for the 480 min after GnRH were not
affected by breed (Figure 4-3). Mean progesterone concentrations for the 480
min after GnRH were 3.1 0.1,3.6 0.1 and 3.2 0.1 ng/mL for Angus,
Brahman x Angus and Brangus heifers, respectively. However, there was a
breed time effect (P < 0.0001) on progesterone concentrations for the 60 min
prior to GnRH until 480 min after GnRH. Further analysis revealed that two


184
breeding season. Body condition score can be used as an indirect indicator of
the cycling status of cattle (Odde, 1990; Yelich et al., 1995; Stevenson et al.,
2000; Moreira et al., 2001), and there Is a positive relationship between BCS and
cycling status in lactating cows (Wettemann, 1994; Lemasteret al., 2001).
However, cycling and noncycling cows had similar BCS in the present study.
Furthermore, BCS did not affect the three-day estrous rates in the present study,
which is contrary to other reports where cows in poor body condition at or near
the start of an estrus synchronization program have decreased estrus rates
(Yelich et al., 1995; Stevenson et al., 2000; Lemaster et al., 2001). One of the
reasons that BCS did not affect many of the variables In the present study Is that
the variation In BCS was very small and the majority of the cows (99%) were
within one condition score value from the overall mean.
With approximately two-thirds of cows cycling at the first GnRH
administration in the present experiment, one would expect estrous rates after
PGF2a to be significantly increased than if the proportion of noncycling cows were
greater (Twagiramungu et al., 1992; Geary and Whittier, 1999; Lemaster etal.,
2001). However, this was not the case in the present experiment. The low
three-day estrous rate (34%) observed in the present experiment is similar to
reports in cycling and noncycling lactating Bos indicus x Bos taurus cows
(Lemaster et al., 2001) but considerably less than those observed in Bos taurus
cows synchronized with GnRH + PGF2a synchronization systems (Twagiramungu
et al., 1992; Geary et al., 1998b; Stevenson et al., 2000). Furthermore, the
overall mean interval to the onset of estrus (64 h) for cows expressing estrus in


138
progesterone exposure may not be completely responsive to a PGF2a Injection.
This could have significant implications on how and when PGF2a is used in a
GnRH + PGF2a systems to induce luteolysis.
The objective of this experiment was to evaluate the effectiveness of a
single Injection of PGF2a administered 7 d after a GnRH treatment, to initiate
luteolysis of an accessory CL, which had been exposed to either high or low
progesterone concentrations before PGF2a in Angus, and Brahman Angus
heifers.
Materials and Methods
The experiment was conducted during the fall (November through January)
of 2001 at Santa Fe Beef Research Unit, Department of Animal Sciences,
University of Florida. Nonpregnant and cycling two-year old Angus (n = 23; Bos
taurus), and Brahman Angus (n = 40) heifers were initially synchronized with a
modified two-injection PGF2a protocol. Heifers were Injected with 25 mg PGF2a
(Lutalyse Sterile Solution, Pharmacia & Upjohn, Kalamazoo, Ml) on d -14 with
the second and third injections of 12.5 mg PGF2a administered on d -3 and -2 of
the experiment (Figure 5-1). Heifers were pastured in one group and body
condition scores (Richards et al., 1986) were recorded. Heifers were also fitted
with an electronic estrus detection device (Heatwatch, DDx, Boulder, CO) on d -4
of the pre-synchronization period to monitor estrus throughout the remainder of
the experiment. Estrus was designated as d 0 of the subsequent estrous cycle.
After expression of estrus, heifers within each breed (Angus n = 7; Brahman x
Angus n = 19) were randomly and equally allotted to either a low progesterone


70
concluded that TAI at 48 or 60 h after PGF in a GnRH-CIDR-PGF protocol was
equally effective and administration of GnRH at TAI improved fertility in all cycling
cows, but not In non-cycling cows.
The same attempt to find the appropriate estrus synchronization and (or)
ovulation system for cattle of Bos indicus breeding, comparing different protocols
has been made by some researchers. Williams et al. (2002) compared the
relative efficacies of three protocols (Syncro-Mate-B, SMB; norgestomet-PGF2a,
NP; and Ovsynch) for synchronizing ovulation for tlmed-AI of predominantly
Brahman-influenced cows and heifers. Animals In the NP treatment were
Implanted with SMB without the Injection of estradiol valerate/norgestomet. In
cows, administration of estradiol (SMB) or GnRH (Ovsynch) to synchronize
follicle development resulted In increased TAI pregnancy rates compared with
the NP group. Conversely, In heifers TAI conception rate was greater In NP
compared with SMB and Ovsynch. Synchronization of ovulation using SMB or
Ovsynch In Bos indicus Influenced beef cows In this study resulted in TAI
conception rates comparable to each other and to those reported for treatments
in Bos taurus cows (Geary et al., 1998b). Fernandes et al. (2001) conducted a
series of experiments to evaluate a 7 d GnRH + PGF2a protocol with or without
GnRH or estradiol benzoate after PGF2Qfor tlmed-AI In cycling or anestrous,
lactating or nonlactating Nelore (Bos indicus) cows. Percentage of animals
detected (44.5 to 70.3%) in heat and pregnancy rates (20 to 42%) varied
according to the number of animals with corpus luteum at the beginning of
treatment. Administration of a second dose of either GnRH or estradiol benzoate


Progesterone, ng/mL
146
Figure 5-3. Progesterone concentrations between GnRH and PGF2a treatment
for heifers in the high progesterone (HP) group including Angus
(diamonds) and Brahman Angus (squares) and heifers in the low
progesterone (LP) group including Angus (triangles) and Brahman *
Angus (X) heifers. Breed (P > 0.10), Day (P < 0.0001), Breed x Group
(P > 0.10), Group x Day (P < 0.0001), Breed x Day (P > 0.10), Breed x
Group x Day (P > 0.10). HP vs. LP (P < 0.0001).


280
Tanabe, T.Y., and R.C. Hann. 1984. Synchronized estrus and subsequent
conception in dairy heifers treated with prostaglandin F2a. I. Influence of
stage of cycle at treatment. J. Anim. Sci. 58: 805-811.
Tanaka, M., M. Yamauchi, T. Kariya, and J. Mori. 1979. Studies of the treatment
of ovarian follicular cysts in the cow by-synthetic LH-RH analog. II. LH
response to synthetic LH-RH analog in the cow with follicular cysts. Jap. J.
Anim. Reprod. 25: 55-60.
Taponen, J., T. Katila, and H. Rodriguez-Martinez. 1999. Induction of ovulation
with gonadotropin-releasing hormone during proestrus in cattle: influence
on subsequent follicular growth and luteal function. Anim. Reprod. Sci. 55:
91-105.
Taylor, C., M. Manikkam, and R. Rajamahendran. 1994. Changes in ovarian
follicular dynamics and luteinizing hormone profiles following different
progestagen treatments in cattle. Can. J. Anim. Sci. 74: 273-279.
Taylor, C., and R. Rajamahendran. 1991. Follicular dynamics, corpus luteum
growth and regression in lactating dairy cattle. Can. J. Anim. Sci. 71: 61-68.
Thatcher, W.W. and J.R. Chenault. 1976. Reproductive physiological responses
of cattle to exogenous prostaglandin F2ct. J. Dairy Sci. 59: 1366-1375.
Thatcher, W.W., R.L. de la Sota, E.J.-P. Schmitt, T.C. Diaz, L. Badinga, F.A.
Simmen, C.R. Staples, and M. Drost. 1996. Control and management of
ovarian follicles in cattle to optimize fertility. Reprod. Frtil. Dev. 8: 203-217.
Thatcher, W.W., M. Drost, J.D. Savio, K.L. Macmilan, K.W. Entwistle, E.J.
Schmitt, R.L. Delasota, and G.R. Morris. 1993. New clinical uses of GnRH
and its analogs in cattle. Anim. Reprod. Sci. 33: 27-49.
Thatcher, W.W., K.L. Macmillan, P.J. Hansen, and M. Drost. 1989. Concepts for
regulation of corpus luteum function by the conceptus and ovarian follicles
to improve fertility. Theriogenology 31: 149-164.
Thatcher, W.W., F. Moreira, S.M. Pancarci, J.A. Bartolom, and J.E.P. Santos.
2002. Strategies to optimize reproductive efficiency by regulation of ovarian
function. Dorn. Anim. Endocrinol. 23: 243-254.
Thatcher, W.W., M. Terqui, J. Thimonier, and P. Mauleon. 1986. Effect of
estardiol-17 beta on peripheral plasma concentration of 15-keto-13,14-
dehydro PGF 2aipha and luteolysis in cyclic cattle. Prostaglandins 31: 745-
761.


24
Deviation marks the completion of follicular selection and recent research
has focused on understanding the endocrine control of deviation. It has been
proposed that the control of deviation involves a two-way functional coupling
between FSH and follicles (Ginther et al., 2000a). Growing follicles of the cohort
attain the capacity to suppress FSH secretion when they are 5 mm in diameter
(Gibbons et al., 1999), and the dominant follicle is 8.5 mm in diameter at the time
of deviation (Ginther et al., 1996b). Estradiol produced by the future dominant
follicle is involved in the suppression of FSH (Ginther et al., 2000b). The
declining concentration of FSH fails to support the continued growth of the
subordinate follicles. However, even with the decreased concentrations of FSH,
it is required and remains at sufficient concentrations for the dominant follicle to
continue to grow (Turzillo and Fortune, 1993). After the decline in FSH, primary
gonadotropic support shifts to LH. Although suppression of LH did not affect the
time of deviation, the diameter of the largest follicle was decreased when LH
decreased after deviation (Ginther et al., 2001). Consequently, an increase in
responsiveness to LH is crucial for continued development of the dominant
follicle. Furthermore, granulosa cells of the dominant follicle express more LH
receptor mRNA than granulosa cells in the largest subordinate follicle before the
time of deviation (Beg et al., 2001). The same authors concluded that acquisition
of LH receptors allows for continued dominant follicle growth after the decline in
FSH. After deviation, the dominant follicle continues to mature in preparation for
ovulation. The most important aspect of maturation of the dominant follicle is its
ability to secrete increasing amounts of estradiol, which ultimately induce a surge


54
10 of the estrous cycle and observed only 50 and 67% estrus expression,
respectively. Additionally, for all heifers treated on d 7 and 10 blood
progesterone concentrations decreased within 12 h after treatment, but In heifers
not expressing estrus the decrease In blood progesterone concentrations were
temporary and progesterone concentrations recovered to pre-treatment
concentrations. However, it should be noted that the rebounding effect of
progesterone observed in heifers falling to undergo CL regression has also been
observed in cattle of Bos taurus breeding (Chenault et al., 1976; Copelln et al.,
1988). Hansen et al. (1987) concluded that In Slmmental x Brahman x Hereford
heifers treated on d 8 to 10 of the estrous cycle required a greater dose of
alfaprostol to elicit luteolysis and estrus than heifers treated on d 11 to 13 of the
estrous cycle.
The aforementioned data demonstrate that luteolysis appears to be
Incomplete and quite variable when PGF2ciis injected between d 6 to 7 of the
estrous cycle In cattle of Bos indicus breeding. Moreover, when cattle of Bos
indicus breeding are administered PGF2a later In the estrous cycle (d 10),
luteolysis and estrous response are improved but still highly variable. In
conclusion, cattle of Bos indicus breeding appear to be less responsive to PGF2a
at all stages of the estrous cycle than cattle of Bos taurus breeding. However,
there are insufficient data to indicate what the difference In magnitude for CL
regression and estrous response are between Bos taurus and Bos indicus cattle
and there are no studies where direct comparisons have been made. If luteolysis
Is significantly compromised in cattle of Bos indicus breeding, this could have


53
appeared to be associated with compromised expression of estrus (Orihuela et
al., 1983; Pinheiro et al., 1998; Rekwot et al., 1999). However, the decreased
estrous response after PGF2a appears to be associated with incomplete
luteolysis, which resulted in blood progesterone concentrations that are great
enough to prevent the expression of estrus (Hardin et al., 1980a, 1980b; Pinheiro
et al., 1998; Rekwot et al., 1999; Mattoni and Ouedraogo, 2000).
In Bos taurus heifers treated with PGF2o! between d 5 and 8 of the estrous
cycle, 85 to 100% of the heifers regressed their CL (King et al., 1982; Tanabe
and Hann 1984; Kiracofe et al., 1985). On the contrary, in Bos taurus x Bos
indicus cattle, Hardin et al. (1980a) observed a 64% estrous response after an
injection of cloprostenol between d 6 and 9 of the estrous cycle. Williams et al.
(1999) reported estrous rates of 54 and 63% in Brahman x Hereford heifers
treated with PGF2aon d 6 and 10 of the estrous cycle, respectively. Likewise,
Cornwell et al. (1985) reported that only 67% of Brahman heifers expressed
1i
estrus after being treated on d 7 of the estrous cycle with PGF2a, while 100% of
the heifers expressed estrus when PGF2awas injected on d 14 of the estrous
cycle. In a second study using Brahman heifers, Cornwell et al. (1985) reported
that only 50% of heifers treated with PGF2c( on d 7 of the estrous cycle expressed
estrus, while 67% injected on d 10 of the estrous cycle expressed estrus. In the
later study, blood progesterone concentrations decreased within 12 h after PGF2a
injection in all heifers; however, for heifers that failed to express estrus, blood
progesterone concentrations began to increase within 48 h after PGF2a. In a
similar study, Santos et al. (1988) treated Brahman heifers with PGF2aon d 7 and


35
especially MHC-II (Penny etal., 1999; Webb et al., 2002). Major
histocompatability complex (MHC) are cell surface markers required for antigen
recognition by T-lymphocytes (Webb et al., 2002). Additionally, tumor necrosis
factor-a (TNF-a) secreted by macrophages Induces programmed cell death
(apoptosis) of small and large luteal cells, and endothelial cells (Nagaosa et al.,
2002).
Other cellular mechanisms are Involved In structural luteolysis. For
Instance, when PGF2o receptors are activated there Is an influx of free calcium
(Ca+t) Into luteal cells, which Is mediated by angiotensln-ll (Pepperell et al.,
1993). The Increased intracellular concentration of free calcium (Ca++) Is
cytotoxic and It Induces apoptosis and degeneration of luteal cells destroying the
structural Integrity of the CL.
The number of PGF2a receptors In bovine luteal cells gradually Increases
from the early to the late luteal stages of the estrous cycle, parallel with the
expression of PGF2a receptor mRNA (Sakamoto et al., 1995; Skarzynski et al.,
2001). Flowever, concentration and affinity of PGF2a receptors were similar
during the early (d 2 4) and mid (d 6 -10) stages of the estrous cycle (Wiltbank
et al., 1995; Mamluk et al., 1998; Skarzynski et al., 2001). The fact that
concentration and affinity of PGF2a receptors were similar during the early and
mid stages of the estrous cycle suggested that PGF2a Induced luteolysis Is
regulated by different mechanisms involved In the regulation of the
responsiveness and activation of PGF2a receptors In the bovine CL during the
entire luteal phase (Skarzynski et al., 2001).


64
et al. (1998a) study tlmed-AI took place 24 h after the second GnRH injection or
48 h after removal of the SMB Implant. In addition, calves were removed from
cows either at SMB implant removal or at PGF2a injection until timed-AI was
completed. The OvSynch protocol resulted In greater tlmed-AI pregnancy rates
(54%) than SMB treated cows (42%). For cows determined to be cyclic at the
start of the treatments, tlmed-AI pregnancy rates were greater for OvSynch
(59%) than SMB (38%). Furthermore, tlmed-AI pregnancy rates for cows
determined to be anestrous at the start of the treatments tended to favor the
OvSynch protocol. The initial GnRH injection of OvSynch system probably
Induced estrous cycles In more cows than the SMB treatment. One
disadvantage of the OvSynch system Is that cattle are handled four times. This
has lead to the development of the Cosynch protocol, where cattle are
inseminated at the time of the second GnRH Injection (Geary et al., 1998b). Both
the OvSynch and CoSynch systems resulted In similar timed-AI pregnancy rates
(48%) In Bos taurus beef cows (Geary and Whittier, 1997).
Stevenson et al. (2000) treated Bos taurus beef cows with SelectSynch,
CoSynch, ora modified SelectSynch system called the HybridSynch system. In
the HybridSynch system, cows were Inseminated after a detected estrus for 54 h
after PGF2a and all cows not exhibiting estrus by 54 h were administered GnRH
and timed-AI. In agreement with a previous study by Thompson et al. (1998),
pregnancy rates of anestrous cows were similar among treatments; however,
pregnancy rates In cycling cows were greater for the SelectSynch (56%)
compared with CoSynch (40%) or HybridSynch (39%).


215
Conception rates were similar among cows classified according to
progesterone concentrations at experimental d -10, 0 and 7 indicating that
regardless of stage of the estrous cycle at GnRH and the response to GnRH
administration, if cows expressed estrus the timing of insemination was optimal
(Rorie et al., 2002), and fertility of the induced estrus is acceptable in cattle of
Bos indicus breeding.
Progesterone concentrations at PGF2a in both cycling and non-cycling cows
had a significant impact not only on the estrous response but also timed-AI
pregnancy rates in experiment 4. Cows with high progesterone concentrations at
PGF2a had a significant reduction in estrous response but increased timed-AI
pregnancy rate compared to cycling cows with low progesterone concentrations
at PGF2a. Although follicular growth and development was not evaluated in the
present study, follicle growth and stage of the estrous cycle at the initial GnRH
appears to be involved in the decreased estrous response and timed-AI
pregnancy rates observed in some cows. This assumption is based on the
different groups of cycling and noncycling cows classified according to
progesterone concentrations at PGF2a. Cycling cows with low progesterone at
PGF2a probably represented cows with advanced follicle development with no CL
to regress resulting in estrous responses that were at least 23% greater than all
other groups. Cows with high progesterone concentrations probably
corresponded to cows that were in the early stages of follicle development and
also had a CL that needed to be regressed by PGF2a resulting in very iow estrous
responses due to lack of a dominant follicle ready to ovulate shortly after CL


140
(LP) or high progesterone (HP) treatments. The LP heifers were injected at 12 h
intervals with 15 mg PGF2cl i.m. on d 4 and 5 of the estrous cycle, followed by an
injection of 25 mg PGF2ai.m. and 100 pg GnRH (Fertagyl; Intervet, Boxmeer,
The Netherlands) i.m. on d 6 of the estrous cycle. Seven days after the GnRH
treatment, LP heifers received 25 mg PGF2ai.m. (Figure 5-1). The HP heifers
were injected at 12 h intervals with saline solution i.m. on d 4 and 5 of the estrous
cycle, followed by an injection of saline solution i.m. and 100 pg GnRH i.m. on d
6 of the estrous cycle. Seven days after the GnRH treatment, HP heifers
received 25 mg PGF2ai.m. (Figure 5-1). On d 6, 8, 13, 15, 17, 19 of the
experiment both ovaries were examined via ultrasonography (Figure 5-1) using a
real time, B-mode ultrasound (Aloka 500V, Corometrics Medical Systems,
Wallingford, ,CT) equipped with a 7.5 MHz transducer. At each ultrasound
examination, follicles greater than 5 mm, height and width of all luteal structures,
diameter of any luteal cavities, and respective locations on the ovaries were
measured with the internal calipers of the ultrasound machine and recorded.
Volume of the CL was calculated by using the formula for the volume of a sphere
(?rd3/6). When a luteal cavity was present, its volume was subtracted from the
volume of the outer sphere resulting in net luteal volume (CL volume)
represented by luteal tissue.
Ovulation was defined as the disappearance of the largest follicle on either
ovary between two consecutive ultrasound examinations. The diameter of the
follicle at the ultrasound exam prior to its disappearance was used as its
ovulatory diameter. Blood samples were collected via jugular venipuncture for


9
LH leads to the maturation of follicles, Induces ovulation, formation of the corpus
luteum (CL), and maintains the synthesis and secretion of progesterone by the
CL (Peters and Lamming, 1983). The secretory nature of FSH and factors
regulating Its control are not as well understood as they are for LH
(Padmanabhan and McNeilly, 2001). Compared to LH (half-life: 30 min), the
longer half-life (4 h) and molecular heterogeneity of FSH makes It difficult to
assess its secretory patterns in the blood stream (Ulloa-Aguirre et al., 1995,
Padmanabhan and Sharma, 2001). Since LH and FSH share a common a-
subunlt (Padmanabhan and McNeilly, 2001) and the gonadotropin a-subunit is
secreted in an episodic pattern (Hall et al., 1990), It Is unclear whether the
pulsatile secretion of FSH is just an Indication of gonadotropin a-subunit
secretion or cross-reactivity with LH. Secretion of FSH appears to be regulated
by two mechanisms. One mechanism controls the basal secretion, which
appears to be the major portion of FSH released, and the other mechanism
controls Its pulsatile release (Padmanabhan and McNeilly, 2001; Padmanabhan
and Sharma, 2001). There is also evidence supporting the presence of a
separate FSH-releasing factor (FSH-RF) in the hypothalamus (Mizunuma et al.,
1983; Lumpkin et al., 1987; McCann et al, 2001). However, a variant form of
GnRH (Montaner et al., 2001) may be the putative FSH-releasing factor
(Padmanabhan and McNeilly (2001); Padmanabhan and Sharma 2001). Along
with this theory, other studies suggest the existence for separate hypothalamic
areas controlling LH and FSH secretion (Chappel and Barraclough, 1976;


159
reason for the decreased fertility is due to incomplete regression of the CL, which
can result after PGF2o, administration during the Ovsynch protocol (Moreira et al.,
2000b). A second reason for the decreased fertility at the breeding following a
low progesterone environment can be due to the luteolytlc response observed
during the subsequent estrous cycle. For instance, Shaham-Albalancy et al.
(1998) reported that low ascending circulating progesterone concentrations
during the luteal phase of one estrous cycle affect the sensitivity of uterine tissue
to luteolytic signals (i.e. oxytocin) in the subsequent estrous cycle. Any
disruption of the hormonal milieu may result In increased embryonic loss.
Both of the previously mentioned scenarios could be applicable to hormone
environments commonly observed in anestrous cattle or cattle at certain stages
of the estrous cycle (Macmillan and Peterson, 1993; Martinez et al., 2000;
Moreira et al., 2000b) when treated with the GnRH + PGF2a synchronization
system. Therefore, It is important to determine management tools to Improve
luteolysis and possibly embryonic loss during the subsequent estrous cycle in
cattle with low progesterone environments prior to PGF2a treatments. Treatment
with exogenous progesterone like the controlled intravaginal progesterone
release device between the GnRH and PGF2cl treatments may improve the
synchronization systems effectiveness in cattle with low progesterone
environments. An increased estrous response has been observed when cycling
and noncycling beef cattle received a CIDR for 7 d and PGF2o at device removal,
which eventually resulted in improved pregnancy rates compared with untreated
control animals and animals treated with PGF2a only at the same day as for the


81
Table 3-1. Corpus luteum (CL) regression and ovarian characteristics (LS means
+ SE) in Angus and Brahman Angus heifers treated with
Breed
Variable
Angus
(n = 26)
Brahman Angus
(n = 31)
CL regression, %a b
96.2
80.6
CL volume, mm3
3231.2 + 226.7
2914.3 + 207.9
Progesterone, ng/mL
3.1 +0.3
3.2 0.3
Largest follicle diameter, mm
10.5 0.4C
12.0 + 0.4d
injection of PGF2a with progesterone < 1 ng/mL.
bBreed with GENMOD (P = 0.04) and with logistic regression (P = 0.10).
c,dValues lacking a common superscript differ (P < 0.01).


18
The luteolytic mechanism of PGF20S described in detail in the section
Ovulation, Corpus Luteum Development, and Luteolysis.
Estrous Cycle in Cattle
The bovine estrous cycle ranges from 18 to 24 d and comprises a
sequence of predictable reproductive events beginning with estrus (period of
sexual receptivity) and ending at the subsequent estrus. Differences in estrous
cycle length between Zebu (Bos indicus) and European breeds (80s taurus)
have been reported. In 80s taurus breeds, the length of the estrous cycle is
typically 21 d (Hansel et al., 1973), with little variation. In contrast, the length of
the estrous cycles in Bos indicus breeds varies considerably, reported to average
28 d in Brahman heifers (Plasse et al., 1970) and 23 d in Boran cows (Llewelyn
et al., 1987). In a study by Moreira-Viana et al. (2000), mean estrous cycle
length was 21.7 d in Gir cows. Alvarez et al. (2000) reported similar estrous
cycle lengths among Senepol (Tropical adapted 80s taurus; 20.4 d), Angus (19.5
d), and Brahman (19.7 d) cows. However, it should be noted that in the later
study, the three breeds were housed as a single group throughout the duration of
the experiment.
Estrus
Estrus is characterized by sexual receptivity and mating and it is the most
identifiable stage of the estrous cycle. Estradiol is the hormone responsible for
inducing estrous behavior in cattle (Short et al., 1973; Randel, 1990). In addition,
estradiol is the main stimulus for induction of the preovulatory surge of LH in the
bovine (Henricks et al., 1971; Christensen et al., 1974; Randel, 1990).
Behavioral estrus is shorter in duration and less evident in 80s indicus breeds


214
report by Hiers et al. (2003). Conception, timed-AI, and overall pregnancy rates
in the present study were acceptable and similar compared to other GnRH +
PGF2o synchronization protocols in Bos indicus influenced cattle (Fernandes et
al., 2001; Lemaster et al., 2001; Hiers et al., 2003). Results observed in the
current experiment regarding synchronized pregnancy rates and those in the
literature are conflicting. The mean synchronized pregnancy rate observed in
this study is greater than a report using the Hybrid Synch protocol (Lemaster et
al., 2001) in crossbred Bos indicus cows, but similar to a report in lactating beef
cows treated with a modified Co-Synch protocol (Geary et al., 2001).
Synchronized pregnancy rates in the present study are also less than results
from an experiment using a GnRH + PGF2tI + short term MGA in lactating beef
cattle (Thundathil et al., 1999). Pregnancy rates were greater for cycling than
noncycling cows in the present study, which is similar to reports in both Bos
taurus (Stevenson et al., 2000; Moreira et al., 2001) and Bos indicus (Fernandez
et al., 2001; Lemaster et al., 2001) cattle synchronized with similar GnRH +
PGF2a synchronization systems. Therefore, it is imperative to have as many
cattle cycling as possible at the initiation of a GnRH + PGF2a synchronization
system to obtain maximum pregnancy rates to the Al. To increase the
percentage of cycling cows in the herd before the implementation of estrous
synchronization and timed-AI systems, appropriate pre- and post-partum
nutritional management is needed to maintain body condition during the
postpartum period.


135
estrous cycle and the newly recruited dominant follicle was In Its early dominant
phase when PGF2a was administered; therefore, they had a mature and fully
developed CL responsive to the luteolytic action of PGF2a. Additionally, Howard
and Britt (1990) and Moreira et al. (2000a) observed that accessory CL, at the
early stages of luteal development, induced during high concentrations of
progesterone responded to an injection of PGF2a. Consequently, results
regarding CL regression observed In the present study suggest that the
accessory CL In the presence of the original CL from the estrous cycle appears
to be very responsive to PGF2a in cattle of Bos indicus breeding. Although, It is
unclear how effective PGF2a induced luteolysis is for early luteal stage CL that
has not been exposed to the extra progesterone.
The decline In progesterone concentrations immediately after PGF2a across
breeds in the present study is in agreement with reports in Bos taurus heifers
(King et al., 1982; Kiracofe et al., 1985; Watts and Fuquay, 1985) and cattle of
Bos indicus breeding (Cornwell et al., 1985; Santos et al., 1988; Plnhelro et al.,
1998) treated with PGF2o or its analogues. In all of these reports, cattle that
regressed their CL had a linear decline in progesterone concentrations within 24
h after PGF2a and progesterone concentrations remained below 1 ng/mL during
the evaluation period. Therefore, breed does not appear to Influence the timing
in which the CL ceases to produce progesterone after a PGF2a induced
luteolysis.
In summary, plasma estradiol concentrations and size of the largest follicle
at GnRH on d 6 of the estrous cycle were similar among Angus, Brangus, and


15
autocrine/paracrine regulation of follicular and luteal function by progesterone.
Similarly, specific membrane binding sites for progesterone In granulosa and
thecal membranes from bovine follicles of different sizes and In luteal cell
membranes have been described (Rae et al., 1998a; 1998b). The secretion of
and possible role of estradlol-17p in the bovine CL are unclear (Schams and
Berisha, 2002). Nevertheless, specific binding sites of estradiol-17|3 are present
In the bovine CL (Kimball and Hansel, 1974). Okuda et al. (2001) reported that
mRNA for the aromatase P450 is present In the bovine CL with a clear up-
regulation during the mid and late luteal phase of the estrous cycle and positive
correlation with the up-regulatlon of LH receptors (Kobayashi et al., 2001;
Schams and Berisha, 2002). Basal release of estradiol within the bovine CL from
an In vitro microdialysis system did not change during the estrous cycle (Okuda
et al., 2001). Okuda et al. (2001) reported that In luteal cell culture, estradiol
stimulated only PGF2a secretion, while It did not affect progesterone and oxytocin
secretion. In contrast, PGF2q did not stimulate estradiol secretion from cultured
bovine luteal cells. The functionality of the early bovine CL Is affected by
progesterone in an autocrine and paracrine manner (Skarzynski and Okuda,
1999; Skarzynski et al., 2001). Secretion of progesterone, oxytocin, and PGF2a
and PGE2 was reduced after treatment with a specific progesterone antagonist
(onapristone) In the early luteal cells (Schams and Berisha, 2002). In addition,
the antagonist Inhibited oxytocin secretion In midcycle luteal cells, even though It
stimulated PGF2a secretion. These results show that progesterone stimulates
secretion of progesterone, oxytocin, and PGs in the early CL. However, In the


167
of the experiment. Half the cows were randomly assigned to receive 25 mg
PGF2ai.m. (single PGF2a; LUTALYSE Sterile Solution, Pharmacia Animal
Health, Kalamazoo, Ml) 7 d later while the remaining cows received 12.5 mg
PGF2a (split PGF2a) on d 7 and 8 of the experiment. Melengestrol acetate
(MGAPremlx Pharmacia Animal Health, Kalamazoo, Ml) was administered In a
high protein cube at a rate of 0.91 kg*head'1* d1 to deliver 0.5 mg MGA*head'1,d
1 and It was fed on the ground to all the cows on d 1 to 6 of the experiment. The
MGA was administered to prevent premature expression of estrus on d 5, 6 and
7 of the experiment which is a problem with the GnRH + PGF2a protocol
(Thatcher et al., 1989; Thompson et al., 1999; Stevenson et al., 2000; DeJarnette
et al., 2001). Moreover, as some cows do not have a functional CL at PGF2a in
the GnRH + PGF2a systems (Twagiramungu et al., 1995; Schmitt et al., 1996a;
Downing et al., 1998; Moreira et al., 2000), MGA was administered until the day
before of PGF2a to prevent early expression of estrus and probably tighten the
'i
synchrony of estrus and ovulation after MGA withdrawal. Furthermore, cows
were not administered MGA on the day of GnRH (d 0), since administering MGA
concomitant with GnRH appears to reduce the effectiveness of GnRH to induce
ovulation in cows with dominant follicles (Pancarci et al., 1999).
Visual detection of estrus was conducted on d 8, 9 and 10 of the
experiment at 0700 and 1700. To assist with estrus detection, cows received a
Kamar patch (Kamar Marketing Group, Steamboat Springs, CO) at the initial
PGF2a treatment. Estrus was defined as any cow standing to be mounted by
another cow or having a Kamar patch that was at least 25% red and cows were


234
0048
-*-0072
0108
-*-0121
-*-0149
-*-0155
Interval from GnRH, min
Figure H-4. Plasma LH concentrations (LS Means) in all Angus heifers receiving
100 gg GnRH on d 6 of the estrous cycle.


195
is affected by the stage of follicular development at the time of treatment
(Presscott et al., 1992; Sllcox et al., 1993; Moreira et al., 2000a); therefore,
follicle turnover Is not Initiated In all animals. However, treatment of
progestogen-implanted cattle with estradiol Induces emergence of a new
follicular wave, regardless of stage of development of the dominant follicle at the
time of treatment (Bo et al., 1993; Martinez et al., 2000); therefore, almost all
animals have follicle turnover. Estradiol at the Initiation of a MGA treatment
resulted In Increased pregnancy rates compared to GnRH at the Initiation of a
MGA based tlmed-AI system (Martinez et al., 2001). However, many of the
estrogens compounds like estradiol benzoate and estradiol-170 are not approved
by the Food and Drug Administration for use as synchronization agents.
Although ECP can be used, it has been reported to result In delayed follicle
turnover when administered at the Initiation of a progestogen treatment due to Its
half-life (Thundathil et al., 1998; Colazo et al., 2003). Alternatively, it has been
hypothesized that induction of a LH surge directly with an Injection of GnRH may
be more effective In noncycling cows compared with ECP because noncycling
cows may not have a functional hypothalamic positive feedback response to
estradiol (Pancarci et al., 2002). This could affect the overall effectiveness of an
estrus synchronization protocol using ECP as part of the treatment when a
significant proportion of animals are noncycling at the start of the synchronization
treatment.
To obtain maximal pregnancy rates when Implementing an estrus
synchronization and timed-AI system In cattle of Bos indicus breeding,


94
reported that the efficacy of PGF2a for causing luteolysis and ovulation in buffalo
cows (Bubalus bubalis) at different stages of the estrous cycle was dependent
upon CL size at treatment. However, results from the present experiment are not
in agreement with Brito et al. (2002), since CL volume did not affect total CL
regression in the current study.
Estrous response of Angus and Brahman x Angus heifers in the present
study was decreased compared to reports in Bos taurus heifers (Tanabe and
Hann, 1984; Kiracofe et al., 1985; Watts and Fuquay, 1985) but similar to reports
in Bos indicus x Bos taurus heifers (Hardin et al., 1980a) and Bos indicus heifers
(Cornwell et al., 1985; Santos et al., 1988) treated with PGF2a between d 5 and 8
of the estrous cycle. Although, Hardin et al. (1980a) reported an estrous
response of 91% in Bos indicus x Bos taurus heifers synchronized with two-
injection PGF2a protocol. However, estrous response among heifers that
regressed their CL was 24% greater in Brahman x Angus than Angus heifers in
the current experiment. The adequate estrous response in Brahman x Angus
heifers that regressed their CL indicates that if these heifers responded to the
luteolytic action of PGF2a, the majority of them would express estrus. The
reason(s) for the decreased estrous response particularly in the Angus heifers in
the present study is unclear. However, in the Brahman x Angus heifers, the
absence of estrous expression is probably due to both physiological and
behavioral effects. A small percentage of cattle of Bos indicus breeding ovulate
without exhibiting visible signs of estrus (Plasse et al., 1970), which is probably
due to strong social interactions amongst heifers (Dobson and Smith, 2000).


100
similar to that reported for Angus heifers synchronized with either Syncromate B
(Rae et al., 1999) or a melengestrol acetate plus PGF2a system (Rohe et al.,
1999). Furthermore, mount data from the present study are in agreement with
other experiments in which differences have been reported between Bos taurus
and Bos indicus cattle (Galina et al., 1982; Galina and Arthur, 1990; Rae et al.,
1999). A significantly increased number of mounts received during estrus in Bos
indicus compared with Bos taurus cattle has been reported (Galina et al., 1982;
Galina and Arthur, 1990). Similarly, Rae et al. (1999) observed a significantly
decreased number of mounts in Angus compared to Bos taurus Bos indicus
heifers synchronized with Syncromate B. Breed differences observed in the total
number of mounts in the present study correspond with the substantial variation
for this variable that has been observed within and among breeds in other
studies (Galina and Arthur, 1982; Gwazdauskas et al., 1983).
The distribution of mounts during estrus in the present experiment was
influenced by breed, contrary to a report by Landaeta-Hernandez et al. (2002)
where the distribution of mounts during estrus was not affected by breed in either
synchronized or a spontaneous estrus in non-lactating Angus, Brahman and
Senepol cows that were managed as a single group. The mount distribution
pattern for Angus heifers in the present study is in agreement with a report by
Landaeta-Flernandez et al. (2002) in which distribution of mounts during a PGF2a
synchronized estrus in Angus, Brahman and Senepol cows peaked 3 to 6 h after
the onset of estrus. In contrast, the distribution of mounts received during estrus
by Brahman x Angus heifers in the present experiment peaked 6 to 9 h after the


42
and it remains on the ovary throughout the duration of the progestogen treatment
(Savio et al., 1993a; Stock and Fortune, 1993). Development of the persistent
follicle is typically observed when exogenous progestagens are administered for
period > 10 d. The persistent dominant follicle is supported by the high
frequency, low amplitude LH secretion during the progestogen treatment in the
absence of a functional CL (Roberson et al., 1989; Kojima et al., 1992). In
addition, fertility of subsequent estrus after progestogen withdrawal is
compromised due to ovulation of the persistent follicle. Oocytes ovulated from
persistent follicles are fertilized but subsequent development is compromised
resulting in increased embryonic mortality (Mihm et al., 1994; Ahmad et al., 1995;
Revah and Butler, 1996). Additionally, due to the increased concentrations of
estradiol produced by the persistent follicle, the altered hormonal environment
effects oviductal protein synthesis and secretion that may contribute to the
decreased fertility (Binelli et al., 1999).
The most common progestogen used for estrus synchronization is
melengestrol acetate (MGA). In most cases, MGA-treated cattle develop
persistent dominant follicles that do not ovulate during the duration of the MGA
treatment (Anderson and Day, 1994; Custer el al., 1994; Yelich et al., 1997)
because MGA blocks the preovulatory surge of LH (Kojima et al., 1995; Imwalle
et al., 2002). While 0.5 mg of MGA per day is enough to block the estradiol-
induced surge of LH, between 1.5 to 5.0 mg of MGA are necessary to suppress
LH pulses (Kojima et al., 1995; Hageleit et al., 2000). However, even at the


Progesterone, ng/mL
82
Days from PGF2a
Figure 3-2. Progesterone concentration profiles in Angus (AN) and Brahman *
Angus (BR x AN) heifers that either regressed or did not regress their
CL after a single prostaglandin F2c, (PGF2o) injection on either d 6 or 7
of the estrous cycle. Breed (P > 0.05), Breed x Time (P > 0.05), CL
regression x Time (P < 0.0001), CL regression vs No-CL regression (P
< 0.0001).


186
estrus detection efficiency could have been compromised. However, the Kamar
heat detectors should have helped prevent missing a visually detected estrus
unless the intensity of the mounting activity associated with estrus was
decreased enough so that the Kamar detectors were not activated. Numerous
studies with cattle of Bos indicus x Bos taurus breeding have observed
decreased estrous responses and extended intervals to a synchronized estrus
(Lemaster et al., 1999, 2001; Bridges et al., 2003) compared to reports for Bos
taurus cattle (Twagiramungu et al., 1992; Geary et al., 1998b; Stevenson et al.,
2000). Additionally, social and behavioral differences between Bos indicus and
Bos taurus cattle can influence the interval to the onset of a synchronized estrus
(Landaeta-Hernandez et al., 2002). Although inadequate estrus detection may
partially explain the decreased estrous rate observed in the present study, other
factors like luteal and follicular status at PGF2a treatment could have influenced
estrous rate.
As previously mentioned, even the cycling cows had a decreased estrous
rate in the present study. This decrease may have been due to a delay in follicle
development after PGF2a, which corresponds with the increased percentage of
cows displaying estrus from 60 to 72 h after the PGF2a (Mihm et al., 1994),
particularly if cows had high progesterone concentrations at PGF2o. When cows
were classified by progesterone concentrations at PGF2l3, cycling cows with low
progesterone concentrations had a significantly greater estrous response than
cycling and noncycling cows with high progesterone concentrations at PGF2o.
Although follicle development and the exact stage of the estrous cycle when


51
Data comparing the induction of LH secretion by GnRH administration
between Bos indicus and Bos taurus females are variable and may be effected
by the dose and (or) reproductive status of the animal. Griffin and Randel (1978)
comparing LH responses to 500 pg GnRH in ovariectomized Brahman and
Hereford cows, and reported that all cows responded with increased serum LH
concentrations within 15 min of administration of GnRH. However, mean LH
response and average peak LH concentrations were decreased in the Brahman
(34 4 ng/ml and 94 + 7 ng/ml, respectively) versus Hereford cows (67 20
ng/ml and 185 + 68 ng/ml, respectively). In contrast, Stahringer et al. (1989)
reported that Brahman cows that were 17 to 34 d post partum and challenged
with a low dose (4.5 ng/lb BW) of GnRH had increased basal LH, increased
mean LH concentrations, increased GnRH-induced LH pulse amplitude, and an
increased GnRH-induced pulse height versus Angus cows. These data suggest
that pituitary function in Bos indicus cattle is different from pituitary function in
Bos taurus animals.
In progesterone-based treatments for the control of ovarian activity, GnRH
has been used to induce turnover of large follicles that would otherwise have the
potential to develop into persistent follicles. Schmitt et al. (1996a) using
nonlactating Holstein cows treated with a norgestomet implant on d 7 of the
estrous cycle and PGF2a at implant withdrawal observed that injection of GnRH
on d 9 of the estrous cycle induced ovulation of dominant follicle and a newly
recruited dominant follicle was induced to grow and ovulate following removal of
the norgestomet implant. Similarly, Ryan et al. (1998) observed that


247
Baker, D.S., W.M. Mackay, J.C. Whittier, M. Osborne, and P.D. Burns. 2002.
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59
significantly reduced when the MGA treatment was initiated late in the estrous
cycle. This is probably due to the development of persistent dominant follicles
(Custer et al. 1994; Kojima et al. 1995), which are associated with reduced
fertility (Patterson et al., 1989; Custer et al., 1994; Ahmad et al., 1995), likely due
to ovulation of aged oocytes (Mlhm et al., 1994). Other researches have used
the PRID and CIDR to deliver progesterone via the vagina for the duration of 7 to
12 d. Luteolysis is achieved by administering PGF2a at or just prior to device
removal. Lucy et al. (2001) reported that treatment with a CIDR for 7 d with
PGF2a on d 6 resulted in a increased synchronized estrous response compared
to PGF2a alone and untreated controls in postpartum beef cows, beef heifers,
and dairy heifers. Differences In fertility between MGA and CIDR treatments are
probably due to differences in circulating levels of progesterone between the two
treatments. Plasma progesterone concentrations have been reported to increase
by 2 h after CIDR-B insertion (Burke et al., 1999). However, progestin levels
'i
may Increase more slowly with an oral preparation such as MGA (Kojima et al.,
1995). Administration of exogenous progesterone to animals lacking a functional
CL, results In the development of large follicles that persist on the ovaries (Sirois
and Fortune, 1990; Savio et al., 1993a; Yellch et al., 1997). The development of
the persistent follicle is associated with an increased LH pulse frequency due to
the lack of negative feedback effects of luteal progesterone (Savio et al., 1993a;
Kojima et al., 1995). This problem may be overcome by strategically
synchronizing follicular wave emergence at the start of the short-term
progesterone treatment. Estrogens and GnRH have been compared in both


25
of LH for ovulation (Fortune, 1994). Estradiol enhances the release of GnRH and
increases sensitivity of the pituitary to GnRH (Kesner et al., 1981), but only if
concentrations of progesterone are relatively low or have declined significantly
(Kesner etal., 1982) since progesterone Inhibits gonadotropin surges (Kesner et
al., 1982). Therefore, if the CL does not regress during the first and some
second wave dominant follicles, an LH surge and ovulation will not occur.
Instead, the dominant follicle undergoes atresia, giving rise to a new wave of
follicular growth. In concordance, Savlo et al. (1993b) proposed that Increased
concentrations of luteal progesterone decreased LH pulse frequency and Initiated
turnover of nonovulatory dominant follicles. If the CL is regressed LH pulse
frequency increases causing the dominant follicle or future dominant follicle to
grow and secrete more estradiol (Auletta and Flint, 1998). Increasing secretion
of estradiol will induce a surge of LH and subsequently ovulation (Kesner et al.,
1982; Ireland et al., 1984).
The length of the estrous cycle in Bos taurus cattle with two follicular waves
averages 20.4 d (Glnther et al., 1989c) comparable to the interovulatory period of
Bos taurus x Bos indicus (Perea et al., 1998; Gomez-Alves et al., 2002) and Glr
(Bos indicus) cows (Morelra-Viana et al., 2000). The first wave dominant follicle
can be Identified on d 3 to 4 of the estrous cycle in Bos taurus (Ginther et al.,
1989b; Savlo et al., 1993b) and Bos taurus x Bos indicus cattle (Perea et al.,
1998; Gomez-Alves et al., 2002). However, the first wave dominant follicle was
Identified earlier (d 1 of the estrous cycle) in Gir cows (Morelra-Viana et al.,
2000). According to Ginther et al. (1989b) and Savlo et al. (1993b), dominance


168
inseminated 8 to 12 h later. Cows that did not exhibit estrus by the morning of d
10 after PGF2a were timed-AI with 100 pg of GnRH i.m. Frozen-thawed semen
from four custom sires was used. Sires were equally distributed across
treatments and randomly assigned to cows as they were inseminated.
Inseminations were performed randomly by five Al technicians. Cows were
placed with bulls 10 d following timed-AI. Pregnancy status was determined
using a real-time B- mode ultrasound (Aloka 500V, Corometrics Medical
Systems, Wallingford, CT) equipped with a 5.0 MHz transducer approximately 50
to 60 d following timed-AI. Because cows were not exposed to bulls until 10 d
after timed-AI, fetal size was used to designate whether the pregnancy was from
Al or natural service (Curran et al., 1986). Any fetus smaller than the determined
size to have become pregnant to either an insemination after a detected estrus or
timed-AI were classified as having become pregnant by natural service sires.
Blood samples were collected via jugular venipuncture on d -10, 0, 7, and 10 of
the experiment to determine progesterone concentrations. After collectidn, blood
samples were immediately placed on ice and centrifuged (3000 rpm). Plasma
was separated and stored at -20"C for later analysis. Progesterone RIA (Seals
et al., 1998) was conducted in multiple assays with intra- and interassay CV of
2.1 and 14.5 %, respectively. Sensitivity of the assay was 0.02 ng/tube.
According to progesterone concentrations on d -10 and 0 of the experiment,
cows were classified as cycling if either of the first two samples had progesterone
concentrations >1 ng/mL typical of luteal phase of the estrous cycle and
noncycling if both of the first two samples had progesterone concentrations <1


62
(47.6%) at sponge insertion, with ovulation rates of 57.1 and 25.0%, respectively.
Very low conception rates at first estrus after these treatments were observed
(26.7% and 10.0% for sponges + GnRH and sponges + estradiol, respectively).
Thatcher et al. (1989) were the first to propose a protocol to synchronize
follicular development using GnRH followed by 7 d later with an injection of
PGF2a to initiate luteal regression. A surge of LH in response to GnRH treatment
causes ovulation when a dominant follicle is present (Thatcher et al., 1989;
Macmillan and Thatcher, 1991) while the PGF2a or its analogues induced
regression of the newly formed CL and other CL present on the ovaries.
Following administration of the GnRH/PGF2a system, cattle can be inseminated
either after a detected estrus ortimed-AI in combination with GnRH in dairy
(Pursley et al., 1995; Schmitt et al., 1996b) and beef cattle (Geary et al., 1998a;
Stevenson et al., 2000) resulting in very acceptable pregnancy rates. The
GnRH/PGF2a systems appear to be more effective in cows of Bos taurus
breeding (Pursley et al., 1995; Geary et al., 1998a) compared to cows of Bos
indicus breeding (Lemaster et al., 2001). Although, no direct breed comparisons
have been made.
Macmillan and Thatcher (1991) synchronized estrus in cattle with GnRH
followed 7 d later with an injection of PGF2a compared to a single injection of
PGF2a. Estrous response and synchrony of estrus were more favorable with the
combination of GnRH and PGF2cl compared to PGF2a alone, with estrus being
expressed 2 to 3 d after PGF2a injection. In addition, there were no differences in
fertility between the two groups. The combination of GnRH and PGF2a, with


95
Because the two breeds used in the present study were managed as a single
group, social interactions (Dobson and Smith, 2000; Landaeta et al., 2002)
between breeds may have also acted to prevent a maximum estrous response.
The effect that social interactions could have had on estrous response could
have been magnified since the Angus heifers had only two weeks to acclimate
not only to the new location but also to being housed together with the Brahman
x Angus heifers. Although there were no significant replication or breed x
replication effects on estrous response in the present study, Angus heifers had a
slightly increased estrous response in the second replication (63.6%) compared
with the first replication (53.3%). This observation suggests that the added
handlings and time spent together may have acclimated the heifers to each other
from physiological and behavioral aspects.
Other reasons for the decreased estrous response could be attributed to
the stress of daily working of cattle or an environmental effect, seasonal in this
case. The effects of stress related hormones (i.e. corticolds and progesterone)
on the actions and (or) concentrations of hormones associated with estrus have
been well documented (Cumming et al., 1976; Pool et al., 1979; Hardin and
Randel, 1982). Increased circulating concentrations of adrenocorticotropic
hormone following an induced stress increased circulating concentrations of both
cortisol and progesterone (Hein and Allrich, 1992; Bolados etal., 1997).
Therefore, stress may lead to increased concentrations of progesterone during
estrus and the consequent loss of the appropriate progesterone to estradiol ratio
resulting in a decreased expression of estrus (Allrich, 1994; Duchens et al.,


83
progesterone concentrations at PGF2a affected (P > 0.10) total CL regression.
Corpus luteum volume and progesterone concentrations at PGF2ct were similar
(P >0.10) between breeds (Table 3-1). Diameter of CL at PGF2a was similar (P >
0.05) between Angus (18.5 0.6 mm) and Brahman x Angus (18.3 0.5 mm)
heifers. Additionally, CL volume and CL diameter at PGF2ct were positively
correlated (Pearson correlation = 0.85; P < 0.0001). Across breeds,
progesterone concentrations at PGF2a were influenced (P < 0.05) by CL volume
(Figure 3-3). For every 1000 mm3 increase in CL volume, there was a 1.28
ng/mL increase in progesterone concentrations.
Estrous response after PGF2a, mean interval from PGF2ato the onset of
estrus, and duration of estrus were not influenced (P > 0.10) by either breed or
replication (Table 3-2). Among heifers that regressed their CL, estrous response
was not affected (P > 0.10) by replication, but more (P < 0.05) Brahman x Angus
heifers expressed estrus compared with Angus heifers (Table 3-2). Across
breeds, 72% (36/50) of heifers that regressed their CL expressed estrus,
whereas 28% (14/50) failed to express estrus. None of the heifers that failed to
regress their CL expressed estrus. Total number of mounts received during
estrus was not affected (P >0.10) by replication, but was greater (P < 0.05) in
Brahman x Angus than Angus heifers (Table 3-2). The distribution of mounting
activity during estrus was affected by breed (P < 0.05), period (P < 0.0001), and
breed x period (P < 0.05; Figure 3-4). Furthermore, the breed response curves
were different (P < 0.05) between the Angus and Brahman x Angus heifers
throughout the duration of estrus. The number of heifers in estrus within a period


232
Figure H-2. Plasma LH concentrations (LS Means) in cycling Brangus heifers
receiving 100 gg GnRH on d 6 of the estrous cycle.


11
McNeilly, 1981, Wallace et al 1988). Moreover, LH concentrations between d 2
and 4 of the estrous cycle did not differ among Brahman (Bos indicus), Senepol
(tropical Bos taurus) and Angus (temperate Bos taurus) cows (Alvarez et al.,
2000). On d 18 to 19 or late luteal phase of the estrous cycle, LH secretion is
variable (Rahe et al., 1980) with an increase in the frequency of LH pulses (Cupp
et al., 1995). Furthermore, the late luteal phase of the estrous cycle is
characterized by a decline in luteal progesterone followed by an Increase In the
concentrations of estradiol, which initiates the preovulatory surge of LH (Kesner
et al., 1981). Moreover, estradiol acts on the hypothalamus to Increase secretion
of GnRH and (or) at the pituitary to increase its sensitivity to GnRH (Parvizi,
2000; Smith and Jennes, 2001) to enhance the preovulatory surge of LH.
Breed differences between Bos taurus and cattle of Bos indicus breeding in
the amplitude and timing of the LH surge have been reported (Randel, 1976).
The interval from the onset of estrus to LH surge Is approximately 0.4 + 3.4 h In
Brahman, 6.8 2.1 h in Brahman x Hereford, and 5.3 1.3 h in Hereford cows.
In addition, the interval between the LH surge and ovulation is 18.5 3.1 h In
Brahman, 22.2 2.6 h in Brahman Hereford, and 23.3 2.1 h in Hereford
cows. Thus, Bos indicus cows have decreased intervals from estrus to the LH
surge and from the LH surge to ovulation than Bos taurus and Bos indicus x Bos
taurus cows.
Estrogens and Progesterone
The preovulatory release of GnRH occurs in the presence of increased
estrogen and decreased progesterone concentrations in the circulation. As


8
many sites in the brain; however, major projections responsible for the control of
the anterior pituitary function end in the median eminence. At this point, GnRH is
released into the fenestrated capillaries of the hypophyseal portal system, which
carries GnRH to the anterior pituitary to regulate gonadotrophin secretion.
Several neurotransmitters, neuropeptide receptor mRNA, and proteins are
expressed by GnRH neurons, which regulate GnRH neuronal activity (for review,
see Parvizi, 2000; Smith and Jennes, 2001). Several neurotransmitters and
neuropeptides including catecholamines, gamma-aminobutyric acid (GABA),
glutamine, neuropeptide Y, neurotensin, vasoactive intestinal polypeptide (Smith
and Jennes, 2001), and nitric oxide (Parvizi, 2000) mediate the stimulatory
effects of estradiol on GnRH secretion. Estradiol may act directly on certain
GnRH neurons through specific nuclear receptors (Roy et al., 1999). However,
most studies have not been able to detect estrogen receptors in GnRH neurons,
or to detect estrogen accumulation in nuclei of GnRH neurons (Smith and
Jennes, 2001). For estradiol to stimulate secretion of GnRH from the GriRH
neurons, estradiol apparently must also stimulate the neurotransmission of other
afferent neuronal systems controlling GnRH neurons (Parvizi, 2000; Smith and
Jennes, 2001). Parvizi (2000) and Smith and Jennes (2001) also suggest that
progesterone may also restrict GnRH secretion by regulating the different
neuronal systems controlling GnRH neurons.
Follicle Stimulating Hormone (FSH) and Luteinizing Hormone (LH)
Gonadotropin releasing hormone released from the hypothalamus
regulates gonadotrophin secretion from the anterior pituitary. Follicle stimulating
hormone is involved in the recruitment and development of follicles. In contrast,


75
maintained at the Santa Fe Beef Research Unit. Heifers were pastured in a
single group and received 25 mg PGF2aim. (LutalyseSterile Solution;
Pharmacia Animal Health, Kalamazoo, Ml) on d -14 and the second and third
injections of 12.5 mg PGF2a i.m. on d -3 and -2 of the experiment (Figure 3-1).
Heifers were fitted with electronic estrus detection devices (HeatWatch, DDx,
Boulder, CO) on d -3 to determine the onset of estrus during the 5 d after PGF2[1
(d -3). The onset of estrus was defined as 3 or more mounts within a 4 h period
(Landaeta et al., 1999; Lorton et al., 1999). After the expression of estrus (d 0),
heifers of each breed type received 25 mg of PGF2a- i.m. on either d 6 or 7 of the
estrous cycle. At PGF2a (d 0) both ovaries were examined via ultrasonography
using a real time, B-mode ultrasound (Aloka 500V, Corometrics Medical
Systems, Wallingford, CT) equipped with a 7.5 MHz transducer. At ultrasound
examination, follicles > 5 mm, height and width of all luteal structures, diameter of
any luteal cavities and their respective locations on the ovaries were measured
with the internal calipers of the ultrasound machine and recorded. Volume of the
CL was calculated using the formula for the volume of a sphere (7id3/6). When a
luteal cavity was present, its volume was subtracted from the volume of the outer
sphere resulting in net luteal volume (CL volume) represented by luteal tissue.
Daily blood samples were collected via jugular venipuncture from PGF2a (d 0)
until heifers either exhibit estrus or for 7 d after PGF2c, for heifers that did not
exhibit estrus. After collection of blood samples, they were immediately placed
on ice and centrifuged (3000 rpm) within 4 h. Plasma was separated and stored
at -20 C


72
two injections of PGF2a were not necessary and that it does not seem to matter
whether a single injection of PGF2a or doprostenol was used.
Summary
In order for estrus synchronization programs to have a high rate of success,
maximizing the synchrony of estrus is essential. Simultaneously, the
synchronization programs must be inexpensive and easy to administer in order
for producers to adopt them. In order to develop such a system, regulation of
follicular development as well as the lifespan of the CL must be undertaken.
Current studies have focused on synchronizing ovulation in conjunction with
timed-AI and eliminating estrus detection. Several estrus synchronization and/or
ovulation protocols include the use of GnRH in combination with PGF2a.
Generally, protocols combining GnRH and PGF2n are effective for synchronizing
estrus in cattle of Bos taurus breeding but less effective in cattle of Sos indicus
breeding. Moreover, few studies have made direct breed comparisons of the
endocrine and ovarian responses of cattle synchronized with the GnRH plus
PGF2a systems. The limited data available and the decreased pregnancy rates
already achieved in Bos indicus x Bos taurus cattle justify additional research.
Therefore, the following experiments were conducted to characterize endocrine,
ovarian and reproductive responses to the administration of GnRH and (or)
PGF2o in Bos indicus x Bos taurus cattle, as well as to make direct comparisons
of these responses between Bos taurus cattle and animals of Bos indicus
breeding.


204
In summary, there was no additional benefit of splitting the injections of
PGF2a on estrous, conception or pregnancy rates, which is supported by the
similar CL regression rates between the single versus the split dose of PGF2().
Timed-AI, synchronized and thirty-day pregnancy rates were greater for cycling
than noncycling cows. Furthermore, initiation of timed-AI at 72 h after PGF2a
resulted in acceptable pregnancy rates in cycling cows with a functional CL at
PGF2a but the timed-AI pregnancy rates were considerably reduced in cycling
and noncycling cows with low progesterone concentrations at PGF2a. The GnRFI
+ PGF2a + MGA protocol used in the present study resulted in high percentage of
cattle pregnant in the first thirty-days of the breeding season, providing an
additional benefit to producers by enhancing the overall reproductive
performance by getting cows pregnant early in the breeding season.
Implications
The split dose of PGF2a did not improve the effectiveness of the GnRFI +
PGF2a + MGA, nor did It enhance the CL regression in lactating Bos tauriis x Bos
indicus cows compared to the single dose of PGF2a. Therefore, the additional
working of cows to administer the second PGF2a does not appear necessary in
mature cows of Bos taurus x Bos indicus breeding. Furthermore, synchronized
pregnancy rates were greatest in cycling cows with increased progesterone
concentrations at PGF2a administration. Therefore, further research needs to be
conducted on modifying existing synchronization systems that allow for increased
progesterone concentrations prior to a PGF2a induced luteolysis to enhance
pregnancy rates in mature cows of Bos indicus breeding.


172
Table 6-1. The effect of treatment and cycling status on estrous, conception and
pregnancy rates of Bos taurus x Bos indicus cows synchronized with a
modified GnRH + prostaglandin F2a (PGF2a) protocol In combination
PGF2a treatment8, % (n)
Cycling statusb, % (n)
Variable
Single
Split
Cycling
Anestrous
Three day estrous
rate, %c
37.2 (145)
30.2(149)
33.7(184)
33.0(103)
Conception rate, %d
61.1 (54)
42.2 (45)
54.8 (62)
47.1 (34)
Timed-AI pregnancy
rate, %e
33.0 (91)
41.4 (104)
46.7h (120)
24.6' (69)
Synchronized
pregnancy rate, %f
43.5(145)
41.6(149)
49.5h (182)
32.0' (103)
30 d pregnancy rate,
%9
79.5 (146)
81.3 (150)
85.3h (184)
73.8' (103)
8 Cows received 100 pg GnRH (Fertagyl) on d 0 of the experiment and either 25
mg PGF2a on d 7 or 12.5 mg PGF2aon d 7 and 8. Cows that exhibited estrus
were Al approximately 8-12 h later and all cows not exhibiting estrus by d 10 of
the experiment were timed-AI with 100 pg GnRH. Cows received 0.5 mg
MGA-day'1 on d 1 to 6 of experiment.
bProgesterone concentrations on d -10, 0 were used to determine cycling status
at GnRH. Cows with plasma progesterone concentrations > 1 ng/mL for either
sample were deemed cycling and cows with progesterone concentrations < 1
ng/mL at both samples were deemed non-cycling.
Percentage of cows displaying estrus 3 d after PGF2a of the total treated.
dPercentage of cows pregnant that exhibited estrus and were Al.
Percentage of cows pregnant of the total that were timed-AI.
Percentage of cows pregnant to estrus Al and timed-AI of the total treated.
Percentage of cows pregnant during the first 30 d of the breeding season of the
total treated.
h,'Values within a category and row without a common superscript differ.
Note: Treatment x Cycling status (P >0.10) for all variables tested.


87
tended (P = 0.08) to affect the distribution of mounting activity during estrus, but it
did not change the significance values for breed, period, and breed period
when included in the model as a covariate. As the number of heifers in estrus
during a 3 h period increased, the number of mounts received increased in a
linear manner (P < 0.0001; Figure 3-5). The Angus and Brahman x Angus
heifers had similar (P > 0.05) mounting activity during the first 6 h of estrus and
during all periods for durations exceeding 15 h. However, Brahman x Angus
heifers had more (P < 0.05) mounting activity between 9 and 15 h after the
initiation of estrus than Angus heifers.
Diameter of the largest follicle on the ovaries at PGF2ci was smaller (P <
0.01) for Angus than Brahman Angus heifers (Table 3-1). Although, largest
follicle size at PGF2a did not influence (P > 0.10) either the expression of estrus
or interval from PGF2a to the onset of estrus. There was no effect (P > 0.10) of
breed on the survival curves for heifers not observed in estrus (Figure 3-6).
Discussion
A PGF2a induced CL regression on either d 6 or 7 of the estrous cycle was
15.6% greater for Angus than Brahman Angus heifers in the present
experiment. Whether the decrease in luteolysis of the Brahman Angus heifers
was significant depends on the method of statistical analysis used. Not
withstanding, results from the present experiment warrant the need for additional
research with larger experimental numbers to provide unequivocal evidence that
the early developing CL (< d 7) in cattle of Bos indicus breeding are less
responsive to administration of PGF2ct.


203
ovulation and the arrival of viable sperm capable of fertilizing the oocyte are more
likely to occur in timed-AI programs where GnRH is injected at the time of
insemination. To reduce the magnitude of this problem, Insemination of cows
could be performed 12 to 16 h following GnRH administration. However, this
involves managing and handling cattle through the working facilities an additional
time and most beef producers would prefer to reduce the total number of cattle
handlings. It is interesting to note that the Al sire that had the lowest conception
rate also had the lowest timed-AI pregnancy rate. Therefore, there may be other
factors besides an asynchrony of the arrival of viable sperm with ovulation that is
having a negative effect on conception rates. This could include abnormal
environmental effects at semen collection, improper freezing and processing of
semen (Ennen et al., 1976), or semen from that particular bull does not freeze
well (Saacke, 2002).
In addition, other factors associated with the sperm itself could also be
influencing the timed-AI pregnancy rates (Macmillan and Watson, 1975; Ennen et
al., 1976; Bellen et al., 1994; Saacke, 2002). Males can differ in the numbers of
sperm required to reach maximum fertilization (Saacke, 2002), and bulls
requiring more sperm would be considered to have compensable seminal
deficiencies. This includes a number of known factors like the presence or
absence of specific proteins on sperm cells (Bellen et al., 1994), the effects of
processing the semen during the freezing and (or) thawing process (Ennen et al.,
1976), and the rate at which the acrosome reaction takes place inside the
reproductive tract (Macmillan and Watson, 1975).


212
ovulatory response to GnRH on d 6 of the estrous cycle in Angus and Brahman x
Angus heifers Is not affected by progesterone concentrations when a large
dominant follicle is present. However, the luteolytic response to PGF2a 7 d after
GnRH is less in heifers with early developing CL with no prior progesterone
exposure compared to heifers with a new developing CL and increased
progesterone concentrations before PGF2o.
Recent research suggests that progesterone acts within the bovine CL as
an autocrine and (or) paracrine regulator of luteal development and activity
(Shams and Berisha, 2002). Some of the rapid hormonal effects on the CL are
clearly mediated by modulating the affinity of PGF2a receptors by progesterone or
Its metabolites (Picard, 1998). Progesterone may be binding the PGF2c, receptor
and inducing a conformational change that prevents PGF2o from binding to Its
own binding site (Picard, 1998). Additionally, progesterone priming of the uterus
Is necessary to stimulate uterine synthesis of PGF2a (Lamming and Mann, 1995;
Staples et a!., 1998; Kieborz-Loos et al., 2003) and the normal changes required
to Induce luteolysis in mature CL (Meyer et al., 1988; Mirando et al., 1993).
Furthermore, progesterone may play an active role in the inhibition of luteal
regression in the early developing CL by preventing apoptosis (Rae et al., 1998;
Friedman et al., 2000; Schams and Berisha, 2002). Further Investigation is
necessary to evaluate If pretreatment with different doses of progesterone and
(or) a progesterone antagonist will differentially affect functionality of PGF2a
receptors of early luteal cells compared to developed luteal cells by either
reducing the ability of PGF2a to bind with its own receptors or decreasing


124
by differences in sensitivities of the pituitary between Angus, Brangus, and
Brahman Angus heifers. Early studies (Rhodes et al., 1978) reported
differences in the responsiveness of the hypothalamic-pituitary axis between Bos
taurus and Bos indicus cows to an exogenous estrogen treatment. Furthermore,
Brahman heifers release less LH during the pre-ovulatory surge of LH and have
an earlier LH surge relative to the onset of estrus than Hereford cows (Randel,
1984). However, Rhodes et al. (1978) did not observe any differences in basal
LH concentrations between ovariectomized Brahman and Hereford cows.
Similarly, Alvarez et al. (2000) reported no differences among Angus, Brahman,
and Senepol cows in either mean concentrations of LH or maximum LH
concentrations between d 2 and 14 of the estrous cycle.
Even though all of the characteristics associated with the GnRH induced LH
response were decreased in Brahman Angus and Brangus heifers compared
with Angus heifers, ovulation rates after GnRH were similar across breeds.
Therefore, even though the Bos indicus heifers had decreased LH concentrations
in response to a GnRH challenge, LH concentrations were still adequate enough
to induce ovulation under the conditions of the present experiment, which are
presumed to be reduced stress conditions due to the acclimation process heifers
were exposed to prior to GnRH treatment. Although, no control or untrained
heifers were used in the present experiment to represent acute stress conditions
it is unclear how much affect the acclimation process had on reducing acute
stress related responses. However, the question remains, will LH release be
compromised due to an acute stress response; thereby, preventing ovulation


APPENDIX
A ABSTRACT FOR EXPERIMENT 1 219
B ABSTRACT FOR EXPERIMENT 2 221
C ABSTRACT FOR EXPERIMENT 3 224
D ABSTRACT FOR EXPERIMENT 4 226
E CORPUS LUTEUM REGRESSION IN CYCLING ANGUS AND
BRAHMAN x ANGUS HEIFERS (EXPERIMENT 1) 228
F ODDS RATIOS AND CONFIDENCE INTERVALS (EXPERIMENT 1) 229
G PRE-SYNCHRONIZATION ESTRUS RESPONSE IN CYCLING ANGUS,
BRAHMAN, BRAHMAN X ANGUS AND BRANGUS HEIFERS
(EXPERIMENT 2) 230
H PLASMA LH CONCENTRATIONS FROM GNRH INJECTION FOR
HEIFERS IN THE ANGUS, BRANGUS, AND BRAHMAN X ANGUS
BREED GROUPS (EXPERIMENT 2) 231
I REGRESSION OF TOTAL CORPUS LUTEUM VOLUME WITH
PROGESTREONE CONCENTRATIONS AT PGF2a INJECTION IN
ANGUS, BRANGUS AND BRAHMAN X ANGUS HEIFERS TREATED
WITH GnRH FOLLOWED BY PGF2o 7 DAYS LATER
(EXPERIMENT 2) 237
J ODDS RATIOS AND CONFIDENCE INTERVALS (EXPERIMENT 3) 238
K ESTROUS, CONCEPTION, AND PREGNANCY RATES OF CYCLING
AND NONCYCLING Bos taurus X Bos indicus COWS AND SURVIVAL
ANALYSIS DESCRIBING THE PROPORTION OF CYCLING AND
NONCYCLING Bos taurus x Bos indicus COWS THAT DID NOT EXHIBIT
ESTRUS AFTER BEING SYNCHRONIZED WITH GNRH AND EITHER
SINGLE OR SPLIT DOSES OF PROSTAGLANDINF2a (PGF2a) IN
COMBINATION WITH MELENGESTROL ACETATE (MGA)
(EXPERIMENT 4) 240
LIST OF REFERENCES 245
BIOGRAPHICAL SKETCH 286
vii


225
PGF2o. Mean progesterone concentrations for the 2 d before GnRH and at
GnRH were greater (P < 0.0001) for HP (3.6 0.1 ng/mL) than LP (0.3 0.1
ng/mL) heifers. Heifers in the HP group had a linear increase in progesterone
concentrations from GnRH until PGF2ct, whereas LP heifers had an ascending
progesterone curve that remained parallel but of decreased concentrations than
the HP group until PGF2ct. Diameter of the largest follicle at GnRH and ovulation
rate after GnRH was similar (P > 0.10) between breeds and progesterone
groups. All heifers had a functional CL at PGF2ol. More (P < 0.001) HP (11/13 =
84.6%) heifers had an accessory CL at PGF2a than LP (0/13 = 0%) heifers. CL
regression was similar (P >0.10) between AN and BA heifers. More (P < 0.05)
HP heifers (13/13 =100%) regressed their CL after PGF2a compared to LP
heifers (10/13 = 76.9%). Progesterone concentrations at PGF2a were greater (P
< 0.0001) for HP (8.9 0.7 ng/mL) than LP heifers (2.9 0.7 ng/mL). There
were no breed effects (P > 0.10) on progesterone concentrations or size of the
largest follicle at PGF2a. Estrous response following PGF2a was greater (P <
0.001) in BA (16/19 = 84.2%) than AN heifers (2/7 = 28.6%), but was not
influenced (P < 0.10) by progesterone group. In conclusion, progesterone
exposure 7 d prior to PGF2ol had no effect on luteolysis.
Key Words: Bos indicus, GnRH, PGF2a, progesterone concentrations.


162
response during the first 3 d of the breeding period compared to animals treated
with only PGF2a on the same day as the former group. There appear to be no
studies in the literature with an experimental design evaluating whether
progesterone priming (either low or high progesterone environments) affects the
ability of cattle to express estrus. However, the first ovulation in postpartum
anestrous beef cows is usually not preceded by a visual estrus (Odde et al.,
1980; Perry et al., 1991; Mackey et al., 2000), which could be due to a low
progesterone environment prior to the first ovulation (Inskeep et al., 1988;
Braden et al., 1989). When an exogenous progestogen is used to induce the first
ovulation in anestrous beef cows, estrus is expressed compared with the
absence of estrus in anestrous cows that did not received progesterone
treatment (Ramirez-Godinez et al., 1981; Murphy etal., 1990; Perry etal., 1991;
Mackey et al., 2000). These observations suggest that progesterone priming
may have an effect on the ability of cattle to express estrus. Results from the
present experiment suggest that even cattle that have low progesterone
concentrations such as the LP heifers still express estrus after a PGF2a induced
luteolysis.
In summary, the experimental approach to inject PGF2a on d 4, 5, and 6 of
the estrous cycle used to induce a low progesterone environment was effective.
The ovulatory response to GnRH treatment on d 6 of the estrous cycle in Angus
and Brahman x Angus heifers was not affected by progesterone concentrations
when a large dominant follicle was present. Seven days after GnRH, HP heifers
had significantly greater progesterone concentrations than LP heifers due to the


169
ng/mL. If cows were determined to be noncycling and had progesterone
concentrations >1 ng/mL on d 7, it was assumed that the GnRH treatment on d 0
Induced ovulation or luteinlzation of a follicle and a formation of a luteal structure
(Stevenson et al., 2000). For further analysis, cows were classified by
progesterone status (H = >1 ng/mL; L = <1 ng/mL) on d -10, 0, and 7 into four
major categories; 1) Cycling and high progesterone concentrations at PGF2a,
which included HFIH, FILH, and LHH. 2) Cycling and low progesterone
concentrations at PGF2c,, which included HHL, FILL, and LHL. 3) Noncycling and
low progesterone concentrations at PGF2a which included LLL, and 4) Noncycling
and high progesterone concentrations at PGF2tJ which included LLFH (Ambrose et
al., 1998; Stevenson et al., 2000). Cows with progesterone concentrations >1
ng/mL on d 7 of the experiment were used to evaluate the response to PGF2a
treatments for cows that were determined to be either cyclic or noncycling on d 0
of the experiment. Corpus luteum (CL) regression was defined by a combination
of three variables. Cows that exhibited estrus from d 8 to 10 of the experiment
were deemed to have had CL regression. Timed-AI CL regression was defined
as the percentage of cows with progesterone concentrations > 1.0 ng/mL at the
initial PGF2athat failed to exhibit estrus and had progesterone concentrations <
1.0 ng/mL at timed-AI. Total CL regression was defined as the percentage of
cows with progesterone concentrations > 1.0 ng/mL at PGF2a and either exhibited
behavioral estrus or had progesterone concentrations < 1.0 ng/mL 72 h following
the initial PGF2tltreatment.


163
presence of the original CL and accessory CL in 85% of the HP heifers
compared to only an accessory CL in the LP heifers. As a result, the PGF2a
Induced CL regression was greater for HP than LP heifers. Additionally,
progesterone concentrations at PGF2a had a negative linear effect on CL
regression rate in LP heifers.
Implications
Results from the present experiment suggest that a PGF2a induced
luteolysis of the early developing CL is compromised in cattle with decreased
progesterone concentrations, whereas luteolysis is enhanced by Increased
progesterone concentrations. The response was similar between the Angus and
Brahman x Angus heifers. These observations suggest a possible reason for the
decreased effectiveness of the GnRH + PGF2a synchronization systems in
anestrous cattle. Furthermore, the experimental model developed in the present
experiment can be used to further evaluate how circulating progesterone
concentrations influence ovarian, endocrine, and uterine responses in cattle.


251
Colazo, M.G., J.P. Kastelic, and R.J. Mapletoft. 2003. Effects of estradiol
cypionate (ECP) on ovarian follicular dynamics, synchrony of ovulation, and
fertility in CIDR-based, fixed-time Al programs In beef heifers.
Theriogenology 60: 855-865.
Coleman, D.A., F.F. Bartol, C.H. Rahe, and-J.R. Chenault. 1988. Endocrine and
reproductive responses of beef cattle to a synthetic gonadotropin-releasing
hormone agonist (fertirelin acetate). Theriogenology 30: 149-157.
Copelln, J.P., M.F. Smith, H.A. Garverick, R.S. Youngquist, W.R. Jr McVey, and
E.K. Inskeep. 1988. Responsiveness of bovine corpora ltea to
prostaglandin F2 alpha: comparison of corpora ltea anticipated to have
short or normal lifespans. J. Anim. Sci. 66: 1236-1245.
Cornwell, D.G., J.F. Hentges Jr., and M.J. Fields. 1985. Lutalyse as a
synchronizer of estrus in Brahman heifers. J. Anim. Sci. 61 (Suppl 1): 416
(Abstr.).
Crowe, M.A., D. Goulding, A. Baguisi, M.P. Boland, and J.F. Roche. 1993.
Induced ovulation of the first postpartum dominant follicle In beef suckler
cows using a GnRPI analogue. J. Reprod. Frtil. 99: 551-555.
Cruz, L.C., E.R. doValle, and D.J. Kesler. 1997. Effect of prostaglandin F2aand
gonadotropin releasing hormone-induced luteinizing hormone releases on
ovulation and corpus luteum function of beef cows. Anim. Reprod. Sci. 49:
135-142.
Cumming, I.A., M.B. White, R. Baxter, and J.K. Findlay. 1976. Effect of oestradiol
benzoate, LH-RH, and stress onto the Intervals between prostaglandin
analogue treatment, the preovulatory LH peak and ovulation in cattle. Proc.
8th Int. Cong. Anim. Reprod. & Al., Krakow, Poland 3: 453.
Cupp, A.S., T.T. Stumpf, F.N. Kojima, L.A. Werth, M.W. Wolfe, M.S. Roberson,
R.J. Kittok, and J.E. Kinder. 1995. Secretion of gonadotropins change
during the luteal phase of the bovine oestrous cycle In the absence of
corresponding changes in progesterone or 17b-oestradiol. Anim. Reprod.
Sci. 37: 109-119.
Cuq, P. 1975. Characteristics of the ovarian function of the female zebu (Bos
indicus) In the Sudano-Sahel zone of tropical West Africa. Bull. Assoc.
Anat. (Nancy) 59: 139-144.
Curran, S., R.A. Pierson, and O.J. Ginther. 1986. Ultrasonographic appearance
of the bovine conceptus from days 20 through 60. J. Am. Vet. Med. 189:
1295-1302.


222
Estrus was monitored using HeatWatchforthe pretreatment synchronization
and treatment phase of the experiment. There was no effect (P > 0.10) of breed
on size of the largest follicle at GnRH, mean plasma estradiol concentrations
before GnRH, ovulation rate after GnRH, or mean CL volume at GnRH. All
heifers had progesterone concentrations > 1 ng/mL at GnRH (P > 0.10) and for
the 480 min after GnRH were not affected (P > 0.10) by breed. There were
breed (P < 0.05), time (P < 0.0001) and breed x time (P < 0.0001) effects on LH
concentrations after the GnRH injection. Mean LH concentrations were greater
(P < 0.05) in AN (7.0 0.8 ng/mL) compared with BR (4.6 0.8 ng/mL) and BA
(2.9 0.8 ng/mL) heifers. Mean LH peak-height in BR (13.9 3.4 ng/mL) heifers
was similar (P >0.10) to that for AN (21.9 + 3.4 ng/mL) and BA (8.0 3.4 ng/mL)
heifers. However, LH peak-height was greater (P < 0.05) in AN compared with
BA heifers. Interval from GnRH to LH peak was similar (P > 0.10) between AN,
BR and BA heifers (overall mean = 74.2 min). Estrous response after PGF2ct,
follicle size at PGF2ot, interval from PGF2cl to the onset of estrus, duratiomof
estrus, and total number of mounts received during estrus were not influenced (P
>0.10) by breed. All heifers had a functional CL and progesterone > 1 ng/mL at
PGF2o. All but the single AN heifer that did not ovulated after GnRH, had an
accessory CL at PGF2a- Mean volume of the original CL and accessory CL, as
well as total CL volume and mean progesterone concentrations at PGF2a were
similar (P > 0.10) between AN, BR and BA heifers. The PGF2a treatment
induced luteolysis in all heifers regardless of breed and the decline in
progesterone concentrations after PGF2a were not influenced (P > 0.10) by breed


55
significant implications on the type of estrus synchronization systems used
relative to how and when PGF2 is used to induce luteolysis.
In general, when CL regression is induced by exogenous PGF2o treatment
at random stages of the estrous cycle, estrus will be displayed within 2 to 7 d.
The large variation for the interval to estrus is likely due to differences in follicle
development at luteolysis (Fogwell et al., 1986; Sirois and Fortune, 1988; 1990).
Sirois and Fortune (1988) observed that size of the dominant follicle at luteolysis
was negatively correlated with the interval to the LH surge, suggesting that the
interval to estrus may be determined by the size of the preovulatory follicle at
luteolysis. The interval to estrus in cows and heifers administered PGF2a early in
the estrous cycle (d 5 to 9) is shorter than in cattle injected late in the estrous
cycle (King et al., 1982; Tanabe and Hann, 1984; Watts and Fuquay, 1985).
Therefore, cattle early in their estrous cycle are likely to have a dominant follicle
from the first follicular wave that is capable of ovulation immediately after
luteolysis. Unlike the first follicular wave, the second and (or) third follicular
waves do not emerge in a synchronous fashion resulting in longer and more
variable intervals from PGF2ct treatment to estrus.
Estrus Synchronization Systems and Time Artificial Insemination
The main purpose for synchronizing estrus in cattle is to decrease the
number of days detecting estrus from approximately 21 d without synchronization
to approximately 5 to 7 d with synchronization so that all cattle have an
opportunity to be artificially inseminated (Al). Decreasing the numbers of days
spent detecting estrus makes Al more practical. An estrus synchronization


63
estrus detection for 5 to 7 d, has been designated as the SelectSynch system
(Geary and Whittier, 1999). Cattle are typically inseminated 8 to 12 h after the
observed estrus. Furthermore, the percentage of synchronized cattle is
increased and the variation in the interval from PGF2a to the onset of estrus is
reduced compared to a single injection of PGF2a (Thatcher et al., 1989;
Twagiramungu et al., 1992). The mean interval from PGF2a injection to estrus is
51 h in Bos taurus cattle (Twagiramungu et al., 1992; Downing et al., 1998).
Conception rates for SelectSynch are similar and (or) increased compared to
cattle inseminated after a single injection of PGF2a (Twagiramungu et al., 1992;
Stevenson et al., 2000).
The SelectSynch system was modified to eliminate the need for estrus
detection. It'Consisted of a GnRH injection followed 7 d later by an injection of
PGF2o. A second dose of GnRH was injected 48 h after the injection of PGF2o
and ail cows are timed-AI 8 to 18 h later, with maximum pregnancy rate achieved
when cows are inseminated 16 h after the second GnRH (Pursley et al., 1998).
The second GnRH injection was administered to synchronize ovulation of the
newly recruited dominant follicle prior to timed-AI. The system was called the
OvSynch system and was first tested in lactating dairy cows and dairy heifers
(Pursley et al., 1995; Burke et al., 1996; Schmitt et al., 1996b). Dairy cows
synchronized with the OvSynch and SelectSynch systems have similar
pregnancy rates (Burke et al., 1996).
Postpartum Bos taurus beef cows synchronized with either SyncroMate-B
(SMB) or OvSynch have also been compared (Geary et al., 1998a). In the Geary


179
and noncycling cows. Cows with low progesterone at PGF2a had similar (P >
0.10) synchronized pregnancy rates, regardless of cycling status. Cycling cows
with high progesterone at PGF2a had greater (P < 0.05) synchronized pregnancy
rates than cows with low progesterone concentrations at PGF2c, regardless of
cycling status. Furthermore, noncycling cows with high progesterone
concentrations at PGF2a had greater (P < 0.05) synchronized pregnancy rates
than noncycling cows with low progesterone at PGF2a.
Timed-AI and total CL regression rates were similar (P >0.10) between
cows in the single and split PGF2a treatments (Figure 6-3). Furthermore, there
were no cycling status or PGF2a treatment x cycling status effects on these
variables. Additionally, timed-AI CL regression rates were numerically greater (P
> 0.10) for the noncycling cows with high progesterone at PGF2a treated with the
split PGF2a (17/18 = 94.4%) compared to those treated with the single PGF2a
(10/12 = 83.3%). Progesterone concentrations at PGF2a had a quadratic effect
(P < 0.05) on total CL regression rate (Y = 0.003x2 0.042x + 1.029; R2 = 0.03).
As progesterone concentrations at PGF2c< increased from 1.0 ng/mL to
proximately 6.0 ng/mL total CL regression rate decreased and total CL
regression rate increased for progesterone concentrations > 7.0 ng/mL.
There was no effect (P > 0.10) of Al technician on conception, timed-AI,
and synchronized pregnancy rates. Conception rate tended (P = 0.06; Table 6-5)
to be affected by Al sire and there was considerable variation as conception
rates ranged from 30.8 to 62.5%. Furthermore, there was an Al sire effect (P <
0.05) on timed-AI pregnancy rates (Table 6-5). Three timed-AI sires had


80
regression. The effect of the number of heifers in estrus during each 3 h period
on the number of mounts received during each period and the effect of CL
volume on progesterone concentrations at PGF20! were analyzed by regression
analysis using the GLM procedure of SAS.
Results
Corpus luteum regression was greater (P = 0.04) for Angus than Brahman
x Angus heifers when the statistical analysis was conducted using GENMOD
(Table 3-1). Using the GENMOD procedure, there was not significant breed x
replication effect on CL regression; therefore, the model was analyzed without
the interaction and breed effect tended to be significant (P = 0.06). However,
when data were analyzed with Logistic Regression there was no breed effect (P
= 0.10).
Mean progesterone concentrations for heifers that did not regress their
CL, including one Angus and six Brahman x Angus heifers after PGF2a, were
different (P < 0.0001) compared to Angus (n = 25) and Brahman x Angus (n =
25) heifers that regressed their CL (Figure 3-2). Mean progesterone
concentrations one day after PGF2a were greater (P < 0.05) for heifers that did
not regress their CL (1.1 0.2 ng/mL) than heifers that regressed their CL (0.5
0.1 ng/mL). For heifers that regressed their CL, progesterone concentrations
after PGF2c! were < 1.0 ng/mL by 24 h after PGF2a and remained <1.0 ng/mL
through 96 h. In contrast, progesterone concentrations of heifers that did not
regress their CL declined to concentrations > 1.0 ng/mL by 24 h after PGF2cl and
remained > 1.0 ng/mL until 96 h (Figure 3-2). Neither CL volume nor


APPENDIX G
PRE-SYNCHRONIZATION ESTRUS RESPONSE IN CYCLING ANGUS,
BRAHMAN, BRAHMAN X ANGUS AND BRANGUS HEIFERS (EXPERIMENT 2)
0
(A
C
o
a
0
i_
(A
3
i
+-
(A
UJ
An Br Br x An An x Br
Breed
Figure G-1. Pre-synchronization estrus response in cycling Angus (An), Brahman
(Br), Brahman x Angus (Br x An) and Brangus (An x Br) heifers.
Heifers were pre-synchronized with 25 mg PGF2a followed 11 d later by
two 12.5 mg PGF2a Injections administered 24 h apart. a,bBreed (P <
0.05).
230


210
Brangus and Brahman Angus heifers. Early studies (Rhodes et al., 1978)
reported differences in the responsiveness of the hypothalamic-pituitary axis
between Bos taurus and Bos indicus cows to an exogenous estrogen treatment,
even though the authors did not observe any differences in basal LH
concentrations between the two breed groups. Likewise, Alvarez et al. (2000)
observed no differences among Angus, Brahman, and Senepol cows in either
mean concentrations of LH or maximum LH concentrations between d 2 and 14
of the estrous cycle. These observations warrant additional research evaluating
the differences between cattle of Bos taurus and Bos indicus breeding in LH
stores and synthesis in the pituitary, and sensitivity of the hypothalamus and
pituitary to hormonal challenges.
Although, it cannot be determined from the present experiment, it would be
interesting if similar endocrine responses would have been observed if the
heifers had not been acclimated to the frequent working conditions and the stress
associated to the workings was not reduced. Acute stress has been reported to
negatively affect hypothalamic function (Dobson et al., 2001). Pulsatile patterns
of GnRH release and frequency and amplitude of LH pulses from the pituitary are
decreased by exposure to acute stressors such as transport (Dobson and Smith,
2000; Dobson et al., 2001). Whether increased stress could have significant
implications on the overall effectiveness of estrus synchronization protocols using
GnRH In cattle of Bos indicus breeding under regular management conditions is
unclear. However, results from Experiment 2 warrant additional research to
determine if the decreased LH response associated with a GnRH administration


mo
,P?52


43
greatest MGA concentrations, there is still not enough suppression of LH pulses
to initiate dominant follicle turnover.
Exogenous progesterone can induce regression of persistent follicles
allowing for recruitment of a new wave of follicular growth (Savio et al., 1993a;
Anderson and Day, 1994; Fike et al., 1997). The mechanism by which
regression of dominant follicles is induced most likely is due to reduction of
pulsatile secretion of LH induced by increasing peripheral concentration of
progesterone or progestagens (Stock and Fortune, 1993; Anderson and Day,
1994; Taylor et al., 1994). Acute administration of progesterone during a period
of progestogen treatment has also been reported to initiate atresia of dominant
follicles and synchronize emergence of a new follicular wave in cattle of Bos
indicus breeding (Cavalieri et al., 1997; Cavalieri et al., 1998a; 1998c).
Combination of Estrogens and Progestagens
Administration of estrogens during progesterone-based treatments at
different stages of follicle development also alters follicle development (Eta et al.,
1995; Tributa et al., 1995; Dlskin et al., 2002). Bo et al., (1995) administered 5
mg of estradiol 170 at the Initiation of a norgestomet implant at varying stages of
dominant follicle development and observed emergence of a new follicle wave
4.3 d after treatment. Emergence of a new follicular wave was similar for heifers
treated on d 3 (growing phase), 6 (early static phase), or 9 (regressing phase) of
the estrous cycle. The authors concluded that treatment with estradiol 170 in
combination with a norgestomet implant is effective in synchronizing follicle
emergence regardless of the stage of dominant follicle development.


74
with PGF2cl appeared to be associated with compromised expression of estrus
(Orihuela et al., 1983; Pinheiro et al., 1998; Rekwot et al., 1999). However, the
decreased expression of estrus may also be associated with incomplete
luteolysls after PGF2a resulting in the blood progesterone concentrations that are
great enough to prevent the expression of estrus (Pinheiro et al., 1998; Rekwot
et al., 1999; Mattoni and Ouedraogo, 2000). In order for cattle producers In
tropical and subtropical environments to Implement the most effective estrus
synchronization systems, a complete understanding of how PGF2a functions In
cattle of Bos indicus breeding is necessary.
The objective of this experiment was to determine the effectiveness of a
single Injection of PGF2a administered during the early estrous cycle (d 6 or 7) to
initiate luteolysis in Angus and Brahman Angus heifers.
Materials and Methods
The experiment was replicated twice during the fall (September to
November) of 2000 at the Santa Fe Beef Research Unit, Department of Animal
Sciences, University of Florida. In replication 1, cycling Angus (Bos taurus; n =
13; mean BW = 379 kg), and Brahman Angus (Bos indicus x Bos taurus 3/8
Brahman x 5/8 Angus and 5/8 Brahman x 3/8 Angus; n=16; mean BW = 451 kg)
heifers that were approximately 18 to 20 months old were Initially synchronized
with a modified two-Injectlon PGF2o protocol. Angus heifers were moved from an
extensively managed cattle operation (Deseret Cattle and Citrus, Deer Park, FL)
to the Santa Fe Beef Research Unit approximately two weeks prior to the start of
the experiment and were co-mlngled with Brahman x Angus heifers, which were


242
Table K-2. Estrous, conception, and pregnancy rates of noncycling Bos taurus x
Bos indicus cows synchronized with GnRH and either a single or split
doses of prostaglandinF2a (PGF2a) in combination with melengestrol
T reatment3
Variable
Single PGF2a
Split PGF2a
Three day estrous rate (%)b
21/58 = 36.2
13/45 = 28.9
Conception rate (%)c
10/21 =47.6
6/13 = 46.2
Timed-AI pregnancy rate (%)d
8/37 = 21.6
9/32 = 28.1
Synchronized pregnancy rate (%)e
18/58 = 31.0
15/45 = 33.3
30 day pregnancy rates (%)f
41/58 = 70.7
35/45 = 77.8
3 Cows received 100 pg GnRFI (Fertagyl) on d 0 of the experiment and either 25
mg PGF2a on d 7 or 12.5 mg PGF2ci on d 7 and 8. Cows that exhibited estrus
were Al apprpximately 8-12 h later and all cows not exhibiting estrus by d 10 of
the experiment were timed-AI with 100 pg GnRFI. Cows received 0.5 mg
MGA*day'1 on d 1 to 6 of experiment.
Percentage of cows displaying estrus 3 d after PGF2a of the total treated.
Percentage of cows pregnant that exhibited estrus and were Al.
Percentage of cows pregnant of the total that were timed-AI.
Percentage of cows pregnant to estrus Al and timed-AI of the total treated.
Percentage of cows pregnant during the first 30 d of the breeding season of the
total treated.
Percentage of cows that regressed their CL after PGF2cl of the total treated.


4
To implement the most effective estrus synchronization system in tropical
and subtropical environments, it is necessary to develop a complete
understanding of how GnRH affects the endocrine and reproductive patterns of
cattle of 60s indicus breeding; and also how cattle of Bos indicus breeding
respond to a luteolytic injection of PGF2a. Therefore, the literature review
discusses the similarities and differences during the estrous cycle including
follicle development and luteolysis in cattle of 60s taurus, Bos indicus, and 80s
taurus x 80s indicus breeding. Furthermore, it discusses factors that influence
the effectiveness of synchronization agents GnRH and PGF2a and effectiveness
of the GnRH and PGF2cc estrus synchronization system in cattle of 80s taurus,
Bos indicus, and 80s taurus x 80s indicus breeding.
The ovprall experimental objective was to evaluate the reproductive
response of 80s taurus, Bos indicus, and 80s taurus x Bos indicus cattle to
exogenous administration of GnRH and PGF2a in order to develop a better
understanding of these compounds for developing effective but practical estrus
synchronization protocols. There are three main objectives of this dissertation.
The first objective was to determine the effectiveness of a single injection of
PGF2c( administered during the early estrous cycle (d 6 or 7) to initiate corpus
luteum regression in cycling Angus and Angus x Brahman (5/8 Angus x 3/8
Brahman and 3/8 Angus x 5/8 Brahman) heifers.
The second objective was to evaluate acute ovarian responses and LH
secretory profiles during and after administration of an exogenous GnRH agonist
on d 6 of the estrous cycle in Angus, Brahman, Brangus, and Brahman x Angus


27
first wave dominant follicle occurred on approximately d 12 to 13 of the estrous
cycle in both Bos taurus and Bos indicus cattle (Ginther et al., 1989c; Moreira-
Viana et al., 2000). The second wave dominant follicle emerged on d 9 and
reached maximal size on d 16 in Bos taurus cattle (Ginther et al., 1989c).
However, in Bos indicus cows the second wave dominant follicle emerged on d 7
and reached its maximal size on d 13 (Moreira-Viana et al., 2000). The third
wave dominant follicle is usually detected on d 16 with continued development
until ovulation in Bos taurus cattle (Ginther et al., 1989c), but it was detected
earlier (d 13) in Bos indicus cows (Gomez-Alves et al., 2002). In any case, the
average diameter of preovulatory follicles range from 12 to 15 mm for Bos taurus
and Bos indicus breeds (Dufour et al., 1971; Figueiredo et al., 1997; Alvarez et
al., 2000).
Ovulation, Corpus Luteum Development, and Luteolysis
Ovulation is defined as the degradation of the follicular basement
membrane and the fragmentation of the extracellular matrix (ECM) at th apex of
the follicle wall, resulting in the release of the oocyte (Richards et al., 1998;
Richards et al., 2000). The vascular, structural and metabolic events leading to
the rupture of the follicle wall share many similarities with tumor formation and an
acute inflammatory reaction (Smith et al., 1994). Remodeling of the follicle wall
involves degradation of extracellular matrix components by proteases and is
associated with prominent changes in vascular architecture and leukocyte
infiltration. Immune cells have been implicated in several physiological
processes occurring in the ovary, and their effects are largely mediated by locally
produced cytokines (Machelon and Emilie, 1997).


217
regressed in response to the PGF2a in the current study. The reason for the
conflicting reports is unclear.
There Is also the possibility that the timed-AI at 72 h after PGF2a in the
present experiment may have been done too late In cows with low progesterone
concentrations at PGF2a In relation to the developmental competence of the
dominant follicle, resulting in the decreased pregnancy rates observed. In beef
cows synchronized with a GnRH + cloprostenol + shortterm MGA protocol
Thundathll et al. (1999) reported that only 35% of the cows were In estrus within
96 h after cloprostenol administration. In the present experiment it Is probably
safe to assume that the GnRH did not turn over all follicles resulting in cows at
different stages of follicle development at PGF2a, which also contributed to the
compromised timed-AI pregnancy rates In this sub group of cows. The use of
estradiol at the initiation of a progestogen treatment may improve the turn over of
almost all large follicles (Martinez et al., 2001), resulting in cows with more
homogeneous follicular development at PGF2a- This may improve the overall
tlmed-AI pregnancy rates.
The GnRH + PGF2ti + MGA protocol used In the present study resulted in a
high percentage of cattle pregnant in the first 30-d of the breeding season,
probably due by an Indirect benefit on enhanced reproductive performance later
In the breeding season. Since the GnRH + PGF2a + MGA protocol Induced
ovulation in some noncycling cows, more cows were cycling early In the breeding
season. Increasing the number of beef cows pregnant early in the breeding
season results in more calves born early in the subsequent calving season,


APPENDIX J
ODDS RATIOS AND CONFIDENCE INTERVALS FOR EXPERIMENT 3
Table J-1. Odds ratio and confidence Intervals using Angus heifers as the
reference for ovulation rate after GnRFI, proportion of heifers with two
corpus luteum (CL) at PGF2ci, CL regression rate and estrous response
after PGF2ol in Angus and Brahman Angus heifers exposed to either
high or low progesterone concentrations before PGF2a
Breed
Variable
Angus
Brahman
x Angus
P
value
Odds
ratio
Confidence
Interval
Ovulation rate after
GnRH, %
6/7 = 85.7
16/19 =
84.2
>0.10
1.13
0.08-10.38
Heifers with two CL at
PGF2a, <34
3/7 = 42.9
8/19 =
42.1
> 0.10
1.03
0.12-8.14
CL regression rate, %
6/7 = 85.7
17/19 =
89.5
>0.10
0.86
0.05-13.48
Estrous response, %a
2/7 = 28.6
16/19 =
<0.05
13.87
1.72-111.65
84.2
aBreed (P < 0.001).
Breed x Progesterone group (P > 0.10).
238


190
to be acceptable, which agrees with an early report of Randel (1994), indicating
that fertility in Brahman or Brahman based cows is similar to European breeds of
cows if they show estrus. Although it should be noted that In cows with
progesterone concentrations >1.0 ng/mL at PGF2a, conception rates were
decreased in the split treatment compared to the single PGF2c, treatment. Further
analysis Indicated an Al technician effect that resulted in the decreased
conception rate in that particular treatment.
Although follicular dynamics were not examined in the present experiment,
it does not appear that stage of follicular development at PGF2a influenced
conception rates. Mihm et al. (1994) reported that cows expressing estrus 60 to
72 h after PGF2a corresponds to cows with a dominant follicle in the growing
phase and with a short duration of dominance at a PGF2tI Induced luteolysis.
Furthermore, fertility was improved for follicles with a shorter duration of
dominance than follicles with longer periods of dominance (Mihm et al., 1994) In
Bos taurus cattle. Results from the present experiment do not support this
observation since interval to estrus, which should reflect follicle development, did
not Influence conception rate. However, it should be noted that the numbers of
cows in many of these categories evaluating conception rates are small and
therefore make it difficult to make adequate comparisons. Other factors that
affect conception rates include embryo mortality (Binelli et al., 2001) or variations
in the interval from estrus to ovulation (Kojima et al., 2000).
As observed with conception rates, there are conflicting reports between
the present study and reports in the literature concerning timed-AI pregnancy


208
based protocols that may prevent incomplete luteal regression (I.e. progesterone-
based protocols).
The objectives of Experiment 2 were to evaluate secretory patterns of LH
and associated ovarian events In response to an administration of GnRH (100
pg) on d 6 of the estrous cycle followed by an Injection of PGF2a 7 d later In
Angus, Brahman, Brangus, and Brahman x Angus heifers. Heifers were
acclimated to experimental conditions for three weeks prior to the experiment.
Because the pre-synchronization estrous response was significantly decreased
in the Brahman heifers, they were removed from the experiment. Angus,
Brangus, and Brahman x Angus heifers had similar dominant follicle size and
plasma estradiol concentrations prior to the GnRH injection. However, as the
percentage of Brahman breeding increased, the amount of LH released in
response to the GnRH injection was decreased on d 6 of the estrous cycle. The
Angus had a greater mean LH concentration and mean LH peak than Brangus
and Brahman x Angus heifers. Despite of the differences in LH release,
ovulation rate to the GnRH was similar between the Angus, Brangus, and
Brahman x Angus heifers. Furthermore, breed had no Influence on the PGF2o
Induced luteolysis that was initiated 7 d after the GnRH treatment. Although the
LH response to GnRH decreased as the percentage of Brahman breeding
Increased, plasma LH concentrations Increased within 15 min following GnRH
and remained greater than baseline concentrations for approximately 180 min for
all breed groups, similar to results in ovariectomized Brahman and Hereford
cows challenged with GnRH (Griffin and Randel, 1978) and Holstein heifers


194
indicated that synchronized pregnancy rates were similar to those observed in
the present experiment. In the former study, no estrus detection was used and
ail the cows were timed-AI 72 h after PGF2a. Therefore, it would be interesting to
evaluate the effects of the MGA treatment on the day of PGF2a administration on
synchrony of estrus in a 7 d GnRH + PGF2a system.
Alternatively, other sources of exogenous progesterone such as the CIDR
could be used in place of the MGA. Duration of CIDR treatment as well as
determining if the PGF2o treatment can be provided the day before CIDR removal
need to be investigated as methods to improve estrus synchrony. In dairy
heifers, Ambrose et al. (2002) administered a CIDR for 8 d plus a PGF2a injection
the day before CIDR removal. Furthermore, animals were treated with either
estradiol cypionate (ECP) or GnRH at CIDR insertion and then with ECP at CIDR
removal or with GnRH 24 h after CIDR removal, resulting in 100% synchronized
ovulation rates and over 60% timed-AI pregnancy rates. These data suggest that
synchronizing follicle development at CIDR insertion and initiating luteolysis the
day prior to CIDR removal appears to result in a very synchronous follicle
development after CIDR removal.
Administration of estradiol at the initiation of a progestogen treatment may
improve follicle turnover and the subsequent timed-AI pregnancy rates. Estradiol
administration has been reported to initiate turnover of follicles < 9 mm (Burke et
al., 2001) whereas GnRH only induces ovulation of follicles greater than 9 mm
(Macmillan and Thatcher, 1991; Vasconcelos et al., 1999; Moreira et al., 2000a).
Additionally, the effectiveness of GnRH to induce ovulation of dominant follicles


23
2000) and 60s taurus breeds (Alvarez et al., 2000). However, Gir (Sos indicus)
cows had a greater incidence of estrous cycles with three (60.0%) and four
(26.7%) waves (Moreira-Viana et al., 2000).
The dynamics of a follicular wave during the estrous cycle consist of
recruitment, selection, dominance, and either atresia or ovulation. Recruitment Is
a process where a cohort of small follicles (2 to 4 mm) reaches the tertiary stage.
Increased concentrations of FSH promote the growth of the cohort of follicles.
The stimulated growth of this cohort of follicles occurs 2 to 4 d after a surge In
FSH (Adams et al., 1992a) resulting in follicles 4 to 5 mm in diameter as
determined by transrectal ultrasound (Adams et al., 1992a). Moreover, mRNA
for FSH receptor was localized in granulosa cells of pre-antral primary follicles
(Xu et al., 1995) supporting the integral role of FSH In follicular recruitment.
Follicles that are recruited, but not selected for continued growth regress in a
process known as follicular atresia. Atresia continues until one follicle remains,
and under a favorable hormonal environment obtains the ability to ovulate. This
process is known as selection. The follicle remaining Is termed dominant, while
all other follicles are termed subordinate. The dominant follicle and the largest
subordinate follicle (second largest follicle) Initially grow in parallel (Ginther et al.,
1996b; Kulick et al., 1999). The point when growth of the largest subordinate
follicle slows, resulting in a difference In the growth rate of the two largest follicles
is known as deviation (Ginther et al., 1996b). The selected follicle exerts Its
dominance through Inhibition of the recruitment of follicles of the next follicular
wave (Lucy et al., 1992).


34
and stimulate luteal PGF20 production (Milvae, 2000). Prostaglandin F2o acts
directly on large luteal cells inducing an acute release of oxytocin, which Increase
endothelln 1 secretion by endothelial cells (Ohtanl et al., 1998). Angiotensin II
inhibits progesterone production In bovine luteal cells (Stirling et al., 1990;
Hayashl and Miyamoto, 1999). Angiotensin II release and the expression of
angiotensin-converting enzyme mRNA In the CL after Injection of PGF2a
analogue have been reported in the cow (Hayashl et al., 2001). Reduction of
progesterone production by the CL results in a processes known as functional
luteolysis.
Functional luteolysis is followed by a process known as structural luteolysis,
which Involves the physical breakdown of the CL. Induction of functional
luteolysis activates cytokine secretion from endothelial cells, which Induces the
recruitment of macrophages (Penny, 2000; Webb et al., 2002). Macrophages
have a phagocytic role on luteal cells during structural regression of the CL
(Webb et al., 2002). Furthermore, endothelln-1 stimulates the production of local
factors including cytokines like tumor necrosis factor-a (TNF-a) and interleukin-p
(IL-(3) not only by endothelial cells, but also by monocytes and T lymphocytes
(Penny, 2000; Webb et al., 2002). Monocyte chemoattractant protein 1 (MCP-1),
a member of the chemoklne family of cytokines Involved in leukocyte physiology
and trafficking, Is a potent chemoattractant for both monocytes and T
lymphocytes (Tsai et al., 1997; Penny et al., 1998). Another Immunological
mechanism that has been hypothesized to potentially regulate luteal regression
Involves the proteins that make up the major histocompatabillty complex (MHC),


283
Vizcarra, J.A., R.P. Wettemann, T.D. Braden, A.M. Turzillo, and T.M. Nett. 1997.
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serum and pituitary concentrations of luteinizing hormone and follicle-
stimulating hormone, GnRH receptors, and messenger ribonucleic acid for
gonadotropin subunits in cows. Endocrinol. 138: 594-601.
Voh Jr., A.A., E.O. Oyedipe, V. Buvanendran, and J. Kumi-Diaka. 1987. Estrus
response of indigenous Nigerian zebu cows after prostaglandin Fin
analogue treatment under continuous observation for two seasons.
Theriogenology 28: 77-99.
Vural, R., S. Kuplulu, B. Guven, and S. Ozsar. 1999. The effect of GnRH
administration on the serum LH response and ovulation in the early
postpartum period of dairy cows. Tur. J. Veter. Anim. Sci. 23 (Suppl. 1): 1-5.
Walczewska, A., W.H. Yu, S. Karanth, and S.M. McCann. 1999. Estrogen and
leptin have differential effects on FSH and LH release in female rats. Proc.
Soc. Exp. Biol. Med. 222:170-177.
Walker, W.L., R.L. Nebel, and M.L. McGilliard. 1996. Time of ovulation relative to
mounting activity in dairy cattle. J. Dairy Sci. 79: 1555 (Abstr.).
Wallace, J.M., G.B. Martin, and A.S. McNeilly. 1988. Changes in the secretion of
LH pulses, FSH and prolactin during the preovulatory phase of the oestrus
cycle of the ewe and during the luteal phase. J. Endocrinol. 116: 123-135.
Walters, D.L., D. Schams, and E. Schallenberger. 1984. Pulsatile secretion of
gonadotrophins, ovarian steroids and ovarian oxytocin during the luteal
phase of the oestrous cycle in the cow. J. Reprod. Frtil. 71: 479-491.
Wathes, D.C., and G.E. Lamming. 1995. The oxytocin receptor, luteolysis and
the maintenance of pregnancy. J. Reprod. Frtil. (Suppl. 1) 49: 53-67.
Watts, T.L., and J.W. Fuquay. 1985. Response and fertility of dairy heifers
following injection with prostaglandin F2n during early, middle, or late
diestrus. Theriogenology 23: 655-661.
Webb, R., K.J. Woad, and D.G. Armstrong. 2002. Corpus luteum (CL) function:
local control mechanisms. Domest. Anim. Endocrinol. 23: 277-285.
Weber, D.M., P.A. Fields, L.J. Romrell, S. Tumwasorn, B.A. Ball, M. Drost, and
M.J. Fields. 1987. Functional differences between small and large luteal
cells of the late pregnant vs. non-pregnant cow. Biol. Reprod. 37: 685-697.
Wehling, M. 1997. Specific, nongenomic actions of steroid hormones. Annual
Rev. Physiol. 59: 365-393.


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Custer, E.E., W.E. Beal, S.J. Wilson, A.W. Meadows, J.G. Berardinelll, and R.
Adair. 1994. Effect of melengestrol acetate (MGA) or progesterone-
releasing Intarvaglnal devices (PRID) on follicular development,
concentrations of estradiol 17-3 and progesterone and luteinizing hormone
release during an artificially lengthened bovine estrous cycle. J. Anlm. Sci.
72: 1282-1289.
Day, M.L., C.R. Burke, V.K. Taufa, A.M. Day, and K.L. Macmillan. 2000. The
strategic use of estradiol to enhance fertility and submission rates of
progestin-based estrus synchronization programs In dairy herds. J. Anim.
Sci. 78: 523-529.
DeBols, C.H.W., and C.J. Blerschwal. 1970. Estrous cycle synchronization In
dairy cattle given a 14-day treatment of melengestrol acetate. Am. J. Vet.
Res. 31: 1545-1548.
Deen, A., S.K. Khar, M.M. Galhotra, and N.K. Khurana. 1995. Effect of GnRH on
reproductive activity of postpartum cows. Indian J. Anlm. Sci. 65: 970-974.
De Fries, C.A., D.A. Nevendorff, and R.D. Randel. 1998. Fat supplementation
Influences postpartum reproductive performance In Brahman cows. J. Anlm.
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DeJarnette, J.M., R.W. Wallace, R.B. House, R.R. Salverson, and C.E. Marshall.
2001. Attenuation of premature estrous behavior in postpartum beef cows
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De Ondlz, A., F. Perea, R. Cruz, G. Portillo, and E. Soto. 2002. Evaluacin
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Diaz, F.J., L.E. Anderson, Y.L Wu, A. Rabot, S.J. Tsai, and M.C. Wiltbank. 2002.
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211-228.
Dobson, H., A.Y. Ribadu, K.M. Noble, J.E. Tebble, and W.R. Ward. 2000.
Ultrasound and hormone profiles of ACTH-Induced persistent ovarian
follicles (cysts) in cattle. J. Reprod. Frertll. 120: 405-410.
Dobson, H., and R.F. Smith. 2000. What is stress, and how does it affect
reproduction? Anlm. Reprod. Sci. 60-61: 743-752.


TABLE OF CONTENTS
page
ACKNOWLEDGMENTS in
LIST OF TABLES vili
LIST OF FIGURES x
ABSTRACT xii
CHAPTER
1 INTRODUCTION 1
2 REVIEW OF LITERATURE 6
Introduction 6
Hypothalamic-Pituitary-Ovarian Axis and Primary Hormones of
Reproduction 7
Gonadotropin Releasing Hormone (GnRH) 7
Follicle Stimulating Hormone (FSH) and Luteinizing Hormone (LH) 8
Estrogens and Progesterone 11
Prostaglandins 17
Estrous Cycle in Cattle 18
Estrus 18
Metestrus 19
Diestrus 20
Proestrus 20
Regulation of Ovarian Follicle Growth, Atretic Demise and the Ovulatory
Response 21
Ovulation, Corpus Luteum Development, and Luteolysis 27
Exogenous Control of Ovarian Follicles and Corpus Luteum Dynamics 38
Estrogens 39
Progesterone and Progestagens 40
Combination of Estrogens and Progestagens 43
Gonadotropin Releasing Hormone (GnRH) 45
Prostaglandin F2a (PGF2a) 52
Estrus Synchronization Systems and Time Artificial Insemination 55
Summary 72


Number of mounts
received within a period
Figure 3-5. Effect of number of heifers in estrus within a 3 h period on number of
mounts received during a period (P < 0.0001).


APPENDIX C
ABSTRACT FOR EXPERIMENT 3
Non pregnant, cycling two-year old Angus (AN, n = 7), and Brahman x
Angus (BA, n = 19) heifers were used to evaluate the effectiveness of a single
injection of PGF2ci (Lutalyse Sterile Solution) administered 7 d after a GnRFI
(Fertagyl) treatment, to initiate luteolysls of an accessory corpus luteum (CL),
which had been exposed to either high or low progesterone concentrations
before PGF2a. Heifers were pre-synchronized with a modified two-injection
PGF2a protocol (25 mg i.m. on d -14 and 12.5 mg i.m. on d -3 and -2 of the
experiment). Estrus was monitored using HeatWatch for all phases of the
experiment. After expression of estrus (d 0), heifers randomly allotted to either a
low progesterone (LP, heifers treated with 15 mg PGF2i.m. at 12 h intervals on
d 4 and 5 of the estrous cycle, followed by 25 mg PGF2ct and 100 pg i.m.-of
GnRFI on d 6) or high progesterone (HP, no PGF2a on d 4, 5 and 6, and 100 pg
i.m. of GnRFI on d 6) treatments. Heifers received 25 mg of PGF2cton d 13. On
d 6, 8,13,15,17,19 of the experiment ovaries were examined via
ultrasonography to determine follicular and CL characteristics. Blood samples
were collected for determination of progesterone concentrations dally from d 4 of
the experiment until either heifers exhibit estrus, or until d 20 of the experiment if
heifers failed to exhibit estrus. There were no breed effects (P > 0.10) on either
mean progesterone concentrations prior to GnRFI, at GnRFI or from GnRFI until
224


50
agonist) administered to crossbred Bos taurus beef heifers 12 h after the onset of
estrus increased plasma concentrations of LH without affecting subsequent luteal
function. Mean LH concentration was 18.6 ng/ml at 120 min after FA; however,
the variation in LH concentrations was significant and ranged form 5.3 to 115.9
ng/ml. Chenault et al. (1990) performed a study to describe the changes in
serum LH and FSH concentrations in Holstein heifers following intramuscular
injection of various dosages of FA and other GnRH agonist at their labeled
dosages. The authors reported that mean LH concentrations increased within 15
min after treatment for the different dosages of FA, and LH concentrations
remained above baseline for approximately 180 to 360 min, dependent on the
dosage of GnRH. The interval to peak LH and the mean LH peak after GnRH for
all products and dosages ranged from 36 to 158 min and 2.5 to 25.5 ng/ml,
respectively. In a study by Cruz et al. (1997), the effect of GnRH on LH secretion
and ovulation in anestrous and cyclic postpartum beef cows were evaluated.
Mean peak LH values were similar (P> 0.20) for the cows that ovulated (107.7
11.0 ng/ml) compared with cows that failed to ovulate (103.6 14.5 ng/ml) after
GnRH administration. Vural et al. (1999) working with Holstein cows injected
with GnRH on d 14 post partum reported that the LH concentrations (0.47 to 24.6
ng/ml) peaked within two hours after GnRH, and returned to preinjection
concentrations within six hours. The variation between animals in LH
responsiveness to exogenous GnRH under standardized physiological conditions
is evident, and has been reported by others (Williams and Stanko, 1996;
Fajersson et al., 1999).


268
Moreira, F., R.L. de la Sota, T. Diaz, and W.W. Thatcher. 2000a. Effect of day of
the estrous cycle at the Initiation of a timed artificial insemination protocol
on reproductive responses in dairy heifers. J. Anim. Sci. 78:1568-1576.
Moreira, F., C. Orlandi, C.A. Risco, R. Mattos, F. Lopes, and W.W. Thatcher.
2001. Effects of presynchronization and bovine somatotropin on pregnancy
rates to a timed artificial insemination protocol in lactating dairy cows. J.
Dairy Sci. 84: 1646-1659.
Moreira, F., C.A. Risco, M.F. Pires, J.D. Ambrose, M. Drost, and W.W. Thatcher.
2000b. Use of bovine somatotropin in lactating dairy cows receiving timed
artificial insemination. J. Dairy Sci. 83: 1237-1247.
Moreira-Viana, J.H., A. de Moraes, W. Ferreira, and L.S. de Almeida. 2000.
Follicular dynamics in zebu cattle. Pesq. Agropec. Bras. 35: 2501-2509.
Moreno, I., G. Galina, F. Escobar, B. Ramirez, and R. Navarro. 1986. Evaluation
of the lytic response of prostaglandin in zebu cattle based on serum
progesterone. Theriogenology 25: 413-421.
Mori, J., T. Endo, T. Takahashi, and J. Masaki. 1974. Release of LH after
administration of an analog of synthetic LH-RH in cattle. Theriogenology 1:
177-180.
Mori, J., J. Masaki, T. Endo, and T. Kariya. 1979. Luteinizing hormone response
to an analog of luteinizing hormone releasing hormone in cows with ovarian
follicular cysts. Jap. J. Anim. Sci. 25: 67-72.
Mori, J., and T. Takahashi. 1978. Effect of an LH-RH analog administerqd at
insemination on conception rate in cows. Jap. J. Anima. Sci. 24:137-138.
Munro, R.K. 1989. The effects of duration and concentration of plasma
progesterone on the fertility of post-partum cows treated with pregnant
mare serum gonadotrophin and intravaginal progesterone. Aust. Vet. J. 66:
43-45.
Murphy, M.G., M.P. Boland, and J.F. Roche. 1990. Pattern of follicular growth
and resumption of ovarian activity in post-partum beef suckler cows. J.
Reprod. Frtil. 90: 523-533.
Murphy, M.G., W.J. Enright, M.A. Crowe, K. McConnell, L.J. Spicer, M.P. Boland,
and J.F. Roche. 1991. Effect of dietary intake on pattern of growth of
dominant follicles during the oestrous cycle in beef heifers. J. Reprod. Frtil.
92: 333-338.


122
concentrations for approximately 180 minutes for all breed groups, similar to
results in ovariectomized Brahman and Hereford cows challenged with GnRH
(Griffin and Randel, 1978) and Holstein heifers receiving different GnRH
analogues (Chenault et al., 1990). The relative differences observed in mean LH
concentrations In the Angus versus Brangus and Brahman x Angus heifers and
mean LH peak-height in Angus versus Brahman Angus heifers observed in the
present experiment are in agreement with Griffin and Randel (1978). Griffin and
Randel (1978) reported that mean serum LH response and average peak LH
concentrations were less In ovariectomized Brahman than Hereford cows
challenged with 500 pg natural GnRH In saline. Mean plasma LH concentrations
after GnRH In Angus, Brangus and Brahman Angus heifers In the present
study were considerably less than In ovariectomized Hereford and Brahman
cows, respectively challenged with 500 pg GnRH (Griffin and Randel, 1978).
These differences are probably due to the larger dose of natural GnRH (500 pg)
administered by Griffin and Randel (1978) compared with the GnRH (100 pg)
administered in the present study. Mean intervals from GnRH to LH peak for all
breed groups (74.17 min) In the current experiment were greater than the time to
LH peak in Holstein heifers (43.09 min) following an injection of 100 pg of
Gonadorelln (Chenault et al., 1990). Additionally, Rhodes et al. (1978) Induced a
LH surge in ovariectomized Brahman, Brahman Hereford, and Hereford cows
with an exogenous estrogen treatment and observed peak LH concentrations
that were smallest In Brahman, intermediate In Brahman x Hereford, and
greatest in Hereford cows. Furthermore, Brahman cows had the smallest area


235
0003
0054
0060
0082
0094
-*0113
Interval from GnRH, min
Figure H-5. Plasma LH concentrations (LS Means) in all Brangus heifers
receiving 100 pg GnRH on d 6 of the estrous cycle.


206
synthesis by the early bovine CL might make the CL refractory to the luteolytic
actions of PGF2a (Mamluk et al., 1998; Levy et al., 2000). Therefore,
endothelium of blood vessels in the CL could be less responsive to the action of
PGF2a during the early luteal phase of the estrous cycle. This is supported by the
observation that there is a positive relationship between vascularization and
mean diameter of the CL (Baumgartner et al., 1998), suggesting that incomplete
vascularization in early bovine CL may be responsible in part for the lack of
luteolytic capacity (Wiltbank et al., 1995). Furthermore, ovaries and corpora
ltea in cattle of Bos indicus breeding are generally smaller than those of Bos
taurus cattle (Moreno et al., 1986; Rentfrow et al., 1987; Soto et al., 1999).
Therefore, these findings suggest that a reduced response of the early CL to the
luteolytic actions of PGF2a in cattle of Bos indicus breeding compared to Bos
taurus cattle may be associated with luteal size and lack of vascularization. The
observation that breed could effect a PGF2a induced luteolysis of the early (d 6 or
7) developing CL could have implications relative to the overall effectiveness of
estrus synchronization protocols using PGF2a were a young corpus luteum (5 to
7 d old) needs to be regressed. Once such synchronization system that falls into
this category is the administration of GnRH followed 7 d later with a PGF2a
treatment.
As mentioned previously, the reduction in luteolysis in the Brahman x
Angus heifers was not statistically significant. However, if further experiments
determine that luteolysis is reduced by even 10% in Bos indicus compared to
Bos taurus heifers, this could still have a major negative impact on pregnancy


76
25 mg
PGF2u
I
Estrus
12.5 mg
PGF2a
-3 -2 0
Daily blood sample collection to
determine if PGF2n regressed
the CL as determined by
progesterone concentrations.
25 mg
PGF2
i
6/7 8 9 10 11 12 13 14
Estrus detection using HeatWatch
Figure 3-1. Experimental protocol evaluating the effectiveness of a single
injection of prostaglandin F2a (PGF2o) administered on d 6 or 7 of the
estrous cycle to initiate corpus luteum (CL) regression in Angus and
Brahman Angus heifers. Heifers were presynchronized with a single
PGF2a injection followed 11 d later by two half injections of PGF2ci
administered 24 h apart.


67
to 4, 5 to 9,10 to 16, and 17 to 21 of the estrous cycle, respectively. The low
ovulation rate during the early (d 1 to 4) and mid (d 10 to 16) estrous cycle was a
result of no dominant follicle available for ovulation (Vasconcelos et al., 1999;
Moreira et al., 2000). Overall, 87% of cows responded to the second GnRH
Injection, which varied according to the response to the first GnRH. For cows
ovulating to the first GnRH, 92% of these ovulated to the second GnRH, whereas
for cows did not ovulate to the first GnRH injection, 79% of these ovulated to the
second GnRH. Consequently, the decreased rate of ovulation to the first GnRH
Injection appears to generate a decreased synchronization rate following the
second Injection of GnRH.
If the Initial GnRH Injection falls to ovulate a follicle in cattle at the late
stages of the estrous cycle, these cattle will exhibit a spontaneous estrus before
the PGF2o( Injection (Thatcher et al., 1996). Approximately 5 to 15% of the cows
may be detected In estrus either on or before the day of PGF2a injection, thus
$
reducing the number of animals inseminated during the synchronized period
(Schmitt et al., 1996b; Downing et al., 1998; Moreira et al., 2000). According to
Geary and Whittier (1999), cows that exhibit estrus early are usually between d
14 and 17 of their estrous cycle at the initial GnRH treatment and do not respond
to GnRH. Several studies have confirmed that the response to the GnRH +
PGF2a protocol differs according to the day of the estrous cycle at the initial
GnRH treatment (Downing et al., 1998; Vasconselos et al., 1999; Moreira et al.,
2000). Downing et al. (1998) reported that 88% of the cows treated with GnRH
at different stages of the estrous cycle responded to PGF2a as noted by complete


185
the present study is longer than reports in Bos taurus cows (53 h) synchronized
with a GnRH + PGF2a followed by either 4 d (Twaglramungu etal., 1992) or 5 d
(Downing et al., 1998) of estrus detection. Therefore, it appears that estrus
occurs approximately 10 to 12 h earlier in Bos taurus than in Bos indicus cattle.
In addition, there was no difference in three-day estrous rates between cycling
and noncycling cows in the present study. This is contrary to other reports where
cycling Bos taurus (Twagiramungu et al., 1992; Geary et al., 1998b; Stevenson
et al., 2000) and Bos taurus x Bos indicus (Lemaster et al., 2001) cows treated
with the Select Synch protocol had greater estrous response than noncycling
cows.
The reason for the decreased estrous rates of the Bos indicus x Bos taurus
cows in the present study could be due to several factors. Behavioral estrus is
shorter in duration and less evident in Bos indicus breeds (Plasse et al., 1970;
Galina et al., 1982; Plnhelro et al., 1998) compared with Bos taurus cattle
(Stevenson et al., 1996). Estrus can also be difficult to detect In cattle of Bos
indicus breeding (Galina et al., 1994) and estrus detection efficiency can
Influence the percentage of cattle detected in estrus (Stevenson et al., 1996).
Furthermore, cattle of Bos indicus breeding can ovulate without any visible signs
of estrus (Plasse et al., 1970), although this number is relatively small. Since
time of ovulation was not determined in the current experiment, it is unclear how
many, if any cows had silent ovulations. Although cows were Intensively
observed during both morning and evening hours for behavioral estrus In the
present experiment, estrus detection may not have been intensive enough and


45
exogenous estradiol from Inducing ovulation when administered during the
follicular phase of the estrous cycle (Bolt et al., 1990).
Treating cattle of Bos indicus breeding (Cavalleri et al., 1997) with a
combination of progesterone and estradiol to regulates ovarian follicular
development similar to the same treatments in Bos taurus cattle (Bo et al., 1994;
Rajamahendran and Manikkam, 1994; Bo et al., 2000). Cavalieri et al. (1997)
treated crossbred Brahman cows with a sub-cutaneous Implant containing
estradiol 17(3 on d 8 of a 10 d CIDR treatment. Estradiol regulated the
emergence of the ovulatory follicle and prevented the ovulation of dominant
follicles present at the time of treatment. The mean interval between initiation of
estradiol treatment and day of emergence of the ovulatory follicle was 3.3 d.
Gonadotropin Releasing Hormone (GnRH)
Administration of GnRH to cattle at various stages of the estrous cycle has
been shown to alter follicular development as evidenced by changes In the
distribution of follicles among different size classes and induction of an L'H surge,
which Induces either ovulation or luteinlzatlon of the dominant follicle (Thatcher et
al., 1989; Macmillan and Thatcher, 1991). The result is emergence and
synchronization of a new follicular wave (Twagiramungu et al., 1995). The GnRH
Initiates an acute secretion of LH and FSH such that circulating concentrations
are elevated for 3 to 5 h, which stimulates ovulation or luteinlzatlon of large
follicles (Thatcher et al., 1993; Vlzcarra et al., 1997; DOcchio and Aspden,
1999). Consequently, removal of the dominant follicle permits an endogenous
surge of FSH, which initiates recruitment of a new follicular wave. The
effectiveness of a GnRH agonist to induce ovulation appears to be affected by


29
with inhibition of CL development and progesterone release (Ferrara et al.,
1998). In the bovine CL, there are increased concentrations of VEGF transcript
and proteins, and increased VEGF receptor-2 (VEGFR-2) expression during the
early luteal phase of the estrous cycle, which coincides with luteal vascularization
(Berisha et al., 2000b). These results suggest an important role of VEGF in
angiogenesis of the newly formed CL. Other angiogenic factors belonging to the
fibroblast growth factor (FGF) family (Redmer and Raynolds, 1996) and ovarian
cell proliferation factors like transforming growth factor p, platelet derived growth
factor, hepatocyte growth factor, and insulin like growth factor biding protein-3
(Smith et al., 1999) are essential for angiogenesis of the developing CL.
Throughout the first third of the ovarian cycle, developing endothelial cells
invade the developing CL and continue to grow (Augustin et al., 1995). The
mature CL is characterized by a dense system of blood vessels with
progressively decreasing blood vessel density.
The initial development of the CL takes approximately 3 d in cattle (d 2 to 5
of the estrous cycle), with angiogenesis playing an important role in development
and maintenance of the CL. Size of the CL increases more than 20 fold during
luteal development (Gottsch et al., 2002) and has one of the highest rates of
blood flow per unit of tissue of any organ in the body (Reynolds and Redmer,
1999). Baumgartner et al. (1998) concluded that luteal vascularization in the
bovine increases during early diestrus and remains constant until luteolysis.
Early studies (Ursely and Leymarie, 1979; Chegini et al., 1984; Rodgers et
al., 1986; Weber et al., 1987) identified two functionally distinct cell populations,


68
regression of the original and (or) induced CL. However, only 14% of the cows
between d 15 and 17 at GnRH injection responded to PGF2a with the majority of
these cows exhibiting estrus prior to PGF2a- Therefore, cows treated with GnRH
between d 15 and 17 of the estrous cycle were most likely undergoing normal
luteal regression prior to PGF2a- In addition, as the day of cycle at initiation of
GnRH treatment progressed, interval to estrus also increased. These data
suggest that the GnRH + PGF2o protocols are effective In synchronizing estrus,
but maximum response is achieved when estrus detection begins 1 to 2 d prior to
PGF2a. The current recommendation is that detection of estrus begin as early as
4 d after GnRH injection and continue through 5 d after PGF2a in cows treated
with GnRH + PGF2a (Kojima et al., 2000). However, this is impractical since it
increases th number of days required to detect estrus. Therefore, alternative
methods such as administering a progestogen between the GnRH and PGF20,
injections would eliminate the need for the extra estrus detection.
Thundathil et al. (1999) conducted an experiment to evaluate the efficacy of
short-term progestogen treatments for estrus synchronization in beef cows.
Cattle were administered MGA for 7 d with 1 or 5 mg of estradiol-170 and 100
mg of progesterone or 100 pg of GnRH at the initiation of MGA with 500 pg of
cloprostenol on 7 d later. Cattle were Al approximately 12 h after detected
estrus. There were no significant differences among treatments for estrous
response or conception rates suggesting short-term progestogen treatments are
effective estrus synchronization systems in lactating cows.


CHAPTER 7
CONCLUSIONS AND IMPLICATIONS
The objective of Experiment 1 was to determine the effectiveness of a
single injection of PGF2o administered during the early estrous cycle (d 6 or 7) to
initiate luteolysis In Angus and Brahman Angus heifers. Corpus luteum (CL)
regression was approximately 16% greater for Angus than for Angus x Brahman
heifers; although, this value was not significant probably due to limited
experimental numbers. Furthermore, heifers with incomplete luteolysis had
progesterone concentrations that remained above 1.0 ng/mL by 48 h after PGF2a
and continued to increase thereafter. In contrast, for heifers with complete
luteolysis, progesterone concentrations decreased below 1.0 ng/mL within 24 h
after PGF2ct and remained below 1 ng/mL. Results from the present experiment
suggest that breed (Angus vs. Brahman x Angus) does not have a statistical
effect on PGF2a Induced luteolysis. However, CL regression rates are still
numerically less in the heifers of Bos indicus breeding than Bos taurus heifers.
The exact reason(s) for a compromised response of the early estrous cycle
CL (< d 7) to a luteolytic dose of PGF2cc are unclear. Incomplete regression of
the early CL occurs in cattle of both Bos taurus (Burke et al., 1996; Moreira et al.,
2000a; Acosta et al., 2002) and Bos indicus breeding (Plnheiro et al., 1998;
Rekwot et al., 1999), but it appears to be of a greater magnitude in cattle of Bos
indicus breeding. Researchers have suggested that lack of endothelin-1
205


Percentage of cows that regressed their CL after PGF2a injection of the total
treated.
^Values with different superscript in the same row differ (P < 0.05).


60
CIDR based programs and short-term (7-d) MGA treatments (Martinez et al.,
2001; 2002) for synchronization of wave emergence and ovulation and therefore,
to regress and prevent the ovulation of a persistent dominant follicle.
As previously discussed in this chapter, administration of exogenous
estrogens initiates turnover of dominant follicles that might develop into
persistent follicles during both long and short-term progestogen treatments.
Recently, researchers have focused on administering estrogens at the initiation
of short-term progestogen treatments to synchronize follicle development, which
leads to a more synchronous estrus and ovulation after progestogen removal.
Tribulo et al. (1995) reported that administration of estradiol-17p either at CIDR
insertion or within 2 d after CIDR insertion resulted in a more synchronous estrus
and ovulation than two injections of PGF2c or CIDR alone. Estrogens
administered after CIDR treatment are very effective in inducing estrus and
ovulation. Administration of estradiol benzoate 24 to 30 h following a 7-d CIDR
treatment in cows (Fike et al., 1997) and heifers (Johnson et al., 1997; Lmaster
et al., 1999) increased the estrous response and the synchrony of estrus
compared to a CIDR treatment alone in cattle of Bos taurus breeding. Similarly,
Lammoglia et al. (1998) reported that over 90% of cows and heifers exhibited
estrus within 48 h after a 7-d CIDR with PGF2a at CIDR removal and estradiol
benzoate administered 24 to 30 h after CIDR removal in heifers of Bos taurus
breeding. Lemaster et al. (1999) also reported that administration of estradiol
benzoate after CIDR removal was effective in inducing a tightly synchronized
estrus and ovulation in Bos indicus x Bos taurus heifers. In addition, exogenous


187
cows received GnRH were not determined in the present study, it is possible to
speculate what stage of the estrous cycle and follicle development cows were
according to progesterone concentrations at PGF20 and the prior progesterone
samples. Cycling cows with low progesterone at PGF2a probably represented
cows with advanced follicle development and no CL to regress, resulting in
estrous responses that were at least 23% greater than all other groups. Both
cycling and noncycling cows with high progesterone concentrations represented
cows that were probably in the early stages of follicle development and also had
a CL that needed to be regressed by PGF2Q resulting in very low three-day
estrous responses due to lack of a dominant follicle ready to ovulate shortly after
CL regression.
It is clear that successful synchronization of estrus involves not only the
control of luteal regression but also the presence of a functional dominant follicle
at the end of the treatment (Mihm et al., 1994). Administration of GnRH has
greater effects on follicular development following divergence and dominance
than following wave emergence (Macmillan et al., 2003). Therefore, follicles of
different maturity will be present about 7 d later and can result in varied intervals
to the onset of estrus following PGF2a. Delayed follicle development after PGF2a
results in a decreased estrous response and (or) increased intervals from PGF2a
to the onset of estrus (Mihm et al., 1994). Therefore, stage of follicle
development and decreased estrous responses appear to be limiting factors in
the present experiment. It appears that GnRH did not turn over all follicles in the
current study, resulting in animals with different stages of follicle development at


96
1995). Reproductive efficiency can be affected negatively by repeated and acute
stressors such as transportation or severe climatic conditions, with a significant
and acute rise of blood cortisol (Stoebel and Moberg, 1982). However, mean
progesterone concentrations for heifers in the present study that regressed their
CL but did not express estrus were 0.7 ng/mL. Therefore, it does not appear that
frequent cattle handlings resulted In an acute stress response that Increased
blood progesterone to concentrations great enough to inhibit the expression of
estrus. Additionally, only one heifer that had CL regression did not develop a
new CL 10 d after PGF2a, indicating that almost all heifers ovulated after PGF2a
administration. These observations suggest that the progesterone to estradiol
ratios were appropriate to initiate ovulation and the decreased estrous response
was probably due to behavioral effects.
There was no effect of breed on interval from PGF2a to onset of estrus in
the present study and the interval Is similar to a report In Bos taurus beef heifers
given PGF2a on d 6 or 7 of the estrous cycle (Klracofe et al., 1985). However,
Landaeta-Hernandez et al. (2002) observed that the onset of estrus occurred
earlier in non-lactating Angus than Brahman cows synchronized with a two-
injection PGF2a system with 11 d between PGF2a injections. In the Landaeta-
Hernandez et al. (2002) study, both breeds were managed in a single group
similar to the present study. Additional comparisons between Bos taurus and
Bos indicus cattle have reported that breed appears to be a major factor
Influencing the Interval form PGF2a treatment to the onset of estrus (Vaca et al.,
1985; Galina and Arthur, 1990; Landaeta-Hernandez et al., 2002). Several


22
Ovarian follicular development in cattle is a dynamic progression of events
that has been described to occur in a wave-llke fashion (Pierson and Glnther,
1984; Savlo etal., 1988; Sunderland et al 1994) and is characterized by waves
of follicular growth and regression during the estrous cycle (Taylor and
Rajamahendran, 1991). In cattle, growth of ovarian antral follicles from
approximately 300 pm In diameter to 3 to 5 mm Is calculated to take more than
30 d (Lussler et al., 1987). Cattle usually have 2 (Ginther et al., 1989c; Taylor
and Rajamahendran, 1991) or 3 waves (Savio et al., 1988; Sirois and Fortune,
1988) of follicular development during an estrous cycle; however, estrous cycles
consisting of either 1 or 4 waves have also been reported (Savlo et al., 1988;
Sirois and Fortune, 1988). The main characteristics of follicular growth and
atresia can change among animals due to factors like energy balance and body
condition score (Rhodes et al., 1995; Burke et al., 1998), reproductive stage
(Roche and Boland, 1991), and breed (Flguelredo etal., 1997). The proportion
of beef heifers having 3 follicular waves was Increased with low dietary intake
compared to 2 follicular waves during normal nutrition (Murphy et al., 1991). In
contrast, parity and negative energy balance during lactation In dairy cows has a
negative effect on the number of waves per estrous cycle (Lucy et al., 1992).
Additionally, follicular waves also occur In prepubertal heifers (Evans et al.,
1994), early postpartum cows (Savio et al., 1990), and throughout pregnancy
(Ginther et al., 1989a; Glnther et al., 1996a). Apparently, there is no difference In
the number of follicular waves during an estrous cycle between cattle of Bos
indicus breeding (Flgueiredo et al., 1997; Gomez-Alves et al., 2002; Henao et al.,


170
Three-day estrous rate was defined as the percentage of cows that
exhibited behavioral estrus during the 72 h after the initial PGF2aof the total cows
treated. Conception rate was defined as the percentage of cows that became
pregnant to the Al after a detected estrus of the total cows expressing estrus.
Timed-AI pregnancy rate was the percentage of cows pregnant following timed-
Al of the total cows timed-AI. Synchronized pregnancy rate was defined as the
percentage of cows pregnant after estrus detection with Al and the timed-AI of
the total cows treated. Thirty-day pregnancy rate was the percentage of cows
pregnant during the first 30 d of the breeding season of the total treated.
For statistical analysis, the effects of PGF2a treatment, cycling status and
the interaction on three-day estrous response, conception, timed-AI pregnancy,
synchronized,pregnancy, and 30 d pregnancy rates were analyzed with logistic
regression modeling computing likelihood ratio test for type 3 contrasts for each
term in the model using GENMOD procedures of SAS (SAS Inst. Inc., Cary, NC).
Furthermore, the same response variables along with timed-AI and total CL
regression rates were analyzed separately for cows with progesterone
concentrations s 1.0 ng/mL and cows with progesterone concentrations <1.0
ng/mL using GENMOD procedures of SAS. Body condition score was included
as a covariate in all models and Al technician and Al sire effects were included
as covariates for the analysis of conception, timed-AI, and synchronized
pregnancy rates. The effect of interval from PGF2a to the onset of estrus on
conception rate was also analyzed as a covariate. The distribution of estrus after
PGF2o for treatment and cycling status were analyzed using the LIFETEST


37
and Okuda, 1999). Thus, luteal oxytocin, progesterone and prostaglandins are
components of an autocrine/paracrine positive feedback in early to mid cycle CL,
which may be responsible for the resistance of the early luteal phase CL to the
exogenous PGF2a-
Progesterone may also have an active role in the inhibition of luteal
regression by preventing apoptosis (Rae et al., 1998; Friedman et al., 2000;
Schams and Berisha 2002). Kobayashi and Miyamoto (2000) reported that
PGF2a is capable of enhancing lipoprotein utilization by luteal cells for
progesterone synthesis in early luteal phase CL, thus supporting the concept that
luteal PGF2a acts as a luteotropic agent in the early luteal phase CL.
The neurotransmitter noradrenaline has been reported to stimulate
progesterone, oxytocin, PGF2a and PGE2 secretion (Sharzynski et al., 2000;
Sharzynski et al., 2001; Kotwlca et al., 2002). Consequently, noradrenaline may
be indirectly involved in resistance of the CL against premature luteolysis by
stimulating these luteotropic factors in cattle. In any case, functional regression
of the CL occurs prior to any morphological change In luteal cells and it Is likely a
reversible step If sufficient luteotropic support is provided (Pate and Townson,
1994). There is growing evidence suggesting that a PGF2crinduced luteolysis
involves altered gene expression in the CL (Tsai et al., 2001). As Indicated by
Tsai and Wiltbank (1998), lack of PGF2a-lnduced luteolysis in the early luteal
phase CL may be due to changes in gene expression, particularly prostaglandin
synthase-2 (COX-2) that probably prevents Intraluteal PGF2a production and
possibly other luteolytic processes.


10
Lumpkin and McCann, 1984; Lumpkin etal., 1989), supporting the existence of a
separate FSH-releasing factor.
Factors other than GnRFI have been implicated for stimulating the secretion
of FSH and LH. In the pituitary, local regulators such as follistatins, activins,
inhibios and other neuroendocrine factors modulate GnRFI and the subsequent
differential secretion of FSH and LH (Padmanabhan and McNeilly, 2001;
Padmanabhan and Sharma, 2001). Additionally, estradiol and inhibin secreted
by the ovaries act via negative feedback to regulate FSH (Walczewska et al.,
1999; Padmanabhan and McNeilly, 2001), with estradiol (Walczewska et al.,
1999) being the central effector. Furthermore, the adipocyte hormone leptin
induces the secretion of FSH and LH (Walczewska et al., 1999; McCann et al.,
2001).
The stage of the estrous cycle differentially influences the secretory pattern
of LH. A GnRH pulse from the hypothalamus precedes each LH pulse (Schams
etal., 1974). In addition, changes in concentrations of circulating progesterone
and estradiol affect the episodic secretion of LH (Wolfe et al., 1992; Stumpf et al.,
1993). In dairy cattle (Rahe et al., 1980), LH pulses are classified as low
amplitude (0.3 -1.8 ng) and high frequency (20 30 pulses/24 h) on d 3 or early
luteal phase of the estrous cycle. In contrast, LH pulses are classified as high
amplitude (1.2 7.0 ng) and low frequency (6 8 pulses/24 h) on d 10 to 11 or
mld-luteal phase of the estrous cycle. A pulse of estradiol follows each LH pulse
during the early and mid-luteal stages of the estrous cycle (Walters et al., 1984).
Similar patterns of LH secretion have also been reported in sheep (Baird and


155
cows. These observations suggest turnover of the first follicular wave to the
second follicular wave in cows with low ascending progesterone during the
estrous cycle. On the contrary, cows with low but constant progesterone
concentrations In the Shaham-Albancy et al. (2000) study, the presence of a first-
wave persistent follicle was evident. These results suggest that low ascending
progesterone concentrations affected follicular development differently from low
and constant progesterone curves. Heifers in the LP group in the current
experiment also have low and ascending progesterone concentrations between
GnRH and PGF2a, and probably still enough progesterone to stimulate follicle
growth, which may explain the similar follicle size at PGF2a observed in heifers in
the LP and HP groups.
In the present study, 11 of 13 HP heifers had both the original and
accessory CL at PGF2o 7 d after GnRH resulting in progesterone concentrations
> 8 ng/mL; whereas, LP heifers had only a new induced CL from the GnRH
induced ovulation resulting in a low progesterone environment of approximately 3
ng/mL. The significant increase in CL regression of the HP compared to LP
heifers in the present study are in agreement with a report by Moreira et al.
(2000a) in which 40% of heifers treated with GnRH on d 18 of the estrous cycle
had Incomplete CL regression when PGF2o was administered 7 d later. In
contrast, 100% of the heifers treated with GnRH on d 5 of the estrous cycle had
CL regression when PGF2a was administered 7 d later. Therefore, results from
the present study and those reported by Howard and Britt (1990) and Moreira et
al. (2000a), indicate that progesterone exposure prior to PGF2ot administration


85
Table 3-2. Estrous response and behavioral estrus characteristics (LS means
SE) In Angus and Brahman Angus heifers treated with prostaglandin
F2a (PGF2a) on either d 6 or 7 of the estrous cycle
Breed
Variable
Angus
Brahman Angus
Estrous response, % a
15/26 = 57.7
21/31 = 67.7
Estrous response In
heifers that regressed
their CL, %b
15/25 = 60.0C
21/25 = 84.0d
Interval from PGF2o
injection to onset of
estrus, h
51.5 3.8 (n = 26)
52.4 3.1 (n = 31)
Duration of estrus, h
10.6+1.4 (n = 26)
12.9 1.1 (n = 31)
Total number of mounts
received during estrus
28.9 9.2c(n = 26)
51.1 7.1d(n = 31)
aThe number of heifers observed In estrus for 7 d after PGF2a divided by the total
treated.
bThe number of heifers observed in estrus for 7 d after PGF2a divided by the
number of heifers that regressed their CL.
c,dValues within a row lacking a common superscript differ (P < 0.05).


13
expression have been located in the bovine follicle (Rosenfeld et al., 1999;
Schams and Berisha, 2002; Van Den Broeck et al., 2002). Additionally, Berisha
et al. (2000a) found expression of progesterone receptor (PR) in the bovine
follicle, which was generally decreased in theca interna cells and granulosa cells
compared to expression of ERs in these cells. Both types of ERs were found In
both types of cells with greater concentrations in theca interna than in granulosa
cells (Berisha et al., 2000a). The significance of these findings Is that the
expression of these steroid receptors may affect folliculogenesls, expression of
other hormone receptors, steroid production in the ovary, gap junctions between
granulosa cells, and apoptosis in granulosa cells (Schams and Berisha, 2002).
Estrogen has been reported to increase expression of FSH and LH
receptors In rat granulosa cells (Richards et al., 1976) and oxytocin receptors in
bovine granulosa cells (Uenoyama and Okuda, 1997). The induction of LH
receptor mRNA in granulosa cells depends on the synergistic effects of estradiol
and FSH in the bovine (Berisha et al., 2000a). Production of androgen and
progesterone in bovine ovaries can be regulated by estradiol-17p and
catecholestrogens (Schams and Berisha, 2002). Some research suggests that a
local feedback regulation may be present in ovarian follicles (Fortune and
Hansel, 1979; Leung and Armstrong, 1980; Fortune, 1986; Roberts and Skinner,
1990). Androgens produced by theca cells undergo aromatizatlon Into estrogens
within granulosa cells, which may feedback to stimulate theca cell production of
androgens. In dominant bovine follicles, the major steroid produced by
granulosa cells is pregnenolone, which is exported to the theca cells for


173
respectively. Although, 18.9% more single PGF2a cows became pregnant to the
Al after an observed estrus than the split PGF2a treatment. Interval from PGF2a
to the onset of estrus did not affect (P > 0.10} conception rates.
There was no effect (P >0.10) of either treatment or cycling status on the
survival curves for heifers not observed in estrus (See appendix for survival
curves). Regardless of treatment or cycling status, for cows that exhibited
estrus, 89% exhibited estrus between 60 and 72 h after PGF2a. However, cows
with progesterone concentrations < 1 ng/mL at PGF2ct tended (P = 0.06; Figure 6-
2) to express estrus earlier (57.1 1.4 h) than cows with progesterone
concentrations 2 1 ng/mL (61.0 + 1.7 h). For cows with progesterone
concentrations < 1 ng/mL at PGF2cl that expressed estrus, 24, 33, and 43%
exhibited estrus at 48, 60, and 72 h after PGF2, respectively. For cows with
progesterone concentrations 2 1 ng/mL at PGF2c, that expressed estrus, 12, 28,
and 60% exhibited estrus at 48, 60, and 72 h after PGF2o respectively. ,
Based on blood progesterone concentrations collected on d -10 and 0 of
the experiment, the proportion of cycling cows were similar between the single
(83/141 = 58.9%) and split PGF2 (101/146 = 69.2%) treatments. Cycling status
at GnRH did not affect (P >0.10) three-day estrous response or conception rate
(Table 6-1). However, cycling cows had greater (P < 0.05) tlmed-AI (Odds ratio,
(OR) = 2.61; 95% confidence interval (Cl) = 1.35 5.04 (P < 0.05), synchronized
(OR = 2.12; 95% Cl = 1.27 -3.53; P < 0.05), and thirty-day pregnancy rates (OR
= 2.07; 95% Cl = 1.13- 3.79; P < 0.05) compared with noncycling cows (Table
6-1).


84
CL volume, mm3
Figure 3-3. The effect of corpus luteum (CL) volume on progesterone
concentrations (P < 0.05) on either d 6 or 7 of the estrous cycle in
Angus and Brahman x Angus heifers.


270
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216
regression. Similarly, decreased timed-AI pregnancy rates observed in cows
with low progesterone concentrations at PGF20 is probably associated with stage
of development of dominant follicles at timed-AI in conjunction with GnRH. Many
of these cows may have had follicles with longer durations of dominance and
aged oocytes, which may have resulted in decreased timed-AI pregnancy rates
observed in this experiment. In contrast, it is likely that cycling cows with high
progesterone at PGF2a administration were animals that responded to the first
GnRH treatment; therefore, GnRH induced the ovulation of the dominant follicle
or luteinization of follicles present at this time resulting in development of a new
cohort of follicles, and consequently more homogeneous preovulatory follicles at
PGF2a treatment. Hence, the second injection of GnRH at timed-AI induced a
fertile ovulation, resulting in improved timed-AI pregnancy rates in cows classified
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227
pregnancy rate (TAIPR), overall Al pregnancy rates (SYNCPR) and 30-day
pregnancy rates (30DPR) between the single or split dose of PGF2a. TAIPR and
SYNCPR were greater (P < 0.05) for Cycling (56/120 = 46.7%; 90/182 = 49.5%)
than for Noncycling (17/69 = 24.6%; 33/103 = 32.0%) cows. In animals with
progesterone concentrations £ 1 ng/mL at PGF201, there were no significant
differences in EST, TAIPR, SYNCPR, 30DPR, and CL regression rates between
the single or split dose of PGF2a- Modifying the delivery of PGF2a from a single
to two consecutive split injections 7 d after GnRH had no effect on subsequent
estrous response and pregnancy rate.
Keywords: Bos indicus, GnRH, Luteinizing hormone.


Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
RESPONSE TO PROSTAGLANDIN F2a (PGF2a) AND GONADOTROPIN
RELEASING HORMONE (GnRH) IN Bos taurus, AND Bos taurus x Bos indicus
CATTLE
By
German E. Portillo
December 2003
Chair: Joel V. Yellch
Major Department: Animal Sciences
A series of experiments were conducted to evaluate the response of Bos
taurus, Bos indicus, and Bos taurus x Bos indicus cattle to PGF2a and GnRH
treatments. Experiment 1 was replicated twice and cycling Angus (AN) and
Brahman x Angus (BA) heifers were used to evaluate the effectiveness of a
single Injection (25 mg i.m.) of PGF2a administered during the early estrous cycle
(d 6 or 7) to Initiate corpus luteum (CL) regression as measured by progesterone
concentrations. Breed had no effect on PGF2a Induced luteolysls on d 6 or 7 of
the estrous cycle. In the second experiment, Angus (AN), Brahman (B), Brangus
(BR), and Brahman x Angus (BA) heifers were used to evaluate secretory
patterns of LH and associated ovarian events In response to administration of
GnRH (100 pg i.m.) on d 6 of the estrous cycle followed by a single Injection of
PGF2a 7 d later. There was a breed-dependent response to a GnRH challenge


267
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in nonlactating Holstein cows. J. Dairy Sci. 74: 2342-2346.


218
which results in heavier and more uniform calves at weaning resulting in
significant economic returns for the producer.
Results from experiment 4 warrant further research extending the duration
of MGA feeding until the day of PGF2a administration in a 7 d GnRH + PGF2a
system to determine if that will improve the synchrony of estrus. Alternatively,
other sources of exogenous progestagens like CIDR could be used to determine
if they further enhance the effectiveness of the system in Bos indicus cattle.
Similarly, the administration of estradiol up front may improve follicle turn over
and therefore timed-AI pregnancy rates. Additionally, in order to reach maximal
pregnancy rates, it is probably necessary to further prolong the interval between
the PGF2a and the second GnRH administration and timed-AI to 96 h, allowing
more cows to be detected in estrus and Al.
Result from the experiments in this dissertation provide some initial data
regarding the reproductive performance of cattle of Bos indicus breeding to many
estrus synchronization products available to beef producers. The slight but
obvious differences in the responses to these products justify additional research
with a greater number of animals to evaluate ovarian and endocrine responses,
as well as their possible association with fertility in cattle of Bos taurus x Bos
indicus breeding.


78
each replication, body condition scores (Richards et al., 1986) and BW were
recorded on each heifer.
For statistical analysis, the effects of breed, replication, and replication x
breed on PGF2 induced CL regression, estrous response and estrous response
in heifers that regressed their CL were analyzed with logistic regression modeling
computing likelihood ratio test for type 3 contrasts for each term in the model
using GENMOD procedure of SAS (SAS Inst. INC., Cary, NC). When there were
no replication and replication x breed effects (P >0.10) data were pooled and
presented as such. Largest follicle size at PGF2a was included as a covariate in
the estrous response model. Progesterone concentrations and CL volume at
PGF2a were included as covariates in the CL regression model. Corpus luteum
regression vyas also analyzed with logistic regression modeling using the Logistic
procedure of SAS. To determine the degree of association between the
independent variables and the outcome variable, odds ratio and Wald 95%
confidence intervals for adjusted odds ratios were calculated. Progesterone
concentrations following PGF2ct were analyzed as repeated measures analysis
using the GLM procedure of SAS. The model included fixed effects of breed,
replication, CL regression, time, and all appropriate interactions with random
effect of heifer nested within breed as the error term. Effects of breed and CL
regression were separated using contrasts for Angus vs. Brahman x Angus, and
CL regression vs. no-CL regression. Effects of breed, replication and breed by
replication interaction on the interval from PGF2a to the onset of estrus, duration
of estrus, total number of mounts received during estrus, size of the largest


263
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estrus characteristics in crossbred Brahman heifers treated with an
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progestagens on the formation and regression of the corpus luteum of the
cyclic cow. Ann. Biol. Anim. Bioch. Biophys. 15: 243-253.
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loop feedback action of estrogen on ovarian androgen production. Life Sci.
27:415-420.
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2000. Administration of prostaglandin F2a during the early bovine luteal
phase does not alter the expression of ET-1 and its type A receptor: A
possible cause for corpus luteum refractoriness. Biol. Reprod. 63: 377-382.
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autocrine progesterone positive feedback loop mediates oxytocin
upregulation in bovine granulosa cells during luteinization. Endocrinology
138: 5059-5062.


Progesterone, ng/mL
APPENDIX I
REGRESSION OF TOTAL CORPUS LUTEUM VOLUME WITH
PROGESTREONE CONCENTRATIONS AT PGF2a INJECTION IN ANGUS,
BRANGUS AND BRAHMAN X ANGUS HEIFERS TREATED WITH GNRH
FOLLOWED BY PGF2a 7 DAYS LATER (EXPERIMENT 2)
CL volume, mm3
Figure 1-1. Regression of total CL volume on progesterone concentrations at
PGF2cl Injection in Angus, Brangus and Brahman x Angus heifers
receiving 100 pg GnRH on d 6 of the estrous cycle (P > 0.10).
237


31
respectively (Perea et al., 1998). The mean diameter of CL was even smaller
(9.3 mm) for Bos indicus cows ranging from 7 to 11.7 mm (Ruiz-Corts and
Olivera-Angel, 1999). Similarly, CL weight on d 8 and 13 of the estrous cycle for
Brahman heifers (2.5 and 2.7 g) was decreased compared to Brahman x
Hereford (4.6 and 3.8) or Hereford heifers (4.0 and 3.6 g), respectively (Irvin et
al., 1978). Likewise, Segerson et al. (1984) reported that CL weight on d 17 of
the estrous cycle was less for Brahman (2.4 g) than Angus cows (4.1 g). In
agreement, progesterone concentrations in CL from Bos indicus heifers and
cows were decreased compared to Bos taurus heifers and cows (Irvin et al.,
1978; Segerson et al., 1984). Related to these findings, progesterone
concentrations are less in cattle of Bos indicus breeding compared to Bos taurus
cattle (Randel, 1977; Segerson et al., 1984). According to Irvin et al. (1978),
progesterone concentrations of CL from Brahman (216.9 pg/CL) and Brahman x
Hereford (217.7 pg/CL) was decreased compared to Hereford heifers (334.6
pg/CL). Similarly, Segerson et al. (1984) observed that CL from Brahman had
decreased progesterone concentrations (190.8 pg/CL) compared to Angus cows
(266.3 pg/CL). Furthermore, Brahman and Brahman x Hereford heifers had
decreased circulating progesterone concentrations from 2 to11 d after estrus
compared to Hereford heifers (Randel et al., 1977). Similar results have been
observed when comparing Brahman and Angus cows from 7 to 17 d after estrus
(Segerson et al., 1984). These observations indicate that both Bos indicus and
Bos indicus x Bos taurus cattle have decreased progesterone concentrations
during diestrus compared with Bos taurus cattle.


152
The progesterone curves following GnRH ad