Response to prostaglandin F₂[subscript alpha] (PGF₂[subscript alpha]) and gonadotropin-releasing hormone (GnRH) in Bos t...

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Response to prostaglandin F₂subscript alpha (PGF₂subscript alpha) and gonadotropin-releasing hormone (GnRH) in Bos taurus, Bos indicus, and Bos taurus x Bos indicus cattle
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xiii, 286 leaves : ill. ; 29 cm.
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Portillo, German E
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Thesis:
Thesis (Ph.D.)--University of Florida, 2003.
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Includes bibliographical references.
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by German E. Portillo.
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Printout.
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Vita.

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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.




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